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Mutations in chromatin regulator ASXL1 are frequently identified in myeloid malignancies, in particular ∼40% of patients with chronic myelomonocytic leukemia (CMML). ASXL1 mutations are associated with poor prognosis in CMML and significantly co-occur with NRAS mutations. Here, we show that concurrent ASXL1 and NRAS mutations defined a population of CMML patients who had shorter leukemia-free survival than those with ASXL1 mutation only. Corroborating this human data, Asxl1−/− accelerated CMML progression and promoted CMML transformation to acute myeloid leukemia (AML) in NrasG12D/+ mice. NrasG12D/+;Asxl1−/− (NA) leukemia cells displayed hyperactivation of MEK/ERK signaling, increased global levels of H3K27ac, upregulation of Flt3. Moreover, we find that NA-AML cells overexpressed all the major inhibitory immune checkpoint ligands: programmed death-ligand 1 (PD-L1)/PD-L2, CD155, and CD80/CD86. Among them, overexpression of PD-L1 and CD86 correlated with upregulation of AP-1 transcription factors (TFs) in NA-AML cells. An AP-1 inhibitor or short hairpin RNAs against AP-1 TF Jun decreased PD-L1 and CD86 expression in NA-AML cells. Once NA-AML cells were transplanted into syngeneic recipients, NA-derived T cells were not detectable. Host-derived wild-type T cells overexpressed programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) receptors, leading to a predominant exhausted T-cell phenotype. Combined inhibition of MEK and BET resulted in downregulation of Flt3 and AP-1 expression, partial restoration of the immune microenvironment, enhancement of CD8 T-cell cytotoxicity, and prolonged survival in NA-AML mice. Our study suggests that combined targeted therapy and immunotherapy may be beneficial for treating secondary AML with concurrent ASXL1 and NRAS mutations.

Bruton tyrosine kinase (BTK) inhibitor is an established treatment for relapsed/refractory (R/R) mantle cell lymphoma (MCL). Zanubrutinib, a highly selective BTK inhibitor, is approved for patients with MCL who have received ≥1 prior therapy. We report the long-term safety and efficacy results from the multicenter, open-label, phase 2 registration trial of zanubrutinib. Patients (n = 86) received oral zanubrutinib 160 mg twice daily. The primary endpoint was the overall response rate (ORR), assessed per Lugano 2014. After a median follow-up of 35.3 months, the ORR was 83.7%, with 77.9% achieving complete response (CR); the median duration of response was not reached. Median progression-free survival (PFS) was 33.0 months (95% confidence interval [CI], 19.4-NE). The 36-month PFS and overall survival (OS) rates were 47.6% (95% CI, 36.2-58.1) and 74.8% (95% CI, 63.7-83.0), respectively. The safety profile was largely unchanged with extended follow-up. Most common (≥20%) all-grade adverse events (AEs) were neutrophil count decreased (46.5%), upper respiratory tract infection (38.4%), rash (36.0%), white blood cell count decreased (33.7%), and platelet count decreased (32.6%); most were grade 1/2 events. Most common (≥10%) grade ≥3 AEs were neutrophil count decreased (18.6%) and pneumonia (12.8%). Rates of infection, neutropenia, and bleeding were highest in the first 6 months of therapy and decreased thereafter. No cases of atrial fibrillation/flutter, grade ≥3 cardiac AEs, second primary malignancies, or tumor lysis syndrome were reported. After extended follow-up, zanubrutinib demonstrated durable responses and a favorable safety profile in R/R MCL. The trial is registered at ClinicalTrials.gov as NCT03206970.

Background Although classic Hodgkin lymphoma (cHL) is highly curable with current treatment paradigms, there is still 20-30% of patients who will experience therapeutic failure (refractory or relapse). Tislelizumab, a monoclonal antibody targeting programmed cell death protein 1, is showing promise in the clinic with obvious response in relapsed/refractory cHL (RRcHL) patients on monotherapy. Further improvements in antitumor efficacy as salvage therapy may require exploration of a tislelizumab-based combination regimen. We conducted a phase II multi-center, open-label, non-randomized study (ChiCTR2000033441) to investigate the efficacy and safety of tislelizumab with gemcitabine plus oxaliplatin (GemOx) in patients with RRcHL. Methods Patients who had at least one previous therapy for cHL and were relapsed or refractory were eligible. Enrolled subjects received up to eight courses of gemcitabine (1g/m 2 on day 1) and oxaliplatin (100 mg/m 2 on day 1) combined with tislelizumab (200 mg on day 2) at 21-day intervals. Then, the cohort was divided into a tislelizumab maintenance group (every 2 months for 2 years) and an autologous hematopoietic stem cell transplantation group based on the choice of the investigators. The primary endpoint of this study was the best complete response rate (CRR), and secondary endpoints included overall response rate (ORR), progression-free survival (PFS) at 12 months, and safety profile. Results As of July 2021, a total of 22 patients (median age of 33 years old) were included. The predominant histologic subtype was nodular sclerosing cHL. Early-stage cHL was found in six patients and late-stage cHL in 16 (two of them with bulky disease). Correspondingly, 59.1%, 18.2%, and 22.7% of patients had 1, 2, or ≥ 3 prior therapies, respectively, and more than half of patients (59.1%) reported refractory to the latest treatment. The efficacy evaluable population comprised 20 patients. The disease control rate was 100% (95% confidence interval [95% CI]: 83-100%) and the ORR was 95% (95% CI: 75-100%). At the time of follow-up cutoff, the best CRR reached 90% (95% CI: 68-99%). Out of these, only one patient demonstrated stable disease and was switched to the other regimen. A total of 126 courses of immunochemotherapy were administered, with a median number of six courses per patient. The follow-up time averaged 191 days (range, 31-345 days), and the estimated PFS rate was 100% at 12 months. After 6-8 courses of tislelizumab plus GemOx, ten subjects received tislelizumab maintenance, and all remain in remission so far, with the longest follow-up of 345 days. Overall, the treatment was well tolerated with the majority of adverse events (AEs) being grade 1-2 in severity. Serious AEs were grade 3 anemia (n=1) and grade 3 thrombocytopenia (n=1). Conclusion Tislelizumab plus GemOx demonstrated promising antitumor activity with manageable toxicities as a salvage treatment for RRcHL. A longer follow-up is needed to demonstrate the durability of the remission after tislelizumab maintenance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Objective: Diffuse large B-cell lymphoma (DLBCL) is a subtype of non-Hodgkin's lymphoma with a higher incidence rate and is highly heterogeneous.The current first-line treatment effect is limited. After entering the relapse/refractory treatment, the second-line treatment lacks an efficient solution. Explore DLBCL Potential therapeutic targets are imperative. The cGAS/STING pathway can promote or inhibit tumorigenesis. The specific tumor type,genetic background,pathway activation level and microenvironment may determine the tumor's response to STING agonists or inhibitors. The disorder of cGAS/STING pathway function can lead to many diseases such as tumors and autoimmune diseases, but it has not been reported in DLBCL. This study found for the first time that STING is heterogeneously expressed in DLBCL and is related to tumor cell proliferation. The STING inhibitor H-151 can kill DLBCL tumor cells. At the same time, multi-omics methods are used to further screen the specific DLBCL population sensitive to H-151, provide a theoretical basis for the improvement of the prognostic model of DLBCL and the choice of targeted therapy drugs. Methods Immunofluorescence staining to detect the expression of cGAS/STING on tissue microarrays in patients with DLBCL; Confocal microscopy imaging technology for localization and quantitative analysis of intracellular cGAS/STING expression; Whole-exome sequencing analysis of mutant genes related to drug sensitivity and pathway;The combination of quantitative proteomics and transcriptome sequencing to compare the biological characteristics of cell lines in different response groups, analyze gene expression differences and related activation pathways;Western Blotting to detect the expression of related proteins;CCK8 and flow cytometry were used to detect cell proliferation and apoptosis;NOD-SCID mice and Hu-PBMC mice were used to construct DLBCL tumor-bearing mouse models to explore the effect of STING inhibitors in vivo. Results The cGAS/STING pathway-related proteins are expressed heterogeneously in real-world clinical DLBCL samples and DLBCL cell lines, and the expression of cGAS/STING pathway-related proteins is positively correlated with the proliferation level of DLBCL cell lines(Figure A). Based on this, we speculate the increased expression of cGAS/STING pathway-related proteins may potentially promote the proliferation of tumor cells. The STING inhibitor H-151 can inhibit the proliferation and induced apoptosis of some DLBCL tumor cells in a concentration-dependent manner.The IC50 of H-151 has a certain positive correlation with the expression of cGAS/STING pathway-related proteins, which suggests that the up-regulation of cGAS/STING pathway-related proteins may be potentially related to the resistance of DLBCL to H-151(Figure B).Whole transcriptome sequencing combined with single-cell transcriptome sequencing (scRNA-seq) analyzes potential subgroups related to H-151 sensitivity and related activation pathways(Figure C-E).. We also use whole exome sequencing to screen potential targets for assessing H-151 sensitivity We found that celllines with ITIH6, NPIPA5, NPY2R and TUBGCP3 mutations are more sensitive to the treatment of H-151, while cell lines with SH2B2 and LILRB5 mutations were less responsive to H-151,while the related gene mutations in the C-MYC pathway may be potentially related to the resistance of DLBCL cells to H-151, so as to screen potentially sensitive target populations for the effective use of H-151(Figure F). Conclusion This study found that the cGAS/STING pathway is heterogeneously expressed in DLBCL and is related to tumor cell proliferation, and the STING inhibitor H-151 has anti-DLBCL effects. The anti-tumor mechanism of STING inhibitor H-151 was further explored by using multi-omics methods, and the sensitivity evaluation system of H-151 was established.Thus providing new ideas for precise and efficient treatment of DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: To evaluate the efficacy and safety of venetoclax, rituximab plus dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, and doxorubicin (VR-DA-EPOCH) in Richter's syndrome (RS), we conducted a single-arm, retrospective, observational, real-world study in our center. Methods: Patients who had history of CLL/SLL were diagnosed as RS by biopsy during treatment or watch and wait strategy. VR-DA-EPOCH was given as follow, venetoclax was administered with accelerated ramp-up from 20 mg per day to 400 mg per day, d1-10 during cycle 1, 400 mg daily on day1-10 of cycle 2-6, rituximab 375 mg/m 2 on day 0 of cycle 1 and 500 mg/m 2 on day 0 of cycle 2-6, plus etoposide (50 mg/m 2,day1-4), vincristine (0.4 mg/m 2 day 1-4) or vindesine 3 mg/m 2 day 1 , doxorubicin (10 mg/m 2 day 1-4), prednisone (60 mg/m 2, day 1-5), cyclophosphamide (750 mg/m 2 day 5), 21 days per cycle，dose adjustment on the basis of nadir ANC and platelet count are as previously reported by Wison WH. Response assessment was conducted after 2 or 3 cycles by enhanced CT or PET/CT and after 6 cycles (EOT) by PET/CT according to 2014 Lugano criteria. Minimal residual disease (MRD) of CLL cell in peripheral blood (PB) and bone marrow (BM) was detected after 2 or 3 and 6 cycles by flow cytometry. uMRD was defined as less than 1 CLL cell per 10 4 leukocytes. Results: 7 RS patients were enrolled in Pukou CLL Center from 10/2019 to 7/2021 and the last follow up was 07/25/2021. The median age was 52 years old. Unmutated IGHV, complex karyotype (CK) and TP53 deletion and/or mutation was detected in 100% (6/6), 20% (1/5) and 40% (2/5) patients, respectively. 5 patients received at least one prior line (range: 1-5) treatment for CLL/SLL, with 4 patients received ibrutinib as last prior therapy and one patient previously exposed to venetoclax. 2 patients were diagnosed as RS during watch and wait. The median duration from diagnoses or previous treatment for CLL/SLL to RS was 12 months (range: 3-14). All patients underwent lymph node (n=6) or bone biopsy(n=1) at the site of SUVmax or secondary SUV uptake (unaccessible for SUVmax) by PET/CT and was confirmed as transformed to non-GCB type of diffuse large B-cell lymphoma (DLBCL). Furthermore, 4 of 4 (100%) available patients were confirmed as clonal-related RS by detecting IGHV gene usage. 3 patients acquired CK, and 2 patients appeared BTK C481S mutation. 7 patients completed at least 2 cycles and were available for efficacy and safety assessment. Overall response rate (ORR) was 100% after 2 or 3 cycles, and CR rate (CRR) was 60% after 6 cycles in 5 patients who completed 6 cycles. 2 patients experience disease progression (PD) after cycle 2 and cycle 4 respectively, with one ceased after the addition of brentuximab and the other received CD20 UCAR-T and progressed 3 months later, transit to allo-hemapoietic stem cell transplant (allo-HCT). One patient received auto-hemapoietic stem cell transplant (auto-HCT) and CD19-CAR-T as consolidation and remains in CR, one patient experience PD after 6 cycles and attained CR with chidamide , programmed death-1 (PD-1) inhibitor Sintilimab and XPO1 inhibitor Selinexor, bridging to allo-HCT and remains in CR. Another patient who achieved CR after 6 cycles progressed 6 months later and ceased within one month. 2 patients transformed without previous treatment wait for final evaluation. 60% (3/5) patients attained MRD negativity both in the PB and BM after 6 cycles. The median tolerable dose was 60% (50%-70%) of standard EPOCH and the median dose of venetoclax was 400mg daily for 7 days each cycle. No tumor lysis syndrome (TLS) happened during venetoclax ramp-up. The most common grade 3 or 4 adverse effects (AEs) was agranulocytic fever (6/7, 85.7%), thrombocytopenia (3/7, 42.9%) and sepsis (2/7, 28.6%). 3 (42.9%) patients discontinued venetoclax due to severe AEs and the median duration of discontinuation was 3 days. 85.7% (6/7) had venetoclax dose reduction or interruption due to grade 3 or 4 neutropenia. Conclusion: VR-DA-EPOCH showed high response rate, impressive CR with uMRD and manageable toxicity in patients with RS, even with previous exposure to new target drugs. VR-DA-EPOCH could be recommended as an effective treatment choice for RS, CAR-T or allo-HCT should be considered as subsequent strategy for long term disease control. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Aims: Diffuse large B-cell lymphoma (DLBCL), the most frequent malignant lymphoma subtype, is a group of highly invasive diseases with great heterogeneity in genomic alterations, clinical characteristics, morphological manifestations, treatment response and prognosis. Although most DLBCL patients can be cured by immunochemotherapy, nearly 40% of DLBCL patients still develop drug resistance and relapse. For relapsed/ refractory (R/R) DLBCL patients, there is still no optimal treatment. The heterogeneity and clonal evolution of tumor cells are the core driving forces for the occurrence and development of DLBCL, and the root causes for their refractory, recurrence and drug resistance. In this study, we screened out a novel small molecule compound effectively killing DLBCL cells, and analyzed its potential mechanism of anti-tumor. Meanwhile, by using single cell sequencing technology, we try to further investigate the heterogeneity and clona evolution and drug resistance mechanism of DLBCL under different drug pressure, explore core driver factors of drug resistance, evaluate and develop new treatment strategies. Methods: In this study, GEXSCOPE microfluidic platform was used for single-cell transcriptome sequencing. Seruat software was used for cell type recognition and clustering analysis. In order to further investigate the molecular mechanism of LAQ824 inducing the apoptosis of DLBCL cells and explore the target of LAQ824, antibody chip was performed to detect the phosphorylation of related signaling pathway. Gene expression was detected by real-time qPCR and Western blot. ChIP, RNA interfering (RNAi) and dual-luciferase activity assay were performed to validate the potential drug resistance targets for LAQ824. Moreover, WES of 21 DLBCL cell lines were performed to map mutations and analyze the correlation between related mutations and LAQ824 resistance. In this study, we established DLBCL animal models using NOD SCID mice transplanted with DLBCL cell lines, by which we could evaluate the tumor inhibition efficiency of LAQ824 alone and/or combination with other small molecular inhibitors. Results: Using GDSC database, we screened out Dacinostat (LAQ824), a novel HDAC inhibitor, was highly sensitive that could effectively induce the apoptosis of most DLBCL cells at low concentrations. Functional assay showed that LAQ824 could inhibit cell proliferation and promote apoptosis of tumor cells. LAQ824 treatment could significantly upregulate the acetylation level of histone H3 within a certain concentration range, and the DNA damage repair function of DLBCL cells was supressed by inhibiting Chk2 expression, thus significantly inducing cell apoptosis and effectively killing DLBCL cells. Meanwhile, through single-cell sequencing analysis, it was found that c-Fos could be activated under certain drug pressure of LAQ824. As a potential drug-resistant core driver gene, the expression level of c-Fos is highly correlated with IC50 of LAQ824 and the prognosis of patients with DLBCL, which can be used as a sensitivity indicator of LAQ824. Treatment with c-Fos inhibitor combined with LAQ824 can significantly improve the tumor inhibition rate, validated both in vitro and in vivo, which is expected to alleviate the recurrence and drug resistance of DLBCL patients. Conclusions: In general, we explores potential therapeutic drugs for DLBCL parients, adjusts and explores new clinical treatment strategies on this basis, and provides theoretical basis and data support for the realization of individualized precise treatment and the solution of DLBCL recurrence and drug resistance. Disclosures No relevant conflicts of interest to declare.

