Synergistic Induction of Apoptosis in Multiple Myeloma Cells by Bortezomib and Hypoxia-Activated Prodrug TH-302, In Vivo and In Vitro

Abstract

Recently, we showed that hypoxia is a critical microenvironmental factor in multiple myeloma, and that the hypoxia-activated prodrug TH-302 selectively targets hypoxic multiple myeloma cells and improves multiple disease parameters in vivo. To explore approaches for sensitizing multiple myeloma cells to TH-302, we evaluated in this study the antitumor effect of TH-302 in combination with the clinically used proteasome inhibitor bortezomib. First, we show that TH-302 and bortezomib synergistically induce apoptosis in multiple myeloma cell lines in vitro. Second, we confirm that this synergism is related to the activation of caspase cascades and is mediated by changes of Bcl-2 family proteins. The combination treatment induces enhanced cleavage of caspase-3/8/9 and PARP, and therefore triggers apoptosis and enhances the cleavage of proapoptotic BH3-only protein BAD and BID as well as the antiapoptotic protein Mcl-1. In particular, TH-302 can abrogate the accumulation of antiapoptotic Mcl-1 induced by bortezomib, and decreases the expression of the prosurvival proteins Bcl-2 and Bcl-xL. Furthermore, we found that the induction of the proapoptotic BH3-only proteins PUMA (p53-upregulated modulator of apoptosis) and NOXA is associated with this synergism. In response to the genotoxic and endoplasmic reticulum stresses by TH-302 and bortezomib, the expression of PUMA and NOXA were upregulated in p53-dependent and -independent manners. Finally, in the murine 5T33MMvv model, we showed that the combination of TH-302 and bortezomib can improve multiple disease parameters and significantly prolong the survival of diseased mice. In conclusion, our studies provide a rationale for clinical evaluation of the combination of TH-302 and bortezomib in patients with multiple myeloma.

TrxR1 inhibition overcomes both hypoxia-induced and acquired bortezomib resistance in multiple myeloma through NF-кβ inhibition

“…For example, the hypoxia-activated prodrug TH-302 induces apoptosis in hypoxic myeloma cells either alone or in combination with bortezomib. 10 In line with this study, our results showed that auranofin-induced TrxR1 inhibition exerted an anti-proliferative effect on hypoxic RPMI8226 and U266 cells (Fig. 2).…”

“…Our results complement another study that showed that TrxR1 inhibition by a similar auranofin concentration (1.5 mM) has increased the bortezomib sensitivity of human colon carcinoma cell line HCT116 overexpressing Bcl ¡ 2 by 2-fold under normoxic conditions. 40 While many therapies targeting the hypoxic myeloma cells are being developed either as a single agent or combinatorial therapies, 8,10 our study provided evidence that targeting hypoxic myeloma cells using TrxR1 inhibitors, such as auranofin, provides a potentially useful and novel treatment strategy for MM.…”

“…9,10,26 We firstly investigated the effect of hypoxia on myeloma cell proliferation in the presence of bortezomib. RPMI8226 and U266 cells were cultured under hypoxic (Hx, 1% O 2 ) and normoxic (Nrx, 21% O 2 ) conditions for 24 hours, and subsequently treated with or without bortezomib (0-40 nM) for 24 hours, and cell proliferation was analyzed.…”

“…8 Together TH-302 and bortezomib synergistically induce apoptosis in myeloma cells growing under hypoxic conditions. 10 Thus, development of therapies that target not only the cell growing under normal oxygen conditions but also under hypoxia, either alone or in combination with conventional anti-myeloma drugs, may provide a better therapeutic approach to treat MM.…”

Hypoxia-inducible KDM3A addiction in multiple myeloma

“…Our findings provide reasons to use a combination of proteasome inhibitors and/or immunomodulatory drugs in addition to hypoxia-targeting drugs. 46 In conclusion, we have shown that KDM3A strongly correlates with HIF-1a dependence in myeloma cells after hypoxic exposure, contributing to the survival of hypoxia-subjected myeloma cells. KDM3A contributes to the maintenance of MALAT1 expression, which may lead to continuous HIF-1a expression during hypoxia.…”