scholarly journals Targeted Inhibition of PI3K α/δ Is Synergistic with BCL-2 Blockade in Genetically Defined Subtypes of DLBCL

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 39-39
Author(s):  
Kamil Bojarczuk ◽  
Kirsty Wienand ◽  
Jeremy A. Ryan ◽  
Linfeng Chen ◽  
Mariana Villalobos-Ortiz ◽  
...  
Keyword(s):  
Tumor Growth ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Research Report ◽  
Genetic Bases

Abstract Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease that is transcriptionally classified into germinal center B-cell (GCB) and activated B-cell (ABC) subtypes. A subset of both GCB- and ABC-DLBCLs are dependent on B-cell receptor (BCR) signaling. Previously, we defined distinct BCR/PI3K-mediated survival pathways and subtype-specific apoptotic mechanisms in BCR-dependent DLBCLs (Cancer Cell 2013 23:826). In BCR-dependent DLBCLs with low baseline NF-κB activity (GCB tumors), targeted inhibition or genetic depletion of BCR/PI3K pathway components induced expression of the pro-apoptotic HRK protein. In BCR-dependent DLBCLs with high NF-κB activity (ABC tumors), BCR/PI3K inhibition decreased expression of the anti-apoptotic NF-κB target gene, BFL1. Our recent analyses revealed genetic bases for perturbed BCR/PI3K signaling and defined poor prognosis DLBCL subsets with discrete BCR/PI3K/TLR pathway alterations (Nat Med 2018 24:679). Cluster 3 DLBCLs (largely GCB tumors) exhibited frequent PTEN deletions/mutations and GNA13 mutations. Cluster 5 DLBCLs (largely ABC tumors) had frequent MYD88L265P and CD79B mutations that often occurred together. These DLBCL subtypes also had different genetic mechanisms for deregulated BCL2 expression - BCL2 translocations in Cluster 3 and focal (18q21.33) or arm level (18q) BCL2 copy number gains in Cluster 5. These observations prompted us to explore the activity of PI3K inhibitors and BCL2 blockade in genetically defined DLBCLs. We utilized a panel of 10 well characterized DLBCL cell line models, a subset of which exhibited hallmark genetic features of Cluster 3 and Cluster 5. We first evaluated the cytotoxic activity of isoform-specific, dual PI3Kα/δ and pan-PI3K inhibitors. In in vitro assays, the PI3Kα/δ inhibitor, copanlisib, exhibited the highest cytotoxicity in all BCR-dependent DLBCLs. We next assessed the transcriptional abundance of BCL2 family genes in the DLBCLs following copanlisib treatment. In BCR-dependent GCB-DLBCLs, there was highly significant induction of the pro-apoptotic HRK. In BCR-dependent ABC-DLBCLs, we observed significant down-regulation of the anti-apoptotic BFL1 protein and another NF-κB target gene, BCLxL (the anti-apoptotic partner of HRK). We then used BH3 profiling, to identify dependencies on certain BCL2 family members and to correlate these data with sensitivity to copanlisib. BCLxL dependency significantly correlated with sensitivity to copanlisib. Importantly, the BCLxL dependency was highest in DLBCL cell lines that exhibited either transcriptional up-regulation of HRK or down-regulation of BCLxL following copanlisib treatment. In all our DLBCL cell lines, PI3Kα/δ inhibition did not alter BCL2 expression. Given the genetic bases for BCL-2 deregulation in a subset of these DLBCLs, we next assessed the activity of the single-agent BCL2 inhibitor, venetoclax, in in vitro cytotoxicity assays. A subset of DLBCL cell lines was partially or completely resistant to venetoclax despite having genetic alterations of BCL2. We postulated that BCR-dependent DLBCLs with structural alterations of BCL2 might exhibit increased sensitivity to combined inhibition of PI3Kα/δ and BCL2 and assessed the cytotoxic activity of copanlisib (0-250 nM) and venetoclax (0-250 nM) in the DLBCL cell line panel. The copanlisib/venetoclax combination was highly synergistic (Chou-Talalay CI<1) in BCR-dependent DLBCL cell lines with genetic bases of BCL2 deregulation. We next assessed copanlisib and venetoclax activity in an in vivo xenograft model using a DLBCL cell line with PTENdel and BCL2 translocation (LY1). In this model, single-agent copanlisib did not delay tumor growth or improve survival. Single-agent venetoclax delayed tumor growth and improved median survival (27 vs 51 days, p<0.0001). Most notably, we found that the combination of copanlisib and venetoclax delayed tumor growth significantly longer than single-agent venetoclax (p<0.0001). Additionally, the combined therapy significantly increased survival in comparison with venetoclax alone (median survival 51 days vs not reached, p<0.0013). Taken together, these results provide in vitro and in vivo pre-clinical evidence for the rational combination of PI3Kα/δ and BCL2 blockade and set the stage for clinical evaluation of copanlisib/venetoclax therapy in patients with genetically defined relapsed/refractory DLBCL. Disclosures Letai: AbbVie: Consultancy, Other: Lab research report; Flash Therapeutics: Equity Ownership; Novartis: Consultancy, Other: Lab research report; Vivid Biosciences: Equity Ownership; AstraZeneca: Consultancy, Other: Lab research report. Shipp:AstraZeneca: Honoraria; Merck: Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

scholarly journals Venetoclax Synergizes with Radiation Therapy for Treatment of B-Cell Lymphomas

