Metabolic reprogramming of oncogene-addicted cancer cells to OXPHOS as a mechanism of drug resistance

Abstract

The ability to selectively eradicate oncogene-addicted tumors while reducing systemic toxicity has endeared targeted therapies as a treatment strategy. Nevertheless, development of acquired resistance limits the benefits and durability of such a regime. Here we report evidence of enhanced reliance on mitochondrial oxidative phosphorylation (OXPHOS) in oncogene-addicted cancers manifesting acquired resistance to targeted therapies. To that effect, we describe a novel OXPHOS targeting activity of the small molecule compound, OPB-51602 (OPB). Of note, a priori treatment with OPB restored sensitivity to targeted therapies. Furthermore, cancer cells exhibiting stemness markers also showed selective reliance on OXPHOS and enhanced sensitivity to OPB. Importantly, in a subset of patients who developed secondary resistance to EGFR tyrosine kinase inhibitor (TKI), OPB treatment resulted in decrease in metabolic activity and reduction in tumor size. Collectively, we show here a switch to mitochondrial OXPHOS as a key driver of targeted drug resistance in oncogene-addicted cancers. This metabolic vulnerability is exploited by a novel OXPHOS inhibitor, which also shows promise in the clinical setting.

Keywords: Metabolic reprogramming; OXPHOS; Oncogene-addiction; STAT3.

Fig. 1

Oncogene-addicted cancer cells with acquired resistance to targeted therapy exhibit elevated aerobic mitochondrial respiration and small molecule OPB inhibits OXPHOS. (A) Basal OCR was measured using Seahorse Analyzer XF^e24 in HCC827, HCC827-GR, A375, A375-VR, H2228, H2228-CR, BaF3WT and BaF3T315I cells. (B) Mitochondrial DNA copy number was measured using qPCR and represented in terms of relative quantification (RQ) with HCC827 and A375 as control. (C) Basal TFAM and PGC1α expression levels were measured as an indication of mitochondrial biogenesis via western blot in HCC827, HCC827-GR, H1975, A375 and A375-VR cells with GAPDH used as a loading control. (D) Mitochondrial supercomplexes was checked via BN-PAGE using individual complex antibodies and HSP70 was used as a loading control. (E) Selleckchem Anti-cancer compound library (414 compounds, #L3000) and Epigenetic library (151 modulators, #L1900) were used for highthroughput chemical screening and cell viability were assayed using Celltiter-Glo luminescent. (F) HCC827, HCC827GR cells were exposed to 1 mM Metformin, 1 μM Elesclomol, 10 μM Stattic and 100 nM OPB for 4hrs before measuring OCR using Seahorse Analyzer XF^e24. (G) Cell viability was measured using crystal violet cell viability assay after 48 h exposure to 1 mM Metformin, 1 μM Elesclomol, 10 μM Stattic and 100 nM OPB in HCC827and HCC827-GR cells. (H) Cell viability was measured using crystal violet cell viability assay after 48 h of OPB treatment on HCC827, HCC827-GR, A375, A375-VR, BaF3WT and BaFT315I cells. (I) 5 × 10⁶ U-937 cells/body inoculated in tumor-bearing mice and allocated to 3 groups of 16 each based on body weight on the day of the inoculation. OPB was orally administered once daily from the day after group allocation until death occurred. Figure shows the survival curve in each group calculated by the Kaplan-Meier method. Statistical differences were determined by log-rank test using the Bonferroni correction (by two independent tests) for multiple comparisons. P values in A, B, F, G and H were calculated by paired Student's t-test using GraphPad Prism. Therapy-resistant oncogene-addicted cell lines are shown in red font in this and subsequent figures.

