Jain, Salvia

Abstract Background: Patients with relapsed or refractory (R/R) peripheral T-cell lymphoma (PTCL) have a poor prognosis with median overall survival of ~3-12 months with current therapies, highlighting an urgent unmet need for novel therapeutics. PTCL is uniquely sensitive to epigenetic modifiers. We have shown that DNMT3 inhibitors like 5-azacytidine have potent immunomodulatory effects, including upregulation of cancer testis antigens, in pre-clinical models of T-cell lymphoma. Similarly, we have shown that pralatrexate (PTX) is potentially synergistic with drugs targeting epigenetic regulation. These data suggest a role for combining the immune checkpoint inhibitor pembrolizumab (pembro) with epigenetic agents in patients with PTCL. Methods: PTCL-002 is a multicenter, multi-arm phase Ib study designed to evaluate the safety and efficacy of pembro in combination with PTX and/or decitabine (DAC) in patients with R/R PTCL and CTCL. The study included three arms: arm A (pembro+PTX), arm B (pembro+PTX+DAC), and arm C (pembro+DAC). All patients received pembro 200 mg by IV infusion every 21 days. Dose selection for DAC and PTX in arms A and C, respectively, was based on a continual reassessment method (CRM) while arm B used a partial order CRM. Primary endpoints included MTD, RP2D, dose-limiting toxicities (DLT) and clinical efficacy as assessed by overall response rate (ORR). Secondary endpoints included progression-free survival, duration of response, and overall survival. Select pharmacokinetic and pharmacodynamic endpoints were also included in order to assess immune modulation and identify potential predictors of response. Results: We enrolled 26 patients, including 9 patients in arm A, 7 in arm B, and 10 in arm C. Median age was 61 (range 27-77) with 13 males and 13 females. Racial demographics were as follows: White 62%, Black 23%, Asian 3%, and Hispanic 1%. Median number of prior treatments was 3 (range 1-6). Histologic subtypes included ATLL (n=1), AITL (n=4), PTCL-NOS (n=10), SS (n=1), MF (n=6), PCALCL (n=1), MEITL (n=1), PCAECTL (n=1), and TFh-PTCL (n=1). Twenty-four of 26 patients received at least one dose of study drug and were evaluable for toxicity. Twenty-one of 26 of patients received at least one full cycle of therapy and were evaluable for response. Reasons for early discontinuation included consent withdrawal (n=1), rapid disease progression during cycle 1 (n=2), adverse event (n=1), and investigator decision (n=1). One DLT was observed each in arms A and B for prolonged grade 3 thrombocytopenia and febrile neutropenia, respectively. Three DLTs were observed in arm C including one patient with grade 3 hyponatremia and rash; one patient with grade 4 thrombocytopenia, neutropenia, and anemia; and one patient with grade 4 neutropenia. There were no treatment related deaths. Based on the observed DLTs, the RP2D for DAC in combination with pembro is 20 mg/m2on days 1-3; the RP2D for PTX in combination with pembro is 30 mg/m2on days 1, 8, and 15; for the triplet combination, the RP2D for DAC is 10 mg/m2given on days 1-5 and the RP2D for PTX is 20 mg/m2given on days 1, 8, and 15. Among 21 evaluable patients, responses were as follows: ORR 19% (4/21), CR 10% (2/21), PR 10% (2/21), and disease control rate (defined as proportion of patients with stable disease or better) 38% (8/21). ORR among evaluable patients in arms A, B, and C were 40% (2/5), 29% (2/7), and 0% (0/9), respectively. Analysis of serum cytokine levels in select patients (4 in Arm A, 4 in Arm B, and 5 in Arm C) revealed significant changes in 5 cytokines when compared to healthy age- and sex-matched controls. Of the 5 cytokines, 3 pro-inflammatory cytokines, TNFα, MIP-3α and IL-10, measured at day 1 pre-treatment were significantly higher in trial patients (p= 0.0001, 0.0008 and 0.003 respectively) than in healthy controls. Trial patients who responded to treatment (PR/CR) in any arms demonstrated a decline in serum levels of TNFα and IL-10 by day 15 of cycle 1, whereas non-responders (PD/SD) showed stable to increased levels of both TNFα and IL-10. Conclusion: We successfully identified the RP2D for DAC and/or PTX in combination with pembro in patients with R/R PTCL and CTCL. We demonstrated that an epigenetic platform incorporating the immune checkpoint inhibitor pembrolizumab is safe in heavily pretreated disease. More work on changes in cytokine profiles is needed before drawing any conclusions on their relationship to response.

Gene fusions involving tumor protein p63 gene (TP63) occur in multiple T and B cell lymphomas and portend a dismal prognosis for patients. The function and mechanisms of TP63 fusions remain unclear, and there is no target therapy for patients with lymphoma harboring TP63 fusions. Here, we show that TP63 fusions act as bona fide oncogenes and are essential for fusion-positive lymphomas. Transgenic mice expressing TBL1XR1::TP63, the most common TP63 fusion, develop diverse lymphomas that recapitulate multiple human T and B cell lymphomas. Here, we identify that TP63 fusions coordinate the recruitment of two epigenetic modifying complexes, the nuclear receptor corepressor (NCoR)—histone deacetylase 3 (HDAC3) by the N-terminal TP63 fusion partner and the lysine methyltransferase 2D (KMT2D) by the C-terminal TP63 component, which are both required for fusion-dependent survival. TBL1XR1::TP63 localization at enhancers drives a unique cell state that involves up-regulation of MYC and the polycomb repressor complex 2 (PRC2) components EED and EZH2. Inhibiting EZH2 with the therapeutic agent valemetostat is highly effective at treating transgenic lymphoma murine models, xenografts, and patient-derived xenografts harboring TP63 fusions. One patient with TP63 -rearranged lymphoma showed a rapid response to valemetostat treatment. In summary, TP63 fusions link partner components that, together, coordinate multiple epigenetic complexes, resulting in therapeutic vulnerability to EZH2 inhibition.

