Triple Negative Breast Cancer Patient-derived Xenograft Models Research Articles

Abstract 3075: Nuclear export inhibitor selinexor (KPT-330) demonstrates anti-tumor efficacy alone and in combination with chemotherapy in multiple breast cancer models

Abstract Background: The nuclear exporter XPO1 (Exportin1 or CRM1), mediates the transport of over 200 proteins, including several tumor suppressors. For this reason, XPO1 is being pursued as a promising target for cancer therapy options. Selinexor (KPT-330), a selective inhibitor of nuclear export, is an oral agent that has been shown to inhibit XPO1 and is currently in phase 2 trials for hematologic and solid tumors. We sought to determine the antitumor effect of selinexor in breast cancer cells in vitro and in vivo Methods: We studied the effects of selinexor in vitro using cell proliferation assays; the half maximal inhibitory concentration (IC50) was calculated using isobologram curves after 3 days of treatment. We also tested the effects in combination with chemotherapy and calculated the combination index by the method of Chou and Talalay. In vivo efficacy was tested in triple negative breast cancer (TNBC) patient derived xenografts (PDXs) with varying levels of paclitaxel sensitivity, as single agent and in combination therapy. T/C ratio was calculated using the formula: [(median tumor volume of treated group)/(median tumor volume of control group) x 100] Results: Selinexor treatment inhibited growth of 12 breast cancer cell lines representing different subtypes (TNBC as well as ER+, HER+) with a median IC50 of 375nM (range 3-750nM). Selinexor significantly reduced tumor growth in vivo in 4 of 5 different TNBC PDX models (with varying status of TP53 and PIK3CA, and gene expression subtypes), with a median T/C score of 48% (range 34-59%) In Multiple TNBC cell lines, selinexor was synergistic with paclitaxel, carboplatin, eribulin and doxorubicin in vitro. In vivo, selinexor in combination with paclitaxel or eribulin had greater antitumor efficacy than either agent alone. Conclusions: Collectively these findings strongly suggest that selinexor is a promising therapeutic option for breast cancer. Citation Format: Natalia Paez Arango, Kurt Evans, Ming Zhao, Erkan Yuca, Stephen Scott, Charissa Kim, Aung Naing, Funda Meric-Bernstam. Nuclear export inhibitor selinexor (KPT-330) demonstrates anti-tumor efficacy alone and in combination with chemotherapy in multiple breast cancer models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3075.

Abstract B14: Targeting the intratumoral heterogeneity of receptor tyrosine kinases in breast cancer

Abstract Breast cancers display a remarkable phenotypic diversity that is exploited to promote both tumor progression and therapeutic resistance. Recent studies in several types of cancer have highlighted the significance of intratumoral heterogeneity on both innate and acquired resistance to tyrosine kinase inhibitors (TKIs). Tumor plasticity is supported by the heterogeneous expression of receptor tyrosine kinases (RTKs) and the robustness that the overlapping signaling networks provide. Therefore a thorough understanding of the intratumoral heterogeneity is necessary for the development of effective therapeutic strategies. The receptor tyrosine kinase MET is overexpressed in 20-30% of breast cancers and correlates with poor patient outcome. Previously, we determined that high MET expression correlated with ER-/ERBB2- and basal like breast cancers. These results and the efficacy of MET inhibitors in other cancers suggest that MET may be an effective clinical target for aggressive breast cancer subtypes. Recent studies have exposed interactions between MET and the ERBB receptor family in the progression and therapeutic resistance of several cancers. Since MET, ERBB2, and EGFR are known to be highly expressed in aggressive breast cancer subtypes, it is critical that we understand the relationships between these receptors in order to develop effective treatment strategies. We are investigating the relationship between MET and ERBB receptor signaling in the progression and resistance of ERBB2+ and triple-negative breast cancer (TNBC). We observe that there is a large subset of ERBB2+ breast cancers that express MET and contain MET+/ERBB2+ subpopulations. In a MET+/ERBB2+ breast cancer cell line, MET depletion results in increased ERBB2 activation whereas, ERBB2 depletion results in increased MET activation. Therefore, ERBB2+ breast cancers with MET+ subpopulations may have an innate resistance to ERBB2 inhibition and may benefit from combined MET and ERBB2 inhibition. In TNBC, we observe heterogeneous expression of MET and EGFR. We have developed patient-derived xenografts (PDX) from primary and metastatic TNBCs that have diverse patterns of MET and EGFR expression/activation. In these TNBC PDX models we observe varied responses to monotherapy with MET inhibitors MGCD265 and crizotinib and the EGFR inhibitor erlotinib. Interestingly, therapeutic response to MET inhibition does not correlate with protein expression levels. In all studies, we observe significantly increased efficacy of combination therapy with MET and EGFR inhibition and a decrease in response variability. Examination of phospho-MET localization in treated tumorgrafts revealed that MET and EGFR inhibition induces distinct phospho-MET localization changes. We also observe that in the residual resistant cells phospho-MET is highly expressed in cells with mitotic bodies. We are currently performing phospho-proteomic analysis to determine the effect of MET and/or EGFR inhibition on RTK signaling networks including ERK and AKT pathways. These results will identify proteomic signatures that represent MET and/or EGFR activation/inhibition and sensitivity or resistance to monotherapy or combined MET and EGFR inhibition. Overall these studies give us a more comprehensive view of TNBC network signaling, the level of RTK heterogeneity within TNBC, and the efficacy of MET and/or EGFR inhibition on TNBC progression. Citation Format: Elizabeth A. Tovar, Erik S. Linklater, Curt J. Essenburg, Zach Madaj, David M. Cherba, Mary E. Winn, Hasan Korkaya, Julie L. Boerner, Carrie R. Graveel. Targeting the intratumoral heterogeneity of receptor tyrosine kinases in 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 B14.

