Negative Breast Cancer Patient-derived Xenografts Research Articles

PROM2 overexpression induces metastatic potential through epithelial-to-mesenchymal transition and ferroptosis resistance in human cancers.

Despite considerable therapeutic advances in the last 20 years, metastatic cancers remain a major cause of death. We previously identified prominin-2 (PROM2) as a biomarker predictive of distant metastases and decreased survival, thus providing a promising bio-target. In this translational study, we set out to decipher the biological roles of PROM2 during the metastatic process and resistance to cell death, in particular for metastatic melanoma. Methods and results: We demonstrated that PROM2 overexpression was closely linked to an increased metastatic potential through the increase of epithelial-to-mesenchymal transition (EMT) marker expression and ferroptosis resistance. This was also found in renal cell carcinoma and triple negative breast cancer patient-derived xenograft models. Using an oligonucleotide anti-sense anti-PROM2, we efficaciously decreased PROM2 expression and prevented metastases in melanoma xenografts. We also demonstrated that PROM2 was implicated in an aggravation loop, contributing to increase the metastatic burden both in murine metastatic models and in patients with metastatic melanoma. The metastatic burden is closely linked to PROM2 expression through the expression of EMT markers and ferroptosis cell death resistance in a deterioration loop. Our results open the way for further studies using PROM2 as a bio-target in resort situations in human metastatic melanoma and also in other cancer types.

Abstract B067: Identification of Gemcitabine-resistant Populations using scRNA-Sequencing in Triple Negative Breast Cancer Patient-Derived Xenografts

Abstract Despite major advances in the treatment of breast cancer (BC), it remains the most diagnosed and second deadliest cancer among American women. BC consists of distinct clinical subtypes, including estrogen receptor or HER2 receptor-positive and triple-negative breast cancer (TNBC) that lacks these receptors preventing the use of established precision therapies and the standard of care remains neoadjuvant chemotherapy (NACT), followed by surgery, radiation and emerging combinations with immunotherapy. While NACT is effective in some patients, up to 50% of patients are intrinsically unresponsive or acquire resistance. These patients demonstrate the highest rates of recurrence and distant metastasis. It is known that chemotherapies can effectively eliminate proliferating tumor cells, but a small population of drug-tolerant persister (DTPs) cells can remain and are thought to play a key role in relapse and resistance. We hypothesized that chemotherapy influences TNBC tumor plasticity by exerting selective pressures leading to the outgrowth of resistant subpopulations with the greatest survival advantage. To evaluate this hypothesis, we generated in vivo models of disease progression and chemotherapy resistance and investigated the plasticity of tumor cell subpopulations by single-cell RNA sequencing. We developed PDX models from a multi-drug resistant primary TNBC treated with standard-of-care chemotherapy and its patient-matched lung metastasis. To model response and resistance at the metastatic stage, we challenged this metastasis PDX with several cycles of Gemcitabine. We obtained residual disease that relapsed and eventually developed resistance, mirroring the patient response. We performed single-cell RNA sequencing (scRNAseq) of these models using a droplet-based technology from 10X Genomics. This scRNAseq data was used to compare the changes in the proportions of cellular subpopulations in each model. Our data show that the rebound model presents greater similarity to the untreated metastasis, while the resistant model significantly differs in cell population expression profiles. Interestingly, the residual disease demonstrates an intermediated state with similarities to both the untreated metastasis and the resistant model. We identified populations with hypoxic signatures in the primary tumor, its matched metastasis and the residual, rebound and resistant models. Immunohistochemistry and Nanostring GeoMx Digital Spatial Profiler validated these populations in these models. In addition, we have identified other varying populations and are currently investigating their cellular mechanisms and gene expression patterns. Using scRNA-sequencing to understand the clonal expansion of resistant subpopulations following chemotherapy reveals distinctive resistant cell features enabling the identification of the vulnerabilities of these tumors. Citation Format: Sandrine Busque, Constanza Martinez Ramirez, Ariel Madrigal Aguirre, Paul Savage, Anie Monast, Anne-Marie Fortier, Gerardo Zapata, Atilla Omeroglu, Jamil Asselah, Nathaniel Bouganim, Sarkis Meterissian, Francine Tremblay, Ari Meguerditchian, Yasser Riaz Alhosseini, Hamed S. Najafabadi, Hellen Kuasne, Morag Park. Identification of Gemcitabine-resistant Populations using scRNA-Sequencing in Triple Negative Breast Cancer Patient-Derived Xenografts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B067.

