Elizabeth A. Tovar

Cabozantinib (XL184) Inhibits Growth and Invasion of Preclinical TNBC Models

Purpose: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is associated with poor clinical outcome. There is a vital need for effective targeted therapeutics for TNBC patients, yet treatment strategies are challenged by the significant intertumoral heterogeneity within the TNBC subtype and its surrounding microenvironment. Receptor tyrosine kinases (RTK) are highly expressed in several TNBC subtypes and are promising therapeutic targets. In this study, we targeted the MET receptor, which is highly expressed across several TNBC subtypes. Experimental Design: Using the small-molecule inhibitor cabozantinib (XL184), we examined the efficacy of MET inhibition in preclinical models that recapitulate human TNBC and its microenvironment. To analyze the dynamic interactions between TNBC cells and fibroblasts over time, we utilized a 3D model referred to as MAME (Mammary Architecture and Microenvironment Engineering) with quantitative image analysis. To investigate cabozantinib inhibition in vivo, we used a novel xenograft model that expresses human HGF and supports paracrine MET signaling. Results: XL184 treatment of MAME cultures of MDA-MB-231 and HCC70 cells (± HGF-expressing fibroblasts) was cytotoxic and significantly reduced multicellular invasive outgrowths, even in cultures with HGF-expressing fibroblasts. Treatment with XL184 had no significant effects on METneg breast cancer cell growth. In vivo assays demonstrated that cabozantinib treatment significantly inhibited TNBC growth and metastasis. Conclusions: Using preclinical TNBC models that recapitulate the breast tumor microenvironment, we demonstrate that cabozantinib inhibition is an effective therapeutic strategy in several TNBC subtypes. Clin Cancer Res; 22(4); 923–34. ©2015 AACR.

Metabolic profiling of triple-negative breast cancer cells reveals metabolic vulnerabilities

BackgroundAmong breast cancers, the triple-negative breast cancer (TNBC) subtype has the worst prognosis with no approved targeted therapies and only standard chemotherapy as the backbone of systemic therapy. Unique metabolic changes in cancer progression provide innovative therapeutic opportunities. The receptor tyrosine kinases (RTKs) epidermal growth factor receptor (EGFR), and MET receptor are highly expressed in TNBC, making both promising therapeutic targets. RTK signaling profoundly alters cellular metabolism by increasing glucose consumption and subsequently diverting glucose carbon sources into metabolic pathways necessary to support the tumorigenesis. Therefore, detailed metabolic profiles of TNBC subtypes and their response to tyrosine kinase inhibitors may identify therapeutic sensitivities.MethodsWe quantified the metabolic profiles of TNBC cell lines representing multiple TNBC subtypes using gas chromatography mass spectrometry. In addition, we subjected MDA-MB-231, MDA-MB-468, Hs578T, and HCC70 cell lines to metabolic flux analysis of basal and maximal glycolytic and mitochondrial oxidative rates. Metabolic pool size and flux measurements were performed in the presence and absence of the MET inhibitor, INC280/capmatinib, and the EGFR inhibitor, erlotinib. Further, the sensitivities of these cells to modulators of core metabolic pathways were determined. In addition, we annotated a rate-limiting metabolic enzymes library and performed a siRNA screen in combination with MET or EGFR inhibitors to validate synergistic effects.ResultsTNBC cell line models displayed significant metabolic heterogeneity with respect to basal and maximal metabolic rates and responses to RTK and metabolic pathway inhibitors. Comprehensive systems biology analysis of metabolic perturbations, combined siRNA and tyrosine kinase inhibitor screens identified a core set of TCA cycle and fatty acid pathways whose perturbation sensitizes TNBC cells to small molecule targeting of receptor tyrosine kinases.ConclusionsSimilar to the genomic heterogeneity observed in TNBC, our results reveal metabolic heterogeneity among TNBC subtypes and demonstrate that understanding metabolic profiles and drug responses may prove valuable in targeting TNBC subtypes and identifying therapeutic susceptibilities in TNBC patients. Perturbation of metabolic pathways sensitizes TNBC to inhibition of receptor tyrosine kinases. Such metabolic vulnerabilities offer promise for effective therapeutic targeting for TNBC patients.