Abstract 3226: Patient-derived xenograft (PDX) models for triple negative breast cancer (TNBC): A pre-clinical platform for drug discovery

Abstract In the United States, TNBC comprises 15-20% of breast cancers and are characterized by the lack of expression of estrogen receptor, progesterone receptor and receptor tyrosine protein kinase ERBB2 or Her-2/neu oncogene amplification. As a result, this type of breast cancer is difficult to treat as most of the chemotherapies target these 3 receptors. TNBC is highly aggressive and is associated with high morbidity, mortality and shorter disease-free survival. Given its heterogeneous clinical presentation, histology and response to therapy, prognosis and management of TNBC is complicated. In this study, we report development and characterization of the TNBC PDX model. We used patient derived TNBC tissues to generate 5 new TNBC models in NOD-SCID mice. Patient tumors were pre-screened for their ER, PR and Her-2/Neu expression by IHC, prior to inoculation in mice. Efficacy of two standard of care drugs, Cisplatin and Vinorelbine in the TNBC PDX model demonstrates the potential utility of the TNBC model in drug discovery effort in oncology for treatment of TNBC. Citation Format: Jayant Thatte, Miguel Meza, Jill Ricono, Cyrus Mirsaidi, Thomas Broudy. Patient-derived xenograft (PDX) models for triple negative breast cancer (TNBC): A pre-clinical platform for drug discovery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3226. doi:10.1158/1538-7445.AM2015-3226

Abstract 4499: Activation of PI3-kinase pathway and tumor response to everolimus in patient-derived xenografts of triple-negative breast cancer

Abstract Purpose: Patients with triple-negative breast cancer (TNBC) have a poor prognosis and targeted therapies are lacking. Recent studies performed on patients tumors showed and increased activity of the phosphatidylinositol 3-kinase (PI3K) pathway in TNBC. PI3K pathway is critical for cell growth, survival, and angiogenesis. Everolimus is a mTOR inhibitor recently showed to increase survival of patients with metastatic luminal breast cancer. The objectives of this work were to analyze the PI3K activation status in a large cohort of patient-derived xenografts (PDX) of TNBC and to investigate the therapeutic potential of mTOR inhibition. Experimental procedures: this study included a panel of 32 TNBC PDX models previously described (Marangoni et al 2007). Expression of AKT, P-AKT, P-mTOR, S6, P-S6, P-4EBP1, PTEN and INPP4B was analyzed by WB and IHC. Mutations of PIK3CA (exons 9 and 20), PIK3R1 (exons 11-15), and AKT1 (exon 4) were detected by sequencing of cDNA fragments obtained by RT-PCR amplification. The efficacy of the mTOR inhibitor everolimus was investigated in vivo on 10 PDX models with different expressions and mutational status of PI3K markers. Results: INPP4B protein expression was lost in 56% of tumors (n=18) and expressed at low levels in 31% of models, while only 2 models displayed a marked expression. PTEN expression was lost in 78% of tumors. Thirteen PDX models (40%) displayed a concomitant loss of both INPP4B and PTEN proteins. In 67% of tumors, the ratio between phosphorylated and unphosphorylated AKT was greater than 1. S6 was found to be phosphorylated in the great majority of tumors. PI3KCA and AKT1 genes were mutated only in 1 and 2 tumors, respectively. On the 10 PDX models treated with everolimus, 6 models responded to treatment with a tumor growth inhibition (TGI) comprised between 60% and 80%. Four models were classified as resistant or low responder (TGI<50%). Preliminary analysis of treated tumors from 6 models indicates increased level of P-AKT (feedback loop) to occur only in responder models, while inhibition of S6 phosphorylation occurred in treated tumors from both responder and resistant models. Finally, expression of INPP4B or PTEN alone did not predict for tumor response, while a P-AKT/AKT ratio greater than 1 predicted response to everolimus (p<0.05, Fisher's exact test). Conclusions: the majority of TNBC PDX models showed loss of PTEN or INPP4B proteins or both, associated with activation of PI3K pathway. Preliminary results obtained from 10 PDX models indicate that mTOR targeting resulted in tumor growth inhibition in several models with AKT phosphorylation. Additional TNBC models will be tested in order to search for robust predictive biomarkers. This large panel of characterized PDX of TNBC models represents a clinical relevant tool to investigate the activity of PI3K-AKT-mTOR inhibitors and identify predictive biomarkers. Citation Format: Elisabetta Marangoni, Rana Hatem, Rania El Botty, Ludmilla De Plater, Dalila Labiod, Sophie Vacher, Sophie Chateau-Joubert, Ivan Bièche. Activation of PI3-kinase pathway and tumor response to everolimus in patient-derived xenografts of triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2014-4499

