Preclinical Triple-negative Breast Cancer Research Articles

Therapeutic potential of Fingolimod in triple negative breast cancer preclinical models

Surgery followed by a chemotherapy agent is the first-line treatment for breast cancer patients. Nevertheless, new targets are required for women with triple-negative breast cancer (TNBC) in order to improve the treatment of this aggressive cancer subtype. Multiple pro-inflammatory molecules including lipid-based substances such as sphingosine-1-phosphate (S1P) promote cancer progression. In this preclinical study, we aim to investigate the efficacy of Fingolimod, an inhibitor of S1P / S1P receptors axis, already approved as an immunomodulator in multiple sclerosis.The impact of Fingolimod was analyzed using in vitro 2D and 3D cell survival analysis and in vivo orthotopic graft models, using mouse and human TNBC cells implanted in immunocompetent or immunodeficient mice, respectively. Resection of the tumor primary mass was also performed to mimic the clinical standard of care.We demonstrated that Fingolimod repressed tumor cell survival in vitro. We also showed in preclinical mouse TNBC models that Fingolimod repressed tumor progression and liver and spleen metastases without apparent adverse effects on the animals. Our data indicate that Fingolimod induces tumor cells apoptosis and thereby represses tumor progression.Globally, our data suggest that Fingolimod merits further evaluation as a potential therapeutic opportunity for TNBC.

Noncanonical pS727 post translational modification dictates major STAT3 activation and downstream functions in breast cancer

Activation of STAT3 via Y705-phosphorylation is well documented across multiple cancer types and thus forms the basis of canonical pathway to judge STAT3 activation. Recently, important roles of two other post translational modification (PTM) sites, i.e. S727-phosphorylation and K685-acetylation, leading to STAT3 activation are reported. However, their critical mode of function in controlling STAT3 dimerization and signaling, independent of canonical activation remains elusive. Therefore, to understand the functional relevance of each STAT3 PTMs in breast cancer (BC), cell models are developed by stable overexpression of PTM-site specific point mutants, i.e. Y705F, S727A or K685R, in a 3′UTR-STAT3 knockdown BC cell background. Results using this model system reveal novel findings showing that phosphorylation at S727 can lead to STAT3 activation independent of phosphoY705. We also demonstrate that loss of pS727 or K685ac significantly affects functional phenotypes such as cell survival and proliferation as well as downstream transcriptional activity (Twist 1, Socs3, c-Myc, Bcl-1 and Mcl-1) of STAT3. Thereafter, by utilizing a BRET biosensor for measuring STAT3 phosphorylation in live cells, a crucial role of pS727 in dictating STAT3 activation and homodimerization formation is uncovered. Further by performing retrospective IHC analysis of total and phospho-forms of STAT3 in a cohort of 76 triple negative breast cancer (TNBC) patient samples, a significant dominant expression of phosphoS727 over phosphoY705 PTM (p < 0.001) is found in STAT3 positive cases. We also focus on validating known STAT3 inhibitor molecules for their action against both pY705 and pS727 activation. This study for the first time demonstrates that an anti-helminth drug compound, Niclosamide, is capable of inactivating both phospho-PTM sites on STAT3 and exhibits excellent anticancer efficacy in preclinical TNBC tumour model.

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 B15: Gut microbiome populations modulate neoadjuvant chemotherapy responsiveness in preclinical triple-negative breast cancer murine model

Abstract Triple-negative breast cancer (TNBC) is a highly aggressive subtype with a 5-year survival rate significantly worse when compared to other breast cancer types. There are no targeted therapy options, limiting these patients to cytotoxic chemotherapy regimens. Our group demonstrated increased tumoral Pseudomonas abundance in breast cancer patients receiving neoadjuvant chemotherapy, suggesting that systemic anticancer therapy administration may modulate microbiota populations. The purpose of our study was to determine whether gut microbiota populations correlate with chemotherapeutic responsiveness and may be a predictive biomarker outcome. We wanted to determine the impact of shifting the microbiome on chemotherapy efficacy. To do so, 8-week old female BALB/c mice were injected with 4T1-luciferase cells in the mammary fat pad. Once tumors reached 100 mm3, mice were either untreated (control group), treated with 1x weekly 2.5 mg/kg doxorubicin (DOX) for 4 weeks, or treated with doxorubicin +antibiotics (mixture of streptomycin, ampicillin, and colistin in the drinking water to ablate the microbiome). Tumor size was monitored. Tumors and lungs were collected after the study. Fecal samples were collected at T0 (before treatment) and T4 (after treatment). Mice receiving DOX were stratified into DOX-responders or DOX-nonresponders based upon tumor size. Mice from DOX-responders and DOX +antibiotics groups displayed reduced tumor weight and decreased lung metastatic burden. 16S-bacterial sequencing indicates elevated fecal Ruminococcus correlates with DOX-nonresponsiveness and increased abundance of Oscillospira and Bacteroidales is associated with better therapeutic outcome. Protein analysis of tumor tissue indicates a significant increase in apoptosis in DOX-responders and DOX +antibiotic groups. To determine whether modulating the gut microbiome influences drug responsiveness, BALB/c mice were stratified into a control group or group receiving lard diet-derived fecal transplant (LDFT) by oral gavage. Mice were injected with 4T1-luciferase cells in the mammary fat pad. Once tumors reached 100 mm3, mice were treated with 1x weekly 2.5 mg/kg DOX for 4 weeks. Tumors and lungs were collected at the end of the study. Fecal samples were collected at T0 (before gavage), T3 (after 3 weeks of LDFT and before DOX-treatment), and T7 (at end of study). All tumors from LDFT-group were larger and displayed reduced chemotherapy responsiveness when compared with control animals, suggesting gut microbiome populations can modulate chemotherapy resistance. Taken together, our data demonstrate that chemotherapy efficacy is modulated by gut microbiome and suggest that modulation of the gut microbiome through dietary or probiotic interventions may affect therapeutic outcomes. Moreover, fecal microbiota populations could be used as a predictive biomarker of chemotherapeutic responsiveness. Citation Format: Kenysha Clear, Adam Wilson, Akiko Chiba, Katherine L. Cook, Alaa Bawaneh. Gut microbiome populations modulate neoadjuvant chemotherapy responsiveness in preclinical triple-negative breast cancer murine model [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr B15.

