The p62-ZZ Domain Inhibitor XRK3F2 Alters Myeloma-Induced Suppression of Osteoblast Differentiation and Is Highly Cytotoxic to Myeloma Cells in Combination with Bortezomib
Authors: Silbermann Rebecca , Zhou Dan , Teramachi Jumpei , Xie Xiang-Qun , Roodman G. David , Kurihara Noriyoshi 
Published: October 2019

Abstract
Multiple myeloma (MM) bone disease results in increased osteoclastic bone destruction and irreversible osteoblast (OB) suppression. Cell-cell interactions between MM cells and bone marrow stromal cells (BMSC), which are OB precursors, activate signaling pathways in BMSC that result in enhanced MM cell growth, osteoclast formation, and inhibition of OB differentiation. OB inhibition results from suppression of key OB transcription factors, such as RUNX2, and induction of Gfi1, a transcriptional repressor of RUNX2. We reported that the ZZ domain of p62 (sequestosome-1) is required for BMSC support of MM cell growth and acts as a signaling hub for the formation of BMSC signaling complexes activated by MM cells and TNFα, resulting in NFκB, p38MAPK, and PI3K activation. We developed a small molecule inhibitor targeting the ZZ domain, XRK3F2 (previously CMPD3) that blocks p62-ZZ domain-mediated protein interactions, including TNFα-mediated signaling in BMSC. Further, we found that XRK3F2 was directly cytoxic to MM cells at high concentrations (5TGM1 murine MM cell IC50 4.35μM). Recently, we reported that administration of XRK3F2 two weeks after intratibial injection of 5TGM1 MM cells induces dramatic new cortical bone formation adjacent to MM cells in an immunocompetent in vivo model of MM bone disease. Additionally, bones from XRK3F2 treated animals not inoculated with tumor did not demonstrate new bone formation. This suggests that XRK3F2 changes the local effect of MM on bone at concentrations that were not sufficient for MM cell death. We now report that XRK3F2 alters MM-induced suppression of OB differentiation, and that the combination of XRK3F2 and bortezomib increases in vitroMM cell cytotoxicity.
To determine if the dramatic bone formation induced by XRK3F2 resulted from reduced production of OB inhibitors by MM cells, enhanced BMSC differentiation to OB, or alteration of MM cell-BMSC interactions, we treated 5TGM1 cells, MC4 (murine stromal) cells, and 5TGM1-MC4 co-cultures with XRK3F2 and evaluated expression of RUNX2, Gfi1, and other key OB differentiation genes by qPCR. XRK3F2 treatment of 5TGM1 cells did not change 5TGM1 production of the OB inhibitors IL-7 or TNFα, and did not alter osteogenic differentiation of MC4 cells. However, XRK3F2 treatment of 5TGM1-MC4 co-cultures performed with a transwell insert increased MC4-derived RUNX2 expression 3-fold, as compared with vehicle treated co-cultures, and fully blocked the induction of Gfi1 in MC4 cells by MM cells (MC4-5TGM1 co-culture resulted in 12.4-fold increase in MC4 cell Gfi1 expression as compared with MC4 expression of Gfi1 without MM cell co-culture.) XRK3F2 treatment of MC4 cells also blocked TNFα-induced upregulation of Gfi1 (TNFα treatment increased MC4 cell Gfi1 expression 16.4-fold compared with control.) These results suggest that XRK3F2 blocks the TNFα-induced suppression of OB differentiation observed in MM cell-BMSC co-cultures through it’s effects on TNFα-mediated signaling facilitated by the p62-ZZ domain.
To determine if we could further enhance the effects of XRK3F2 on bone formation and tumor burden, we evaluated if bortezomib, an anti-MM drug with potential bone anabolic effects, enhanced XRK3F2-induced MM cell cytotoxicity. Human MM cell lines were treated with combinations of XRK3F2 and bortezomib at concentrations below the IC50 of each drug for each cell line. H929, MM1.S, U266, ANBL6, and RPMI8226 cells were treated for 48 hours with XRK3F2, bortezomib (1nM), or the combination, and analyzed by MTT assay for viability. All cell lines demonstrated a highly significant decrease in viability in response to treatment with the XRK3F2-bortezomib combination as compared with either alone (p < 0.01). These results suggest that XRK3F2 or its derivatives, in combination with bortezomib or other proteasome inhibitors, should have a profound therapeutic effect on MM tumor burden and bone disease.

Disclosures
Silbermann: Amgen: Consultancy; Celgene: Research Funding. Roodman:Eli Lilly and Co.: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees.