Immunotherapy-resistant acute lymphoblastic leukemia cells exhibit reduced CD19 and CD22 expression and BTK pathway dependency.

While therapies targeting CD19 by antibodies, chimeric antigen receptor T cells (CAR-T), and T cell engagers have improved the response rates in B cell malignancies, the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19 immunotoxin. Single-cell RNA-Seq (scRNA-Seq) showed an increase in transcriptionally distinct CD19lo populations among resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19lo-resistant cells. An assay for transposase-accessible chromatin with sequencing (ATAC-Seq) showed decreased chromatin accessibility at promoters of both CD19 and CD22 in the resistant cell populations. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with B cell acute lymphoblastic leukemia (B-ALL) that relapsed after CD19 CAR-T-targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19lo resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both Bruton's tyrosine kinase (BTK) and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.

Combined epigenetic and immunotherapy for blastic and classical mantle cell lymphoma.

Classical MCL (cMCL) constitutes 6-8% of all B cell NHL. Despite recent advances, MCL is incurable except with allogeneic stem cell transplant. Blastic mantle cell lymphoma (bMCL) is a rarer subtype of cMCL associated with an aggressive clinical course and poor treatment response, frequent relapse and poor outcomes. We treated 13 bMCL patients with combined epigenetic and immunotherapy treatment consisting of vorinostat, cladribine and rituximab (SCR). We report an increased OS greater than 40 months with several patients maintaining durable remissions without relapse for longer than 5 years. This is remarkably better then current treatment regimens which in bMCL range from 14.5-24 months with conventional chemotherapy regimens. We demonstrate that the G/A870 CCND1 polymorphism is predictive of blastic disease, nuclear localization of cyclinD1 and response to SCR therapy. The major resistance mechanisms to SCR therapy are loss of CD20 expression and evasion of treatment by sanctuary in the CNS. These data indicate that administration of epigenetic agents improves efficacy of anti-CD20 immunotherapies. This approach is promising in the treatment of MCL and potentially other previously treatment refractory cancers.

Triple MAPK inhibition salvaged a relapsed post-BCMA CAR-T cell therapy multiple myeloma patient with a BRAF V600E subclonal mutation.

BACKGROUND: Multiple Myeloma (MM) is a progressive plasma cell neoplasm characterized by heterogeneous clonal expansion. Despite promising response rates achieved with anti-BCMA CAR-T cell therapy, patients may still relapse and there are currently no clear therapeutic options in post-CAR-T settings. In this report, we present a case of a post-BCMA CAR-T relapsed/refractory (RR) MM patient with skin extramedullary disease (EMD) in which a novel MAPK inhibition combinatorial strategy was implemented based on next-generation sequencing and in vitro experiments. CASE PRESENTATION: A 61-year-old male with penta-refractory MM penta- (IgA lambda), ISS stage 3 with hyperdiploidy, gain of 1q21 and del13 was treated with anti-BCMA CAR-T cell therapy, achieving a best response of VGPR. He progressed after 6 months and was salvaged for a short period with autologous stem cell transplantation. Eventually, he progressed with extramedullary disease manifested as subcutaneous nodules. Based on whole-exome sequencing, we identified a BRAF (V600E) dominant subclone in both bone marrow and cutaneous plasmacytoma. Following in vitro experiments, and according to our previous studies, we implemented a triple MAPK inhibition strategy under which the patient achieved a very good partial response for 110 days, which allowed to bridge him to subsequent clinical trials and eventually achieve a stringent complete response (sCR). CONCLUSION: Here, we show the applicability, effectiveness, and tolerability the triple MAPK inhibition strategy in the context of post-BCMA CAR-T failure in specific subset of patients. The triple therapy could bridge our hospice bound RRMM patient with BRAF (V600E) to further therapeutic options where sCR was achieved. We will further evaluate triple MAPK inhibition in patients with BRAF V600E in a precision medicine clinical trial launching soon.

A Three-Gene Signature Predicts Response to Selinexor in Multiple Myeloma.

