Triple Negative Breast Cancer Patient-derived Xenograft Models Research Articles

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.

Abstract P5-17-04: Combined PI3K and NOS inhibition enhances efficacy of taxane-based chemotherapy in metaplastic breast cancer

Abstract Background: Metaplastic breast cancer (MpBC) is a therapeutically chemoresistant, aggressive, and heterogeneous breast cancer variant accounting for &amp;lt;5% of all breast cancers. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis and decreased survival compared to TNBC. Despite its chemorefractory nature, the current mainstay of treatment for MpBC is surgery and systemic chemotherapy. Common molecular alterations found in MpBC associated with poor prognosis and worse overall survival include 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced production of nitric oxide via inducible nitric oxide synthase (iNOS). In MpBC, both the PI3K and NOS signaling pathways may synergistically work together to enhance chemoresistance. We propose that combined inhibition of PI3K and iNOS will enhance the efficacy of taxane-based chemotherapy in MpBC. Methods: For in vitro and in vivo studies, we used MpBC cell lines (Hs578T and BT549) and TNBC/MpBC Patient-Derived Xenograft (PDX) models, respectively. For all studies, we used pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA, L), PI3K inhibitor alpelisib (A), and docetaxel (D). Immunohistochemistry (IHC), Western Blotting (WB), Cell Proliferation Assays, Flow Cytometry (to evaluate cell death and cell cycle distribution analysis), and HIV reverse transcriptase-based dNTP assay to quantify dNTPs were performed. For in vivo studies, five MpBC PDX models were implanted into the mammary fat pad of NSG mice and they received single therapy (vehicle control, L, A, D), dual therapy (D+A, D+L), or triple combination therapy (D+A+L). Tumor volumes were recorded twice weekly. Results: 66% (4/6) MpBC and 33% (7/21) TNBC PDX models had double-positive IHC staining of both iNOS and p-Akt (Ser473), supporting the concept that both signaling pathways are typically activated in MpBC tumors, relative to non-metaplastic TNBC tumors. Apoptosis and cell proliferation analysis found that MpBC cell lines treated with triple-combination (D+A+L) had an increased number of apoptotic cells and decreased cell proliferation relative to MpBC cells treated with dual combination (L+A), or single treatment (vehicle, D, A, or L). Cell cycle distribution analysis of treated MpBC cells found that in a time-dependent manner, there was a substantial decrease in the % of MpBC cells in S-phase and an increase in the % cells in G2/M cell cycle arrest due to dual and triple combination. This result was supported by dNTP quantification analysis revealing that combined PI3K and NOS inhibition induced greater nucleotide depletion within 8 hours of treatment relative to single treatment in MpBC cells. WB analysis revealed that dual/triple combination therapy in MpBC cells resulted in an enhanced DNA damage response signaling relative to single treatment, as indicated with increased expression of γ-H2AX, p-Chk1, p-Chk2, p-P53 (Ser15 and 20), and p21. Pro-survival PI3K signaling pathway was activated in response to docetaxel treatment alone in MpBC cells, but its activation was significantly reduced when docetaxel was coupled with PI3K and NOS inhibition. In vivo studies revealed that triple combination therapy significantly reduced tumor volume and improved survival proportions compared to dual/single therapy and vehicle control. Conclusions: The present data suggest that combined PI3K and NOS inhibition enhances docetaxel-mediated DNA damage by depleting nucleotide pools, leading to enhanced DNA damage response, growth arrest, and apoptosis. Ongoing studies are investigating how docetaxel coupled with PI3K and NOS inhibition influences DNA repair signaling and MpBC metastatic capacity. The addition of PI3K and NOS inhibitors to taxane-based chemotherapy may be a novel therapeutic strategy for aggressive MpBCs. Citation Format: Tejaswini P Reddy, Bijan Mahboubi, Roberto R. Rosato, Liliana Guzman-Rojas, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Jenny C. Chang. Combined PI3K and NOS inhibition enhances efficacy of taxane-based chemotherapy in metaplastic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-04.

Abstract P5-05-01: Metabolite profiling and RNA-seq identifies novel metabolomic-genomic biomarker and therapeutic options for rapidly proliferating breast cancers

Abstract Metabolic dysregulation is one of the distinctive features of breast cancer, yet remains under-characterized in different subtypes of breast cancer. In this study, we performed full metabolome profiling and RNA-seq gene expression analyses on patient samples comprised of Triple Negative Breast Cancer (TNBC) and Estrogen Receptor (ER) positive breast cancers, as well as TNBC patient-derived xenografts (PDX). We identified two major metabolic groups using hierarchical clustering analysis of global metabolite levels: a Nucleotide/Carbohydrate-Enriched group and a Lipid/Fatty Acid-Enriched group. The Nucleotide/Carbohydrate-Enriched group is populated by the majority of TNBCs and all TNBC PDX samples, and shows high levels of energy consumption and nucleotide biosynthesis related metabolites, while the Lipid/Fatty Acid-Enriched group contained all ER+ cancers (and normal breast tissues) and showed high levels of lipids and fatty acids. Using these two metabolite-defined groups, we compared metabolic signatures to RNA-seq expression data and identified gene expression signatures that correlated with each metabolic group. This novel integrated signature identified classic proliferation-associated genes as highly expressed in the Nucleotide/Carbohydrate-Enriched metabolomics group. We next sought to therapeutically target the Nucleotide/Carbohydrate-Enriched group through targeting of nucleotide metabolism using a pyrimidine biosynthesis inhibitor (DHODH inhibitor, Brequinar), and/or a glutaminase inhibitor (CB-839), and observed tumor volume reductions and improved survival times using the TNBC PDX models. Notably,. when the DHODH inhibitor Brequinar was tested on four different immune-competent TNBC mouse models, it showed significant single agent activity, and in 3/4 cases had better activity than carboplatin/paclitaxel combination, and with less toxicity. Our study reveals a new molecularly targeted therapy for rapidly proliferating TNBCs, guided by a novel set of metabolic-genomic biomarkers, which might be translated quickly as DHODH inhibitors are already FDA approved for use in other diseases. Citation Format: Cherise R Glodowski, Chengheng Liao, Cheng Fan, Juan Liu, Kevin R Mott, Akash Kaushik, Hieu Vu, Jason W Locasale, Samuel K McBrayer, Ralph J DeBerardinis, Charles M Perou, Qing Zhang. Metabolite profiling and RNA-seq identifies novel metabolomic-genomic biomarker and therapeutic options for rapidly proliferating breast cancers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-01.