Abstract P2-17-03: Preclinical study of trastuzumab deruxtecan (T-Dxd; DS-8201a) in combination with DNA damage response pathway inhibitors in HER2-low/Hormone receptor negative breast cancer patient-derived xenograft models

Abstract Background: Triple negative breast cancer (TNBC), defined by negative hormone receptor (HR) status and the lack of HER2 gene amplification, is a significant clinical challenge because of its aggressive clinical course and association with a poor prognosis. Recent studies demonstrated that approximately a third of TNBC has low expression of HER2, so called HER2-low TNBC, which could be targeted by the new generation HER2-directed antibody-drug conjugate (ADC). Trastuzumab deruxtecan (also known as T-Dxd, DS-8201a) is a novel HER2-directed ADC with a topoisomerase 1 inhibitor payload and a tetrapeptide-based cleavable linker, which has been FDA approved for patients with metastatic HER2-positive, or amplified breast cancer. T-Dxd has also shown to improve survival outcomes when compared to physicians’ choice chemotherapy in patients with metastatic HR-negative/HER2-low breast cancer in the Phase 3 DESTINY-Breast04 trial. However, resistance is invariable. We hypothesize that small molecule inhibitors against DNA damage response (DDR) pathways could potentiate the DNA damaging effect of T-Dxd and improve its anti-tumor activity in HER2-low TNBC. Method: We conducted preclinical experiments to assess the anti-tumor activity of T-Dxd and each of the DDR pathway inhibitors, including the ATR inhibitor AZD6738, the ATM inhibitor AZD1390, and the PARP inhibitor olaparib, as well as the Wee 1 inhibitor AZD1775, either alone or in combination, compared to the vehicle treatment, in a panel of HER2-low TNBC PDX models in vivo, as well as mechanistic studies using HER2-low TNBC cell lines in vitro. HER2-low was defined as 1+ or 2+ on HER2 immunohistochemistry. Results: Four HER2-low TNBC PDX models, including WHIM2 (TP53 mutated, basal-like), WHIM6 (TP53 WT, basal-like), WHIM12 (TP53, PIK3CA, and PTEN mutated, claudin-low), and WHIM30 (BRCA1 and TP53 mutated, basal-like), as well as a panel of 4 TNBC cell lines with variable HER2 expression were included in the study. We demonstrated that the addition of DDR pathway inhibitors to T-Dxd led to further tumor shrinkage and delayed tumor progression in HER2-low TNBC PDX models in vivo and induced synergistic anti-tumor effects in vitro. Enhanced DNA damaging effect and apoptosis induction were observed with combination therapies in vitro. Biomarker analysis on xenograft tumors harvested 3 days following treatment with either Vehicle, T-Dxd, olaparib, or T-Dxd plus olaparib in WHIM6, was also performed, which demonstrated that the addition of olaparib enhanced DNA damage (increased pH2AX), and promoted apoptosis (increased cleaved PARP). Further mechanistic studies in HER2-low TNBC cell lines demonstrated the formation of DNA topoisomerase 1 covalent complexes (TopoIcc) following treatment with T-Dxd. Interestingly, while the formation of Topolcc was reduced with the addition of either ATR or ATM inhibitor, no reduction was observed with the addition of olaparib. Additionally, we observed that combination therapies reduced cell migration in vitro. The combination of olaparib with T-Dxd, in particular, reduced mammosphere formation and Epithelial Mesenchymal Transition (EMT) modulation markers, including SNAIL, SLUG and/or TWIST. Conclusion: Our study demonstrated that the combination of T-Dxd and DDR pathway inhibitors induced additive or synergistic anti-tumor effect in HER2-low TNBC. Our data from the studies of cell lines and PDX models provided preclinical rationale for the combination of T-Dxd and DDR pathway inhibitors in patients with HER2-low TNBC. The olaparib combination showed unique mechanistic characteristics compared with other DDR inhibitors, which are being further examined in additional HER2-low TNBC models. Citation Format: Adrian Gonzalez-Gonzalez, Zhanfang Guo, Alice Meroni, Emily Cybulla, Jeremy Hoog, Alessandro Vindigni, Cynthia Ma. Preclinical study of trastuzumab deruxtecan (T-Dxd; DS-8201a) in combination with DNA damage response pathway inhibitors in HER2-low/Hormone receptor negative breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-17-03.

Abstract A033: Identification of gemcitabine-resistant populations using scRNA-sequencing in triple negative breast cancer patient-derived xenograft