PURPOSE: Selinexor is the first selective inhibitor of nuclear export to be approved for the treatment of relapsed or refractory multiple myeloma (MM). Currently, there are no known genomic biomarkers or assays to help select MM patients at higher likelihood of response to selinexor. Here, we aimed to characterize the transcriptomic correlates of response to selinexor-based therapy. METHODS: We performed RNA sequencing on CD138+ cells from the bone marrow of 100 patients with MM who participated in the BOSTON study, followed by differential gene expression and pathway analysis. Using the differentially expressed genes, we used cox proportional hazard models to identify a gene signature predictive of response to selinexor, followed by validation in external cohorts. RESULTS: The three-gene signature predicts response to selinexor-based therapy in patients with MM in the BOSTON cohort. Then, we validated this gene signature in 64 patients from the STORM cohort of triple-class refractory MM and additionally in an external cohort of 35 patients treated in a real-world setting outside of clinical trials. We found that the signature tracks with both depth and duration of response, and it also validates in a different tumor type using a cohort of pretreatment tumors from patients with recurrent glioblastoma. Furthermore, the genes involved in the signature, WNT10A, DUSP1, and ETV7, reveal a potential mechanism through upregulated interferon-mediated apoptotic signaling that may prime tumors to respond to selinexor-based therapy. CONCLUSION: In this study, we present a present a novel, three-gene expression signature that predicts selinexor response in MM. This signature has important clinical relevance as it could identify patients with cancer who are most likely to benefit from treatment with selinexor-based therapy.

Patient similarity network of newly diagnosed multiple myeloma identifies patient subgroups with distinct genetic features and clinical implications.

The remarkable genetic heterogeneity of multiple myeloma poses a substantial challenge for proper prognostication and clinical management of patients. Here, we introduce MM-PSN, the first multiomics patient similarity network of myeloma. MM-PSN enabled accurate dissection of the genetic and molecular landscape of the disease and determined 12 distinct subgroups defined by five data types generated from genomic and transcriptomic profiling of 655 patients. MM-PSN identified patient subgroups not previously described defined by specific patterns of alterations, enriched for specific gene vulnerabilities, and associated with potential therapeutic options. Our analysis revealed that co-occurrence of t(4;14) and 1q gain identified patients at significantly higher risk of relapse and shorter survival as compared to t(4;14) as a single lesion. Furthermore, our results show that 1q gain is the most important single lesion conferring high risk of relapse and that it can improve on the current International Staging Systems (ISS and R-ISS).

SOX11 Inhibitors Are Cytotoxic in Mantle Cell Lymphoma.

PURPOSE: Mantle cell lymphoma (MCL) is a fatal subtype of non-Hodgkin lymphoma. SOX11 transcription factor is overexpressed in the majority of nodal MCL. We have previously reported that B cell-specific overexpression of SOX11 promotes MCL pathogenesis via critically increasing BCR signaling in vivo. SOX11 is an attractive target for MCL therapy; however, no small-molecule inhibitor of SOX11 has been identified to date. Although transcription factors are generally considered undruggable, the ability of SOX11 to bind to the minor groove of DNA led us to hypothesize that there may exist cavities at the protein-DNA interface that are amenable to targeting by small molecules. EXPERIMENTAL DESIGN: Using a combination of in silico predictions and experimental validations, we report here the discovery of three structurally related compounds (SOX11i) that bind SOX11, perturb its interaction with DNA, and effect SOX11-specific anti-MCL cytotoxicity. RESULTS: We find mechanistic validation of on-target activity of these SOX11i in the inhibition of BCR signaling and the transcriptional modulation of SOX11 target genes, specifically, in SOX11-expressing MCL cells. One of the three SOX11i exhibits relatively superior in vitro activity and displays cytotoxic synergy with ibrutinib in SOX11-expressing MCL cells. Importantly, this SOX11i induces cytotoxicity specifically in SOX11-positive ibrutinib-resistant MCL patient samples and inhibits Bruton tyrosine kinase phosphorylation in a xenograft mouse model derived from one of these subjects. CONCLUSIONS: Taken together, our results provide a foundation for therapeutically targeting SOX11 in MCL by a novel class of small molecules.

A phase II study of pomalidomide, daily oral cyclophosphamide, and dexamethasone in relapsed/refractory multiple myeloma.