Metabolic stress induces GD2+ cancer stem cell-like phenotype in triple-negative breast cancer.

Metabolic stress resulting from nutrient deficiency is one of the hallmarks of a growing tumour. Here, we tested the hypothesis that metabolic stress induces breast cancer stem-like cell (BCSC) phenotype in triple-negative breast cancer (TNBC). Flow cytometry for GD2 expression, mass spectrometry and Ingenuity Pathway Analysis for metabolomics, bioinformatics, in vitro tumorigenesis and in vivo models were used. Serum/glucose deprivation not only increased stress markers but also enhanced GD2+ BCSC phenotype and function in TNBC cells. Global metabolomics profiling identified upregulation of glutathione biosynthesis in GD2high cells, suggesting a role of glutamine in the BCSC phenotype. Cueing from the upregulation of the glutamine transporters in primary breast tumours, inhibition of glutamine uptake using small-molecule inhibitor V9302 reduced GD2+ cells by 70-80% and BCSC characteristics in TNBC cells. Mechanistic studies revealed inhibition of the mTOR pathway and induction of ferroptosis by V9302 in TNBC cells. Finally, inhibition of glutamine uptake significantly reduced in vivo tumour growth in a TNBC patient-derived xenograft model using NSG (non-obese diabetic/severe combined immunodeficiency with a complete null allele of the IL-2 receptor common gamma chain) mice. Here, we show metabolic stress results in GD2+ BCSC phenotype in TNBC and glutamine contributes to GD2+ phenotype, and targeting the glutamine transporters could complement conventional chemotherapy in TNBC.

FGFR-inhibitor-mediated dismissal of SWI/SNF complexes from YAP-dependent enhancers induces adaptive therapeutic resistance.

How cancer cells adapt to evade the therapeutic effects of drugs targeting oncogenic drivers is poorly understood. Here we report an epigenetic mechanism leading to the adaptive resistance of triple-negative breast cancer (TNBC) to fibroblast growth factor receptor (FGFR) inhibitors. Prolonged FGFR inhibition suppresses the function of BRG1-dependent chromatin remodelling, leading to an epigenetic state that derepresses YAP-associated enhancers. These chromatin changes induce the expression of several amino acid transporters, resulting in increased intracellular levels of specific amino acids that reactivate mTORC1. Consistent with this mechanism, addition of mTORC1 or YAP inhibitors to FGFR blockade synergistically attenuated the growth of TNBC patient-derived xenograft models. Collectively, these findings reveal a feedback loop involving an epigenetic state transition and metabolic reprogramming that leads to adaptive therapeutic resistance and provides potential therapeutic strategies to overcome this mechanism of resistance.

Abstract 1952: CDK4/6 inhibition radiosensitizes RB1 wild type triple negative breast cancers through impaired homologous recombination