Abstract Despite major advances in the treatment of breast cancer (BC), it remains the most diagnosed and second most deadly cancer among American women. BC is a heterogeneous disease consisting of distinct subtypes, including hormone receptor-positive, HER2 receptor-positive and cancers that lack these receptors categorized as triple-negative breast cancers (TNBC). The TNBC subtype presents the worst outcome and the highest rates of recurrence and metastasis. The absence of targets prevents the use of established precision therapies in TNBC, and the standard of care remains neoadjuvant chemotherapy. While this is effective in some patients, about 50% develop resistance, leading to the development of metastasis. It is known that selective pressures exerted by chemotherapy treatment can promote the outgrowth of resistant tumor subclones. However, the diverse intra-tumoral population and the mechanisms that lead to chemotherapy resistance in TNBC are still poorly understood. We hypothesized that therapeutic regimens influence tumor plasticity by exerting selective pressures leading to the outgrowth of resistant subpopulations with the greatest survival advantage. To evaluate this hypothesis, we aimed to generate in vivo models of chemotherapy resistance and to investigate the plasticity of tumor cell subpopulations challenged with standard-of-care chemotherapies. To this end, we selected a multi-drug resistant (Doxorubicin, Cyclophosphamide, Cisplatin, and Paclitaxel) BC patient and developed a patient-derived xenograft (PDX) from the primary tumor and the lung metastasis. The metastasis PDX model was initially responsive to Gemcitabine (as observed in the BC patient) but eventually developed resistance. We challenged this metastasis PDX with several cycles of Gemcitabine and obtained residual, rebound, and resistant tumor samples. We performed single-cell RNA sequencing (scRNAseq) of these models using a droplet-based technology from 10X Genomics. This scRNAseq data was used to compare the changes in the proportions of cellular subpopulations in each model. Interestingly, our data shows that the rebound model presents greater similarity to the untreated metastasis, while the resistant model has significant differences in cell population expression profiles. We identified a hypoxic population in the primary tumor and its matched metastasis. This population was validated in these models by Nanostring GeoMx Digital Spatial Profiler. Our recent analyses have identified that this hypoxic population persists in the residual, rebound and resistant models. In addition, we have identified other populations that vary in these models. We are currently investigating their cellular mechanisms and gene expression patterns. Using scRNA-sequencing to understand the clonal expansion of resistant subpopulations following chemotherapy reveals distinctive resistant cell features enabling the identification of the vulnerabilities of these tumors. Citation Format: Sandrine Busque, Constanza Martinez Ramirez, Hellen Kuasne, Paul Savage, Anne-Marie Fortier, Anie Monast, Atilla Omeroglu, Jamil Asselah, Nathaniel Bouganim, Sarkis Meterissian, Claudia Kleinman, Mark Basik, Morag Park. Identification of gemcitabine-resistant populations using scRNA-sequencing in triple negative breast cancer patient-derived xenograft [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A033.

Docosahexaenoic acid enrichment of tumor phospholipid membranes increases tumor necroptosis in mice bearing triple negative breast cancer patient-derived xenografts

Docosahexaenoic acid (DHA) reduces breast cancer tumor growth in preclinical models. To better understand how DHA amplifies the actions of docetaxel (TXT) chemotherapy, we examined the effects of two doses of dietary DHA on tumor size, membrane DHA content and necroptosis using a drug resistant triple negative breast cancer (TNBC) patient derived xenograft (PDX) model. Female NOD.Cb-PrkdcscidIl2rg mice bearing TNBC PDXs were randomized to one of three nutritionally complete diets (20% w/w fat): control (0% DHA), high DHA (3.8% HDHA), or low DHA (1.6% LDHA) with or without intraperitoneal injections of 5 mg/kg TXT, twice weekly for 6 weeks (n=8 per group). Tumors from mice fed either HDHA+TXT or LDHA+TXT were similar in size to each other, but were 36% and 32% smaller than tumors from mice fed control+TXT, respectively (P<.05). A dose effect of DHA incorporation was observed in plasma total phospholipids and in phosphatidylethanolamine and phosphatidylinositol. Both doses of DHA resulted in similarly increased necrotic tissue and decreased NFκB protein expression compared to control tumors, however only the HDHA+TXT had increased expression of necroptosis related proteins: RIPK1, RIPK3 and MLKL (P<.05). Increased MLKL was observed in the lipid raft portion of HDHA+TXT tumor extracts. This work confirms the efficacy of a combination therapy consisting of DHA supplementation and TXT chemotherapy using two doses of DHA as indicated by reduced tumor growth in a TNBC PDX model. Moreover, the results suggest that decreased growth may occur through increased DHA incorporation into tumor phospholipid membranes and necroptosis.

Secreted indicators of androgen receptor activity in breast cancer pre-clinical models

PurposeAccumulating evidence has attracted attention to the androgen receptor (AR) as a biomarker and therapeutic target in breast cancer. We hypothesized that AR activity within the tumor has clinical implications and investigated whether androgen responsive serum factors might serve as a minimally invasive indicator of tumor AR activity.MethodsBased on a comprehensive gene expression analysis of an AR-positive, triple negative breast cancer patient-derived xenograft (PDX) model, 163 dihydrotestosterone (DHT)-responsive genes were defined as an androgen responsive gene set. Among them, we focused on genes that were DHT-responsive that encode secreted proteins, namely KLK3, AZGP1 and PIP, that encode the secreted factors prostate specific antigen (PSA), zinc-alpha-2-glycoprotein (ZAG) and prolactin induced protein (PIP), respectively. Using AR-positive breast cancer cell lines representing all breast cancer subtypes, expression of candidate factors was assessed in response to agonist DHT and antagonist enzalutamide. Gene set enrichment analysis (GSEA) was performed on publically available gene expression datasets from breast cancer patients to analyze the relationship between genes encoding the secreted factors and other androgen responsive gene sets in each breast cancer subtype.ResultsAnti-androgen treatment decreased proliferation in all cell lines tested representing various tumor subtypes. Expression of the secreted factors was regulated by AR activation in the majority of breast cancer cell lines. In GSEA, the candidate genes were positively correlated with an androgen responsive gene set across breast cancer subtypes.ConclusionKLK3, AZGP1 and PIP are AR regulated and reflect tumor AR activity. Further investigations are needed to examine the potential efficacy of these factors as serum biomarkers.