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 467-467
Author(s):  
Shyril O'Steen ◽  
Amelia Waltman ◽  
Garrett Booth ◽  
Aimee L Kenoyer ◽  
Margaret Nartea ◽  
...  
Keyword(s):  
B Cell ◽  
Survival Time ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Combination Group ◽  
Mean Survival Time

Abstract Introduction: An estimated 19,970 Americans died of non-Hodgkin lymphoma (NHL) in 2015, with diffuse large B-cell lymphoma (DLBCL) accounting for roughly 30% of newly diagnosed NHL. Our study focuses on three NHL subtypes: germinal center (GCB)-DLBCL, the most common DLBCL subtype; activated (ABC)-DLBCL, a particularly aggressive and high-risk subtype; and mantle cell lymphoma (MCL), considered incurable. Constitutive B-cell receptor signaling is implicated in the pathogenesis of ABC-DLBCL and MCL and may couple with aberrant apoptotic BCL-2 pathway proteins. The BCL-2 inhibitor venetoclax is a promising targeted agent that promotes apoptosis in a variety of NHL subtypes, but is almost never curative as a single agent. Radiotherapy promotes apoptosis by creating DNA strand breaks, and we hypothesized that the combination of radiotherapy and venetoclax would act synergistically in NHL to increase the probability of cures. Methods: We tested in vitro killing efficacy of sublethal 137Cesium irradiation combined with venetoclax in 15 cell lines, representing a diversity of NHL subtypes. Cells were treated with 137Cesium and venetoclax in 8 x 8 dose combination matrices, incubated 72-120 hrs, then assayed for viability with Celltiter-Glo (Promega). The degree of treatment antagonism, additivity, or synergism was determined using isobolographic analyses. For in vivo studies, we tested combinations of venetoclax with either 137Cesium total body irradiation (TBI), or CD20 pre-targeted radioimmunotherapy (PRIT), in threetumor models chosen for divergent single agent sensitivities. Tumor xenografts of Rec-1 (MCL), U2932 (ABC-DLBCL), and SU-DHL-6 (GCB-DLBCL) were produced by subcutaneous flank injection of 10 x 106 cells in male and female NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) mice. When tumor volumes were 50 mm3, mice (n = 8-12/group) were treated with either venetoclax (100-200mg/kg daily for 10-30 days), diluent control, TBI (single dose, 6-10 Gy 137Cesium), or a combination of venetoclax and TBI. In PRIT studies, mice were coinjected with 300µg unlabeled streptavidin-conjugated anti-CD20 antibody (murine IgG2a) and 400µg HB8181 (IgG2a isotype control to block non-specific binding) in place of TBI. Twenty-one hours later, 5.8 nmol biotin-galactose "clearing agent" was administered, followed in 3 hours by 1.2 nmol DOTA-biotin labeled with 400, 800, or 1200 µCi of 90Y (14.8, 29.6, or 44.4 MBq, respectively). Results: In vitro, 10 of 15 lymphoma cell lines responded synergistically to combined radiotherapy and venetoclax, including GCB-DLBCL, ABC-DLBCL and MCL lines (p < .04 in 10 cell lines). In vivo, each of 3 lymphoma models responded synergistically to combination therapy. In mice bearing Rec-1 xenografts, venetoclax alone did not affect mean survival time (p = .32), 8 Gy TBI lengthened survival by 44% compared to controls (p < .0001), but TBI combined with venetoclax tripled survival time compared to controls (p < .0001, combination group > TBI alone). The SU-DHL-6 model produced similar results. In the U2932 model, tumors disappeared during venetoclax monotherapy, but recurred in all mice, such that mean survival time doubled compared to controls (p = .0001). Six Gy TBI had no effect (p = .73), but combining TBI with venetoclax tripled survival time compared to controls (p = .0003, combination group > venetoclax alone). Using PRIT in place of TBI produced yet greater efficacy. In Rec-1 bearing mice, venetoclax had no effect alone (p = .12), 800µCi PRIT lengthened survival time 111% beyond controls (p = .0001), while the combination extended survival 483% beyond controls and cured 40% (p = .001, combination group > PRIT alone). In the U2932 xenograft model, venetoclax alone doubled survival time compared to controls (p < .0001) and 800µCi PRIT alone doubled survival and cured 30% (Fig. 1, p < .0001). Combination treatments cured 100% (Fig. 1). Conclusion: In vitro and in vivo results support our hypothesis that radiotherapy combines effectively with venetoclax to treat NHL. Despite differences in single agent sensitivity, xenograft models of GCB-DLBCL, ABC-DLBCL and MCL all responded synergistically to combinations of either TBI or PRIT with venetoclax. PRIT combinations with venetoclax produced cures (Fig. 1) without detectable toxicity, and merit clinical preference. Ongoing studies examine predictive biomarkers and optimal treatment protocols for therapeutic efficacy. Disclosures Gopal: Paid Consultancy- Gilead, Janssen, Seattle Genetics, Spectrum, Research funding- Gilead, Janssen, Pfizer, BMS, Merck, Teva, Takeda, Spectrum, Seattle Genetics: Consultancy, Honoraria, Research Funding.

