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Purpose: This global phase I trial investigated the safety, efficacy, pharmacokinetics, and pharmacodynamics of lisaftoclax (APG-2575), a novel, orally active, potent selective B-cell lymphoma 2 (BCL-2) inhibitor, in patients with relapsed or refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (R/R CLL/SLL) and other hematologic malignancies (HMs). Patients and Methods: Maximum tolerated dose (MTD) and recommended phase II dose were evaluated. Outcome measures were safety and tolerability (primary) and pharmacokinetic variables and antitumor effects (secondary). Pharmacodynamics in patient tumor cells were explored. Results: Among 52 patients receiving lisaftoclax, MTD was not reached. Treatment-emergent adverse events (TEAEs) included diarrhea (48.1%), fatigue (34.6%), nausea (30.8%), anemia and thrombocytopenia (28.8% each), neutropenia (26.9%), constipation (25.0%), vomiting (23.1%), headache (21.2%), peripheral edema and hypokalemia (17.3% each), and arthralgia (15.4%). Grade ≥ 3 hematologic TEAEs included neutropenia (21.2%), thrombocytopenia (13.5%), and anemia (9.6%), none resulting in treatment discontinuation. Clinical pharmacokinetic and pharmacodynamic results demonstrated that lisaftoclax had a limited plasma residence and systemic exposure and elicited rapid clearance of malignant cells. With a median treatment of 15 (range, 6–43) cycles, 14 of 22 efficacy-evaluable patients with R/R CLL/SLL experienced partial responses, for an objective response rate of 63.6% and median time to response of 2 (range, 2–8) cycles. Conclusions: Lisaftoclax was well tolerated, with no evidence of tumor lysis syndrome. Dose-limiting toxicity was not reached at the highest dose level. Lisaftoclax has a unique pharmacokinetic profile compatible with a potentially more convenient daily (vs. weekly) dose ramp-up schedule and induced rapid clinical responses in patients with CLL/SLL, warranting continued clinical investigation.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.Significance:Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.This article is highlighted in the In This Issue feature, p. 101

These are all the Supplementary Figures: S1, S2, S3, S4, S5, S6, and S7

Summary of BH3 profiling for PBMC samples collected at baseline.

Representativeness of study participants.

Purpose:This global phase I trial investigated the safety, efficacy, pharmacokinetics, and pharmacodynamics of lisaftoclax (APG-2575), a novel, orally active, potent selective B-cell lymphoma 2 (BCL-2) inhibitor, in patients with relapsed or refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (R/R CLL/SLL) and other hematologic malignancies (HMs).Patients and Methods:Maximum tolerated dose (MTD) and recommended phase II dose were evaluated. Outcome measures were safety and tolerability (primary) and pharmacokinetic variables and antitumor effects (secondary). Pharmacodynamics in patient tumor cells were explored.Results:Among 52 patients receiving lisaftoclax, MTD was not reached. Treatment-emergent adverse events (TEAEs) included diarrhea (48.1%), fatigue (34.6%), nausea (30.8%), anemia and thrombocytopenia (28.8% each), neutropenia (26.9%), constipation (25.0%), vomiting (23.1%), headache (21.2%), peripheral edema and hypokalemia (17.3% each), and arthralgia (15.4%). Grade ≥ 3 hematologic TEAEs included neutropenia (21.2%), thrombocytopenia (13.5%), and anemia (9.6%), none resulting in treatment discontinuation. Clinical pharmacokinetic and pharmacodynamic results demonstrated that lisaftoclax had a limited plasma residence and systemic exposure and elicited rapid clearance of malignant cells. With a median treatment of 15 (range, 6–43) cycles, 14 of 22 efficacy-evaluable patients with R/R CLL/SLL experienced partial responses, for an objective response rate of 63.6% and median time to response of 2 (range, 2–8) cycles.Conclusions:Lisaftoclax was well tolerated, with no evidence of tumor lysis syndrome. Dose-limiting toxicity was not reached at the highest dose level. Lisaftoclax has a unique pharmacokinetic profile compatible with a potentially more convenient daily (vs. weekly) dose ramp-up schedule and induced rapid clinical responses in patients with CLL/SLL, warranting continued clinical investigation.

Transformation to aggressive disease histologies generates formidable clinical challenges across cancers, but biological insights remain few. We modeled the genetic heterogeneity of chronic lymphocytic leukemia (CLL) through multiplexed in vivo CRISPR-Cas9 B-cell editing of recurrent CLL loss-of-function drivers in mice and recapitulated the process of transformation from indolent CLL into large cell lymphoma [i.e., Richter syndrome (RS)]. Evolutionary trajectories of 64 mice carrying diverse combinatorial gene assortments revealed coselection of mutations in Trp53, Mga, and Chd2 and the dual impact of clonal Mga/Chd2 mutations on E2F/MYC and interferon signaling dysregulation. Comparative human and murine RS analyses demonstrated tonic PI3K signaling as a key feature of transformed disease, with constitutive activation of the AKT and S6 kinases, downmodulation of the PTEN phosphatase, and convergent activation of MYC/PI3K transcriptional programs underlying enhanced sensitivity to MYC/mTOR/PI3K inhibition. This robust experimental system presents a unique framework to study lymphoid biology and therapy.Significance:Mouse models reflective of the genetic complexity and heterogeneity of human tumors remain few, including those able to recapitulate transformation to aggressive disease histologies. Herein, we model CLL transformation into RS through multiplexed in vivo gene editing, providing key insight into the pathophysiology and therapeutic vulnerabilities of transformed disease.This article is highlighted in the In This Issue feature, p. 101

Supplementary file contains a PDF version of supplementary figures S1-S7 and associated figure legends.