Wee1 Inhibition Enhances the Anti-Tumor Effects of Capecitabine in Preclinical Models of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive subtype defined by lack of hormone receptor expression and non-amplified HER2. Adavosertib (AZD1775) is a potent, small-molecule, ATP-competitive inhibitor of the Wee1 kinase that potentiates the activity of many DNA-damaging chemotherapeutics and is currently in clinical development for multiple indications. The purpose of this study was to investigate the combination of AZD1775 and capecitabine/5FU in preclinical TNBC models. TNBC cell lines were treated with AZD1775 and 5FU and cellular proliferation was assessed in real-time using IncuCyte® Live Cell Analysis. Apoptosis was assessed via the Caspase-Glo 3/7 assay system. Western blotting was used to assess changes in expression of downstream effectors. TNBC patient-derived xenograft (PDX) models were treated with AZD1775, capecitabine, or the combination and assessed for tumor growth inhibition. From the initial PDX screen, two of the four TNBC PDX models demonstrated a better response in the combination treatment than either of the single agents. As confirmation, two PDX models were expanded for statistical comparison. Both PDX models demonstrated a significant growth inhibition in the combination versus either of the single agents. (TNBC012, p < 0.05 combo vs. adavosertib or capecitabine, TNBC013, p < 0.01 combo vs. adavosertib or capecitabine.) An enhanced anti-proliferative effect was observed in the adavosertib/5FU combination treatment as measured by live cell analysis. An increase in apoptosis was observed in two of the four cell lines in the combination when compared to single-agent treatment. Treatment with adavosertib as a single agent resulted in a decrease in p-CDC2 in a dose-dependent manner that was also observed in the combination treatment. An increase in γH2AX in two of the four cell lines tested was also observed. No significant changes were observed in Bcl-xL following treatment in any of the cell lines. The combination of adavosertib and capecitabine/5FU demonstrated enhanced combination effects both in vitro and in vivo in preclinical models of TNBC. These results support the clinical investigation of this combination in patients with TNBC, including those with brain metastasis given the CNS penetration of both agents.

Abstract A105: PARP inhibition modulates the infiltration, phenotype, and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the antitumor potential of TAMs

Abstract Patients with BRCA-associated triple-negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies the greatest infiltration of immune cells into the tumor was macrophages. Given that objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high-dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wild-type TNBC. Using a preclinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing costimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an antitumor phenotype. We also identified a novel negative feedback mechanism that limits the functionality of the anti-tumor TAMs and is consistent with induction of an immune-suppressive macrophage population. Utilizing transcriptomic, proteomic, and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biologic processes associated with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the preclinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by protumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Anita K. Mehta, Emily M. Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Christina A. Hartl, Nathan T. Johnson, William M. Oldham, Marian Kalocsay, Sarah A. Boswell, Olmo Sonzogni, Constantia Pantelidou, Brett P. Gross, Shawn Johnson, Deborah A. Dillon, Sandro Santagata, Judy E. Garber, Nadine Tung, Elizabeth A. Mittendorf, Gerburg M. Wulf, Geoffrey I. Shapiro, Peter K. Sorger, Jennifer L. Guerriero. PARP inhibition modulates the infiltration, phenotype, and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the antitumor potential of TAMs [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A105.

Abstract P5-05-07: Foxo3a activation contributes to the synergy of imipridone and radiation in triple-negative breast cancer

