Search

To identify the prognostic factors that are useful to improve central nervous system (CNS) control in children with acute lymphoblastic leukemia (ALL), we analyzed the outcome of 7640 consecutive patients treated on Chinese Children’s Cancer Group ALL-2015 protocol between 2015 and 2019. This protocol featured prephase dexamethasone treatment before conventional remission induction and subsequent risk-directed therapy, including 16 to 22 triple intrathecal treatments, without prophylactic cranial irradiation. The 5-year event-free survival was 80.3% (95% confidence interval [CI], 78.9-81.7), and overall survival 91.1% (95% CI, 90.1-92.1). The cumulative risk of isolated CNS relapse was 1.9% (95% CI, 1.5-2.3), and any CNS relapse 2.7% (95% CI, 2.2-3.2). The isolated CNS relapse rate was significantly lower in patients with B-cell ALL (B-ALL) than in those with T-cell ALL (T-ALL) (1.6%; 95% CI, 1.2-2.0 vs 4.6%; 95% CI, 2.9-6.3; P < .001). Independent risk factors for isolated CNS relapse included male sex (hazard ratio [HR], 1.8; 95% CI, 1.1-3.0; P = .03), the presence of BCR-ABL1 fusion (HR, 3.8; 95% CI, 2.0-7.3; P < .001) in B-ALL, and presenting leukocyte count ≥50×109/L (HR, 4.3; 95% CI, 1.5-12.2; P = .007) in T-ALL. Significantly lower isolated CNS relapse was associated with the use of total intravenous anesthesia during intrathecal therapy (HR, 0.2; 95% CI, 0.04-0.7; P = .02) and flow cytometry examination of diagnostic cerebrospinal fluid (CSF) (HR, 0.2; 95% CI, 0.06-0.6; P = .006) among patients with B-ALL. Prephase dexamethasone treatment, delayed intrathecal therapy, use of total intravenous anesthesia during intrathecal therapy, and flow cytometry examination of diagnostic CSF may improve CNS control in childhood ALL. This trial was registered with the Chinese Clinical Trial Registry (ChiCTR-IPR-14005706).

Three proteasome inhibitors have garnered regulatory approvals in various multiple myeloma settings; but drug resistance is an emerging challenge, prompting interest in blocking upstream components of the ubiquitin-proteasome pathway. One such attractive target is the E1 ubiquitin-activating enzyme (UAE); we therefore evaluated the activity of TAK-243, a novel and specific UAE inhibitor. TAK-243 potently suppressed myeloma cell line growth, induced apoptosis, and activated caspases while decreasing the abundance of ubiquitin-protein conjugates. This was accompanied by stabilization of many short-lived proteins, including p53, myeloid cell leukemia 1 (MCL-1), and c-MYC, and activation of the activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE-1), and protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) arms of the ER stress response pathway, as well as oxidative stress. UAE inhibition showed comparable activity against otherwise isogenic cell lines with wild-type (WT) or deleted p53 despite induction of TP53 signaling in WT cells. Notably, TAK-243 overcame resistance to conventional drugs and novel agents in cell-line models, including bortezomib and carfilzomib resistance, and showed activity against primary cells from relapsed/refractory myeloma patients. In addition, TAK-243 showed strong synergy with a number of antimyeloma agents, including doxorubicin, melphalan, and panobinostat as measured by low combination indices. Finally, TAK-243 was active against a number of in vivo myeloma models in association with activation of ER stress. Taken together, the data support the conclusion that UAE inhibition could be an attractive strategy to move forward to the clinic for patients with relapsed and/or refractory multiple myeloma.

Leukemia is the most common malignant tumor in children under 15 years old, which is divided into several subtypes, including acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphoblastic leukemia (CLL) and chronic myeloid leukemia (CML), based on the disease phases and effected cells. Each subtype has its specific molecular feature and key regulation factors. In our previous studies, we reported that β-arrestin1 (ARRB1), the pivotal scaffold protein to transduce various cellular signals, could bind with EZH2 to increase Bcr/Abl H4 acetylation level and thus promote CML progression (Brit J Cancer 2014, 111(3): 568-76). ARRB1 could enhance DNMT1 activity and PTEN methylation, decrease PTEN expression and promote self-renew of B-ALL leukemia initiating cells (LICs) (Cancer Lett 2015, 357(1): 170-8.). ARRB1 could increase P300 to bind with SP1 to hTERT promoter, and thus increase hTERT transcription/expression, telomerase activity, telomere length and cell senescence in B-ALL LICs (Cell Death Diff 2017, 8(4): e2756.). However, little is known in the T-ALL, which about 70% have the mutations of NOTCH1 gene. Here, we unveil ARRB1 could curb the progression of T-ALL cells in vitro and in vivo, while the expression of ARRB1 was suppressed by the aberrant increased miR-223. Mechanistically, ARRB1 could recruit DTX1, the E3 ubiquitin ligase, to promote the ubiquitination and degradation of NOTCH1 protein in T-ALL. Furthermore, Overexpression of ARRB1-derived miR-223 sponge BUTR was incompatible with cell proliferation and induces apoptosis in T-ALL cells. Collectively, our results for the first time revealed that ARRB1 acted as a tumor suppressor by promoting NOTCH1 degradation in T-ALL cells where miR-223 effectively antagonized ARRB1 functions. This provides that miR-223 may serve as a valid drug target for developing novel and efficacious T-ALL therapeutics. Disclosures No relevant conflicts of interest to declare.

Studies in mice have shown that proinflammatory Th17 cells can cause acute graft-versus-host disease (aGVHD) related tissue damage; however, whether they play a role in human aGVHD remains unclear. In a prospective study, we measured the proportion of Th17 cells in the blood and skin of patients at the onset of aGVHD. We found no difference in the proportion or amount of IL-17 produced by T cells in the blood of patients with aGVHD (n = 20) compared with time-matched patients without GVHD (n = 14). Moreover, Th17 cells were not increased in the skin of patients with cutaneous aGVHD (n = 7) compared with healthy controls (n = 10). In contrast, we found significantly more interferon-γ–producing T cells in the skin of patients with aGVHD compared with controls. These data support the long-standing paradigm that tissue localized interferon-γ–producing cells are the perpetrators of aGVHD.

