Abstract 971: PRMT5 modulates splicing for genome integrity and preserves proteostasis of hematopoietic stem cells

Protein arginine methyltransferase 5 (PRMT5) has recently been identified as a promising therapeutic target in various solid and hematologic cancers, while PRMT5 inhibitors are currently in clinical development. Functionally, PRMT5 has an established role in regulating splicing, and is essential for hematopoiesis. However, the mechanisms underlying PRMT5 dependency in HSCs, particularly PRMT5-mediated splicing, remain unclear. Moreover, how pharmacological inhibition of PRMT5 affects HSCs remains undetermined. To clarify the function of PRMT5 in HSCs, we adopted a loss-of-function approach, coupled with functional and transcriptome profiling. We report that depletion of PRMT5 activity severely perturbs HSC maintenance. HSCs from Mx1-Cre Prmt5fl/fl mice (PRMT5Δ/Δ), which exhibit severe bone marrow (BM) failure, failed to reconstitute lethally irradiated wild-type recipients. Notably, this was associated with rapid loss of Prmt5Δ/Δ HSCs. Similarly, EPZ015666-mediated PRMT5 inhibition led to a dose-dependent decrease in the growth and viability of EML cells (murine BM-derived hematopoietic precursor cell line). Interestingly, this was associated with increased HSC size and PI3K/AKT/mTOR pathway activity. In addition, protein synthesis rate was also elevated in an mTOR-dependent manner. Thus, we confirm the cell-intrinsic requirement for PRMT5 activity in HSCs, and demonstrate a potential role for PRMT5 in regulating HSC proteostasis. We also analyzed the splice variant landscape of PRMT5Δ/Δ HSCs, and observed substantial upregulation of intron retention and exon skipping events; which were overrepresented for processes associated with maintenance of genomic integrity. Notably, we show that splicing of key genes involved in base excision repair, telomere maintenance and the Fanconi anemia pathway were dysregulated in PRMT5Δ/Δ HSCs, as well as EPZ015666-treated EML cells. Thus, suggesting that HSCs depleted of PRMT5 activity are potentially deficient in DNA repair. In agreement, PRMT5Δ/Δ HSCs exhibited elevated γH2A.X levels, replicative stress-associated oxidative DNA lesions and DNA strand breaks. Likewise, EPZ015666-treated EML cells exhibited dose-dependent increments in γH2A.X levels, and increased sensitivity to DNA damaging agents. Corroborating these observations, loss of PRMT5 activity in both PRMT5Δ/Δ HSCs and EPZ015666-treated EML cells led to elevated reactive oxygen species (ROS) levels, p53 activation and induction of apoptosis; which was significantly rescued by antioxidant treatment or co-depletion of p53. Therefore, depletion of PRMT5 activity renders HSCs vulnerable to DNA damage. Collectively, our findings uncover a critical role for PRMT5 in maintaining genomic integrity, and highlight a novel link between PRMT5 and proteostasis in HSCs. Citation Format: Darren Qiancheng Tan, Ying Li, Chong Yang, Jia Li, Shi Hao Tan, Desmond W. Chin, Ayako Nakamura-Ishizu, Henry Yang, Toshio Suda. PRMT5 modulates splicing for genome integrity and preserves proteostasis of hematopoietic stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 971.