Triple Negative Breast Cancer Patient-derived Xenograft Models Research Articles

Abstract 2181: Functionalizing intratumoral signaling heterogeneity in triple negative breast cancer

Abstract Tumors are highly heterogeneous populations of cells, and measures of intratumoral heterogeneity (ITH) and diversity correlate with worse prognosis in many cancers, including breast cancer. Emerging studies are highlighting functional interactions between subclones, as well as among subclones and components of the tumor microenvironment. However, these studies have largely focused on soluble factors without interrogating the spatial distribution of subclones defined by activated signaling pathways. Previous work in this area has been severely limited by technical restrictions – existing techniques allowing measurement of many biomarkers simultaneously lose all information about the tissue architecture, while those that do retain spatial information can only assay a handful of markers at once. We will circumvent these limitations by undertaking imaging mass cytometry (IMC), which allows for simultaneous measurement of 30-40 antigens while retaining the spatial organization of the sample. Our objective is to dissect the signaling heterogeneity in tumors from patients and patient-derived xenograft (PDX) models of triple negative breast cancer (TNBC), through two main approaches: (1) characterization of signaling heterogeneity in human tumors and PDX models of TNBC through the use of imaging mass cytometry (IMC) and (2) modeling cell signaling heterogeneity in cell line-based models to determine mechanisms of cell-cell interaction and communication. We have constructed an IMC panel of antibodies that combines markers for tissue architecture, cell phenotyping, and signaling pathway activation. Profiling of a panel of TNBC PDX models, including matched sets of pre- and post-chemotherapy treatment, indicates emerging patterns of pathway activation. We will utilize these results to inform functional studies in our isogenic cell line models. As it is becoming more clear that tumors are composed of heterogeneous subclones, it is crucial that we understand the communication between subclones and with components of the microenvironment. Ultimately, we plan to use these results to identify novel vulnerabilities in subclonal interactions that can be targeted therapeutically in TNBC. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Xiaomei Zhang, Helen Piwnica-Worms. Functionalizing intratumoral signaling heterogeneity in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2181.

Abstract 5172: A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis

Abstract Metastases are responsible for the vast majority of deaths due to breast cancer. Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high rates of metastasis and poor prognosis. We are employing patient-derived xenograft (PDX) models of TNBC to identify drivers of metastasis. Tumor samples are obtained from the breast tumors of patients with TNBC and engrafted immediately into the humanized mammary fat pads of immune-compromised mice. Lentiviral transduction was employed to express bioluminescent and fluorescent markers in two independent PDX models of TNBC. Using these models, we demonstrated that human breast tumors are capable of completing all stages of the metastatic cascade in mice, and metastatic lesions are observed in organs normally found in patients with metastatic breast cancer including lung, liver, bone, brain, and lymph nodes. Dynamic and reversible shifts in the epithelial-to-mesenchymal transition (EMT) were observed as tumors metastasized to lung and were re-passaged to recipient mouse mammary fat pads (MFPs). Lung metastases were isolated using bioluminescence imaging and lung metastasis gene expression signatures were generated. Metastasis signatures from two independent PDX models were compared to identify genes that were commonly deregulated in lung metastases relative to corresponding mammary tumors. Comprehensive gain-of-function screens were then conducted in vivo to identify functional drivers of TNBC metastasis. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver in this screen. CEACAM5 mRNA and protein levels were elevated in lung metastases relative to corresponding MFP tumors in mice. In addition, we demonstrated that CEACAM5 expression was upregulated in the lung metastases of breast cancer patients, and its expression inversely correlated with patient survival. Our data indicate that the metastatic function of CEACAM5 is to promote growth of breast tumors in the lung by inducing MET (mesenchymal-to-epithelial transition). Citation Format: Emily Powell, Jiansu Shao, Hector Picon, Christopher Bristow, Zhongqi Ge, Caitlin Grzeskowiak, Michael Peoples, Frederick Robinson, Sabrina Jeter-Jones, Christopher Schlosberg, Fei Yang, Yun Wu, Ignacio Wistuba, Stacy Moulder, Fraser Symmans, Kenneth Scott, John Edwards, Han Liang, Timothy Heffernan, Helen Piwnica-Worms. A functional genomic screen in vivo identifies CEACAM5 as a clinically relevant driver of breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5172.