MLN4924 is a potent and selective small molecule NEDD8-activating enzyme (NAE) inhibitor. In most cancer cells tested, inhibition of NAE leads to induction of DNA rereplication, resulting in DNA damage and cell death. However, in preclinical models of activated B cell–like (ABC) diffuse large B-cell lymphoma (DLBCL), we show that MLN4924 induces an alternative mechanism of action. Treatment of ABC DLBCL cells with MLN4924 resulted in rapid accumulation of pIκBα, decrease in nuclear p65 content, reduction of nuclear factor-κB (NF-κB) transcriptional activity, and G1 arrest, ultimately resulting in apoptosis induction, events consistent with potent NF-κB pathway inhibition. Treatment of germinal-center B cell–like (GCB) DLBCL cells resulted in an increase in cellular Cdt-1 and accumulation of cells in S-phase, consistent with cells undergoing DNA rereplication. In vivo administration of MLN4924 to mice bearing human xenograft tumors of ABC- and GCB-DLBCL blocked NAE pathway biomarkers and resulted in complete tumor growth inhibition. In primary human tumor models of ABC-DLBCL, MLN4924 treatment resulted in NF-κB pathway inhibition accompanied by tumor regressions. This work describes a novel mechanism of targeted NF-κB pathway modulation in DLBCL and provides strong rationale for clinical development of MLN4924 against NF-κB–dependent lymphomas.

Background: The ubiquitin-proteasome system (UPS) has been validated as a target in multiple myeloma (MM) through the success of proteasome inhibitors such as bortezomib, but drug resistance is an emerging challenge. Targeting some of the upstream components of the UPS, such as the E1 ubiquitin activating enzyme (UAE), could therefore be a promising alternative. TAK-243 (MLN7243) specifically blocks the ubiquitin conjugation cascade through the formation of a TAK-243-ubiquitin adduct, thereby inhibiting the UAE. Our aim was to explore the effectiveness of TAK-243 against pre-clinical myeloma models, and to understand some its mechanisms of action. Methods: We performed pre-clinical studies in myeloma cell lines and mouse models using TAK-243. Downstream effects were evaluated using viability, apoptosis assays, western blotting, gene expression profiling (GEP), and Reverse Phase Protein Array (RPPA) techniques. Results: MM1.S and MOLP-8 TP53 wild-type cell lines were sensitive to TAK-243, with median inhibitory concentrations (IC50) of 25 nM at 24 hours based on viability assays. In otherwise isogenic cell lines in which TP53 was suppressed using genome editing techniques, the IC50 was ~40 nM, but higher TAK-243 concentrations of 100 nM overcame resistance due to TP53 inactivation. Similarly, TAK-243 was able to overcome resistance to both conventional (dexamethasone) and novel (bortezomib, lenalidomide) drugs in paired sensitive and resistant cell line models. After treatment with TAK-243, Annexin V and TO-PRO3 staining determined that viable MM1.S cells were induced into early or late apoptosis. This was accompanied by a significant increase in cleaved caspase-3, -8, and -9 as detected by flow cytometry, and in cleaved caspase-7 detected by RPPA and western blot. Exposure to TAK-243 reduced the cellular content of ubiquitin-protein conjugates, and did not enhance expression levels of a fusion protein degraded by the proteasome in a ubiquitin-independent manner, indicating the lack of direct proteasome inhibition. GEP analysis and RPPA detected enhanced expression of p53-pathway related proteins, including MDM2, TP53, and p21 in TAK-243 treated MM1.S cells. Several mRNAs and proteins in the ER stress pathway, including ATF6, ATF4, IRE1a and XBP1 were also elevated, as were many non-coding RNAs and DNA-damage related genes. Combination experiments in MM cell lines demonstrated synergy between TAK-243 and lenalidomide, pomalidomide, panobinostat, melphalan and doxorubicin. Finally, TAK-243 demonstrated in vivo antitumor activity against MM1.S and MOLP-8 xenograft models when dosed at 12.5 mg/kg IV twice-weekly for 2 weeks (tumor growth inhibition of 60% and 73%, respectively). Elevation of BiP, ATF4, XBP1s and cleaved-caspase 3 was detected within the first 24 hrs after dosing in the sensitive MM1.S xenografts. In contrast, RPMI 8226 cells, which showed a 2000 nM IC50 in cell culture, were also resistant to TAK-243 in vivo, with no tumor growth inhibition detected. Conclusions: TAK-243 is a UAE inhibitor that is active against myeloma cells in vitro and in xenograft models in vivo, overcomes conventional and novel drug resistance, and its action is associated with stimulation of the TP53 and ER stress pathways. Thus, it may deserve further evaluation as an anti-myeloma agent. Disclosures Berger: Takeda Pharmaceuticals: Employment. Hyer:Takeda Pharmaceuticals: Employment. Chattopadhyay:Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Shi:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Shinde:Takeda Pharmaceuticals: Employment. Kreshock:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Menon:Takeda Pharmaceuticals: Employment. Orlowski:Takeda Pharmaceuticals: Research Funding.

