Targeting the T-Cell Lymphoma Epigenome Induces Cell Death, Cancer Testes Antigens, Immune-Modulatory Signaling Pathways.

The peripheral T-cell lymphomas (PTCL) could be considered the prototypical epigenetic disease. As a disease, they are uniquely sensitive to histone deacetylase (HDAC) and DNA methyltransferase (DNMT) inhibitors, both alone and in combination, are characterized by a host of mutations in epigenetic genes, and can develop spontaneously in genetically engineered murine models predicated on established recurring mutations in (RHOAG17V) and TET2, an epigenetic gene governing DNA methylation. Given the clinical benefit of HDAC inhibitors (HDACi) and hypomethlyation agents alone and in combination in PTCL, we sought to explore a mechanistic basis for these agents in PTCL. Herein, we reveal profound class synergy between HDAC and DNMT inhibitors in PTCL, and that the combination induces degrees of gene expression that are substantially different and more extensive than that observed for the single agents. A prominent signature of the combination relates to the transcriptional induction of cancer testis antigens and genes involved in the immune response. Interestingly, TBX21 and STAT4, master regulators of TH1 differentiation, were among the genes upregulated by the combination, suggesting the induction of a TH1-like phenotype. Moreover, suppression of genes involved in cholesterol metabolism and the matrisome were also identified. We believe that these data provide a strong rationale for clinical studies, and future combinations leveraging an immunoepigenetic platform.

Combined oral 5-azacytidine and romidepsin are highly effective in patients with PTCL: a multicenter phase 2 study.

Peripheral T-cell lymphomas (PTCLs) are uniquely vulnerable to epigenetic modifiers. We demonstrated in vitro synergism between histone deacetylase inhibitors and DNA methyltransferase inhibitors in preclinical models of T-cell lymphoma. In a phase 1 trial, we found oral 5-azacytidine and romidepsin to be safe and effective, with lineage-selective activity among patients with relapsed/refractory (R/R) PTCL. Patients who were treatment naïve or who had R/R PTCL received azacytidine 300 mg once per day on days 1 to 14, and romidepsin 14 mg/m2 on days 8, 15, and 22 every 35 days. The primary objective was overall response rate (ORR). Targeted next-generation sequencing was performed on tumor samples to correlate mutational profiles and response. Among 25 enrolled patients, the ORR and complete response rates were 61% and 48%, respectively. However, patients with T-follicular helper cell (tTFH) phenotype exhibited higher ORR (80%) and complete remission rate (67%). The most frequent grade 3 to 4 adverse events were thrombocytopenia (48%), neutropenia (40%), lymphopenia (32%), and anemia (16%). At a median follow-up of 13.5 months, the median progression-free survival, duration of response, and overall survival were 8.0 months, 20.3 months, and not reached, respectively. The median progression-free survival and overall survival were 8.0 months and 20.6 months, respectively, in patients with R/R disease. Patients with tTFH enjoyed a particularly long median survival (median not reached). Responders harbored a higher average number of mutations in genes involved in DNA methylation and histone deacetylation. Combined azacytidine and romidepsin are highly active in PTCL patients and could serve as a platform for novel regimens in this disease. This trial was registered at www.clinicaltrials.gov as #NCT01998035.

ATM inhibition overcomes resistance to histone deacetylase inhibitor due to p21 induction and cell cycle arrest.

The antiproliferative effect induced by histone deactylase inhibitors (HDACi) is associated with the up-regulated expression of the cyclin-dependent kinase inhibitor p21. Paradoxically, the increased expression of p21 correlates with a reduced cell killing to the drug. The direct targeting of p21 is not feasible. An alternate approach could selectively target factors upstream or downstream of p21 that affect one or more specific aspects of p21 function. HDAC inhibitors appear to activate p21 expression via ataxia telangiectasia mutated (ATM) activity. KU60019, a specific ATM inhibitor, has shown to decrease the p21 protein levels in a concentration dependent manner. We explored the potential synergistic interaction of the ATM inhibitor with romidepsin, given the potential complementary impact around p21. A synergistic cytotoxic effect was observed in all lymphoma cell lines examined when the HDACi was combined with KU60019. The increase in apoptosis correlates with decreased expression of p21 due to the ATM inhibitor. KU60019 decreased expression of the cyclin-dependent kinase inhibitor at the transcriptional level, compromising the ability of HDACi to induce p21 and cell cycle arrest and ultimately facilitating a shift toward the apoptotic phase. Central to the increased apoptosis observed when romidepsin is combined with KU60019 is the reduced expression of p21 and the absence of a G2/M cell cycle arrest that would be exploited by the tumor cells to evade the cytotoxic effect of the HDAC inhibitor. We believe this strategy may offer a promising way to identify rational combinations for HDACi directed therapy, improving their activity in malignant disease.