Supplementary file contains all supplementary tables S1-S16 in Excel format in the order in which they appear in the text.

Supplementary methods, Table S1, supplementary figures 1-9

Pathological analysis of human BPDCN growing as a patient-derived xenograft (PDX) in mice. Splenic tissue is shown stained with H&E (at 40x and 100x magnification), and human BCL-2, CD45, CD4, CD56, and CD123 (all at 40x) from animals bearing a representative BPDCN PDX.

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics.Significance:Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution.See related commentary by Hilton and Scott, p. 2965.This article is highlighted in the In This Issue feature, p. 2945

Supplementary Figure Legends

Supplementary Table 2 - %CLL cells marked by mtDNA mutations

Response of BPDCN and AML cells to navitoclax and A-1331852. (A) MTT assay results for the indicated cell lines after 72 hours of exposure to the indicated doses of navitoclax (combined BCL-2/BCL-XL inhibitor). (B) MTT assay results for the indicated cell lines after 72 hours of exposure to the indicated doses of A-1331852 (BCL-XL only inhibitor). In both panels, data points represent mean of 3 replicates +/- SEM, line represents non-linear regression (curve fit).

Time and dose-dependent response of primary BPDCN to venetoclax in vitro. (A) Cell death measurement by flow cytometry at 4 and 8 hours after addition of venetoclax at the indicated doses in a primary BPDCN, represented as change in Annexin V positivity relative to vehicle. (B) Dose response of primary BPDCNs and AMLs to venetoclax in vitro measured as in (A).

Venetoclax induces dose-dependent apoptotic cell death in BPDCN and AML cell lines. (A) Representative Annexin V-FITC vs propidium iodide (PI) flow cytometry staining to measure apoptosis induction in CAL1 cells after 24 hours of exposure to vehicle, or 10 nM or 100 nM venetoclax are shown. (B) Quantitation of cell death by Annexin V and PI flow cytometry after 24 hours of exposure to the indicated concentrations of venetoclax. Graphs represent mean +/- SEM of 3 independent experiments, *P

BPDCN expresses BCL-2 protein. Photomicrographs of H&E stains and BCL-2, CD123, and TCL-1 immunohistochemistry for BPDCN and AML biopsies from bone marrow and skin are shown. Each of the biopsies (each row) is from a different patient. The BPDCN from Figure 1A is re-presented here for comparison to other BPDCNs and AMLs.

Additional pharmacodynamic data showing in vivo PDX response to venetoclax. PD assessment of percent human CD45+CD123+ cells in the peripheral blood (PB), spleen, and bone marrow (BM) of six animals bearing PDX1 after 21 days of treatment with vehicle (n=3) or venetoclax (n=3).

Genetic analysis of the primary BPDCNs and AMLs, and PDXs.

Figure S2C displays probability of progression-free survival over time in subgroups of patients with and without bulky disease

Supplementary Data from Long-term Follow-up of Patients with Relapsed or Refractory Non–Hodgkin Lymphoma Treated with Venetoclax in a Phase I, First-in-Human Study

Figure S1. Individual and mean AUC0-24 ({plus minus} standard deviation) in patients with solid tumors (37) (n = 14) or lymphoma (n = 9) on cycle 1, day 28 after treatment with pilaralisib (SAR245408) 600 mg capsules once daily.

Purpose:In the phase III DUO trial, duvelisib, an oral dual PI3K-δ,γ inhibitor, demonstrated significantly improved efficacy versus ofatumumab [median (m) progression-free survival (PFS), 13.3 vs. 9.9 months (HR, 0.52; P < 0.0001); overall response rate [ORR], 74% vs. 45% (P < 0.0001)], with a manageable safety profile in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). We report results from patients with progressive disease (PD) after ofatumumab who crossed over to duvelisib in the DUO trial.Patients and Methods:Patients with radiographically confirmed PD after ofatumumab received duvelisib 25 mg twice daily in 28-day cycles until PD, intolerance, death, or study withdrawal. The primary endpoint was ORR per investigator. Secondary endpoints included duration of response (DOR), PFS, and safety.Results:As of December 14, 2018, 90 ofatumumab-treated patients in the DUO trial prior to crossover had an ORR of 29%, mDOR of 10.4 months, and mPFS of 9.4 months. After crossover, 77% of patients (69/90) achieved a response, with an mDOR of 14.9 months and mPFS of 15.7 months. Patients with del(17p) and/or TP53 mutations had similar outcomes [ORR, 77% (20/26); mPFS, 14.7 months]. Notably, 73% of patients (47/64) with disease previously refractory to ofatumumab achieved a response. The most frequent any-grade/grade 3/4 treatment-emergent adverse events were diarrhea (47%/23%), neutropenia (26%/23%), pyrexia (24%/4%), cutaneous reactions (23%/4%), and thrombocytopenia (10%/6%).Conclusions:Duvelisib demonstrated high response rates with good durability and a manageable safety profile in patients with R/R CLL/SLL who progressed on ofatumumab, including patients with high-risk disease and disease previously refractory to ofatumumab.