Abstract 2586: PI3K/AKT signaling pathway is transcriptionally elevated in prexasertib-resistant TNBC PDX models

Abstract Triple-negative breast cancer (TNBC) is a heterogeneous disease characterized by poor clinical outcomes and limited treatment options. Transcriptional analysis has divided TNBC into different molecular subtypes (BL1, BL2, M and LAR) that were characterized with different signaling pathways and differed in response to similar neoadjuvant chemotherapies [1]. In a sporadic TNBC phase II study, prexasertib monotherapy showed modest single agent activity, which suggested that combination therapy is needed to maximize the benefit from prexasertib (LY2606368), a selective CHK1 inhibitor [2]. Here we leveraged a large cohort of TNBC patient-derived xenograft (PDX) models developed with nude mice to find biomarker/pathways/combination strategies related with prexasertib. Parametric Gene Set Enrichment Analysis (PGSEA) has been shown as a powerful sample-level analysis to generate enrichment scores for pathways [3]. First we confirmed that our collection of PDX models captured the heterogeneities of TNBC disease, with similar compositions of molecular subtypes as the TCGA dataset. The different molecular subtypes of PDX models also showed similar pathway changes as TNBC disease, such as increase of cell-cycle pathway in BL1-subtype, Glycolysis in BL2-subtype and ECM pathway in M-subtype. Molecular subtype analysis of a single-mouse (n=1) PDX trial evaluating antitumor activity of prexasertib showed that majority of M-subtype showed resistance to prexasertib and the other subtypes showed differential sensitivity. Within the BL1-subtype, the resistant models also showed high expression of stem-cell like features. Further PGSEA analysis based on KEGG signaling pathways revealed that the PI3K/mTOR pathway was transcriptionally elevated in resistant models, which was consistent with the additive effect of the combination therapy of prexasertib and PI3K/mTOR inhibitor (LY3023414) in this PDX trial. These data provide rationale for the development of combination therapy of prexasertib and PI3K/mTOR inhibitor for TNBC. 1. Lehmann, B.D., et al., Refinement of Triple-Negative Breast Cancer Molecular Subtypes: Implications for Neoadjuvant Chemotherapy Selection. PLoS One, 2016. 11(6): p. e0157368. 2. Karzai, F., et al., A phase II study of the cell cycle checkpoint kinases 1 and 2 (CHK1/2) inhibitor (LY2606368; prexasertib) in sporadic triple negative breast cancer (TNBC). Annals of Oncology, 2016. 27. 3. Kim, S.Y. and D.J. Volsky, PAGE: parametric analysis of gene set enrichment. BMC Bioinformatics, 2005. 6: p. 144. Citation Format: Yan Ding, Jiangang Liu, John N. Calley, Hui-Rong Qian, Philip W. Iversen, Philip J. Ebert, Richard P. Beckmann, Gregory P. Donoho, Ricardo Martinez, Wenjuan Wu, Aimee Bence Lin, Emma Bowden, Amit Aggarwal. PI3K/AKT signaling pathway is transcriptionally elevated in prexasertib-resistant TNBC PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2586.

Capecitabine Efficacy Is Correlated with TYMP and RB1 Expression in PDX Established from Triple-Negative Breast Cancers.

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments.Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX.Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment.Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605-15. ©2018 AACR.

Mass Spectrometry-Based Proteomics Reveals Potential Roles of NEK9 and MAP2K4 in Resistance to PI3K Inhibition in Triple-Negative Breast Cancers.

Activation of PI3K signaling is frequently observed in triple-negative breast cancer (TNBC), yet PI3K inhibitors have shown limited clinical activity. To investigate intrinsic and adaptive mechanisms of resistance, we analyzed a panel of patient-derived xenograft models of TNBC with varying responsiveness to buparlisib, a pan-PI3K inhibitor. In a subset of patient-derived xenografts, resistance was associated with incomplete inhibition of PI3K signaling and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified novel candidates functionally important to buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAP2K4 reduced both baseline and feedback MAPK/MEK signaling and showed synthetic lethality with buparlisib in vitro A complex in/del frameshift in PIK3CA decreased sensitivity to buparlisib via NEK9/MAP2K4-dependent mechanisms. In summary, our study supports a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased omic analyses in uncovering resistance mechanisms to targeted therapy.Significance: Integrative phosphoproteogenomic analysis is used to determine intrinsic resistance mechanisms of triple-negative breast tumors to PI3K inhibition. Cancer Res; 78(10); 2732-46. ©2018 AACR.