Abstract Purpose/Objective: Imipridone (IMIP) is a small molecule DRD2 antagonist that has shown efficacy in preclinical and early phase clinical studies in advanced cancers. Antitumor mechanisms of IMIP appears to be cell-type dependent, and may include Foxo3a-dependent TRAIL upregulation, ATF4/CHOP integrated stress response pathway, and upregulation of death receptors (DR4/DR5). Our previous work has demonstrated therapeutic synergy of combination treatment with IMIP and radiation (RT) in preclinical triple-negative breast cancer (TNBC) models. In this study, we investigated the potential mechanism of synergy in combination treatment with IMIP and RT in TNBC. Methods: In vitro experiments were performed in six TNBC cell lines (MB-468, BT-549, BT-20, MB-231, MB-436, and HCC1937). Cells were treated with IMIP alone, RT alone, or combination of IMIP and RT. RT (0-8 Gy) was delivered via a Gamma-cell 40 exactor and IMIP was given at doses of 0-2 µM. To evaluate the AKT-Foxo3a axis function, we examined the phosphorylation status of AKT and Foxo3a as a surrogate for their functional activities. Cells were treated with IMIP, followed by a single dose of RT (0, 4 or 8 Gy), and cells were harvested at 72 hours and 96 hours post-RT. Whole cell lysates were collected and Western blotting was performed with phosphorylation-specific antibodies of AKT and Foxo3a. Downstream Foxo3a target gene expression was evaluated via RT-qPCR, including TRAIL, PUMA, and various targets. To determine the dependency of Foxo3a, knockdown experiment by transfection of Foxo3a siRNA was performed. Results: Of the six TNBC cell lines tested, MB-468 and BT-20 cells were found to exhibit synergistic cell death from combination treatment with IMIP (1 µM) and RT (4 or 8 Gy). Western blotting of whole cell lysates of both MB-468 and BT-20 revealed a mild decrease in the inhibitory Foxo3a phosphorylation at S253 (Foxo3a-P S253) with RT alone at 96 hours post-RT, no effect with IMIP alone, but a marked reduction of Foxo3a-P S253 with combination of IMIP and RT, without significant changes in the total Foxo3a protein levels. No effect was observed with AKT phosphorylation at S473. In addition, cleaved caspase 3 levels were increased at 96-hours post-RT with combination treatment. RT-qPCR of extracted mRNA revealed Foxo3a target upregulation of apoptotic PUMA gene in both MB-468 and BT-20 cells, as well as death ligand TRAIL in BT-20 cells. Finally, transient depletion of Foxo3a level via siRNA demonstrated a partial rescue of treatment-induced cell death in MB-468 cells, but not in BT-20 cells. Conclusion: We demonstrated therapeutic synergy of IMIP and RT in select TNBC cell lines (MB-468 and BT-20). Synergistic decrease in the inhibitory S253 Foxo3a phosphorylation with combination treatment, independent of upstream AKT activity, correlated with increased cell death, increased cleaved caspase-3 levels, and activation of Foxo3a targets PUMA and TRAIL. Foxo3a knockdown partially rescued MB-468 cells, but not BT-20 cells, from combination treatment, suggesting that other cellular pathways may also contribute to the observed synergy of IMIP and RT in select TNBC cells. Citation Format: Shang-Jui Wang, Devora Schiff, Hao Wu, Sachin Jhawar, Bruce G Haffty. Foxo3a activation contributes to the synergy of imipridone and radiation in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-05-07.

Abstract P5-04-01: PARP inhibition modulates the infiltration, phenotype and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the anti-tumor potential of TAMs

Abstract Patients with BRCA-associated triple negative breast cancer (TNBC) have few effective treatment options. PARP inhibitors are promising, and we recently showed they induce an influx of white blood cells, including CD8+ T-cells and macrophages into the tumor. The influx of CD8+ cells, mediated by activation of the STING pathway in tumor cells, contributes substantially to efficacy of PARP inhibition in mice. Strikingly, in these studies, the greatest infiltration of immune cells into the tumor was macrophages. Given objective responses to PARP inhibition have been observed in clinical trials but the benefits are transitory, we hypothesized that this was presumably due to a suppressive tumor microenvironment, driven by tumor macrophages. To better understand the molecular basis of resistance to PARP inhibitors, we used high dimensional single-cell immune profiling on human TNBC. We observed a ≥10-fold increase in TAMs in BRCA-associated TNBC compared to BRCA-wildtype TNBC. Using a pre-clinical model of BRCA1-deficient triple-negative breast cancer, we found that PARP inhibitors not only further increased TAM abundance but also induced functional and phenotypic changes associated with STING pathway activation, antigen presentation, and chemokine and cytokine signaling. PARP inhibitors increased the frequency of TAMs expressing co-stimulatory molecules CD80 and CD86 as well as the activation and maturation marker CD40, which are indicative of an anti-tumor phenotype. We also identified a novel negative feedback mechanism which limits the functionality of the anti-tumor TAMs, and is consistent with induction of an immune suppressive macrophage population. Utilizing transcriptomic, proteomic and metabolic profiling of ex vivo cultured human myeloid cells, we identified multiple biological processes associate with PARP inhibition, showing that these drugs directly affect macrophage states and phenotypes. Remarkably, in the pre-clinical BRCA1-deficient TNBC model, the novel combination of PARP inhibition with macrophage modulation significantly extended remissions obtained with PARP inhibitor therapy only, and this advantage persisted when treatment was discontinued, suggestive of a durable reprogramming of the tumor microenvironment. Moreover, CD8+ cells were required for the extension of PARP inhibitor-induced remissions, suggesting that targeting macrophages lifted the constraints imposed by pro-tumor macrophages on CD8+ T cell-mediated tumor cell killing. We identify mechanisms related to macrophage and T-cell activation that increase PFS and provide evidence that TAMs may serve as targets for new therapeutic interventions designed to overcome PARP inhibitor resistance in BRCA-associated TNBC. Citation Format: Jennifer L Guerriero, Anita K Mehta, Emily M Cheney, Jessica A. Castrillon, Jia-Ren Lin, Mateus de Oliveira Taveira, Olmo Sonzogni, Constantia Pantelidou, Christina A Hartl, William M Oldham, Nathan T Johnson, Sarah A Boswell, Marian Kalocsay, Matthew J Berberich, Sholin Mei, Dan Wang, Shawn Johnson, Brett Gross, Deborah A Dillon, Mikel Lipschitz, Evisa Gjini, Scott Rodig, Sandro Santagata, Judy E Garber, Nadine Tung, Peter Sorger, Geoffrey I Shapiro, Gerburg M Wulf, Elizabeth A Mittendorf. PARP inhibition modulates the infiltration, phenotype and function of tumor-associated macrophages (TAMs) in BRCA-associated breast cancer and can be augmented by harnessing the anti-tumor potential of TAMs [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-01.