Purpose: This phase I expansion-cohort study evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of the pan-PI3K inhibitor pilaralisib (SAR245408/XL147) in patients with chronic lymphocytic leukemia (CLL) or relapsed or refractory lymphoma.Patients and Methods: Patients were treated with the maximum tolerated dose of pilaralisib previously determined in patients with solid tumors (600 mg capsules once daily). Adverse events (AE) and response were evaluated. Plasma pharmacokinetics and pharmacodynamic effects on cytokines and chemokines were also assessed.Results: Twenty-five patients were included in the study: 10 with CLL and 15 with lymphoma. The most frequent AEs of any grade were diarrhea (92.0%), pyrexia (52.0%), and fatigue (44.0%). The most frequent grade ≥3 AEs were neutropenia (32.0%), diarrhea (20.0%), and anemia (16.0%). Pilaralisib exposure on cycle 1 day 28 was similar to exposure in patients with solid tumors. In patients with CLL, pilaralisib significantly reduced plasma levels of several cytokines and chemokines involved in B-cell trafficking. Five patients (50.0%) with CLL and 3 patients (20.0%) with lymphoma had a partial response. Six patients (60.0%) with CLL had nodal shrinkage ≥50%. Overall, 14 patients (56.0%; 7 patients with CLL and 7 patients with lymphoma) had progression-free survival ≥6 months.Conclusions: Pilaralisib demonstrated an acceptable safety profile in patients with CLL and lymphoma, generally consistent with findings in patients with solid tumors. Single-agent pilaralisib showed preliminary clinical activity in patients with CLL and lymphoma, supporting further development. Clin Cancer Res; 21(14); 3160–9. ©2015 AACR.

Supplemental Figure Legend

Purpose: The oral BCL-2 inhibitor venetoclax is an effective therapy for patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL), including disease with high-risk genomic features such as chromosome 17p deletion [del(17p)] or progressive disease following B-cell receptor pathway inhibitors.Patients and Methods: We conducted a comprehensive analysis of the safety of 400 mg daily venetoclax monotherapy in 350 patients with CLL using an integrated dataset from three phase I/II studies.Results: Median age was 66 years and 60% had del(17p). Patients had received a median of three prior therapies (range: 0–15); 42% previously received ibrutinib or idelalisib. Median duration of exposure to venetoclax was 16 months (0–56). In the pooled analysis, the most common adverse events (AE) of any grade were diarrhea (41%), neutropenia (40%), nausea (39%), anemia (31%), fatigue (28%), and upper respiratory tract infection (25%). The most common grade 3/4 AEs were neutropenia (37%), anemia (17%), and thrombocytopenia (14%). With the current 5-week ramp-up dosing, the incidence of laboratory TLS was 1.4% (2/166), none had clinical sequelae, and all of these patients were able to ramp-up to a daily dose of 400 mg. Grade 3/4 neutropenia was manageable with growth factor support and dose adjustments; the incidence of serious infections in these patients was 15%. Ten percent of patients discontinued venetoclax due to AEs and 8% died while on study, with the majority of deaths in the setting of disease progression.Conclusions: Venetoclax as a long-term continuous therapy is generally well tolerated in patients with R/R CLL when initiated with the current treatment algorithm. Clin Cancer Res; 24(18); 4371–9. ©2018 AACR.

Purpose:To develop a prognostic model and cytogenetic risk classification for previously treated patients with chronic lymphocytic leukemia (CLL) undergoing reduced intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT).Experimental Design:We performed a retrospective analysis of outcomes of 606 patients with CLL who underwent RIC allogeneic HCT between 2008 and 2014 reported to the Center for International Blood and Marrow Transplant Research.Results:On the basis of multivariable models, disease status, comorbidity index, lymphocyte count, and white blood cell count at HCT were selected for the development of prognostic model. Using the prognostic score, we stratified patients into low-, intermediate-, high-, and very-high-risk [4-year progression-free survival (PFS) 58%, 42%, 33%, and 25%, respectively, P < 0.0001; 4-year overall survival (OS) 70%, 57%, 54%, and 38%, respectively, P < 0.0001]. We also evaluated karyotypic abnormalities together with del(17p) and found that del(17p) or ≥5 abnormalities showed inferior PFS. Using a multivariable model, we classified cytogenetic risk into low, intermediate, and high (P < 0.0001). When the prognostic score and cytogenetic risk were combined, patients with low prognostic score and low cytogenetic risk had prolonged PFS (61% at 4 years) and OS (75% at 4 years).Conclusions:In this large cohort of patients with previously treated CLL who underwent RIC HCT, we developed a robust prognostic scoring system of HCT outcomes and a novel cytogenetic-based risk stratification system. These prognostic models can be used for counseling patients, comparing data across studies, and providing a benchmark for future interventions. For future study, we will further validate these models for patients receiving targeted therapies prior to HCT.

