PIM Kinases Regulate Super-Enhancer-Dependent Gene Expression in Diffuse Large B-Cell Lymphoma

The PIM kinase family consists of 3 proto-oncogenic proteins: PIM1, PIM2 and PIM3, expressed in numerous cancers, including diffuse large B-cell lymphoma (DLBCL). PIM kinases regulate crucial processes, such as proliferation, apoptosis, metabolism, and migration, therefore their inhibition is of great interest as a potential therapeutic strategy. However, the detailed mechanisms of PIM's oncogenic effects and the consequences of their inhibition in DLBCL remain insufficiently understood. Our earlier studies (Szydłowski et al., Blood 2017, Szydłowski et al., Cancer Res. 2021) highlighted immunomodulatory functions of PIM kinases in lymphoid malignancies, but also indicated that their inhibition has a profound impact on gene expression. Although earlier studies indicated that PIMs regulate certain transcription factors and/or regulate gene expression via epigenetic mechanisms through histone H3S10 phosphorylation, the detailed genomic functions of PIM kinases in modulating transcription in DLBCL remains unclear. In order to investigate the mechanisms and consequences of PIM kinase inhibition on gene expression in DLBCL cells, we first performed RNA sequencing-based transcriptome profiling in DLBCL cells treated with the pan-PIM and FLT3 inhibitor MEN1703, which is currently in clinical development. MEN1703 markedly downregulated the expression of transcripts controlled by super-enhancers (SE), i.e. large regulatory regions responsible for high levels of transcript expression of the key essential genes for the cell, including oncogenes. The analysis also suggested new biological processes controlled by PIMs, including the reduction of non-coding RNAs generated from the SE regions. To understand the mechanisms linking PIM inhibition with decreased SE-driven gene expression, we assessed PIM-dependent H3S10 phosphorylation in lymphoma cells. Since H3S10 phosphorylation facilitates MYST family KAT8 (MOF) acetyltransferase recruitment to certain promoters/enhancers, we evaluated the histone H3 and H4 acetylations in DLBCL cells following PIM inhibition. MEN1703 markedly reduced the H3K9Ac, H3K14Ac and H4 pan-acetylation, which was associated with reduced BRD4 binding to the SE regions and reduced RNAPII serine 5 (S5) phosphorylation, but not S3 or S7 phosphorylations. Together, these observations suggest the participation of PIM in the elongation phase of SE-dependent gene transcription via H3S10-dependent epigenetic mechanism. The effects of PIM inhibition on histone modifications and proliferation were confirmed by genetic silencing or CRISPR-Cas9 deletion of individual PIM kinases. To better characterize the function of H3S10 phosphorylation in DLBCL, non-phosphorylatable H3S10A mutants were introduced to DLBCL cells. Compared to wild type counterparts, cells with the mutated H3S10 exhibited decreased proliferation, indicating a significant biological role of this modification in DLBCL. The effect was potentiated in cells exposed to metabolic stress (starvation) conditions. Together, these studies confirm the epigenetic role of PIM kinases, especially in the regulation of SE-dependent gene transcription and suggest that inhibition of PIM activity leads to disturbances of SE integrity and DLBCL transcription. The results document new, epigenetic function of PIM kinases and present a novel cytotoxicity mechanism of their inhibition in DLBCL. They also indicate that PIM inhibition with MEN1703 can be a therapeutic option in DLBCL. Study supported aby Foundation For Polish Science TEAM Grant # POIR.04.04.00-00-5C84/17 and Polish National Science Centre Grant # 2018/29/B/NZ5/01471.