Abstract P1-19-25: Wee1 inhibition enhances the anti-tumor effects of capecitabine in preclinical models of triple negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype defined by lack of hormone receptor expression and non-amplified HER2. TNBC accounts for approximately 15% of breast cancer cases, however, is associated with an increased risk of cancer recurrence, brain metastasis, and death due to metastatic breast cancer. Mutations in p53 are common in TNBC, occurring in approximately 85% of tumors. While a number of promising targeted therapies are on the horizon in TNBC including immunotherapy, there remains an unmet need for active targeted therapies where chemotherapy remains the standard treatment for metastatic disease and results in a median survival of 12-18 months. Adavosertib (AZD1775) is a potent, small molecule, ATP-competitive inhibitor of the Wee1 kinase that potentiates the activity of many DNA-damaging chemotherapeutics and is currently in clinical development for multiple indications. AZD1775 potentiates the activity of DNA-damaging and antimetabolite chemotherapeutics in preclinical models without TP53-deficiency, possibly due to baseline replicative stress or compromised DNA repair proficiency. A previous unbiased screen of CTEP compounds in TNBC PDX models demonstrated that the combination of adavosertib and capecitabine/5FU had greater anti-proliferative effects than either of the single agents. The purpose of this study was to further investigate the combination of adavosertib and capecitabine/5FU in preclinical TNBC models. Methods: HCC1937, CAL51, MDA-MB-231 and MDA-MB-468 cells were plated in 96-well plates and exposed to increasing concentrations of adavosertib, 5FU, or the combination. Cellular proliferation was assessed in real-time using IncuCyte® Live Cell Analysis. Apoptosis was assessed via the Caspase-Glo 3/7 assay system. Western blotting was used to assess changes in expression of CDC2, phospho-CDC2, H2AX, and Bcl-xL. TNBC PDX models CU_TNBC_012 and CU_TNBC_013 were treated with vehicle, adavosertib, capecitabine, or the combination and assessed for tumor growth inhibition. Results: From the initial PDX screen, two of the four TNBC PDX models demonstrated a better response in the combination treatment than either of the single agents. As confirmation, two PDX models were expanded for statistical comparison. Both PDX models demonstrated a significant growth inhibition in the combination versus either of the single agents. (TNBC012, p&amp;lt;0.05 combo vs adavosertib or capecitabine, TNBC013, p&amp;lt;0.01 combo vs adavosertib or capecitabine ). An enhanced antiproliferative effect was observed in the adavosertib/5FU combination treatment as measured by live cell analysis. An increase in apoptosis was observed in two of the four cell lines in the combination when compared to single agent treatment. Treatment with single agent adavosertib resulted in an increase in p-cdc2 in a dose dependent manner that was also observed in the combination treatment. Similar results were observed with γH2AX in two of the four cell lines tested. No significant changes were observed in Bcl-xL following treatment in any of the cell lines. Conclusions: The combination of adavosertib and capecitabine/5-FU demonstrated enhanced combination effects both in vitro and in vivo in preclinical models of TNBC. These results support the clinical investigation of this combination in patients with TNBC, including those with brain metastasis given the CNS penetration of both agents. Citation Format: Todd M Pitts, Dennis M Simmons, Kyrie Dailey, Stacey M Bagby, Sarah J Hartman, Betelehem W Yacob, Brian Gittleman, John J Tentler, Diana Cittely, D. Ryan Ormond, Wells A Messersmith, S Gail Eckhardt, Jennifer R Diamond. Wee1 inhibition enhances the anti-tumor effects of capecitabine in preclinical models of triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-19-25.

Regulation of hypoxia-inducible factor functions in the nucleus by sphingosine-1-phosphate.

Sphingosine kinase 2 (SphK2) is known to phosphorylate the nuclear sphingolipid metabolite to generate sphingosine-1-phosphate (S1P). Nuclear S1P is involved in epigenetic regulation of gene expression; however, the underlying mechanisms are not well understood. In this work, we have identified the role of nuclear S1P and SphK2 in regulating hypoxia-responsive master transcription factors hypoxia-inducible factor (HIF)-1α/2α, and their functions in breast cancer, with a focus on triple-negative breast cancer (TNBC). We have shown SphK2 is associated with HIF-1α in protein complexes, and is enriched at the promoters of HIF target genes, including vascular endothelial growth factor (VEGF), where it enhances local histone H3 acetylation and transcription. S1P specifically binds to the PAS domains of HIF-1α. SphK2, and HIF-1α expression levels are elevated in metastatic estrogen receptor-positive (ER+) and TNBC clinical tissue specimens compared to healthy breast tissue samples. To determine if S1P formation in the nucleus by SphK2 is a key regulator of HIF functions, we found using a preclinical TNBC xenograft mouse model, and an existing selective SphK2 inhibitor K-145, that nuclear S1P, histone acetylation, HIF-1α expression, and TNBC tumor growth were all reduced in vivo. Our results suggest that S1P and SphK2 in the nucleus are linked to the regulation of HIF-1α/2α functions associated with breast cancer progression, and may provide potential therapeutic targets.

Exploiting DNA repair defects in triple negative breast cancer to improve cell killing.