Figure S3 displays probability of overall survival over time in the study population

Supplementary Material

•Table S1 shows a summary of prior anticancer therapies received by all patients in the study •Table S2 shows best overall response, overall response rate, and duration of response by investigator in the study population of patients while receiving ofatumumab in the parent DUO study before crossover •Table S3 shows rates of all serious TEAEs and individual hematologic and non-hematologic serious TEAEs in {greater than or equal to} 2% of patients in the study population • Table S4 shows rates of all TEAEs leading to death in patients in the study population •Table S5 shows a summary of patient disposition information

Figure S1 displays probability of progression-free survival over time in the study population and in the subgroup of patients with del(17p) and/or TP53 mutations that received ofatumumab while participating in the parent DUO study before crossover

Purpose:We previously reported a 44% overall response rate (ORR) with the oral BCL-2 inhibitor venetoclax in a phase I study of relapsed/refractory non–Hodgkin lymphoma (NHL). Complete response (CR) was observed in patients with mantle cell lymphoma [(MCL), 21%, n = 6/28] and follicular lymphoma [(FL), 17%, n = 5/29], and partial response (PR) noted in several patients with Waldenström macroglobulinemia (WM), and marginal zone lymphoma (MZL). Here, we report the long-term outcomes of these four cohorts.Patients and Methods:All patients (n = 106) received venetoclax monotherapy in dose cohorts of 200 to 1,200 mg daily until disease progression or unacceptable toxicity. ORR, progression-free survival (PFS), duration of response (DoR), and adverse events (AEs) were evaluated.Results:At a median follow-up of 38.5 months (range, 30.0–46.5), the median PFS for all 106 patients was 5.4 [95% confidence interval (CI), 3.5–8.4] months (FL, 10.8; MCL, 11.3; MZL, 21.2; and WM, 30.4). The median DoR was 14.9 (95% CI, 9.7–27.6) months (FL, 26.6; MCL, 15.7; MZL, 20.1; and WM, 25.3). Achievement of CR versus PR predicted longer DoR in both MCL (31.5 vs. 10.1 months) and FL (37.6 vs. 9.7 months). All grade hematologic AEs were infrequent: neutropenia (19%), anemia (19%), and thrombocytopenia (17%), with no new cytopenias after 2 years on therapy. Nonhematologic AEs included nausea (49%), diarrhea (46%), fatigue (44%), with decreased incidence after 1 year.Conclusions:Venetoclax monotherapy has a manageable safety profile and achieves durable responses in a subset of patients with FL, MCL, WM, and MZL, particularly in those who achieve CR. Further research is warranted on combination strategies to enhance the durability of response to venetoclax.

Supplementary Data

PDF file - 12K, Sensitivity of lung cancer cell lines to the PARP1 inhibitor AG014699

PDF file - 135K, 293T cells were transfected with empty pcDNA3.1 or pcDNA3.1-HA-BCL2. After 24 hours, cells were harvested and lysates were separated into nuclear (nuc) and cytoplasmic (cyto) fractions. Western blotting was performed using indicated antibodies. Untransfected 293T, OCI-LY1 and OCI-LY8 cells were collected while in exponential culture

PDF file - 19K, Peptides recovered from immunprecipitation of BCL2 from the chromatin fraction of irradiated cells that matched PARP1 with at least 99% confidence

PDF file - 108K

PDF file - 18K, Clinical characteristics for the 14 patients outlined in Figure 6A. The majority of cases were previously untreated and harbored 13q deletion, the most common cytogenetic abnormality in CLL. Several higher risk patients with unmutated IGHV, positive ZAP-70, or adverse cytogenetics were also assessed

While cancers evolve during disease progression and in response to therapy, temporal dynamics remain difficult to study in humans due to the lack of consistent barcodes marking individual clones in vivo. We employ mitochondrial single-cell assay for transposase-accessible chromatin with sequencing to profile 163,279 cells from 9 patients with chronic lymphocytic leukemia (CLL) collected across disease course and utilize mitochondrial DNA (mtDNA) mutations as natural genetic markers of cancer clones. We observe stable propagation of mtDNA mutations over years in the absence of strong selective pressure, indicating clonal persistence, but dramatic changes following tight bottlenecks, including disease transformation and relapse posttherapy, paralleled by acquisition of copy-number variants and changes in chromatin accessibility and gene expression. Furthermore, we link CLL subclones to distinct chromatin states, providing insight into nongenetic sources of relapse. mtDNA mutations thus mirror disease history and provide naturally occurring genetic barcodes to enable patient-specific study of cancer subclonal dynamics.Significance:Single-cell multi-omic profiling of CLL reveals the utility of somatic mtDNA mutations as in vivo barcodes, which mark subclones that can evolve over time along with changes in accessible chromatin and gene expression profiles to capture dynamics of disease evolution.See related commentary by Hilton and Scott, p. 2965.This article is highlighted in the In This Issue feature, p. 2945

The management of chronic lymphocytic leukemia (CLL) has undergone unprecedented changes over the last decade. Modern targeted therapies are incorporated into clinical practice. Unfortunately, patients have begun to develop resistance or intolerance to multiple classes. Symptomatic patients previously treated with a BTK inhibitor (BTKi) and venetoclax represent a new and rapidly growing unmet need in CLL. Here, we define unmet needs in a modern treatment context. We also critically review the literature for PI3K inhibitors and chemoimmunotherapy and lack of data to support their utility following BTKis and venetoclax. Finally, we suggest opportunities to ensure the continued innovation for patients with CLL.