PKD1 is a potential biomarker and therapeutic target in triple-negative breast cancer.

Protein Kinase D1 (PKD1) is a serine/threonine kinase encoded by the PRKD1 gene. PKD1 has been previously shown to be a prognostic factor in ERα+ tamoxifen-resistant breast tumors and PKD1 overexpression confers estrogen independence to ERα+ MCF7 cells. In the present study, our goal was to determine whether PKD1 is a prognostic factor and/or a relevant therapeutic target in breast cancer. We analyzed PRKD1 mRNA levels in 527 primary breast tumors. We found that high PRKD1 mRNA levels were significantly and independently associated with a low metastasis-free survival in the whole breast cancer population and in the triple-negative breast cancer (TNBC) subtype specifically. High PRKD1 mRNA levels were also associated with a low overall survival in TNBC. We identified novel PKD1 inhibitors and assessed their antitumor activity in vitro in TNBC cell lines and in vivo in a TNBC patient-derived xenograft (PDX) model. Pharmacological inhibition and siRNA-mediated depletion of PKD1 reduced colony formation in MDA-MB-436 TNBC cells. PKD1 inhibition also reduced tumor growth in vivo in a TNBC PDX model. Together, these results establish PKD1 as a poor prognostic factor and a potential therapeutic target in TNBC.

Pharmacological Inhibition of NOS Activates ASK1/JNK Pathway Augmenting Docetaxel-Mediated Apoptosis in Triple-Negative Breast Cancer.

Purpose: Chemoresistance in triple-negative breast cancer (TNBC) is associated with the activation of a survival mechanism orchestrated by the endoplasmic reticulum (EnR) stress response and by inducible nitric oxide synthase (iNOS). Our aim was to determine the effects of pharmacologic NOS inhibition on TNBC.Experimental Design: TNBC cell lines, SUM-159PT, MDA-MB-436, and MDA-MB-468, were treated with docetaxel and NOS inhibitor (L-NMMA) for 24, 48, and 72 hours. Apoptosis was assessed by flow cytometry using Annexin-V and propidium iodide. Western blot was used to assess ER stress and apoptosis, and rtPCR was used to evaluate s-XBP1. TNBC patient-derived xenografts (PDX) were treated either with vehicle, docetaxel, or combination therapy (NOS inhibition + docetaxel). Mouse weight and tumor volumes were recorded twice weekly. Docetaxel concentration was determined using mass spectrometry. To quantify proliferation and apoptosis, PDX tumor samples were stained using Ki67 and TUNEL assay.Results:In vitro, L-NMMA ameliorated the iNOS upregulation associated with docetaxel. Apoptosis increased when TNBC cells were treated with combination therapy. In TNBC PDXs, combination therapy significantly reduced tumor volume growth and increased survival proportions. In the BCM-5998 PDX model, intratumoral docetaxel concentration was higher in mice receiving combination therapy. Coupling docetaxel with NOS inhibition increased EnR-stress response via coactivation of ATF4 and CHOP, which triggered the pASK1/JNK proapoptotic pathway, promoting cleavage of caspases 3 and 9.Conclusions: iNOS is a critical target for docetaxel resistance in TNBC. Pharmacologic inhibition of NOS enhanced chemotherapy response in TNBC PDX models. Combination therapy may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Clin Cancer Res; 24(5); 1152-62. ©2018 AACR.

Abstract GS6-06: Identifying metastatic drivers in patient-derived xenograft models of triple negative breast cancer

