Search

Supplementary Figure Legends

Supplementary Table 4: Combination Index (CI) Values. CI values calculated using the Chou and Talalay method from SRB proliferation data for varying concentrations of Nutlin and MLN8237 in combination. CI < 1 indicates synergy; CI = 1 additivity; CI > 1 antagonism

Supplementary Figure 2. Quantification of total cell counts and average cellular size at Day 15 as depicted in Fig. 4A.

Supplementary Table 3: Quantification of total cell counts and average cellular size under conditions depicted in Fig. 4A.

Supplementary Figure 4. Effect of MLN8237 on Cleaved Caspase-3 in TNBC xenografts in vivo.

Supplementary Figure 1. Treatment with MLN8237 induces markers of cellular senescence in two additional CAL-51 p53 and p73 shRNA knockdown clones (p53-12, p73-26).

Supplementary Table 2: Sensitivity of CAL-51 KD clones to MLN8237 in vitro.

Purpose:The purpose of this study was to evaluate the rational combination of TORC1/2 inhibitor TAK-228 and Aurora A kinase inhibitor alisertib in preclinical models of triple-negative breast cancer (TNBC) and to conduct a phase I dose escalation trial in patients with advanced solid tumors.Experimental Design:TNBC cell lines and patient-derived xenograft (PDX) models were treated with alisertib, TAK-228, or the combination and evaluated for changes in proliferation, cell cycle, mTOR pathway modulation, and terminal cellular fate, including apoptosis and senescence. A phase I clinical trial was conducted in patients with advanced solid tumors treated with escalating doses of alisertib and TAK-228 using a 3+3 design to determine the maximum tolerated dose (MTD).Results:The combination of TAK-228 and alisertib resulted in decreased proliferation and cell-cycle arrest in TNBC cell lines. Treatment of TNBC PDX models resulted in significant tumor growth inhibition and increased apoptosis with the combination. In the phase I dose escalation study, 18 patients with refractory solid tumors were enrolled. The MTD was alisertib 30 mg b.i.d. days 1 to 7 of a 21-day cycle and TAK-228 2 mg daily, continuous dosing. The most common treatment-related adverse events were neutropenia, fatigue, nausea, rash, mucositis, and alopecia.Conclusions:The addition of TAK-228 to alisertib potentiates the antitumor activity of alisertib in vivo, resulting in increased cell death and apoptosis. The combination is tolerable in patients with advanced solid tumors and should be evaluated further in expansion cohorts with additional pharmacodynamic assessment.

Pharmacodynamic effects of alisertib and TAK-228 in patient tissue samples

Studies of biological function demand probes that can report on processes in real time and in physiological environments. Bioluminescent tools are uniquely suited for this purpose, as they enable sensitive imaging in cells and tissues. Bioluminescent reporters can also be monitored continuously over time without detriment, as excitation light is not required. Rather, light emission derives from luciferase-luciferin reactions. Several engineered luciferases and luciferins have expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Conventional approaches image one luciferase at a time. Consequently, short-term changes in cell growth or gene expression cannot be easily captured. Here, we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale.

Bioluminescent tools can illuminate cellular features in whole organisms. Multi-component tracking remains challenging, though, owing to a lack of well-resolved probes and long imaging times. To address the need for more rapid, quantitative, and multiplexed bioluminescent readouts, we developed an analysis pipeline featuring sequential substrate administration and serial image acquisition. Light output from each luciferin is layered on top of the previous image, with minimal delay between substrate delivery. A MATLAB algorithm was written to analyze bioluminescent images generated from the rapid imaging protocol and deconvolute (i.e., unmix) signals from luciferase-luciferin pairs. Mixtures comprising three to five luciferase reporters were readily distinguished in under 50 min; this same experiment would require days using conventional workflows. We further showed that the algorithm can be used to accurately quantify luciferase levels in heterogeneous mixtures. Based on its speed and versatility, the multiplexed imaging platform will expand the scope of bioluminescence technology.

