Small Molecule Inhibition of SIRT Activity: A Novel Therapeutic Approach for Refractory Multiple Myeloma

Multiple myeloma (MM) is the second most common adult hematologic malignancy in the United States and is characterized by a unique form of progressive bone destruction. Although new treatments such as bortezomib (BZ), carfilzomib and IMIDs have provided benefit to many patients with MM, drug resistance remains a very significant problem. Patients diagnosed with high-risk MM in particular are in urgent need of more effective therapeutic strategies in order to improve their survivorship. The sirtuin deacetylases (SIRTs, SIRT1-7) are critical epigenetic regulators that control longevity, cell growth, tumor suppression, and apoptosis. Elevated SIRT expression has been reported in several types of cancer and may promote pathogenesis and drug resistance by increasing the lifespan and survival capacity of malignant cells. Our preliminary analysis of SIRT expression indicated that SIRT1 was consistently expressed at significantly higher levels in primary MM cells from patients with high-risk disease than those with favorable risk MM or normal CD138+ controls. We investigated the role of SIRT1 as a potential regulator of MM pathogenesis and evaluated the benefit of SIRT inhibition as a novel strategy for MM therapy. A series of experiments established a mechanistic relationship between MYC and SIRT1 in that SIRT1 increased MYC protein stability and transcriptional activity. Disruption of SIRT1 function through targeted knockdown or with the small molecule inhibitor tenovin-6 (T6) directly led to the reduction in the expression of key MYC transcriptional targets that play important roles in MM pathogenesis including cyclin D and MAF. Subsequent analyses revealed that U266 and RPMI-8226 cells with acquired BZ resistance exhibited significantly higher basal SIRT1 expression levels (p<0.05) than their BZ-sensitive parental counterparts despite their very different cytogenetic backgrounds, suggesting that SIRT1 upregulation may be a frequent event that occurs during the process of developing drug resistance. Studies focused on T6 demonstrated that BZ-resistant U266 and RPMI-8226 and primary MM cells (n=10) displayed high sensitivity to T6 as quantified by its effects on cell viability, clonogenic survival, and apoptosis.  Comprehensive kinetic metabolic profiling of MM cells treated with T6 showed that its pharmacodynamic (PD) effects were related to the induction of severe oxidative stress characterized by the significant time-dependent accumulation of cystine (formed by the oxidation of 2 cysteine molecules covalently linked by a disulfide bond, p<0.001), progressive disruption of the TCA cycle, reduced glutaminolysis, and inhibition of branched chain amino acid catabolism. Notably, resistant cells exhibited a modest increase in sensitivity to T6 compared to parental cells or BZ-sensitive primary MM cells. This indicates that there is unlikely to be cross-resistance between BZ-based regimens and T6. Additional assays showed that T6 synergistically augmented the activity of BZ in BZ-sensitive cells and achieved greater than additive effects in BZ-resistant cells. Administration of the combination of BZ and T6 to mice bearing RPMI-8226 xenografts yielded significantly greater anti-MM activity than either single agent (p<0.01). Our collective findings demonstrate that SIRT1 is a promising novel therapeutic target in MM. Further investigation aimed to elucidate the safety, efficacy, and mechanism of action of T6 is warranted.