Targeting MET and EGFR crosstalk signaling in triple-negative breast cancers.

There is a vital need for improved therapeutic strategies that are effective in both primary and metastatic triple-negative breast cancer (TNBC). Current treatment options for TNBC patients are restricted to chemotherapy; however tyrosine kinases are promising druggable targets due to their high expression in multiple TNBC subtypes. Since coexpression of receptor tyrosine kinases (RTKs) can promote signaling crosstalk and cell survival in the presence of kinase inhibitors, it is likely that multiple RTKs will need to be inhibited to enhance therapeutic benefit and prevent resistance. The MET and EGFR receptors are actionable targets due to their high expression in TNBC; however crosstalk between MET and EGFR has been implicated in therapeutic resistance to single agent use of MET or EGFR inhibitors in several cancer types. Therefore it is likely that dual inhibition of MET and EGFR is required to prevent crosstalk signaling and acquired resistance. In this study, we evaluated the heterogeneity of MET and EGFR expression and activation in primary and metastatic TNBC tumorgrafts and determined the efficacy of MET (MGCD265 or crizotinib) and/or EGFR (erlotinib) inhibition against TNBC progression. Here we demonstrate that combined MET and EGFR inhibition with either MGCD265 and erlotinib treatment or crizotinib and erlotinib treatment were highly effective at abrogating tumor growth and significantly decreased the variability in treatment response compared to monotherapy. These results advance our understanding of the RTK signaling architecture in TNBC and demonstrate that combined MET and EGFR inhibition may be a promising therapeutic strategy for TNBC patients.

Glesatinib Exhibits Antitumor Activity in Lung Cancer Models and Patients Harboring MET Exon 14 Mutations and Overcomes Mutation-mediated Resistance to Type I MET Inhibitors in Nonclinical Models

Purpose: MET exon 14 deletion (METex14 del) mutations represent a novel class of non–small cell lung cancer (NSCLC) driver mutations. We evaluated glesatinib, a spectrum-selective MET inhibitor exhibiting a type II binding mode, in METex14 del–positive nonclinical models and NSCLC patients and assessed its ability to overcome resistance to type I MET inhibitors. Experimental Design: As most MET inhibitors in clinical development bind the active site with a type I binding mode, we investigated mechanisms of acquired resistance to each MET inhibitor class utilizing in vitro and in vivo models and in glesatinib clinical trials. Results: Glesatinib inhibited MET signaling, demonstrated marked regression of METex14 del-driven patient-derived xenografts, and demonstrated a durable RECIST partial response in a METex14 del mutation-positive patient enrolled on a glesatinib clinical trial. Prolonged treatment of nonclinical models with selected MET inhibitors resulted in differences in resistance kinetics and mutations within the MET activation loop (i.e., D1228N, Y1230C/H) that conferred resistance to type I MET inhibitors, but remained sensitive to glesatinib. In vivo models exhibiting METex14 del/A-loop double mutations and resistance to type I inhibitors exhibited a marked response to glesatinib. Finally, a METex14 del mutation-positive NSCLC patient who responded to crizotinib but later relapsed, demonstrated a mixed response to glesatinib including reduction in size of a MET Y1230H mutation-positive liver metastasis and concurrent loss of detection of this mutation in plasma DNA. Conclusions: Together, these data demonstrate that glesatinib exhibits a distinct mechanism of target inhibition and can overcome resistance to type I MET inhibitors. Clin Cancer Res; 23(21); 6661–72. ©2017 AACR.