Relapsed/refractory multiple myeloma patients treated with pomalidomide and dexamethasone have an overall response rate (ORR) of ∼30% and median progression-free survival (PFS) of 4-5 months. Previous studies explored addition of weekly cyclophosphamide, but we hypothesized that daily dosing allows for better synergy. We report the open-label, single-center phase II study of pomalidomide, daily cyclophosphamide and weekly dexamethasone (PCD). Thirty-three patients were evaluable for efficacy and underwent 28-day cycles of pomalidomide (4 mg/day, D1-21), cyclophosphamide (50 mg b.i.d., D1-21) and weekly dexamethasone. All were lenalidomide-refractory and 55% were refractory to lenalidomide and proteasome inhibitor. ORR was 73%; median PFS and overall survival were 13.3 months and 57.2 months respectively. Grade 3/4 toxicities were primarily hematologic but manageable with dose reductions. Early disease progression correlated with MYC expression and flow cytometry demonstrates an activated microenvironment post-PCD. Addition of metronomic cyclophosphamide to pomalidomide and dexamethasone is a cost-effective, oral regimen with encouraging PFS.

Discovery of a first-in-class EZH2 selective degrader.

The enhancer of zeste homolog 2 (EZH2) is the main enzymatic subunit of the PRC2 complex, which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3) to promote transcriptional silencing. EZH2 is overexpressed in multiple types of cancer including triple-negative breast cancer (TNBC), and high expression levels correlate with poor prognosis. Several EZH2 inhibitors, which inhibit the methyltransferase activity of EZH2, have shown promise in treating sarcoma and follicular lymphoma in clinics. However, EZH2 inhibitors are ineffective at blocking proliferation of TNBC cells, even though they effectively reduce the H3K27me3 mark. Using a hydrophobic tagging approach, we generated MS1943, a first-in-class EZH2 selective degrader that effectively reduces EZH2 levels in cells. Importantly, MS1943 has a profound cytotoxic effect in multiple TNBC cells, while sparing normal cells, and is efficacious in vivo, suggesting that pharmacologic degradation of EZH2 can be advantageous for treating the cancers that are dependent on EZH2.

Mutation-derived Neoantigen-specific T-cell Responses in Multiple Myeloma.

PURPOSE: Somatic mutations in cancer cells can give rise to novel protein sequences that can be presented by antigen-presenting cells as neoantigens to the host immune system. Tumor neoantigens represent excellent targets for immunotherapy, due to their specific expression in cancer tissue. Despite the widespread use of immunomodulatory drugs and immunotherapies that recharge T and NK cells, there has been no direct evidence that neoantigen-specific T-cell responses are elicited in multiple myeloma. EXPERIMENTAL DESIGN: Using next-generation sequencing data we describe the landscape of neo-antigens in 184 patients with multiple myeloma and successfully validate neoantigen-specific T cells in patients with multiple myeloma and support the feasibility of neoantigen-based therapeutic vaccines for use in cancers with intermediate mutational loads such as multiple myeloma. RESULTS: In this study, we demonstrate an increase in neoantigen load in relapsed patients with multiple myeloma as compared with newly diagnosed patients with multiple myeloma. Moreover, we identify shared neoantigens across multiple patients in three multiple myeloma oncogenic driver genes (KRAS, NRAS, and IRF4). Next, we validate neoantigen T-cell response and clonal expansion in correlation with clinical response in relapsed patients with multiple myeloma. This is the first study to experimentally validate the immunogenicity of predicted neoantigens from next-generation sequencing in relapsed patients with multiple myeloma. CONCLUSIONS: Our findings demonstrate that somatic mutations in multiple myeloma can be immunogenic and induce neoantigen-specific T-cell activation that is associated with antitumor activity in vitro and clinical response in vivo. Our results provide the foundation for using neoantigen targeting strategies such as peptide vaccines in future trials for patients with multiple myeloma.

SOX11 augments BCR signaling to drive MCL-like tumor development.

Mantle cell lymphoma (MCL) is characterized by increased B-cell receptor (BCR) signaling, and BTK inhibition is an effective therapeutic intervention in MCL patients. The mechanisms leading to increased BCR signaling in MCL are poorly understood, as mutations in upstream regulators of BCR signaling such as CD79A, commonly observed in other lymphomas, are rare in MCL. The transcription factor SOX11 is overexpressed in the majority (78% to 93%) of MCL patients and is considered an MCL-specific oncogene. So far, attempts to understand SOX11 function in vivo have been hampered by the lack of appropriate animal models, because germline deletion of SOX11 is embryonically lethal. We have developed a transgenic mouse model (Eµ-SOX11-EGFP) in the C57BL/6 background expressing murine SOX11 and EGFP under the control of a B-cell-specific IgH-Eµ enhancer. The overexpression of SOX11 exclusively in B cells exhibits oligoclonal B-cell hyperplasia in the spleen, bone marrow, and peripheral blood, with an immunophenotype (CD5+CD19+CD23-) identical to human MCL. Furthermore, phosphocytometric time-of-flight analysis of the splenocytes from these mice shows hyperactivation of pBTK and other molecules in the BCR signaling pathway, and serial bone marrow transplant from transgenic donors produces lethality with decreasing latency. We report here that overexpression of SOX11 in B cells promotes BCR signaling and a disease phenotype that mimics human MCL.