Background Cancer metastasis is a complex process involving the spread of malignant cells from a primary tumor to distal organs. Understanding this cascade at a mechanistic level could provide critical new insights into the disease and potentially reveal new avenues for treatment. Transcriptome profiling of spontaneous cancer models is an attractive method to examine the dynamic changes accompanying tumor cell spread. However, such studies are complicated by the underlying heterogeneity of the cell types involved. The purpose of this study was to examine the transcriptomes of metastatic breast cancer cells using the well-established MMTV-PyMT mouse model. Methods Organ-derived metastatic cell lines were harvested from 10 female MMTV-PyMT mice. Cancer cells were isolated and sorted based on the expression of CD44low/EpCAMhigh or CD44high/EpCAMhigh surface markers. RNA from each cell line was extracted and sequenced using the NextSeq 500 Illumina platform. Tissue-specific genes were compared across the different metastatic and primary tumor samples. Reads were mapped to the mouse genome using STAR, and gene expression was quantified using RSEM. Single-cell RNA-seq (scRNA-seq) was performed on select samples using the ddSeq platform by BioRad and analyzed using Seurat v3.2.3. Monocle2 was used to infer pseudo-time progression. Results Comparison of RNA sequencing data across all cell populations produced distinct gene clusters. Differential gene expression patterns related to CD44 expression, organ tropism, and immunomodulatory signatures were observed. scRNA-seq identified expression profiles based on tissue-dependent niches and clonal heterogeneity. These cohorts of data were narrowed down to identify subsets of genes with high expression and known metastatic propensity. Dot plot analyses further revealed clusters expressing cancer stem cell and cancer dormancy markers. Changes in relevant genes were investigated across pseudo-time and tissue origin using Monocle2. These data revealed transcriptomes that may contribute to sub-clonal evolution and treatment evasion during cancer progression. Conclusions We performed a comprehensive transcriptome analysis of tumor heterogeneity and organ tropism during breast cancer metastasis. These data add to our understanding of metastatic progression and highlight targets for breast cancer treatment. These markers could also be used to image the impact of tumor heterogeneity on metastases.

Studies of biological function demand probes that can report on processes in real time and in physiological environments. Bioluminescent tools are uniquely suited for this purpose, as they enable sensitive imaging in cells and tissues. Bioluminescent reporters can also be monitored continuously over time without detriment, as excitation light is not required. Rather, light emission derives from luciferase-luciferin reactions. Several engineered luciferases and luciferins have expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Conventional approaches image one luciferase at a time. Consequently, short-term changes in cell growth or gene expression cannot be easily captured. Here we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale.

Purpose: The purpose of this study was to evaluate the rational combination of TORC1/2 inhibitor TAK-228 and Aurora A kinase inhibitor alisertib in preclinical models of triple-negative breast cancer (TNBC) and to conduct a phase I dose escalation trial in patients with advanced solid tumors. Experimental Design: TNBC cell lines and patient-derived xenograft (PDX) models were treated with alisertib, TAK-228, or the combination and evaluated for changes in proliferation, cell cycle, mTOR pathway modulation, and terminal cellular fate, including apoptosis and senescence. A phase I clinical trial was conducted in patients with advanced solid tumors treated with escalating doses of alisertib and TAK-228 using a 3+3 design to determine the maximum tolerated dose (MTD). Results: The combination of TAK-228 and alisertib resulted in decreased proliferation and cell-cycle arrest in TNBC cell lines. Treatment of TNBC PDX models resulted in significant tumor growth inhibition and increased apoptosis with the combination. In the phase I dose escalation study, 18 patients with refractory solid tumors were enrolled. The MTD was alisertib 30 mg b.i.d. days 1 to 7 of a 21-day cycle and TAK-228 2 mg daily, continuous dosing. The most common treatment-related adverse events were neutropenia, fatigue, nausea, rash, mucositis, and alopecia. Conclusions: The addition of TAK-228 to alisertib potentiates the antitumor activity of alisertib in vivo, resulting in increased cell death and apoptosis. The combination is tolerable in patients with advanced solid tumors and should be evaluated further in expansion cohorts with additional pharmacodynamic assessment.