Approximates 80% of essential thrombocythemia (ET) patients carrying JAK2, CALR, MPL mutations. However, there is still 20% of ET patients are negative for all three mutations. Our previous studies have demonstrated that platelet-derived growth factor (PDGF) and its receptor (PDGFR) has a potential effect in the regulation of hematopoiesis and megakaryopoiesis. Therefore, we speculated that PDGF/PDGFR may plays an important role in megakaryocyte/platelet related disease, such as ET. In this study, we found an increase of PDGF-BB and PDGFR expression in ET patients comparing to the normal persons. We then used the adjusted dosage of PDGF-BB (1.5ug/kg/d) to establish an ET mimic mouse model. We found that PDGF-BB can enhance platelet production in vivo significantly, while PDGFR inhibitor imatinib can block this effect. Bone marrow histology data also confirmed the megakaryopoiesis effect of PDGF, as shown by an increased number of megakaryocytes in PDGF-treated mouse. The addition of imatinib can block the effect, evident by a decrease number of megakaryocytes and an increase of apoptosis. Furthermore, we demonstrated that PDGF-BB activated theP-JAK2, the P-STAT3 and the P-AKT expression in the megakaryocytic cell line CHRF-288, while addition of imatinib can reduce the phosphorylation level of all three genes. Our results suggested that the overexpression of PDGF-BB and PDGFR may be important in the pathogenesis of ET, especially in the JAK2-mutation negative ET. The elevated PDGF-BB activates PDGFR with subsequently activation of the JAK2/ STAT3 and PI3K/AKT pathway. Imatinib may have an effect on treating ET via blocking PDGFR and the following JAK2/STAT3 and PI3K/AKT pathway. This study suggests that PDGF/PDGFR may be involved in the pathogenesis of essential thrombocythemia, and the expression of PDGF-BB and PDGFR is potential to be a diagnostic index of ET. Blockage of PDGFR by imatinib may be a new therapeutic target for ET. Figure 1 Effect of PDGF-BB on Jak2/Stat3 and PI3K/Akt pathway of megakaryocytes. Figure 1. Effect of PDGF-BB on Jak2/Stat3 and PI3K/Akt pathway of megakaryocytes. Figure 2 The platelet numbers and the megakaryocytes of each group mice. Figure 2. The platelet numbers and the megakaryocytes of each group mice. Figure 3 Potential mechanisms for imatinib in treating essential thrombocythemia. Figure 3. Potential mechanisms for imatinib in treating essential thrombocythemia. Disclosures No relevant conflicts of interest to declare.

Epstein-Barr virus (EBV) is associated with the development of several cancers including Burkitt's Lymphoma, Hodgkin Lymphoma, Nasopharyngeal and Gastric Carcinomas. EBV-encoded Latent Membrane Protein 2A (LMP2A), a BCR mimic, is expressed in cancer cells and is essential for transformation, migration and inhibition of differentiation. LMP2A/λ-MYC double transgenic mice develop splenomegaly and lymphoma much faster than mice that express Myc only. LMP2A induces cancer phenotype by recruiting and activating BCR signaling components such as Lyn and Syk kinases to the SH2 motifs found on its N terminal cytoplasmic domain. In this study, we explored inhibition of Syk kinase activity with a novel Syk/FLT-3 inhibitor TAK-659, in lymphomas from these transgenic mice to assess SYK inhibition as a potential therapeutic strategy for EBV-associated malignancies. The cell lines developed from LMP2A/Myc transgenic mice derived lymphomas showed higher basal pSYK levels than Myc lymphoma cell lines. TAK-659 treatment resulted in early apoptosis within 8hrs in LMP2A/Myc lymphoma cells in comparison to 24 hrs in Myc lymphoma cells. In both pre-tumor and tumor transfer models in vivo, compared with placebo and Myc tumors, LMP2A/MYC mice treated with TAK-659 did not develop splenomegaly and tumor development was totally inhibited in these mice indicating SYK signaling is essential for tumor development in LMP2A/Myc mice. In addition, metastasis of tumor cells into BM was abrogated. TAK-659 killed tumor cells but not host cells in spleen and tumors. Taken together our data show that TAK-659, even in low nanomolar concentrations, inhibits pSYK and induces apoptosis in LMP2A/Myc tumor cells. It also inhibits splenomegaly and tumor development in our autochthonous and syngeneic tumor transfer models of EBV-associated lymphoma by targeting tumor cells while sparing non-tumor cells. Therefore, TAK-659 may provide an effective therapeutic option for the treatment of EBV-associated malignancies and should be explored further in clinical trials. Disclosures Kannan: Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Huck:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Zhang:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. Gordon:Northwestern University: Patents & Royalties: Patent for gold nanoparticles pending.

High mobility group AT-hook 2 (HMGA2) is an architectural transcriptional factor that is negatively regulated by Let-7 microRNA. Dysregulated HMGA2 has been shown to exhibit a myeloproliferative phenotype in engineered mice. To decipher the Let-7-HMGA2 axis in myeloproliferative neoplasms (MPN), we studied a cohort of 151 MPN patients. Overexpressed HMGA2 was detected in about one-fifth of the cases, and it was more commonly seen in ET (26.9%, vs. 12.7% in PV, p=0.044). Compared to their counterparts, HMGA2-overexpressing patients had higher platelet counts, increased thromboembolic risk, and inferior thrombosis-free survival. Fluorescence in situ hybridizationanalysis showed that chromosomal translocation was not a major cause of HMGA2 overexpression in MPN patients, yet there was an inverse correlation between the expression levels of let-7a and HMGA2. Furthermore, up-regulation of HMGA2 was significantly associated with MPN patients carrying JAK2V617F mutation. In vitro studies showed that Ba/F3 cells carried JAK2V617F (Ba/F3-JAK2V617F) had decreased let-7a and up-regulated HMGA2. Silencing of HMGA2 in Ba/F3-JAK2V617F cells resulted in growth inhibition coupled with a significant increase in apoptosis. Our findings suggest that, in a subset of JAK2-mutated MPN patients, Let-7-HMGA2 axis plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. Disclosures Hsu: Ministry of Science and Technology (Taiwan): Research Funding; Chang-Gung Memorial Hospital: Employment, Research Funding. Chen:Ministry of Science and Technology (Taiwan): Research Funding; Chang Gung Memorial Hospital, Chiayi branch: Research Funding. Gau:Taipei Veterans General Hospital, Taipei, Taiwan: Employment. Huang:Chang-Gung Memorial Hospital: Employment, Research Funding. You:Lotung Poh-Ai Hospital, Yilan, Taiwan: Employment. Lung:Chang-Gung Memorial Hospital: Employment, Research Funding. Chen:Chang-Gung Memorial Hospital: Employment. Ho:Chang-Gung Memorial Hospital: Employment, Research Funding. Li:Chang-Gung Memorial Hospital: Employment, Research Funding. Lu:Chang Gung Memorial Hospital, Chiayi, Taiwan: Employment. Lee:Chang-Gung Memorial Hospital: Employment, Research Funding.