Background:The lack of molecular targets for triple negative breast cancer (TNBC) has limited treatment options and reduced survivorship. Identifying new molecular targets may help improve patient survival and decrease recurrence and metastasis. As DNA repair defects are prevalent in breast cancer, we evaluated the expression and repair capacities of DNA repair proteins in preclinical models.Methods:DNA repair capacity was analyzed in four TNBC cell lines, MDA-MB-157 (MDA-157), MDA-MB-231 (MDA-231), MDA-MB-468 (MDA-468), and HCC1806, using fluorescence multiplex host cell reactivation (FM-HCR) assays. Expression of DNA repair genes was analyzed with RNA-seq, and protein expression was evaluated with immunoblot. Responses to the combination of DNA damage response inhibitors and primary chemotherapy drugs doxorubicin or carboplatin were evaluated in the cell lines.Results:Defects in base excision and nucleotide excision repair were observed in preclinical TNBC models. Gene expression analysis showed a limited correlation between these defects. Loss in protein expression was a better indicator of these DNA repair defects. Over-expression of PARP1, XRCC1, RPA, DDB1, and ERCC1 was observed in TNBC preclinical models, and likely contributed to altered sensitivity to chemotherapy and DNA damage response (DDR) inhibitors. Improved cell killing was achieved when primary therapy was combined with DDR inhibitors for ATM, ATR, or CHK1.Conclusion:Base excision and nucleotide excision repair pathways may offer new molecular targets for TNBC. The functional status of DNA repair pathways should be considered when evaluating new therapies and may improve the targeting for primary and combination therapies with DDR inhibitors.

Abstract B144: Lack of immunotherapy efficacy in a syngeneic bone metastasis model of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive cancer with treatment options limited to standard-of-care chemotherapy. TNBC patients develop metastases in high incidence, and almost all patients have bone metastases at end-stage disease. Bone metastases are mainly osteolytic, causing severe bone loss that can be prevented by bone-targeted agents. Currently, there is no efficient cure for bone metastases. Activation of the patient’s own immune system by immunotherapies is widely studied in various cancers, but their efficacy on bone metastases is not well established. In this study, we aimed to evaluate the efficacy of immunotherapies in comparison to standard-of-care compounds in a preclinical TNBC bone metastasis model. Materials and methods: Mouse 4T1-GFP TNBC cells were inoculated intracardially to immunocompetent female Balb/c mice to model bone metastasis. Treatment of single agents of chemotherapy (cyclophosphamide, 100 mg/kg), bone-targeting agent (zoledronic acid, 0.1 mg/kg), and immunotherapies programmed cell death 1 antibody (mouse anti-PD-1, 10 mg/kg) and indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor (epacadostat, 100 mg/kg), and the combination of anti-PD-1 and epacadostat were started on the day following the cancer cell inoculations. The study was terminated 12-13 days after the inoculations. Tumor burden was evaluated by GFP imaging and tumor-induced bone loss by X-ray imaging at sacrifice. Tumor infiltrating lymphocytes (TILs) were evaluated by immunohistochemical stainings of CD3+, CD4+ and CD8+ T cells. Results: Cyclophosphamide decreased skeletal tumor burden and the area of tumor-induced osteolytic bone lesions. Zoledronic acid decreased the area of osteolytic lesions but had no effect on skeletal tumor burden. Anti-PD-1, epacadostat and their combination had no effect on skeletal tumor burden or in the development of osteolytic lesions. Low number or no TILs were observed in the tumors growing in bone obtained from vehicle-treated mice. Conclusions: Immunotherapies did not prevent tumor growth in bone or tumor-induced bone changes in the syngeneic TNBC model. This is probably caused by the low number of TILs in the tumors growing in bone. Further studies are needed to evaluate the effects of treatment combinations and to increase the responsiveness to immunotherapies especially in bone metastases. Citation Format: Tiina E Kähkönen, Mari I Suominen, Jenni HE Mäki-Jouppila, Jussi M Halleen, Jenni Bernoulli. Lack of immunotherapy efficacy in a syngeneic bone metastasis model of triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B144. doi:10.1158/1535-7163.TARG-19-B144

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.