Studies of biological function demand probes that can report on processes in real time and in physiological environments. Bioluminescent tools are uniquely suited for this purpose, as they enable sensitive imaging in cells and tissues. Bioluminescent reporters can also be monitored continuously over time without detriment, as excitation light is not required. Rather, light emission derives from luciferase-luciferin reactions. Several engineered luciferases and luciferins have expanded the scope of bioluminescence imaging in recent years. Multicomponent tracking remains challenging, though, due to a lack of streamlined methods to visualize combinations of bioluminescent reporters. Conventional approaches image one luciferase at a time. Consequently, short-term changes in cell growth or gene expression cannot be easily captured. Here, we report a strategy for rapid, multiplexed imaging with a wide range of luciferases and luciferins. Sequential addition of orthogonal luciferins, followed by substrate unmixing, enabled facile detection of multiple luciferases in vitro and in vivo. Multicomponent imaging in mice was also achieved on the minutes-to-hours time scale.

RX-5902 is a first-in-class anticancer agent targeting phosphorylated-p68 and attenuating nuclear shuttling of β-catenin. The purpose of this study was to evaluate the efficacy of RX-5902 in preclinical models of triple-negative breast cancer (TNBC) and to explore effects on β-catenin expression. A panel of 18 TNBC cell lines was exposed to RX-5902, and changes in proliferation, apoptosis, cellular ploidy, and effector protein expression were assessed. Gene expression profiling was used in sensitive and resistant cell lines with pathway analysis to explore pathways associated with sensitivity to RX-5902. The activity of RX-5902 was confirmed in vivo in cell line and patient-derived tumor xenograft (PDX) models. RX-5902 demonstrated potent antiproliferative activity in vitro against TNBC cell lines with an average IC50 of 56 nmol/L in sensitive cell lines. RX-5902 treatment resulted in the induction of apoptosis, G2–M cell-cycle arrest, and aneuploidy in a subset of cell lines. RX-5902 was active in vivo against TNBC PDX models, and treatment resulted in a decrease in nuclear β-catenin. RX-5902 exhibited dose-proportional pharmacokinetics and plasma and tumor tissue in nude mice. Pathway analysis demonstrated an increase in the epithelial-to-mesenchymal transformation (EMT), TGFβ, and Wnt/β-catenin pathways associated with sensitivity to RX-5902. RX-5902 is active against in vitro and in vivo preclinical models of TNBC. Target engagement was confirmed with decreases in nuclear β-catenin and MCL-1 observed, confirming the proposed mechanism of action. This study supports the continued investigation of RX-5902 in TNBC and combinations with immunotherapy.

Introduction: Cancer metastasis is a complex process involving the spread of malignant cells from a primary tumor to distal organs. Understanding this cascade at a mechanistic level could provide critical new insights into the disease and potentially reveal new avenues for treatment. Transcriptome profiling of spontaneous cancer models is an attractive method to examine the dynamic changes accompanying tumor cell spread. However, such studies are complicated by the underlying heterogeneity of the cell types involved. The purpose of this study was to examine the transcriptomes of metastatic breast cancer cells using the well established MMTV PyMT mouse model. Methods: A suite of organ derived metastatic cell lines were harvested from 10 female PyMT mice and used for transcriptome profiling. The cells were first extracted from mouse organs with metastatic disease. Tissues were processed into single cell suspensions. Cancer cells were isolated and sorted based on the expression of specific cell surface markers. Cells exhibiting CD44low EpCAMhigh or CD44high EpCAMhigh profiles were collected. RNA from each cell line was extracted in biological replicates for bulk RNA sequencing. Samples were sequenced using the NextSeq 500 Illumina platform. Reads were mapped to the mouse genome using STAR and gene expression was quantified using RSEM. Tissue specific genes were compared across the different metastatic and primary tumor samples. Results: Cell lines harvested from PyMT mice were able to recapitulate the metastatic cascade in vivo. Expression of the oncogenic PyMT transgene was observed in all cell lines. Comparison of RNA sequencing data across all cell populations produced distinct gene clusters. Differential gene expression patterns related to organ tropism and immunomodulatory signatures were observed. Furthermore, the sequencing results identified expression profiles based on tissue dependent niches and clonal heterogeneity. These cohorts of data were narrowed down to identify a subset of genes with high expression and known metastatic propensity. These key genes are being used to image specific stages of the metastatic cascade. Conclusion: The collection of organ derived cancer cell lines enabled comprehensive transcriptomic analysis of different metastatic niches. This transcriptome pattern could contribute to subclonal evolution during cancer progression. In future work, we aim to investigate intratumoral heterogeneity by preforming single cell RNA sequencing on key metastatic samples and compare them to the primary tumor. The markers will also be used to image the impact of tumor heterogeneity on metastases. Citation Format: Anastasia A. Ionkina, Gabriela Balderrama-Gutierrez, Steve Huy Phan, Ali Mortazavi, Jennifer A. Prescher. Comparative transcriptome analysis of metastatic heterogeneity in a mouse model of 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 2496.