Introduction Positron emission tomography (PET) positivity before autologous stem cell transplantation (ASCT) predicts poor prognosis in relapsed/refractory (rel/ref) classical Hodgkin lymphoma (cHL). However, there is limited data regarding the value of pre-ASCT residual metabolic tumor volume (rMTV) assessed by PET to predict post-ASCT outcomes. We aimed to evaluate the role of pre-ASCT Deauville score (DS) combined with rMTV in patients with rel/ref cHL who underwent salvage ASCT, and also to establish a risk model integrating pre-ASCT DS, rMTV, and other clinical risk factors. Methods This is a retrospective cohort study using clinical data of patients with rel/ref cHL identified from the lymphoma database and the ASCT database of the Princess Margaret Cancer Center, Toronto, Canada. Following criteria were required: (1) patients (≥18 yrs) with histologically confirmed rel/ref cHL between January 2014 to March 2019; (2) treatment with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) or ABVD-equivalent regimens; (3) chemosensitive disease after salvage therapy; (4) restaging PET before ASCT clearance. Pre-ASCT PET scans were assessed using the Deauville 5-point scale, and rMTV was computed in the scans with DS 4 or 5 using the 41% maximum standardized uptake value threshold method. Receiver operating characteristics analysis was used to determine an optimal cutoff of rMTV 41% for event prediction. The primary endpoint was event-free survival (EFS), defined as time form ASCT to first documented disease progression, any new lymphoma therapy, death from any cause, or last follow-up. Results A total of 106 patients fulfilled the eligibility criteria and were included. Median age was 33.5 yrs (range, 18-70), and 72 (68%) were male. The cohort characteristics at time of progression were 55 (52%) patients with advanced stage, 28 (26%) with B symptoms, 39 (37%) with extranodal disease, and 39 (37%) with primary refractory disease, defined as achieving less than a complete response, disease progression during first-line therapy, or time to relapse (TTR) 3 months or less. All patients received gemcitabine, dexamethasone, cisplatin (GDP) regimen as salvage therapy. Based on the response to GDP therapy, 24 patients required second (N=20) or third (N=4) salvage regimens before ASCT (brentuximab vedotin [BV] alone or combination with bendamustine [N=16], pembrolizumab [N=9], and mini-BEAM [N=3]). 32 patients (30%) received planned pre- or post-ASCT involved field radiation for DS 4-5 lesions. 4 patients received post-ASCT BV consolidation. With a median follow-up of 26.2 months (interquartile range [IQR], 14.4-49.2), 2-year EFS and overall survival rates were 72.1% and 93.1%, respectively. Pre-ASCT DS was determined as 1-3 (N=63, 59%) and 4-5 (N=43, 41%). None of the patients with DS 5 had new lesions. Patients with pre-ASCT DS 1-3 had significantly better EFS than those with DS 4-5 (2-year; 80.8% vs 59.2%; P=0.002; Fig 1A). Median rMTV 41% of the 43 patients with DS 4-5 was 5.1 cm 3 (IQR, 2.0-24.7). The optimal cutoff of rMTV 41% was 4.4 cm 3. Patients with rMTV low (< 4.4cm 3, N=21) had better EFS than those with rMTV high (N=22; 2-year, 75.0% vs 45.4%; P=0.009), but had similar 2-year EFS when compared with patients with DS 1-3 (80.8%, P=0.280; Fig 1B) In the multivariable analysis for EFS, a combined assessment of pre-ASCT DS/rMTV 41% and rel/ref status at time of progression were independently associated with EFS (for DS 4-5/rMTV low, hazard ratio [HR] 1.58; for DS 4-5/rMTV high, HR 4.72; P 3 months] and either pre-ASCT DS 1-3 or DS 4-5/rMTV low, N=54), intermediate-risk (refractory disease and either pre-ASCT DS 1-3 or DS 4-5/rMTV low, N=30), and high-risk (pre-ASCT DS 4-5/rMTV high irrespective of rel/ref status, N=22) groups. The risk model was significantly associated with EFS (intermediate vs low, HR 3.26, 95%CI 1.20-8.83, P=0.020; high vs low, HR 7.61, 95% CI 2.89-20.01, P Conclusion We propose a risk stratification model integrating a combination of DS and rMTV 41% on pre-ASCT PET and rel/ref status at time of progression, which allow discrimination of post-ASCT outcomes in patients with rel/ref cHL. The model may serve as a tool to facilitate risk-stratified treatment decisions. Figure 1 Figure 1. Disclosures Metser: POINT Biopharm Inc: Consultancy. Prica: Astra-Zeneca: Honoraria; Kite Gilead: Honoraria. Crump: Roche: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Epizyme: Research Funding; Kyte/Gilead: Membership on an entity's Board of Directors or advisory committees. Kuruvilla: Seattle Genetics: Honoraria; Amgen: Honoraria; Antengene: Honoraria; AbbVie: Honoraria; Janssen: Honoraria, Research Funding; BMS: Honoraria; Medison Ventures: Honoraria; TG Therapeutics: Honoraria; AstraZeneca: Honoraria, Research Funding; Gilead: Honoraria; Incyte: Honoraria; Karyopharm: Honoraria, Other: Data and Safety Monitoring Board; Roche: Honoraria, Research Funding; Novartis: Honoraria; Merck: Honoraria; Pfizer: Honoraria. Kridel: Gilead Sciences: Research Funding.

Objective: Ibrutinib is currently the most widely used BTK inhibitor that approved for the treatment of both initially diagnosed and relapsed and refractory chronic lymphocytic leukemia (CLL) patients. Although ibrutinib shows high response rates in clinical practice, it has certain limitations. There are still a certain number of patients who have to discontinue treatment due to drug-resistance or side effects. The ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies. Methods: Here, we examined the heterogeneity of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-sensitive (IBS) and -resistant (IBR) CLL by analyzing bulk and single-cell level gene expression profiles, clinical features, biological properties, and phenotypes. Seven distinct ibrutinib-resistant subpopulations were identified and two candidate genes LGALS1 (galectin 1, Gal-1) and LAG3 (lymphocyte-activating gene 3, CD223) were screened that contribute toward ibrutinib-resistance and poor survival in CLL patients. These results were validated in primary cells from CLL patients and also in ibrutinib-resistant CLL cell line (MEC1-IR) which was generated by culturing the parental cell line in vitro with progressively increasing concentrations of ibrutinib. Marker-gene expression was detected using qRT-PCR, western blotting, and ELISA, while functional analyses including CCK8, flow cytometry and trypan blue staining were conducted with or without OTX008, a selective Gal-1 inhibitor. Results: ScRNA-seq revealed that cells from IBR and IBS samples were distributed in different clusters and suggested that IBR cells display a unique transcriptional pattern (Fig A). IBR-B cells have higher stemness scores and are enriched in some energy metabolism Pathways (Fig B). According to the proportion of B cells from IBR samples, we classified each B-cell cluster into three main subgroups, i.e., IBR, IBS, and shared cluster (Fig C). IBR-B cells displayed more interactions with monocytes, NK, T, and dendritic cells than IBS B cells, suggesting that IBR B cells may actively build connections with other immune cells to reshape the protective niche (Fig D). A close correlation between LGALS1 and LAG3 expression was observed and both of them were found to be highly expressed in IBR CLL patients (Fig E), their expression level gradually increased along the trajectory of B cells from IBS to IBR (Fig F). Diagnosis and prognosis stratification of CLL with receiver operating characteristic (ROC) curves revealed that patients with higher expression of both LGALS1 and LAG3 showed the poorest overall survival, indicating that LGALS1 and LAG3 are associated with ibrutinib-resistance and poor prognosis in CLL (Fig G). Concordantly, acquired resistance following chronic exposure to ibrutinib leads to upregulation of LGALS1 and LAG3 (Fig H). LGALS1 inhibitor OTX008 effectively inhibits the growth of ibrutinib-resistant CLL cells, particularly for IBR patients (Fig I). Conclusion: In conclusion, our findings demonstrate that ibrutinib-resistant CLL cells exhibit a unique transcriptional pattern. The combination of LGALS1 and LAG3 expression could serve as an indicator of the sensitivity of ibrutinib and prognosis of CLL patients. LGALS1 inhibitor OTX008 helps to overcome ibrutinib-resistance of CLL cells. Our findings may expand the current knowledge regarding ibrutinib-resistant CLL patients, identify improved biomarkers for patient selection, and offer a promising combinatorial therapeutic strategy for IBR CLL patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Chronic lymphocytic leukemia(CLL), characterized by monoclonal CD5+ B cells appearing in blood, marrow and lymphoid tissues, is the most common chronic leukemia in western countries. Until now CLL remains incurable with a heterogeneous clinical course, some patients developing progression rapidly while the others presenting a relatively indolent course. It has been reported that higher percentage of prolymphocytes was associated with increasing refractoriness to treatment and worse prognosis. In this study we were trying to explore the effect of prolymphocytes on the prognosis of CLL. Methods Two hundred and fifty-one treatment naïve CLL patients in Jiangsu Province Hospital were retrospectively enrolled in our study from April 2014 to November 2019. The median time from diagnosis to peripheral-blood smear examination was 0.13 month and median follow-up time was 30.87 months. A total of 200 lymphocytes and prolymphocytes were counted. The percentage of prolymphocytes was collected. Basic clinical characteristics and other prognostic markers including age, sex, Rai and Binet stage, B2-microglobulin(B2MG), lactate dehydrogenase(LDH) level, CD38 and ZAP70 expression level, hemoglobulin(HB), platelets count(PLT), absolute lymphocyte count(ALC), thymidine kinase-1(TK-1), albumin(ALB), IGHV mutation status and TP53 status were also put in the analysis. The time to first treatment(TTFT)was defined as the time from sampling to first treatment and the overall survival(OS) time was the time from sampling to death. An X-tile analysis provided the optimal prolymphocyte cutoff point. Wilcoxon rank sum test was used to compare the distribution of prolymphocytes percentage in different subgroups. The Kaplan-Meier method was used to construct the survival curves and the log-rank test was used to compare the difference. Multivariate analysis was conducted based on the Cox-regression model. All tests were two-sided and P Results Among 252 patients, most of them were male(67.7%) and 35.9% were older than 65 years old. We found significantly different distribution of prolymphocytes percentage in patients with different B2MG, Rai stage, LDH, CD38 expression, TK1, IGHV mutation status and TP53 status(Table 1). The optimal cutoff of prolymphocytes percentage provided by X-tile analysis was 1%. Then all the patients were divided into two groups based on the prolymphocytes percentage and obvious survival difference between the two groups was shown in both TTFT(P Discussion In this study we found the optimal cutoff of prolymphocytes percentage was 1% which is different with previous study(Oscier et al. 2016). That may be due to that we choose TTFT as the endpoint. Besides, about prolymphocytes percentage was not statistically significant in multivariate analysis, we guess that may be attributed to that the sample was too little or there were only Chinese patients in our cohort. In conclusion, prolymphocytes percentage has certain prognostic significance in CLL patients and we think multivariate analysis would be different after we expanded our cohort. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background Waldenstrom's Macroglobulinemia (WM) is a B-cell disorder characterized primarily by bone marrow infiltration with lymphoplasmacytic cells, along with immunoglobulin M (IgM) monoclonal gammopathy. Bruton's tyrosine kinase (BTK) plays a key role in signaling pathways for the survival of WM clone, particular in patients harboring MYD88 L265P mutations. However, due to target selectivity issue, Clinical uses of early BTK inhibitors are still compromised with off-target activities to many other kinases besides BTK. Orelabrutinib is a novel, highly potent small molecule inhibitor of BTK with superior selectivity for B-cell malignancies and autoimmune diseases. Preliminary efficacy and safety data of ICP-CL-00105 in relapsed/refractory WM patients are presented here. Methods ICP-CL-00105 is a single arm, multiple centers, open label, phase 2 study in clinical and histopathological confirmed patients with R/R WM requiring treatment per IWWM-7. MYD88 and CXCR4 mutations were assessed in bone marrow samples at baseline. Orelabrutinib at a daily dose of 150mg was administered orally until disease progression or unacceptable toxicity. Blood samples for IgM were assessed at baseline and every cycle for 6 cycles and every 3 cycles thereafter by central lab. Responses were assessed in accordance with IWWM-6 and NCCN guidelines. The primary endpoint was major response rate (MRR) as assessed by IRC. Key secondary endpoints were MRR as assessed by investigator, overall response rate (ORR), duration of major response (DOMR), progression-free survival (PFS), OS, changes in IgM levels from baseline, improvement on hemoglobin levels and safety. Treatment-emergent adverse events (TEAEs) and treatment-related adverse events (TRAEs) were assessed according to NCI CTCAE v4.03. Results As of June 1, 2021, for the 47 patients the median follow-up duration was 10.5 months. The median age was 63 years (range, 56-68 years), 40 patients (85.1%) were male. 87.2% of patients were at intermediate or high risk according to the Prognostic Scores (IPSS). The proportion of patients with MYD88 L265PCXCR4 wildtype was 83% at baseline. With a median duration of treatment of 9.2 months, MRR was 74.5% as assessed by investigator. ORR was 87.2% with 97.9% patients achieved disease control. The estimated 12-month DOMR were 89.5%. The estimated 12-month PFS and OS were 88.0% and 92.3%, respectively. The median PFS and median OS have not been reached. The MRR was higher in patients with MYD88 L265PCXCR4 wildtype (79.5%). The median IgM level was 30.3g/L at baseline. The decline in the serum IgM levels from baseline were observed with a median reduction by 79.0% (IQR: -89.4, -57.2). The median hemoglobin level at baseline was 102g/L. Durable improvement in hemoglobin levels was found in 83% of patients with a median maximal improvement of 40g/L (IQR: 24.0, 62.0). Safety data were summarized by the cutoff date of June 1, 2021. The most commonly reported AEs were thrombocytopenia (27.7%)，neutropenia (14.9%), leukopenia (10.6%), upper respiratory infection (14.9%)，weight increased (14.9%), influenza-like disease (12.8%) and rash (10.6%). Most reported AEs (89.5%) were grade 1-2. 16 patients (34.0%) reported grade ≥ 3 TEAE while 9 patients (19.1%) reported grade ≥ 3 TRAE. There was no reported grade ≥3 atrial fibrillation and/or atrial flutter, or grade ≥3 diarrhea. Only One TRAE (2.1%) resulted in drug discontinuation. Conclusion Orelabrutinib has demonstrated substantial efficacy in treating r/r WM patients under short-term follow-up. It has shown favorable safety and tolerability profile with limited off-target adverse effects. It has the potential to be a promising treatment option for r/r WM patients. Disclosures Hu: Astellas Pharma, Inc.: Research Funding. Tian: Innocare pharma: Current Employment. Zhu: Innocare pharma: Current Employment. Zhang: Innocare pharma: Current Employment. Zhao: Innocare pharma: Current Employment. Zhang: Innocare pharma: Current Employment.