Abstract Metastases are responsible for the vast majority of deaths due to breast cancer. Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high rates of metastasis and poor prognosis. We are employing patient derived xenograft (PDX) models of TNBC to identify drivers of metastasis. Tumor samples are obtained from the breast tumors of patients with TNBC and engrafted immediately into the humanized mammary fat pads of immune compromised mice. Lentiviral transduction was employed to express bioluminescent and fluorescent markers in two independent PDX models of TNBC. Using these models, we demonstrated that human breast tumors are capable of completing all stages of the metastatic cascade in mice, and metastatic lesions are observed in organs normally found in patients with metastatic breast cancer including lung, liver, bone, brain, and lymph nodes. Dynamic and reversible epithelial to mesenchymal transition (EMT) was observed as tumors metastasized to lung and were re-passaged to recipient mouse mammary glands. Lung metastases were isolated using bioluminescence imaging and lung metastasis gene expression signatures were generated. Metastasis signatures from two independent PDX models were compared to identify genes that were commonly de-regulated in lung metastases relative to corresponding mammary tumors. Comprehensive gain-of-function screens were then conducted in vivo to identify functional drivers of TNBC metastasis. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a metastatic driver in this screen. CEACAM5 mRNA and protein levels were elevated in lung metastases relative to corresponding mammary gland tumors in mice. In addition, we demonstrated that CEACAM5 expression was upregulated in the lung metastases of breast cancer patients, and its expression inversely correlated with patient survival. Our data indicate that the metastatic function of CEACAM5 is to promote growth of breast tumors in the lung by inducing MET (mesenchymal to epithelial transition). Citation Format: Powell E, Shao J, Picon HM, Ge Z, Echeverria GV, Peoples M, Bristow C, Cai S, Tu Y, McCoy AM, Piwnica-Worms D, Draetta G, Edwards JR, Moulder SL, Symmans WF, Heffernan TP, Liang H, Piwnica-Worms H. Identifying metastatic drivers in patient-derived xenograft models of 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 GS6-06.

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

Triple-negative breast cancer (TNBC) subtypes are clinically aggressive andcannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derivedxenograft methods. We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole andintact breast tumor, specific cell populations within the tumor, andthe extracellular matrix. Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Preclinical PET imaging of glycoprotein non-metastatic melanoma B in triple negative breast cancer: feasibility of an antibody-based companion diagnostic agent.

High levels of expression of glycoprotein non-metastatic B (gpNMB) in triple negative breast cancer (TNBC) and its association with metastasis and recurrence make it an attractive target for therapy with the antibody drug conjugate, glembatumumab vedotin (CDX-011). This report describes the development of a companion PET-based diagnostic imaging agent using 89Zr-labeled glembatumumab ([89Zr]DFO-CR011) to potentially aid in the selection of patients most likely to respond to targeted treatment with CDX-011. [89Zr]DFO-CR011 was characterized for its pharmacologic properties in TNBC cell lines. Preclinical studies determined that [89Zr]DFO-CR011 binds specifically to gpNMB with high affinity (Kd = 25 ± 5 nM), immunoreactivity of 2.2-fold less than the native CR011, and its cellular uptake correlates with gpNMB expression (r = 0.95). In PET studies at the optimal imaging timepoint of 7 days p.i., the [89Zr]DFO-CR011 tumor uptake in gpNMB-expressing MDA-MB-468 xenografts had a mean SUV of 2.9, while significantly lower in gpNMB-negative MDA-MB-231 tumors with a mean SUV of 1.9. [89Zr]DFO-CR011 was also evaluated in patient-derived xenograft models of TNBC, where tumor uptake in vivo had a positive correlation with total gpNMB protein expression via ELISA (r = 0.79), despite the heterogeneity of gpNMB expression within the same group of PDX mice. Lastly, the radiation dosimetry calculated from biodistribution studies in MDA-MB-468 xenografts determined the effective dose for human use would be 0.54 mSv/MBq. Overall, these studies demonstrate that [89Zr]DFO-CR011 is a potential companion diagnostic imaging agent for CDX-011 which targets gpNMB, an emerging biomarker for TNBC.

Selinexor (KPT-330) demonstrates anti-tumor efficacy in preclinical models of triple-negative breast cancer

BackgroundSelinexor (KPT-330) is an oral agent that has been shown to inhibit the nuclear exporter XPO1. Given the pressing need for novel therapies for triple-negative breast cancer (TNBC), we sought to determine the antitumor effects of selinexor in vitro and in vivo.MethodsTwenty-six breast cancer cell lines of different breast cancer subtypes were treated with selinexor in vitro. Cell proliferation assays were used to measure the half-maximal inhibitory concentration (IC50) and to test the effects in combination with chemotherapy. In vivo efficacy was tested both as a single agent and in combination therapy in TNBC patient-derived xenografts (PDXs).ResultsSelinexor demonstrated growth inhibition in all 14 TNBC cell lines tested; TNBC cell lines were more sensitive to selinexor (median IC50 44 nM, range 11 to 550 nM) than were estrogen receptor (ER)-positive breast cancer cell lines (median IC50 > 1000 nM, range 40 to >1000 nM; P = 0.017). In multiple TNBC cell lines, selinexor was synergistic with paclitaxel, carboplatin, eribulin, and doxorubicin in vitro. Selinexor as a single agent reduced tumor growth in vivo in four of five different TNBC PDX models, with a median tumor growth inhibition ratio (T/C: treatment/control) of 42% (range 31 to 73%) and demonstrated greater antitumor efficacy in combination with paclitaxel or eribulin (average T/C ratios of 27% and 12%, respectively).ConclusionsCollectively, these findings strongly suggest that selinexor is a promising therapeutic agent for TNBC as a single agent and in combination with standard chemotherapy.