Genomic Status of MET Potentiates Sensitivity to MET and MEK Inhibition in NF1-Related Malignant Peripheral Nerve Sheath Tumors

Malignant peripheral nerve sheath tumors (MPNST) are highly resistant sarcomas that occur in up to 13% of individuals with neurofibromatosis type I (NF1). Genomic analysis of longitudinally collected tumor samples in a case of MPNST disease progression revealed early hemizygous microdeletions in NF1 and TP53, with progressive amplifications of MET, HGF, and EGFR To examine the role of MET in MPNST progression, we developed mice with enhanced MET expression and Nf1 ablation (Nf1fl/ko;lox-stop-loxMETtg/+;Plp-creERTtg/+ ; referred to as NF1-MET). NF1-MET mice express a robust MPNST phenotype in the absence of additional mutations. A comparison of NF1-MET MPNSTs with MPNSTs derived from Nf1ko/+;p53R172H;Plp-creERTtg/+ (NF1-P53) and Nf1ko/+;Plp-creERTtg/+ (NF1) mice revealed unique Met, Ras, and PI3K signaling patterns. NF1-MET MPNSTs were uniformly sensitive to the highly selective MET inhibitor, capmatinib, whereas a heterogeneous response to MET inhibition was observed in NF1-P53 and NF1 MPNSTs. Combination therapy of capmatinib and the MEK inhibitor trametinib resulted in reduced response variability, enhanced suppression of tumor growth, and suppressed RAS/ERK and PI3K/AKT signaling. These results highlight the influence of concurrent genomic alterations on RAS effector signaling and therapy response to tyrosine kinase inhibitors. Moreover, these findings expand our current understanding of the role of MET signaling in MPNST progression and identify a potential therapeutic niche for NF1-related MPNSTs.Significance: Longitudinal genomic analysis reveals a positive selection for MET and HGF copy number gain early in malignant peripheral nerve sheath tumor progression. Cancer Res; 78(13); 3672-87. ©2018 AACR.

NF1 deficiency correlates with estrogen receptor signaling and diminished survival in breast cancer

AbstractThe key negative regulatory gene of the RAS pathway, NF1, is mutated or deleted in numerous cancer types and is associated with increased cancer risk and drug resistance. Even though women with neurofibromatosis (germline NF1 mutations) have a substantially increased breast cancer risk at a young age and NF1 is commonly mutated in sporadic breast cancers, we have a limited understanding of the role of NF1 in breast cancer. We utilized CRISPR–Cas9 gene editing to create Nf1 rat models to evaluate the effect of Nf1 deficiency on tumorigenesis. The resulting Nf1 indels induced highly penetrant, aggressive mammary adenocarcinomas that express estrogen receptor (ER) and progesterone receptor (PR). We identified distinct Nf1 mRNA and protein isoforms that were altered during tumorigenesis. To evaluate NF1 in human breast cancer, we analyzed genomic changes in a data set of 2000 clinically annotated breast cancers. We found NF1 shallow deletions in 25% of sporadic breast cancers, which correlated with poor clinical outcome. To identify biological networks impacted by NF1 deficiency, we constructed gene co-expression networks using weighted gene correlation network analysis (WGCNA) and identified a network connected to ESR1 (estrogen receptor). Moreover, NF1-deficient cancers correlated with established RAS activation signatures. Estrogen-dependence was verified by estrogen-ablation in Nf1 rats where rapid tumor regression was observed. Additionally, Nf1 deficiency correlated with increased estrogen receptor phosphorylation in mammary adenocarcinomas. These results demonstrate a significant role for NF1 in both NF1-related breast cancer and sporadic breast cancer, and highlight a potential functional link between neurofibromin and the estrogen receptor.Genetics: Mutant tumor suppressor linked to estrogen receptor signalingMutations in a tumor suppressor gene called NF1 may be an important prognostic indicator for women with breast cancer and a therapeutic target for tumors resistant to hormone therapy. A team led by Carrie Graveel and Matthew Steensma from the
Van Andel Research Institute in Grand Rapids, Michigan, USA, studied a large dataset of well-characterized breast cancer cases. They showed that 25% harbored mutations in NF1, a genetic alteration that correlated with diminished survival. Gene network analyses revealed links between NF1 deficiency, RAS oncogene activity, and signaling through the estrogen receptor, including with genes known to mediate resistance to hormone therapy. The researchers also describe a newly created rat model of NF1-mutant breast cancer that they say could help further interrogate the importance of these genetic connections.