Purpose: We previously reported a 44% overall response rate (ORR) with the oral BCL-2 inhibitor venetoclax in a phase I study of relapsed/refractory non–Hodgkin lymphoma (NHL). Complete response (CR) was observed in patients with mantle cell lymphoma [(MCL), 21%, n = 6/28] and follicular lymphoma [(FL), 17%, n = 5/29], and partial response (PR) noted in several patients with Waldenström macroglobulinemia (WM), and marginal zone lymphoma (MZL). Here, we report the long-term outcomes of these four cohorts. Patients and Methods: All patients (n = 106) received venetoclax monotherapy in dose cohorts of 200 to 1,200 mg daily until disease progression or unacceptable toxicity. ORR, progression-free survival (PFS), duration of response (DoR), and adverse events (AEs) were evaluated. Results: At a median follow-up of 38.5 months (range, 30.0–46.5), the median PFS for all 106 patients was 5.4 [95% confidence interval (CI), 3.5–8.4] months (FL, 10.8; MCL, 11.3; MZL, 21.2; and WM, 30.4). The median DoR was 14.9 (95% CI, 9.7–27.6) months (FL, 26.6; MCL, 15.7; MZL, 20.1; and WM, 25.3). Achievement of CR versus PR predicted longer DoR in both MCL (31.5 vs. 10.1 months) and FL (37.6 vs. 9.7 months). All grade hematologic AEs were infrequent: neutropenia (19%), anemia (19%), and thrombocytopenia (17%), with no new cytopenias after 2 years on therapy. Nonhematologic AEs included nausea (49%), diarrhea (46%), fatigue (44%), with decreased incidence after 1 year. Conclusions: Venetoclax monotherapy has a manageable safety profile and achieves durable responses in a subset of patients with FL, MCL, WM, and MZL, particularly in those who achieve CR. Further research is warranted on combination strategies to enhance the durability of response to venetoclax.

Despite the use of high-dose chemotherapy and HSCT, less than 70% of adult AML patients enter complete remission and most patients relapse in less than two years. These poor outcomes have motivated intensive searches for effective and safe targeted agents. Asp-Glu-Ala-Asp (DEAD)-box protein DDX5 was found to be highly expressed in several solid cancers. DDX5, especially its phosphorylated form at Y593, has been reported to promote β-catenin cytoplasm-to-nucleus shuttling and subsequently facilitate the transcriptional activities of the β-catenin/T-cell factor (TCF) complex leading to enhanced expression of genes that are critical for growth, including MYC and CCND1. Knockdown of DDX5 has also been reported to reduce the growth of AML cells, indicating that DDX5 is a potential therapeutic target in AML. However, it remains unknown whether the growth dependence of AML on DDX5 is mainly mediated by β-catenin signaling. Importantly, drugs that selectively target DDX5 have not been tested in AML. In this study, we first analyzed the TCGA AML dataset and found that DDX5 gene is significantly overexpressed in the AML cohort compared to normal bone marrow samples. High DDX5 expression is associated with worse overall survival in AML patients. Moreover, DDX5 correlates with CTNNB1 which encodes β-catenin, at transcript levels. We further found that DDX5 is constitutively phosphorylated at Y593 and predominantly present in the nucleus of 10 AML cell lines tested. These results indicate the clinical relevance of DDX5 whose functional impact may link to β-catenin signaling in AML. We then determined the therapeutic efficacy of targeting DDX5 in human AML using RX-5902, a quinoxalinyl-piperazine compound. RX-5902 was previously shown to interfere with the interaction between phosphorylated DDX5 and β-catenin, inhibiting β-catenin signaling in some solid tumors. We found that RX-5902 attenuated phosphorylation of DDX5 at Y593, led to accumulation of β-catenin in the cytoplasm and consequently downregulated c-MYC in human AML cells within 24 hours. Treatment with RX-5902 dramatically increased cellular ROS level, induced cleaved-caspase3 mediated apoptosis and eventually inhibited cell growth of most human AML cell lines and primary AML cells in culture. These results mirrored what we observed following DDX5 depletion by shRNAs in AML cell lines. Also, dosing of RX-5902 in vivo significantly suppressed the growth of AML xenotransplants in immunocompromised mice and prolonged survival. Finally, we performed dynamic BH3 profiling and showed that RX-5902 increased apoptotic priming and BCL-2 dependence in AML cells. Therefore, we reasoned that simultaneous targeting DDX5 and BCL-2 may improve the therapeutic efficacy in AML. Indeed, concurrent treatment using RX-5902 and Venetoclax, a BCL-2 inhibitor, synergistically induced apoptosis in AML cells. Collectively, therapeutic targeting of DDX5 may be a novel and effective approach in AML and warrants further study. Citation Format: WEI NI, Swati Garg, Fen Zhu, Ellen L. Weisberg, Basudev Chowdhury, Martin Sattler, Amulya Jakkani, Suiyang Liu, Dana M. Sanchez, Lizi Wu, Richard M. Stone, Matthew S. Davids, James D. Griffin. Inhibiting β-catenin in AML by targeting DDX5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6248.