Fig. 2

OPB-51602 inhibits aerobic mitochondrial respiration and induces mitochondrial dysfunction.(A) HCC827, HCC827GR, A375, A375VR and BaF3 WT/T315I/M315T/H396R cells were exposed to 50 nM (HCC827/HCC827-GR), 20 nM (A375/A375-VR), and 10 nM (BaF3 cells) of OPB respectively for 1 h before measuring OCR using Seahorse Analyzer XF^e24. (N = 3) (B) Maximum OCR was measured using Seahorse Analyzer XF^e24 with FCCP in HCC827, HCC827GR, A375 and A375VR cells after exposing to 50 nM and 20 nM of OPB respectively for 1 h. (N = 3) (C) Maximum ECAR was measured using Seahorse Analyzer XF^e24 in HCC827, HCC827GR, A375, A375VR and H1975 cells after exposing to 50 nM (HCC827 and HCC827-GR) and 20 nM (A375, A375-VR and H1975) of OPB respectively for 1 h. (N = 3) (D) Lactate level was measured using lactate measurement kit in H1975 cells after exposing to different doses of OPB for 1 h. (N = 3) (E) OCR measurement was done on treated (20 nM OPB) and non-treated H1975 cells using Seahorse Analyzer XF^e24. Oligomycin (1 μM), FCCP (1 μM) and Rotenone/Antimycin A (Rot/AA) (1/0.5 μM) was used in injector to check different mitochondrial complexes (F) Maximum OCR was measured using Seahorse Analyzer XF^e24 with FCCP in HK-1 and C666-1 cells after exposing to 30 nM OPB for 1 h. (N = 3) (G) Maximum ECAR was measured using Seahorse Analyzer XF^e24 in HK-1 and C666-1 cells after exposing to 30 nM OPB for 1 h. (N = 3) (H) OCR measurement was done on treated (30 nM OPB) and non-treated HK-1 and C666-1 cells using Seahorse Analyzer XF^e24. Oligo (1 μM), FCCP (1 μM) and Rot/AA (1 μM/0.5μM) was used in injector to check different mitochondrial complexes (I) ATP level was detected in HK and C666-1 cells after exposing to 30 nM OPB in normoxic (21%O₂) and hypoxic (4%O₂) condition. Error bars shown as SD, P values in A were calculated by two ways ANOVA and C-G were calculated by paired Student's t-test.

Fig. 3

OPB-51602 specifically inhibits mitochondrial complex I activity.(A) OXPHOS regulated genes were analyzed using RT² PCR Profiler Array in H1975 cells after exposing the cells to 10 nM and 20 nM OPB for 48 h. (N = 3) (B) 2D gel electrophoresis was done after running BN-PAGE with mitochondrial lysates of untreated Jurkat cells and Jurkat cells treated with 20 nM OPB. Membranes were probed with mitochondrial complex cocktail antibody (Abcam) to detect different mitochondrial respiratory complexes. (C) Mitochondrial complex I activity was measured using Complex I activity assay kit on bovine heart mitochondria (BHM) after exposing mitochondria with different concentrations of OPB and rotenone was used as a positive control. (N = 3) (D) Mitochondrial complex I activity was measured in H1975 cells using complex I activity assay after treating the cells with 10 nM and 20 nM OPB for 1 h. (N = 3) (E) NAD⁺/NADH ratio was measured using NAD⁺/NADH kit in H1975 cells treated and non-treated for 1 h with 20 nM OPB. (N = 3) (F) In-gel complex I activity was measured using BN-PAGE after H1975 cells were exposed to 20 nM OPB. (G) Time kinetics OCR was measured in HK-1 and C666-1 cells with separate injection of rotenone and OPB. (H) Time kinetics OCR was measured in HK-1 and C666-1 cells with separate injection of rotenone and different concentrations of OPB. Time interval between 2 measurements in the Y axis is 9 min. Error bars shown as SD, P values in A, D and F were calculated by paired Student's t-test.

Fig. 4

OPB inhibits cancer stemness in increased CSC population in therapy-resistant oncogene-addicted cells.(A) Microarray analysis was done in HCC827, HCC827-GR, A375 and A375-VR cells using Agilent platform. (B) ALDH level was detected using flow cytometry in A375 basal level and in A375-VR after exposing to 20 nM OPB for 48hrs. (C) & (D) ALDH⁺ and ALDH^- populations were sorted in A375VR cells using FACS and the sorted cells were plated and OCR was measured in both populations after exposing the cells to 20 nM OPB for 1 h. (N = 3) (E) 2000 sorted cells of ALDH⁺ and ALDH^- population of A375VR were plated for spheroid formation, untreated and treated spheroids with 20 nM OPB for 7 days; Spheroids were imaged using ZEISS microscope. (F) Microarray analysis was done in MCF-7 and MDA-MB-231 cells using Agilent platform. (G) CD44⁺/CD24^- cells were sorted using flow cytometry, sorted cells were plated and OCR was measured in CD44⁺/CD24^- after exposing with 100 nM OPB. (H) CD44 and CD24 levels were measured by flow cytometry in MDA-MB-231 cells after cells were exposed to 200 nM of OPB for 48hrs. (I) 2000 sorted cells of CD44⁺/CD24^- +ve and CD44⁺/CD24^- -ve population of MDA-MB-231 were plated for spheroid formation, spheroids were treated 100 nM OPB for 7days and imaged using ZEISS microscope. (J) CD44 and CD24 level was measured by flow cytometry in MCF10A cells after cells were exposed to 100 nM OPB for 48hrs. Error bars shown as SD, P values in D were calculated by paired Student's t-test.