Thymoglobulin (Polyclonal Rabbit Anti Thymocyte Globulin) Has In Vivo Activity in a Mouse Model of Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3487-3487
Author(s):  
Michael Timm ◽  
Linda Wellik ◽  
Teresa Kimlinger ◽  
Jessica Haug ◽  
Michael Kline ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Growth ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Solid Organ ◽  
Polyclonal Rabbit

Abstract Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in myeloma. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines. Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure). Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan. Figure Figure

The Addition of the BTK Inhibitor Ibrutinib to Anti-CD19 Chimeric Antigen Receptor T Cells (CART19) Improves Engraftment and Antitumor Responses Against Mantle Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 704-704
Author(s):  
Marco Ruella ◽  
Saad S Kenderian ◽  
Olga Shestova ◽  
Joseph A. Fraietta ◽  
Sohail Qayyum ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
University Of Pennsylvania ◽  
Tumor Control

Abstract Introduction: The bruton tyrosine kinase (BTK) inhibitor ibrutinib demonstrates considerable activity in mantle cell lymphoma (MCL). However, approximately 30% of patients do not respond to this treatment and the therapy invariably leads to drug resistance with a median response of 17.5 months. Infusion of autologous T cells transduced with chimeric antigen receptors (CAR) against the B-cell specific CD19 antigen (CART19) leads to dramatic clinical responses in the majority of patients with acute lymphoblastic leukemia and the activity in B cell lymphoma is currently being evaluated in clinical trials. Bulky disease, as sometimes seen in MCL, may impair T cell infiltration. The features of ibrutinib that make it an interesting addition to CART19 include its efficacy in reducing tumor masses and its ability to mobilize neoplastic B cells into the peripheral blood, thereby potentially exposing them to the killing activity of CART19. Therefore, we sought to investigate the combination of the two novel targeted therapies, ibrutinib and CART19 in MCL. Results: In vitro studies with established MCL cell lines and with a novel cell line (MCL-RL) showed a range of responses to ibrutinib with an IC50 ranging from 10 nM to 10 µM; MCL-RL was the most sensitive cell line evaluated with an IC50 of 10nM, similar to primary MCL. Both ibrutinib-sensitive and ibrutinib-resistant cell lines strongly activated CART19 in an antigen-specific manner as detected by CD107a degranulation, cytokine production and CFSE proliferation assays. Importantly, in vitro assays with MCL cell lines co-cultured with increasing doses of CART19 (E:T= 2:1, 1:1, 0.5:1, 0.25:1) combined with increasing concentrations of ibrutinib (0, 10, 100, 1000 nM) demonstrated strong additive tumor killing (Figure 1). Notably, supra-therapeutic doses of Ibrutinib (>/=1 uM) impaired cytokine production and T cell proliferation in vitro. In order to test this combination in vivo we established a novel MCL model, injecting i.v. luciferase-positive MCL-RL cells into NSG mice. This resulted in 100% MCL engraftment in liver and spleen, with eventual dissemination into lymph nodes and bone marrow. Treatment with three different doses of CART19 (0.5, 1 and 2 million cells/mouse) led to a dose dependent anti-tumor effect. A similar dose response to CART19 was also observed in the ibrutinib-resistant Jeko-1 cell line. We also treated MCL-RL xenografts with different doses (0, 25 and 125 mg/Kg/day) of ibrutinib, with a median overall survival respectively of 70, 81 and 100 days (p<0.001). Importantly, a direct in vivo comparison of the highest ibrutinib dose (125 mg/kg) and CART19 showed a significantly improved tumor control for mice treated with CART19. However, treatment with either CART19 or ibrutinib as single agents invariably led to late relapse. Therefore we sought to treat MCL-RL xenografts with the combination of CART19 and ibrutinib and compare it to the single agent activity. The combination resulted in significant improvement in tumor control compared to mice treated with the single agents with 80% of mice achieving long-term disease-free survival ( p=0.007 at day 110, representative mice shown in Figure 2A). Intriguingly, we found that mice treated with ibrutinib had higher numbers of circulating CART19 cells (Figure 2B). Conclusions: Combining CART19 with ibrutinib represents a rational way to incorporate two of the most recent therapies in MCL. Our findings pave the way to a two-pronged therapeutic strategy in patients with MCL and other types of B-cell lymphoma. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Ruella: Novartis: Patents & Royalties, Research Funding. Kenderian:Novartis: Patents & Royalties, Research Funding. Maus:Novartis: Consultancy, Patents & Royalties, Research Funding. Milone:Novartis: Patents & Royalties, Research Funding. Lacey:Novartis: Patents & Royalties, Research Funding. Mato:Genentech: Consultancy; Pronai Pharmaceuticals: Research Funding; Celgene Corporation: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; TG Therapeutics: Research Funding; AbbVie: Consultancy, Research Funding; Janssen: Consultancy. Schuster:Genentech: Consultancy; Pharmacyclics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Hoffman-LaRoche: Research Funding; Janssen: Research Funding; Gilead: Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Kalos:Novartis: Patents & Royalties, Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Gill:Novartis: Patents & Royalties, Research Funding. Wasik:Janseen and Novartis: Research Funding.