Abstract Purpose: Cyclin-dependent kinase 4&amp;6 (CDK4/6) inhibitors have been approved for the treatment of metastatic, estrogen receptor positive (ER+) breast cancers, but there is growing interesting in CDK4/6 inhibition as a therapeutic strategy in other breast cancer subtypes, including triple negative breast cancer (TNBC). Previous studies have shown that CDK4/6 inhibition radiosensitizes ER+ breast cancers, but the interaction between CDK4/6 inhibition and radiation (RT) in TNBC is incompletely understood. Methods: Cellular viability was quantified 72 hours after drug treatment (in the absence of RT) to calculate a half maximal inhibitory concentration (IC50) value of proliferation. Radiation enhancement ratios (rER) and surviving fractions of cells after RT were calculated using clonogenic survival assays in RB1 wild type and mutant TNBC cell lines. Homologous recombination (HR) was assessed using RAD51 foci formation and a stable HR reporter system. G1 cell cycle arrest was quantified using propidium iodide-based flow cytometry. CRISPR-induced knockout of RB1 and transient siRNA-mediated knockdown of RB1 in TNBC cell lines was used in both clonogenic survival assays and immunofluorescence experiments. In vivo efficacy of CDK4/6 inhibition + RT was assessed using TNBC patient-derived xenograft models (PDX4664). Results: Although most TNBC cell lines are resistant to CDK4/6 inhibitor monotherapy (IC50 &amp;gt; 250nM) compared to ER+ cells, treatment with 250nM-1μM palbociclib radiosensitized RB1 wild type TNBC (MDA-MB-231, CAL-51, SUM-159, CAL-120; rER 1.08 – 2.22) but failed to radiosensitize RB1 mutant TNBC (CAL-851, MDA-MB-468; rER: 0.84 – 1.00). Radiosensitization of TNBC cell lines also occurred with short term ribociclib or abemaciclib pretreatment. At 6 and 16 hours following RT, significant suppression of RT-induced homologous recombination (HR) activity (RAD51 foci) was observed in RB1 wild type (p &amp;lt; 0.001) but not RB1 mutant (p &amp;gt; 0.05) TNBC cell lines. Cell cycle arrest after short term CDK4/6 inhibition was dependent on the presence of RB1. In addition, genetic knockdown of RB1 in RB1 wild type TNBC lead to a loss of CDK4/6 inhibitor-mediated HR suppression (p &amp;gt; 0.05) and diminished radiosensitization. Conclusions: In TNBC, CDK4/6 inhibition and RT leads to suppression of HR activity in an RB1-dependent manner. While ongoing studies seek to elucidate the role of RB1 in HR suppression in the context of CDK4/6 inhibitor-mediated radiosensitization of TNBC, our data suggests that CDK4/6 inhibition + RT could be a valuable clinical strategy to radiosensitize a wide range of breast cancer subtypes, including RB1 wild type TNBC. Citation Format: Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Benjamin C. Chandler, Kari Wilder-Romans, Meilan Liu, Lori J. Pierce, James M. Rae, Corey W. Speers. CDK4/6 inhibition radiosensitizes RB1 wild type triple negative breast cancers through impaired homologous recombination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1952.

Abstract 1237: Preclinical evaluation of ERX-41 in triple negative breast cancer

Abstract Background: Women with triple-negative breast cancer (TNBC) have a more aggressive clinical course, with a higher propensity to metastasize and a worse outcome due to a lack of effective therapies and significant intratumoral and intertumoral heterogeneity. Development of novel therapeutic strategies represents a clear unmet need. We have developed a first-in-class compound, an oligobenzamide, ERX-41, that has anti-proliferative activity against all six molecular subtypes of TNBC. Methods: In vitro activity of ERX-41 on TNBC cell lines was tested using CellTiter glo, MTT, and apoptosis assays. Efficacy of ERX-41 was tested using TNBC patient derived explants (PDEs) ex vivo, cell line-derived xenografts (CDXs), and patient derived xenografts (PDX) in vivo. To examine the mechanism, we conducted mass spec analyses using total lysates of TNBC cells treated with vehicle or ERX-41. Results: ERX-41 demonstrated potent activity in both blocking proliferation and inducing apoptosis in 30 distinct cell line models of TNBC, (representing all six molecular subtypes of TNBC), with an IC50 that ranges from 50-250nM. Incubation of ERX-41 with PDEs from primary TNBC patient tumors ex vivo caused a significant reduction in proliferation indices, as measured by Ki67 staining. ERX-41 also decreased proliferation and increased apoptosis in explants from TNBC CDX and PDX tumors cultured ex vivo. Oral administration of ERX-41 (10 mg/kg/daily) was shown to be non-toxic and dramatically limited the growth of CDX tumors derived from MDA-MB-231, SUM-159 or D2A1 syngeneic tumors. Importantly, ERX-41 treatment also significantly reduced tumor progression in four TNBC PDX (PDX-1, PDX-89, PDX-96 and PDX-98) models compared to the vehicle treated control group. Our ultrastructural and molecular studies indicate that ERX-41 induces significant endoplasmic reticulum (ER) stress within TNBC cells but not in primary epithelial cells. Global mass spectrometry studies indicated that ERX-41 treatment resulted in the alteration [down regulation (265 proteins) or upregulation (218 proteins)] of 483 proteins out of ~4000 proteins quantified with two or more peptides. Reactome pathway analysis indicated that the top pathways modulated by ERX-41 included Intra-Golgi and retrograde Golgi-to-ER traffic, membrane trafficking and TP53 mediated apoptosis. ER stress induced by ERX-41 blocks de novo protein synthesis, and triggers ER-assisted degradation (ERAD) pathways, causing TNBC apoptotic cell death. Conclusions: ERX-41 is orally bioavailable, non-toxic, and demonstrated activity in primary PDEs, CDXs and PDXs. The ability of ERX-41 to induce ER stress and apoptotic cell death in multiple types of TNBC suggests that this drug targets a fundamental weakness in TNBC cells (the high basal level of ER stress) and can effectively overcome the heterogeneity of TNBC. These studies strongly support the further clinical translation of ERX-41. Citation Format: Suryavathi Viswanadhapalli, Xihui Liu, Shi-Hong Ma, Tae-Kyung Lee, Mengxing Li, Weiwei Tang, Junhao Liu, Xiaonan Li, Gangadhara R. Sareddy, Rajeshwar Rao Tekmal, Jung-Mo Ahn, Ratna K. Vadlamudi, Ganesh V. Raj. Preclinical evaluation of ERX-41 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1237.

Clonal fitness inferred from time-series modelling of single-cell cancer genomes.