Abstract 1151: SY-1365, a potent and selective CDK7 inhibitor, exhibits promising anti-tumor activity in multiple preclinical models of aggressive solid tumors

Abstract CDK7 has recently emerged as an attractive target in cancer since its inhibition decreases the transcript levels of oncogenic transcription factors, especially those driven by super-enhancers (SEs). Cancers have been hypothesized to be addicted to SE regulated genes and simultaneous suppression of multiple SE associated genes through CDK7 inhibition might represent a novel, powerful way to selectively kill cancer cells. Previously, we reported that SY-1365, a highly selective covalent CDK7 inhibitor, induces apoptosis in leukemia cells, but not in non-malignant cells, and demonstrates anti-tumor activity in in vivo models of leukemia. In this study, we extend these findings to the identification of multiple solid tumors that are susceptible to SY-1365 and compare the effects of SY-1365 on gene expression to other gene control agents. SY-1365 was screened in a panel of solid tumor cell lines, revealing activity in breast, ovarian, colorectal and lung cancer cells with low nM EC50 and rapid induction of apoptosis. In breast cancer, a subset of triple negative breast cancer (TNBC) cell lines were found to be more sensitive than luminal breast cancer cell lines, so we extended our studies to in vivo models and showed substantial tumor growth inhibition in multiple patient-derived xenograft (PDX) models of TNBC. Since other compounds have been reported to modulate the expression of SE regulated genes, we compared the transcriptional effects of SY-1365 treatment with those of a pan-CDK inhibitor (flavopiridol), a CDK9 inhibitor (NVP2) and a BRD4 inhibitor (JQ1) using microarray analysis. Applying principal component analysis, we observed a unique transcriptional response elicited by SY-1365 compared to the other inhibitors. NVP2 and flavopiridol inhibited an overlapping and much larger gene set than either JQ1 or SY-1365. Interestingly, SY-1365 treatment decreased the expression of oncogenic transcription factors, cell cycle checkpoint regulators and DNA damage response genes. The downregulation of transcripts involved in apoptosis and DNA damage response by SY-1365 suggests that CDK7 inhibition might synergize with targeted agents that affect these processes. Indeed, we observed that SY-1365 was synergistic with the PARP inhibitor niraparib and the Bcl-2 inhibitor venetoclax in triple negative breast cancer and AML cell lines, respectively. In summary, we have identified TNBC, ovarian and small cell lung cancer cell lines as highly sensitive to SY-1365 in vitro and have observed substantial tumor growth inhibition in PDX models of TNBC. SY-1365 induced a distinct transcriptional response compared with other transcriptional inhibitors, with apoptotic and DNA damage pathways being central. Finally, these mechanism of action studies support a rationale for investigating combinations of SY-1365 with inhibitors of PARP and Bcl-2. Citation Format: Shanhu Hu, Nan Ke, Yixuan Ren, Sofija Miljovska, Nisha Rajagopal, Michael McKeown, David Orlando, Kevin Sprott, Yoon J. Choi, Eric Olson, Christian C. Fritz. SY-1365, a potent and selective CDK7 inhibitor, exhibits promising anti-tumor activity in multiple preclinical models of aggressive solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1151. doi:10.1158/1538-7445.AM2017-1151