Background Approximately 15% of children newly diagnosed for acute lymphoblastic leukemia (ALL) present with the T cell lineage (T-ALL), which historically has had inferior outcomes compared to the children with precursor-B cell ALL. In this study, we analyzed the efficacy of the Chinese Children Leukemia Group 2008 (CCLG-ALL 2008) protocol on pediatric T-ALL patients enrolled from 4 centers located at north, east, and southwest of China, to identify specific prognostic factors. Patients and methods Patients enrolled on the CCLG-ALL 2008 Protocol came from four hospitals in China: two hospitals in the southwest, one in the east, and one in the north, with 33, 37, 34 and 48 patients respectively. The study was approved by the hospitals' Ethical Committee, with informed consent obtained from patients' patents or legal guardians. Definitions: Failures of relapse-free survival (RFS) included relapse and death of any cause. M1: Blast 25%. Results Outcome disparity is observed across the four hospitals (Table 1 and Figure 1). Response to prednisone window treatment (Good vs. Poor) was highly prognostic and its prognostic value held universally across the centers despite the outcome disparity across the centers and abandonment: log-rank test stratified by centers (hospitals) P value = 0.0311, and by abandonment (Y vs. N) P = 0.0004. Multivariable Cox regression analysis showed that response to prednisone window treatment was an independent early (before end of remission induction) prognostic factor (Table 2). The hazard ratio of prednisone response (Poor vs. Good) was 2.6 (95% CI 1.49 to 4.57) after accounting for RFS disparities across centers and abandonment. When further adjusted for post-induction prognostic markers by Day-35 and Month-three bone marrow status (M1, M2 or M3), response to predisone window remained boarder-line significant (P=0.0683). Prednisone response was only significantly associated with Day-15 bone-marrow status, with poor responders tended to likely have M2 and M3 marrow status at Day 15 (Table 3). Day-15 and Day-35 marrow status was not significantly associated with RFS, but patients with Day-35 M2 and M3 status tended to fail early. Marrow status before intensification (Month 3) seemed highly associated with RFS, with all 4 M3 patients failed within 1 year. The percentage of abandonment in the southwest 1, the southwest 2, the east, and the north hospital was 21.6%, 33.3%, 8.8% and 25% respectively; the difference was not significant (P=0.1078). Nonetheless the east hospital seemed to have distinctly lower percentage of abandonment. The percentage of non-urban patients in the southwest1, the southwest2, the east and the north is 52.8%, 75.8%, 61.8% and 68.8% respectively; this difference was not significant (P=0.2171), although the percentage seemed higher in southwest. Patients from urban area tended to fair better in RFS but the difference was not statistically significant (P=0.1785). There was a weak trend that abandonment rate tended to be higher among non-urban patients, but not statistically significant (P=0.3361). Patients' Day-35 bone marrow status (with M2 and M3 marrow related to higher likelihood of abandonment), abandonment and socio-economic status might be relevant factors for the inter-center disparity in RFS. With the current data however, we are unable to identify any strong factor that can explain the inter-center disparity of treatment outcome. Conclusion Response to prednisone window treatment is a universal early prognostic marker in Chinese children with T lineage ALL despite outcome disparity. Disclosures No relevant conflicts of interest to declare.

The ubiquitin-proteasome system (UPS) represents the primary mechanism by which cells degrade proteins. Proteasome inhibition results in accumulation of proteasome substrates, leading to cell cycle disruption, endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and, ultimately, the activation of apoptotic pathways and cell death. Ixazomib (NINLARO) is the first FDA-approved oral proteasome inhibitor, indicated in combination with lenalidomide and dexamethasone for treatment of patients with multiple myeloma who have received at least one prior therapy. Ixazomib has been evaluated as a single agent in relapsed/refractory lymphoma, but additional data is needed to identify combination agents for lymphoma trials. Brentuximab vedotin (ADCETRIS) is an anti-CD30 antibody drug conjugate (ADC) that is protease cleavable linker with monomethyl auristatin E (MMAE), a highly potent antimicrotubule agent. Brentuximab vedotin binds specifically to CD30, a cell surface marker expressed at high levels on Hodgkin lymphoma (HL) and anaplastic large cell lymphoma (ALCL) tumor cells and induces cell death by direct cytotoxicity and also antibody-dependent cellular phagocytosis (ADCP). Brentuximab vedotin is approved in patients with relapsed/refractory HL, relapsed/refractory ALCL, and for consolidation in in the post autologous stem cell transplant (ASCT) setting of HL. Preclinical evidence suggests that resistance to anti-CD30 therapy can be mediated via activation of the NFkB pathway, which can be reversed by proteasome inhibitor treatment, suggesting a rationale for the combination of ixazomib and brentuximab vedotin. In the current study we evaluated this combination in three CD30+ ALCL xenograft models, SR-786, Karpas-299 and SUDHL-2 grown in immunocompromised mice. In SR-786 and Karpas-299, the combination treatment using dose levels of ixazomib and brentuximab vedotin with minimal single-agent activity resulted in synergistic effect with complete tumor regression. In most animals, the tumor did not regrow as long as 60 days after the last dose of combination drug treatment. Unlike in SR-786 and Karpas-299, no combination benefit was observed in the SUDHL-2 xenograft model. Pharmacodynamic studies are ongoing to understand the mechanism of synergy. Our data suggest that combination of ixazomib and brentuximab vedotin warrants clinical evaluation in CD30+ lymphoma patients. Disclosures Chattopadhyay: Takeda Pharmaceuticals: Employment. Syed:Takeda Pharmaceuticals: Employment. Zhang:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals International Co, Cambridge, MA: Employment. He McDougall:Takeda Pharmaceuticals: Employment. Yu:Takeda Pharmaceuticals: Employment. Tirrell:Takeda Pharmaceuticals: Employment. Berger:Takeda Pharmaceuticals: Employment. van de Velde:Millennium Pharmaceuticals Inc., Cambridge, MA, USA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Tremblay:Takeda Pharmaceuticals: Employment. Huebner:Takeda Pharmaceuticals International Co.: Employment, Equity Ownership.