Progress in defining genomic fitness landscapes in cancer, especially those defined by copy number alterations (CNAs), has been impeded by lack of time-series single-cell sampling of polyclonal populations and temporal statistical models1-7. Here we generated 42,000 genomes from multi-year time-series single-cell whole-genome sequencing of breast epithelium and primary triple-negative breast cancer (TNBC) patient-derived xenografts (PDXs), revealing the nature of CNA-defined clonal fitness dynamics induced by TP53 mutation and cisplatin chemotherapy. Using a new Wright-Fisher population genetics model8,9 to infer clonal fitness, we found that TP53 mutation alters the fitness landscape, reproducibly distributing fitness over a larger number of clones associated with distinct CNAs. Furthermore, in TNBC PDX models with mutated TP53, inferred fitness coefficients from CNA-based genotypes accurately forecast experimentally enforced clonal competition dynamics. Drug treatment in three long-term serially passaged TNBC PDXs resulted in cisplatin-resistant clones emerging from low-fitness phylogenetic lineages in the untreated setting. Conversely, high-fitness clones from treatment-naive controls were eradicated, signalling an inversion of the fitness landscape. Finally, upon release of drug, selection pressure dynamics were reversed, indicating a fitness cost of treatment resistance. Together, our findings define clonal fitness linked to both CNA and therapeutic resistance in polyclonal tumours.

AR Is Not an Independent Marker for TNBC: The Lesson We Learn From Two PDX Models

Extensive efforts, through cell line-based models, have been made to characterize the androgen receptor (AR) signaling pathway in triple-negative breast cancer (TNBC). However, these efforts have not yet reached a consensus with regards to the mechanism of AR in TNBC. On the other hand, patient-derived xenografts (PDXs) are generally considered more appropriate than cell line-based models for recapitulating the structural and molecular features of a patient’s tumor, but only a few have been reported to be AR-positive TNBC. In our study, we identified and molecularly characterized two new, AR-positive TNBC PDX models and assessed the impacts of AR agonist (DHT) and antagonist (enzalutamide) on tumor growth and gene expression profiles by utilizing immunohistochemistry (IHC), western blots, and RNA-Seq and TNBC subtyping analyses. Two PDX models, termed TN1 and TN2, were derived from two grade 3 TNBC tumors, each containing 1~5% of AR positive tumor cells. DHT activated AR in both PDX tumors by increasing AR nuclear localization and protein levels. However, the endpoint tumor volume of DHT-treated TN1 was 3-folds smaller than that of non-treated TN1 tumors. Conversely, the endpoint tumor volume of DHT-treated TN2 was 2-folds larger than that of non-treated TN2. Moreover, enzalutamide failed to antagonize DHT-induced tumor growth in TN2. The RNA-Seq analyses revealed that DHT suppressed gene expression in TN1 (961 down-regulated genes versus 149 up-regulated genes), while the DHT promoted gene expression in TN2 (673 up-regulated genes versus192 down-regulated genes). TNBC subtyping analyses based on RNA-Seq data predicted distinct molecular subtypes of TN1 and TN2: TN1 correlated to a basal-like 1 (BL1) subtype, and TN2 correlated to a basal-like 2 (BL2) subtype. These analyses suggest that TN1 and TN2, which both express functional AR, are two molecularly distinct PDX models that expand our current knowledge of AR-positive TNBC. Our results do not support that AR is a suitable therapeutic target in TNBC. To our best knowledge, the molecular mechanisms of AR in TNBC are equivocal and should be evaluated using clinically relevant models, considering both the heterogeneous expression of AR in TNBC and the general complexities of AR signaling.

Antibody-drug conjugate MORAb-202 exhibits long-lasting antitumor efficacy in TNBC PDx models.

The antibody‐drug conjugate (ADC) MORAb‐202, consisting of farletuzumab paired with a cathepsin B–cleavable linker and eribulin, targets folate receptor alpha (FRA), which is frequently overexpressed in various tumor types. MORAb‐202 was highly cytotoxic to FRA‐positive cells in vitro, with limited off‐target killing of FRA‐negative cells. Furthermore, MORAb‐202 showed a clear in vitro bystander cytotoxic effect in coculture with FRA‐positive/negative cells. In vivo antitumor efficacy studies of MORAb‐202 were conducted with a single administration of MORAb‐202 in triple‐negative breast cancer (TNBC) patient–derived xenograft (PDx) models expressing low and high levels of FRA. MORAb‐202 exhibited durable efficacy proportional to tumor FRA expression. Toxicology studies (Q3Wx2) in nonhuman primates suggested that the major observed toxicity of MORAb‐202 is hematologic toxicity. Overall, these findings support the concept that MORAb‐202 represents a promising investigational ADC for the treatment of TNBC patients.

Activity of docetaxel, carboplatin, and doxorubicin in patient-derived triple-negative breast cancer xenografts

Triple-negative breast cancer (TNBC) is highly responsive to neoadjuvant polychemotherapy regimens including anthracyclines, taxanes, and, more recently, carboplatin. However, there is inadequate information on the individual contribution of each of these agents to the global activity of the combinations, and the use of combinations of up to four of these drugs is associated with relevant toxicity. Identifying single-drug activity in the clinical neoadjuvant setting is challenging. We developed patient-derived xenografts (PDXs) from several chemotherapy-naïve TNBC samples to assess the antitumor activity of single drugs and combinations of drugs. PDXs were established from chemotherapy-naïve TNBC samples. Nine TNBC PDX models (all of which corresponded to a basal-like phenotype according to the PAM50 classifier) were treated with carboplatin, docetaxel, and doxorubicin and the combination of docetaxel and carboplatin. Only one of nine PDX models showed sensitivity to doxorubicin, while eight of nine PDX models showed sensitivity to docetaxel and carboplatin as single agents. The 3 PDX models derived from patients with gBRCA-1 or gPALB2 mutations were very sensitive to carboplatin single agent. All 6 PDX models from patients without hereditary germ-line mutations showed increased sensitivity to the combination of docetaxel and carboplatin. In the present study, docetaxel and carboplatin single agents were active drugs against basal-like TNBC, while doxorubicin monotherapy showed low activity. The combination of docetaxel and carboplatin was more effective than the drugs used as single agents, except in the PDX from patients with gBRCA1/PALB2 mutations.

