Runx2 Deficiency in Osteoblasts Promotes Myeloma Resistance to Bortezomib By Increasing TSP-1-Dependent TGF-β1 Activation in Bone Marrow

Multiple myeloma (MM) is a plasma cell malignancy that thrives in the bone marrow (BM). The proteasome inhibitor bortezomib (BTZ) is one of the most effective front-line chemotherapeutic drugs for MM; however, 15-20% of high-risk patients do not respond to or become resistant to this drug and the mechanisms of chemoresistance remain unclear. We previously demonstrated that MM cells inhibit Runt-related transcription factor 2 (Runx2) in pre- and immature osteoblasts (OBs). In the current study, we investigated the impact of OB-Runx2 deficiency on the outcome of BTZ treatment using our syngeneic mouse model of MM in which Runx2 is specifically deleted in the immature OBs of C57BL6/KaLwRij mice (OB-Runx2 ^-/- mice) and OB-Runx2 ^+/+ mice as control.

Five-week-old OB-Runx2 ^+/+ mice and OB-Runx2 ^-/- mice were i.v. injected with 5TGM1-Luc murine MM cells (2×10 ⁶). On day 8, after the tumor injection, mice were randomly assigned to treatment for 4 weeks, with either BTZ (i.p. injection, 0.5 mg/kg body weight, twice/week) or PBS. Bioluminescence imaging and serum IgG2bκ (a soluble marker of 5TGM1 MM cells) ELISA showed that BTZ significantly inhibited tumor growth in OB-Runx2 ^+/+ mice, but not in OB-Runx2 ^-/- mice. Cytokine array and ELISA showed that in PBS-treated, tumor-bearing OB-Runx2 ^-/- mice, BM levels of thrombospondin-1 (TSP-1), a matricellular protein that converts latent TGF-β1 to its active form, and active TGF-β1 were significantly higher than levels in tumor-bearing OB-Runx2 ^+/+ counterparts. Interestingly, BTZ treatment further increased the levels of both TSP-1 and active TGF-β1 in the BM of tumor-bearing OB-Runx2 ^-/- mice, but it did not affect the level of either TSP-1 or TGF-β1 in the BM of tumor-bearing OB-Runx2 ^+/+ mice. These results suggest that OB-Runx2 deficiency increases TGF-β1 activation via TSP-1 in BM and BTZ treatment further enhances this effect.

SRI31277 is a tripeptide antagonist that blocks TSP-1-mediated activation of TGF-β1 and it has been shown to reduce tumor burden, TGF-β signaling, and osteolytic bone disease in multiple mouse models of MM. To confirm the involvement of TSP-1/TGF-β1 activation in MM BTZ resistance induced by OB-Runx2 deficiency and to test whether blocking TSP1-mediated TGF-β1 activation can alleviate BTZ resistance, 5TGM1-Luc MM tumor-bearing OB-Runx2 ^+/+ and OB-Runx2 ^-/- mice were treated with PBS, BTZ, SRI31277 (osmotic pump, 30 mg/kg body weight per day), or BTZ + SRI31277 for 4 weeks. While SRI31277 did not further increase BTZ-induced MM inhibition in OB-Runx2 ^+/+ mice, SRI31277 treatment significantly reduced the tumor burden in OB-Runx2 ^-/- mice. Furthermore, treatment with BTZ + SRI31277 augmented the reduction in tumor burden induced by SRI31277 treatment alone. Flow cytometry and Western blot analyses demonstrated that SRI31277 treatment enhanced apoptosis, reduced Ki-67 expression as well as the activity of both canonical (SMAD2/3) and non-canonical (ERK1/2) signaling pathways of TGF-β1 in MM cells in OB-Runx2 ^-/- mice; and BTZ+SRI31277 treatment augmented these effects. Furthermore, TGF-β has immunosuppressive effects in MM and our data show that SRI31277 overcomes this by reducing myeloid derived suppressor cells, checkpoint, and T cell exhaustion markers and by increasing cytotoxic T cells.

In conclusion, OB-Runx2 deficiency, induced by MM cells, promotes MM resistance to BTZ through the upregulation of TGF-β1 activation in the BM and TGF-β1 signaling in MM cells. Importantly, blocking TSP-1-mediated TGF-β1 activation with SRI31277 can reverse this resistance and immune dysfunction. Our findings demonstrate a novel mechanism for BTZ resistance in MM and identify a new target and strategy for overcoming immune dysregulation and chemoresistance in MM.