Several recent reports have suggested that in vitro exposure of CD4+ T cells to rabbit antithymocyte globulin (rATG), which is commonly used to prevent and treat graft-versus-host disease and allograft rejection, is an effective method to induce CD4+CD25+FOXP3+ T regulatory cells (Tregs). We and others, however, have shown that FOXP3 is also expressed in activated T cells. We therefore investigated whether the induction of FOXP3 expression by rATG resulted in a stable population of suppressive Tregs. We found that exposure of peripheral blood mononuclear cells (PBMCs) or conventional T cells to rATG resulted in induction of transient rather than stable expression of CD25 and FOXP3. Furthermore, rATG-treated T effector cells acquired neither an immunosuppressive profile of cytokine production nor suppressive capacity, even at the time of maximal FOXP3 expression. These findings indicate that the notion that rATG can be used to induce Tregs in vitro for cellular therapy in vivo should be re-evaluated.

Epithelial-mesenchymal transition (EMT) is a critical process for inducing stem-like properties of epithelial cancer cells. However, the role of EMT inducers in hematologic malignancies is unknown. Twist1, an EMT inducer necessary for cell migration, has recently been found to have transcriptionally regulatory activity on the expression of Bmi1, and these two are capable of promoting tumorigenesis in a synergized manner. Knowing that Bmi1 expression is essential for maintenance of leukemic stem cells, we speculate that Twist1 might govern the pathogenesis of acute myeloid leukemia (AML) development as well. We used AML cell lines KG1a and THP1 cells, which had low and high expression levels of Twist1, respectively to demonstrate that upregulated Twist1 increased Bmi1 expression in AML. Bmi1 overexpression endued leukemic cells a higher proliferative potential and increased resistance to apoptosis. In primary AML samples, there was strong positive correlation between the expression levels of Twist1 and Bmi1. AML Patients whose leukemic blasts harbored overexpressed Twist1 had a more aggressive clinical phenotype, but they were more likely to have a better clinical outcome after standard therapy. In vitro studies confirmed that Twist1-overexpressing leukemic cells were more susceptible to cytarabine, but not daunorubicin, cytotoxicity. Our findings suggest that, in a subset of AML patients, Twist1 plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. The result indicates that Twist1 could represent a powerful biomarker that serves as a prognostic as well as therapeutic guide for patients with AML. Disclosures No relevant conflicts of interest to declare.

Background Leukemia is the most common malignant tumor in children under 15 years old. The main subtype of children leukemia is acute lymphoblastic leukemia (ALL), and B-lineage ALL (B-ALL) accounts for approximately 70%. The leukemia-initiating cells (LICs) are cancer stem cells with long-term repopulating potential and propagation ability, to maintain the leukemia cell phenotype, and possess leukemia-initiating activity. However, the regulation of LICs for the leukemia progression is poorly understood. The multifunctional scaffold proteins β-arrestins are proven to mediate H4 acetylation and gene expression. And β-arrestin2 is found to regulate the initiation and progression of chronic myeloid leukemia (CML). However, the role of β-arrestin1 in B-ALL is still unknown. Our preliminary data showed that both the high expression of β-arrestin1 and high proportion of CD34+CD38- cells are positively correlated with risk stratification and poor prognosis of childhood B-ALL. And β-arrestin1 binds with EZH2 to increase BCR/ABL H4 acetylation and thus promotes CML cell progression in vitro and in vivo. The aim of study is to investigate the essential function of β-arrestin1 in LICs from B-ALL. Materials and Methods The bone marrow (BM) and periphery blood (PB) of children B-ALL patients were collected, isolated and identified LICs by Magnetic-activated cell sorting (MACS) and flow cytometry. The total RNA and protein were purified for gene and protein expression by real-time RT-PCR and Western blot. The leukemia cells (LICs, Raji, and Reh) of β-arrestin1 depletion were constructed by transient or stable screening si-β-arrestin1 (siβ1) lentivirus vector. The serial cell colony formation and NSG mice survival analysis was measured the LICs self-renewal ability. The CCK8 and MTS assays were used to detect the cell proliferation, and annexin V-FITC and PI staining for cell apoptosis. The DNA methylation of gene promoter region was detected by methylation-specific PCR and the methltransferase activity by ELISA. The telomere length was indicated by Southern blot and FISH, and telomerase activity by TRAP. Electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay were applied to explain gene transcription. Student’s t test and Log-Rank test were used in the corresponding statistical significance and P Results The expression of β-arrestin1 was elevated in LICs from B-ALL patients, and the high level of β-arrestin1 was negatively correlated with the survival of these patients. Further study showed that the loss of β-arrestin1 in B-ALL LICs attenuates their self-renewal capacity and promotes their senescence in vitro and in vivo. The mRNA expression level of β-arrestin1 is negatively correlated with that of PTEN in LICs. Moreover, the DNA methylation of the PTEN promoter region, the activity and the expression of DNMTs were enhanced in the LICs. The inhibition of DNMT1 activity impaired the self-renewal and increased the expression of PTEN of LICs. In addition, depletion of β-arrestin1 significantly decreased DNMT1 activity and PTEN methylation, and consistently increased PTEN expression in LICs. For B-ALL cell senescence, the mRNA expression level of β-arrestin1 is negatively related with the length of telomere, positively related with the activity of telomerase and the mRNA expression of hTERT in B-ALL LICs and engrafted NSG mice. Moreover, the weakened effect of β-arrestin1 on telomere, telomerase and the gene of hTERT were observed by injected the inhibitor of telomerase in leukemic mice. In addition, depletion of β-arrestin1 significantly decreased the binding of SP1 to the promoter of hTERT and thus reduced the transcription of hTERT in B-ALL Raji and Reh cells. Furthermore, β-arrestin1 interacted with P300 to bind with SP1 in the -104bp to -113bp of hTERT core promoter region in B-ALL cells. Conclusions β-arrestin1 could regulate the self-renewal and senescence of LICs from B-ALL, by partially mediating DNMT1 activity and hTERT transcription respectively, indicating that β-arrestin1 is a potential therapeutic target for B-ALL. Disclosures No relevant conflicts of interest to declare.