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

Abstract Purpose: Triple negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer with limited therapeutic options. Development of molecularly targeted agents for TNBC is an unmet clinical need. The phosphoinositide 3-kinase (PI3K) pathway, which is a major cell growth and survival pathway, is frequently activated in TNBC as a result of genetic aberrations such as loss of the negative regulator PTEN or gain of function mutations in PIK3CA, therefore an attractive therapeutic target. However, single agent PI3K inhibitors have shown limited anti-tumor activity in both preclinical models and in clinical trials. One of the most important mechanisms of resistance to PI3K inhibitor is amplification of Myc. Dinaciclib, a potent inhibitor of cyclin-dependent kinases (CDKs)1, 2, 5 and 9, has shown to be particularly effective in Myc dependent tumors in preclinical studies and in TNBC. We therefore hypothesized that dinaciclib could overcome tumor cell resistance to PI3K inhibitors and improve the therapeutic efficacy in TNBC. In this study, we evaluated the anti-tumor and molecular effect of dinaciclib and BKM120 (Pan-PI3K inhibitor), either alone or in combination, in a panel of patient derived xenograft (PDX) models of TNBC. Methods: Four TNBC PDXs models were selected for the study for in-vivo and ex-vivo response to vehicle, BKM120, dinaciclib, or the combination of BKM120 and dinaciclib. Tumor volume changes over time in each group were documented to calculate the percentage of tumor growth inhibition by either agent alone or in combination. Tumor tissues harvested post treatment were examined by immunohistochemistry for cleaved PARP to determine the extent of apoptosis and phospho-Histone H3 for G2 to M phase cell cycle progression. Western blot analysis and reverse protein phase array (RPPA) were also performed to determine treatment effect on PI3K downstream targets and cell cycle molecules. Results: The combination of BKM120 and dinaciclib induced significantly greater growth inhibitory effect on tumor growth than either single agent alone in TNBC PDX models. This is accompanied by an enhanced apoptotic induction and reduced cell cycle progression. In addition, the combination of dinaciclib and BKM120 significantly reduced the level of cyclin B and the key anti-apoptotic protein survivin as well as significant downregulation of pAKT, and pS6. Conclusions: These data suggest that dinaciclib and BKM120 combination is an effective approach in treating TNBC. Additional mechanistic investigation for the efficacy of this combination is underway. Citation Format: Sandeep Rajput, Fang Guo, Li Shun, Cynthia Ma. Dinaciclib overcomes resistance to BKM120 in triple negative breast cancer patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2017-1078

Efficacy and Molecular Mechanisms of Differentiated Response to the Aurora and Angiogenic Kinase Inhibitor ENMD-2076 in Preclinical Models of p53-Mutated Triple-Negative Breast Cancer.

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. 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. 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. 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.

Abstract P3-03-02: Inhibition of NOS promotes ER stress response and augments docetaxel-mediated apoptosis in TNBC

Abstract Introduction: Chemoresistance in triple negative breast cancer (TNBC) is related to an activation of a survival response orchestrated by endoplasmic reticulum (ER) stress. We hypothesize that attenuation of nitric oxide (NO) signaling pathway can overcome treatment resistance, preventing relapse, ultimately improving survival of TNBC patients. Here, we aimed to investigate the effects of pharmacological iNOS (inducible nitric oxide synthase) inhibition by L-NMMA on docetaxel-meditated ER stress response and to determine whether the therapeutic NOS inhibition may improve chemotherapy-based response. Methods: BT-549, SUM-149, MDA-MB-436, and MDA-MD-468 TNBC cell lines were treated with docetaxel (D; 5 nm)/ L-NMMA (L; 4mM)/ amlodipine (A; 5 µm) daily for 48 and 72 hours. Cell death and proliferation were assayed by Annexin V and ATP quantification, respectively. Western Blot (WB) was used to measure ER stress markers. In vivo regimen treatment followed three 2-weekscycles of D (20 mg/kg intraperitoneal [IP] on day 1) and L (200 mg/kg oral gavage on day 2-6); A (10 mg/kg IP on day 2-6) A was administered together with L to counteract the well-known effects of L on blood pressure (hypertension). TNBC Patient derived xenograft (PDX) models #2147, #5998, #3107 and #4664 were transplanted into the mammary fat pad of SCID Beige mice. PDX #2147 received either, single drug (vehicle, L, A, D), double (L+A, D+L, D+A), or triple drug combination (L+A+D). Models #4664, #3107 and #5998 received only vehicle, D or D+ L+A. Mice weight and tumor volumes were recorded twice weekly. D concentration was measured by mass spectrometry. Results: Studies on SUM-159 cell line showed that, when compared to the docetaxel-treated group, D+L+A increased cell death significantly, as indicated by a rise in annexin V/propidium iodide-positive cells. Increase in cell death by D+L+A was further demonstrated by accumulation of mitochondrial cleaved BAX. The enhanced apoptotic effects of D+L+A in MDA MD 468, BT 549 and MDA MD TNBC cell lines were confirmed by a decrease in ATP levels compared to D alone. WB revealed a survival stress response activated by docetaxel. When it was coupled with NOS inhibition, ER stress response showed higher expression of ATF4 and CHOP, triggering a proapoptotic response by pASK1/JNK pathway and cleaved caspases (CC3 and CC9). PDX #2147 showed that L, A and L+A treatment groups had similar tumor volume growth as the untreated group. However, combination therapy, D+L+A, significantly reduced the tumor volume and increased survival proportions compared with vehicle and docetaxel. Combination therapy also dramatically reduced tumor size on TNBC #4664 and #3107, and significantly improved response on #5998 compared with docetaxel alone. Intratumoral docetaxel concentration was 5.3-fold higher in mice receiving D+L+A than in those receiving docetaxel alone (#5998). In both groups, docetaxel was not detected in the plasma one week after injection. Conclusion: The present data suggest that iNOS may be a critical target for docetaxel resistance in TNBC. iNOS inhibition enhanced chemotherapy response in TNBC PDX models indicating that addition of iNOS inhibitor may improve prognosis and prevent relapse in TNBC patients who have failed conventional chemotherapy. Citation Format: Davila-Gonzalez D, Choi DS, Kuhn J, Granados SM, Rosato RR, Dave B, Chang JC. Inhibition of NOS promotes ER stress response and augments docetaxel-mediated apoptosis in TNBC [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-02.