Introduction Peripheral T-cell lymphoma (PTCL) is a group of hematological malignancies originating from mature T/NK cells. Most of the subtypes are associated with aggressive clinical features and dismal outcomes. Routine first-line chemotherapy has low efficiency and a high recurrence rate, so there is an urgent need for new drugs. Monotherapy or combination therapy of epigenetic inhibitors have been shown to be effective in several hematologic malignancies. Here, we report the interim efficacy of an epigenetic priming regimen with azacytidine and chidamide prior to salvage chemotherapy for relapsed or refractory (R/R) PTCL. Methods The prospective phase II study (ChiCTR2000037232) enrolled pts were pathologically confirmed T/NK cell non-Hodgkin's lymphoma with at least one imaging measurable lesion. Pts needed to have received at least one systemic chemotherapy regimen including hematopoietic stem cell transplantation, radiotherapy, or a single epigenetic drug. Pts received AZA hypodermically at a dose of 100 mg on days 1 to 7, chidamide of 20 mg orally twice per week; the combined chemotherapy regimens included but were not limited to GemOx (gemcitabine, oxaliplatin); CPT (cyclophosphamide, prednisone, thalidomide) , etc. Treatment was performed for up to eight cycles of each 21 days. Pts who achieved partial response (PR) and better remission began maintenance therapy every two months with double epigenetic inhibitors for two years. The trial aimed to explore the efficacy and safety of AZA and chidamide combined chemotherapy in the treatment of R/R PTCL. The primary objective was investigator-assessed best overall response rate (ORR). Secondary objectives included duration of response (DOR), complete response rate (CRR), progression-free survival (PFS), overall survival (OS), and safety profiles. Results A total of 24 pts have been enrolled, baseline characteristics are shown in Table 1. Pathological subtypes included angioimmunoblastic T-cell lymphoma（AITL, n=15）, PTCL-not otherwise specified (PTCL-NOS, n=4), extranodal NK/T-cell lymphoma (ENKTCL, n=3) and mycosis fungoides(MF, n=2). The median age was 57 (range,38-72) years with male predominance. Ann Arbor Classification ≥ stage III in 20 pts. Twelve pts had B symptoms at the time of diagnosis, five pts had performance status ≥ 3 before treatment. The median number of previous systemic treatment regimens was two. Autologous hematopoietic stem cell transplantation in two pts, radiation in three pts and prior treatments containing chidamide in eight pts. At the time of data cutoff, the median number of treatments for all pts was four cycles (range,1-13). Among 16 pts evaluable for response, the best ORR was 68.8% (11/16) with five pts achieved CR, six achieved PR. In subgroup analysis, eleven AITL pts achieved an objective response. The best ORR was 72.7% (8/11) with four pts attained CR, four attained PR (Table 2). The median follow-up was 12.4 (range, 0.1-18.7) months. For all pts, the median PFS was 6.7 months (95% CI,5.8-7.6), the median OS was 8.4 months (95% CI,0.0-18.3) (Figure 1). And the median DOR was 10.2 months (95% CI,4.9-15.5). For AITL pts, the median PFS was 14.6 months (95% CI,3.6-25.6), and the median OS was not reached (Figure 2). The OS between AITL and other subtypes pts was statistically significant (1-year OS: 76.2% vs 13.9%; p=0.003, Figure 3). Almost all pts had experienced at least one adverse event (AE). The most common grade 3 or 4 AEs were anemia, leukopenia, neutropenia, thrombocytopenia, and infections. Conclusions Epigenetic priming regimen with azacitidine plus chidamide with salvage chemotherapy is effective and tolerable. The best ORR of all enrolled pts with AITL were 68.8% and 72.7%, respectively. Compare to other subtypes, patients with AITL subtype benefit more obviously from our regimen with durable remission. And further studies will focus on patients with AITL and follicular helper T-cell originated. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction To evaluate the safety and efficacy of zanubrutinib, lenalidomide plus R-CHOP (ZR 2-CHOP) as the first-line treatment for DLBCL patients, we conducted this single-arm retrospective observational study. Methods Treatment-naïve patients with DLBCL(including but not limited to double-hit, double expression) aged 18 to 75 years were enrolled.ZR 2-CHOP was given as follows, Oral zanubrutinib was given continuously (160 mg twice daily) from Day 0, lenalidomide 25 mg daily Day 1-7. Patients were administered intravenously rituximab (375 mg/m 2 Day 0), cyclophosphamide (750 mg/m 2 Day 1), doxorubicin (50 mg/m 2 Day 1), vincristine (1.4 mg/m 2 Day 1), and oral prednisone (100 mg Day 1-5). All patients were recommended to receive 6 cycles of ZR 2-CHOP (R-CHOP or R 2-CHOP were allowed in cycle 1-2 due to poor physical condition at treatment) and patients older than 70 years old were administered ZR 2-miniCHOP (Figure 1). CT or PET-CT scans were applied to mid-term efficacy and PET-CT scan was conducted after 6 cycles. ctDNA was dynamically detected before treatment, after 3 and 6 cycles to evaluate tumor mutational burden. The primary endpoint was complete response ratio (CRR) at mid-term and after 6 cycles. The secondary endpoint was overall response rate (ORR), ctDNA dynamics and adverse events (AE). AEs were graded based on CTCAE (version 5.0). Results 12 treatment-naïve DLBCL patients diagnosed in Pukou CLL Center were enrolled in this cohort between July 2020 and June 2021. The median age was 56 years old and all patients had ECOG-PS ≤2. 1 patient (1/12) was diagnosed as double-hit DLBCL and 9 patients (9/12) as double-expression. 10 patients were non-GCB and 2 were GCB. 7 patients were classified as high-intermediate and high-risk group according to NCCN-IPI (Table 1). At data cutoff (1st July, 2021), the median follow-up was 8 months (3-12 months) with all patients have completed at least 3 cycles and mid-term assessment has been conducted. The ORR was 100.0%, with 10 patients achieved CR and 2 patients achieved PR (both two patients received R-CHOP regimen in cycle 1/cycle 1-2 due to poor physical condition at initial treatment, Figure 1). 10 patients have received 6 cycles, all of them achieved CR (Figure 2). ctDNA was dynamically detected in six patients. The median number of detectable ctDNA mutation among six patients was 8 (0-12) with two patients classified as MCD subtype and one patients as EZB subtype. All six patients showed undetectable ctDNA after 3 cycles. During end of treatment follow-up, one patient (triple-hit, EZB) who scheduled to receive auto-SCT underwent disease progression 4 months later and reemergence of ctDNA showed previous homologous clones. The most common hematological toxicity events were lymphocytes count decreased, neutrophil count decreased, thrombocytopenia and anemia, with 3-4 level occurrence rate was 66.7%, 25.0%, 25.0% and 16.7%. The most common non-hematological toxicity events were nausea, fatigue and anorexia. One patients discontinued oral zanubrutinib and lenalidomide in last two cycles due to drug rash. Conclusion ZR 2-CHOP could attain high CR rate and ctDNA clearance in TN DLBCL, including patients with DEL and/or high-risk. The overall tolerability was manageable. ZR 2-CHOP could be one of the promising choice for TN DLBCL. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Zanubrutinib was used in the initial treatment of high-risk DLBCL.

Background: Chidamide, as a novel subtype-selective histone deacetylase inhibitors (HDACi), can directly inhibit tumor cell cycle progression and induce tumor cell apoptosis, inhibit phenotypic transformation of tumor cells and pro-drug resistance/pro-metastasis activity of the microenvironment, induce differentiation of tumor stem cells, and reverse epithelial-mesenchymal transformation of tumor cells, thereby restoring the sensitivity of drug-resistant tumor cells to drugs and enhancing the effect of chemotherapy agents through loosening chromatin and exposing DNA. The efficacy and safety of chidamide-BEAC (carmustine, etoposide, cytarabine, and cyclophosphamide, Chi-BEAC) conditioning regimen combined with autologous stem cell transplantation (ASCT) were evaluated in a current phase II clinical trial for the treatment of high-risk and relapsed/refractory aggressive lymphoma. Methods: A total of 70 patients with high-risk diffuse large B-cell lymphoma (DLBCL), peripheral T-cell lymphoma (PTCL), or mantle cell lymphoma (MCL) who had achieved complete remission (CR) or partial remission (PR) after first-line therapy or second-/third-line therapy were recruited from January 2018 to June 2021. Three of the patients were not evaluated after ASCT and two patients withdrew from the study; 65 patients were then evaluated for the effectiveness of Chi-BEAC treatment. Results: DLBCL and MCL are referred as B-cell non-Hodgkin lymphoma (B-NHL) and PTCL is referred as T and NK-cell non-Hodgkin lymphoma (T&NK-NHL) in the study. The median neutrophil engraftment time was 10 d (7-13 d) and median platelet engraftment time was 11 d (8-13 d). The CR rate at 3-6 months after transplantation was 75.4% in the 65 evaluable patients. The median progression free survival (PFS) and overall survival (OS) had not reached at the end of the follow-up period (median 18.1 month; range 1.8-42.0 months). The estimated PFS and OS at 24 months was 78.5% and 84.2%, respectively. Stratified analyses showed that in patients with B-NHL who previously received first-line treatment, the CR rate at 3-6 months after transplantation was 77.8%, and 2-year PFS was 74.8%. The survival rate might be better than previously reported for SWOG9704 (Stiff et al., 2013; PMID 24171516)-a 2-year PFS of 69%-and for DLCL04 (Chiappella et al., 2017; PMID 28668386)-a 2-year failure-free survival of 71%. In our patients with B-NHL who previously received second-/third-line treatments, the CR rate at 3-6 months after transplantation was 71.4%, and 2-year PFS was 77.1%. The survival rate might also be higher than previously reported for the CORAL trial (Gisselbrecht et al., 2010; PMID: 20660832), which showed a 2-year PFS of approximately 65%. In our patients with T&NK-NHL the CR rate at 3-6 months after transplantation was 73.3%, and 2-year PFS was 93.3%, The survival rate might be higher than previously reported for the NLG-T-01 trial (Francesco d'Amore et al., 2012; PMID: 22851556), which showed a 2-year PFS of approximately 55%. PFS stratification analysis showed that previous treatment outcomes affected the PFS: patients who had achieved CR before transplantation demonstrated better PFS than patients who had achieved PR (P = 0.199), while there was no significant difference between patients with different pathological subtypes (B or T&NK-NHL) or different risk groups. Most non-hematological adverse events (AEs) were of grade 1/2. Grade 4 AE only occurred in one patient, i.e., γ-glutamyl transpeptidase (GGT) elevation. Grade 3 AEs that occurred in ≥ 5% of the patients included febrile agranulocytosis (37.7%), hypokalemia (24.7%), hyponatremia (23.4%), GGT elevation (9.1%), and diarrhea (5.2%), which were well tolerated. Conclusions: This study showed that inclusion of the HDACi chidamide in conditioning regimen for ASCT greatly increased the PFS and OS, especially in patients with T&NK-NHL, and revealed an acceptable safety profile for refractory and relapsed lymphoma patients. Therefore, chidamide-containing conditioning regimen may be a great choice for patients with refractory and relapsed lymphoma, and awaits further confirmation by additional large-scale multi-center investigations. Disclosures No relevant conflicts of interest to declare.