Introduction The combination of HDAC inhibitors and proteasome inhibitors has demonstrated preclinical benefit in several settings, including multiple myeloma and lymphoma, and is being explored in clinical trials testing various HDAC inhibitors in combination with proteasome inhibitors. ACY-1215 is an investigational, orally available HDAC6-selective inhibitor that has demonstrated preclinical combination benefit with bortezomib in vitro and in vivo (Santos et al, Blood 2012; 119: 2579). These preclinical studies also support the hypothesis that the improved selectivity of ACY-1215 for HDAC6 over class I HDACs (HDAC1,2,3) may provide an improved tolerability profile compared to pan-HDAC inhibitors, while still providing the anti-myeloma effect of other HDACi/proteasome inhibitor combinations. ACY-1215 is currently in a Phase I/II trial in multiple myeloma with bortezomib (VELCADE) and dexamethasone to test this hypothesis (NCT01323751). Ixazomib citrate (MLN9708) is an investigational oral proteasome inhibitor in Phase III clinical trials in multiple myeloma (NCT01850524, NCT01564537). To examine the potential efficacy of the all-oral combination of ixazomib citrate and ACY-1215, we evaluated the combination of these agents in cell lines and xenograft models of multiple myeloma. Results In vitro viability experiments in 2 multiple myeloma cell lines (RPMI-8226 and MM.1S) using a dose matrix format demonstrated a combination benefit of ACY-1215 and ixazomib over a range of concentrations, very similar to the previously reported benefit of ACY-1215 plus bortezomib. Likewise, the combination benefit of the selective HDAC6 inhibitor ACY-1215 with ixazomib was similar to the combination effect observed with the pan-HDAC inhibitor SAHA (vorinostat). Together, these in vitro studies support the hypothesis that the combination of ACY-1215 and ixazomib provides similar levels of benefit as do combinations including other HDACi/proteasome inhibitors. Furthermore, experiments in MM.1S xenograft-bearing mice demonstrated an in vivo combination benefit of ACY-1215 and ixazomib. An all-oral regimen was well tolerated when ACY-1215 was dosed at 100 mg/kg PO twice daily for 5 days per week in combination with ixazomib dosed at 5 mg/kg PO twice weekly, and the combination regimen demonstrated additive antitumor activity (Figure 1). The in vivo combination benefit of ACY-1215 and ixazomib was further demonstrated in MM.1S xenograft-bearing mice using alternate routes of administration (IV dosing of ixazomib and IP dosing of ACY-1215). The combination of ACY-1215 dosed at 30 mg/kg IP once daily for 5 days per week with ixazomib dosed IV at 1.5 mg/kg twice-weekly was also well tolerated and had striking antitumor activity. This combination regimen in fact caused regression of the MM.1S xenograft tumors below the starting volumes, and this level of activity was maintained throughout the entire 17 day dosing period (Figure 2). In an accompanying pharmacodynamic (PD) study of the PO and IP doses of ACY-1215, we confirmed selective HDAC6 inhibition in MM.1S xenograft tumors as evidenced by elevated acetylation levels of the HDAC6 substrate tubulin, with little if any change in the levels of acetylated histone H3, a class I HDAC substrate. In vivo experiments in a second xenograft model, RPMI-8226, also demonstrated a combination benefit of ACY-1215 (30 mg/kg IP for 5 days per week) with ixazomib (0.75 mg/kg IV twice-weekly). Conclusion The combination benefit of ACY-1215 and ixazomib observed here in preclinical experiments utilizing in vitro and in vivo models of multiple myeloma provides rationale for clinical evaluation of this first all-oral combination of a proteasome inhibitor with an HDAC inhibitor. Disclosures: Berger: Takeda Pharmaceutical Company Ltd: Employment. Bannerman:Takeda Pharmaceutical Company Ltd: Employment. Quayle:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yu:Takeda Pharmaceutical Company Ltd: Employment. Garcia:Takeda Pharmaceutical Company Ltd: Employment. Ciavarri:Takeda Pharmaceutical Company Ltd: Employment. Tamang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Yang:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc: Employment, Equity Ownership.