Abstract P6-01-08: Identifying metastatic drivers in patient derived xenograft models of triple negative breast cancer

Abstract Metastases are responsible for the vast majority of deaths due to breast cancer. Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high rates of metastasis and poor response to chemotherapy. We are using patient derived xenograft (PDX) models of TNBC to identify drivers of TNBC metastasis. Using these models, we demonstrated that human breast tumors are capable of completing all stages of the metastatic cascade in mice, and metastatic lesions are observed in organs normally found in patients with metastatic breast cancer including lung, liver, bone, brain and lymph nodes. Lentiviral transduction was employed to express both bioluminescent and fluorescent proteins in three distinct PDX models of TNBC. In this way, metastatic lesions can be isolated using bioluminescent imaging and circulating tumor cells (CTCs) are isolated by flow cytometry. A lung metastasis gene expression signature was generated and comprehensive gain-of-function screens are being conducted in vivo to validate this signature and identify functional drivers of TNBC metastasis. Citation Format: Powell E, Shao J, Tieu T, Peoples M, Bristow C, Manyam G, Cai S, Tu Y, Edwards JR, Heffernan TP, Piwnica-Worms D, Liang H, Piwnica-Worms H. Identifying metastatic drivers in patient derived xenograft models of triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-08.

Abstract LB-112: PLK1 inhibition in genetically characterized triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) models results in robust antitumor activity including significant and durable tumor regression

Abstract Background: PLK1 plays multiple critical cell cycle roles and numerous preclinical studies have validated the impact of PLK1 inhibition in tumors. A Phase II trial of a PLK1 inhibitor in AML showed significantly increased ORR and OS (Döhner et al 2014), but results in solid tumors have been uniformly modest. However, recent studies indicate that PLK1 inhibition may yield greater responses in specific genetic subtypes. Maire et al (2012) reported impressive response and survival data in TNBC PDX models, and PLK1 has been implicated in promoting growth and survival in cooperation with MYC, PTEN-loss, the PI3K/mTOR pathway and AR signaling in prostate cancer and other models (Zhang et al 2014, Hou et al 2013, Liu et al 2011 and Tan et al 2013). We therefore tested whether the highly selective PLK1 inhibitor NMS-P937 could differentially inhibit the growth of a diverse panel of genetically-characterized TNBC PDX models, enabling selection of a potentially higher responding patient population. Methods: Ten genetically-characterized TNBC PDX models containing a broad set of mutations were tested for sensitivity to NMS-P937. After implantation and establishment in SCID mice (n = 3 per group), NMS-P937 was administered at different doses and schedules over a 24-day treatment period. Body weight, tumor growth and growth delay after cessation of treatment were measured. Results: Nine models were evaluable and significant tumor growth inhibition (50-90%) was noted in four. Mutations in the responding models have been proposed to enhance response to PLK1 inhibition in other tumor types, but not previously in TNBC. All lines with sensitivity-enhancing mutations responded while lines without did not. In the most sensitive model, NMS-P937 caused uniform, near-complete regressions that were stable for 21 days after dosing, with minimal body weight loss. This model harbors an amplified AR locus; though Hou et al proposed a connection between PLK1 and AR signaling, the extent and durability of the response was unanticipated. Further characterization of all models will be provided. Conclusion: This study appears to be the first report of a PLK1 inhibitor evaluation in a diverse panel of genetically-characterized models in any tumor type. Our results demonstrate that differentially sensitive molecular subtypes of TNBC can be identified and that underlying genetic profiles can be exploited to identify TNBC patients who may benefit most from PLK1 inhibitors. We are continuing to explore additional predictive genetic markers of NMS-P937 sensitivity in TNBC as well as other solid tumors, and planning for a clinical trial in selected TNBC patients. Citation Format: Benjamin Palleiko, Mark R. Miglarese, Michael King, Sandra L. Silberman. PLK1 inhibition in genetically characterized triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) models results in robust antitumor activity including significant and durable tumor regression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-112.

