Abstract
Drug combination therapies remain pivotal for the treatment of heterogeneous malignancies, such as glioblastomas. Here, we show a novel lethal interaction between Bcl-xL and c-myc inhibition accomplished by bromodomain protein inhibitors. Established, patient-derived xenograft and stem cell-like glioma cells were treated with the novel bromodomain protein inhibitors, JQ1 and OTX015, along with BH3-mimetics, ABT263 or Obatoclax. Synergy was assessed by calculation of CI values. Small interfering RNAs (siRNAs) were used for gene silencing and mechanistic studies. In vivo experiments were performed in a glioblastoma xenograft model. Single treatments with JQ1 and OTX015 had only moderate effects on the reduction of cellular viability. However, the combination treatment of BH3-mimetics along with JQ1 or OTX015 resulted in a highly synergistic reduction of cellular viability in a broad range of different model systems of malignant glioma. Similarly, knockdown of c-myc sensitized glioma cells for ABT263 mediated cell death. The enhanced loss of cellular viability in the combination treatment was mediated by activation of apoptosis with dissipation of mitochondrial membrane potential and caspase cleavage. The combination treatment led to a modulation of anti- and pro-apoptotic Bcl-2 family members with an increase in pro-apoptotic Noxa mediated by ATF4. Small interfering RNA mediated knockdown of Bak and Noxa protected glioma cells from ABT263/JQ1 mediated apoptosis. Finally, the combination treatment of ABT263 and OTX015 resulted in a regression of tumors and a significantly smaller tumor size as compared to single or vehicle treated tumors. Thus, these results warrant clinical testing for the drug combination of BH3-mimetics along with bromodain protein inhibitors.
Highlights
Glioblastoma and malignant gliomas remain incurable diseases [1]
In order to assess the effects of JQ1 on the proliferation of glioblastoma cells, we treated established glioblastoma cells of different genetic backgrounds (U87, LN229 and T98G), patient derived xenograft cells (GBM6, GBM14 and GBM39) and stem cell-like glioma cells (NCH644 and NCH421k) with increasing concentrations of JQ1 (Figure 1A-1C)
Our results show that zVAD-fmk protects cells from DNA – fragmentation induced by ABT263+JQ1 (Supplementary Figure 2D), suggesting that apoptosis and caspases play a role in the death mediated by the combination treatment
Summary
Glioblastoma and malignant gliomas remain incurable diseases [1]. It is for that reason that new therapeutic approaches need to be elucidated. Many oncogenic pathways are simultaneously active in one tumor [2] The activation of these pro-survival pathways results in dysregulation of apoptosis with high levels of inhibitor of apoptosis proteins, such as XIAP, cIAP-1 and survivin, and anti-apoptotic Bcl-2 family members, which in turn block physiological cell death and promote unrestrained growth. Bcl-2 family members, such as Bcl-2, Bcl-xL and Mcl-1, are expressed at high levels in gliomas, rendering these molecules potential targets for therapy. This has led to the discovery and synthesis of a certain class of molecules, which are known as BH3-mimetics due to their high affinity binding to antiapoptotic Bcl-2 family members
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