Introduction To evaluate the safety and efficacy of BTK inhibitor ibrutinib or zanubrutinib plus bendamustine and rituximab (iBR or zBR) as the initial treatment for chronic lymphocytic leukemia (CLL)/ small lymphocytic leukemia (SLL) patients, we conducted this single-arm retrospective observational study. Methods We enrolled patients over 18 years old with CLL/SLL who required treatment. iBR or zBR were administrated as following, ibrutinib 420 mg daily or zanubrutinib 160 mg twice daily from day 0, rituximab 375 mg/m 2 on day 0 of cycle 1 and 500 mg/m 2 on day 0 of cycle 2-6, bendamustine (70 mg/m 2, days 1-2), 28 days per cycle, ibrutinib or zanubrutinib was maintained at least 2 years and planned to discontinue in patients with undetectable minimal residual disease (uMRD) in both peripheral blood (PB) and bone marrow (BM). Response assessment was conducted after 3 cycles and 2 months after 6 cycles (EOT) according to 2018 iwCLL criteria in patients with CLL and 2014 Lugano criteria in patients with SLL. Minimal residual disease (MRD) in PB was detected after each cycle and MRD in BM was detected after 3 and 6 cycles by flow cytometry. uMRD was defined as less than 1 CLL cell per 10 4 leukocytes by flow cytometry. R esults At data cut-off (15 th June 2021), 10 treatment-naïve patients in the First Affiliated Hospital of Nanjing Medical University were enrolled, with 9 CLL and 1 SLL. All patients had completed planned six cycles of iBR(n=8) or zBR(n=2). The median age was 56 years old. Unmutated IGHV was detected in 30.0% (3/10) patients, one patient (1/8, 12.5%) had both del(17p) and TP53 mutation. Among 9 patients with CLL, 3 (33.3%) was classified as low-risk group according to CLL-IPI, 4(44.4%) in intermediate-risk, 1(11.1%) in high-risk group and 1(11.1%) in very-high risk group (Table 1). After 3 cycles, the overall response rate (ORR) was 100%, and complete remission (CR) rate was 20.0%, one patient achieved CRi (CR with incomplete bone marrow recovery). 50% (5/10) and 37.5% (3/8) patients achieved uMRD in PB and BM respectively. The ORR was 100% (10/10) and CR rate was 60.0%, 3 patients achieved CRi (30.0%) at EOT. 60.0% (6/10) and 50.0% (5/10) patients achieved uMRD in PB and BM respectively (Table 2, Figure 1). The most common hematological toxicity events were neutropenia and thrombocytopenia, and the non-hematological toxicity events were malaise, pruritus, nausea, vomiting and hematuria. The occurrence rate of grade 3 to 4 neutropenia and agranulocytic fever were 40.0% (4/10) and 10.0% (1/10) respectively. The most common ibrutinib-related AE were purpura, rash, diarrhea and hematuria. The occurrence rate of AE induced discontinued ibrutinib or zanubrutinib were 30.0% (3/10). Conclusion Here, we first reported the efficacy and tolerance of BTK inhibitor plus BR as initial treatment in a small cohort of CLL/SLL patients. BTK inhibitor plus BR could achieved high response rate and high proportion of undetectable MRD，with manageable toxicity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Objective: Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder that mainly affects the elderly and is characterized by the expansion of small mature B-cells. New targeted drugs, such as the BTK inhibitor ibrutinib, have greatly improved patient survival but have also posed the challenge of drug resistance. The three-dimensional (3D) spatial structure of chromatin is highly dynamic and varies greatly between cell types and developmental stages, with the maintenance of chromatin homeostasis being of major significance in disease prevention. Accumulating evidence has suggested that changes in 3D genomic structures play an important role in cell development and differentiation, disease progression, as well as drug resistance. Nevertheless, the characteristics and functional significance of chromatin conformation in the resistance of CLL to ibrutinib remain unclear. In this study, we aimed to investigate the mechanism underlying ibrutinib resistance through multi-omics profiling, including the study of chromatin conformation. Thus, we would be able to demonstrate the importance of chromatin spatial organization in CLL and highlight the oncogenic factors contributing to CLL development and mediating ibrutinib resistance. Methods: An ibrutinib-resistant cell line was established by exposing cells to increasing doses of ibrutinib. High-throughput chromosome conformation capture (Hi-C), assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), bulk RNA sequencing (RNA-seq), and Tandem Mass Tag (TMT) were performed to explore differences between ibrutinib-resistant and parental cells. Peripheral blood mononuclear cells (PBMCs) from 53 CLL patients were collected for RNA-seq. Mitochondrial respiration and glycolysis were assessed via Seahorse analysis. The growth-inhibitory effects of tested drugs were evaluated via a CCK8 assay, and the combination index (CI), indicating synergy, was calculated using CompuSyn software. Apoptosis was detected via annexin V staining. Results: Between ibrutinib-resistant and parental cells changes in some chromosomes, including chr11 were observed (Figure 1A). p21-activated kinase 1 (PAK1), which is located on chr11 and frequently overexpressed or excessively activated in almost all cancer types and involved in almost every stage of cancer progression, was first explored for its role in CLL progression and drug resistance. The oncogene PAK1 was observed locate in a region where B-to-A compartment switching occurred (Figure 1B). Consistent with the results of ATAC-seq, RNA-seq, and TMT, Hi-C analysis revealed a transcriptional upregulation of PAK1 in ibrutinib-resistant CLL cells (Figure 1C). Functional analysis demonstrated that PAK1 overexpression significantly promoted cell proliferation, while knockdown markedly suppressed cell viability (Figure 1D). Cell viability assays indicated that the depletion of PAK1 increased ibrutinib sensitivity (Figure 1E). In addition, PAK1 positively regulates glycolysis and oxidative phosphorylation in CLL cells (Figure 1F and G). To verify the results of sequencing and further explore the role of PAK1 in CLL, B-cells from healthy volunteers and PBMCs from CLL patients were collected. The level of PAK1 mRNA expression was significantly higher in CLL primary cells than in B-cells from healthy volunteers (Figure 1H). Kaplan-Meier survival analysis of qRT-PCR data confirmed that patients with high PAK1 expression had a significantly lower OS (Figure 1I). IPA-3, the small molecular inhibitor of PAK1 suppressed the proliferation of ibrutinib-resistant and parental CLL cells in a dose-dependent manner. The combination of IPA-3 and ibrutinib exerted potent cell growth inhibition (Figure 1J), and the combination index (CI) calculated using the CompuSyn software confirmed the synergistic effect (CI Conclusions: In the current study, we have provided a genome-wide view of alterations in 3D chromatin organization between ibrutinib-resistant and parental CLL cells and confirmed the oncogenic role of PAK1 in CLL. Most importantly, our research provides promising therapeutic targets for overcoming ibrutinib resistance. In particular, the treatment of CLL patients with a combination of IPA-3 and ibrutinib may improve clinical outcomes. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Purpose: To evaluate the safety and efficacy of ibrutinib plus fludarabine, cyclophosphamide, and rituximab (iFCR) as the treatment for chronic lymphocytic leukemia (CLL)/ small lymphocytic leukemia (SLL) patients, we conducted time-limited, minimal residual disease (MRD)-driven real world study in The First Affiliated Hospital of Nanjing Medical University Methods: We enrolled patients aged 18 to 65 years old with CLL/SLL who required treatment. Oral Ibrutinib was given continuously (420 mg/day) from day 0, and patients were administered intravenously rituximab (375mg/m2 in day 0 of cycle 1; 500mg/m2 in day 0 of cycle 2-3), fludarabine (25mg/m2, days 1-3) and cyclophosphamide (250mg/m2, days 1-3), every 28-day cycles. All the patients received 3 cycles of iFCR in the first stage. Interim analysis was done according to the 2018 iwCLL criteria and MRD was detected by flow cytometry. Patients who achieved complete response (CR) or partial response (PR) with low MRD level (L-MRD, 10-4-10-2) or undetectable MRD (uMRD) level (10-2) in PB or bone marrow (BM), or with diameter of residual lymph node larger than 3cm were administered another 3 cycles of iFCR (6 in all) and followed by ibrutinib maintenance. The primary endpoint was the proportion of patients who achieved uMRD in the PB after 3 cycles of iFCR. The secondary endpoint was overall response rate (ORR), complete response rate (CR) and the number of adverse events (AE). Results: Between May 2019 and June 2020, 29 CLL/SLL patients were enrolled in this cohort with 27 CLL and 2 SLL. 26 of patients were the previously untreated and 3 of patients were relapse or treated before. The median age of patients was 53 years old. Unmutated IGHV was detected in 14 (50.0%) of 29 patients, 4 patients had del(17p) and/or TP53 mutation (13.8%), 7 patients had del(11q) (24.1%), 7 had NOTCH1 mutation (24.1%) and 7 had MYD88 mutation (24.1%). 8 patients (30.8%) were classified as low-risk group according to CLL-IPI, with 9 in intermediate-risk group (34.6%), 5 in high-risk group (19.2%) and 4 in very-high risk group (15.4%). At data cutoff (1st July, 2020), the median follow-up and treatment time was seven months (1-13 months) with 23 patients had completed at least two cycles of iFCR. At interim analysis, the ORR was 100% with 8 patients achieved CR (34.8%), 3 achieved CRi (13.0%) and 12 achieved PR (52.2%). 57.1% (12/21) achieved uMRD in both PB and BM, among which 6 of them (28.6%) achieved CR/CRi and 6 achieved PR (28.6%). All four patients with TP53 abnormalities achieved PR and two of them (50.0%) achieved uMRD in both PB and BM. One of 6 patients with del(11q) achieved CR (16.7%) and five achieved PR (83.3%), with 2 achieved uMRD in both PB and BM (33.3%). Among 13 assessable patients with unmutated IGHV, four of them achieved CR (30.8%) and nine achieved PR (69.2%), with 5 achieved uMRD in both PB and BM (38.5%). 18 patients had turned to ibrutinib maintenance with 12 of them finished 4th cycle of iFCR or 3 additional cycles of rituximab and could be assessed further. 10 of the patients achieved CR/CRi (83.3%) and 2 achieved PR (16.7%), with 8 achieved uMRD in both PB and BM (66.7%). The most common hematological toxicity events were neutropenia and thrombocytopenia and the non-hematological toxicity events were nausea and vomiting. The occurrence rate of grade 3 to 4 neutropenia and agranulocytic fever were 51.9% (15/29) and 17.2% (5/29) respectively. The most common ibrutinib-related AE were purpura, rash and diarrhea. The occurrence rate of AE induced discontinued ibrutinib more than 7 days, discontinued ibrutinib fewer than 7 days and ibrutinib decrement were 20.7%(6/29), 17.2%(5/29) and 20.7%(6/29) respectively. Conclusion: MRD-driven Ibrutinib plus FCR could achieve high response rate with uMRD in yonger fit patients with CLL/SLL, with manageable toxicity. Ibrutinib plus FCR could be one of the most promising choice as a time-limited regimen in the new drug era. Disclosures No relevant conflicts of interest to declare.

Background: ENKTL is a highly aggressive NHL related to Epstein-Barr virus, with a higher incidence in Asia. Patients with r/r-ENKTL have a poor prognosis after failing asparaginase based regimen, and there is lack of effective treatment. Anti-PD-1 antibody represented a favorable treatment for rr-ENKTL. ORIENT-4 trial demonstrated Sintilimab was effective and well tolerated in r/r-ENKTL. The efficacy of Chidamide, an oral HDACi was reported on rr-ENKTL(CHIPEL trial, 2017 ASH). Here we present preliminary results of Sintilimab plus Chidamide for r/r-ENKTL. Methods: This trial enrolled eligible patients with histologically confirmed r/r-ENKTL failing from asparaginase-based regimen; ECOG performance status ≤ 2; adequate organ function and bone marrow function; and at least one measurable or evaluable lesion. This study consisited of a Phase Ib (dose escalation) portion followed by a Phase II expansion portion. In the Phase Ib, a standard "3+3" design was utilized to identify the MTD, DLT and recommended Phase II dosage (RP2D) of Chidamide plus Sintilimab. In the phase II portion, patients received 6 cycles of Sintilimab (200 mg) plus Chidamide (RP2D) every 3 weeks. Patients with complete response (CR) or partial response (PR) received up to 1 year. The primary endpoint was objective response rate (ORR) assessed by investigators per RECIL 2017 criteria. Key secondary endpoints included TTR, DOR,PFS,OS and safety. Adverse events (AEs) were defined according to CTCAE 5.0. Pretreatment formalin-fixed, paraffin-embedded (FFPE) tumor samples were obtained. Samples were tested using Dako PD-L1 22C3. PD-L1 protein expression was determined using tumor proportion score (TPS) and combined positive score (CPS). CPS ≥1 was considered positive. All patients' blood samples were collected for ctDNA assessment before treatment in each of cycles. ctDNA samples were analyzed by capture-based NGS targeting 475 lymphoma- and cancer- relevant genes. This trial was registered at ClinicalTrials.gov (NCT 03820596) Results: From March 2019 to May 2020, 41 patients were screened and 37 eligible patients were enrolled from 5 institutions. Median age 48 years (range, 20-72), 27 (73.0%) male, 26 (70.3%) patients with Stage IV of disease at screening, 20 (54.1%) patients with PINK-E score ≥ 3 points, 16 (43.2%) patients received ≥ 2 lines of prior systemic therapy. In Phase Ib , no MTD or DLT events were observed, RP2D of chidamide was 30 mg twice a week. Of 36 response evaluable patients, 21 (58.3%) achieved response including 16 (44.4%) patients with CR and 5 (13.9%) with PR(Figure 1). The median time to initial response was 6.0 weeks (5.0-12.4w). The median DOR time was 9.2+ months (1.3 -14.5+m) (Figure 2). The median follow-up time was 7.3 (0.9-16.1) months. Estimated 1-year OS rate was 79.1%, 1-year PFS rate was 66.0%. 19 (51.3%) patients remained on treatment,18 (48.7%) patients had discontinued from study treatment (16 for PD, 1 for AEs, 1 for personal reasons). The OS and PFS of patients with CR/PR is significantly superior to patients with SD/PD. Estimated 1-year OS rate for CR/PR and SD/PD was 95.0% and 51.9% (P Conclusion: Sintilimab plus Chidamide showed manageable safety profile and yielded effective antitumor activity, durable response in patients with r/r-ENKTL for the first time. It is a promising therapeutic option for this population, especially for those with CPS≥30. Epigenetic strategies synergize with anti-PD-1 antibody maybe enhanced antitumor responses to r/r-ENKTL, further investigation is warranted. Disclosures No relevant conflicts of interest to declare.

