XRK3F2 Inhibition of p62-ZZ Domain Signaling Rescues Myeloma-Induced GFI1-Driven Epigenetic Repression of the Runx2 Gene in Pre-osteoblasts to Overcome Differentiation Suppression

Multiple myeloma bone disease (MMBD) is characterized by non-healing lytic bone lesions that persist even after a patient has achieved a hematologic remission. We previously reported that p62 (sequestosome-1) in bone marrow stromal cells (BMSC) is critical for the formation of MM-induced signaling complexes that mediate OB suppression. Importantly, XRK3F2, an inhibitor of the p62-ZZ domain, blunted MM-induced suppression , and induced new bone formation and remodeling in the presence of tumor . Additionally, we reported that MM cells induce the formation of repressive chromatin on the gene in BMSC via direct binding of the transcriptional repressor GFI1, which recruits the histone modifiers, histone deacetylase 1 (HDAC1) and Enhancer of zeste homolog 2 (EZH2). In this study we investigated the mechanism by which blocking p62-ZZ domain-dependent signaling prevents MM-induced suppression of in BMSC. XRK3F2 prevented MM-induced upregulation of and repression of the gene when present in MM-preOB co-cultures. We also show that p62-ZZ-domain blocking by XRK3F2 also prevented MM conditioned media and TNF plus IL7-mediated mRNA upregulation and the concomitant repression, indicating that XRK3F2's prevention of p62-ZZ domain signaling within preOB is involved in the response. Chromatin immunoprecipitation (ChIP) analyses revealed that XRK3F2 decreased MM-induced GFI1 occupancy at the promoter and prevented recruitment of HDAC1, thus preserving the transcriptionally permissive chromatin mark H3K9ac on and allowing osteogenic differentiation. Furthermore, treatment of MM-exposed preOB with XRK3F2 after MM removal decreased GFI1 enrichment at P1 and rescued MM-induced suppression of mRNA and its downstream osteogenic gene targets together with increased osteogenic differentiation. Further, primary BMSC (hBMSC) from MM patients (MM-hBMSC) had little ability to increase H3K9ac on the promoter in osteogenic conditions when compared to hBMSC from healthy donors (HD). XRK3F2 treatment enriched gene H3K9ac levels in MM-hBMSC to the level observed in HD-hBMSC, but did not alter HD-hBMSC H3K9ac. Importantly, XRK3F2 treatment of long-term MM-hBMSC cultures rescued osteogenic differentiation and mineralization. Our data show that blocking p62-ZZ domain-dependent signaling with XRK3F2 can reverse epigenetic-based mechanisms of MM-induced suppression and promote osteogenic differentiation.