scholarly journals Establishment of B-Cell Lymphoma Cell Lines Persistently Infected with Hepatitis C Virus In Vivo and In Vitro: the Apoptotic Effects of Virus Infection

2003 ◽  
Vol 77 (3) ◽  
pp. 2134-2146 ◽  
Author(s):  
Vicky M.-H. Sung ◽  
Shigetaka Shimodaira ◽  
Alison L. Doughty ◽  
Gaston R. Picchio ◽  
Huong Can ◽  
...  
Keyword(s):  
Hepatitis C ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Culture System ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Hcv Rna

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Studies of HCV replication and pathogenesis have so far been hampered by the lack of an efficient tissue culture system for propagating HCV in vitro. Although HCV is primarily a hepatotropic virus, an increasing body of evidence suggests that HCV also replicates in extrahepatic tissues in natural infection. In this study, we established a B-cell line (SB) from an HCV-infected non-Hodgkin's B-cell lymphoma. HCV RNA and proteins were detectable by RNase protection assay and immunoblotting. The cell line continuously produces infectious HCV virions in culture. The virus particles produced from the culture had a buoyant density of 1.13 to 1.15 g/ml in sucrose and could infect primary human hepatocytes, peripheral blood mononuclear cells (PBMCs), and an established B-cell line (Raji cells) in vitro. The virus from SB cells belongs to genotype 2b. Single-stranded conformational polymorphism and sequence analysis of the viral RNA quasispecies indicated that the virus present in SB cells most likely originated from the patient's spleen and had an HCV RNA quasispecies pattern distinct from that in the serum. The virus production from the infected primary hepatocytes showed cyclic variations. In addition, we have succeeded in establishing several Epstein-Barr virus-immortalized B-cell lines from PBMCs of HCV-positive patients. Two of these cell lines are positive for HCV RNA as detected by reverse transcriptase PCR and for the nonstructural protein NS3 by immunofluorescence staining. These observations unequivocally establish that HCV infects B cells in vivo and in vitro. HCV-infected cell lines show significantly enhanced apoptosis. These B-cell lines provide a reproducible cell culture system for studying the complete replication cycle and biology of HCV infections.

Targeting the PI3K/mTOR Pathway in Lymphoma with PQR309 and PQR620: Single Agent Activity and Synergism with the BCL2 Inhibitor Venetoclax

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3017-3017
Author(s):  
Chiara Tarantelli ◽  
Eugenio Gaudio ◽  
Petra Hillmann ◽  
Filippo Spriano ◽  
Ivo Kwee ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Apoptosis Induction ◽  
In Vivo Experiments

