Bim-Targeting Therapy Circumvents Adaptive Bortezomib-Resistance In Myeloma Through a Novel Cross-Link Between Autophagy and Apoptosis

In diverse human cancers, oncogene-driven pathways antagonize the lethal effects of Bim, a major apoptosis executioner implicated in the activity of various anti-cancer agents including bortezomib (btz). As loss of Bim plays an important role in tumor development and growth as well as in acquisition of drug resistance, Bim represents an attractive therapeutic target, particularly for efforts to circumvent resistance to standard chemotherapy and diverse novel therapies. In the present study, the role of Bim in acquired resistance of multiple myeloma (MM) cells to btz was investigated. Bcl-2 family member expression profiles revealed high basal levels of Bim in most MM cell lines (Bim^hi) and in primary CD138⁺ MM samples. However, Bim^low cells (e.g., H929) were highly susceptible to btz, arguing against the possibility that endogenous Bim levels correlate with btz sensitivity. In sharp contrast, shRNA Bim knockdown in Bim^hi cells conferred pronounced resistance to btz. Interestingly, btz-resistant cells (PS-R) generated by exposure of U266 cells (Bim^hi) to progressively increasing btz concentrations (to 20 nM) displayed markedly diminished Bim expression, a phenomenon validated in btz-resistant cells derived from other Bim^hi cell lines (e.g., OPM2, RPMI8226). Notably, Bim up-regulation by histone deacetylase inhibitors (HDACIs e.g., SBHA), when combined with BH3-mimetics (e.g., ABT-737) that release Bim from binding to Bcl-2/Bcl-xL, effectively killed btz-resistant MM cells. Moreover, this strategy was highly active against primary CD138⁺ cells isolated from relapsed MM patients. In view of recent evidence that Bim negatively regulates autophagy (Luo et al., Mol Cell 47:359-70, 2012), the possibility that autophagy might also play a role in Bim-targeting strategies was investigated. Whereas drug-naïve and btz-resistant cells exhibited little difference in expression of autophagy-inducing proteins or autophagy initiation (e.g., by thapsigargin or tunicamycin), resistant cells displayed deficiencies in autophagosome maturation, reflected by delayed removal of GFP-LC3 puncta. Interestingly, SBHA co-administration largely abrogated ABT-737-induced autophagy and markedly increased the association between Beclin-1 and Bcl-2, accompanied by diminished binding of Bim to Bcl-2. Significantly, shRNA Bim knockdown strikingly increased autophagy in Bim^hi cells. Moreover, SBHA was unable to inhibit autophagy in such cells due to failure of Bim up-regulation, resulting in a pronounced attenuation of SBHA/ABT-737-induced apoptosis. In Bim^low H929 cells, SBHA also failed to up-regulate Bim and potentiate ABT-737 lethality. However, disruption of autophagy by chloroquine (CQ) dramatically restored the sensitivity of these cells to this Bim-targeting regimen, an event associated with a striking increase in Bim expression. In contrast, CQ only modestly increased lethality in Bim^hi cells in which autophagy was already disabled by SBHA-mediated Bim up-regulation. Moreover, compared to parental drug-naïve cells (Bim^hi), btz-resistant cells displaying acquired loss of Bim were significantly more susceptible to potentiation of SBHA/ABT-737 lethality by CQ (e.g., 50% vs 22% increase in apoptosis; P < 0.01). Finally, in bim^-/- MEFs derived from gene knockout mice, SBHA/ABT-737 co-treatment, with or without the addition of CQ, was unable to induce apoptosis, arguing that the presence of the bim gene is required for restoration of drug sensitivity by Bim-targeting therapy. Together, these findings argue that Bim deficiency represents a novel mechanism of adaptive (rather than intrinsic) btz-resistance in MM cells, and that simultaneous up-regulation of Bim (e.g., by HDACIs) and unleashing of Bim from binding to anti-apoptotic Bcl-2 family members (e.g., by BH3-mimetics such as ABT-737) can circumvent this form of resistance. They also raise the possibility that in addition to direct induction of apoptosis, Bim-mediated suppression of autophagy contributes to the lethality of Bim-targeting strategies, and that disruption of the latter process (e.g., by CQ) may be particularly beneficial in cells in which Bim up-regulation is disabled. Collectively, these findings suggest that Bim-targeting therapy may represent a novel and effective strategy in overcoming btz resistance in MM. This study was supported by the M. D. Anderson Cancer Center SPORE in Multiple Myeloma.