Generation of pralatrexate resistant T-cell lymphoma lines reveals two patterns of acquired drug resistance that is overcome with epigenetic modifiers.

While pralatrexate (PDX) has been successfully developed for the treatment of T-cell lymphoma, the mechanistic basis for its T-cell selectivity and acquired resistance remains elusive. In an effort to potentially identify synergistic combinations that might circumnavigate or delay acquired PDX resistance, we generated resistant cells lines over a broad concentration range. PDX-resistant cell lines H9-12 and H9-200 were developed, each exhibiting an IC50 of 35 and over 1000 nM, respectively. These lines were established in vitro from parental H9 cells. Expression analysis of the proteins known to be important determinants of antifolate pharmacology revealed increase expression of dihydrofolate reductase (DHFR) due to gene amplification, and reduced folate carrier1 downregulation, as the putative mechanisms of resistance in H9-12 and H9-200 cells. Cross resistance was only seen with methotrexate but not with romidepsin, azacitidine (AZA), decitabine, gemcitabine, doxorubicin, or bortezomib. Resistance to PDX was reversed by pretreatment with hypomethylating agents in a concentration-dependent fashion. Comparison of gene expression profiles of parental and resistant cell lines confirmed markedly different patterns of gene expression, and identified the dual specificity phosphatase four (DUSP4) as one of the molecular target of PDX activity. Reduced STAT5 phosphorylation following exposure to PDX was observed in the H9 but not in the H9-12 and H9-200 cells. These data suggest that combination with hypomethylating agents could be potent, and that DUSP4 and STAT5 could represent putative biomarkers of PDX activity.

The anti-tumor activity of pralatrexate (PDX) correlates with the expression of RFC and DHFR mRNA in preclinical models of multiple myeloma.

Multiple myeloma (MM) is the second most common hematologic malignancy. While major advances have been made in the disease, it is still incurable. Although antifolate-based drugs are not commonly used to treat myeloma, new generation analogs with distinct patterns of preclinical and clinical activity may offer an opportunity to identify new classes of potentially active drugs. Pralatrexate (PDX), which was approved for the treatment of relapsed or refractory peripheral T-cell lymphoma in 2009, may be one such drug. Pralatrexate exhibits a potency and pattern of activity distinct from its predecessors like methotrexate (MTX). We sought to understand the activity and mechanisms of resistance of multiple myeloma to these drugs, which could also offer potential strategies for selective use of the drug. We demonstrate that PDX and MTX both induce a significant decrease in cell viability in the low nanomolar range, with PDX exhibiting a more potent effect. We identified a series of myeloma cell lines exhibiting markedly different patterns of sensitivity to the drugs, with some lines frankly resistant, and others exquisitely sensitive. These differences were largely attributed to the basal RFC (Reduced Folate Carrier) mRNA expression levels. RFC mRNA expression correlated directly with rates of drug uptake, with the most sensitive lines exhibiting the most significant intracellular accumulation of pralatrexate. This mechanism explains the widely varying patterns of sensitivity and resistance to pralatrexate in multiple myeloma cell lines. These findings could have implications for this class of drugs and their role in the treatment of multiple myeloma.

Oral 5-azacytidine and romidepsin exhibit marked activity in patients with PTCL: a multicenter phase 1 study.

The peripheral T-cell lymphomas (PTCLs) are uniquely sensitive to epigenetic modifiers. Based on the synergism between histone deacetylase inhibitors and hypomethylating agents that we established in preclinical PTCL models, we conducted a phase 1 study of oral 5-azacytidine (AZA) and romidepsin (ROMI) in patients with advanced lymphoid malignancies, with emphasis on PTCL. According to a 3 + 3 design, patients were assigned to 1 of 7 cohorts with AZA doses ranging from 100 mg daily on days 1 to 14 to 300 mg daily on days 1 to 21, ROMI doses ranging from 10 mg/m2 on days 8 and 15 to 14 mg/m2 on days 8, 15, and 22, with cycles of 21 to 35 days. Coprimary end points included maximum tolerated dose (MTD) and dose-limiting toxicity (DLT). We treated a total of 31 patients. The MTD was AZA 300 mg on days 1 to 14 and ROMI 14 mg/m2 on days 8, 15, and 22 on a 35-day cycle. DLTs included grade 4 thrombocytopenia, prolonged grade 3 thrombocytopenia, grade 4 neutropenia, and pleural effusion. There were no treatment-related deaths. The combination was substantially more active in patients with PTCL than in those with non-T-cell lymphoma. The overall response rate in all, non-T-cell, and T-cell lymphoma patients was 32%, 10%, and 73%, respectively, and the complete response rates were 23%, 5%, and 55%, respectively. We did not find an association between response and level of demethylation or tumor mutational profile. This study establishes that combined epigenetic modifiers are potently active in PTCL patients. This trial was registered at www.clinicaltrials.gov as NCT01998035.