Abstract PS8-20: Bc mps a novel breast cancer microphysiological system

Abstract Breast cancer (BC) is among the most commonly diagnosed cancers in women and is the leading cause of malignant death in U.S. women. The triple negative breast cancer (TNBC) subtype is more aggressive and has a poorer prognosis compared to other subtypes. Few treatments exist for TNBC, partially due to limitations of current preclinical models. Current approaches to drug development in TNBC rely on simple, in vitro models. However, TNBCs are marked by cellular heterogeneity and complex interactions with the tumor microenvironment. Preclinical data from models that do not capture this complexity have yielded poor translational results. Patient derived xenografts (PDX), human tumors transplanted and grown in mice, are a newer, better model of TNBC. However, there are barriers when using mouse models: mice stroma can take over the PDX tumors, using immunocompromised mice can prevent immune responses and site-specific interaction, and mice are expensive. To overcome these barriers, a translational microphysiological system (MPS) was developed that is capable of maintaining the primary, human breast microenvironment in vitro. By seeding these MPS with breast cancer cell lines or tumor explants, we produce breast cancer MPS (BC-MPS).Here, we demonstrate BC-MPS’ stability ex vivo; the models remain healthy and viable for at least 2 weeks. This allows for long term studies on breast cancer and human breast tissue interactions. Different BC cell lines have been studied in the system. Initial comparisons between TNBC and ER+ cell lines showed a more aggressive remodeling of the human breast tissue (HBT) ECM by MDA-231 compared to MCF7. These cell lines have remained viable up to 14 days within the system. To examine the ability of BC-MPS to support PDX explant viability ex vivo, the TNBC PDX models 4QAN and 4IC were excised and seeded alone or in the BC-MPS system. After 6 days of incubation, viability was assessed by flow cytometry. Live/dead staining demonstrated that the 4QAN and the4IC PDX tumor explants have better viability in the BC-MPS model compared to being cultured in only media.After demonstrating the model’s capabilities of supporting PDX tumor viability, assays were performed to test experimental capabilities of BC-MPS. In addition to tumor architectural changes, cells in the system were treated with paclitaxel, and their response was monitored by luciferase imaging, demonstrating that BC-MPS can be used for drugstudies. BC-MPS is a promising new translational microphysiological system that facilitates studying long term interactions between real human breast tissue and cancer cells as well as the native tumor environment in HBT. The BC-MPS system’s ability to support the growth of established cell lines as well as PDXs has been demonstrated. Future studies will focus on developing the model for drug discovery studies. Citation Format: Katherine Hebert, Loren Brown, Khoa Nguyen, Madlin Alzoubi, Margarite Matossian, Bridgette Collins-Burow, Matthew Burow, Frank Lau. Bc mps a novel breast cancer microphysiological system [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-20.

Functional characterization of androgen receptor in two patient-derived xenograft models of triple negative breast cancer

Extensive efforts, through cell line-based models, have been made to characterize the androgen receptor (AR) signaling pathway in triple-negative breast cancer (TNBC). However, these efforts have not yet reached a consensus with regards to the mechanism of AR in TNBC. Considering that patient-derived xenografts (PDXs) are more appropriate than cell line-based models for recapitulating the structural and molecular features of a patient’s tumor, we have identified and molecularly characterized two new AR-positive TNBC PDX models and assessed the impacts of AR agonist [dihydrotestosterone (DHT)] and antagonist (enzalutamide) on tumor growth and gene expression profiles by utilizing immunohistochemistry, western blots, and RNA-Seq analyses. Two PDX models, termed TN1 and TN2, were derived from two grade-3 TNBC tumors, each harboring 1∼5% of AR nuclear positive cancer cells. DHT activated AR in both PDX tumors by increasing nuclear localization and AR protein levels. However, the endpoint tumor volume of DHT-treated TN1 was 3-folds smaller than that of non-treated TN1 tumors. Conversely, the endpoint tumor volume of DHT-treated TN2 was 2-folds larger than that of non-treated TN2. Moreover, enzalutamide failed to antagonize DHT-induced tumor growth in TN2. The RNA-Seq analyses revealed that DHT mainly suppressed gene expression in TN1 (961 down-regulated genes versus 149 up-regulated genes), while DHT promoted gene expression in TN2 (673 up-regulated genes versus 192 down-regulated genes). RNA-Seq data predicted distinct TNBC molecular subtypes for TN1 and TN2. TN1 correlated to a basal-like 1 (BL1) subtype, and TN2 correlated to a basal-like 2 (BL2) subtype. These analyses suggest that TN1 and TN2, which both express functional AR, are two molecularly distinct PDX models. The molecular characterization of these PDX models expands our current knowledge on AR-positive TNBC. Our results do not support that AR is a suitable therapeutic target in TNBC. To our best knowledge, the molecular mechanisms of AR in TNBC are equivocal and should be evaluated using clinically relevant models, considering both the heterogeneous expression of AR in TNBC and the general complexities of AR signaling.

