Cooperative Targets of Combined mTOR/HDAC Inhibition Promote MYC Degradation

Abstract

Cancer treatments often require combinations of molecularly targeted agents to be effective. mTORi (rapamycin) and HDACi (MS-275/entinostat) inhibitors have been shown to be effective in limiting tumor growth, and here we define part of the cooperative action of this drug combination. More than 60 human cancer cell lines responded synergistically (CI<1) when treated with this drug combination compared to single agents. In addition, a breast cancer patient-derived xenograft, and a BCL-XL plasmacytoma mouse model both showed enhanced responses to the combination compared to single agents. Mice, bearing plasma cell tumors lived an average of 70 days longer on combination treatment compared to single agents. A set of 37 genes cooperatively affected (34 down-regulated; 3 up-regulated) by the combination responded pharmacodynamically in human myeloma cell lines, xenografts, and a P493 model, and were both enriched in tumors, and correlated with prognostic markers in myeloma patient datasets. Genes down-regulated by the combination were overexpressed in several untreated cancers (breast, lung, colon, sarcoma, head and neck, myeloma) compared to normal tissues. The MYC/E2F axis, identified by upstream regulator analyses and validated by immunoblots, was significantly inhibited by the drug combination in several myeloma cell lines. Furthermore, 88% of the 34 genes downregulated have MYC binding sites in their promoters, and the drug combination cooperatively reduced MYC half-life by 55% and increased degradation. Cells with MYC mutations were refractory to the combination. Thus, integrative approaches to understand drug synergy identified a clinically actionable strategy to inhibit MYC/E2F activity and tumor cell growth in vivo.

Chemical and structural studies provide a mechanistic basis for recognition of the MYC G-quadruplex

“…1a, b , and Supplementary Table 1 ) 31 , 32 . Most compounds were evaluated for affinity and effects on cell viability, and selected compounds also for effects on MYC protein levels in L363 cells (a MYC-driven multiple myeloma cell line 33 ). Affinities were generated by measuring K D values determined by the compound-induced change in fluorescence of an Alexa Fluor® 647 tag conjugated to the 5′ end of Pu27.…”

mTOR Cross-Talk in Cancer and Potential for Combination Therapy

“…Simmons and his colleagues demonstrated synergistic interactions between rapamycin and entinostat (small molecule inhibitor of Class 1 HDAC) in B cells neoplasia, in both in vitro and in vivo models ( Table 2 ) [ 83 ]. Subsequently, the same group demonstrated that this combination is synergistic in other cancer cell lines with different histological origin (i.e., BC and plasmacytoma) and hypothesized that Myc could be a central player in this combination strategy [ 106 ]. Combination approaches include different epigenetic drugs, such as azacitidine, a chemical analog of cytidine which inhibits DNA methyltransferase, causing hypomethylation of DNA; a phase Ib/II study to assess the effects of everolimus in combination with azacitidine in AML patients has been reported [ 107 ].…”

Snail determines the therapeutic response to mTOR kinase inhibitors by transcriptional repression of 4E-BP1

“…In this study, we found that, similar to genetic depletion of Snail, pharmacological inhibition of HDAC activity significantly repressed Snail activity by inducing 4E-BP1 expression, and co-targeting HDAC and mTOR resulted in a marked suppression of tumor growth. Several recent studies 55 – 57 also show that the combination of mTOR and HDAC inhibitors exert a synergistic antitumor activity in a large panel of human cancer cell lines, and the patient-derived xenograft and transgenic mouse models. Interestingly, the synergistic growth inhibiting consequence of combined mTOR/HDAC inhibition is likely attributed to their mechanistic convergences on the mTOR/4E-BP1 signaling axis and impaired polysome formation 56 .…”