Proteomic Resistance Biomarkers for PI3K Inhibitor in Triple Negative Breast Cancer Patient-Derived Xenograft Models.

Simple SummaryThe objective of this study is to identify potential proteomic biomarkers in triple negative breast cancer (TNBC) that associate with response to PI3K inhibitors which are in clinical trials. We tested a panel of TNBC patient-derived xenograft (PDX) models for their tumor growth response to a pan-PI3K inhibitor, BKM120. Proteomic analyses by reverse phase protein array (RPPA) of 182 markers were performed on baseline and post short-term treatment PDX samples, to correlate with tumor growth response. We identified several baseline and treatment induced proteomic biomarkers in association with resistance. These results provide important insights for the development of PI3K inhibitors in TNBC. PI3K pathway activation is frequently observed in triple negative breast cancer (TNBC). However, single agent PI3K inhibitors have shown limited anti-tumor activity. To investigate biomarkers of response and resistance mechanisms, we tested 17 TNBC patient-derived xenograft (PDX) models representing diverse genomic backgrounds and varying degrees of PI3K pathway signaling activities for their tumor growth response to the pan-PI3K inhibitor, BKM120. Baseline and post-treatment PDX tumors were subjected to reverse phase protein array (RPPA) to identify protein markers associated with tumor growth response. While BKM120 consistently reduced PI3K pathway activity, as demonstrated by reduced levels of phosphorylated AKT, percentage tumor growth inhibition (%TGI) ranged from 35% in the least sensitive to 84% in the most sensitive model. Several biomarkers showed significant association with resistance, including elevated baseline levels of growth factor receptors (EGFR, pHER3 Y1197), PI3Kp85 regulatory subunit, anti-apoptotic protein BclXL, EMT (Vimentin, MMP9, IntegrinaV), NFKB pathway (IkappaB, RANKL), and intracellular signaling molecules including Caveolin, CBP, and KLF4, as well as treatment-induced increases in the levels of phosphorylated forms of Aurora kinases. Interestingly, increased AKT phosphorylation or PTEN loss at baseline were not significantly correlated to %TGI. These results provide important insights into biomarker development for PI3K inhibitors in TNBC.

QPI Allows in vitro Drug Screening of Triple Negative Breast Cancer PDX Tumors and Fine Needle Biopsies

The development of resistance to initially successful cancer therapies is a major cause of the morbidity and mortality associated with cancer. Identifying evolving resistance at an early stage could inform clinical decision making to adapt therapies before resistant cancer cell phenotypes have become clonally dominant or metastasized. This goal of early detection has prompted heavy investments in liquid biopsy, organoid, and high-throughput screening methodologies. Recently, High-Speed Live-Cell Interferometry (HSLCI), a quantitative phase imaging (QPI) methodology, was shown to predict triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) sensitivity to carboplatin only 40 hours after tumor removal from a mouse. Before HSLCI can be tested in the clinic, it must be adapted for minimally invasive sample acquisition techniques, throughput must be increased to enhance drug screening capacity, and, like any screening method, protocols must be adjusted to reflect drug-specific effects. To overcome these barriers, the system’s hardware was redesigned to increase throughput six-fold and enable the simultaneous screening of multiple therapeutics, and experiments were expanded to include several new classes of drugs. Additionally, the updated system was then incorporated into fine needle biopsy-compatible protocols that were developed for HSLCI and yielded data concordant with recently-published in vivo screens.

Abstract 357: Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2, and HER3, is highly effective in triple negative breast cancer patient-derived xenografts

Abstract As a heterogeneous disease, breast cancer is clinically classified taking into account the expression of estrogen receptor alpha (ERα), progesterone (PR), and the presence/amplification status of the oncogenic human epidermal growth factor receptor 2 (HER2). Triple negative breast cancer (TNBC) represents ~10-20% of all cases, and displays poor prognosis and aggressive clinical course. EGFR is more often overexpressed in TNBC than in other breast cancer subtypes, providing cancer cells with compensatory signals that greatly contribute to the development of resistance and survival in response to therapy. This characteristic has made HER family members potentially important therapeutic targets. The present study was designed to investigate the effects of Pan-HER, a novel mixture of six monoclonal antibodies directed against members of the human epidermal growth factor receptor (HER) family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs). Fifteen low passage TNBC PDX tumor samples were transferred and engrafted into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 150-200 mm3, mice were randomized (n ≥ 3 per group), and treated following three, one-week cycles, consisting of 3 times/week intraperitoneally (IP) injection of either formulation buffer (Vehicle control) or Pan-HER (50 mg/kg). At the end of treatment, tumors were collected for western blot, RNA and immunohistochemistry analyses. All 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor down-modulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways including AKT, ERK and NF-κB. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation. This observation may provide a plausible explanation for the response of TNBC tumors to Pan-HER as an indication of the potential use of EGFR and associated signaling expression as biomarkers for selecting patients that may benefit of targeting these proteins. In summary, this preclinical TNBC PDX trial with Pan-HER provides strong justification for further biomarker-guided studies in TNBC. The finding that tumors were affected rapidly and effectively, with long-lasting results, offers exciting perspectives to treat this aggressive form of breast cancer, for which available treatment options are currently limited. Citation Format: Roberto R. Rosato, Don S. Choi, Wei Qian, Wen Chen, Johan Lantto, Ivan D. Horak, Michael Kragh, Jenny C. Chang. Pan-HER, an antibody mixture simultaneously targeting EGFR, HER2, and HER3, is highly effective in triple negative breast cancer patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 357.

Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model

BackgroundTriple-negative breast cancer (TNBC) represents an aggressive subtype with limited therapeutic options. Experimental preclinical models that recapitulate their tumors of origin can accelerate target identification, thereby potentially improving therapeutic efficacy. Patient-derived xenografts (PDXs), due to their genomic and transcriptomic fidelity to the tumors from which they are derived, are poised to improve the preclinical testing of drug-target combinations in translational models. Despite the previous development of breast and TNBC PDX models, those derived from patients with demonstrated health-disparities are lacking.MethodsWe use an aggressive TNBC PDX model propagated in SCID/Beige mice that was established from an African-American woman, TU-BcX-2 K1, and assess its metastatic potential and drug sensitivities under distinct in vitro conditions. Cellular derivatives of the primary tumor or the PDX were grown in 2D culture conditions or grown in mammospheres 3D culture. Flow cytometry and fluorescence staining was used to quantify cancer stem cell-like populations. qRT-PCR was used to describe the mesenchymal gene signature of the tumor. The sensitivity of TU-BcX-2 K1-derived cells to anti-neoplastic oncology drugs was compared in adherent cells and mammospheres. Drug response was evaluated using a live/dead staining kit and crystal violet staining.ResultsTU-BcX-2 K1 has a low propensity for metastasis, reflects a mesenchymal state, and contains a large burden of cancer stem cells. We show that TU-BcX-2 K1 cells have differential responses to cytotoxic and targeted therapies in 2D compared to 3D culture conditions insofar as several drug classes conferred sensitivity in 2D but not in 3D culture, or cells grown as mammospheres.ConclusionsHere we introduce a new TNBC PDX model and demonstrate the differences in evaluating drug sensitivity in adherent cells compared to mammosphere, or suspension, culture.

1894PD - Single-cell RNA sequencing of triple negative breast cancer patient-derived xenograft reveals distinct cellular populations spatially mapped to histological sections

1894PD - Single-cell RNA sequencing of triple negative breast cancer patient-derived xenograft reveals distinct cellular populations spatially mapped to histological sections

Adipose-derived stem cells enhance human breast cancer growth and cancer stem cell-like properties through adipsin.

Tumor microenvironment plays a key role for tumor development and progression. Although adipose tissue is a predominant component of stroma in mammary tissues and secretes various cytokines, chemokines and growth factors, roles of adipocytes in breast cancers remain to be elucidated. In this study, we found that adipsin, an adipokine secreted from mammary adipose tissues, enhanced proliferation and cancer stem cell (CSC)-like properties of human breast cancer patient-derived xenograft (PDX) cells. Adipsin was predominantly expressed in both adipose tissues of the surgical specimens of breast cancer patients and adipose-derived stem cells (ADSCs) isolated from them, and its expression level was significantly higher in obese patients. ADSCs significantly enhanced the sphere-forming ability of breast cancer PDX cells derived from both estrogen receptor-positive and -negative breast cancer PDX cells. Suppression of adipsin-mediated signaling by a specific inhibitor or adipsin knockdown in ADSCs significantly decreased the sphere-forming ability and the expression of CSC markers in co-cultured breast cancer PDX cells. Growth of breast cancer PDX tumors was significantly enhanced by co-transplantation with ADSCs in vivo, and it was weakened when co-transplanted with the adipsin knocked-down ADSCs. These results suggest that adipsin is an important adipokine secreted from mammary adipose tissue that functions as a component of tumor microenvironment and a CSC niche in breast cancers.