Thalassaemia companied with iron-overload is common in Hong Kong. Iron overload induced cardiomyopathy is the commonest cause of morbidity and mortality in b-thalassaemia patients. One of the causes of cardiac failure is chronic iron overload of blood transfusion. Some studies showed that iron overload can cause toxic effect in heart cells. Iron-overload induced cardiomyopathy damages are the major complications in patients with beta-thalassaemia major. Iron-overload may induce apoptosis in cardiomyocytes. Our previous study showed TPO has cardiac protective effect (Li et al, Circulation, 2007). In this study, we demonstrated firstly that iron induced oxidative stress can cause apoptosis in cardiomyocytes. By using H9C2 cells, we showed that iron increased reactive oxygen species (ROS) production (n=3) and reduced cell viability in a dose-dependent manner (0-0.6 mM) (n=6). Apoptotic cells were found to be significantly increased under iron treatment (0.3 mM, 72 hrs) in the AnnexinV/PI assay (n=6). The expression of active caspase-3 significantly increased in iron-treated cells. Furthermore, iron treatment increased the proportion of cells containing JC-1 monomers, indicating a trend in the drop of mitochondrial membrane potential (n=6). Secondly, we found that TPO exerted cardio-protective effect on iron-induced apoptosis. H9C2 cells were cultured in the presence of iron (0.3 mM) with or without TPO (50 ng/mL). The ROS production was significantly decreased with the addition of TPO at 50 ng/mL (n=3). Dot-plot analysis of AnnexinV/PI staining demonstrated that TPO significantly reduced the population of apoptotic cells (n=6). Incubation with TPO also decreased the iron-induced caspase-3 expression (n=6). Flow cytometric dot-plot analysis also showed trends of amelioration of the increase in JC-1 monomers in the iron plus TPO group (n=6), indicating a trend in attenuation of the drop of mitochondrial membrane potential. Our findings suggest that iron-overload lead to generation of ROS which further induces apoptosis in cardiomyocytes via mitochondrial pathways and TPO might exert a protective effect on iron-overload induced apoptosis via inhibiting oxidative stress and mitochondrial pathway in cardiomyocytes. Disclosures: No relevant conflicts of interest to declare.

Thrombopoietin (TPO) is a growth factor for platelet/megakaryocytic lineage. Our previous study showed TPO has cardiac protective effect(Li et al, Circulation, 2006). TPO may also has a protective effect in neural cells. Tanshinone IIA (TIIA), isolated from Danshen (Radix Salviae Miltiorrhiza Bge), is a derivative of phenanthrenequinone, which has been widely used as anti-platelet and neural protection drug for stroke for many years in China. In this study, a hypoxic–ischemic encephalopathy model was established, followed by 2 hrs of hypoxia in 7-day-old neonatal rat pups. TPO (12.5 ug/kg) or/and TIIA (10 mg/kg) were administrated daily (IP) for two weeks. The severity of brain injury was estimated by the weight reduction of the ipsilateral cerebral hemisphere as compared to the contralateral hemisphere. There was significant less atrophy in TPO alone or TPO plus TIIA treated animals compared with that in controls at 7 and 21 days (P Disclosures: No relevant conflicts of interest to declare.

Abstract 2709 Poster Board II-685 Introduction: The ubiquitin-proteasome system processes the majority of cellular proteins and is the principal manner by which cells regulate protein homeostasis. The successful development of bortezomib for multiple myeloma and previously treated mantle cell lymphoma has validated the proteasome as a therapeutic target for hematological malignancies. MLN9708 was identified in screens for a proteasome inhibitor with greater antitumor activity than bortezomib in preclinical xenograft models. MLN9708 immediately hydrolyzes to MLN2238, the biologically active form, upon exposure to aqueous solutions or plasma. MLN2238 was used for all preclinical studies described below. Results: MLN2238 inhibited the 20S proteasome b5 site (IC50 of 3.4 nM), an engineered proteasome substrate (4x ubiquitin-luciferase reporter) and blocked TNFa-induced activation of the NFkB pathway. Cell viability studies confirmed that MLN2238 has potent activity against both myeloma and lymphoma cell lines. The proteasome dissociation half life of MLN2238 was determined to be approximately 6-fold faster than bortezomib, consistent with data generated from Proteasome-Glo wash out experiments where proteasome activity recovered more quickly in MLN2238-treated cells compared to bortezomib. In immunocompromised mice MLN2238 achieved exposures that resulted in significant blood and tumor proteasome inhibition and had increased plasma and tumor exposure compared to bortezomib (when dosed at MTD). In Sprague-Dawley rats, MLN2238 had improved plasma exposure, lower plasma clearance, higher blood Vdss, better oral F% and higher plasma protein binding than bortezomib. In WSU-DLCL2 xenografts, a model of diffuse large B cell lymphoma, greater antitumor activity was seen in mice treated with IV or SC doses of MLN2238 compared to bortezomib. For example, MLN2238 dosed SC QD at its MTD resulted in a T/C of 0.29 compared to bortezomib dosed SC QD at its MTD which resulted in a T/C of 0.79. Pharmacodynamic (PD) responses (in mice) were assessed by evaluating tumor 20S b5 site-specific activity and expression levels of GADD34, an unfolded protein response (UPR) pathway gene shown to be upregulated in response to proteasome inhibition. Consistent with the efficacy difference between MLN2238 and bortezomib in WSU-DLCL2 xenografts, MLN2238 demonstrated an improved PD response compared to bortezomib, showing higher levels of tumor proteasome inhibition and pathway marker elevation. MLN2238 and bortezomib were then evaluated for their ability to reduce tumor burden and improve overall survival in a disseminated model of lymphoma. Tumor burden was tracked over time via bioluminescent scans in NOD-SCID mice inoculated with OCI-Ly7-luciferase cells. The strongest antitumor response was seen in mice treated with MLN2238 SC QD at its MTD, and this dosing regimen also significantly prolonged overall survival compared to vehicle treated controls (median survival was 54 vs. 33 days). Weaker antitumor responses were seen following treatment with bortezomib (either at its SC QD MTD or weekly IV MTD) and these dose regimens did not significantly prolong survival in this study. Conclusions: MLN2238 is a potent, reversible and orally bioavailable proteasome inhibitor with improved pharmacokinetics, pharmacodynamics and antitumor activity in preclinical xenograft models compared to bortezomib. MLN9708 is currently in clinical development for a variety of oncology indications. Disclosures: Kupperman: Milllennium: Employment. Lee:Milllennium: Employment, Equity Ownership. Cao:Milllennium: Employment, Equity Ownership. Bannerman:Milllennium: Employment. Fitzgerald:Millennium Pharmaceuticals: Employment. Berger:Millennium Pharmaceuticals: Employment. Yu:Millennium Pharmaceuticals: Employment. Zhang:Millennium Pharmaceuticals: Employment. Hales:Millennium Pharmaceuticals: Employment. Bruzzese:Millennium Pharmaceuticals: Employment. Liu:Millennium Pharmaceuticals: Employment. Blank:Millennium Pharmaceuticals: Employment. Garcia:Millennium Pharmaceuticals: Employment. Tsu:Millennium Pharmaceuticals: Employment. Dick:Millennium Pharmaceuticals: Employment. Fleming:Millennium Pharmaceuticals: Employment. Yu:Millennium Pharmaceuticals: Employment. Manfredi:Millennium Pharmaceuticals: Employment. Rolfe:Millennium Pharmaceuticals: Employment. Bolen:Millennium Pharmaceuticals: Employment.