Fig. 5

Greater OPB sensitivity in oncogene-addicted cancer cells can be exploited for resensitization to targeted therapies(A) 2000 cells were plated in 6-wells plate for long term colony formation assay after H1975, HCC827, HCC827-GR, A375 and A375-VR cells were exposed to different concentrations of OPB for 1 h followed by 5 μM gefitinib (H1975), 0.5 μM gefitinib (HCC827 and HCC827-GR) and 0.4 μM vemurafinib (A375 and A375-VR) respectively for a total of 48hrs. Colonies were stained with crystal violet after 10 days of plating. (B) 2000 cells were plated in 96-wells Corning® spheroids plates after H1975, HCC827, HCC827-GR, A375 and A375-VR cells were exposed to different concentrations of OPB for 1 h followed by 5 μM gefitinib (H1975), 0.5 μM gefitinib (HCC827 and HCC827-GR) and 0.4 μM vemurafinib (A375 and A375-VR) respectively for a total of 48hrs. Spheroids were imaged using ZEISS microscope. (C) Cell viability was determined using MTT cell viability assay in H1975, HCC827, HCC827-GR, A375 and A375-VR cells after exposing to OPB for 1 h followed by 5 μM gefitinib (H1975), 0.5 μM gefitinib (HCC827 and HCC827-GR) and 0.4 μM vemurafinib (A375 and A375-VR) respectively for a total of 48hrs. (N = 3) Error bars shown as SD, P values in C were calculated by paired Student's t-test.

Fig. 6

Patients harboring heavily pre-treated or metastatic cancers respond positively to OPB-51602 treatment.(A) CT scan shows a right-sided pleural nodule (arrow, left panel) in a lady with EGFR TKI-resistant NSCLC. After 1 cycle of OPB-51602 treatment, the pleural nodule has shown significant reduction in size (right panel). (B) An ¹⁸F-fluorodeoxyglucose–positron-emission tomographic (FDG–PET) scan showing FDG-avid right paratracheal lymph node (SUVmax 10.9; arrow, left panel) and a right lower lobe lung nodule (SUVmax 13.4; arrowhead, left panel) in a 61 year old man with non-small cell lung cancer (NSCLC). After 3 weeks of OPB-51602 treatment, there was significant reduction in metabolic activity of both lesions [SUVmax of the right paratracheal lymph node was 6.8 (arrow); SUVmax of the right lower lobe lung nodule was 8.3 (arrowhead), right panel]. (C) An FDG–PET scan showing an FDG-avid peritoneal nodule (SUVmax 18.8; arrow, left panel) in a 42 year old man with C-KIT mutation positive imatinib-refractory gastrointestinal stromal tumor (GIST). After 8 weeks of OXPHOS inhibitor treatment, there was significant reduction in SUVmax of the lesion to 11.3 (arrow, right panel). (D) An FDG–PET scan showing multiple FDG-avid liver metastases (SUVmax 13.6, arrow, left panel) in a young lady with heavily pre-treated NPC. After 3 weeks of OPB-51602 treatment, there was significant reduction in metabolic activity of the liver lesions (SUVmax 7.2, arrow, right panel). (E) Schematic representation of increased OXPHOS dependence of oncogene-addicted TKI-resistant tumors. Left panel shows tumor cells depend on aerobic glycolysis. Middle panel shows prolonged exposure to TKI results in metabolic reprogramming and switch to OXPHOS dependent bioenergetics, which drives the development of therapy resistance. Right panel shows pretreatment with OXPHOS inhibitor OPB-51602 target this vulnerability and restores sensitivity to TKI.