Violetta V. Leshchenko

Genomewide DNA methylation analysis reveals novel targets for drug development in mantle cell lymphoma

Mantle cell lymphoma (MCL) is a mostly incurable malignancy arising from naive B cells (NBCs) in the mantle zone of lymph nodes. We analyzed genomewide methylation in MCL patients with the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay and found significant aberrancy in promoter methylation patterns compared with normal NBCs. Using biologic and statistical criteria, we further identified 4 hypermethylated genes CDKN2B, MLF-1, PCDH8, and HOXD8 and 4 hypomethylated genes CD37, HDAC1, NOTCH1, and CDK5 when aberrant methylation was associated with inverse changes in mRNA levels. Immunohistochemical analysis of an independent cohort of MCL patient samples confirmed CD37 surface expression in 93% of patients, validating its selection as a target for MCL therapy. Treatment of MCL cell lines with a small modular immunopharmaceutical (CD37-SMIP) resulted in significant loss of viability in cell lines with intense surface CD37 expression. Treatment of MCL cell lines with the DNA methyltransferase inhibitor decitabine resulted in reversal of aberrant hypermethylation and synergized with the histone deacetylase inhibitor suberoylanilide hydroxamic acid in induction of the hypermethylated genes and anti-MCL cytotoxicity. Our data show prominent and aberrant promoter methylation in MCL and suggest that differentially methylated genes can be targeted for therapeutic benefit in MCL.

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.

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. Clin Cancer Res; 18(9); 2534–44. ©2012 AACR.

High-resolution chromatin immunoprecipitation (ChIP) sequencing reveals novel binding targets and prognostic role for SOX11 in mantle cell lymphoma

Sex determining region Y-box 11 (SOX11) expression is specific for mantle cell lymphoma (MCL) as compared with other non-Hodgkin’s lymphomas. However, the function and direct-binding targets of SOX11 in MCL are largely unknown. We used high-resolution chromatin immunoprecipitation sequencing to identify the direct target genes of SOX11 in a genome-wide, unbiased manner and elucidate its functional significance. Pathway analysis identified WNT, PKA and TGF-beta signaling pathways as significantly enriched by SOX11-target genes. Quantitative chromatin immunoprecipitation sequencing and promoter reporter assays confirmed that SOX11 directly binds to individual genes and modulates their transcription activities in these pathways in MCL. Functional studies using RNA interference demonstrate that SOX11 directly regulates WNT in MCL. We analyzed SOX11 expression in three independent well-annotated tissue microarrays from the University of Wisconsin (UW), Karolinska Institute and British Columbia Cancer Agency. Our findings suggest that high SOX11 expression is associated with improved survival in a subset of MCL patients, particularly those treated with intensive chemotherapy. Transcriptional regulation of WNT and other biological pathways affected by SOX11-target genes may help explain the impact of SOX11 expression on patient outcomes.

Epigenetic therapy overcomes treatment resistance in T cell prolymphocytic leukemia

Epigenetic drugs can overcome resistance to monoclonal antibodies in T-PLL and activate therapeutic targets for antibody-drug conjugates. A second chance for leukemia therapy T cell prolymphocytic leukemia (T-PLL) is a rare and aggressive type of leukemia, which is usually difficult to treat. Alemtuzumab, a monoclonal anti-CD52 antibody, is currently used to treat this disease, but patients quickly develop resistance to alemtuzumab or do not respond to it altogether. Now, Hasanali et al. show that epigenetic therapy can help overcome drug resistance in T-PLL patients. In addition, the epigenetic drugs frequently up-regulated CD30 on the surface of leukemia cells, which allowed subsequent successful treatment with brentuximab vedotin, an antibody-drug conjugate targeting CD30. The findings suggest a new treatment regimen for this challenging disease and demonstrate a potential approach to successfully combining epigenetic and immunotherapy. 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.

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-β/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. Clin Cancer Res; 20(2); 382–92. ©2013 AACR.

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.

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.

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.

Abstract LB-140: Combined epigenetic and immunotherapy produces dramatic responses in 100% of newly diagnosed mantle cell lymphoma patients.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Mantle cell lymphoma (MCL) is an incurable disease in which there is no accepted standard of care. While dose intensive approaches are effective, we and others have shown that cladribine and rituximab (RTX) combination therapy is well tolerated and can induce durable remissions. The synergistic effect observed with the combination of cladribine, a purine analogue with known but cryptic epigenetic activity as a hypomethylating agent, and a monoclonal antibody, RTX, prompted us to add a histone deacetylase inhibitor, vorinostat (SAHA), to this combination. We describe a phase II study that uses SAHA and a unique hypomethlating agent, cladribine, that potentially can inhibit both DNA and histone methylation through SAM. In combination with RTX, dramatic durable responses are observed in newly diagnosed MCL (ORR 100%, CR rate 70%) as well as in relapsed MCL and other B cell malignancies. Objectives: The primary objective is overall response rate (ORR). Secondary objectives include progression free survival (PFS) and overall survival (OS) as well as correlative epigenetic, transcriptional and pharmacogenomic studies. Methods: Dosing is as follows: starting dose was vorinostat 400 mg po (days 1-14) combined with cladribine 5mg/m2 IV (days 1-5), and RTX 375 mg/m2 IV (weekly x 4 for cycle 1 and 1x/month) every 28 days for up to 6 cycles. Responses were evaluated after 2 and 6 cycles. The majority of complete responders have received maintenance RTX. Phase II eligibility includes relapsed NHL and previously untreated MCL. Scientific correlatives included epigenetic and gene expression assays. Results: 32 previously untreated MCL patients have been enrolled on the phase II portion of the study. 2 have not yet completed the planned 6 cycles; however, 30 patients have completed ≥ 2 cycles and are evaluable for response. The ORR is 100% (30/30) with 70% (21/30) achieving CR. Of those patients not attaining CR, 3 had blastic MCL and 2 have died, 2 have not yet completed treatment, and 1 withdrew consent after two cycles. At a median follow up of 14.7 months (.07 - 25 months) 4 patients have relapsed and 3 have died. Of the relapsing patients, two had blastic MCL. No patient achieving a CR has relapsed. The estimated PFS curve did not reach the 0.5 level. Toxicities using CTCAE 4 included neutropenia, thrombocytopenia, fatigue, anorexia, and dehydration. We found that cladribine hypomethylates DNA in vivo (6/6 pts) and inhibits histone methylation in MCL cell lines. Upregulated genes after treatment included DUSP2 (3 pts), FOXO3 (2 pts), NOXA1 (2 pts), CEBPβ (2 pts) and p53 (3 pts). Dramatic responses and CRs were also seen in fewer patients with low grade NHLs, but not in DLBCL. Conclusion: Combined epigenetic and immunotherapy (SCR) is nontoxic and effective in the treatment of newly diagnosed mantle cell lymphoma. This therapeutic approach potentially has broader applications in other malignancies. Citation Format: Zainul Hasanali, Kamal Sharma, Stephen Spurgeon, Craig Okada, August Stuart, Sara Shimko, Violetta Leshchenko, Samir Parekh, Yiyi Chen, Mark Kirschbaum, Elliot M. Epner. Combined epigenetic and immunotherapy produces dramatic responses in 100% of newly diagnosed mantle cell lymphoma patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-140. doi:10.1158/1538-7445.AM2013-LB-140