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1575 Runx2 Deficiency in Osteoblasts Promotes Myeloma Resistance to Bortezomib By Increasing TSP-1-Dependent TGF-β1 Activation in Bone Marrow

Program: Oral and Poster Abstracts
Session: 651. Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational: Poster I
Hematology Disease Topics & Pathways:
Fundamental Science, Translational Research
Saturday, December 11, 2021, 5:30 PM-7:30 PM

Chao Zhang, PhD1,2*, Xiaoxuan Xu, MD1*, Timothy Trotter, PhD1*, Amjad Javed, PhD3,4*, Joanne E. Murphy-Ullrich, PhD1*, Juan Li, PhD2 and Yang Yang, MD1,4

1Department of Pathology, University of Alabama at Birmingham, Birmingham, AL
2First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
3Department of Oral and Maxillofacial Surgery, School of Dentistry, University of Alabama at Birmingham, Birmingham, AL
4Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL

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 (2×106). 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.

Disclosures: Murphy-Ullrich: Millipore Sigma: Patents & Royalties.

*signifies non-member of ASH