Background: We have previously demonstrated the efficacy of pharmacologic targeting of Aurora Kinase A (AurA) in cell line and patient-derived xenograft (PDX) models of triple-negative breast cancer (TNBC). We observed that inherent or acquired resistance to AurA inhibitors was associated with a senescence phenotype and up-regulation of the PI3K/Akt/mTOR pathway. This provided the rationale to assess whether this resistance could be overcome with the combination of the AurA inhibitor alisertib with the TORC1/2 inhibitor TAK-228. In select TNBC PDX models, we found that there were highly significant anti-tumor effects of the combination compared to single agents. The purpose of the current study was to evaluate the potential role for alterations in tumor metabolic profiles in anti-tumor activity after dual inhibition of AurA and TORC1/2, as both pathways are known to mediate cellular metabolism and energy utilization in normal and cancer cells. Methods: Three TNBC PDX models were used for these studies. Tumor sections were implanted subcutaneously on bilateral flanks of athymic nude mice. When tumors reached ~150 mm3 mice were randomized into four treatment arms: vehicle; alisertib (30 mg/kg); TAK-228 (0.75 mg/kg); or the combination and dosed daily by oral gavage for 30 days (n=10 tumors per arm). At end of study, tumors were harvested and polar metabolites extracted for global ultra-high pressure liquid chromatography-mass spectrometry (UHPLC-MS)-based metabolomics with a focus on central carbon and nitrogen metabolism. Metabolites were separated using a high throughput gradient-based C18 method and identified and validated manually against an in-house library of 800+ compounds. Results: Global metabolomics analysis of PDX tumors revealed a significant impact of TAK-228 treatment on fatty acid oxidation, resulting in increased levels of saturated fatty acids (C8, C10, C12) and palmitoleic acid, an increase in arginine, proline, metabolism and a decrease in glutamine metabolism. Alisertib treatment impacted tryptophan metabolism; initial results suggest a disruption of metabolite balance downstream of tryptophan. These disruptions in nitrogen and carbon metabolism occurred in the combination treatment arm as well. Of note, glucose utilization via glycolysis and the pentose phosphate pathway (PPP) were decreased with treatments, with the most profound decrease in the combination arm. Conclusions: In addition to increased anti-tumor growth activity, TNBC PDX tumor metabolic profiles were altered in mice treated alisertib, TAK-228 and the combination. Global metabolomics analysis revealed that several key metabolic pathways, including fatty acid oxidation, amino acid metabolism, glycolysis and PPP activity were significantly dysregulated, likely contributing to the observed tumor growth inhibition. Importantly, the effects on several pathways by the single agents persisted in the combination, while others, including the decrease in glycolysis and PPP activity, were more pronounced in the combination arm. These results support the hypothesis that dual targeting of AurA and TORC1/2 may exert potent anti-tumor activity in TNBC through several mechanisms, including the disruption of tumor cell energetics. This combination is currently being evaluated in a Phase I clinical trial with planned expansion cohorts with serial tumor biopsies (NCT02719691). These clinical samples may provide a means for validation of these preclinical observations and allow continued elucidation of the potential antitumor metabolic reprogramming resulting from this dual targeting strategy and the clinical setting most likely to benefit from this combination. Citation Format: John J Tentler, Angelo D'Allesandro, Julie A Reisz, Kyrie L Dailey, Anna Capasso, Anastasia Ionkina, Stacey M Bagby, Todd M Pitts, Jennifer R Diamond. Combined aurora kinase A and mTORC 1/2 inhibition results in disruption of tumor cell energetics 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 P3-02-04.