Abstract Background. The PI3K/AKT/mTOR pathway is an important therapeutic target in lymphomas. PQR309 is a dual PI3K/mTOR inhibitor that has shown in vitroanti-lymphoma activity (Tarantelli et al, ASH2015) and is in phase 2 trial (NCT02249429, , NCT02723877, NCT02669511). PQR620 is a novel mTORC1/2 inhibitor that has shown preclinical activity in solid tumor models (Beaufils et al, AACR 2016). Here, we present the in vitro and in vivo anti-lymphoma activity of PQR620 as single agent and also the in vivo results of PQR620 or PQR309 containing combinations with the BCL2 inhibitor venetoclax. Materials and Methods. The drug concentration causing 50% inhibition of cell proliferation (IC50) was obtained in lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), no.=26; mantle cell lymphoma (MCL), no.=8; anaplastic large T-cell lymphoma, no.=5; others, no=5] exposed to increasing doses of PQR620 for 72h using a Tecan D300e Digital Dispenser on 384well plates. For in vivo experiments, NOD-Scid (NOD.CB17-Prkdcscid/J) mice were subcutaneously inoculated with 10 x106 (RIVA) or with 5 x106(SU-DHL-6) cells. Results. PQR620 had a median IC50 of 250 nM (95%CI, 200-269 nM) when tested on 44 lymphoma cell lines. Activity was higher in B cell (no.=36) than in T cell tumors (no.=8) (median IC50s: 250 nM vs 450 nM; P=0.002). At 72h, anti-tumor activityof PQR620 was mostly cytostatic and apoptosis induction was seen only in 6/44 cell lines (13%), Sensitivity to PQR620 or apoptosis induction did not differ between DLBCL and MCL, and they were not affected by the DLBCL cell of origin, by TP53 status or by the presence of MYC or BCL2 translocations. The activity of PQR620 as single agent underwent in vivo evaluation in two DLBCL models, the germinal center B cell type DLBCL (GCB-DLBCL) SU-DHL-6 and the acivated B cell-like DLBCL (ABC-DLBCL) RIVA. Treatments with PQR620 (100mg/kg dose per day, Qdx7/w) started with 100-150 mm3 tumors and were carried for 14 (SU-DHL-6) or 21 days (RIVA). In both models, PQR620 determined a 2-fold decrease of the tumor volumes in comparison with control, with significant differences in both SU-DHL-6 (D7, D9, D11, D14; P < 0.005) and RIVA (D14, D16, D19, D21; P < 0.005). Based on the previously reported synergy between the dual PI3K/mTOR inhibitor PQR309 and venetoclax (Tarantelli et al, ASH 2015), we evaluated the combination of the PQR620 or PQR309 with the BCL2 inhibitor venetoclax (100 mg/kg, Qdx7/w) in the SU-DHL-6 model. Both the venetoclax combination with the dual PI3K/mTOR inhibitor and the venetoclax combination with mTORC1/2 inhibitor were superior to the compounds given as single agents, leading to the eradication of the xenografts. The combination of PQR620 with venetoclax showed highly significant differences either versus control or single agents during all days of the experiment (D4, D7, D9, D11, D14; P < 0.001). Similarly, the combination of PQR309 with venetoclax showed highly significant differences versus venetoclax (D7, D9, D11, D14; P < 0.001) and PQR309 (D7, D9, D11; P < 0.005) alone. Conclusions. The novel mTORC1/2 inhibitor PQR620 had in vitro and in vivo anti-lymphoma activity as single agent. In vivo experiments showed that both PQR620 and the dual PI3K/mTOR inhibitor PQR309 can strongly benefit from the combination with the BCL2 inhibitor venetoclax. Disclosures Hillmann: PIQUR Therapeutics AG: Employment. Fabbro:PIQUR Therapeutics AG: Employment. Cmiljanovic:PIQUR Therapeutics AG: Employment, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Pharmacologic Inhibition of the Histone Methyltransferase SETD2 with EZM0414 As a Novel Therapeutic Strategy in Relapsed or Refractory Multiple Myeloma and Diffuse Large B-Cell Lymphoma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1142-1142
Author(s):  
Jennifer Totman ◽  
Dorothy Brach ◽  
Vinny Motwani ◽  
Selene Howe ◽  
Emily Deutschman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Publicly Traded ◽  
The Past ◽  
Molecule Inhibitor

Abstract Introduction: SETD2 is the only known histone methyltransferase (HMT) capable of catalyzing H3K36 trimethylation (H3K36me3) in vivo. It plays an important role in several biological processes including B cell development and maturation, leading to the hypothesis that SETD2 inhibition in these settings could provide anti-tumor effects. The normal process of B cell development/maturation renders B cells susceptible to genetic vulnerabilities that can result in a dysregulated epigenome and tumorigenesis, including in multiple myeloma (MM) and diffuse large B-cell lymphoma (DLBCL). For example, 15%-20% of MM harbors the high risk (4;14) chromosomal translocation, resulting in high expression of the multiple myeloma SET domain (MMSET) gene. MMSET is an HMT that catalyzes H3K36me1 and H3K36me2 formation and extensive scientific work has established overexpressed MMSET as a key factor in t(4;14) myeloma pathogenesis. To the best of our knowledge MMSET has eluded drug discovery efforts, however, since t(4;14) results in high levels of the H3K36me2 substrate for SETD2, inhibiting SETD2 offers promise for targeting the underlying oncogenic mechanism driven by MMSET overexpression in t(4;14) MM patients. In addition, SETD2 loss of function mutations described to date in leukemia and DLBCL are always heterozygous, suggesting a haploinsufficient tumor suppressor role for SETD2. This observation points to a key role for SETD2 in leukemia and lymphoma biology and suggests that therapeutic potential of SETD2 inhibition may also exist in these or similar settings. EZM0414 is a first-in-class, potent, selective, orally bioavailable small molecule inhibitor of the enzymatic activity of SETD2. We explored the anti-tumor effects of SETD2 inhibition with EZM0414 in MM and DLBCL preclinical studies to validate its potential as a therapy in these tumor types. Methods: Cellular proliferation assays determined IC 50 values of EZM0414 in MM and DLBCL cell line panels. Cell line-derived xenograft preclinical models of MM and DLBCL were evaluated for tumor growth inhibition (TGI) in response to EZM0414. H3K36me3 levels were determined by western blot analysis to evaluate target engagement. Combinatorial potential of SETD2 inhibition with MM and DLBCL standard of care (SOC) agents was evaluated in 7-day cotreatment in vitro cellular assays. Results: Inhibition of SETD2 by EZM0414 results in potent anti-proliferative effects in a panel of MM and DLBCL cell lines. EZM0414 inhibited proliferation in both t(4;14) and non-t(4;14) MM cell lines, with higher anti-proliferative activity generally observed in the t(4;14) subset of MM cell lines. The median IC 50value for EZM0414 in t(4;14) cell lines was 0.24 μM as compared to 1.2 μM for non-t(4;14) MM cell lines. Additionally, inhibitory growth effects on DLBCL cell lines demonstrated a wide range of sensitivity with IC 50 values from 0.023 μM to &gt;10 μM. EZM0414 resulted in statistically significant potent antitumor activity compared to the vehicle control in three MM and four DLBCL cell line-derived xenograft models. In the t(4;14) MM cell line-derived xenograft model, KMS-11, robust tumor growth regressions were observed at the top two doses with maximal TGI of 95%. In addition, two non-t(4;14) MM (RPMI-8226, MM.1S) and two DLBCL xenograft models (TMD8, KARPAS422) demonstrated &gt; 75% TGI; with two additional DLBCL models (WSU-DLCL2, SU-DHL-10) exhibiting &gt; 50% TGI in response to EZM0414. In all models tested, the antitumor effects observed correlated with reductions in intratumoral H3K36me3 levels demonstrating on-target inhibition of SETD2 methyltransferase activity in vivo. In vitro synergistic antiproliferative activity was also observed when EZM0414 was combined with certain SOC agents for MM and DLBCL. Conclusions: Targeting SETD2 with a small molecule inhibitor results in significantly reduced growth of t(4;14) MM, as well as non-t(4;14) MM and DLBCL cell lines, in both in vitro and in vivo preclinical studies. In addition, in vitro synergy was observed with EZM0414 and certain SOC agents commonly used in MM and DLBCL, supporting the combination of SETD2 inhibition with current MM and DLBCL therapies. This work provides the rationale for targeting SETD2 in B cell malignancies such as MM, especially t(4;14) MM, as well as DLBCL, and forms the basis for conducting Phase 1/1b clinical studies to evaluate the safety and activity of EZM0414 in patients with R/R MM and DLBCL. Disclosures Totman: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Brach: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Motwani: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Howe: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Deutschman: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Lampe: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Riera: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Tang: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Eckley: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Alford: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Duncan: Epizyme, Inc.: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Farrow: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Dransfield: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Raimondi: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Thomeius: Foghorn Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cosmopoulos: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company. Kutok: Epizyme, Inc.: Current Employment, Current equity holder in publicly-traded company.