Precision Medicine for Relapsed Multiple Myeloma on the Basis of an Integrative Multiomics Approach.

PURPOSE: Multiple myeloma (MM) is a malignancy of plasma cells, with a median survival of 6 years. Despite recent therapeutic advancements, relapse remains mostly inevitable, and the disease is fatal in the majority of patients. A major challenge in the treatment of patients with relapsed MM is the timely identification of treatment options in a personalized manner. Current approaches in precision oncology aim at matching specific DNA mutations to drugs, but incorporation of genome-wide RNA profiles has not yet been clinically assessed. METHODS: We have developed a novel computational platform for precision medicine of relapsed and/or refractory MM on the basis of DNA and RNA sequencing. Our approach expands on the traditional DNA-based approaches by integrating somatic mutations and copy number alterations with RNA-based drug repurposing and pathway analysis. We tested our approach in a pilot precision medicine clinical trial with 64 patients with relapsed and/or refractory MM. RESULTS: We generated treatment recommendations in 63 of 64 patients. Twenty-six patients had treatment implemented, and 21 were assessable. Of these, 11 received a drug that was based on RNA findings, eight received a drug that was based on DNA, and two received a drug that was based on both RNA and DNA. Sixteen of the 21 evaluable patients had a clinical response (ie, reduction of disease marker ≥ 25%), giving a clinical benefit rate of 76% and an overall response rate of 66%, with five patients having ongoing responses at the end of the trial. The median duration of response was 131 days. CONCLUSION: Our results show that a comprehensive sequencing approach can identify viable options in patients with relapsed and/or refractory myeloma, and they represent proof of principle of how RNA sequencing can contribute beyond DNA mutation analysis to the development of a reliable drug recommendation tool.

A phase 2 study of panobinostat with lenalidomide and weekly dexamethasone in myeloma.

Phase 3 studies combining histone deacetylase inhibitors with bortezomib were hampered by gastrointestinal (GI) intolerance, which was not observed when combined with immunomodulatory drugs. This study is a single-center phase 2 study of panobinostat with lenalidomide and dexamethasone (FRD). Twenty-seven relapsed multiple myeloma patients were enrolled. Twenty-two patients (81%) were lenalidomide refractory and 9 (33%), 14 (52%), and 7 (26%) were refractory to pomalidomide, bortezomib, and carfilzomib, respectively. High-risk molecular findings were present in 17 (63%) patients. Responses included 2 complete responses (CRs), 4 very good partial responses (VGPRs), 5 partial responses (PRs), and 9 minimal responses (MRs) for an overall response rate of 41%, clinical benefit rate of 74%, and a disease control rate of 96%. The median progression-free survival (PFS) was 7.1 months. In the 22 lenalidomide-refractory patients, there were 1 CR, 4 VGPRs, 3 PRs, and 7 MRs, with a median PFS of 6.5 months. Median overall survival was not reached. Grade 3/4 toxicities were primarily hematologic. Gene expression profiling of enrollment tumor samples revealed a set of 1989 genes associated with short (<90 days) PFS to therapy. MAGEA1 RNA and protein expression were correlated with short PFS, and laboratory studies demonstrated a role for MAGE-A in resistance to panobinostat-induced cell death. FRD demonstrates durable responses, even in high-risk, lenalidomide-refractory patients, indicating the essential role of panobinostat in attaining responses. MAGEA1 expression may represent a functional biomarker for resistance to panobinostat. In contrast to PANORAMA 1, there were no significant GI toxicities and primarily expected hematologic toxicities. This trial was registered at www.clinicaltrials.gov as #NCT00742027.

Dual Targeting of CDK4 and ARK5 Using a Novel Kinase Inhibitor ON123300 Exerts Potent Anticancer Activity against Multiple Myeloma.

