Session: 652. Myeloma: Pathophysiology and Pre-Clinical Studies, excluding Therapy: Poster I
Hematology Disease Topics & Pathways:
Diseases, multiple myeloma, Biological, Therapies, Biological Processes, enzyme inhibitors, Plasma Cell Disorders, Lymphoid Malignancies, molecular interactions
Histone deacetylases (HDACs) are generally accepted to be therapeutic targets for MM treatment. However, clinical application of currently available pan-HDAC inhibitors is limited with their adverse effects induced by a non-selective HDAC inhibition. To develop safe and effective HDAC inhibitor-based treatment, the therapeutic roles of HDAC isoform-specific inhibition should be elucidated. In this regard, we have recently reported therapeutic impacts on MM cells of inhibition of class-I HDACs, especially HDAC1 and HDAC3. HDAC3-selective inhibitor BG45 induces anti-MM activity in combination with DNA methyltransferase (DNMT) inhibitor azacytidine (Leukemia 2017). In the present study, we aimed to clarify the underlying mechanisms for impairment of MM cell growth and survival by HDAC1 inhibition.
We first referenced the expression of class-I HDACs using a publicly available GSE6691 data set. Among class-I HDACs, HDAC1 and HDAC3 were highly expressed in MM cells. We then knockdowned HDAC1 gene using lentiviral shRNA system in MM cell lines. The HDAC1 gene silencing induced MM cell death with caspase-3 activation, indicating the critical role of HDAC1 in MM cell growth and survival. To determine target molecules of HDAC1, we carried out RNA-sequencing with and without the HDAC1 gene silencing in RPMI 8226 cells. Among genes whose expression significantly changed by the HDAC1 knockdown (adjusted P values < 0.05, log fold change > 0.5), we focused on IRF4 together with PIM2, because MM cell has been demonstrated to addict to aberrant IRF4-c-Myc regulatory network (Nature 2008). Downregulation of IRF4 and Pim-2 by the HDAC1 knockdown was further confirmed by quantitative PCR (Q-PCR) and immunoblotting in RPMI 8226 and MM.1S cells. Treatment with the class I HDAC-selective inhibitor MS-275 (entinostat) also induced MM cell death along with reduction of IRF4 and Pim-2 expression.
Since previous study has shown that IRF4 binds to PIM2 promoter in MM cells (Nature 2008), we examined whether IRF4 regulates PIM2 expression. We found that IRF4 binds to the PIM2 promoter region by analyzing ChIP-Seq data in KMS-12 cells (GSE22901). We further confirmed the binding of IRF4 on PIM2 promoter by ChIP-Q-PCR. Indeed, the IRF4 knockdown downregulated Pim-2 expression in RPMI 8226 cells. These results suggest that HDAC1 inhibition downregulates IRF4 expression, thereby transcriptionally reducing PIM2 expression in MM cells.
Pim-2 expression can also be augmented by multiple signaling pathways, including HIF-1a, JAK-STAT and NF-kB-mediated ones in MM cells through the interaction with BM microenvironment. Interestingly, the Pim inhibitor SMI-16a and MS-275 cooperatively induced apoptotic cell death in MM cell lines and CD138-positive primary MM cells even in the presence of BMSCs.
Taken together, our results demonstrate the critical role of the HDAC1-IRF4-Pim-2 axis in MM cell growth and survival, and provoke the novel treatment strategy targeting the HDAC1-IRF4-Pim-2 axis in MM cells.
Disclosures: Anderson: Takeda Millennium: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership; Celgene: Consultancy; Bristol Myers Squibb: Consultancy.
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