Abstract P2-06-15: Rational combination of Wee1 and BCL-2 inhibition in preclinical models of triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive subtype distinguished by its lack of expression of receptors for estrogen, progesterone, and normal human epidermal growth factor 2 expression. TNBC is difficult to treat and is associated with a high risk of recurrence and mortality. In order to effectively treat TNBC, alternative therapeutic targets need to be identified. Wee1 is a tyrosine kinase that phosphorylates CDC2 to pause the cell cycle at the G2 checkpoint as a means to delay mitosis while DNA damage undergoes repair. Inactivation of Wee1 via adavosertib (AZD1775, MK1775), a highly selective inhibitor of Wee1, allows CDC25 to dephosphorylate the CDC2/cyclin B complex resulting in premature initiation of mitosis and, ultimately, mitotic catastrophe and apoptosis. An unbiased screen of adovosertib in combination with other targeted compounds in TNBC patient-derived xenograft (PDX) models demonstrated that the combination of adavosertib and navitoclax, an inhibitor of anti-apoptotic BCL-2 and BCL-XL proteins, had greater efficacy than the single agents alone. The purpose of this study was to investigate the combination of adavosertib and navitoclax in preclinical TNBC models, both in vitro and in vivo. Methods: HCC1937, CAL51, MDA-MB-231 and MDA-MB-468 cells were plated in 96-well plates and exposed to increasing concentrations of adavosertib (125nM – 1000nM), navitoclax (2500nM – 10000nM), or the combination. Cellular proliferation was assessed in real-time using IncuCyte Live Cell Analysis, followed by endpoint sulforhodamine B (SRB) assay. Combination effects were analyzed using Calcusyn to determine combination indexes (CI). Apoptosis was assessed via the Caspase 3/7 assay. Western blotting was used to assess changes in expression of CDC2, phospho-CDC2, and BCL2. TNBC PDX models CU_TNBC_013 and CU_TNBC_014 were treated with vehicle, adavosertib (50mg/kg), navitoclax (100mg/kg), or the combination and assessed for tumor growth inhibition. Results: The combination of adavosertib and navitoclax resulted in greater antiproliferative effects in vitro compared to either single agent (p&amp;lt; 0.05). This effect was classified as synergistic with CI values &amp;lt;1. We observed a significant increase in apoptosis with the combination treatment as measured by Caspase 3/7 (p &amp;lt;0.005). The combination of adavosertib and navitoclax treatment resulted in a decrease in phospo-CDC2, and BCL2 in cell lines. In vivo, the combination treatment resulted in greater tumor growth inhibition as compared to adavosertib or navitoclax alone in the CU_TNBC_013 and CU_TNBC_014 PDX models. Conclusions: The combination of adavosertib and navitoclax is active in preclinical TNBC models and induces apoptosis and tumor growth inhibition. This data supports the continued development of this combination in TNBC with investigation of potential selective markers. Citation Format: Simmons DM, Tse TE, Dailey K, Hartman SJ, Bagsby S, Pitts TM, Tentler JJ, Diamond JR. Rational combination of Wee1 and BCL-2 inhibition in preclinical models of triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-15.

Nuclear Imaging Study of the Pharmacodynamic Effects of Debio 1143, an Antagonist of Multiple Inhibitor of Apoptosis Proteins (IAPs), in a Triple-Negative Breast Cancer Model.

Background Debio 1143, a potent orally available SMAC mimetic, targets inhibitors of apoptosis proteins (IAPs) members and is currently in clinical trials. In this study, nuclear imaging evaluated the effects of Debio 1143 on tumor cell death and metabolism in a triple-negative breast cancer (TNBC) cell line (MDA-MB-231)-based animal model. Methods Apoptosis induced by Debio 1143 was assessed by FACS (caspase-3, annexin 5 (A5)), binding of 99mTc-HYNIC-Annexin V, and a cell proliferation assay. 99mTc-HYNIC-Annexin V SPECT and [18F]-FDG PET were also performed in mice xenografted with MDA-MB-231 cells. Results Debio 1143 induced early apoptosis both in vitro and in vivo 6 h after treatment. Debio 1143 inhibited tumor growth, which was associated with a decreased tumor [18F]-FDG uptake when measured during treatment. Conclusions This imaging study combining SPECT and PET showed the early proapoptotic effects of Debio 1143 resulting in a robust antitumor activity in a preclinical TNBC model. These imaging biomarkers represent valuable noninvasive tools for translational and clinical research in TNBC.

Leptin Signaling Mediates Obesity-Associated CSC Enrichment and EMT in Preclinical TNBC Models.

Obesity is associated with poor prognosis in triple-negative breast cancer (TNBC). Preclinical models of TNBC were used to test the hypothesis that increased leptin signaling drives obesity-associated TNBC development by promoting cancer stem cell (CSC) enrichment and/or epithelial-to-mesenchymal transition (EMT). MMTV-Wnt-1 transgenic mice, which develop spontaneous basal-like, triple-negative mammary tumors, received either a control diet (10% kcal from fat) or a diet-induced obesity regimen (DIO, 60% kcal from fat) for up to 42 weeks (n = 15/group). Mice were monitored for tumor development and euthanized when tumor diameter reached 1.5 cm. Tumoral gene expression was assessed via RNA sequencing (RNA-seq). DIO mice had greater body weight and percent body fat at termination than controls. DIO mice, versus controls, demonstrated reduced survival, increased systemic metabolic and inflammatory perturbations, upregulated tumoral CSC/EMT gene signature, elevated tumoral aldehyde dehydrogenase activity (a CSC marker), and greater leptin signaling. In cell culture experiments using TNBC cells (murine: E-Wnt and M-Wnt; human: MDA-MB-231), leptin enhanced mammosphere formation, and media supplemented with serum from DIO versus control mice increased cell viability, migration, invasion, and CSC- and EMT-related gene expression, including Foxc2, Twist2, Vim, Akt3, and Sox2 In E-Wnt cells, knockdown of leptin receptor ablated these procancer effects induced by DIO mouse serum. These findings indicate that increased leptin signaling is causally linked to obesity-associated TNBC development by promoting CSC enrichment and EMT.Implications: Leptin-associated signals impacting CSC and EMT may provide new targets and intervention strategies for decreasing TNBC burden in obese women. Mol Cancer Res; 16(5); 869-79. ©2018 AACR.