Abstract 3182: Kindlin-1 expression is associated with EGFR/RAS/MAPK activation and response to MEK inhibitors in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) accounts for ~15% of all invasive breast cancers. This heterogeneous group of tumors is the most aggressive and difficult-to-treat subtype of breast cancer. We previously demonstrated that Kindlin-1 is overexpressed in TNBC and patients with high Kindlin-1 have a worse prognosis. We determined that Kindlin-1 plays a major role in the epithelial to mesenchymal transition, tumor growth and breast cancer metastasis. TNBC have also been reported to express high levels of EGFR, in ~70% of the cases. Unfortunately, anti-EGFR therapy in TNBC fails to show a clinical benefit. In this study, we aimed to investigate a potential link between EGFR/RAS/MAPK pathway and Kindlin-1 in TNBC, and to propose new therapeutic strategies based on our findings. Methods: First, a gene set enrichment analysis was performed in 58 breast cancer cell lines (CCLE dataset) to detect the signaling pathways differentially enriched in cells expressing high levels of Kindlin-1. We next evaluated whether Kindlin-1 expression could be associated to an increased MAPK signaling by testing the mutational status and phosphorylation of key effectors of the pathway. To determine whether Kindlin-1 could be a predictive biomarker for the response to MEK inhibitors, we assessed its expression in sensitive versus resistant cell lines. Finally, we examined in vivo the anti-tumor efficacy of Selumetinib in a series of 27 triple negative breast cancer patient derived xenografts (PDX), highly representative of the originating tumor in terms of biology and therapeutic sensitivity. These PDX models have been characterized for Kindlin-1 expression (at RNA and protein levels). The sensitivity to MEKi of the TNBC PDX models was examined with regard to their Kindlin-1 expression. Results: Gene set enrichment analysis put in evidence that EGFR/RAS/MAPK pathway is upregulated in breast cancer cells with high Kindlin-1 expression. Although mutations are infrequent in breast cancer, an increased Kindlin-1 expression was observed in a subset of EGFR/RAS-activated cell lines (mutated or amplified). Moreover, TNBC expressing high levels of Kindlin-1 showed an increased phosphorylation of key effectors (ERK, MEK). We also demonstrated that Kindlin-1 mRNA expression was significantly increased in sensitive cell lines to different MEK inhibitors such as Trametinib (p=0.007) or Selumetinib (p=0.005) compared with resistant cells. In addition, in preclinical TNBC PDX models treated with Selumetinib there was a correlation between tumor growth inhibition and Kindlin-1 protein expression (r=0.57; p=0.017). Conclusion: Our findings indicate that Kindlin-1 expression may be a promising predictive biomarker of MEK inhibitors response in TNBC and may offer new therapeutic opportunities for patients with limited treatment options and refractory cancer types to current treatments. Citation Format: Paula Azorin, Florian Bonin, Zakia Tariq, Florence Coussy, Elisabetta Marangoni, Rosette Lidereau, Keltouma Driouch. Kindlin-1 expression is associated with EGFR/RAS/MAPK activation and response to MEK inhibitors in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3182.

Abstract 562: Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors

Abstract Background: Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BC patients. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elicit feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. The objective of this study is to examine the therapeutic efficacy of combination therapy using preclinical and patient-derived xenograft (PDX) models. Methods: We tested utility of combination therapy using multiple HDACIs that are currently in clinical trails along with EC359. The effect of combination therapy was evaluated using MTT, invasion, colony formation, and Caspase3/7 assays. Mechanistic studies were performed using Western blotting, qRT-PCR, and STAT3 reporter assays. The efficacy of combination therapy in vivo was examined using xenograft, PDX, and patient-derived explant (PDEx) models. Results: Immunohistochemical analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC tissues. Treatment of TNBC model cells with four different HDACIs increased the expression of LIFR. LIFR inhibitor EC359 at nM concentration is additive to HDACIs in reducing cell viability. Knockdown of LIFR or treatment with EC359 significantly enhanced the efficacy of HDACIs in reducing the cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy in TNBC model cells. On the contrary, treatment with STAT3 inhibitor requires µM concentrations to reduce the cell viability of TNBC cells and is not additive to HDACIs. Mechanistic studies utilizing STAT3 reporter gene assays and biochemical studies using multiple TNBC model cells exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359 therapy. Treatment of human TNBC utilizing PDEx assays showed that EC359 enhanced the ability of HDACIs to decrease proliferation (Ki-67 positivity) compared to monotherapy. Using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDACIs in treating TNBC. Supported by DOD BCRP grant W81XWH-18-1-0016 (R.K. Vadlamudi; K.J. Nickisch) Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P. Pratap, Yiliao Luo, Junhao Liu, Kristin A. Altwegg, Xiaonan Li, Ahmed Gulzar, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R. Sareddy, Manjeet K. Rao, Rajeshwar R. Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 562.