Introduction Zanubrutinib is a highly specific, potent BTK inhibitor with minimal off-target inhibition of other kinases such as EGFR, JAK3, TEC and ITK. Zanubrutinib has shown 100% BTK occupancy, sustained over 24-hours, in both the peripheral blood and lymph node biopsies from patients treated at 160 mg twice daily and achieves durable responses in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) (Tam 2019). In a phase 2 study conducted in patients with relapsed/refractory (R/R) CLL/SLL, treatment with zanubrutinib resulted in an overall response rate (ORR) of 85%. In addition, duration of response (DOR), progression free survival (PFS) and overall survival (OS) of zanubrutinib monotherapy at 12 months were 93%, 87% and 96%(Xu 2020). Here, we present the pooled analysis to evaluate the impact of number of prior lines of therapy on outcomes of zanubrutinib treatment for CLL/SLL patients. Methods Our analysis was based on a pooled data including CLL/SLL patients treated with zanubrutinib monotherapy in two phase 1 studies (ClinicalTrials.gov NCT02343120, and ClinicalTrials.gov NCT03189524) and one phase 2 study (ClinicalTrials.gov NCT03206918), with median study follow-up time of 29.2, 21.1 and 15.1 months, respectively. Firstly, efficacy and safety outcomes were compared between the treatment naïve (TN) and relapsed/refractory (R/R) groups. Secondly, patients with 1 prior therapy were compared to patients with ≥ 2 prior therapies within the R/R setting. To control confounding in each analysis, entropy balancing was used to create a weighted sample where the baseline covariates were balanced between groups (Hainmueller 2012). In each weighted sample, the efficacy outcomes of zanubrutinib included complete response rate (CRR), ORR (defined as the achievement of complete response [CR], or CRi, partial response [PR], nodular PR, PR with lymphocytosis), PFS and OS. The difference between groups in CRR and ORR was investigated by logistic regression, and those in PFS and OS by Cox proportional hazards models and log-rank test. The 24-month PFS and OS rates were calculated by the Kaplan-Meier method. The extent of exposure and safety profile of each group were summarized. Results The analysis data consisted of 19 TN patients, 93 patients with 1 prior therapy, and 99 patients with ≥ 2 prior therapies. Seven patients were excluded due to missing baseline covariates. In the weighted samples, all baseline covariates were balanced between groups. After weighting, the effective sample sizes were 19 and 31 for the TN and the R/R groups respectively. The median follow-up times were 31.3 and 20.9 months for the TN and R/R group, respectively; 54.4%, 18.8% and 26.8% of the patients in the R/R group had 1, 2 and >2 prior lines of therapy. The ORR and CRR were higher in TN group, compared with R/R groups (100% vs. 92.1% in ORR [p After weighting, the effective sample sizes were 77 and 85 for the 1 prior therapy and the ≥ 2 prior therapies groups respectively. The median follow-up times were 17.1 and 15.8 months for the 1 prior therapy and the ≥ 2 prior therapies groups; 56.5%, 20.6% and 22.9% of the patients in the ≥ 2 prior therapies group were treated with 2, 3 and >3 prior lines of therapy. The ORR was numerically higher in the 1 prior therapy group, compared with ≥ 2 prior therapies group (97.0% vs. 88.3%; p=0.05). The CRR was comparable in two groups (9.8% vs. 8.4%; p=0.75). The PFS of 1 prior therapy group was significantly longer than that in ≥ 2 prior therapies group (p Conclusion Zanubrutinib administered in the early lines, including treatment of naïve patients and patients with 1 prior therapy, led to higher overall response rates and greater durability of therapeutic benefit. Safety profile was similar across all lines of therapy. References Tam CS, et al. Blood. 2019; 134 (11): 851-859. Xu W, et al. J Hematol Oncol. 2020; 13 (1): 48. Hainmueller, J. Political Analysis. 2012; 20(1): 25-46. Disclosures Tam: BeiGene: Honoraria; AbbVie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Seymour:Morphosys: Consultancy, Honoraria; Mei Pharma: Consultancy, Honoraria; Gilead: Consultancy; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nurix: Honoraria. Zhou:Henan Cancer Hospital: Current Employment. Opat:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BeiGene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZenca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Epizyme: Research Funding. Trotman:Celgene: Research Funding; Takeda: Research Funding; BeiGene: Research Funding; F. Hoffmann-La Roche: Research Funding; PCYC: Research Funding. Huang:BeiGene: Current Employment, Current equity holder in publicly-traded company. Lu:BeiGene: Current Employment, Current equity holder in publicly-traded company. OffLabel Disclosure: Zanubrutinib in treatment-naive CLL/SLL

Orelabrutinib (ICP-022) is a novel and highly selective irreversible BTK inhibitor. We previously reported that orelabrutinib had high bioavailability with ~100% BTK occupancy at 24 hours at 150 mg daily dosing regimen and demonstrated excellent safety and efficacy profiles at median follow-up of 8.7 months in a Phase I/II trial of refractory/relapsed (r/r) CLL/SLL (Chronic Lymphocytic Leukemia/Small Cell Leukemia). Here we present an updated analysis of efficacy and safety results from the clinical study of Orelabrutinib in Chinese patients with r/r CLL/SLL following extended treatment. This is an open-label, multicenter, phase II study with objectives to evaluate its safety and efficacy following an oral daily administration. The primary endpoint was objective response rate (ORR). The duration of response (DOR), progression-free survival (PFS) and safety were chosen as secondary endpoints. Response was assessed per 2008 IWCLL criteria with modification for partial remission (PR) with lymphocytosis (PR-L) (Hallek 2012) or the Lugano Classification (Cheson, 2014) for CLL and SLL, respectively. Results: A total of 80 patients with r/r CLL (n=70)/SLL (n=10) were enrolled. Eligible patients had relapsed after or were refractory to ≥1 prior treatment with median age of 60.0 (range, 36.0-78.0 years). There are 70% of patients for Rai stage 3-4 disease, 22.5% for 17p13.1 deletion [del(17p)] and/or TP53 mutation, 41.3% for unmutated immunoglobulin heavy chain variable region (IGHV) and 23.8% for 11q22.3 deletion [del(11q)]. The median follow-up time was 14.3 months (range, 0.2-21.6 months), and the last patient completed a minimum of 12 cycles of orelabrutinib treatment. Efficacy: The efficacy results presented here were evaluated by IRC (Independent review committee). Following a minimum of 12 cycles treatment, the ORR (PR-L or above) was 91.3% (95% confidence interval [CI]: 82.80 ~ 96.41%) with 10.0% of patients having complete response (CR), 63.8% PR and 17.5% PR-L. Median time for achieving first response was 1.87 months (range, 1.84 - 1.94 months). The median DOR and PFS were not reached. The estimated 12-month DOR was 77.1% (95% CI: 62.50-86.60%), PFS 81.1% (95% CI: 70.53 - 88.13%) and OS 86.3% (95% CI: 76.55 - 92.14%). Patients with Del(17p) and/or TP53 mutation achieved 100% ORR. The ORR is 94.7% for Del(11q)) and 93.9% for unmutated IGHV. Comparing to the first analysis results of which the median follow-up was 8.7 months, the CR rate had increased from 3.8% to 10.0%, and 8 patients with PR-L had converted to a deeper response. So, orelabrutinib showed a significant higher CR rate comparing to other BTK inhibitors at a similar treatment period and we anticipate a further increase of CR rate with longer duration of treatment. Safety: Most adverse events (AEs) were mild to moderate, similar to the first reported safety profiles at median follow-up of 8.7 months. Extended follow-up analysis did not reveal new safety nor toxicity concerns. The most frequent adverse events (AEs) of any cause/any grade were well characterized as hematological toxicities: thrombocytopenia, neutropenia, and anemia; upper respiratory tract infection, pneumonia and hypokalemia. No case of atrial fibrillation nor secondary malignancy was reported, no patient was observed for ≥3 hypertension and only one patient had ≥3 grade diarrhea. Major hemorrhage was reported in 2 patients, one with intracranial hemorrhage (65-year-old male patient with more than 10 years hypertension) and the other with vitreous hemorrhage which were resulted from posterior vitreous detachment that was assessed as unlikely related to the treatment of orelabrutinib. Once again, it has been further confirmed that orelabrutinib has excellent safety profiles following extended treatment. Conclusion: This updated and extended study further confirms that orelabrutinib is efficacious in treating r/r CLL patients with a higher CR rate, durable response and improved safety profiles. As a highly selective BTK inhibitor with favorable pharmacokinetic and pharmacodynamic properties, orelabrutinib provides a favorable therapeutic choice for patients with r/r CLL/SLL and a potential best candidate for combination therapy. Disclosures Zhu: Beijing InnoCare Pharma Tech Co., Ltd: Current Employment. Zhao:Beijing InnoCare Pharma Tech Co., Ltd: Current Employment. Zhang:Beijing InnoCare Pharma Tech Co., Ltd: Current Employment. Xu:Beijing InnoCare Pharma Tech Co., Ltd: Current Employment.

Through a clustered regularly insterspaced short palindromic repeats (CRISPR) screen to identify mitochondrial genes necessary for the growth of acute myeloid leukemia (AML) cells, we identified the mitochondrial outer membrane protein mitochondrial carrier homolog 2 (MTCH2). In AML, knockdown of MTCH2 decreased growth, reduced engraftment potential of stem cells, and induced differentiation. Inhibiting MTCH2 in AML cells increased nuclear pyruvate and pyruvate dehydrogenase (PDH), which induced histone acetylation and subsequently promoted the differentiation of AML cells. Thus, we have defined a new mechanism by which mitochondria and metabolism regulate AML stem cells and gene expression.

Background: In spite of years of effort, Mantle Cell Lymphoma (MCL) remains a clinical challenge. The clinical significance of Bruton's Tyrosine Kinase (BTK) inhibitor has been validated with the approvals by FDA for the treatment of multiple subtypes of NHL including refractory and relapse (r/r) MCL. However, some serious adverse events (AEs) due to poor target selectivity (inhibition of EGFR, TEC, BMX and others), such as diarrhea, sever bleeding and atrial fibrillation, remain as challenges in clinic. Orelabrutinib is a novel, potent irreversible BTK inhibitor with high selectivity for BTK. Results of early clinical study showed that it has excellent safety/tolerability profiles and favorable pharmacokinetic/pharmacodynamic properties. Sustained ~100% BTK occupancy at 24 hours was achieved with once daily dosing regimen of 50 mg and above. In this presentation, we will report an updated analysis of orelabrutinib in Chinese patients with r/r MCL with minimum of 12 cycles of treatment. Aims: To evaluate the sustained efficacy and long-term safety of orelabrutinib in Chinese patients with r/r MCL. Methods: This is an open-label, multicenter, two stages, phase II study. The primary endpoint was objective response rate (ORR) assessed per Lugano criteria (2014). Safety and other efficacy (DOR, PFS, OS) evaluations were chosen as secondary endpoints. Results: A Total 106 patients, were enrolled in this study with median follow up time of 15.0 months. 79.2% of the patients were male and median age of 62.0 years old. Most patients were at advanced stage (73.6% were at stage IV and 20.8% were at stage III). According to per protocol analysis, 87 (87.9%) patients achieved ORR and 93.9% patients achieved disease control. The median duration of response (DOR) was not reached, the DOR rate at 12 months was 73.7%, as expected the median PFS and OS were not reached, the PFS and OS rates at 12-month were 70.8% and 88.7% respectively. Comparing to the results of previous analysis, the CR rate, by conventional CT method, increased to 27.4% and it was expected a higher rate of in depth response may occur with prolonged treatment. Further analysis showed orelabrutinib was efficacious in all subgroups (age, gender, status, stage, prior therapy, etc.). Orelabrutinib demonstrated excellent safety profile in r/r MCL patients. The frequently reported treatment related adverse events (TRAE) were primarily hematological toxicities including thrombocytopenia, neutropenia, leukopenia, and hypertension. The frequently reported grade 3 or higher AEs of any cause was thrombocytopenia . No treatment related ≥grade 3 GI and cardio toxicity, nor severe bleeding, were observed. Of the 106 patients, 32 experienced serious AEs; and 17 of them, mainly hematological toxicities and / or infections were treatment-related. Comparing to the safety data of median follow up of 10.5 months, there was only a mild increase of adverse events rate after extended treatments; the safety profiles were essentially the same. These results suggested that safety events primarily occurred during early treatment and it appeared less eventful with orelabrutinib continue treatment. Conclusion: Orelabrutinib showed continuous efficacious in treating patients with r/r MCL. In addition, orelabrutinib is safe and well tolerated with no treatment related grade 3 or higher diarrhea, atrial fibrillation/flutter or severe bleeding in this study. Results of prolong treatment expected to produce a higher rate of in depth response without altering its safety profiles support orelabrutinib being a better selection for BTKi therapy. The improved safety as a resulting of high target selectivity, and the convenience of daily dosing regimen provides orelabrutinib as preferred therapeutic choice for B cell malignancy. Disclosures Zhang: Beijing InnoCare Pharma Tech Co., Ltd: Current Employment. Zhao:Beijing InnoCare Pharma Tech Co., Ltd: Current Employment. Xu:Beijing InnoCare Pharma Tech Co., Ltd: Current Employment.

Background: Bruton tyrosine kinase (BTK) is one of the key kinases implicated in the pathogenesis of multiple B cell malignancies. Orelabrutinib is a novel, highly selective and potent irreversible BTK inhibitor with minimal activities against other kinases (ITK, EGFR, ERBB2, etc.). As thus orelabrutinib may avoid off-target related adverse events and shall have improved safety profiles comparing to other BTK inhibitors. Here we present the safety profile of orelabrutinib analyzed based on data from 5 ongoing clinical studies in B cell malignancies (Table 1). Methods: Safety data of 266 patients (pts) from 5 ongoing orelabrutinib monotherapy studies were pooled and analyzed. All pts have been treated with at least one dose of oral orelabrutinib at ≥150 mg daily. The analysis includes the frequency and severity of adverse events (AEs), AEs of special interest, and AEs leading to treatment discontinuation or dose modifications. Results: Safety data were pooled from 266 pts with median age of 60 years (range 35.0-79.0, 69.2% males). The median duration of exposure was 11.0 months (range 0.2-22.0). The most common (occurring in ≥15% of pts) AEs were neutropenia (28.6%), thrombocytopenia (25.9%), Upper respiratory tract infection (24.4%), leukopenia (18.0%), anemia (16.2%) and rash (15.8%). Treatment related serious AEs (SAEs) were reported in 14.7% pts. The most common treatment related SAEs included thrombocytopenia (3.0%), lung infection (3.0%), pneumonitis (1.9%), anemia (1.1%) and lymphadentis (0.8%), The safety profiles were comparable in pts with various subtypes of B cell malignancies. It's noted that orelabrutinib has much less frequency of BTK off-target related adverse events, such as atrial fibrillation, diarrhea, major hemorrhage etc. Among all 266 pts, only one patient was reported with one episode of transient grade 1 atrial fibrillation, and no grade ≥3 atrial fibrillation was observed. Diarrhea of any grade was 7.1% and only one case (0.4%) was reported as grade 3. The major hemorrhage, defined as serious or ≥ G3 bleeding of any site, or central nervous system bleeding of any grade, was rarely observed; as only one case of cerebral hemorrhage, in 65-year-old male patient with more than 10 years hypertension was reported; the other three cases were subcutaneous hemorrhage, vitreous hemorrhage and vitreous hemorrhage/retinal hemorrhage. The later 2 cases of vitreous hemorrhage and/or retinal hemorrhage were resulted from posterior vitreous detachment and macular degeneration and both events were assessed as unlikely related to the treatment. Among 266 pts, the second primary malignancies were reported in only one pt with r/r MCL during orelabrutinib treatment. Grade ≥3 infection occurred in 41 pts (15.4%); most common infections were upper respiratory tract infection and lung infection. Most of the AEs were occurred during the early treatment, the frequency of the new event occurrence was significantly decreased during the later cycles. Dose reductions due to AEs occurred in 15 pts (5.6%), and treatment discontinuation due to AEs in 5.3% of pts with 2.3% related to orelabrutinib. Conclusions: Orelabrutinib shows excellent safety profiles and tolerability across various B-cell malignancies in long-term treatment. These data suggested orelabrutinib as a favorable treatment choice including the combinational therapy for B-cell malignancies. . Disclosures Zhu: Beijing InnoCare Pharma Tech Co., Ltd: Current Employment. Zhao:Beijing InnoCare Pharma Tech Co., Ltd: Current Employment. Xu:Beijing InnoCare Pharma Tech Co., Ltd: Current Employment.

