p62-ZZ Domain Signaling Inhibition Rescues MM-Induced Epigenetic Repression at the Runx2 promoter and Allows Osteoblast Differentiation of MM Patient Pre-Osteoblasts In Vitro

Multiple myeloma (MM) bone disease is characterized by non-healing lytic bone lesions with highly suppressed or absent osteoblast (OB) function. These lesions persist in the absence of active disease, significantly contributing to patient morbidity and mortality after patients achieve hematologic remission. We previously reported that MM cells induce epigenetic changes at the Runx2 promoter in bone marrow stromal cells (BMSC), which are preOB. We also demonstrated that Gfi1, a transcriptional repressor of the key OB differentiation factor, Runx2, is induced in BMSC by MM. Gfi1 is highly expressed in the MM bone marrow microenvironment and directly binds the Runx2 promoter, recruiting chromatin corepressors such as HDAC1 in preOB to induce epigenetic repression of Runx2 and prevent OB differentiation. Importantly, we recently showed that primary MM patient BMSC cultured in the absence of MM cells have decreased levels of the chromatin activation mark H3K9Ac at the Runx2 gene promoter when compared to healthy donor BMSC, suggesting that MM cells induce persistent epigenetic changes in patient BMSC.

We reported that p62 (sequestosome-1) in BMSC is critical for the formation of MM-induced signaling complexes that mediate OB suppression and identified XRK3F2, an inhibitor of the p62 ZZ domain. XRK3F2 blunts MM cell-induced Runx2 suppression and Gfi1 induction in murine preOB in vitro, and induced new bone formation and remodeling in the presence of tumor in vivo. In addition, coculture experiments using human MM cells and murine preOB showed that XRK3F2 both prevents and reverses Gfi1 upregulation. Recently, we demonstrated that XRK3F2 prevents the epigenetic suppression of Runx2 in murine preOB cocultured (48h) with MM cells. Using ChIP-qPCR analysis we found that XRK3F2 prevented MM-induced Gfi1 occupancy at the Runx2 promoter, recruitment of the chromatin corepressor HDAC1, and histone de-acetylation.

We now report that XRK3F2 restores OB suppression in persistently suppressed BMSC from MM patients. ChIP analysis of primary MM patient-derived BMSC cultured in the presence of XRK3F2 showed that XRK3F2 rescues H3K9ac levels at the Runx2 promoter. XRK3F2 did not alter H3K9ac levels in healthy donor BMSC or enhance OB differentiation. Importantly, XRK3F2 treatment of long-term primary MM patient BMSC cultures allowed osteogenic differentiation and mineralization, as evidenced by alizarin red staining. Further analysis of these cultures demonstrated that XRK3F2 treatment reduced Gfi1 protein expression.

We conclude that XRK3F2 blocks MM-induced signaling in BMSC, resulting in decreased Gfi1 levels, thereby reducing recruitment of Gfi1 and HDAC1 to the Runx2 promoter, and reversing MM-induced epigenetic suppression of Runx2. These results suggest that targeting the p62 ZZ domain may reverse Gfi1 upregulation and rescue MM-induced epigenetic suppression of Runx2 in BMSC, allowing restoration of OB function in patients with MM bone disease.