Richter syndrome (RS), an aggressive lymphoma that develops in patients with chronic lymphocytic leukemia (CLL), is a striking example of histologic transformation. While recent therapeutic advances have transformed the treatment landscape of CLL and lymphoma, RS remains associated with dismal overall survival. Despite an advanced genomic and molecular characterization of CLL over the past decade, the current understanding of the genetic factors driving evolution of CLL to RS is limited. To decipher the genetics underlying this transformation, we have performed an integrative analysis of exome, genome and transcriptome data generated from matched RS and CLL samples from a discovery cohort of 53 patients with newly diagnosed RS of DLBCL histology. Through computational deconvolution of CLL and RS clones, we constructed phylogenetic relationships and traced evolution of CLL to RS, confirming both clonal related (87%) and unrelated cases (13%). In addition to identifying recognized RS-risk genetic lesions, we discovered novel RS-specific alterations, including 5 putative somatic driver genes (IRF2BP2, SRSF1, B2M, DNMT3A and EZH2), frequent copy number alterations beyond del(9p21)(CDKN2A/B), (including amp(7q21.2) (CDK6), amp(9p24) (PDL1/L2), and amp(1q23)(MCL1)), and recurrent whole genome duplication and chromothripsis. Integration of exome and genome sequencing data led to the identification of distinct molecular subtypes of RS with prognostic importance. To confirm these molecular subtypes, a validation cohort of 47 RS cases has been assembled with paired exome and transcriptome data. To further investigate the stepwise clonal evolution of CLL to RS, we performed single-cell RNA-sequencing on biopsy samples obtained at diagnosis from 5 individuals with clonally related transformation. Using a novel tool, CNVSingle, we inferred allele specific single-cell copy number alterations that enabled identification of the single-cell clusters representing distinct CLL and RS genetic subclones as well as intermediate, or transitional, evolutionary states. RS cells displayed gene expression enriched in pathways of MYC targets and cell cycle, in line with similar analysis on bulk transcriptomes. Finally, by ultra-low pass (ULP)-WGS sequencing of plasma samples from RS patients, we demonstrate detection of RS tumor DNA in plasma months prior to initial clinical diagnosis (n=3 of 6) or post-allogeneic stem cell transplant relapse (n=2 of 2). cfDNA is thus a promising tool for early detection of emerging RS and RS relapse as well as for non-invasive detection surrounding diagnosis. Altogether, our study defines RS-specific alterations and provides a molecular definition of RS, identifies distinct genetic subtypes of RS with prognostic significance, traces the evolutionary path to RS and suggests future strategies for improved detection. Citation Format: Erin M Parry, Ignaty Leshchiner, Romain Guieze, Connor Johnson, Eugen Tausch, Sameer A Parikh, Camilla K Lemvigh, Conor Messer, Filippo Utro, Chaya Levovitz, Kahn Rhrissorrakrai, Matthew S Davids, Julien Broseus, Shuqiang Li, Ziao Lin, Binyamin A Knisbacher, Christof Schneider, Laura Z Rassenti, Thomas J Kipps, Nitin Jain, William Wierda, Florence Cymbalista, Neil E Kay, Kenneth J Livak, Brian P Danysh, Chip Stewart, Donna Neuberg, Jennifer R Brown, Laxmi Paridi, Stephan Stilgenbauer, Gaddy Getz, Catherine Wu. Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A13.

Interaction between CLL and its microenvironment is a major factor in drug resistance. Bone marrow derived stroma cells provide a protective and supportive niche to allow CLL cells to survive drug-induced apoptosis. Therefore, a current challenge is to develop new strategies to target the microenvironment. To understand the role of tumor microenvironment in CLL survival, we studied conditioned media (CM) derived from NKTert cells. Human NKTert stroma cells provide a survival advantage to primary CLL cells by protecting them from spontaneous apoptosis in ex-vivo culture. We identified three cytokines (IL6, IL8 and CCL2) from CM as candidates for mediating survival. We exposed CLL cells to neutralizing antibodies against each cytokine and found significant decrease in CLL viability in presence of anti-CCL2 suggesting its role in CLL survival. BH3 profiling revealed an increase in apoptotic priming with anti-CCL2 treatment as well. Using dynamic BH3 profiling (DBP), we found that anti-CCL2 increased dependence on BCL-2 as well as BCL-xL in primary CLL cells suggesting a potential role of CCL2 on CLL survival. To our surprise, we also identified a CLL group (CM independent, CMI) that were resilient to cell death in the absence of stroma support in ex-vivo culture. In addition to this, anti-CCL2 antibody showed no effect on their survival. Next, we exposed CMI CLL cells to venetoclax and found that these CLL cells were less sensitive to apoptosis compared to the CLL samples that are stroma dependent (CM dependent, CMD). BH3 profiling showed that CMI CLL cells are less primed for apoptosis. Consistent with our findings, DBP with anti-CCL2, venetoclax or the combination didn’t increase any drug induced apoptotic priming in CMI CLL samples. In our study, 80% of CMI harbor unmutated IgHV. To provide additional insight to the molecular mechanism, we performed western blot analyses for BCL-2 family proteins and found decreased BAX ad BCL-2 expression in CMI samples. This result suggests altered BCL-2 family proteins expression might be a mechanism for differential priming. Bulk RNA seq analysis revealed upregulation of focal adhesion genes and Ras signaling pathway in CMI samples. In summary, our study suggest that in the CMD subset of CLL, anti-CCL2 therapy enhances apoptotic priming and venetoclax efficacy. Furthermore, we have also functionally defined two biologically distinct CLL subgroups based on stroma dependence. Citation Format: Salma Parvin, Aditi Aryal, Shanye Yin, Matthew S. Davids, Geoffrey G. Fell, Catherine J. Wu, Anthony Letai. Targeting tumor-microenvironmental dependencies as a therapeutic approach in Chronic Lymphocytic Leukemia (CLL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A39.

