Stephen M. Scott

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.

Abstract Purpose: Neratinib is an irreversible, pan-HER tyrosine kinase inhibitor that is FDA approved for HER2-overexpressing/amplified (HER2+) breast cancer. In this preclinical study, we explored the efficacy of neratinib in combination with inhibitors of downstream signaling in HER2+ cancers in vitro and in vivo. Experimental Design: Cell viability, colony formation assays, and Western blotting were used to determine the effect of neratinib in vitro. In vivo efficacy was assessed with patient-derived xenografts (PDX): two breast, two colorectal, and one esophageal cancer (with HER2 mutations). Four PDXs were derived from patients who received previous HER2-targeted therapy. Proteomics were assessed through reverse phase protein arrays and network-level adaptive responses were assessed through Target Score algorithm. Results: In HER2+ breast cancer cells, neratinib was synergistic with multiple agents, including mTOR inhibitors everolimus and sapanisertib, MEK inhibitor trametinib, CDK4/6 inhibitor palbociclib, and PI3Kα inhibitor alpelisib. We tested efficacy of neratinib with everolimus, trametinib, or palbociclib in five HER2+ PDXs. Neratinib combined with everolimus or trametinib led to a 100% increase in median event-free survival (EFS; tumor doubling time) in 25% (1/4) and 60% (3/5) of models, respectively, while neratinib with palbociclib increased EFS in all five models. Network analysis of adaptive responses demonstrated upregulation of EGFR and HER2 signaling in response to CDK4/6, mTOR, and MEK inhibition, possibly providing an explanation for the observed synergies with neratinib. Conclusions: Taken together, our results provide strong preclinical evidence for combining neratinib with CDK4/6, mTOR, and MEK inhibitors for the treatment of HER2+ cancer.

PURPOSE: Datopotamab deruxtecan (Dato-DXd) is a humanized anti-trophoblast cell-surface antigen-2 (TROP2) IgG1 mAb linked to a potent topoisomerase I inhibitor payload (DXd). Dato-DXd has already shown antitumor activity in breast cancer; however, the determinants of response, including the importance of TROP2 expression, remain unclear. We tested the activity of Dato-DXd in a panel of breast cancer patient-derived xenografts (BCX) varying in TROP2 expression. EXPERIMENTAL DESIGN: The antitumor activity of Dato-DXd and isotype-control-DXd (IgG-DXd) was assessed against 11 BCXs varying in TROP2 expression, 10 representing tumors postneoadjuvant chemotherapy. Pharmacodynamic effects were assessed at 24 and 72 hours. The effects of TROP2 expression on Dato-DXd activity was assessed in vitro and in vivo using viral overexpression in BCX-derived cell lines. RESULTS: Models differed in their sensitivity to both Dato-DXd and IgG-DXd. Dato-DXd (10 mg/kg) led to objective response in 4 (36%) models and statistically significant prolongation of event-free survival in 8 (73%) models, whereas IgG-DXd (10 mg/kg) led to response in 1 (9%) and prolonged event-free survival in 3 (27%) models. TROP2 RNA and protein were significantly higher in Dato-DXd-sensitive models. In isogenic cell lines derived from Dato-DXd-resistant BCXs, overexpression of TROP2 conferred Dato-DXd antitumor activity in vitro and in vivo. Dato-DXd increased γH2AX and phospho-KAP1 in the two Dato-DXd-sensitive BCXs but not in a Dato-DXd-resistant BCX. In Dato-DXd-sensitive models, antitumor activity was enhanced in combination with a PARP inhibitor, olaparib. CONCLUSIONS: Dato-DXd is active in breast cancer models. Dato-DXd has TROP2-dependent and -independent mediators of activity; however, high TROP2 expression enhances Dato-DXd antitumor activity.