Triple-negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. Advances in the treatment of TNBC have been hampered by the lack of novel effective targeted therapies. The primary goal of this study was to evaluate the efficacy of targeting Aurora kinase A (AurA), a key regulator of mitosis, in TNBC models. A secondary objective was to determine the role of the p53 family of transcriptional regulators, commonly mutated in TNBC, in determining the phenotypic response to the AurA inhibitor alisertib (MLN8237). Alisertib exhibited potent antiproliferative and proapoptotic activity in a subset of TNBC models. The induction of apoptosis in response to alisertib exposure was dependent on p53 and p73 activity. In the absence of functional p53 or p73, there was a shift in the phenotypic response following alisertib exposure from apoptosis to cellular senescence. In addition, senescence was observed in patient-derived tumor xenografts with acquired resistance to alisertib treatment. AurA inhibitors are a promising class of novel therapeutics in TNBC. The role of p53 and p73 in mediating the phenotypic response to antimitotic agents in TNBC may be harnessed to develop an effective biomarker selection strategy in this difficult to target disease. Mol Cancer Ther; 14(5); 1117–29. ©2015 AACR.

Purpose Triple-negative breast cancer (TNBC) is a subtype associated with poor prognosis and for which there are limited therapeutic options. The purpose of this study was to evaluate the efficacy of ENMD-2076 in p53-mutated TNBC patient-derived xenograft (PDX) models and describe patterns of terminal cell fate in models demonstrating sensitivity, intrinsic resistance, and acquired resistance to ENMD-2076. Experimental design p53-mutated, TNBC PDX models were treated with ENMD-2076 and evaluated for mechanisms of sensitivity or resistance to treatment. Correlative tissue testing was performed on tumor tissue to assess for markers of proliferation, apoptosis, senescence, and pathways of resistance after treatment and at the time of acquired resistance. Results Sensitivity to ENMD-2076 200 mg/kg daily was associated with induction of apoptosis while models exhibiting intrinsic or acquired resistance to treatment presented with a senescent phenotype. Response to ENMD-2076 was accompanied by an increase in p53 and p73 levels, even within the background of mutant p53. Treatment with ENMD-2076 resulted in a decrease in pAurA and an increase in pHH3. We observed a TNBC subtype switch from the luminal androgen receptor to the basal-like subtype at acquired resistance. Conclusion ENMD-2076 has antitumor activity in preclinical models of p53-mutated TNBC. Increased levels of p53 and p73 correlated with sensitivity whereas senescence was associated with resistance to ENMD-2076. The novel finding of a TNBC subtype switch at time of acquired resistance may provide mechanistic insights into the biologic effects of selective pressure of anticancer treatments on TNBC. ENMD-2076 is currently being evaluated in a Phase 2 clinical trial in patients with metastatic, previously treated TNBC where these biologic correlates can be further explored.