Multiple myeloma is a fatal plasma cell neoplasm accounting for over 10,000 deaths in the United States each year. Despite new therapies, multiple myeloma remains incurable, and patients ultimately develop drug resistance and succumb to the disease. The response to selective CDK4/6 inhibitors has been modest in multiple myeloma, potentially because of incomplete targeting of other critical myeloma oncogenic kinases. As a substantial number of multiple myeloma cell lines and primary samples were found to express AMPK-related protein kinase 5(ARK5), a member of the AMPK family associated with tumor growth and invasion, we examined whether dual inhibition of CDK4 and ARK5 kinases using ON123300 results in a better therapeutic outcome. Treatment of multiple myeloma cell lines and primary samples with ON123300 in vitro resulted in rapid induction of cell-cycle arrest followed by apoptosis. ON123300-mediated ARK5 inhibition or ARK5-specific siRNAs resulted in the inhibition of the mTOR/S6K pathway and upregulation of the AMPK kinase cascade. AMPK upregulation resulted in increased SIRT1 levels and destabilization of steady-state MYC protein. Furthermore, ON123300 was very effective in inhibiting tumor growth in mouse xenograft assays. In addition, multiple myeloma cells sensitive to ON123300 were found to have a unique genomic signature that can guide the clinical development of ON123300. Our study provides preclinical evidence that ON123300 is unique in simultaneously inhibiting key oncogenic pathways in multiple myeloma and supports further development of ARK5 inhibition as a therapeutic approach in multiple myeloma.

Epigenetic therapy overcomes treatment resistance in T cell prolymphocytic leukemia.

T cell prolymphocytic leukemia (T-PLL) is a rare, mature T cell neoplasm with distinct features and an aggressive clinical course. Early relapse and short overall survival are commonplace. Use of the monoclonal anti-CD52 antibody alemtuzumab has improved the rate of complete remission and duration of response to more than 50% and between 6 and 12 months, respectively. Despite this advance, without an allogeneic transplant, resistant relapse is inevitable. We report seven complete and one partial remission in eight patients receiving alemtuzumab and cladribine with or without a histone deacetylase inhibitor. These data show that administration of epigenetic agents can overcome alemtuzumab resistance. We also report epigenetically induced expression of the surface receptor protein CD30 in T-PLL. Subsequent treatment with the anti-CD30 antibody-drug conjugate brentuximab vedotin overcame organ-specific (skin) resistance to alemtuzumab. Our findings demonstrate activity of combination epigenetic and immunotherapy in the incurable illness T-PLL, particularly in the setting of previous alemtuzumab therapy.

Harnessing Noxa demethylation to overcome Bortezomib resistance in mantle cell lymphoma.

Bortezomib (BZM) is the first proteasome inhibitor approved for relapsed Mantle Cell Lymphoma (MCL) with durable responses seen in 30%-50% of patients. Given that a large proportion of patients will not respond, BZM resistance is a significant barrier to use this agent in MCL. We hypothesized that a subset of aberrantly methylated genes may be modulating BZM response in MCL patients. Genome-wide DNA methylation analysis using a NimbleGen array platform revealed a striking promoter hypomethylation in MCL patient samples following BZM treatment. Pathway analysis of differentially methylated genes identified molecular mechanisms of cancer as a top canonical pathway enriched among hypomethylated genes in BZM treated samples. Noxa, a pro-apoptotic Bcl-2 family member essential for the cytotoxicity of BZM, was significantly hypomethylated and induced following BZM treatment. Therapeutically, we could demethylate Noxa and induce anti-lymphoma activity using BZM and the DNA methytransferase inhibitor Decitabine (DAC) and their combination in vitro and in vivo in BZM resistant MCL cells. These findings suggest a role for dynamic Noxa methylation for the therapeutic benefit of BZM. Potent and synergistic cytotoxicity between BZM and DAC in vitro and in vivo supports a strategy for using epigenetic priming to overcome BZM resistance in relapsed MCL patients.

Integrative genomic analysis of temozolomide resistance in diffuse large B-cell lymphoma.