MLN4924 is a first-in-class, small molecule inhibitor of the Nedd8 Activating Enzyme (NAE) in Phase I clinical trials in hematological malignancies. Inhibition of NAE by MLN4924 leads to decreased neddylation and inhibition of cullin-dependent ubiquitin ligase (CDL) activity. CDLs are enzyme complexes which control the ubiquitination and degradation of proteins with important roles in cell cycle progression and cell survival. CDL-mediated degradation of pIkBa regulates NF-kB signaling by freeing cytoplasmic NF-kB transcription factors to translocate to the nucleus promoting cell proliferation and survival. In tumors dependent on the NF-kB pathway for growth and survival, we hypothesized that MLN4924 inhibition of CDL activity would prevent pIkBa degradation and inhibit NF-kB signaling. We utilized models of ABC-like Diffuse Large B-cell Lymphoma (ABC-like DLBCL, OCI-Ly10 and OCI-Ly3 cells) dependent on NF-kB signaling for survival and Germinal Center B-cell like DLBCL (GCB-like DLBCL, OCI-Ly19 and OCI-Ly7 cells) that are not dependent on NF-kB signaling for survival. In vitro, we show that NAE inhibition by MLN4924 in ABC-like DLBCL produces marked stabilization of pIkBa, inhibits p65 nuclear translocation and NF-KB gene transcription demonstrating an inhibition of NF-kB signaling. The inhibition of NF-KB signaling in Ly10 cells results in a G1 phenotype and an acute induction of apoptosis. In contrast, in GCB-like DLBCL we observed an elevation of multiple substrates of the CDLs, an accumulation of cells with increased DNA content (>4N) followed by a DNA damage response and induction of cell death. This mechanism of action in GCB-like DLBCL cells is observed in other tumor cell lines that are not dependent on NF-kB signaling for survival. In vivo administration of MLN4924 to mice bearing xenograft tumors of OCI-Ly10 and OCI-Ly19 resulted in a pharmacodynamic response of NAE pathway inhibition. In both models, a single dose of MLN4924 resulted in time and dose-dependent inhibition of total neddylated cullin levels and stabilization of CDL substrates including the CDL3Keap1 substrate, Nrf-2. Notably, in the OCI-Ly10 model, a single dose of MLN4924 resulted in a marked elevation of pIkBa levels, indicative of NF-kB pathway inhibition, and induction of apoptosis. In both OCI-Ly10 and OCI-Ly19 xenograft models, inhibition of the NAE pathway following repeated daily and intermittent dosing of MLN4924 translated into significant tumor growth inhibition. In the OCI-Ly10 model tumor regressions were observed showing this model to be particularly sensitive to MLN4924 treatment, reflecting the addiction of these tumors to NF-kB signaling. Additionally we demonstrate an inhibition of the NAE pathway and NF-KB signaling in a primary human tumor DLBCL xenograft model (PHTX-22L) resulting in tumor regressions following MLN4924 treatment. In summary, in tumors dependent on NF-kB signaling for growth and survival, MLN4924 inhibition of CDL activity provides a novel mechanism for targeted NF-kB pathway modulation and therapeutic intervention. In addition, these data demonstrate that MLN4924 is a novel agent that has broad activity in pre-clinical models of lymphoma.

Background Arsenic trioxide provides significant benefits in newly diagnosed and relapsed acute promyelocytic leukemia (APL) respectively. However, the high relapsed rate is still threatened the life of APL patients. Which regimen should be used to overcome or reduce the relapse in consolidated treatment is a key problem at present. We performed a pilot study about that. Objective To Compare the effectiveness and security of cocktail therapy with single arsenic trioxide therapy in APL consolidated treatment. Methods Sixty-Five APL patients, who once received arsenic trioxide treatment and obtained complete remission, were enrolled in this study. Patients were divided into two groups according to the different consolidated regimens. After reinforced treated with DA (daunomycin and cytarabine) or HOAP (harringtonine, vincristin, cytarabine and prednisone) for two course, Group A involved twenty cases received single arsenic trioxide consolidated, Group B included forty-five cases treated with the cocktail therapy, alternatively treated with arsenic trioxide, all trans-retinoic acid and chemotherapy (DA or HOAP). The relapse rates, the survival rates and the central nervous system infiltration rates in 3 years followed up were compared. Results The relapsed rate of Group A was 55%, which was higher than that of Group B(17.8%). The re-remission rate after the first relapse in Group A was 22%, which was lower than that of Group B(42.8%). The central nervous system infiltration rate of Group A was 28%, which was higher than that of Group B(6%). The average survival time of Group A was 10.5±4.2months, which was shorter than that of Group B (22.5±5.5 months). The three-year survival rate of Group A was 15%, which was less than that of Group B (65.8%). Conclusions Cocktail therapy —alternatively treated with arsenic trioxide, all trans-retinoic acid and chemotherapy will be the reasonable regimen for APL consolidated treatment. Which provided benefited on inhibiting relapse and central nervous system infiltration of APL.