The Selective ATR Inhibitor VX-970 Enhances the Therapeutic Effects of Radiation Therapy in Triple Negative Breast Cancer Patient-Derived Xenografts and is a Novel Strategy to Overcome Therapeutic Resistance

TNBC represents an aggressive subtype characterized by earlier relapse and worse survival compared to non-TNBC. TNBC patients with pathologic complete response after neoadjuvant chemotherapy (NAC) have excellent long term prognoses with low rates of local regional relapse whereas patients with residual disease after NAC (i.e chemoresistant disease) have high rates of locoregional and distant relapse despite adjuvant RT. Thus, new approaches to address microscopic residual disease are needed in patients with residual TNBC after NAC. VX-970 is a novel potent inhibitor of ATM and RAD3-related (ATR), a phosphoinositide 3-kinase which plays a key role in the cellular response to replication stress and DNA damage. We hypothesized that VX-970 would sensitize TNBC to RT by inhibiting DNA repair. TNBC PDXs generated in the context of the prospective neoadjuvant Breast Cancer Genome Guided Therapy Study from either pre-NAC biopsy specimens or chemotherapy resistant residual tumor samples were studied. For in vitro experiments, TNBC PDX explants were treated with VX-970, RT, or the combination and DNA damage signaling was assessed using immunoblot and immunofluorescence. For in vivo studies, 1 x 106 tumor cells from TNBC PDX lines were injected into the hind-leg of athymic nude mice. After the tumors reached 75 mm3, mice were randomized and treated in the following arms: 1) Vehicle + Sham RT 2) RT 3) VX-970 4) RT + VX-970. RT was delivered in 5 daily fractions of 2 Gy. VX970 was administered by oral gavage daily 1 hour prior to RT at a dose of 60 mg/kg. The primary endpoint was time to tumor tripling. VX-970 administered 1 hour prior to RT reduced phospho(p)-Ser428-ATR and the downstream ATR target, p-Ser345-Chk1, whereas p-thr68-Chk2 was not impacted. Pre-treatment with VX-970 delayed resolution of RT-induced p-Ser139-H2AX foci, suggesting that VX-970 decreased DNA repair. VX-970 also attenuated the recruitment of RAD51, which catalyzes the homology search and strand invasion steps in homologous recombination (HR), into RT-induced foci suggesting that inhibition of HR was a cause of unrepaired DNA damage. The combination of VX-970 and RT was well tolerated and significantly increased or trended towards an increase in the median time to tumor tripling compared with RT alone in two PDX lines established from pre-NAC TNBC biopsy specimens (TNBC1 49 vs 26 days, p=0.02; TNBC3 88 vs 49, p=0.29). Median time to tumor tripling was also increased in a PDX line established from a post-NAC resistant residual tumor sample (TNBC2 43 vs 25, p=0.01). The combination of VX-970 and RT resulted in promising antitumor activity in TNBC resistant to standard NAC. We recommend that this combination be studied in patients with residual TNBC after NAC.

Abstract 1078: Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models

Abstract Purpose: Triple negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer with limited therapeutic options. Development of molecularly targeted agents for TNBC is an unmet clinical need. The phosphoinositide 3-kinase (PI3K) pathway, which is a major cell growth and survival pathway, is frequently activated in TNBC as a result of genetic aberrations such as loss of the negative regulator PTEN or gain of function mutations in PIK3CA, therefore an attractive therapeutic target. However, single agent PI3K inhibitors have shown limited anti-tumor activity in both preclinical models and in clinical trials. One of the most important mechanisms of resistance to PI3K inhibitor is amplification of Myc. Dinaciclib, a potent inhibitor of cyclin-dependent kinases (CDKs)1, 2, 5 and 9, has shown to be particularly effective in Myc dependent tumors in preclinical studies and in TNBC. We therefore hypothesized that dinaciclib could overcome tumor cell resistance to PI3K inhibitors and improve the therapeutic efficacy in TNBC. In this study, we evaluated the anti-tumor and molecular effect of dinaciclib and BKM120 (Pan-PI3K inhibitor), either alone or in combination, in a panel of patient derived xenograft (PDX) models of TNBC. Methods: Four TNBC PDXs models were selected for the study for in-vivo and ex-vivo response to vehicle, BKM120, dinaciclib, or the combination of BKM120 and dinaciclib. Tumor volume changes over time in each group were documented to calculate the percentage of tumor growth inhibition by either agent alone or in combination. Tumor tissues harvested post treatment were examined by immunohistochemistry for cleaved PARP to determine the extent of apoptosis and phospho-Histone H3 for G2 to M phase cell cycle progression. Western blot analysis and reverse protein phase array (RPPA) were also performed to determine treatment effect on PI3K downstream targets and cell cycle molecules. Results: The combination of BKM120 and dinaciclib induced significantly greater growth inhibitory effect on tumor growth than either single agent alone in TNBC PDX models. This is accompanied by an enhanced apoptotic induction and reduced cell cycle progression. In addition, the combination of dinaciclib and BKM120 significantly reduced the level of cyclin B and the key anti-apoptotic protein survivin as well as significant downregulation of pAKT, and pS6. Conclusions: These data suggest that dinaciclib and BKM120 combination is an effective approach in treating TNBC. Additional mechanistic investigation for the efficacy of this combination is underway. Citation Format: Sandeep Rajput, Fang Guo, Li Shun, Cynthia Ma. Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models [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 1078. doi:10.1158/1538-7445.AM2017-1078

Abstract 1117: Triple negative breast cancer patient-derived xenografts as a translational model for discovery of novel therapeutic targets

