Targeting PRMT5-Mediated DNA Repair Pathway in Extra-Nodal NK/T-Cell Lymphoma (ENKTL)

Extra-Nodal (NK)/T-cell Lymphoma (ENKTL) is the most common subtype of NK lymphoma derived from EBV-infected NK cells. ENKTL is incurable and the 5-year survival is around 40% (<10% if disseminated) highlighting the unmet need for novel therapies. It has been shown that upregulation of DNA damage repair (DDR) plays a pivotal role in ENKTL survival by promoting genomic stability and resistance to lymphoma conventional DNA damaging therapies as reflected by the limited response rate (<50%). We identified protein arginine methyltransferase (PRMT5) to be oncogenic in many malignancies by supporting the cell cycle, WNT, and PI3K/AKT pathways. PRMT5, also promotes DDR through RNA splicing of TIP60/KAT5 and stabilization of 53BP1, essential for homologous recombination (HR) and non-homologous end joining (NHEJ), respectively. PRMT5-deficiency leads to DNA damage accumulation, cell cycle arrest, and apoptosis. Unbiased analysis of our RNA-seq data on ENKTL patient samples (n=22) show significant upregulation of DDR genes compared to normal NK cells. Interestingly, we also detected PRMT5 overexpression in 95% of this ENKTL cohort by RNA-seq, suggesting that PRMT5 may also be playing a role in the DDR within ENKTL. We confirmed these RNA-seq data and detected PRMT5 overexpression in primary ENKTL patients' samples (n=10) by immunohistochemical (IHC) staining compared to normal tonsils (n=5), in ENKTL-cell lines (NKL, NK92, and YT) and in a patient-derived xenograft (PDX) mouse model of ENKTL (ENKTL1) by western-blot. We hypothesize that PRMT5 inhibition 1) targets DNA repair in ENKTL cells leading to increased DNA damage-induced cell death and 2) sensitizes ENKTL cells to other DNA damage-inducing agents used in the current treatment of ENKTL leading to substantial increases in DNA damage-mediated cell death with combination therapy. To address this hypothesis, we used selective PRMT5-inhibitors, PRT382 and PRT808 (Prelude-Therapeutics) and detected cytotoxicity (IC50 25 - 500nM) in ENKTL-cell lines and ENKTL1. Furthermore, PRT382 significantly prolonged overall survival (OS) in our in vivo PDX mouse model of ENKTL1 (median OS: control 43 days vs PRT382 65 days; p=0.0088). Mechanistically, accumulation of DNA-damage was evident by the increase in phosphorylated forms of 1) H2AX (H2A histone family member X) at Ser139, and 2) Chk1/Chk2 (major DNA check points control) at Ser317/Ser345 and Thr68, respectively, in PRT808-treated cells and upon PRMT5 knock-down. Lastly, PRT808 sensitized NK-lymphoma cells to killing by doxorubicin (DNA-damage agent) and Olaparib (PARP1/2-inhibitor, key regulator of HR). In conclusion, PRMT5-inhibition results in an unrepairable level of DNA damage/subsequent apoptosis and sensitizes NK-lymphoma to other DNA damage-inducing agents. All together, these data justify targeting PRMT5 in ENKTL, as a single agent and in combination with other DNA damage agents which are an essential part of the current ENKTL regimen (Doxorubicin and Etoposide) representing a novel approach for better outcome. Further mechanistic studies investigating the role of PRMT5 in DNA repair and DNA damage in ENKTL cells are ongoing.

Chan:Xellera Therapeutics: Consultancy; Cellperior Process LLC: Current equity holder in private company. Bhagwat:Prelude Therapeutics: Current Employment. Vaddi:Prelude Therapeutics: Current Employment. Scherle:Prelude Therapeutics: Current Employment. Baiocchi:Prelude Therapeutics: Other: Advisory Board, Research Funding; Codiak Biosciences: Research Funding; Agenus: Other: Involved in supply of drug (vaccine) and product development; Viracta Therapeutics: Consultancy, Current holder of stock options in a privately-held company, Other: Advisory Board; ATARABio: Consultancy, Other: Advisory Board.