TY - JOUR
T1 - Combined HDAC and bromodomain protein inhibition reprograms tumor cell metabolism and elicits synthetic lethality in glioblastoma
AU - Zhang, Yiru
AU - Ishida, Chiaki Tsuge
AU - Ishida, Wataru
AU - Lo, Sheng Fu L.
AU - Zhao, Junfei
AU - Shu, Chang
AU - Bianchetti, Elena
AU - Kleiner, Giulio
AU - Sanchez-Quintero, Maria J.
AU - Quinzii, Catarina M.
AU - Westhoff, Mike Andrew
AU - Karpel-Massler, Georg
AU - Canoll, Peter
AU - Siegelin, Markus D.
N1 - Funding Information:
This work was supported by NIH NINDS grants R01NS095848, R01NS102366, and K08NS083732; the Louis V. Gerstner, Jr. Scholars Program (2017–2020); and American Brain Tumor Association Discovery Grant 2017 (DG1700013) (M.D. Siegelin).
Funding Information:
The combination of panobinostat and OTX015 resulted in morphological signs of apoptosis. In support of this finding, GSEA showed that the combination treatment activated a transcriptional proapoptotic state (Fig. 2C). Therefore, we determined as to whether or not features of apoptotic cell death can be confirmed biochemically. To this purpose, LN229, T98G, and U87 GBM cells or stem-cell like GBM cells, NCH421k, and NCH644 were treated with panobinostat (or vorinostat), OTX015, or the combination of both and stained with Annexin V/propidium iodide and analyzed by multiparametric flow cytometric analysis. Consistently, we found that the combination treatment of OTX015 and Panobinostat led to more apoptotic cells than control or single treatments. Similar findings were made when vorinostat was used in lieu of panobinostat (Fig. 2A and B; Supplementary Fig. S2A–S2C). Intrinsic apoptosis is accompanied by loss of mitochondrial membrane potential. Consistently, the combination treatments (panobinostat+OTX015; vorinostat+OTX015) reduced the amount of TMRE positive cells stronger than single treatments or control in LN229, T98G, and U87 cells (Fig. 2D and E; Supplementary Fig. S2D). To assess whether or not caspases are involved in the death, we treated GBM cells in the presence or absence of pan-caspase inhibitor, zVAD-fmk. We found that zVAD-fmk partially protected the cells from DNA fragmentation induced by the combination treatment, suggesting that caspases are involved in the death (Supplementary Fig. S2E). This finding was also supported by enhanced cleavage of PARP by the combination treatment (Fig. 2F; Supplementary Fig. S2H).
Publisher Copyright:
© 2018 American Association for Cancer Research.
PY - 2018/8/15
Y1 - 2018/8/15
N2 - Purpose: Glioblastoma remains a challenge in oncology, in part due to tumor heterogeneity. Experimental Design: Patient-derived xenograft and stem-like glioblastoma cells were used as the primary model systems. Results: Based on a transcriptome and subsequent gene set enrichment analysis (GSEA), we show by using clinically validated compounds that the combination of histone deacetylase (HDAC) inhibition and bromodomain protein (BRD) inhibition results in pronounced synergistic reduction in cellular viability in patient-derived xenograft and stem-like glioblastoma cells. Transcriptome-based GSEA analysis suggests that metabolic reprogramming is involved with synergistic reduction of oxidative and glycolytic pathways in the combination treatment. Extracellular flux analysis confirms that combined HDAC inhibition and BRD inhibition blunts oxidative and glycolytic metabolism of cancer cells, leading to a depletion of intracellular ATP production and total ATP levels. In turn, energy deprivation drives an integrated stress response, originating from the endoplasmic reticulum. This results in an increase in proapoptotic Noxa. Aside from Noxa, we encounter a compensatory increase of antiapoptotic Mcl-1 protein. Pharmaco-logic, utilizing the FDA-approved drug sorafenib, and genetic inhibition of Mcl-1 enhanced the effects of the combination therapy. Finally, we show in orthotopic patient-derived xenografts of GBM, that the combination treatment reduces tumor growth, and that triple therapy involving the clinically validated compounds panobinostat, OTX015, and sorafenib further enhances these effects, culminating in a significant regression of tumors in vivo. Conclusions: Overall, these results warrant clinical testing of this novel, efficacious combination therapy.
AB - Purpose: Glioblastoma remains a challenge in oncology, in part due to tumor heterogeneity. Experimental Design: Patient-derived xenograft and stem-like glioblastoma cells were used as the primary model systems. Results: Based on a transcriptome and subsequent gene set enrichment analysis (GSEA), we show by using clinically validated compounds that the combination of histone deacetylase (HDAC) inhibition and bromodomain protein (BRD) inhibition results in pronounced synergistic reduction in cellular viability in patient-derived xenograft and stem-like glioblastoma cells. Transcriptome-based GSEA analysis suggests that metabolic reprogramming is involved with synergistic reduction of oxidative and glycolytic pathways in the combination treatment. Extracellular flux analysis confirms that combined HDAC inhibition and BRD inhibition blunts oxidative and glycolytic metabolism of cancer cells, leading to a depletion of intracellular ATP production and total ATP levels. In turn, energy deprivation drives an integrated stress response, originating from the endoplasmic reticulum. This results in an increase in proapoptotic Noxa. Aside from Noxa, we encounter a compensatory increase of antiapoptotic Mcl-1 protein. Pharmaco-logic, utilizing the FDA-approved drug sorafenib, and genetic inhibition of Mcl-1 enhanced the effects of the combination therapy. Finally, we show in orthotopic patient-derived xenografts of GBM, that the combination treatment reduces tumor growth, and that triple therapy involving the clinically validated compounds panobinostat, OTX015, and sorafenib further enhances these effects, culminating in a significant regression of tumors in vivo. Conclusions: Overall, these results warrant clinical testing of this novel, efficacious combination therapy.
UR - http://www.scopus.com/inward/record.url?scp=85051694427&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85051694427&partnerID=8YFLogxK
U2 - 10.1158/1078-0432.CCR-18-0260
DO - 10.1158/1078-0432.CCR-18-0260
M3 - Article
C2 - 29764852
AN - SCOPUS:85051694427
SN - 1078-0432
VL - 24
SP - 3941
EP - 3954
JO - Clinical Cancer Research
JF - Clinical Cancer Research
IS - 16
ER -