Abstract Triple negative breast cancers (TNBCs) constitute 12% of all breast cancers, and is approximately twice more prevalent in African-American populations. Louisiana has a high proportion of African-American residents (32.5% in 2015), and thus hosts a higher population of TNBC patients. TNBCs have an aggressive phenotype that is elusive to the targeted therapeutics used to treat other breast cancer subtypes. The claudin-low molecular subtype has higher rates of metastases and recurrence. Certain kinase families have been extensively studied as regulators of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Discovery of novel kinase targets within the subset of uncharacterized kinases could provide important insight into future targeted therapies. However, current models utilized in target discovery research are limited by the inability to accurately recapitulate the complex stromal architecture and heterogenous genetic and molecular composition of breast cancer. Furthermore, immortalized cell lines are limited to a 2D environment and over time acquire mutations that may not reflect the primary tumor. Recently, our laboratory has successfully established two TNBC patient-derived xenograft (PDX) models derived from African-American patients, and generated cell lines (TU-BCx-2K1, TU-BCx-2O0) and mammospheres. One of these models, 2O0, presents tumor architecture, cellular composition, genomic (qRT-PCR) and protein (western blot) expressions that are concordant with a claudin-low subtype. Furthermore, we show that both TNBC models metastasize to the lungs, and exhibit molecular characteristics consistent with mesenchymal phenotypes. We utilized these translational PDX models to screen a library of small molecule inhibitors that represent a variety of kinase pathways to identify novel therapeutic targets and/or pathways that are specific to TNBC subtypes. We found in a preliminary cell morphology screen using three TNBC cell lines (MDA-MB-231, BT549, MDA-MB-157), two small molecule inhibitors that increased epithelial marker (CDH1) gene expression, suppressed mesenchymal (VIM, c-FOS, SNAI1, ZEB1) expression and/or suppressed cellular motility in transwell migration assays. We observed after ex vivo treatments with our PDX tumors the two compounds increase the epithelial marker CDH1 expression, and suppress mesenchymal markers (VIM, MMP2) expressions. We confirm these findings in the TU-BCx-2K1 cell line. Kinase array data revealed candidate kinases responsible for the observed EMT changes in the two compounds of interest (NEK5, NEK9, NEK1 potentially affect cell motility; SRC-family kinases, TAOK2, STK10 potentially affect EMT gene changes); we plan to utilize the PDX cell lines to characterize these kinases in EMT. We aim to ultimately discover novel therapeutic targets specific to different TNBC molecular subtypes. Citation Format: Margarite D. Matossian, Hope E. Burks, Annie C. Bowles, Rachel A. Sabol, Van T. Hoang, Bruce Bunnell, William J. Zuercher, David H. Drewry, Carrow Wells, Krzysztof Moroz, Matthew E. Burow, Bridgette Collins-Burow. Triple negative breast cancer patient-derived xenografts as a translational model for discovery of novel therapeutic targets [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 1117. doi:10.1158/1538-7445.AM2017-1117

Abstract P4-06-04: Triple negative breast cancer patient-derived xenografts: Molecular characteristics and sensitivity to emerging therapies

Abstract Background: Patients with triple negative breast cancer (TNBC) that have significant residual disease following neoadjuvant therapy have a poorer prognosis. The difficulty in developing improved treatment strategies for these patients is compounded by the molecular diversity of this pathology. Thus, preclinical efficacy testing with models representing the molecular heterogeneity of TNBC may not only improve efficacy testing but also has the potential to facilitate biomarker discovery to inform patient selection. Methods: We generated 28 hormone receptor-low/HER2- patient-derived xenografts (PDXs) from 26 patients; the majority from residual disease following neoadjuvant chemotherapy. We characterized the tumors from the patients and the matched PDXs by targeted exome sequencing, RNA sequencing, and Reverse Phase Protein Arrays. We established single agent sensitivity for a set of PDXs to standard TNBC therapies as well as emerging targeted therapies targeting PARP, mTOR, PI3K and MEK1/2. Results: PDXs maintained DNA/RNA profiles similar to the patients' tumors. Our PDX set exhibits a diverse set of potential driver alterations and is comprised of PDXs representative of all TNBC molecular subtypes. The PDXs varied in their sensitivity to standard chemotherapeutics. PI3K, mTOR and MEK inhibition caused tumor growth inhibition in several PDX models but were unable to cause tumor regression as single agent. In contrast, talazoparib caused dramatic regression in 5 out of 12 PDXs tested, including 3 PDXs from patients whose tumors had progressed on neoadjuvant anthracyclines and/or paclitaxel. Four of these talazoparib sensitive PDXs were derived from patients not harboring BRCA1/2 germline alterations; three had somatic alterations in genes linked to DNA damage repair (ATM, BRCA2, PTEN). The comparison of PARP sensitivity with molecular profiles will be presented. Conclusions: TNBC PDXs represent molecular tumor heterogeneity as well as heterogeneity in response to targeted therapeutics. PDXs represent an opportunity for biomarker discovery as well as for rationale combination therapies to enhance the efficacy targeted therapies. Citation Format: Evans KW, Yuca E, Akcakanat A, Scott SM, Paez Arango N, Zheng X, Chen K, Tapia C, Tarco E, Eterovic AK, Black DM, Jennifer LK, Tripathy D, Mills GB, Meric-Bernstam F. Triple negative breast cancer patient-derived xenografts: Molecular characteristics and sensitivity to emerging therapies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-06-04.