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

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

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

Abstract Breast cancers display a remarkable phenotypic diversity that is exploited to promote both tumor progression and therapeutic resistance. Recent studies in several types of cancer have highlighted the significance of intratumoral heterogeneity on both innate and acquired resistance to tyrosine kinase inhibitors (TKIs). Tumor plasticity is supported by the heterogeneous expression of receptor tyrosine kinases (RTKs) and the robustness that the overlapping signaling networks provide. Therefore a thorough understanding of the intratumoral heterogeneity is necessary for the development of effective therapeutic strategies. The receptor tyrosine kinase MET is overexpressed in 20-30% of breast cancers and correlates with poor patient outcome. Previously, we determined that high MET expression correlated with ER-/ERBB2- and basal like breast cancers. These results and the efficacy of MET inhibitors in other cancers suggest that MET may be an effective clinical target for aggressive breast cancer subtypes. Recent studies have exposed interactions between MET and the ERBB receptor family in the progression and therapeutic resistance of several cancers. Since MET, ERBB2, and EGFR are known to be highly expressed in aggressive breast cancer subtypes, it is critical that we understand the relationships between these receptors in order to develop effective treatment strategies. We are investigating the relationship between MET and ERBB receptor signaling in the progression and resistance of ERBB2+ and triple-negative breast cancer (TNBC). We observe that there is a large subset of ERBB2+ breast cancers that express MET and contain MET+/ERBB2+ subpopulations. In a MET+/ERBB2+ breast cancer cell line, MET depletion results in increased ERBB2 activation whereas, ERBB2 depletion results in increased MET activation. Therefore, ERBB2+ breast cancers with MET+ subpopulations may have an innate resistance to ERBB2 inhibition and may benefit from combined MET and ERBB2 inhibition. In TNBC, we observe heterogeneous expression of MET and EGFR. We have developed patient-derived xenografts (PDX) from primary and metastatic TNBCs that have diverse patterns of MET and EGFR expression/activation. In these TNBC PDX models we observe varied responses to monotherapy with MET inhibitors MGCD265 and crizotinib and the EGFR inhibitor erlotinib. Interestingly, therapeutic response to MET inhibition does not correlate with protein expression levels. In all studies, we observe significantly increased efficacy of combination therapy with MET and EGFR inhibition and a decrease in response variability. Examination of phospho-MET localization in treated tumorgrafts revealed that MET and EGFR inhibition induces distinct phospho-MET localization changes. We also observe that in the residual resistant cells phospho-MET is highly expressed in cells with mitotic bodies. We are currently performing phospho-proteomic analysis to determine the effect of MET and/or EGFR inhibition on RTK signaling networks including ERK and AKT pathways. These results will identify proteomic signatures that represent MET and/or EGFR activation/inhibition and sensitivity or resistance to monotherapy or combined MET and EGFR inhibition. Overall these studies give us a more comprehensive view of TNBC network signaling, the level of RTK heterogeneity within TNBC, and the efficacy of MET and/or EGFR inhibition on TNBC progression. Citation Format: Elizabeth A. Tovar, Erik S. Linklater, Curt J. Essenburg, Zach Madaj, David M. Cherba, Mary E. Winn, Hasan Korkaya, Julie L. Boerner, Carrie R. Graveel. Targeting the intratumoral heterogeneity of receptor tyrosine kinases in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B14.

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

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