scholarly journals Paics Inhibition Is a Potential Therapeutic Strategy for MYC-Positive DLBCL

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 396-396
Author(s):  
Kohta Miyawaki ◽  
Takuji Yamauchi ◽  
Takeshi Sugio ◽  
Kensuke Sasaki ◽  
Hiroaki Miyoshi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cell ◽  
Cell Lines ◽  
Research Funding ◽  
Poor Prognosis ◽  
De Novo ◽  
Lymphoma Cells ◽  
Purine Synthesis

Diffuse large B-cell lymphoma (DLBCL) is among the most common hematological malignancies with varying prognosis. As many as forty percent of patients eventually experience relapsed/refractory disease after combinatorial chemo-immunotherapies, R-CHOP, and prognosis after relapse is dismal. MYC is among the most established prognostic factors and associated with clinically-distinct subsets of DLBCL with poor prognosis: double-expressor lymphoma (DEL) and double-hit lymphoma (DHL). MYC is co-expressed with BCL2 in DEL, which consists of 60% of activated B-cell type DLBCL (ABC-DLBCL) cases, while DHL, defined by coexistence of MYC and BCL2/BCL6 rearrangements, were reportedly observed in 15% of germinal center B-cell like DLBCL (GCB-DLBCL). Considering that MYC-positive DLBCLs exhibit dismal outcomes, pharmacological inhibition of MYC activity is highly demanded; however, direct targeting of MYC has been proven challenging. Here we show that PAICS (phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase), which catalyzes a critical step in de novo purine synthesis, functions downstream of MYC in DLBCL cells. We further show MRT252040, a newly-developed PAICS inhibitor, effectively suppresses proliferation of MYC-driven DLBCL cells in vitro and in vivo. Through the nCounter-based transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) tissues from 170 untreated DLBCL patients, we found that MYC and PAICS were co-expressed and their mRNA levels were among the most predictive for poor prognosis after standard R-CHOP therapy. Their expression levels were particularly high in a subset of ABC-DLBCL and extranodal DLBCL, namely in DEL and DHL cases. Importantly, these findings were validated using three independent cohorts (Schmitz et al. NEJM, 2018). MYC and PAICS expression levels were high in most DLBCL lines and low in normal B cells in the lymph nodes, while they were variable in primary DLBCL tissues, revealed by nCounter and immunofluorescence. This trend was more evident in PAICS due presumably to active de novo purine biosynthesis in highly-proliferative cell lines and a subset of DLBCLs, including MYC-positive DLBCLs. These findings were also validated using the DepMap, a publicly-available genome-wide CRISPR/Cas9 dropout screen datasets. PAICS was among the top-ranked essential genes for the survival of DLBCL cell lines. Since co-expression of MYC and PAICS in a subset of DLBCL were indicative of a functional relationship between the two factors, we explored publicly-available ChIP-seq datasets to see if MYC directly regulates PAICS expression. As expected, MYC ChIP-seq signals were highly enriched near the PAICS promoter in a series of cancer cell lines. Furthermore, shRNA-mediated MYC knockdown led to reduced levels of PAICS mRNA in MYC-positive DLBCL cells and significantly slowed their growth. Collectively, these data suggest that PAICS is a direct transcriptional target of MYC, playing a key role in proliferation of MYC-positive DLBCL cells. To assess the feasibility of PAICS-inhibition as a therapeutic option for MYC-positive DLBCLs, we tested MRT252040 for its anti-lymphoma activity in vitro and in vivo. To do so, we first assessed cell cycle status and Annexin positivity upon MRT252040 treatment using a series of DLBCL cell lines. As expected, MRT252040-mediated PAICS inhibition induced cell cycle arrest and apoptosis. Furthermore, MRT252040 treatment significantly delayed proliferation of DLBCL cell lines, namely those harboring MYC rearrangements. Finally, to assess anti-lymphoma activity of MRT252040 in vivo, we tested MRT252040 efficacy using patient-derived xenograft DLBCL. After xenotransplantation, proportions of lymphoma cells per total mononuclear cells in peripheral blood were examined over time by FACS, and MRT252040 (or vehicle) treatment was initiated once lymphoma cells constituted &gt;0.1%. MRT252040-treated mice survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of MRT252040 monotherapy against DLBCL in vivo. Our data suggest that MYC regulates the de novo purine synthesis pathway via directly transactivating PAICS expression. We propose that MRT252040, a newly-developed PAICS inhibitor, warrants attention as a novel therapeutic approach for MYC-positive DLBCLs, which otherwise exhibit poor clinical outcomes. Disclosures Ohshima: SRL, Inc.: Consultancy; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Celgene Corp.: Honoraria, Research Funding; NEC Corp.: Research Funding. Akashi:Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

