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639 The Tumor Associated Antigen PRAME Exhibits Dualistic Functions That Are Targetable in Diffuse Large B-Cell Lymphoma

Program: Oral and Poster Abstracts
Type: Oral
Session: 621. Lymphoma—Genetic/Epigenetic Biology: Clinical implications of biological insights in lymphoma
Hematology Disease Topics & Pathways:
Biological, Diseases, Therapies, DLBCL, B-Cell Lymphoma, Lymphoid Malignancies
Monday, December 7, 2020: 11:45 AM

Katsuyoshi Takata, MD, PhD1*, Lauren C. Chong, MSc2*, Daisuke Ennishi, MD, PhD1*, Avinash Thakur, Ph.D.3*, Shannon Healy, PhD4*, Elena Viganò, PhD4, Tao Dao, MD, PhD5*, Julie S Nielsen6*, Susana Ben-Neriah, BSc2*, Tomohiro Aoki, MD, PhD4*, Ethan Tse7*, Stacy Hung8*, Michael Y. Li, BS7*, Merrill Boyle, BSc4*, Sung Soo Mun5*, Christopher Bourne9*, Bruce W Woolcock4*, Adele Telenius4*, Allen Zhang7*, Ali Bashashati10*, Saeed Saberi11*, Gianluca D' Antonio6*, Brad H Nelson6*, Sohrab P Shah12*, Pamela Hoodless3*, Ari Melnick, MD13, Randy D. Gascoyne, MD4*, Joseph M Connors, MD14*, David A. Scheinberg, MD, PhD5, Wendy Béguelin, PhD15, David W. Scott, MBChB, PhD16 and Christian Steidl, MD4

1British Columbia Cancer Centre for Lymphoid Cancer, Vancouver, BC, Canada
2Centre for Lymphoid Cancer, British Columbia Cancer, Vancouver, BC, Canada
3Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada
4Centre for Lymphoid Cancer, BC Cancer, Vancouver, BC, Canada
5Memorial Sloan Kettering Cancer Center, New York, NY
6Trev and Joyce Deeley Research Centre, British Columbia Cancer Agency, Victoria, BC, Canada
7BC Cancer, Centre for Lymphoid Cancer, Vancouver, BC, Canada
8Lymphoid Cancer Research, BC Cancer Agency, Vancouver, BC, Canada
9Memorial Sloan Kettering Cancer Center, New York
10Molecular Oncology, British Columbia Cancer, Vancouver, BC, Canada
11Department of Molecular Oncology, British Columbia Cancer, Vancouver, BC, Canada
12Department of Epidemiology and Biostatistics, Memorial Sloan Kettering Cancer Center, New York, NY
13Division of Hematology/Oncology, Department of Medicine, Weill Cornell Medical College, New York, NY
14BC Cancer Centre for Lymphoid Cancer, Vancouver, BC, Canada
15Department of Medicine, Division of Hematology & Medical Oncology, Weill Cornell Medicine, New York, NY
16British Columbia Cancer, Vancouver, Canada

Background: With the goal of translating biological discovery into clinical actionability, deciphering crosstalk in the cellular ecosystem of the tumor microenvironment (TME) has emerged as a research focus. Although comparatively little is known about the immune biology of diffuse large B-cell lymphoma (DLBCL), as reflected in clonal selection of specific somatic gene mutations in response to immune system pressure and the specific composition of the TME, PRAME has emerged as a prominent member of the cancer germline antigen/ tumor associated antigen (TAA) family of proteins. It is expressed in various types of cancers, but generally not in normal tissues, apart from male germinal cells, and triggers autologous T-cell mediated immune responses. PRAME is highlighted as a new cancer therapeutic target of T-cell or antibody-based immunotherapies with promising anti-tumor responses in early phase clinical trials and pre-clinical models for several types of cancers. In the context of developing new immunotherapies, targeting TAAs that are presented by major histocompatibility complexes on tumor cells is a promising therapeutic strategy for patients that experience treatment failure.

Material and methods: We performed integrative genomic analysis of whole-transcriptome RNAseq, targeted genomic sequencing, and high-resolution copy number analysis in 347 de novo DLBCL tumors from patients uniformly treated with R-CHOP. Findings were correlated with pathological and clinical parameters, as well as TME composition. Using DLBCL-derived cell lines (7 EZH2 mutated and 5 wt) and CRISPR-Cas9 genome editing, we performed in vitro functional studies to characterize cell-intrinsic effects of PRAME knockout. Moreover, we studied EZH2 inhibition in a murine model of Ezh2 mutant lymphoma with a focus on mechanistic links between EZH2 activity and PRAME expression, as well as TME composition (cell-extrinsic effects).

Results: We discovered recurrent, and highly focal deletions of 22q11.22 including the PRAME gene, which were associated with poor treatment outcome, independent of pathological and clinical risk factors. To explore PRAME-deletion-associated phenotypes and interaction with the tumor microenvironment (TME), we analyzed corresponding RNAseq, immunohistochemistry (IHC), and flow-cytometry data from our DLBCL cohort and utilized enzyme-linked immunospot (ELISPOT) assays using patient-derived peripheral blood mononuclear cells. PRAME deletions contributed to an immunologically “cold” TME, representing a somatically acquired mechanism to evade anti-tumor T-cell response (cell-extrinsic effect). Using PRAME knock out by CRISPR-Cas9 in vitro, TRAIL-mediated apoptotic signaling was impaired. In addition, PRAME down-modulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. Using proximity ligation assays and co-IP, we demonstrated that PRAME directly interacted with EZH2 as a negative regulator, establishing a link between PRAME deletions and EZH2 mutations with anti-apoptotic signaling in DLBCL (cell-intrinsic effect). An in vivo murine model of Ezh2 mutant lymphoma showed decreased T-cell infiltration in the TME and Ezh2 inhibition induced PRAME restoration as compared to vehicle controls. IHC for CD3, CD4, FOXP3, and GZMB revealed significant increases in various T-cell populations of the TME, including Tregs and cytotoxic T cells. EZH2 inhibition with EPZ-6438 abrogated these dualistic effects leading to increased immune cell infiltration in the tumor microenvironment and acceleration of apoptosis via PRAME restoration. Moreover, restoration of PRAME antigen presentation by EZH2 inhibition resulted in enhancement of PRAME binding using a T-cell receptor mimic PRAME antibody (Pr20), suggesting immunotherapeutic potential.

Conclusion: Our findings highlight multiple functions of PRAME during lymphomagenesis. PRAME restoration by EZH2 inhibition provides a preclinical rationale for synergistic therapies combining epigenetic re-programming with PRAME-targeted therapies.

Disclosures: Dao: Eureka Therapeutics: Consultancy. Melnick: Jubilant: Consultancy; Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding. Scheinberg: Lantheus: Current equity holder in private company; Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Enscyse: Current equity holder in private company; Arvenas: Current equity holder in private company; Pfizer: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Oncopep: Consultancy. Scott: Roche/Genentech: Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; AstraZeneca: Consultancy; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl: Curis Inc: Consultancy; Roche: Consultancy; AbbVie: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy.

*signifies non-member of ASH