Abstract 676A: Overcoming PARP inhibitor resistance with molecularly guided rational combinations in triple-negative breast cancer patient-derived xenograft models

Abstract Introduction: A constant challenge in the field of cancer therapeutics is the presence of intrinsic resistance or the development of acquired resistance to chemotherapeutic and targeted agents. One of the approaches to overcome drug resistance is the utilization of combination therapies that simultaneously target several molecules involved in cancer development, progression, and metastasis. Numerous methods are utilized to guide the selection of the appropriate combination therapy. We propose a molecularly guided approach for therapy selection to overcome PARP inhibitor resistance. Methods: We used a multipronged strategy to characterize triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) models. We identified copy number alterations and pathogenic germline and somatic variants obtained from genomic sequencing. Proteomic and transcriptomic data were analyzed to evaluate gene expression affected by PARP inhibitor therapy. ATM expression status was also determined by immunohistochemistry. Additionally, a comprehensive literature review was performed focused on PARP inhibitor resistance mechanisms to support our rationale for combination therapy selections. We tested selected combination therapies using a cell viability screening assay in PARP inhibitor-resistant TNBC cell lines. Synergistic combinations were tested in vivo in PARP inhibitor-resistant TNBC PDX models. Results: Model specific pathogenic genomic alterations in PARP inhibitor-resistant TNBC PDX models included genes in the PI3K/AKT/mTOR, DNA damage response, MAPK, and apoptosis signaling pathways. Reverse phase protein array analysis demonstrated differential expression and upregulation of proteins involved in the PI3K/AKT/mTOR and MAPK signaling pathways. Differential gene expression data obtained by RNA sequencing analysis was aggregated to further enhance the individual molecular characterization of PDX models. Subsequently, the antitumor efficacy of selected rational combination therapies was tested in vitro and in vivo. Conclusions: Our study underscores the importance of a detailed analysis of key molecular biomarkers for therapy selection and proposal of rational combinations to overcome resistance to PARP inhibitors. The translational relevance of this method relies on the potential to recapitulate this approach in patient tumor samples and the opportunity to generate and expand PDX resistant models to test the antitumor efficacy of several rational combinations. Our ultimate goal is to identify the right personalized therapy for the individual cancer patient. Citation Format: Christian X. Cruz Pico, Kurt W. Evans, Ken Chen, Xiaofeng Zheng, Argun Akcakanat, Ming Zhao, Coya Tapia, Stephen M. Scott, Erkan Yuca, Funda Meric-Bernstam. Overcoming PARP inhibitor resistance with molecularly guided rational combinations in triple-negative breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 676A.

Dual TGF-β and AURKA targeting enhances chemosensitivity in triple-negative breast cancer.

e13106 Background: Chemotherapy resistance remains a significant barrier in the effective treatment of patients with triple negative breast cancer (TNBC). TGF-β signaling is a well characterized oncogenic pathway in TNBC that promotes chemoresistance by inducing epithelial to mesenchymal transition (EMT) and tumor stemness. Aurora-A kinase (AURKA) is a serine/threonine kinase responsible for chromosomal stability during mitosis. Significantly, aberrant expression of AURKA induces breast cancer progression. We hypothesized that aberrant activation of TGF-β and AURKA signaling pathways contributes to chemoresistance in TNBC by promoting EMT and tumor stemness and that dual-targeting of these oncoproteins will enhance chemosensitivity. Methods: RNA-Seq data were analyzed from patient derived xenografts (PDx) established from patients with Stage I-III TNBC who received pre-operative taxane and anthracycline based chemotherapy. Chemoresistance was defined as an RCB score of 1-3, and chemosensitivity was defined as an RCB score of 0. Unique TNBC cell lines developed from brain metastasis PDxs (TNBC M14, TNBC M25) were treated in vitro with 10nM docetaxel (DTX), 50nM LY2157299 (TGF-β inhibitor) and 50nM MLN8237 (AURKA inhibitor). EMT reprogramming was determined by measuring the expression of vimentin, ALDH activity and mammosphere growth. Apoptotic cells following drug treatment were labeled with Red Annexin-V marker and monitored in real time using the IncuCyte instrument. Results: RNA-Seq revealed that there was no baseline difference in expression of AURKA between chemoresistant and chemosensitive TNBC PDxs. However, there was a 2.7-fold increase in AURKA expression in the post-treatment PDx models compared to pre-treatment PDx models. In vitro treatment of the M14 and M25 cell lines with DTX demonstrated no significant increase in apoptotic cells compared to control, whereas treatment with the combination of DTX, LY2157299 and MLN8237 resulted in a two-fold increase in apoptosis compared to treatment with DTX alone (p = 0.0068 M14, p = 0.003 M25). Dual-targeting with LY2157299 and MLN8237 reduced the expression of vimentin and ALDH activity. Conclusions: TGF-β and AURKA play a central role inducing EMT and a stem-cell-like phenotype in TNBC that confers chemoresistance. AURKA is up-regulated after exposure to chemotherapy in chemoresistant TNBC PDx models. LY2157299 and MLN8237 reduce TNBC stemness in M14 and M25 cell lines and enhance DTX chemosensitivity. Dual-targeting of TGF-β and AURKA is a potentially promising approach in chemoresistant TNBC.