scholarly journals Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1442-1442
Author(s):  
Xiangmeng Wang ◽  
Po Yee Mak ◽  
Wencai Ma ◽  
Xiaoping Su ◽  
Hong Mu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Single Agent ◽  
Advisory Committees ◽  
Equity Ownership ◽  
Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

The Novel Proteasome Inhibitor CEP-18770 Inhibits Myeloma Tumor Growth In Vitro and In Vivo and Enhances the Anti-MM Effects of Melphalan

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 843-843
Author(s):  
Eric SancheZ ◽  
Richard A Campbell ◽  
Jeffrey A Steinberg ◽  
Mingjie Li ◽  
Haiming Chen ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Tumor Volume ◽  
Single Agent ◽  
Human Tumor ◽  
The Novel

Abstract Proteasome inhibitors (PI) have been shown to be effective agents for the treatment of multiple myeloma (MM) and enhance the anti-tumor effects of a variety of chemotherapeutic drugs including melphalan and doxorubicin as well as arsenic trioxide (ATO). The novel proteasome inhibitor CEP-18770 has recently been shown to induce cytotoxic effects across a broad panel of human tumor cell lines including MM in vitro. However, little data exists on the in vivo anti-MM effects of this PI either alone or in combination with other active anti-MM drugs. First, we examined the anti-proliferative effects of treating MM cell lines in vitro with CEP-18770 alone and in combination with melphalan, arsenic trioxide (ATO) and doxorubicin. MM cell lines were cultured without fetal bovine serum and incubated in the presence of CEP-18770 alone and in combination with these agents for 48 hours. Cell growth was then measured using an MTS assay. First, RPMI8226 and U266 cells were tested in vitro using a constant concentration of melphalan or doxorubicin in combination with varying concentrations of CEP-18770 or varying concentrations of the chemotherapeutic agent with constant CEP-18770. Although single agent treatment showed marked anti-proliferative effects, combination indexes as calculated by the Chou-Talalay method showed synergistic anti- MM effects of CEP-18770 with either melphalan or doxorubicin in these MM cell lines. In addition, similar experiments were carried out evaluating the combination of ATO plus CEP-18770 in RPMI8226 cells and also showed synergism with this combination. Next, a series of in vivo studies were conducted using our SCID-hu models of MM including LAGλ-1, LAGκ-1A and LAGκ-1B. Mice receiving CEP-18770 at 0.1, 0.3, 1, and 3 mg/kg were injected twice weekly via intravenous injection throughout the study. CEP-18770 dosed at 10 mg/kg was administered via oral gavage twice weekly and mice dosed with melphalan received injections once weekly via intraperitoneal injection. Mice bearing intramuscularly implanted LAGλ-1 were treated with CEP-18770 or vehicle alone. Mice treated with the PI inhibited tumor growth as determined by human immunoglobulin (hIg) G levels and measurement of tumor volume (P = 0.0008) compared to mice receiving vehicle. A significant inhibition of both human paraprotein secretion and reduction of tumor growth was also observed in LAGk-1A-bearing mice treated with CEP-18770 at 1, 3 and 10 mg/kg (hIgG: P = 0.0001, P = 0.0002 and P = 0.0001, respectively; tumor volume: P = 0.0001, P = 0.0001 and P = 0.0001, respectively) and LAGk-1B-bearing mice treated with CEP-18770 at 3 and 10 mg/kg (hIgG: P = 0.0008 and P = 0.0034, respectively; tumor volume: P = 0.0008 and P = 0.0028, respectively) compared to mice receiving vehicle. Finally, the combination of CEP-18770 (1 mg/kg) plus melphalan (3 mg/kg) was tested in LAGk-1B-bearing mice. Mice treated with the combination showed markedly smaller tumors compared to treatment with vehicle (P = 0.0008) or melphalan alone (P = 0.0204). Mice treated with the PI alone or in combination with melphalan did not show any observed toxicity. Thus, these studies provide promising preclinical data to suggest the potent anti-MM effects of CEP-18770 both in vitro and in vivo and also suggest that this new PI may enhance the anti-MM effects of several active anti-MM agents including melphalan, doxorubicin and ATO.

