Oxidative Phosphorylation Is a Metabolic Vulnerability in Chemotherapy-Resistant Triple-Negative Breast Cancer

Abstract

Oxidative phosphorylation (OXPHOS) is an active metabolic pathway in many cancers. RNA from pretreatment biopsies from patients with triple-negative breast cancer (TNBC) who received neoadjuvant chemotherapy demonstrated that the top canonical pathway associated with worse outcome was higher expression of OXPHOS signature. IACS-10759, a novel inhibitor of OXPHOS, stabilized growth in multiple TNBC patient-derived xenografts (PDX). On gene expression profiling, all of the sensitive models displayed a basal-like 1 TNBC subtype. Expression of mitochondrial genes was significantly higher in sensitive PDXs. An in vivo functional genomics screen to identify synthetic lethal targets in tumors treated with IACS-10759 found several potential targets, including CDK4. We validated the antitumor efficacy of the combination of palbociclib, a CDK4/6 inhibitor, and IACS-10759 in vitro and in vivo. In addition, the combination of IACS-10759 and multikinase inhibitor cabozantinib had improved antitumor efficacy. Taken together, our data suggest that OXPHOS is a metabolic vulnerability in TNBC that may be leveraged with novel therapeutics in combination regimens. SIGNIFICANCE: These findings suggest that triple-negative breast cancer is highly reliant on OXPHOS and that inhibiting OXPHOS may be a novel approach to enhance efficacy of several targeted therapies.

Figure 1.

We performed RNA-seq on pretreatment biopsies from 43 patients with operable TNBC who received sequential taxane- and anthracycline-based neoadjuvant chemotherapy. At greater than 5-year median follow-up, 14 patients recurred and of those all but two patients had died. At a FDR of 0.05, 33 genes were differentially expressed between patients who did and did not have a subsequent recurrence. IPA demonstrated that one of the top canonical pathways that differed was higher expression of oxidative phosphorylation signature (P < 0.001).

Figure 2.

Inhibiting OXPHOS results in broad tumor in a range of TNBC PDXs developed from residual disease. Ten TNBC PDXs were treated with IACS-10759 (5 mg/kg, orally, 5 days on 2 days off). Cohorts of 2–4 mice were used for this initial screening. IACS-10759 stabilized disease (<20% median change from baseline) for at least 21 days in four PDXs and one PDX regressed to be immeasurable disease. Only one of five BL1 PDXs grew more than >20% in 21 days. Means ± SEM are shown.

Figure 3.

Predictor of response to IACS-10759 in TNBC. A, Using baseline RNA-seq analysis for PDX with known IACS-10759 sensitivity, we found that protein-coding mitochondrial genes are expressed significantly higher in PDXs more sensitive to IACS-10759. B, AXL protein as determined by RPPA is higher in PDXs less sensitive to IACS-10759. C and D, A highly sensitive PDX was treated for >90 days (individual tumors shown) and sporadic tumors began growing, and a reformed tumor had increased AXL1 mRNA expression compared with the control. E–G, We treated a select set of PDXs with ranges of responses to IACS-10759 (5 mg/kg, orally, 5 days on 2 days off) and collected the tumors after 12 days of treatment. We analyzed the samples for CC3 (E), PCNA (F), and phosphohistone H3 (G). *, P < 0.05; **, P < 0.01.

Figure 4.

Targeting AXL1-expressing TNBC with combination of cabozantinib and IACS-10759. A and B, Two AXL1 high TNBC PDXs (A, BCX.010; B, BCX.084) were treated with cabozantinib (20 mg/kg, orally, daily) and IACS-10759 (5 mg/kg, orally, 5 days on 2 days off), which prolonged tumor stability compared with either single agent alone. C, A low AXL1-expressing PDX that is relatively more sensitive to IACS-10759 was treated with cabozantinib (20 or 5 mg/kg, orally, daily) and IACS-10759 (5 mg/kg, orally, 5 days on 2 days off) and both combinations resulted in tumor regression from baseline. Data shown mean ±SEM. D, In the PI3KCA-mutant PDX (BCX.010), the combination of cabozantinib and IACS-10759 significantly inhibited PI3K/mTOR pathway to a greater extent than either single agent alone as evidenced by decreased phosphorylation of ribosomal protein S6 on RPPA. *, P < 0.05; **, P < 0.01.

Figure 5.

Identification of combination partners for IACS-10759 using in vivo synthetic lethality screen. A, Illustration of method used to identify genes (from gene panel linked to FDA-approved agents) whose suppression led to increased cell loss in presence of IACS-10759 in mice. B, List of genes identified in screen. We chose to validate CDK4, PARP1, and HDAC3 using palbociclib, talazoparib, and entinostat, respectively. C and D, IACS-10759 was synergistic (CI < 1) with palbociclib, talazaparib, and entinostat in BCX.010-CL and MDA-MB-468 cells as assessed by cell growth assays. Ave CI, average combination index.

Figure 6.

Validation of functional shRNA screen to identify potential IACS-10759 combination partners. A, We tested palbociclib (50 mg/kg daily) in BCX.010 PDX in vivo. The combination of IACS-10759 and palbociclib showed clear improvement over both single agents similar to in vitro. B, We further validated palbociclib + IACS-10759 in an additional retinoblastoma gene positive less sensitive TNBC PDX (BCX.080). Data are shown as mean ± SEM. **, P < 0.001. C, We also tested palbociclib + IACS-10759 using a PDX (T141–003) generated from a patient with breast cancer who had received palbociclib and progressed. Data are shown as mean ± SEM. *, P < 0.01. D, We tested talazaparib (0.3 mg/kg daily) + IACS-10759 combination in BCX.010 PDX in vivo but found with a more limited combination efficacy. E and F, We next tested talazoparib in combination with IACS-10759 in PDXs with acquired resistance to talazoparib (BCX.022 talazoparib resistant 1 and BCX.024 talazoparib resistant 1). These models were created by treating talazoparib–sensitive PDXs until tumors were not palpable and then collecting and serial passaging the grown tumors. The combination did not have improved efficacy in these models, but IACS-10759 did have significantly greater inhibition versus talazoparib (BCX.022 talazoparib resistant, P < 0.001; BCX.024 talazoparib resistant, P < 0.01).