PURPOSE: Despite advances, there is an urgent need for effective therapeutics for relapsed diffuse large B-cell lymphoma, particularly in elderly patients and primary central nervous system (CNS) lymphoma. Temozolomide (TMZ), an oral DNA-alkylating agent routinely used in the therapy of glioblastoma multiforme, is active in patients with primary CNS lymphoma but the response rates are low. The mechanisms contributing to TMZ resistance are unknown. EXPERIMENTAL DESIGN: We undertook an unbiased and genome-wide approach to understand the genomic methylation and gene expression profiling differences associated with TMZ resistance in diffuse large B-cell lymphoma cell lines and identify mechanisms to overcome TMZ resistance. RESULTS: TMZ was cytotoxic in a subset of diffuse large B-cell lymphoma cell lines, independent of MGMT promoter methylation or protein expression. Using Connectivity Map (CMAP), we identified several compounds capable of reversing the gene expression signature associated with TMZ resistance. The demethylating agent decitabine (DAC) is identified by CMAP as capable of reprogramming gene expression to overcome TMZ resistance. Treatment with DAC led to increased expression of SMAD1, a transcription factor involved in TGF-ß/bone morphogenetic protein (BMP) signaling, previously shown to be epigenetically silenced in resistant diffuse large B-cell lymphoma. In vitro and in vivo treatment with a combination of DAC and TMZ had greater antilymphoma activity than either drug alone, with complete responses in TMZ-resistant diffuse large B-cell lymphoma murine xenograft models. CONCLUSIONS: Integrative genome-wide methylation and gene expression analysis identified novel genes associated with TMZ resistance and demonstrate potent synergy between DAC and TMZ. The evidence from cell line and murine experiments supports prospective investigation of TMZ in combination with demethylating agents in diffuse large B-cell lymphoma.

Akt inhibitors MK-2206 and nelfinavir overcome mTOR inhibitor resistance in diffuse large B-cell lymphoma.

PURPOSE: The mTOR pathway is constitutively activated in diffuse large B-cell lymphoma (DLBCL). mTOR inhibitors have activity in DLBCL, although response rates remain low. We evaluated DLBCL cell lines with differential resistance to the mTOR inhibitor rapamycin: (i) to identify gene expression profile(s) (GEP) associated with resistance to rapamycin, (ii) to understand mechanisms of rapamycin resistance, and (iii) to identify compounds likely to synergize with mTOR inhibitor. EXPERIMENTAL DESIGN: We sought to identify a GEP of mTOR inhibitor resistance by stratification of eight DLBCL cell lines with respect to response to rapamycin. Then, using pathway analysis and connectivity mapping, we sought targets likely accounting for this resistance and compounds likely to overcome it. We then evaluated two compounds thus identified for their potential to synergize with rapamycin in DLBCL and confirmed mechanisms of activity with standard immunoassays. RESULTS: We identified a GEP capable of reliably distinguishing rapamycin-resistant from rapamycin-sensitive DLBCL cell lines. Pathway analysis identified Akt as central to the differentially expressed gene network. Connectivity mapping identified compounds targeting Akt as having a high likelihood of reversing the GEP associated with mTOR inhibitor resistance. Nelfinavir and MK-2206, chosen for their Akt-inhibitory properties, yielded synergistic inhibition of cell viability in combination with rapamycin in DLBCL cell lines, and potently inhibited phosphorylation of Akt and downstream targets of activated mTOR. CONCLUSIONS: GEP identifies DLBCL subsets resistant to mTOR inhibitor therapy. Combined targeting of mTOR and Akt suppresses activation of key components of the Akt/mTOR pathway and results in synergistic cytotoxicity. These findings are readily adaptable to clinical trials.

HDAC inhibitors and decitabine are highly synergistic and associated with unique gene-expression and epigenetic profiles in models of DLBCL.

Interactions between histone deacetylase inhibitors (HDACIs) and decitabine were investigated in models of diffuse large B-cell lymphoma (DLBCL). A number of cell lines representing both germinal center B-like and activated B-cell like DLBCL, patient-derived tumor cells and a murine xenograft model were used to study the effects of HDACIs and decitabine in this system. All explored HDACIs in combination with decitabine produced a synergistic effect in growth inhibition and induction of apoptosis in DLBCL cells. This effect was time dependent, mediated via caspase-3 activation, and resulted in increased levels of acetylated histones. Synergy in inducing apoptosis was confirmed in patient-derived primary tumor cells treated with panobinostat and decitabine. Xenografting experiments confirmed the in vitro activity and tolerability of the combination. We analyzed the molecular basis for this synergistic effect by evaluating gene-expression and methylation patterns using microarrays, with validation by bisulfite sequencing. These analyses revealed differentially expressed genes and networks identified by each of the single treatment conditions and by the combination therapy to be unique with few overlapping genes. Among the genes uniquely altered by the combination of panobinostat and decitabine were VHL, TCEB1, WT1, and DIRAS3.