Abstract 204: Understanding breast cancer resistance to chemotherapy: Characterization of cancer cell sub-populations in residual and relapsed tumors

Abstract Tumor recurrence fueled by residual tumor cells having survived chemotherapy represents the principal cause of breast cancer treatment failure. Triple-negative breast cancer (TNBC) is a heterogeneous disease at both molecular and cellular level, and the presence of different tumor cell sub-populations is likely the reason for this heterogeneity and for the incomplete response to neoadjuvant chemotherapy observed for most TNBC. To identify and isolate tumor cell sub-populations that resist to chemotherapy, we used a panel of 45 antibody fluorochrome conjugates in combination with multi-parameter flow cytometry to screen for the expression of a set of cell surface markers during the course of tumor chemotherapy. This set of markers represented both proteins involved in stem cell function and proteins known to be over-expressed in stem cells or cancer stem cell sub-populations. As a source of tumor samples, we used a panel of TNBC patient-derived xenografts (PDXs). These tumor models are known to preserve the morphology, molecular characteristics and drug response profile of the original patient tumors. We used TNBC PDX models to reproduce in vivo chemotherapy-induced tumor regression and relapse. Tumor dissociation, depletion of mouse cells and multi-parameter flow cytometry allowed us to measure the percentage of marker expression only in the fraction of human tumor cells. We found that the expression of most of markers was very heterogeneous among the different TNBC models analyzed. In residual tumors, we observed enrichment or depletion of several cell populations expressing stem and cancer stem cell markers such as CD44+ and CD133/1+. Interestingly, in the majority of relapsed tumors, the percentage of these marker-positive cells shifted back to pre-treatment levels. Moreover, the percentage of certain tumor cell sub-populations was higher in untreated chemotherapy-resistant tumors compared to untreated chemotherapy-sensitive ones. To study the properties of enriched tumor cells sub-populations, we coupled cell separation of marker-positive tumor cells to large scale molecular analysis. Microarray-based gene expression analysis of sorted tumor cells revealed cell sub-populations with up-regulated signaling pathways related to stem cell function, cell migration or EMT. Further in vivo and in vitro assays with sorted sub-populations are currently being performed to search for possible functional role of selected markers during tumor resistance to chemotherapy and initiation of tumor relapse. In summary, by coupling PDX and cell surface marker screening technologies, we have identified distinct tumor cell sub-populations which are associated with tumor resistance to chemotherapy. We believe that this approach will help gaining knowledge of tumor cell adaptation and selection in response to chemotherapy, which will ultimately lead to design tailored treatments for TNBCs. Citation Format: Andrea Aloia, Evgeniya Petrova, Olivier Deas, Sophie Banis, Enora Le Ven, Andreas Bosio, Olaf Hardt, Stefano Cairo, Jean-Gabriel Judde. Understanding breast cancer resistance to chemotherapy: Characterization of cancer cell sub-populations in residual and relapsed tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 204. doi:10.1158/1538-7445.AM2014-204