scholarly journals Chemical Genomics Reveals JAK STAT Activation As a Mechanism of Resistance to HDAC Inhibitors in B Cell Lymphomas

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 271-271
Author(s):  
Matthew S. McKinney ◽  
Anne W Beaven ◽  
Andrea Moffitt ◽  
Jason Landon Smith ◽  
Eric Lock ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Hdac Inhibitors ◽  
Resistant Cell ◽  
Hdac Inhibition ◽  
Stat Pathway

Abstract Background: HDAC inhibitors (HDACi) are being investigated as treatment for relapsed/refractory non Hodgkin lymphoma (NHL) and other cancers. However, the mechanisms underlying sensitivity and resistance to HDAC inhibition in lymphomas have not been fully characterized. We probed the cellular and molecular response to HDACi in vitro and in vivo in order to determine factors that dictate the response to HDACi and to enable design of approaches to incorporate HDACi into novel combination therapeutics. Methods: High-throughput cytotoxicity screening was performed using two different HDAC inhibitors, LBH589 (panobinostat) and SAHA (vorinostat) in 52 lymphoid cell lines characterized through RNA-seq and microarray gene expression profiling. This screen revealed a greater than 50-fold range in concentration needed to induce cytotoxicity for the 2 different HDAC inhibitors and there was moderate correlation between the 2 compounds in this panel (Pearson correlation r = 0.76, p < 0.01). By pairing this chemosensitivity data with gene expression profiles of the screened cell lines, we developed a gene expression classifier for LBH589 that identified resistant and sensitive cell line groups. This predictor was applied to B-cell NHL cell lines tested with LBH589 in the Cancer Cell Line Encyclopedia (CCLE) and we found that the sensitive and resistant cell line groups distinguished by this method differed more than 5-fold in IC50 (0.021 vs. 1.24 nM, P < 0.01 by Wilcoxon rank sum), thus validating the ability of this approach to distinguish HDACi resistant cell lines. We further initiated a clinical trial of LBH589 in relapsed/refractory diffuse large B cell lymphoma patients combined with RNAseq profiling of their tumors prior to embarking on treatment. We treated nine patients with LBH589, and application of our response predictor to scaled RNAseq gene expression data revealed 4 predicted responders and 5 predicted non-responders. Two of the predicted responders had a clinical response to LBH589, whereas none of the predicted non-responders had a clinical response, thus our classifier was able to identify all of the LBH589-responsive patients from this cohort (P = 0.08 by Fisher's exact test). Analysis of differentially expressed molecular pathways in HDACi sensitive and resistant samples by gene set enrichment revealed the JAK-STAT pathway as the most differentially expressed pathway associated with HDACi resistance (at P < 0.001 and FDR < 0.20). We further identified a number of distinct mutations including STAT3, SOCS1 and JAK1 that were associated with activation of the JAK-STAT pathway by gene expression signatures and the LBH589 response signature in DLBCL cell lines and patient samples by analysis of RNA-seq data. Phosphoprotein analysis by Western blot and Sis-inducible-element (SIE) luciferase reporter assays were used to confirm JAK-STAT activation in these samples and we found that overexpression of STAT3 Src-homology domain mutations activated JAK-STAT3 signaling in isogenic cell lines and fostered resistance to LBH589 in vitro. Conversely, using in vivo DLBCL xenograft models, we found that combining JAK-STAT and HDAC inhibition by treatment with LBH589 and ruxolitinib resulted in synergistic reduction of tumor cell viability and tumor growth with tolerable toxicity in mice. Conclusions: Sustained JAK-STAT activation appears to mediate resistance to HDAC inhibition in DLBCL and other NHLs and several recurrent genetic lesions drive JAK-STAT activation in these diseases. This process can be overcome by JAK 1/2 inhibition with ruxolitinib and these findings demonstrate a role for combination therapy with HDAC inhibitors and small molecules targeting the JAK-STAT pathway in lymphoid malignancies. Disclosures No relevant conflicts of interest to declare.

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