As the SARS-CoV-2 (COVID-19) pandemic spreads and the number of Bruton's tyrosine kinase inhibitor (BTKi)-treated COVID-19-affected patients grows, we must consider the pros and cons of BTKi discontinuation for our patients. In favor of BTKi continuation, BTK plays an active role in macrophage polarization. By modulating key transcription factors, BTK may regulate macrophage polarization downstream of classic M1 and M2 polarizing stimuli and mitigate the hyperinflammatory state associated with COVID-19. In favor of BTKi discontinuation, we note a potentially increased risk of secondary infections and impaired humoral immunity. We hypothesize that the potential benefit of blunting a hyperinflammatory response to SARS-CoV-2 through attenuation of M1 polarization outweighs the potential risk of impaired humoral immunity, not to mention the risk of rapid progression of B-cell malignancy following BTKi interruption. On the basis of this, we suggest continuing BTKi in patients with COVID-19.

Purpose: To develop a prognostic model and cytogenetic risk classification for previously treated patients with chronic lymphocytic leukemia (CLL) undergoing reduced intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT). Experimental Design: We performed a retrospective analysis of outcomes of 606 patients with CLL who underwent RIC allogeneic HCT between 2008 and 2014 reported to the Center for International Blood and Marrow Transplant Research. Results: On the basis of multivariable models, disease status, comorbidity index, lymphocyte count, and white blood cell count at HCT were selected for the development of prognostic model. Using the prognostic score, we stratified patients into low-, intermediate-, high-, and very-high-risk [4-year progression-free survival (PFS) 58%, 42%, 33%, and 25%, respectively, P < 0.0001; 4-year overall survival (OS) 70%, 57%, 54%, and 38%, respectively, P < 0.0001]. We also evaluated karyotypic abnormalities together with del(17p) and found that del(17p) or ≥5 abnormalities showed inferior PFS. Using a multivariable model, we classified cytogenetic risk into low, intermediate, and high (P < 0.0001). When the prognostic score and cytogenetic risk were combined, patients with low prognostic score and low cytogenetic risk had prolonged PFS (61% at 4 years) and OS (75% at 4 years). Conclusions: In this large cohort of patients with previously treated CLL who underwent RIC HCT, we developed a robust prognostic scoring system of HCT outcomes and a novel cytogenetic-based risk stratification system. These prognostic models can be used for counseling patients, comparing data across studies, and providing a benchmark for future interventions. For future study, we will further validate these models for patients receiving targeted therapies prior to HCT.

Introduction: Despite recent advances in the therapy of diffuse large B cell lymphoma (DLBCL), many patients are still not cured, and therefore new therapeutic combinations are needed. The anti-apoptotic B cell lymphoma 2 (BCL-2) gene is commonly dysregulated in DLBCL due to various mechanisms such as chromosomal translocation t(14;18)(q32.3;q21.3), copy number alterations and gene amplifications; however, targeting BCL-2 with a selective inhibitor, venetoclax, led to response in only a minority of patients. Thus, we sought to identify a novel combination partner for venetoclax to improve response to therapy. Previously with dynamic BH3 profiling, we found that DNA hypomethylating agents (HMA) could increase BCL2 dependence in DLBCL cells. Here, we investigate the activity of the HMA decitabine with venetoclax in DLBCL. Methods: We utilized BH3 profiling, a functional assay to assess the mitochondrial priming of cells for apoptosis and the dependence of cells on various anti-apoptotic BCL-2 family proteins. Other standard techniques included Western blot, cell cycle (BrdU/7-AAD), CellTiter-Glo, and Seahorse XF cell mito stress test. The DLBCL cell lines (TMD8, HBL1, OCI-Ly3, OCI-Ly1, Karpas 422, SUDHL4, Toledo, OCI-Ly7) and double hit lymphoma (DHL) patient-derived xenograft cell lines were used to investigate the in vitro anti-cancer activity of decitabine and venetoclax. Results: Through BH3 profiling, we found heterogenous dependence of DLBCL, consistent with the variable expression of multiple anti-apoptotic proteins in DLBCL assessed by Western blot. DLBCL cells were less sensitive to single BH3 mimetic treatment (venetoclax, S63 (MCL-1i) and A133 (BCL-xLi)). Using dynamic BH3 profiling (which assesses the change in priming induced by ex vivo drug treatment), we found that decitabine primes DLBCL cells for apoptosis and increases BCL-2 dependence. Consequently, decitabine increases the sensitivity of DLBCL cells to venetoclax, as the combination of decitabine and venetoclax synergistically induces apoptosis and suppresses cell proliferation. Decitabine has previously been shown to increase TGF-β signaling in DLBCL by restoring the expression of a downstream target, SMAD1. Interestingly, we found that inhibition of TGF-β signaling only partially antagonizes the anti-cancer activity of decitabine, implicating other anti-cancer mechanisms of decitabine. Indeed, we found that decitabine also induces DNA damage and leads to cell cycle arrest in DLBCL cells. Furthermore, decitabine promotes activation of the key apoptotic effector proteins, BAK and BAX. Additionally, we found that this combination also suppresses oxidative phosphorylation, thus restricting the energy supply for DLBCL cells. Conclusions: The HMA decitabine sensitizes DLBCL cells to venetoclax in vitro through increasing apoptotic priming and BCL-2 dependence. This combination is worthy of further study in in vivo model systems. Citation Format: Matthew S. Davids, Fen Zhu, Stephen J. Chong, Jennifer Crombie, Liam Hackett, May C. Collins. Decitabine sensitizes diffuse large B cell lymphoma cells to venetoclax [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5377.