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. TNBC carries an increased risk of developing distant metastatic disease and cancer-related death. TNBC is heterogeneous in its underlying biology, however, mutations in p53 are found in approximately 80% of tumors. The purpose of this study was to utilize TNBC cell line models to investigate the role of p53 and p73 in mediating sensitivity to MLN8237, a selective Aurora kinase A inhibitor, in the setting of mutant or wildtype p53. Additionally, we used p53 and p73 knock-down models to determine the role of p53 and p73 in mediating induction of apoptosis and cellular senescence in response to Aurora kinase inhibition. Methods: Eighteen TNBC cell lines were exposed to MLN8237 and the effects on proliferation, apoptosis, and cell cycle distribution were evaluated. Proliferation was assessed using an SRB assay, apoptosis was analyzed using a caspase 3/7 assay and cell cycle was measured using flow cytometry. Proliferation was confirmed using a cell counting technique to control for an increase in cell size following MLN8237 exposure. The p53 wildtype TNBC cell line CAL51 was transduced with several clones of shRNA constructs targeting p53 and p73 with > 80% knockdown by RT-PCR. These clones were exposed to escalating doses of MLN8237 and the effect on proliferation was determined using an SRB assay and the effect on cellular senescence was determined using a senescence associated beta-galactosidase (SA b-gal) assay. Results: In vitro exposure to MLN8237 resulted in robust inhibition of proliferation in TNBC cell lines which was associated with dose-dependent G2/M cell cycle arrest and induction of caspase-dependent apoptosis in a subset of sensitive cell lines. Knock-down of p53 and p73 in the CAL51 cell line resulted in an increase in the absolute 50% inhibitory concentration (IC50) calculated from the SRB proliferation assay curves from 0.04 μM to > 2 μM, 1.8 μM, > 2 μM and 1.5 μM in the CAL51scr, CAL51p5310, CAL51p5312, CAL51p7326, CAL51p7355, respectively. The sensitive p53 mutated MDA-MB-468 cell line (IC50 40 nM) and the sensitive p53 wild-type CAL51 cell line (IC50 45 nM) were selected for further experimentation. Exposure to MLN8237 at concentrations of 50 nM and 100 nM for 7 days resulted in induction of cellular senescence as detected by the SA b-gal assay in the CAL51 p53 wild-type cell line, but not in the MDA-MB-468 cell line where induction of apoptosis at 48 hours was observed using the caspase 3/7 assay. In the CAL51 p73 knock-down clones, induction of cellular senescence was not observed following exposure to MLN8237. Conclusions: MLN8237 exhibited robust anticancer activity towards preclinical models of p53 mutated and p53 wild-type TNBC, supporting future clinical investigation of Aurora kinase inhibitors in TNBC. In p53 wild-type TNBC, both p53 and p73 mediate sensitivity to MLN8237 and p73 is essential for induction of cellular senescence following exposure to MLN8237. Biomarkers predictive of response to MLN8237 in p53 mutated TNBC are being developed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-06.

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.

Background Triple-Negative Breast Cancer (TNBC) is an aggressive disease with a poor prognosis. Clinically, TNBC patients have limited treatment options besides chemotherapy. The goal of this study was to determine the kinase dependency in TNBC cell lines and to predict compounds that could inhibit these kinases using integrative bioinformatics analysis. Results We integrated publicly available gene expression data, high-throughput pharmacological profiling data, and quantitative in vitro kinase binding data to determine the kinase dependency in 12 TNBC cell lines. We employed Kinase Addiction Ranker (KAR), a novel bioinformatics approach, which integrated these data sources to dissect kinase dependency in TNBC cell lines. We then used the kinase dependency predicted by KAR for each TNBC cell line to query K-Map for compounds targeting these kinases. Wevalidated our predictions using published and new experimental data. Conclusions In summary, we implemented an integrative bioinformatics analysis that determines kinase dependency in TNBC. Our analysis revealed candidate kinases as potential targets in TNBC for further pharmacological and biological studies.

Background: Despite advances in targeted therapies for cancer, TNBC remains an aggressive breast cancer subtype with limited treatment options. Mutations in p53 are common in TNBC, however, the exact contribution of individual p53 mutations to response to therapy and mechanisms of acquired resistance are unknown. The purpose of this study was to characterize the activity of ENMD-2076, a multi-target Aurora kinase A and angiokinase inhibitor, against p53 mutated TNBC patient-derived tumor xenografts (PDX) and to identify differences in molecular pathways determining cellular fate in sensitive and resistant models. Methods: TNBC PDX models harboring different p53 mutations were used for ENMD-2076 treatment studies. Athymic nude mice were injected with tumor tissue and tumor volumes were measured twice a week. When the mean tumor volumes reached 150 mm3, mice were randomized and treated with vehicle control or ENMD-2076 200 mg/kg by oral gavage daily. A subset of animals were sacrificed at Day 4, 30, and at the time of acquired resistance for correlative tissue testing which included: immunofluorescence (IF) for p53, p73, BCL2, BAX, p16, phospho Aurora A (pAA) and phospho histone H3 (pHH3); immunohistochemistry (IHC) for cleaved caspase 3 (CC3) and Ki67; H&E and staining for senescence associated beta-galactosidase (SA -βgal) activity. Tumor growth inhibition (TGI) was calculated at Day 30 and sensitive models were treated until resistance when additional correlative tissue samples were obtained. Results: ENMD-2076 had significant anti-tumor activity against the CU_002 and CU_005 TNBC PDX models (TGI 71.3%, p value Conclusions: ENMD-2076 has pro-apoptotic anti-cancer activity in a subset of p53 mutated TNBC PDX models. Sensitivity was associated with the induction of p73, which may mediate the response in the absence of functional p53. Intrinsic and acquired resistance to ENMD-2076 in TNBC PTX models was associated with loss of p73 expression and an increase in markers associated with senescence, including p16 expression and SA β-gal activity. These data support the role of senescence as a potential mechanism of resistance to Aurora kinase inhibitors in p53 mutated TNBC and support the continued development of combination therapies including with inhibitors of pathways that mediate senescence. Citation Format: Anastasia A. Ionkina, S. Gail Eckhardt, Todd M. Pitts, Carol Sartorius, Peter Kabos, Jiyhe Kim, Aik Choon Tan, John J. Tentler, Jennifer R. Diamond. Senescence as a mechanism of resistance to the Aurora kinase and angiokinase inhibitor, ENMD-2076, in p53 mutated triple-negative breast cancer (TNBC) models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C34.

Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of ER, PR and HER2 over-expression. TNBC is heterogeneous in its biology, however, mutations in p53 are found in approximately 80% of tumors. The purpose of this study was to utilize TNBC cell line models to investigate the role of p53 and p73 in mediating sensitivity to ENMD-2076, a selective Aurora kinase A inhibitor (AurKi). p53 and p73 knockdown (KD) models were developed to determine their role in mediating induction of apoptosis and senescence in response to AurK inhibition. Additionally, the effects of combining ENMD-2076 with the p53 modulator nutlin-3 were assessed. Methods: TNBC cell lines, MDA-MB-468 (p53 mut) and CAL51 (p53 WT) were transduced with shRNA constructs targeting p53 and p73 with > 80% knockdown as determined by RT-PCR. These clones were exposed to escalating doses of ENMD-2076 and the effect on proliferation was determined using an SRB assay. Proliferation was assessed using an SRB assay, and confirmed using a direct cell counting to control for increase in cell size following ENMD-2076 exposure. In the CAL51 KD lines, the effects of ENMD-2076 treatment on cellular senescence were determined using a senescence associated beta-galactosidase (SA β-gal) assay. Combination studies with nutlin-3 in MDA-MB-468 and HCC1937 were assessed by SRB and synergy measured using Calcusyn software. Results: KD of p53 and p73 in the MDA-MB-468p5310, MDA-MB-468p7355 cell lines resulted in an increase in the IC50 from 0.625 μM to 1.5 μM compared to scrambled. KD of CAL51 p53 and p73 increased from 0.16 µM to 0.41 µM, and 1.3 µM, in the CAL51scr, CAL51p5310, CAL51p7326, respectively. The sensitive MDA-MB-468 cell line (IC50 40 nM) and the sensitive CAL51 cell line (IC50 45 nM) were selected for further experimentation. Exposure to ENMD-2076 at concentrations of 0.5 μM and 1 μM for varying time points resulted in induction of senescence in the CAL51 cell line, but not in the MDA-MB-468 cell line, where induction of apoptosis at 48 hours was observed. In CAL51 p53 and p73 KD clones, induction of senescence was observed following exposure to ENMD-2076. Combination treatment with nutlin-3 showed synergistic activity. Conclusions: ENMD-2076 exhibited robust anticancer activity towards preclinical models of TNBC, in both p53 mutated and p53 WT cell lines, supporting future clinical investigation of AurKi in TNBC. In p53 WT TNBC, both p53 and p73 mediate sensitivity to ENMD-2076 and p73 is essential for induction of senescence following exposure to ENMD-2076. Synergistic anticancer activity of ENMD-2076 and nutlin reinforces the role of p53 in Aurora kinase inhibition of TNBC. Biomarkers predictive of response to ENMD-2076 in p53 mutated TNBC are being developed. These results with p53 KD cell lines are currently being confirmed in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A286. Citation Format: Anastasia A. Ionkina, John J. Tentler, Timothy P. Newton, Kelsey L. Brunkow, Jared S. Johnson, Aik Choon Tan, Todd M. Pitts, S. Gail Eckhardt, Jennifer R. Diamond. The role of p53 family tumor suppressors in mediating response to the Aurora and angiogenic kinase inhibitor ENMD-2076 in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A286.