Abstract Activation by amplification or overexpression of the proto-oncogene HER2 (also known as ERBB2) is associated with the development and progression of breast cancer. Neratinib is a novel, irreversible, pan-HER tyrosine kinase inhibitor which selectively inhibits EGFR, HER2 and HER4. In this preclinical study, we explored the efficacy of neratinib in combination with other clinically relevant targeted agents for optimal treatment of HER2-positive breast cancer in in vitro and in vivo models. Western blot (WB) analysis of a panel of breast cancer cell lines showed that BT-474, SKBR-3, and HCC-1954 cells displayed elevated HER2 expression levels (here denoted as HER2+), while MDA-MB-361, MDA-MB-453, and CAMA-1 breast cancer cells expressed relatively lower HER2 expression levels. Interestingly, neratinib inhibition positively correlated with HER2 expression levels as assessed by cell survival assays. The IC50 of neratinib in the HER2+ cell lines was in the 3.6 - 113nM range. Consistent with the in vitro data, treatment of a HER2+ breast cancer PDX model with neratinib single agent (p.o. 5 days/week) led to a significant dose-dependent tumor growth inhibition with tumor regressions observed at a low dose of 10 mg/kg (p<0.0001). The activity of neratinib in combination with other targeted agents was then evaluated in vitro in the HER2+ breast tumor cell lines using a Chou-Talalay method. Neratinib showed synergistic anti-proliferative activity in combination with the PI3Kα inhibitor BYL719 in all the HER2+ cell lines tested. By contrast, synergy with mTOR inhibitors (everolimus and TAK228) and with the MEK inhibitor trametinib was only observed in BT-474 and HCC-1954 cells. HCC-1954 cells had the best response to neratinib combinations among all HER2+ cell lines tested, with combination index values <0.5. Immunoblot analysis demonstrated that neratinib treatment displayed complete inhibition of HER2 phosphorylation in these cells. Substantial inhibition of downstream signaling pathways following single agent neratinib treatment was evident by reduced phosphorylation levels of AKT, S6K1, S6, 4EBP1, ERK1/2 and MEK1/2. Consistently with the cell viability data, the combination of neratinib with PI3K/Akt/mTOR and MAPK inhibitors showed a synergy on signaling blockage in these two downstream pathways respectively. Taken together this data shows that neratinib has efficacy as single agent in preclinical models of HER2+ breast tumors and its anti-proliferative activity in vitro was enhanced when combined with PI3K/Akt/mTOR and MAPK pathway inhibitors. Further studies of neratinib-based combination treatments are underway in HER2+ breast cancer xenograft and PDX models to further corroborate these findings. Citation Format: Ming Zhao, Stephen Scott, Kurt Evans, Erkan Yuca, Rashmi Murthy, Francesca Avogadri-Connors, Richard Cutler, Alshad S. Lalani, Sarina Piha-Paul, Funda Meric-Bernstam. Exploring optimal targeted combination therapies with neratinib for HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4038. doi:10.1158/1538-7445.AM2017-4038

<div>AbstractPurpose:<p>Datopotamab deruxtecan (Dato-DXd) is a humanized anti–trophoblast cell-surface antigen-2 (TROP2) IgG1 mAb linked to a potent topoisomerase I inhibitor payload (DXd). Dato-DXd has already shown antitumor activity in breast cancer; however, the determinants of response, including the importance of TROP2 expression, remain unclear. We tested the activity of Dato-DXd in a panel of breast cancer patient-derived xenografts (BCX) varying in TROP2 expression.</p>Experimental Design:<p>The antitumor activity of Dato-DXd and isotype-control-DXd (IgG-DXd) was assessed against 11 BCXs varying in TROP2 expression, 10 representing tumors postneoadjuvant chemotherapy. Pharmacodynamic effects were assessed at 24 and 72 hours. The effects of TROP2 expression on Dato-DXd activity was assessed <i>in vitro</i> and <i>in vivo</i> using viral overexpression in BCX-derived cell lines.</p>Results:<p>Models differed in their sensitivity to both Dato-DXd and IgG-DXd. Dato-DXd (10 mg/kg) led to objective response in 4 (36%) models and statistically significant prolongation of event-free survival in 8 (73%) models, whereas IgG-DXd (10 mg/kg) led to response in 1 (9%) and prolonged event-free survival in 3 (27%) models. TROP2 RNA and protein were significantly higher in Dato-DXd–sensitive models. In isogenic cell lines derived from Dato-DXd–resistant BCXs, overexpression of TROP2 conferred Dato-DXd antitumor activity <i>in vitro</i> and <i>in vivo</i>. Dato-DXd increased γH2AX and phospho-KAP1 in the two Dato-DXd–sensitive BCXs but not in a Dato-DXd–resistant BCX. In Dato-DXd–sensitive models, antitumor activity was enhanced in combination with a PARP inhibitor, olaparib.</p>Conclusions:<p>Dato-DXd is active in breast cancer models. Dato-DXd has TROP2-dependent and -independent mediators of activity; however, high TROP2 expression enhances Dato-DXd antitumor activity.</p></div>