Introduction: BCL2 family protein dysfunction is an important mediator of chemoresistance in lymphoid malignancies. Venetoclax (VEN), a BH3 mimetic that selectively targets the anti-apoptotic protein BCL2, is FDA approved for treatment of chronic lymphocytic leukemia (CLL), with clinical trials underway for other lymphoid malignancies. Despite the clinical efficacy of VEN monotherapy, complete remission and progression free rates are still relatively low, thereby indicating the existence of inherent or acquired mechanisms of resistance. Preclinically, increases in anti-apoptotic BCL2 and MCL1 phosphorylation (BCL2-P/MCL1-P) and MCL1 protein level have been implicated in chemoresistance. We therefore question if phosphorylation of these proteins may drive VEN resistance and if removing this modification may re-sensitize resistant cells to VEN induced cell death. Methodology: BH3 profiling (a functional assay that assesses mitochondrial apoptotic priming and anti-apoptotic protein dependence), western blot and cell death assays were performed. Result: We report that VEN acquired resistant DLBCL cell line (OCI-Ly1R) has increased BCL2-P, MCL1-P and MCL1 protein levels as compared to sensitive parental cell line (OCI-Ly1S), while inherently resistant DHL cell line (Su-DHL4) possesses higher MCL1-P and MCL1 protein levels as compared to OCI-Ly1S. We further show that this increase in phosphorylation status contributes to the induction of VEN resistance. Using BH3 profiling, we demonstrate that anti-apoptotic protein phosphorylation-driven VEN resistance involves a change in sensitivity to pro-apoptotic proteins, with an increase in sensitivity to MCL1 inhibition and reciprocal decrease in sensitivity to BCL2 inhibition. We further show that BCL2 and MCL1 dephosphorylation by PP2A activating drugs (PADs) re-sensitizes resistant cells to VEN treatment. Mechanistically, PADs such as the fingolimod (FTY720) not only dephosphorylate BCL2-P and MCL1-P, but also dissociate BAX from BCL2 and destabilize MCL1 protein. These changes lead to the rewiring of cellular sensitivity to BCL2 inhibition, thereby re-sensitizing both VEN acquired and inherent resistant cells to VEN induced cell death. Importantly, the PP2A activating effects of FTY720 were recapitulated in primary cells from the peripheral blood of 16 CLL patients, where FTY720 treatment also reduced BCL2-P, MCL1-P and MCL1 protein levels and dissociated BAX from BCL2. This led to an increased sensitivity to BCL2 inhibition and enhanced cell death upon combined treatment with FTY720 and VEN. Conclusion: Our work defines a new targetable mechanism of VEN resistance in lymphoid malignancies due to the phosphorylation of anti-apoptotic BCL2 family proteins. PADs re-sensitize resistant malignant cells to VEN treatment via dephosphorylation of anti-apoptotic proteins, suggesting a promising combination approach to explore further. Citation Format: Stephen J. Chong, Fen Zhu, Jolin X. Lai, Liam Hackett, Mary C. Collins, Shazib Pervaiz, Jean-Philippe Coppe, Matthew S. Davids. Targeting BCL2 family protein phosphorylation in venetoclax resistant lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3959.

The synchronous activation of BCR and CD40 signaling via B-T cell interactions is required for proliferation of normal (Luo et al, 2018) and some malignant B cells, especially in chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). In CLL/FL cells, proliferation occurs mainly in lymph nodes, but not in bone marrow or peripheral blood which lack access to proper B-T cell interactions. Here we have analyzed the mRNA and miRNAs profile in the proliferative intraclonal CLL cell subpopulation that has recently exited lymph node niches (CXCR4dimCD5bright cells; Calissano et al,2011; Pavlasova et al,2016) to reveal molecules potentially participating in synchronous regulation of CD40 and BCR pathway. This has identified 36 miRNAs and 1370 mRNAs differentially expressed in CLL cells exiting lymph nodes as compared to resting non-proliferative CLL cells (CXCR4brightCD5dim cells). Next, we overlapped the 36 miRNAs with their predicted target mRNAs with putative function in CD40/BCR (TargetScan, KEGG) which were anti-correlated to at least one miRNA (miRNAs typically decrease mRNA stability). This revealed among others anti-correlation of lower miR-29 with higher TRAF4 levels in immune niches, which was validated in paired lymph node biopsies vs peripheral blood CLL cells (P Altogether, we have described a novel MYC-miR-29-TRAF4 axis that regulates CD40 signaling in B cells, and acts to synchronize BCR activation with CD40 pathway. Supported by: The ERC under the European Union's Horizon 2020 research and innovation program (grant agreement No 802644), MH CZ, grant nr. NV18-03-00054 and NU20-03-00292. All rights reserved. Czech Science Foundation (20-02566S), MEYS CZ under the project CEITEC 2020 (LQ1601), MH CZ-DRO (FNBr,65269705) and MUNI/A/1595/2020. Citation Format: Sonali Sharma, Vaclav Seda, Eva Vojackova, Gabriela Mladonicka Pavlasova, Daniel Filip, Laura Ondrisova, Veronika Sandova, Lenka Kostalova, Pedro F. Zeni, Marek Borsky, Sarka Pospisilova, Medhat Shehata, Laura Z. Rassenti, Ulrich Jaeger, Michael Doubek, Matthew S. Davids, Jennifer R. Brown, Jiri Mayer, Thomas J. Kipps, Marek Mraz. miR-29-TRAF4 axis is a novel regulator of CD40 signaling in malignant B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2368.