Ruxolitinib is the first available JAK inhibitor (JAKi) therapy for amelioration of constitutional symptoms or splenomegaly in myelofibrosis (MF). Duration of response varies, as patients discontinue ruxolitinib due to disease progression or drug toxicity. Ruxolitinib failure is a poorly defined clinical entity, but may include suboptimal or lost spleen response, worsening cytopenias, accelerated or blast phase (AP/BP) progression, or non-hematologic side effects. It is not clear what other clinical parameters should be considered indicative of JAKi failure. Leukocytosis is a known prognostic factor in MF and is included in various prognostic models. We have observed that some patients on stable doses of JAKi therapy develop progressive leukocytosis in the absence of other signs of MF progression. The significance of this event is not known, and it is not clear whether the onset of leukocytosis should prompt changes in clinical management. To assess the clinical significance of emergent leukocytosis, we evaluated leukocyte counts in our database of MF patients receiving Ruxolitnib or Momelotinib as first-line JAKi therapy. We defined emergent leukocytosis as any of:New onset of WBC ≥25 x 109/L in patients with WBC ≤12.5 x 109/L at JAKi start.Doubling of WBC from the nadir value in patients with WBC >12.5 x 109/L at JAKi start and nadir WBC >12.5 x 109/L.WBC ≥25 x 109/L in patients with WBC > 12.5 x 109/L at JAKi start after attaining a nadir WBC ≤12.5 x 109/L. Leukocytosis had to be sustained over consecutive blood counts at least one month apart and had to occur in the absence of infection, steroid therapy, AP/BP transformation, splenectomy or JAKi dose reduction. Exclusion criteria included concurrent hematologic diagnoses, and splenectomy or AP/BP MF preceding JAKi initiation. Of 290 patients with MF receiving JAKi therapy, 217 met study criteria. Of these 217 patients, 27 developed emergent leukocytosis while receiving JAKi. The cumulative incidence of leukocytosis was 4%, 10% and 15% at 1, 3, and 5 years from the start of JAKi therapy, respectively. Transformation to AP/BP, splenectomy, bone marrow transplant, or death from any cause were considered as competing risks in the calculation of cumulative incidence. In a multivariate analysis, clinical parameters associated with emergent leukocytosis included presence of baseline anemia (HR 4.94 [95% CI, 1.13-21.53]; p = 0.03) or leukocytosis (HR 5.01 [95% CI, 1.44-17.41]; p = 0.01) at JAKi start and female gender (HR 0.21 for male [0.08-0.06]; p=0.002). Baseline leukocytosis as WBC ≥25 x 109/L, and anemia was as a hemoglobin In patients with available targeted sequencing performed prior to JAKi therapy (n=141, 65%), TET2 mutations were associated with increased risk of leukocytosis (HR 5.48 [95% CI, 1.73-17.35]; p=0.004), but JAK2, CALR, MPL or ASXL1 mutations were not. Among 27 patients who experienced emergent leukocytosis, 21 were deceased while 6 are in ongoing follow-up. Median overall survival calculated from the date of leukocytosis was 14 months [95% CI, 8.6-27.9]. Overall, 7 patients transformed to AP/BP, 7 died from progressive MF, 4 underwent transplant, one underwent splenectomy, 6 died of miscellaneous causes and two remain on first-line JAKi without any complicating events. Among 27 patients experiencing leukocytosis, paired samples for molecular analysis at JAKi initiation and at the time of leucocytosis were available in 21 (78%). Results from targeted sequencing of a panel of 49 myeloid genes were available from 4 patients with paired samples (Table 1). New mutations at leukocytosis were seen in NRAS and CEBPA in the first patient, KRAS and SH2B3 in the second, and CBL in the third. The fourth patient acquired no new mutations but demonstrated marked expansion of a dominant clone. The remaining 17 paired samples have been submitted for sequencing with results pending. In summary, MF patients with baseline anemia, leukocytosis, or female gender are at higher risk for emergent leucocytosis while on JAKi therapy. The median overall survival of 14 months in patients who experience emergent leukocytosis is comparable to that observed in patients following ruxolitinib failure. Our data also suggest that onset of leukocytosis may be indicative of underlying clonal evolution, which will need to be confirmed in a larger number of patients. These findings suggest consideration of emergent leukocytosis as a criterion for JAKi failure in MF. Disclosures Maze: Novartis: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. Gupta:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Incyte: Honoraria, Research Funding.

Introduction: Patients with diffuse large B cell lymphoma (DLBCL) arising from the testis have a relatively poor outcome. Age, stage and use of radiation and surgery are important prognostic factors in patients with primary testicular DLBCL. Additionally, the study by Gundrum et al suggested that laterality was also an important predictor of outcomes in patients with primary testicular DLBCL, with left side involvement being associated poorer prognosis. However, most patients included in the study by Gundrum et al were diagnosed in the pre-rituximab era, therefore, the role of laterality in the prognostification of patients with primary testicular DLBCL remains to be explored. In this study, the Surveillance, Epidemiology, and End Results (SEER) database was used to evaluate the prognostic roles of laterality in patients with primary testicular DLBCL. Methods: Data from the SEER 18 Registries were used to conduct this study. Cases with newly-diagnosed testicular (International Classification of Diseases for Oncology, 3rd Edition [ICDO-3] codes 9823, sites: C62.0-C63.2) in the time period between 1973 and 2015 were included. Exclusion criteria included history of cancer, unknown laterality, unknown survival data and unknown cause of death. For each case we included age at the time of diagnosis, laterality (left, right, bilateral), SEER cause-specific death classification, survival months and vital status. Overall survival (OS) was defined as time from diagnosis to death or last follow-up and cancer-specific survival (CSS) was calculated as time from diagnosis to death from DLBCL or last follow-up. Survival curves were plotted by the Kaplan-Meier method and the log-rank test was used for comparison. P value was 2-sided and P Results: A total of 1213 patients were included in this analysis. The median follow-up was 43 months (interquartile range[IQR]: 13-90 months). Of these patients, 372 patients were diagnosed from 1973-2000 (pre-rituximab era) and 841 patients were diagnosed from 2001-2015 (rituximab era). We found that patients with bilateral testis involvement had a significantly decreased CSS (median CSS: 53 vs. 142 months, P=0.0035) and OS (median OS: 32 vs. 77 months, P=0.0008) compared with those with unilateral involvement. Patients with left-side involvement had a similar CSS (median CSS: 136 vs. 153 months, P=0.2997) and OS (median OS: 76 vs. 80 months, P=0.7360) compared to those with right-side involvement. For patients with left-side involvement, patients diagnosed in the rituximab era had a significantly longer CSS(hazards ratio[HR]: 0.4140, 95% confidence interval[CI]: 0.3065 to 0.5593, P Conclusion: our study demonstrated that laterality was not a prognostic factor for patients with primary testicular DLBCL. And the improvement in the prognosis from pre-rituximab era to rituximab era was more remarkable in primary testicular DLBCL patients with left-side involvement than those with right-side involvement. These data suggest primary testicular DLBCL from different sides had different responses to therapy and may have different biological characteristics. Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction : PI3Kδ, one of the class I PI3Ks, is found expressed primarily in leukocytes and plays an essential role in B-cell development and function. Here, we comprehensively evaluated the in vitro and in vivo antitumor activity and the underlying mechanism of SHC014748M, an oral selective inhibitor of PI3Kδ under Phase I clinical evaluation. Methods : Biochemical and cell-based assays were used to measure compound potency and selectivity in lymphoma cell lines as well as primary CLL cells, and PI3K/AKT pathway was measured by Western blot assay, Alphalisa and Elisa. Xenograft model was carried out to validate in-vivo antitumor potency of the compound. Besides, chemokines and cytokines derived from blood samples of patients were also detected. Results: SHC014748M was 125- to 306-fold more selective for PI3Kδ inhibition relative to other class I PI3K enzymes and showed in vitro activity in most of 23 B lymphoma cell lines. We identified that SHC014748M treatment resulted in a 3.1- to 5.5-fold increase in annexin V/7-ADD staining, indicating a significant apoptosis induction. SHC014748M inhibited phosphorylation of AKT, targets downstream of PI3Kδ, in lymphoma cells. Among the 15 primary CLL cells, the 50% inhibitory concentration (IC50) of SHC014748M varies from 850 nM to 37040 nM respectively and expression of phosphorylation AKT decreased to the normal levels in the presence of SHC014748M or positive control, Idelalisib. In-vivo study revealed that SHC014748M significantly reduced lymphoma cell growth in the treatment group compared with control mice. CCL4, CCL17, CCL22 and CXCL13 derived from patients decreased sharply after SHC014748M treatment. Conclusion: According to the results, SHC014748M appeared to be a novel promising compound in the treatment of B cell lymphomas and CLL. Disclosures Wang: Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment. Wang:Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment. Zhang:Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment.

Objective Chronic lymphocytic leukemia (CLL) is a heterogeneous disease and characterized by the accumulation of monoclonal B lymphocytes in blood, bone marrow and lymphoid tissues. Although diagnosis and therapy have been developing, several high-risk features for CLL patients have been identified, only few of them are well-characterized and clinically actionable. IGHV mutation status has been confirmed as a valuable independent prognostic factor. However, it is of vital importance to explore alternative markers because the detection method of IGHV mutation is expensive and time consuming, which limits its application in the routine laboratory diagnosis process. Thus, further exploration of effective diagnostic biomarkers for CLL are worthy and important. Circular RNAs (circRNAs), a novel family of non-coding RNAs, have been described as critical regulators of gene expression and are stable in exosomes. However, the compelling role of circRNAs in mediating CLL progression was still poorly understood. In this study, we attempted to identify a novel circRNA which could serve as a plasma biomarker and explore its molecular mechanism and clinical significance in CLL. Methods To analyse circRNAs expression profiles in CLL, plasma samples from three untreated CLL patients and five normal donors were collected for circRNA microarray. Then, we validated and analysed the clinical characteristics of candidate circRNAs in a larger cohort of patients (137 CLL patients vs. 50 healthy donors, age- and sex-matched). The receiver-operating characteristic (ROC) curve analysis was performed to evaluate the potential diagnostic value of circ-RPL15 as a biomarker for CLL, plasma and PBMC samples from multiple lymphomas were collected for qRT-PCR detection as well. The oncogenic functions of circ-RPL15 were further measured in CLL cell lines (MEC-1 and JVM-3) by performing RNA interfering (RNAi), immunofluorescence (IF) staining, CCK8 assay and flow cytometry. Moreover, we explored the molecular mechanisms of circ-RPL15 and verified the interactions among POLR2A, circ-RPL15, miR-146b-3p and RAF1 by performing RNA-FISH, ChIP, RIP, dual-luciferase reporter assay and Western blotting. Results A total of 3,656 circRNAs were identified, among which 71 circRNAs were dysregulated in CLL patients ( p≤0.05) and circ-RPL15 showed the most significantly high expression level and was finally selected for further investigation. Circ-RPL15 was abundantly present in exosomes and was significantly higher expressed in CLL compared with normal and other lymphomas. Moreover, we found that circ-RPL15 was highly relevant to IGHV region mutation status, which has long been considered as an important prognostic indicator of CLL. Patients without IGHV mutation presented a higher circ-RPL15 levels and the ROC curve showed that circ-RPL15 could be a good alternative to IGHV status with AUC values of 0.86. Kaplan-Meier analysis showed that CLL patients with circ-RPL15 high expression had a significantly poorer OS than those with low expression of circ-RPL15 (Figure A-F). Next, we unraveled an original mechanism in CLL that up-regulated circ-RPL15 was mainly enriched in the cytoplasm, acted as a "sponge" of miR-146b-3p. CCK8 assay and flow cytometry analysis showed that the cell viability of CLL cell lines were evidently decreased after RNAi of circ-RPL15 and this result could be reversed by miR-146b-3p inhibitor transfection. Western blot suggested a critical role of circRPL15- sponged miR-146b-3p in the regulation of RAS/RAF1/MEK/ERK pathway (Figure G-L). Furthermore, we identified circ-RPL15, a circRNA derived from exon 2 and partial exon 3 of RPL15 pre-mRNA which is located on chromosome 3p24.2, was negatively regulated by transcription factor POLR2A and delivered by exosomes into CLL patient plasma (Figure M,N). Conclusion In this study, we uncovered a novel oncogenic biomarker for CLL patients, which contributes to promoting CLL progression and is associated with poor outcome. The higher expression of circ-RPL15 was positively correlated with IGHV mutation status which is crucial in the evaluation of CLL prognosis. The miR-146b-3p mediated suppression of RAS/RAF1/MEK/ERK pathway could be alleviated by circ-RPL15 up-regulation in CLL. Our results revealed that circ-RPL15 may represent a promising plasma biomarker for the diagnosis and prognosis of CLL as well as a reliable surrogate of IGHV mutation status. Figure Disclosures No relevant conflicts of interest to declare.

Background Circular RNAs (circRNAs), a novel family of non-coding RNAs, have crucial roles in cancer progression. Conventional research is mainly focus on nuclear genome derived circRNAs (nu-circRNAs). The biological and clinical characteristics of mitochondrial genome derived-circRNAs (mt-circRNA) remains largely unknown, especially in chronic lymphocytic leukemia (CLL), the most prevalent incurable B-cell neoplasm in western countries. Lack of convenient and reliable clinical biomarkers is a hindrance in monitoring the progression of CLL. It is in urgent need of screening effective new biomarkers and exploring potential therapeutic targets associated with CLL initiation and progression. Recent studies have shown that circRNAs are enriched and stable in serum exosomes. However, the biological mechanisms of exosomal circRNAs remain unclear. In this study, we attempted to identify the novel characteristics of a mt-circRNA mc-COX2, which was abundant in plasma exosomes and could be involved in the progression of CLL. Since CLL patients have specific expression features of exosomal circRNAs in plasma, the function of the circRNAs and their clinical significance is urged to be explored. Methods Firstly, to unveil circRNAs expression profiles in CLL, plasma samples from five treatment-naive CLL patients and five age-/sex-matched healthy donors (HDs) were collected for circRNA microarray analyses. Northern blot and RNA- FISH were conducted to verify the existence of circRNAs in mitochondria. qPCR and other functional analysis such as RNase R, actinomycin D and RIP experiments were carried out to demonstrated the clinical and biological characteristics of mc-COX2, one of the mt-circRNAs. Cell apoptosis ability was determined by FCM. Moreover, electron microscope, partical size analysis, FCM and Western blot were used to explore the existence of exosomes and q-PCR analysis was performed to detect the expression of mc-COX2. Results Mt-circRNAs were highly expressed in CLL patients plasma (Figure A, B). Herein, we reported a novel circRNA named as mc-COX2 which was generated from the COX2 gene on mitochondrial genome by back-splicing and closely related to prognosis of CLL patients (Figure C). The enrichment of mc-COX2 in the mitochondria was further confirmed by RNA-FISH (Figure D). Northern blot was performed using head-to-tail probe of mc-COX2 and the results showed that mc-COX2 was detectable within the splice sites (Figure E). Notably, obviously different from nu-circRNAs, mc-COX2 showed lower stability with lower tolerance to RNase R and actinomycin D, but it was much more stable compared with linear RNAs (Figure G, F). And mc-COX2 cannot bind to AGO2 protein, suggesting that it probably function via other mechanisms instead of acting as ceRNA (Figure H). Furthermore, the up-regulated expression of mc-COX2 was positively associated with leukemogenesis and worse survival of CLL patients (Figure I, J). CLL patients with TP53 deletion rather than mutation displayed higher expression of mc-COX2 (Figure K). The endogenous reduction of mc-COX2 could induce cell apoptosis (Figure L). In addition, we indicated that mc-COX2 was highly enriched in exosomes, by which circRNAs could enter the circulation and be readily measured in the serum (Figure M). Moreover, the existence of mc-COX2 in plasma suggests that mc-COX2 may serve as a potentially novel prognosis biomarker for CLL patients, guiding targeted therapy in clinic. Conclusions In summary, we demonstrated the existence and clinical significance of mc-COX2, a novel class of circRNA species abundant in CLL plasma exosomes for the first time, which was distinct from nu-circRNAs. Furthermore, the specific high expression of mc-COX2 in CLL plasma which was strongly correlated with P53 deletion, can indicate worse prognosis of CLL patients. Taken together, our study not only identifies a novel circRNA which may serve as a new "liquid biopsy" biomarker for CLL prognosis but also expands the current knowledge regarding molecular mechanisms of circRNAs, providing potential diagnostic and therapeutic implications for CLL. It would be of great interest to explore the biogenesis of mt-circRNAs and their impact on mitochondrial function in future studies. Figure Disclosures No relevant conflicts of interest to declare.

