Loss of METTL3 Attenuates BPDCN Response to PRMT5 Inhibition Via Interferon Signaling

Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) is an aggressive hematologic cancer that stems from plasmacytoid dendritic cells (pDCs) and predisposes to leukemic transformation. Other than Tagraxofusp-erzs, a recently approved targeted therapy, treatment options for BPDCN remain inadequate. Recently, it was reported that BPDCN patient samples were enriched for the PRMT5 gene signature, suggesting a role for PRMT5 in BPDCN. Pursuing the hypothesis that targeting PRMT5 represents a potential therapeutic option in BPDCN, we first clarified the implications of inhibiting PRMT5's activity using a small molecule inhibitor - GSK3326595 (GSK595). Our previous findings revealed that perturbations to PRMT5's activity dysregulated the splicing landscape and mitigated tumor growth. Additionally, we identified RNA methylation as a process affected by PRMT5 inhibition.

Presently, to follow up on RNA methylation's role in BPDCN we focused on METTL3 - a key RNA modifying enzyme. CRISPR-Cas9 mediated knockout (KO) of METTL3 in CAL-1 (BPDCN patient-derived cell line) was found to affect CAL-1 cell growth, similar to PRMT5 inhibition. To ascertain if PRMT5 inhibition in these KO cells exacerbates the effects on cell growth, we treated METTL3 KO cells with GSK595. Unexpectedly, GSK595-treated cells displayed reduced sensitivity to PRMT5 inhibition. This was supported by the lack of decreased cell growth and diminished apoptosis activation, despite dose-dependent decrease in SDMA expression, signifying that GSK595 still perturbs PRMT5 activity in these cells. To uncover the mechanism underlying this unique finding, we performed transcriptomic analyses of untreated and treated METTL3 KO cells. RNA-seq results showed that Type 1 interferon (IFN) signaling was highly upregulated in KO cells upon GSK595 treatment. Volcano plot analysis then identified Interferon Induced Protein with Tetratricopeptide Repeats 1 (IFIT1), an Interferon-stimulated gene (ISG) to be highly upregulated post treatment and this finding was validated in vitro as well. This now highlights the possibility that increased Type 1 IFN signaling might confer resistance against PRMT5 inhibition in BPDCN cells with low/no METTL3 expression. To gain further insight into this observation, we assessed the expression of Interferon Regulatory Factor 7 (IRF7), the transcription factor regulating expression of ISGs like IFIT1. Upon PRMT5 inhibition in WT CAL-1 cells, IRF7 displayed dysregulated splicing (intron retention), similar to METTL3. In METTL3 KO cells, like IFIT1, the expression of IRF7 increased significantly post GSK595 treatment. This implies that the IRF7-IFIT1 axis is controlled by the expression status of METTL3 and this in turn affects response to PRMT5 inhibition. To verify this functionally, we added TLR7 agonists to CAL-1 cells to induce Type 1 IFN signaling and concurrently treated these cells with GSK595. Comparable to METTL3 KO cells, these TLR7-induced cells showed rescue in cell growth post treatment and had very weak apoptosis activation.

Overall, the findings from this study shows that the status of METTL3 affects BPDCN cells' response to pharmacologic modulation of PRMT5's activity by regulating IFN signaling via the IRF7-IFIT1 axis. Thus, implying that stratification based on METTL3 status may help select BPDCN patients who might respond optimally to PRMT5-targted therapies.