Genetic Ablation or Pharmacological Inhibition of PRMT5 Affects Maintenance of Genomic Integrity in Hematopoietic Stem Cells

Protein arginine methyltransferase 5 (PRMT5) has been implicated in diverse processes, including spliceosome assembly. In addition, the therapeutic potential of PRMT5-selective inhibitors has recently been demonstrated in several hematological malignancies. PRMT5 was also recently reported to be essential for hematopoiesis, and required for splicing in hematopoietic stem/progenitor cells. However, the role of PRMT5, particularly its importance in splicing regulation, in hematopoietic stem cells (HSCs) has not been extensively studied. Moreover, how pharmacological inhibition of PRMT5 affects the HSC compartment remains undetermined. To do so, we employed a loss-of-function approach, coupled with functional and transcriptome profiling.

We report that PRMT5 is essential for HSCs. Mx1-Cre Prmt5^fl/fl mice (PRMT5^Δ/Δ) exhibited bone marrow (BM) failure; characterized by rapid reduction in BM cellularity, severe pancytopenia and lethality within 17.5 days. Importantly, PRMT5^Δ/Δ HSCs failed to reconstitute lethally irradiated wild-type recipients. This was associated with transient expansion of the PRMT5^Δ/Δ HSC compartment, which was less quiescent and had higher reactive oxygen species (ROS) levels, followed by significant loss of HSCs a week after. Correspondingly, pharmacological inhibition (using EPZ015666) of PRMT5 in EML cells (murine BM-derived hematopoietic precursor cell line) lead to a dose-dependent increase in the proportion of lineage-negative EML cells, increased ROS levels and decreased cell viability. Thus, confirming the cell-intrinsic requirement for PRMT5 in HSCs.

We also analyzed the splice variant landscape in PRMT5^Δ/Δ HSCs, and identified differential splicing events (DSEs) across several splicing classes; of which intron retention and exon skipping events were substantially elevated. Interestingly, DSEs were overrepresented for processes associated with maintenance of genome integrity. Notably, we show that splicing of key genes involved in base excision repair, telomere maintenance and the Fanconi anemia pathway were dysregulated in PRMT5^Δ/Δ HSCs. We also detected dose-dependent dysregulated splicing of these genes in EPZ015666-treated EML cells. Further analysis in-silico determined that these dysregulated splicing events generated premature termination codons. Thereby suggesting that loss of PRMT5 activity in HSCs potentially leads to deficient DNA repair.

In agreement, PRMT5^Δ/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. Corroborating these observations, apoptosis was increased among PRMT5^Δ/Δ HSCs and EPZ015666-treated EML cells. This was associated with aberrant splicing of MDM4 (negative regulator of p53) and p53 activation; which involves upregulation of several pro-apoptotic genes and CDKN1A . Therefore, loss of PRMT5 activity renders HSCs vulnerable to DNA damage. Collectively, our study highlights a novel link between PRMT5 and maintenance of genome integrity in HSCs.