Investigation of the intrinsic mechanism of drug resistance in multiple myeloma

Abstract

The focus of this thesis was to evaluate the mechanisms whereby myeloma cells develop intrinsic resistance with a focus on resistance in the context of bortezomib treatment. The aims of this thesis were to examine multidrug resistance pumps as a mechanism of resistance in MM, to investigate the contribution of p53 signalling perturbations in resistance mechanism in MM, to study the AMPK pathway as an alternative target to overcome MM resistance and finally to characterise myeloma resistance to bortezomib treatment using 2D-DIGE analysis. Focussing on bortezomib resistance models, we found that that overexpression of P-gp attenuates bortezomib activity. Bortezomib is a P-gp substrate and a combination of P-gp inhibitor and bortezomib is able to overcome resistance. Bortezomib is also able to downregulate the expression and function of P-gp. Our findings therefore suggest that combination of a P-gp inhibitor and bortezomib in P-gp positive myeloma would be a reasonable treatment combination to extend use of the drug. We have shown that p53 apoptotic signalling pathways can be accentuated when bortezomib is combined with a Mdm2 inhibitor. In p53 WT cells, nutlin-3 in combination with bortezomib generates additive toxicity in MM cells but is highly synergistic in epithelial models and p53-mutated cell lines. This synergy persists in the presence of BMSCs. This observation has implications more so in epithelial cancers and p53 mutated cancers where single agent bortezomib activity is mild. We have also shown that bortezomib-treated patients who had high expression of nutlin-3-suppressed genes had significantly shorter progression-free (p=0.001, log-rank test) and overall survival (p=0.002, log-rank test) compared to those with low expression levels. AMPK activation is promising as an anticancer pathway and may also be a chemoprevention target. Metformin and AICAR, which activate this pathway, both have demonstrated useful preclinical anticancer properties and have a good therapeutic index in patients. We explored mechanism of cell death and showed that AICAR was able to activate the apoptotic pathway. These agents also synergise with glycolysis inhibitors to further increase cytotoxicity in cancer cells. Identification of proteins whose expression is altered in differing states of sensitivity and resistance provides candidates for better understanding of resistance mechanisms so we also investigated bortezomib resistance in cellular models using proteomic techniques and isolated and identified several novel proteins which may play a role in this phenomenon. Our findings are mechanistically consistent since two of the identified proteins Hsp70 and caspase-3 are known in the literature to be affected by bortezomib treatment