Oncogenic RAS-MET signal interactions are modulated by P53 status in NF1-related MPNSTs

We previously reported that cooperative RAS-MET signaling drives disease progression in NF1-related MPNSTs, and that MET inhibition results in downstream inhibition of RAS/MAPK in the context of MET amplification. This study revealed that response to MET inhibition appeared to be modulated by P53 gene status. It is currently unclear how P53 function affects kinome signaling and response to kinase inhibition. Here we utilized genetically engineered mouse models with variable levels of Met and Hgf amplification and differential p53 status (NF1fl/KO;lox-stop-loxMETtg/+;Plp-creERTtg/+; NF1+/KO;p53R172H;Plp-creERTtg/+; and NF1+/KO;Plp-creERTtg/+t). These NF1-MPNST models were used to assess a novel MET/MEK (i.e. RAS-MET) inhibition strategy and investigate the adaptive kinome response to MET and MEK inhibition. We demonstrate that combination MET (capmatinib) and MEK (trametinib) inhibition fully suppresses MET, RAS/MAPK, and PI3K/AKT activation in P53 wild type tumors, whereas P53-mutant tumors demonstrated sustained CRAF, BRAF, and AKT activation in the presence of combined MET and MEK inhibition. Interestingly, trametinib therapy alone strongly activates MET signaling in MET and HGF-amplified tumors regardless of P53 status, an effect that was abrogated by the addition of capmatinib. We conclude that P53 alters RAS-MET signaling interactions that drive therapy resistance in NF1-related MPNSTs.

Genomic MET amplification occurs early in NF1-related malignant peripheral nerve sheath tumor (MPNST) progression and is a potent therapeutic target

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are highly resistant sarcomas that occur in up to 13% of individuals with Neurofibromatosis Type 1 (NF1). Genomic analysis of longitudinally collected tumor samples in a case of MPNST disease progression revealed early hemizygous microdeletions in NF1 and TP53, with concomitant amplifications of MET, HGF, and EGFR. To examine the role of MET in MPNST progression, we developed mice with enhanced MET expression and NF1 ablation (NF1fl/KO;lox-stop-loxMETtg/+;Plp-creERTtg/+; referred to as NF1-MET). NF1-MET mice express a robust MPNST phenotype in the absence of additional mutations. A comparison of NF1-MET MPSNTs with MPNSTs derived from NF1KO/+;p53R172H;Plp-creERTtg/+ (NF1-P53) and NF1KO/+;Plp-creERTtg/+ (NF1) mice revealed unique Met, Ras, and PI3K signaling patterns. To investigate the therapeutic potential of MET inhibition among tumorgrafts derived from the respective MPNST models, we tested the highly selective MET inhibitor, capmatinib. NF1-MET MPNSTs were uniformly sensitive to MET inhibition whereas only a small subset of NF1-P53 and NF1 MPNSTs were inhibited. These results confirm that MET activation is sufficient for Schwann cell dedifferentiation into MPNSTs in the context of NF1 deficiency. RAS-MET signal interactions may be an important driver of MPSNT disease progression.

In vivo Efficacy Studies in Cell Line and Patient-derived Xenograft Mouse Models

[Abstract] In vivo xenograft models derived from human cancer cells have been a gold standard for evaluating the genetic drivers of cancer and are valuable preclinical models for evaluating the efficacy of cancer therapeutics. Recently, patient-derived tumorgrafts from multiple tumor types have been developed and shown to more accurately recapitulate the molecular and histological heterogeneity of cancer. Here we detail the procedures for developing patient-derived xenograft models from breast cancer tissue, cell-based xenograft models, serial tumor transplantation, tumor measurement, and drug treatment.

