Bone disease occurs in over 80% of Multiple Myeloma (MM) patients and is characterized by severe osteolytic bone destruction accompanied by profoundly suppressed or absent bone formation that has devastating consequences for patients These consequences include pathologic fractures, severe bone pain and derangements in mineral metabolism that negatively impact survival, performance status and quality of life. Adhesive interactions between bone marrow stromal cells (BMSC) and MM cells play a key role in pathogenesis of MMBD because they activate multiple signaling pathways and induce potent mediators that regulate osteoclast (OCL) and osteoblast (OB) activity as well as promote tumor growth. Although new MM therapies have greatly improved progression-free and overall survival, MM still remains incurable for most patients. Thus, novel treatments that effectively target the multiplicity of mediators and signaling pathways induced by the interactions of MM cells with their microenvironment are needed if we are to cure MM. In studies leading to this proposal, we identified Sequestosome-1 (p62), as a potential target to block the effects of key signal pathways in MMBD. p62 is a multidomain adapter protein that serves as a signaling hub for formation of multiple signaling complexes induced in BMSC by adhesive interactions with MM cells resulting in activation of NF-kB, p38 MAPK and JNK. Activation of these signaling pathways increases MM cell growth, OCL activity, and production of IL-6, RANKL and TNF? in BMSC as well as suppresses OB differentiation of patient BMSCs (MM-BMSC). Importantly, knockdown (KD) of p62 in MM-BMSC markedly decreases the capacity of MM cells to upregulate VCAM1, IL-6, TNF? and RANKL production, and enhances MM growth and OCL formation. Recently, we showed that when mice with established MM were treated with a small molecule antagonist to the p62-ZZ domain, the domain that mediates many of the effects of enhanced p62-mediated signaling in MM-BMSC, dramatic new bone formation occurred exclusively in MM-involved bones. In addition we found that: 1) the transcriptional repressor Gfi1 (growth factor independence1) is increased in MM-BMSC and MM cells from a majority of patients; 2) direct co-culture of MM cells with wild type (WT), but not p62 knockout (KO) BMSC, as well as IL-6 treatment markedly increased Gfi1 expression in MM cells and BMSC; 3) upregulation of Gfi1 in MM-BMSC inhibits OB differentiation by repressing transcription of the critical OB transcription factor Runx2; 4) knock-down (KD) of Gfi1 allows MM-BMSC to differentiate to OB and 5) KD of Gfi1 in MM cells decreased their growth and induced apoptosis. However, the role of the p62/Gfi1 signaling axis in MMBD is not established. It is our hypotheses that adhesive interactions between BMSC and MM cells enhance p62-mediated signaling in BMSC, which increases Gfi1, IL-6, RANKL, VCAM1, and TNF? in BMSC and results in suppressed OB differentiation, increased bone destruction and Gfi1 upregulation in MM cells. Increased Gfi1 expression in MM cells suppresses transcription of pro- apoptotic genes and increases STAT3 signaling in MM cells to promote MM survival and growth. To test these hypotheses, we will use novel ex vivo and in vivo approaches to determine:
Aim 1) the contributions of the p62/Gfi1 axis in MM-BMSC to the suppression of OB differentiation, MM-BMSC enhancement of MM cell growth and MMBD and the mechanisms mediating these effects in vitro and in vivo.
Aim 2) the contributions of increased Gfi1 expression in MM cells to MM cell growth and survival and the mechanisms mediating these effects in vitro and in vivo. Results of these studies should provide important new information on the role of the p62/Gfi1 axis in MM, and provide a basis for development of novel mechanism-based therapies that target the p62/Gfi1 axis as treatment for Veterans with MM.
This Merit Review proposal will use novel ex vivo and in vivo approaches to assess the mechanisms responsible for p62/Gfi1's enhancement of Myeloma Bone Disease (MMBD) and MM cell growth and survival, and the potential of targeting the p62/Gfi1 axis as a novel treatment for Veterans with MM. The project will test the hypotheses that adhesive interactions between BMSC and MM cells enhance p62-mediated signaling in bone marrow stromal cells (BMSC) and increase bone destruction and Gfi1 expression in MM cells. Increased Gfi1 expression in MM cells suppresses transcription of pro-apoptotic genes and increases STAT3 signaling by blocking PIAS3 binding to STAT3, to promote MM survival and growth. The study will determine: 1) the contributions of the p62/Gfi1 axis in MM-BMSC to MM cell growth and MMBD and the mechanisms mediating these effects in vitro and in vivo and 2) the contributions of increased Gfi1 expression in MM cells to MM cell growth and survival and the mechanism mediating these effects in vitro and in vivo.