Introduction:Mantle cell lymphoma is an uncommon subtype of B-cell non-Hodgkin lymphoma with variable initial treatment strategies defined by disease risk factors, patient preferences and access to medical care. There is a paucity of reported data on treatment patterns and clinical outcomes in Chinese patient populations managed in the rituximab era. The study aim was to evaluate and compare outcome in relation to prognostic factors and first-line treatment in patients with MCL managed in real-world academic medical centers in China. Methods:Retrospective data were collected from 5 major Chinese Hematology Centers in Beijing, Guangzhou, Nanjing, Shanghai and Suzhou from the period of 2007 to 2017. Diagnosis for MCL was based on characteristic immuno-phenotype and CyclinD1 immunohistochemistry staining. Overall Survival (OS) time was calculated from date of diagnosis to date of death or date of last follow-up, whichever comes first. Kaplan-Meier estimator was used to estimate survival probability. Median follow-up time was estimated on overall survival by reverse Kaplan-Meier method. Survival difference between groups was evaluated by log-rank test for statistical significance. Results:A total of 605 patients with newly diagnosed MCL were included in the analysis. The median follow-up time of the whole group was 38 months. The median age was 58 years (range 28-83) with 59% patients under age 60; the M:F ratio was 3.5:1; 76% presented with ECOG PS of 0-1. Eighty-three percent patients had stage 3-4 disease, 38% had intermediate and high risk MIPI scores, and 30% had IPI scores of 3-5. Ki-67 was 30 (p=0.003). OS correlated with MIPI and IPI scores. The 3-year OS rates were 39.7%, 67.7% and 86.7% respectively for high-, intermediate- and low-risk MIPI scores (p Conclusions:This large retrospective dataset of MCL patients who received contemporaneous real-world management in Chinese Hematology Centers confirmed the survival advantage afforded by rituximab-containing chemo-immunotherapy and consolidative autologous stem-cell transplant in first-line setting. The majority of patients seeking care at major medical centers were younger than 60 years of age with good performance status and lower risk MIPI/IPI scores, which correlated with more favorable survival. Incorporation of novel agents signaled infrastructural readiness to explore novel agents and combination in both first-line and relapsed settings. Disclosures Ruan: Celgene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie company: Research Funding; Juno: Consultancy; Kite: Consultancy.

Introduction : Ibrutinib, an oral, selective inhibitor of Bruton's tyrosine kinase (BTK), dramatically improved Progression-free survival (PFS) and Overall survival (OS) compared with immunochemotherapy in CLL both in first line and relapsed/refractory patients. However, some patients did progress on ibrutinb with dismal outcome. The underlying mechanism remains to be investigated beyond evolving of BTK and/or PLCg2 mutation, the dysfunction of apoptotic protein and mitochondrial apoptotic dependencies may be involves in ibrutinib resistance. PTBP1 (Polypyrimidine tract binding protein 1), a splicing factor, was found to be necessary for B cell selection in germinal centers. Knocking out PTBP1 in B cell resulted in impaired BCR-mediated B-cell activation and antibody production. Here, we investigate the regulation of PTBP1 on alternative splicing of apoptotic protein and its implications in CLL and ibrutinib resistance. Methods: Eighty-one CLL patients and 5 healthy controls were enrolled in this study from January 2010 to May 2018. The PTBP1 mRNA expression was measured by real-time polymerase chain reaction (RT-PCR) and Western-blot. We analyzed the PTBP1 expression with established CLL prognostic factors such as p53 and IGHV mutation status, and treatment to first treatment (TTFT). Resistant MEC-1 cell line was established by intermittently incubating with ibrutinib at a low concentration for short intervals and then gradually increased to 2-fold of IC50 value. Cells were allowed to recover every time after washing off the drug. RT-PCR was performed for both long and short isoform of MCL-1 by using specific primer in both parent and resistant cell lines and series ibrutinb-treated (both sensitive and resistant) patients' primary cells. Resistant MEC-1 cell line was cultured in RPMI 1640 without ibrutinib for 48hrs before transfection, siRNA targeting with PTBP1 mRNA and non-targeting siRNA were transfected into cells by using lipofectamine 3000. The transfection efficiency were verified by Western blot after 24 h and ibrutinib was added into resistant cell line. Apoptosis was then analyzed using flow cytometry (FCM) after 24 hrs. Receiver operating characteristic curve (ROC) and area under the ROC curve (AUC) were established to verify the best cut-off value in differentiating the high or low expression of PTBP1 mRNA. Time-to-first-treatment (TTFT) interval was defined as interval from diagnosis to first treatment. All statistical analyses were performed using the SPSS software program. Results: The expression of PTBP1 in CLL primary patients was significantly increased than 5 healthy donors (p < 0.01)(A). Patients with IGHV-mutated had higher level of PTBP1 as compared with patients with IGHV-unmutated (p < 0.05). Furthermore, Higher level of PTBP1 was associated with shorter TTFT in whole cohort, also in IGHV-mutated and unmutated subgroup (p < 0.05)(B). We further demonstrated that PTBP1 was aberrant expressed in ibrutinib resistant MEC-1 cell line or ibrutinib resistant primary patients' samples, as compared with parent cell line or patients' baseline samples. We also found the dysregulation of alternative splicing of MCL-1 in ibrutinib resistant models, presented with increased anti-apoptotic MCL-1L and decreased pro-apoptotic MCL-1S(C). Moreover, knocking down of PTBP1 sensitized CLL to ibrutinib via switching alternative splicing of MCL-1 to its pro-apoptotic isform MCL-1S(D). Conclusions: The splicing factor PTBP1 is involved in the pathogenesis of CLL. Its aberrant expression may lead to the dysregulation of alternative splicing of MCL-1, resulted in increased MCL-1L/s ratio. PTBP1 can be as a promising target for the treatment of CLL patients progressed on ibrutinib. Figure Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) cells and stem cells have unique mitochondrial characteristics with an increased reliance on oxidative phosphorylation (OXPHOS). To identify new biological vulnerabilities in the mitochondrial proteome of AML cells, we conducted an shRNA screen and identified neurolysin (NLN), a zinc metalloprotease whose mitochondrial function is not well understood and whose role in AML has not been previously reported. To begin our investigation into the role of NLN in AML, we analyzed NLN gene expression in a database of 536 AML and 73 normal bone marrow samples. NLN was overexpressed in 41% of AML samples. Overexpression of NLN in primary AML cells compared to normal hematopoietic cells was confirmed by immunoblotting. To validate the results of the screen and to determine whether NLN is required for AML growth and viability, we knocked down NLN in the leukemia cell lines OCI-AML2, MV4-11, NB4, and TEX with shRNA. NLN knockdown reduced leukemia growth and viability by 50-70%. Moreover, knockdown of NLN in AML cells reduced the clonogenic growth of leukemic cells in vitro and the engraftment of AML cells into mouse marrow after five weeks by up to 80% and 85%, respectively. The mitochondrial function of NLN is largely unknown, so we identified NLN's mitochondrial protein interactors in T-REx HEK293 cells using proximity-dependent biotin labeling (BioID) coupled with mass spectrometry (MS). This screen identified 73 mitochondrial proteins that preferentially interacted with NLN and were enriched for functions including respiratory chain complex assembly, respiratory electron transport, and mitochondrion organization. Therefore, we assessed the effects of NLN knockdown on OXPHOS. NLN knockdown reduced basal and maximal oxygen consumption, but there were no changes in the levels of individual respiratory chain complex subunits. To understand how NLN influences OXPHOS, we examined the formation of respiratory chain supercomplexes (RCS). Respiratory chain complexes I, III, and IV assemble into higher order quaternary structures called RCS, which promote efficient oxidative metabolism. NLN knockdown significantly impaired RCS formation in T-REx HEK293, OCI-AML2, and NB4 cells, which was rescued by overexpressing wild-type shRNA-resistant NLN. RCS have not been previously studied in leukemia. Therefore, we analyzed their levels in primary AML patient samples and normal hematopoietic cells. RCS assembly was increased in a subset of AML patient samples and positively correlated with NLN protein expression (R2 = 0.83, p < 0.05), suggesting that NLN mediates RCS assembly in AML. To investigate how NLN may be regulating RCS assembly, we analyzed our BioID results to identify NLN interactors that are known regulators of supercomplex formation. Among the top interactors was the known RCS regulator, LETM1. Knockdown of NLN in AML cells impaired LETM1 assembly. Of note, knockdown of LETM1 also reduced growth and oxygen consumption of AML cells. As a chemical approach to evaluate the effects of NLN inhibition on AML cells, we used the allosteric NLN inhibitor R2, (3-[(2S)-1-[(3R)-3-(2-Chlorophenyl)-2-(2-fluorophenyl)pyrazolidin-1-yl]-1-oxopropan-2-yl]-1-(adamantan-2-yl)urea), whose anti-cancer effects have not been previously reported. R2 reduced viability of AML cells, as well as two primary AML culture models, 8227 and 130578. R2 impaired RCS formation in OCI-AML2, NB4, 8227, and primary AML cells. Moreover, R2 reduced the CD34+CD38- stem cell enriched population in 8227 cells, reduced LETM1 complex assembly, and impaired OXPHOS in OCI-AML2 and 8227 cells. Finally, we assessed the effects of inhibiting NLN in mice engrafted with primary AML and normal hematopoietic cells in vivo. Treatment of mice with R2 reduced the leukemic burden in these mice without toxicity. Moreover, inhibiting NLN targeted the AML stem cells as evidenced by reduced engraftment in secondary experiments. In contrast, inhibiting NLN did not reduce the engraftment of normal hematopoietic cells. Collectively, these results demonstrate that inhibition of NLN preferentially targets AML cells and stem cells as compared to normal hematopoietic cells. In summary, we defined a novel role for NLN in RCS formation. We show that RCS are necessary for oxidative metabolism in AML and highlight NLN inhibition as a potential therapeutic strategy. Disclosures Minden: Trillium Therapetuics: Other: licensing agreement. Chan:Agios: Honoraria; AbbVie Pharmaceuticals: Research Funding; Celgene: Honoraria, Research Funding. Schimmer:Medivir Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Consultancy; Otsuka Pharmaceuticals: Consultancy.

Patients with relapsed AML (Acute Myeloid Leukemia) have a poor prognosis and few therapeutic options. Therefore, it remains critical to identify biological vulnerabilities in relapsed AML cells. To identify such vulnerabilities, we analyzed mRNA expression by RNA sequencing of 11 paired diagnosis and relapsed AML samples and identified 34 genes that were upregulated at FDR IPO11 is a member of the importin-β family of proteins functions that facilitate the import of protein cargo into the nucleus. To date, 10 importin-β family members have been identified, but only IPO11 was upregulated at AML relapse, although, importin β1, TNPO3 AND IPO13 were also hits in the CRISPR screen and necessary for AML growth and viability. Therefore, we focused our study on IPO11. Using independent databases of gene expression, we confirmed that IPO11 mRNA was upregulated in LSC+ (engrafting) vs. LSC- (non-engrafting) samples (rank 125, FDR To determine whether IPO11 is necessary for AML growth and viability, we knocked down IPO11 in OCI-AML2, TEX and NB4 leukemia cells with shRNA in lentiviral vectors. Knockdown of IPO11 induced cell cycle arrest with reduced AML growth and viability by 80-90%. In contrast, knockdown of another importin-β family member, IPO5, that was not a hit in our CRIPSR screen did not reduce AML growth and viability. In addition, we demonstrated that knockdown of IPO11 increased differentiation of AML cells as evidenced by increased CD11b expression and staining with non-specific esterase. Finally, knockdown of IPO11 reduced the clonogeneic growth of the above-mentioned AML cell lines and engraftment of TEX cells and the low passage primary AML sample 8227 cells into immune deficient mice by over 90%. To identify cargos of IPO11 whose nuclear import is necessary for AML survival, we compared IPO11 cargo identified through a Bio-ID mass spectrometry protein-protein interaction screen with hits identified from our CRISPR screen. Through this analysis, we identified 7 hits in common to both screens, including RFC5 (replication fork complex unit 5). RFC5 is an essential DNA polymerase accessory protein, involved in telomere maintenance, DNA replication and mismatch repair. Knockdown of IPO11 decreased the nuclear localization of RFC5 in AML cells. In addition, knockdown of RFC5 in AML cells decreased AML growth and decreased clonogeneic growth. Thus, in summary, comparing genes that are upregulated in paired diagnosis and relapse AML samples to our CRISPR screen we identified IPO11 as a single hit. IPO11 knockdown reduced growth and viability, promoted differentiation, reduced CFU generation of several AML cells lines. In a mouse model we show that IPO11 knockdown dramatically reduced engraftment of leukemic cells. We further identify RFC5, a protein involved in replication and DNA damage response as one of the cargos of IPO11. Our study suggests IPO11 supports LSC survival and relapse and provides a new target for novel therapeutic intervention. Disclosures Schimmer: Otsuka Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy.

Antibody-based immunotherapy represents a promising strategy to target chemo-resistant leukemic cells. However, current antibody-based approaches are restricted to cell lineage surface antigens. Targeting intracellular antigens enables to enlarge the number of suitable tumor-associated target antigens with a more restricted expression profile. In this study we evaluated a 2+1 T Cell Bispecific (TCB) antibody for immunotherapy of acute myeloid leukemia (AML). The T cell receptor (TCR)-like TCB targets the intracellular tumor antigen Wilms tumor 1 (WT1) by bivalent recognition of the peptide RMFPNAPYL in the context of human leukocyte antigen allele A*02 (HLA-A2). Complementary binding to CD3ε recruits T cells irrespective of their TCR-specificity. We further analyzed enhancement of TCB-mediated T cell cytotoxicity through combination with the immune-modulatory drug lenalidomide. WT1 expression levels in cancer cell lines and primary AML patient samples at different time points during course of the disease were determined by quantitative real-time PCR, western blot and immunohistochemical staining. WT1-TCB-mediated cytotoxicity was analyzed by co-cultivation of WT1-expressing HLA-A2+ cancer cell lines with T cells from healthy donors. Specific lysis was assessed by flow cytometry. TCR downstream signaling was measured by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCB-mediated cytotoxicity against primary AML cells and combination with 10 μM lenalidomide was evaluated in our pre-established feeder layer-based ex vivo long-term culture system. For in vivo testing, NSG mice (NOD.Cg-Prkdcscid-Il2rgtm1Wjl/SzJ) were humanized with human HLA-A2+ CD34+ cord blood cells. After successful engraftment and development of human T cells, WT1-expressing HLA-A2+ SKM-1 tumor cells were subcutaneously inoculated followed by weekly administration of the WT1-TCB. In accordance with previous reports, we observed WT1 expression in 79% (n=38) of cancer cell lines and in 92% (n=65) of AML patient samples at the time of initial diagnosis. Moreover, WT1 expression levels correlated with the percentage of AML blasts: no significant WT1 expression was observed at time of CR (n=26), whereas WT1 was expressed again at time of relapse (n=21). WT1-TCBs elicited antibody-mediated T cell cytotoxicity against peptide-pulsed T2 cells and AML cell lines in a WT1 and HLA-restricted manner. Equally, TCR downstream signaling was observed in a WT1-restrictive manner by co-cultivation of primary AML cells with NFAT Luciferase Reporter Jurkat cells. WT1-TCBs further mediated specific lysis of primary AML cells upon addition of allogenic T cells from healthy donors (mean specific lysis: 67±6% after 13-14 days; ±SEM; n=18). Correspondingly, up-regulation of T cell activation and surrogate exhaustion markers was observed (MFI fold change CD69: 9.3±1.5, PD-1: 5.1±0.7, TIM-3: 4.7±0.6; ±SEM; n=22). WT1-TCBs also mediated killing of primary AML cells in an autologous setting (mean specific lysis: 38±13% after 13-14 days; ±SEM; n=5). In comparison with WT1RMF-specific T cells, only bivalent binding by WT1-TCB induced efficient lysis of primary AML cells. Interestingly, combination of WT1-TCB with lenalidomide further enhanced antibody-mediated T-cell cytotoxicity against primary AML cells (mean specific lysis on day 3-4: 32±10% vs 59±9%; p=0.0017; ±SEM; n=13). This was accompanied by an increased secretion of the proinflammatory cytokines IL-2, IFN-γ and TNF-α and promoted the differentiation of naïve T cells towards a memory phenotype characterized by a downregulation of CD45RA. Furthermore, WT1-TCB-treated humanized mice bearing SKM-1 tumors showed a dose dependent and significant reduction in tumor growth resulting in tumor control. TCR-like TCBs targeting intracellular tumor antigens are a promising tool for cancer immunotherapy. Notably, the 2+1 TCB molecular format for bivalent binding facilitates potent in vitro, ex vivo and in vivo killing of AML cell lines and primary AML samples which present low numbers of the RMF peptide-MHC complex on the cell surface validating WT1-TCB as a promising therapeutic agent for the treatment of AML. Our results further indicate that the combinatorial approach with lenalidomide leads to increased TCB-mediated T cell cytotoxicity. Disclosures Klein: Roche: Employment, Equity Ownership, Patents & Royalties. Xu:Roche: Employment, Equity Ownership, Patents & Royalties. Heitmüller:Roche: Employment. Hanisch:Roche: Employment, Equity Ownership, Patents & Royalties. Sam:Roche: Employment, Equity Ownership, Patents & Royalties. Pulko:Roche: Employment, Equity Ownership, Patents & Royalties. Schönle:Roche: Employment, Equity Ownership, Patents & Royalties. Challier:Roche: Employment, Equity Ownership, Patents & Royalties. Carpy:Roche: Employment, Equity Ownership, Patents & Royalties. Lichtenegger:Roche: Employment. Umana:Roche: Employment, Equity Ownership, Patents & Royalties. Subklewe:Roche: Consultancy, Research Funding; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Morphosys: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AMGEN: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy.

Background Acute erythroleukemia (AEL) is defined as an distinct subtype ( Method In our study, we collected blood samples of 11 AEL patient, 20 other types of AML patients and 20 normal patients. qPCR and Western blot analysis were performed to examine the expression of KEL and circ-KEL. We set up the erythroid differentiation models in vitro using two AEL cells lines (K562 and HEL) , induced with 50 μM hemin from day1 to day 7. The oncogenic functions of KEL were further measured in AEL cell lines by performing RNA interfering (RNAi), immunofluorescence (IF) staining, CCK8 assay and flow cytometry. The expression levels of γ-globin and FUT1 as well as the erythroid surface markers TER119 and GPA were examined to evaluate their function in erythroid differentiation. The proliferative ability of AEL cells was determined by CCK-8 assay. AEL animal models were established using NCG mice transplanted with K562 (NC and KEL over-expression). The tumor formation rate, body weight and survival curve were investigated to evaluate the oncogenic functions of KEL in-vivo. Protein antibody microarray was used to analyze the activation of related pathways after KEL over-expression in AEL cells. Moreover, we explored the molecular mechanisms of KEL and verified the interactions among GATA1 and H3K27ac as well as the PKC/RSK2/CREB pathway by performing ChIP, RIP, dual-luciferase reporter assay and Western blotting. Result KEL was specifically higher expressed in AEL clinical samples/cells lines, consistent with the results of the database. The over-expression of KEL could up-regulate the expression of GATA1, thus promoting the erythroid differentiation of AEL cells. Moreover, protein antibody microarray results showed that the up-regulation of KEL in AEL cells leads to the activation of multiple proliferation and PDL1 related pathways. After subsequent verification, it was verified that PKC/RSK2/CREB pathway was significantly activated. Meanwhile, the expression of PDL1 was highly correlated with KEL. We found that the higher expression of KEL in AEL cells could up-regulate PDL1 expression through activating the phosphorylation of relative transcript factors on PDL1 DNA promoter. In-vivo experiment showed that KEL over-expression may accelerate the leukemia progression. In addition, we discovered that JQ1 could be used as a inhibitor of KEL, which could effectively decrease the expression of GATA1 and PDL1. A number of circRNAs have been reported to act as microRNA (miRNA) sponges, which regulate target gene expression by interacting with miRNA. Our results revealed that circ-KEL could serve as a sponge for miR-671 and up-regulate miR-617 functional target KEL, thus promote the erythroid differentiation. Conclusion Our study reveals the oncogenic role and a novel regulatory mechanism of KEL in AEL progression. For the first time, we demonstrated that KEL was specifically highly expressed in AEL cells, leading to the dysregulation of erythroid differentiation through up-regulating GATA1, and on the other hand, promoting cell proliferation via PKC/RSK2/CREB pathway. Circ-KEL which also had a high expression in AEL, may play a role of coordinate regulation with KEL. These findings may provide a promising strategy for future diagnosis, biomarker discoveries and treatment for AEL patients. Figure Disclosures No relevant conflicts of interest to declare.

Introduction: The phosphatidylinositol-3-kinase (PI3K) pathway plays an important role in multiple cellular functions, including proliferation, differentiation, and trafficking, and deregulation of PI3K is a hallmark of many malignancies. The preferential expression of the PI3Kδ isoform on leukocytes provides an attractive target for hematologic malignancies. SHC014748M is a novel, potent, and highly selective PI3Kδ inhibitor. This study is a phase I, first-in-human study to assess the safety, tolerability and pharmacokinetics of SHC014748M in patients with relapsed or refractory indolent B-cell lymphomas (NCT03588598). Methods: Dose escalation was performed in a 3+3 design in sequential cohorts at doses of 50-250 mg QD, and then dose expansion was conducted at the selected cohorts to find recommended Phase II dose (RP2D). Eligible patients were those with relapsed or refractory indolent B-cell lymphomas, having at least 1 measurable lesion, and with ECOG performance status 0-2. Patients received a single dose on day 1, and then after 3 days washout multiple dosing once daily was initiated until disease progression or unacceptable toxicity. Primary endpoints included safety, tolerability and pharmacokinetics. Secondary endpoints included overall response rate (ORR), lymph node response (LNR), time to response (TTR), progression-free survival (PFS) and duration of response (DOR). Results: As of Jul 16, 2019, 38 patients with relapsed or refractory indolent B-cell lymphomas were enrolled in the study, including 23 with follicular lymphoma (FL), 11 with chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL), 1 with marginal zone lymphoma (MZL) and 3 with Waldenstrom's macroglobulinemia (WM). 15 patients were treated in the dose escalation phase and 23 were treated in the dose expansion phase (12 in 150 mg cohort, 11 in 200 mg cohort). The average number of prior therapies was 2.2 (range 1-8), and 84 percent of patients had received rituximab before. No dose-limiting toxicities occurred. The most common adverse reactions were neutropenia (39.5%), fatigue (21.1%), thrombocytopenia (18.4%), diarrhea (13.2%), pneumonia (13.2%), cough (13.2%), lymphopenia (13.2%), anemia (13.2%) and rash (13.2%). Grade ≥ 3 adverse reactions were neutropenia (23.7%), pneumonia (13.2%), rash (7.9%) and diarrhea (5.3%). Among 14 FL patients who were available for efficacy evaluation, ORR was 57.1% (8 PR, 5 SD, 1 PD). Of 9 CLL/SLL evaluable patients, 5 (55.6%) had partial response, 3 (33.3%) had partial response with lymphocytosis, and 1 had stable disease. Of 3 WM patients, 1 had partial response and 2 had minor response. Conclusion: SHC014748M was well tolerated and demonstrated promising clinical activity in patients with relapsed or refractory indolent B-cell lymphomas. Disclosures Wang: Nanjing Sanhome Pharmaceutical Co., Ltd.: Employment.

Bruton's Tyrosine Kinase (BTK) plays a critical role in B-cell receptor (BCR) signaling, which mediates B-cell proliferation, migration, and adhesion. As a therapeutic target, its clinical significance has been well established in multiple subtypes of non-Hodgkin's lymphoma (NHL). Orelabrutinib (ICP-022) is a novel, potent irreversible BTK inhibitor with much improved target selectivity in comparison to Ibrutinib and Acalabrunitib, which leads to improved safety profiles. With a proprietary formulation, Orelabrutinib achieves high bioavailability comparing to other BTK inhibitors. Results of Phase I study (another presentation on Orelabrutinib) demonstrated favorable pharmacokinetic and pharmacodynamic properties for Orelabrutinib. Sustained BTK occupancy at 24 hr was achieved with daily dosing regimen. In this presentation, we will report results from clinical study of Orelabrutinib in Chinese patients with r/r CLL/SLL. This is an open-label, multicenter, Phase II study with objectives to evaluate its safety, tolerability and efficacy following oral daily administration. The primary endpoint was objective response rate (ORR). The duration of response (DOR), progression-free survival (PFS) and safety were chosen as secondary endpoints. Response was assessed per 2008 IWCLL criteria with modification for PR with lymphocytosis (PR-L) (Cheson, Hallek 2012). This study is composed of two stages. The stage I was to assess the safety and tolerability of Orelabrutinib at 150 mg, QD in pts with r/r CLL/SLL, while the stage II was to evaluate its therapeutic benefits (N=80, 150 mg, QD). Total of 80 patients with r/r CLL (n=70)/SLL (n=10) were enrolled. As of 31 May 2019, 40 pts had completed six cycles of treatment (28 days/cycle). The Median follow-up was 6.3 months (range, 0.4-13.7 months). Safety: The most frequent (≥15%) any grade adverse events (AEs) of any cause were well characterized hematological toxicities: thrombocytopenia, neutropenia, and anemia; and respiratory system infections as well as purpura. No case of atrial fibrillation or secondary malignancy was reported. The most frequently (≥10%) reported ≥ Grade 3 AEs of any cause were neutropenia, thrombocytopenia, lung infection. Twenty-five pts experienced at least one serious AE. Of those, 13 were considered related to Orelabrutinib treatment, including platelet count decrease (3 pts), pneumonitis, pyrexia (2 pts each) and herpes zoster etc. (1 pt each). Efficacy: Of the 80 enrolled pts, seventy-eight pts were evaluable for response (by 31 of May 2019), the ORR was 88.5% (69/78). Among them, one patient was reported as CR, 39 pts were PR and 29 pts were PR-L. Stable disease was seen in 6/78 (7.7%). Total disease control rate is 96.2%. The median DOR was not reached, 6 months DOR rate was 89.8%. Subgroup analysis (age, disease stage, previous treatment, 17p deletion, 11q deletion, IGHV mutation) did not reveal significant differences. Conclusion: Orelabrutinib is safe and well tolerated. No significant adverse events like atrial fibrillation/flutter or secondary malignancies were reported. Orelabrutinib is efficacious to treat pts with r/r CLL/SLL. The improved safety, resulting from high target selectivity, and daily dosing regimen enable Orelabrutinib to be a valuable therapeutic choice both as monotherapy and likely in combination with other agents to treat B-cell malignancies. Disclosures Xu: Beijing InnoCare Pharma Tech Co., Ltd: Employment. Zhao:Beijing InnoCare Pharma Tech Co., Ltd: Employment. Zhang:Beijing InnoCare Pharma Tech Co., Ltd: Employment. Zhu:Beijing InnoCare Pharma Tech Co., Ltd: Employment.

Introduction: Socioeconomic factors including insurance and marital status have impacts on the outcomes of cancer patients. Until now, there are few data regarding whether insurance status and marital status have effects on the outcomes of patients with chronic lymphocytic leukemia (CLL). In this study, the Surveillance, Epidemiology, and End Results (SEER) database was used to evaluate the prognostic roles of insurance and marital status in patients with CLL. Methods: Data from the SEER 18 Registries were used to conduct this study. Cases with newly-diagnosed CLL/small lymphocytic lymphoma (CLL/SLL) (International Classification of Diseases for Oncology, 3rd Edition [ICDO-3] codes 9823) in the time period between 2008 and 2015 were included. Exclusion criteria included history of cancer, unknown insurance status, unknown marital status, unknown survival data, unknown cause of death and survival months documented as 0. For each case we included age at the time of diagnosis, sex, marital status (married, divorced, single, widowed, unmarried or domestic partner, or separated), insurance status (Medicaid, insured, or uninsured), SEER cause-specific death classification, survival months and vital status. Survival curves were plotted by the Kaplan-Meier method and the log-rank test was used for comparison. P value was 2-sided and P Results: Atotal of 23,611 patients with CLL/SLL were included into the current analysis. The median follow-up was 34 months. We found that insurance status (uninsured or Medicaid) was significantly associated with decreased cancer-specific survival (CSS) (hazards ratio[HR]: 1.378, 95% confidence interval [CI]:1.251-1.664, P Conclusion: Insurance status and marital status have significant impacts on survival outcomes of patients with CLL/SLL. A 3-points prognostic index comprising insurance status, marital status and age could be used for risk stratification for patients with CLL/SLL. Figure 1 Disclosures No relevant conflicts of interest to declare.