Abstract B19: MET and IL6 signaling in triple-negative breast cancer

Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancers and is associated with advanced stage at diagnosis and poorer outcome compared to other breast cancer subtypes. There is an unmet need for targeted therapeutic strategies for TNBC patients since current treatment options are restricted to standard chemotherapy. Both receptor tyrosine kinase (RTK) and inflammatory signaling have been shown to promote cancer progression and are promising therapeutic targets. Our laboratory was the first to demonstrate that the MET receptor tyrosine kinase is highly expressed in TNBC. Hepatocyte growth factor (HGF), the MET ligand, is highly expressed in breast carcinoma and breast carcinoma-associated fibroblasts (CAFs) and is able to induce paracrine or autocrine MET signaling. MET/HGF signaling is also connected with the pro-inflammatory cytokine interleukin 6 (IL6). HGF and IL6 have been shown to interact to enhance invasion of lung cancer cells and progression of multiple myeloma. In breast cancer patients, high serum expression of HGF and IL6 distinguishes metastatic breast cancers. Nonetheless, there is a gap in knowledge as to whether MET and IL6 signaling pathways directly or indirectly interact and how MET/IL6 activation promotes TNBC progression. We are examining the novel concept that MET and IL6 signaling pathways act through a positive signaling feedback loop to drive TNBC progression. By understanding the interactions between these signaling networks, we will be able to design therapeutic strategies that target critical signaling nodes in TNBC. Analysis of gene expression profiles in the four molecular TNBC subtypes defined by Burstein et al. revealed that MET, HGF, and IL6 are expressed in each of the TNBC subtypes. Immunohistochemical analysis of HGF and IL6 expression in breast cancer tissues revealed significantly higher HGF and IL6 expression in TNBC compared to ER+ breast cancers. To determine the effect of MET and IL6 inhibition, we established Mammary Architecture and Microenvironment Engineering (MAME) 3D co-culture models of TNBC cells ± fibroblasts. In these models, TNBC cells have high MET expression, moderate to high IL6 expression, and minimal IL6 receptor (IL6R) expression; whereas the CAF cells have high HGF expression and moderate IL6R expression. Our preliminary studies revealed that an IL6 neutralizing antibody (siltuximab) reduced TNBC structure volumes relative to IL6 expression in the TNBC cells, whereas an IL6 receptor (IL6R) neutralizing antibody (tocilizumab) had no effect. These results correlate with IL6 and IL6R expression levels in TNBC cell lines. We evaluated the efficacy of MET inhibition using XL184 (cabozantinib) and observed that XL184 significantly inhibited TNBC growth, proliferation, and invasion of diverse TNBC cell lines, yet was ineffective against MET-negative breast cancer cells. We are currently evaluating the effect of HGF-mediated MET activation on IL6 signaling and inhibition in our 3D TNBC models. To evaluate the effect of MET and/or IL6 inhibition in vivo we utilized a novel xenograft mouse model that expresses human HGF (hHGFtg SCID). In TNBC cell lines MDA-MB-231 and HCC70, IL6R inhibition slowed tumor progression marginally, whereas MET inhibition with XL184 and the combination of XL184 + anti-IL6R drastically inhibited tumor growth. In a model of established tumor growth, we started treatment when tumor reached 500 mm3. Again we observed a significant decrease in tumor growth with XL184 treatment (p Citation Format: Elizabeth A. Tovar, Mansoureh Sameni, Curt J. Essenburg, Anita Chalasani, Erik S. Linklater, David M. Cherba, Aruselvi Anbalagan, Mary E. Winn, Bonnie F. Sloane, Carrie R. Graveel. MET and IL6 signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B19.