Simultaneous targeting of HER family pro-survival signaling with Pan-HER antibody mixture is highly effective in TNBC: a preclinical trial with PDXs

BackgroundThe human epidermal growth factor receptor (HER) family, notably EGFR, is overexpressed in most triple-negative breast cancer (TNBC) cases and provides cancer cells with compensatory signals that greatly contribute to the survival and development of resistance in response to therapy. This study investigated the effects of Pan-HER (Symphogen, Ballerup, Denmark), a novel mixture of six monoclonal antibodies directed against members of the HER family EGFR, HER2, and HER3, in a preclinical trial of TNBC patient-derived xenografts (PDXs).MethodsFifteen low passage TNBC PDX tumor samples were transferred into the right mammary fat pad of mice for engraftment. When tumors reached an average size of 100–200 mm3, mice were randomized (n ≥ 6 per group) and treated following three 1-week cycles consisting of three times/week intraperitoneal (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.ResultsAll 15 TNBC PDXs were responsive to Pan-HER treatment, showing significant reductions in tumor growth consistent with Pan-HER-mediated tumor downmodulation of EGFR and HER3 protein levels and significantly decreased activation of associated HER family signaling pathways AKT and ERK. Tumor regression was observed in five of the models, which corresponded to those PDX tumor models with the highest level of HER family activation.ConclusionsThe marked effect of Pan-HER in numerous HER family-dependent TNBC PDX models justifies further studies of Pan-HER in TNBC clinical trials as a potential therapeutic option.

Targeting triple-negative breast cancers with the Smac-mimetic birinapant

Smac mimetics target inhibitor of apoptosis (IAP) proteins, thereby suppressing their function to facilitate tumor cell death. Here we have evaluated the efficacy of the preclinical Smac-mimetic compound A and the clinical lead birinapant on breast cancer cells. Both exhibited potent in vitro activity in triple-negative breast cancer (TNBC) cells, including those from patient-derived xenograft (PDX) models. Birinapant was further studied using in vivo PDX models of TNBC and estrogen receptor-positive (ER+) breast cancer. Birinapant exhibited single agent activity in all TNBC PDX models and augmented response to docetaxel, the latter through induction of TNF. Transcriptomic analysis of TCGA datasets revealed that genes encoding mediators of Smac-mimetic-induced cell death were expressed at higher levels in TNBC compared with ER+ breast cancer, resulting in a molecular signature associated with responsiveness to Smac mimetics. In addition, the cell death complex was preferentially formed in TNBCs versus ER+ cells in response to Smac mimetics. Taken together, our findings provide a rationale for prospectively selecting patients whose breast tumors contain a competent death receptor signaling pathway for the further evaluation of birinapant in the clinic.

Abstract A49: A dual blockade of N-CoR2- and immune checkpoints induces complete remissions in treatment-refractory tumors

Abstract Background: The majority of human malignant tumors are resistant or only partially respond to conventional chemotherapy (C/T) or immunotherapy (I/T) such as immune checkpoint inhibitors (ICIs). Irrespective of the treatments, efficient tumor-cell killing requires amplification of inflammatory signaling, which, however, is tightly regulated by various “checkpoint” mechanisms evolved by epithelial cells to prevent excessive tissue damage induced by virus and immune attack. Novel approaches to disable theses conserved and cell-intrinsic inflammation checkpoints may provide breakthrough and “tumor-agnostic” strategies to circumvent the innate treatment resistance to unleash the full potential of C/T and I/T in treatment-refractory and highly lethal malignant tumors, such as triple-negative breast cancer (TNBC), pancreatic ductal adenocarcinoma (PDAC), and glioblastoma multiform (GBM). Materials and Methods: We designed and conducted integrated genomic and proteomic screening combined with molecular and functional studies to identify conserved anti-inflammatory pathways that mediate innate and cell-intrinsic resistance to C/T and I/T agents. Preclinical studies were used to validate a gene therapy strategy to disable the inflammation-checkpoint identified from this process. Results: We uncovered that the cytotoxic and immunogenic death induced by C/T and I/T agents is constrained by repression of a toll-like receptor-2 (TLR-2)/TLR-3- and NF-kB-induced interferon regulatory factor-1 (IRF-1) and interferon (IFN)-gamma anti-viral response program in various malignant tumor cells, including breast cancer, PDAC and GBM. Loss- and gain-of-function studies implicated that co-repressor-2 (N-CoR2) co-translocated with NF-kB p50 into cell nuclei in response to therapy, wherein it serves as an epigenetic checkpoint of this inflammation program by mediating a histone deacetylase-dependent chromatin remodeling and repression of a specific panel of proinflammatory and proapoptotic genes. Thus, high N-CoR2 expression predicts treatment refractoriness and poor prognosis in neoadjuvant or adjuvant-treated breast cancer patients. Blockade of the epigenetic checkpoint function of N-CoR2 by a small decoy of N-CoR2 hypersensitized malignant cells to assorted C/T agents, death ligands, and IFN-gamma. Consistently, intratumoral delivery of the N-CoR2 checkpoint blockade dramatically potentiated systemic C/T and ICI therapies, including anti-PD-1 and anti-CTLA-4 antibodies, and completely halted tumor growth or induced remissions in orthotopic and patient-derived xenograft models of TNBC and GBM. Conclusion: Our findings suggest that malignant tumors can access intrinsically conserved anti-inflammatory mechanisms that enable them to escape from C/T and I/T. As such, strategies that can override this defense program constitute novel antitumor gene therapies that may be applied to overcome resistance in treatment-refractory tumors and improve patient prognosis. Citation Format: Kelvin Tsai, Valerie M. Weaver. A dual blockade of N-CoR2- and immune checkpoints induces complete remissions in treatment-refractory tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A49.