Abstract
Protein arginine methylation regulates several cellular functions, including RNA splicing, translation and DNA damage repair. Protein arginine N-methyltransferase (PRMT) dysregulation is often seen in malignant hematopoiesis. PRMT7, the only type III PRMT, catalyzes monomethyl arginine (MMA) modification, but its role in leukemogenesis is elusive.
Re-analysis of a previous genome-wide CRISPR/Cas9 screen revealed PRMT7 to be a crucial negative-regulator of MHC-I, prompting us to ask whether PRMT7 inhibition might promote anti-AML immunity. Our analysis of human TCGA AML dataset as well as normal healthy dataset (GTEX) revealed that AML cases exhibited higher PRMT7 expression than did those from healthy donors. Among cytogenetic subgroups, PRMT7 levels were significantly higher in KMT2A-rearranged (MLL-r) AML. Moreover, correlation analysis across combined datasets (MDACC, BeatAML and TCGA) demonstrated that PRMT7 levels negatively correlate with expression of MHC-I associated genes, including HLA-A (r = -0.33, p<0.001), HLA-C (r = -0.37, p<0.001), B2M (r = -0.45, p<0.001) and NLRC5 (r = -0.25, p<0.001), but not MHC-II genes, such as HLA-DR, HLA-DQ, HLA-DP and CIITA. PRMT7 levels were also negatively correlated with Cytotoxic T Lymphocyte (CTL) scores in the same combined datasets, highlighting its immunosuppressive role in leukemogenesis.
To assess the potential PRMT7 function in MHC-I presenting, we knocked out PRMT7 by electroporating human AML lines including THP-1 and Molm13 with ribonucleoprotein (RNP) complexes formed by Cas9 protein and anti-PRMT7 sgRNA and assessed MHC-I levels. Relative to controls, PRMT7 KO remarkably upregulated MHC-I (HLA-A/B/C) expression in both lines. We also observed MHC-I upregulation was also observed in both cell lines after treatment with the PRMT7 inhibitor SGC3027 or administration of a targeted degrader (PRMT7 PROTAC) ex-vivo at relatively low concentrations, while the same dose of either compound spared normal hematopoietic stem/progenitor (CD34+) cells. To confirm MHC-I dynamics in an MLL-AF9 mouse model, we generated a Prmt7 KO mouse model by introducing MLL-AF9 retrovirus into Kit+ cells from hematopoietic-specific Prmt7 KO mice (Prmt7fl/fl;Vav1-Cre) and observed 2-fold upregulation of H-2Kb expression, based on antibody staining and flow cytometry analyses, relative to WT MLL-AF9 cells.
Given the critical role of MHC-I in CD8+ T cell activation, we next asked whether PRMT7 deletion would enhance antigen-specific CD8+ T cell responses. To do so, we evaluated human T cell killing effects in PRMT7-KO/-WT THP-1 or Molm13 cells cocultured with activated CD8+ T cells derived from healthy donors. Post-coculture, we calculated T cell killing effects by flow cytometry using Count-bright absolute counting beads and found that PRMT7 KO AML cells were more susceptible to human T cell-mediated killing. In agreement, we confirmed that relative to PRMT7-WT murine AML cells, PRMT7 KO murine AML cells were more sensitive to mouse T cell-mediated killing using a coculture model of MA9 and syngeneic active CD8+ T cells. Moreover, following PRMT7 PROTAC pretreatment, THP1 cells were more sensitive to human T cells mediated killing in a coculture system of THP1 cells and CD8+ T cells.
Next, to assess whether PRMT7 deletion impairs normal hematopoiesis, we analyzed total bone marrow cellularity and lineage frequency in Prmt7 KO (Vav1-Cre+) versus Prmt7-WT (Vav1-Cre-) mice via Cytek full-spectrum flow cytometry. While total BM cellularity was comparable, PRMT7 KO slightly increased the number of CD4⁺ or CD8⁺ T cells. Further analysis revealed increased frequencies of CD25⁺CD4⁺ T and CD69⁺CD8⁺ T cells. These results suggest that although PRMT7 function is likely dispensable for normal hematopoiesis, PRMT7 loss may have a modest effect on T cell proliferation or activation. In future studies, we will confirm whether PRMT7 inhibition promotes anti-tumor T cell activity in-vivo, and whether the underlying mechanism is via MHC-I regulation.
Overall, we have shown that PRMT7 depletion or pharmacological inhibition enhances T cell function in part by upregulating MHC-I. These findings suggest that combining PRMT7 inhibitors with immunotherapy could be a promising strategy to overcome AML's immune-cold properties.