Abstract Recurrent chromosomal rearrangements are a hallmark of hematologic malignancies and play critical roles in pathogenesis. The TP53 analog TP63 is rearranged in 5-10% of diverse subtypes of both aggressive T- and B-cell lymphomas. Patients with TP63-rearranged lymphomas have dismal outcomes, with 5-year overall survival rates between 0-17%, depending on cohorts. The function and mechanisms of TP63 rearrangements and TP63 fusion proteins in tumorigenesis are poorly understood. As a result, attempts to treat these patients to date have been largely empiric. Thus, there is an urgent need to understand how TP63 fusions contribute to tumorigenesis and to translate the findings into novel therapeutic options for these patients. Here, we demonstrated that TP63 fusions are essential for the propagation of T-cell lymphomas (TCLs). Knockdown of TP63 fusions with specific shRNAs in TCL cell lines harboring TP63 fusions suppressed both cell growth in vitro and tumor growth in vivo. Retroviral expression of TBL1XR1-TP63, the most common TP63 fusion, conferred cytokine independence in Ba/F3 cells, consistent with its role as an oncogene. To investigate the role of TP63 fusions in T- and B-cell lymphomagenesis, we engineered a CAG-Loxp-Stop-Loxp-TBL1XR1-TP63 conditional knock-in mouse model and crossed with hCD2-Cre mice. This results in expression beginning during early lymphoid development. As observed in patients, transgenic mice developed multiple subtypes of both T- and B-cell lymphoma. To define the effects and mechanisms of TP63 fusions within T cells, we performed CRISPR scanning, transcriptomic, epigenomic, and proteomic analyses. Our data showed that domains within both the N-terminal TBL1XR1 and C-terminal TP63 portions contribute to the function of this fusion. We found that the N-terminal component of TP63 fusions interacts with components of the NCOR/SMRT complex. At the same time, the C-terminal portion of TP63 (which recapitulates the deltaN-p63 isoform expressed in some carcinomas) interacts with the enhancer modifier KMT2D and its complex members. TBL1XR1-TP63 binds to a novel distal enhancer to drive MYC expression, and thus upregulates the expression of the histone H3K27 methylase EZH2. Finally, we assessed whether EZH2 is a vulnerability of TP63-rearranged lymphomas. We found that knockdown of EZH2 in TP63-rearranged lines significantly impaired cell growth, as did treatment with the EZH2 and 1 dual inhibitor valemetostat. Valemetostat, which is now being tested in patients with lymphoma, counteracted the oncogenic effects of TP63 fusions in multiple preclinical models in vivo. Together, our results identify the TP63 fusion as a highly unique oncogenic driver in lymphomagenesis capable of recruiting multiple epigenetic modifier complexes and inducing a targetable dependence on EZH2. Citation Format: Gongwei Wu, Noriaki Yoshida, Jihe Liu, Xiaoyang Zhang, Yuan Xiong, Tayla Heavican-Foral, Huiyun Liu, Geoffrey Nelson, Lu Yang, Renee Chen, Katherine Donovan, Marcus Jones, Mikhail Roshal, Yanming Zhang, Ran Xu, Ajit Nirmal, Salvia Jain, Catharine Leahy, Kristen Jones, Kristen Stevenson, Natasha Galasso, Nivetha Ganesan, Tiffany Chang, Wen-Chao Wu, Abner Louissaint, Lydie Debaize, Hojong Yoon, Paola Dal Cin, Wing Chan Chan, Shannan Ho Sui Ho Sui, Samuel Ng, Andrew Feldman, Steven M. Horwitz, Mathew Meyerson, Karen Adelman, Eric Fischer, Chun-Wei Chen, David Weinstock, Myles Brown. TP63 fusions drive enhancer rewiring, lymphomagenesis, and dependence on EZH2. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5755.

8049 Background: Our group has demonstrated that combinations of epigenetic modifiers produce potent synergy in pre-clinical models of PTCL and induce the expression of cancer testis antigen, suggesting a role in the addition of the immune-checkpoint inhibitor, pembrolizumab. Methods: This is a phase 1b study of pembrolizumab combined with pralatrexate alone (Arm A), with pralatrexate + decitabine (Arm B), or decitabine alone (Arm C) in patients with relapsed and refractory PTCL and CTCL. A standard 3+3 dose-escalation is applied in the triplet Arm (Arm B) while in the doublet Arms (A and C) de-escalation is applied in case of toxicity. Pharmacokinetic and pharmacodynamic studies are ongoing. Results: We treated a total of 12 patients with 4 patients in each Arm. All patients that received at least one dose of drug were evaluable for toxicity. There was a dose limiting toxicity (DLT) in each arm including prolonged grade 3 thrombocytopenia (Arm A), febrile neutropenia (Arm B), grade 3 hyponatremia, and rash (Arm C). There were no treatment-related deaths. Six patients out of 12 were evaluable for response at the time of this analysis. One patient achieved a complete remission, 2 had partial remission, 1 had stable disease, and 2 experienced progression of disease. Interestingly, all of the responses were seen in the triple combination of pralatrexate, decitabine, and pembrolizumab. Table summarizes the patient characteristics, toxicities, and response rates. Conclusions: These preliminary clinical data suggest that the integration of pembrolizumab on an epigenetic backbone is safe and demonstrates encouraging responses in patient with PTCL and CTCL. Clinical trial information: 03240211 . [Table: see text]