Abstract OT2-07-07: ATR inhibitor M6620 (formerly VX-970) with cisplatin in metastatic triple-negative breast cancer: Preliminary results from a phase 1 dose expansion cohort (NCT02157792)

Abstract Background: ATR is a critical regulator of the cellular response to replication stress; it signals DNA damage repair, mediated through homologous recombination. Many cancers depend on ATR to survive DNA damage. M6620 is a potent, selective inhibitor of ATR that augments the anticancer activity of cisplatin in preclinical triple-negative breast cancer (TNBC) models. Given the high prevalence of TP53 mutations in TNBC and limited platinum responsiveness in patients lacking a BRCA1/2 mutation, this study was designed to evaluate the safety and efficacy of M6620 in combination with cisplatin in an expansion cohort of patients with BRCA1/2 wild-type advanced/metastatic TNBC. Methods: Eligible patients had advanced/metastatic ER-, PR-, and HER2- breast cancer with 0-2 prior non–platinum-based therapies and measurable disease per RECIST 1.1. First line patients were eligible if relapse occurred ≥3 months after prior (neo)adjuvant chemotherapy. Of a maximum 50 patients planned for enrollment, ≥30 were required to have BRCA1/2 germline wild-type status and basaloid molecular subtype tumors on central testing. Patients received intravenous cisplatin 75 mg/m2 on day 1 with intravenous M6620 140 mg/m2 on days 2 and 9 of each 21-day cycle. In patients intolerant of cisplatin or at investigator's discretion, cisplatin could be switched to carboplatin AUC 5 with M6620 90 mg/m2. Results: At the time of abstract submission, 35 female patients were enrolled in this study; 18 patients with confirmed BRCA1/2 wild-type and basaloid metastatic TNBC who received ≥1 cycle of study drug and had ≥1 baseline scan and ≥1 on-treatment scan at the time of the data cut were included in the primary efficacy analysis. Median progression-free survival (PFS) was 4.1 months (90% CI, 1.6-6.9 months). PFS was ≥ 6 months in 2 patients and ≥ 3 months in 8 patients. Preliminary unconfirmed objective response [complete response or partial response (PR)] was observed in 38.9% (90% CI, 19.9%-60.8%) of patients. All 7 patients with preliminary objective response had PR as best overall response; the longest duration of response was 183 days. Response was ongoing in 4 patients with PR at the time of data cutoff. Grade ≥3 related treatment-emergent adverse events occurred in 16 of 35 patients: neutropenia (n=8), anemia (n=5), vomiting (n=4), nausea (n=3), and, in 1 patient each, thrombocytopenia, neutrophil count decreased, platelet count decreased, hypokalemia, generalized weakness, rigors, and acute kidney injury. Conclusions: Combination of M6620 and cisplatin shows encouraging antitumor activity and tolerability in patients with advanced/metastatic TNBC. The study is ongoing; updated safety and efficacy results will be presented. Citation Format: Telli ML, Lord S, Dean E, Abramson V, Arkenau H-T, Murias C, Becerra C, Tang R, Penney MS, Pollard J, Conboy G, Fields SZ, Shapiro G, Tolaney SM. ATR inhibitor M6620 (formerly VX-970) with cisplatin in metastatic triple-negative breast cancer: Preliminary results from a phase 1 dose expansion cohort (NCT02157792) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT2-07-07.

Abstract PD3-16: Clinical safety and efficacy of the aurora and angiogenic kinase inhibitor ENMD-2076 in previously treated, locally advanced or metastatic triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of the estrogen and progesterone receptors and lack of HER2 over-expression. ENMD-2076 is an orally bioavailable small molecule inhibitor of Aurora and angiogenic kinases with pro-apoptotic and antiproliferative activity in preclinical models of TNBC. Methods: This two institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC refractory to 1-3 prior lines of chemotherapy in the advanced setting. Patients had ECOG PS ≤ 1, measureable disease by RECIST 1.1 and no evidence of brain metastasis. Patients were treated with ENMD-2076 250 mg PO daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary end point was 6-month clinical benefit rate (6-CBR) and secondary endpoints included time to progression (TTP), PK profile, safety and biologic correlatives in archival and fresh serial tumor biopsies in a subset of patients. Results: Between July 2012 and October 2016, 41 patients were enrolled (median age 54; range 30-73; female 40; male 1). Patients received a mean 1.7 prior lines of chemotherapy for locally advanced unresectable or metastatic disease and 80.5% received prior neoadjuvant or adjuvant chemotherapy (N=33). Thirty-six patients were evaluable per protocol for the primary efficacy analysis. Five patients (12.2%) were not included in the efficacy analysis due to: adverse events (AE) leading to discontinuation prior to objective efficacy assessment (N=3), not meeting eligibility criteria on day 1 (N=1) and withdraw of consent in cycle 1 (N=1). The study proceeded to the second stage of enrollment based on observing three 6-CBR events in Stage 1 (N=18 patients). The 6-CBR in the overall trial was 16.7% (95% exact CI: 6%-32.8%; 2 patients with PR and 4 patients with SD &amp;gt; 6 mos). The median duration of response or clinical benefit in these patients was 32 weeks (8 cycles). 4-CBR was 27.8% (95% exact CI: 14%-45.2%). Dose reduction occurred in 8 patients (20%) for fatigue, hypertension and proteinuria. The most common grade 3 treatment-related adverse events were hypertension (37.5%) and fatigue (10%). One patient experienced grade 4 hypertension. Analysis of serial tumor biopsies prior to and following 2 weeks of ENMD-2076 (N=8 patients), demonstrated a treatment-induced decrease in cellular proliferation (Ki-67) and microvessel density (CD34) as assessed by IHC. Immunofluorescence performed on a subset of samples demonstrated an increase in p53-family member expression following treatment, consistent with changes observed in preclinical TNBC patient-derived tumor xenograft models. Conclusions: ENMD-2076 has durable clinical activity in a subset of patients with pretreated, advanced or metastatic triple-negative breast cancer. Predictive biomarker development using archival and fresh tumor tissue is underway. Exploration of lower doses of ENMD-2076 in future clinical trials may improve tolerability. Citation Format: Diamond JR, Eckhardt SG, Pitts TM, van Bokhoven A, Aisner D, Gustafson DL, Capasso A, Elias AD, Storniolo AM, Schneider BP, Gao D, Tentler JJ, Borges VF, Miller KD. Clinical safety and efficacy of the aurora and angiogenic kinase inhibitor ENMD-2076 in previously treated, locally advanced or metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-16.

