Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients’ plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) β. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II+ CAFs. TGFβ mediates bort-induced autophagy, and its blockade by LY2109761, a selective TβRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.
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This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC), Investigator Grant (no. 14095 to AV); Special Program Molecular Clinical Oncology 5 per 1000 (no. 9965 to AV), Milan, IT; and the European Commission’s Seventh Framework Programme (EU FPT7) OVER-MyR (no. 278706 to AV and KV) and OPTATIO (no. 278570 to DR). Abstract was awarded by Società Italiana di Citometria with “Francesco Mauro” prize. The sponsors of this study are public or no-profit organizations that support science in general; they had no role in gathering, analyzing or interpreting the data.
MAF and AV designed the research, analyzed data and wrote the manuscript. VD and LDM performed research. KDV, EM and KV performed animal studies. DV and MM performed proteomic studies. SR, TA, BN and DR performed confocal dual immunofluorescence studies. IC, RR, VR, EA and DD provided reagents and materials. RF, FD, KDV and KV read and made comments on manuscript.
The authors declare no conflict of interest.
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Frassanito, M., De Veirman, K., Desantis, V. et al. Halting pro-survival autophagy by TGFβ inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients. Leukemia 30, 640–648 (2016). https://doi.org/10.1038/leu.2015.289
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