Vandetanib as a potential new treatment for estrogen receptor-negative breast cancers.

The receptor tyrosine kinase RET is implicated in the progression of luminal breast cancers (BC) but its role in estrogen receptor (ER) negative tumors is unknown. Here we investigated the expression of RET in breast cancer patients tumors and patient-derived xenografts (PDX) and evaluated the therapeutic potential of Vandetanib, a tyrosin kinase inhibitor with strong activity against RET, EGFR and VEGFR2, in ER negative breast cancer PDX. The RT-PCR analysis of RET expression in breast tumors of 446 patients and 57 PDX, showed elevated levels of RET in ER+ and HER2+ subtypes and in a small subgroup of triple-negative breast cancers (TNBC). The activity of Vandetanib was tested in vivo in three PDX models of TNBC and one model of HER2+ BC with different expression levels of RET and EGFR. Vandetanib induced tumor regression in PDX models with high expression of RET or EGFR. The effect was associated with inhibition of RET/EGFR phosphorylation and MAP kinase pathway and increased necrosis. In a PDX model with no expression of RET nor EGFR, Vandetanib slowed tumor growth without inducing tumor regression. In addition, treatment by Vandetanib decreased expression of murine Vegf receptors and the endothelial marker Cd31 in the four PDX models tested, suggesting inhibition of tumor vascularization. In summary, these preclinical results suggest that Vandetanib treatment could be useful for patients with ER negative breast cancers overexpressing Vandetanib's main targets.

Abstract 221: Cancer stem cell-selected patient-derived xenograft models with sensitivity to FAK and Pan-PI3K/mTOR inhibitors .

Abstract In this study, Tumor-initiating (Creighton et al, 2009) and EMT gene expression signatures (Gupta et al., 2009, Taube et al, 2009) were used as classifiers. Tumors from 25 human triple negative breast cancer Patient-Derived Xenografts (PDX) were examined with Affymetrix U133Plus2 RNA arrays. Datasets were RMA normalized, and samples scored with the TI or EMT signature based on the Pearson Correlation Coefficient of a vector of mean-centered expression values of signature genes and a vector of indicators for signature genes such that 1 (up-regulated, correlated) genes and -1 (down-regulated, anti-correlated) genes formed a TI or EMT score. A high EMT score is also representative of mesenchymal tumors. Several of the triple negative breast cancer PDX models had both high TI and high EMT scores, indicated by p&amp;lt;0.05 of a one-sided Pearson Correlation Coefficient test. Two of the PDX models with high positive TI and EMT scores, 4664 and 3887, were evaluated for sensitivity to a FAK inhibitor (VS-4718), a pan-PI3K/mTOR inhibitor (VS-5584), and chemotherapy (docetaxel). FAK and PI3K/mTOR are implicated in tumorigenesis, including tumor initiation, growth, angiogenesis and metastasis. Both 4664 and 3887 tumors were N-cadherin-positive (mesenchymal), and strongly positive for CD44 (TI marker) by immunohistochemistry. These tumors were also weakly positive for E-cadherin and highly heterogeneous for pan-cytokeratin, two epithelial marker patterns suggesting a mesenchymal phenotype. Whereas 3887 was Notch3-positive, 4664 was Notch3-negative. VS-4718-treated 4664 and 3887 models showed diminished tumor growth over 21 days compared with vehicle-treated animals. VS-4718-treated animal tumors showed decreased FAK autophosphorylation (pY397) by immunohistochemistry. Likewise, the VS-5584-treated 4664 and 3887 animals blocked tumor growth over 21 days. In these models, the PI3K/mTOR pathways activate downstream signaling phosphorylation of pS6 pSer240/244, p4EBP1 pThr70, and pPRAS40 pThr246. In the VS-5584-treated 3887 tumors, pS6 pSer240/244 levels were significantly diminished relative to vehicle- and docetaxel-treated tumors. As predicted by TI and EMT gene expression indicators, both models were docetaxel-resistant. These findings and our previous work on these agents for the inhibition of CSCs support the clinical development of the FAK and the pan-PI3K/mTOR inhibitors to target CSCs for treatment of breast, mesothelioma, ovarian and other cancers. Citation Format: David T. Weaver, Daniel Paterson, Mahesh Padval, Jonathan Pachter, Michael Lewis. Cancer stem cell-selected patient-derived xenograft models with sensitivity to FAK and Pan-PI3K/mTOR inhibitors . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 221. doi:10.1158/1538-7445.AM2013-221