Abstract B175: Rational combination of mTOR and Aurora kinase A inhibition in preclinical models of triple-negative breast cancer

Abstract Background: Alisertib is a highly selective inhibitor of Aurora kinase A and has antiproliferative and proapoptotic activity in a subset of triple-negative breast cancer (TNBC) cell lines and patient-derived tumor xenograft (PDX) models. Cellular senescence and increased expression of genes in the PI3K/AKT/mTOR pathway has been observed in TNBC models following treatment with alisertib that demonstrate de novo or acquired resistance. The purpose of this study was to investigate the combination of alisertib and the TORC1/2 inhibitor TAK-228 in preclinical TNBC models. Methods: MDA-MB-468, HCC1187, HCC1937, CAL51, and BT20 cells were plated in 96 well plates and exposed to increasing concentrations of alisertib (25nM-125nM), TAK-228 (25nM-125nM), or the combination and proliferation was assessed using the Cell Titer-Glo (CTG) assay. Apoptosis was assessed using long-term live cell microscopy and caspase 3/7 staining. Western blotting was used to assess changes in pS6, p4EBP1, and survivin expression. TNBC p53 wildtype CAL51 cells were transfected with the fluorescent ubiquitin cell cycle indicator (FUCCI) reporters and exposed to increasing concentrations of alisertib, TAK-228, or the combination to evaluate the effect on cell cycle progression, growth, and apoptosis. TNBC PDX models CU_TNBC_004 and CU_TNBC_007 were treated with vehicle, alisertib (30mg/kg), TAK-228 (0.5mg/kg), or the combination and assessed for tumor growth inhibition and translational markers by immunofluorescence (IF) and senescence-associated- ß-galactosidase (SA-ß-gal) staining. Results: A combination effect was observed for alisertib and TAK-228 in vitro with a decrease in cellular proliferation with the combination as measured by CTG. We observed an increase in cell death with the combination, as opposed to cell cycle arrest with single-agent treatment. Alisertib treatment was associated with an increase in survivin not observed with combination treatment. TAK-228 treatment was associated with a decrease in pS6 and p4EBP1 as a single agent or in combination. The combination of TAK-228 and alisertib resulted in greater tumor growth inhibition in vivo as compared to either single agent alone, accompanied by an increase in apoptosis as measured by BAX and DR5 expression and a decrease in senescence as evaluated by SA-ß-gal and phenotypic changes. Single agents in the CAL51 FUCCI system resulted in a dose-dependent effect on cell cycle progression and apoptosis by live cell microscopy. The combination, however, led to a complete block of cell growth and simultaneous apoptosis, leading to no expansion of cells after treatment and a gradual loss of the cell population. Conclusions: The combination of alisertib and TAK-228 in vitro and in vivo in TNBC models resulted in greater antiproliferative and proapoptotic activity. This combination is currently being investigated in a phase I dose escalation trial in patients with advanced solid tumors with a planned expansion cohort in metastatic TNBC to further evaluate the mechanism of the combination (NCT02719691). Citation Format: Jennifer R. Diamond, James D. Orth, Anastasia Ionkina, Kyrie Dailey, Todd M. Pitts, Anna Capasso, Joshua M. Marcus, Russell T. Burke, Sarah L. Davis, Jiyhe Kim, Aik-Choon Tan, Sue G. Eckhardt, John J. Tentler. Rational combination of mTOR and Aurora kinase A inhibition in preclinical models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B175.

In vivo magnetic resonance imaging investigating the development of experimental brain metastases due to triple negative breast cancer.

Triple negative breast cancer (TNBC), when associated with poor outcome, is aggressive in nature with a high incidence of brain metastasis and the shortest median overall patient survival after brain metastasis development compared to all other breast cancer subtypes. As therapies that control primary cancer and extracranial metastatic sites improve, the incidence of brain metastases is increasing and the management of patients with breast cancer brain metastases continues to be a significant clinical challenge. Mouse models have been developed to permit in depth evaluation of breast cancer metastasis to the brain. In this study, we compare the efficiency and metastatic potential of two experimental mouse models of TNBC. Longitudinal MRI analysis and end point histology were used to quantify initial cell arrest as well as the number and volume of metastases that developed in mouse brain over time. We showed significant differences in MRI appearance, tumor progression and model efficiency between the syngeneic 4T1-BR5 model and the xenogeneic 231-BR model. Since TNBC does not respond to many standard breast cancer treatments and TNBC brain metastases lack effective targeted therapies, these preclinical TNBC models represent invaluable tools for the assessment of novel systemic therapeutic approaches. Further pursuits of therapeutics designed to bypass the blood tumor barrier and permit access to the brain parenchyma and metastatic cells within the brain will be paramount in the fight to control and treat lethal metastatic cancer.