Preclinical Models Of Triple-negative Breast Cancer Research Articles

Abstract P1-07-04: Unique overlapping subtypes of triple-negative breast and ovarian cancers and sensitivity of “mesenchymal-like” cancers to HSP90 inhibition is revealed by integrated gene expression and drug sensitivity profiling

Abstract Next-generation sequencing and gene expression signature analysis of primary tumors from The Cancer Genome Atlas (TCGA) revealed striking similarities between triple-negative breast cancer (TNBC) and ovarian cancer (OVCA). We hypothesized that these similarities may reveal transcriptionally-identifiable subgroups within a mix of TNBC and OVCA that are uniquely sensitive to certain drugs. To test this hypothesis, gene expression profiles for TNBC and OVCA cell lines in the Cancer Cell Line Encyclopedia were analyzed by unsupervised hierarchical clustering, which revealed two major unique subgroups containing both TNBC and OVCA cell lines that clustered according to the previously defined Mesenchymal and Basal subclasses of TNBC. Differential gene expression between “Mesenchymal-like” and “Basal-like” TNBC/OVCA cell lines was used to generate a gene signature that was subsequently validated using gene expression data in the Genomics of Drug Sensitivity in Cancer (GDSC) database. Drug sensitivity data from GDSC was then utilized to profile differential sensitivity of “Mesenchymal-like” and “Basal-like” cell lines to the 99 anti-cancer drugs with coverage of >50 breast and ovarian cell lines. Mesenchymal-like TNBC/OVCA cells were uniquely sensitive to HSP90 inhibition compared to Basal-like TNBC/OVCA cells for both HSP90 inhibitors in GDSC: CCT018159 (p<0.001) and 17-AAG (p=0.012). Strikingly, Mesenchymal-like TNBC/OVCA cells were most sensitive to HSP90 inhibition among all 33 solid tumor cancer lineages in GDSC, as well as other subgroups of breast and ovarian cancers. Differential sensitivity of Mesenchymal-like and Basal-like TNBC/OVCA cells to HSP90 inhibition with 4 agents (CCT018159, 17-AAG, AT13387, PU-H71) was validated using growth assays for 12 cell lines (6 of each subgroup) previously characterized in GDSC. To further validate the predictive value of our gene signature, gene expression data for 6 TNBC/OVCA cell lines with uncharacterized drug sensitivity was used to classify cell lines as “Mesenchymal-like” (n=4) or “Basal-like” (n=2). Our gene signature successfully predicted differential sensitivity to HSP90 inhibition, with the 4 Mesenchymal-like cell lines displaying greater sensitivity to HSP90 inhibition. The gene signature has been used to select PDX models with predicted differential sensitivity to HSP90 inhibition, and treatment studies are ongoing. In summary, we developed a novel approach to generate a gene expression signature that robustly predicts sensitivity to HSP90 inhibitors in preclinical models of TNBC and OVCA. HSP90 thus represents a therapeutic opportunity in an overlapping subpopulation of TNBC and OVCA. Our approach to identify novel combinations of drugs and histology/lineage-independent cancer subgroups may be used to discover new therapeutic opportunities in other cancer types. Citation Format: Shee K, Ung MH, Cheng C, Miller TW. Unique overlapping subtypes of triple-negative breast and ovarian cancers and sensitivity of “mesenchymal-like” cancers to HSP90 inhibition is revealed by integrated gene expression and drug sensitivity profiling [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 P1-07-04.

Abstract P6-11-05: Phase 1 study of CB-839, a small molecule inhibitor of glutaminase (GLS), in combination with paclitaxel (Pac) in patients (its) with triple negative breast cancer (TNBC)

Abstract Background: CB-839 is a first-in-class highly selective inhibitor of GLS, a key enzyme in the utilization of glutamine by cancer cells. TNBC has high GLS expression and is very dependent upon GLS-mediated conversion of glutamine to glutamate for tumor cell growth. CB-839 has antitumor activity in vitro and in vivo in preclinical models of TNBC. Recent studies demonstrate that glutamine utilization can contribute to resistance to paclitaxel, a therapy frequently used to treat TNBC patients. Paclitaxel sensitivity is dependent on down-regulation of the glutamine transporter, SLC1A5, and over-expression of SLC1A5 causes paclitaxel resistance. Consistent with these observations, inhibition of glutamine metabolism with CB-839 has demonstrated strong antitumor activity in combination with paclitaxel. CX-839-001 is an ongoing Phase 1 trial of CB-839 as monotherapy and in combination with approved agents. We previously reported pharmacodynamic studies demonstrating robust inhibition of GLS in pt blood and tumors and excellent tolerability of CB-839 monotherapy in a variety of tumor types including TNBC. In light of the preclinical rationale and monotherapy tolerability a combination arm was opened testing CB-839 with paclitaxel (Pac-CB) in patients with advanced TNBC. We report here updated results on the Pac-CB dose escalation and expansion cohorts. Methods: Patients with refractory advanced/metastatic TNBC (prior taxane therapy allowed) received escalating doses of CB-839 (400-800 mg BID) in combination with a fixed weekly Pac dose of 80 mg/m2 Days 1, 8, 15 of a 28 day cycle. Upon demonstration of safety and tolerability, an expansion cohort of TNBC pts was opened. Results: To date, 15 pts have received Pac-CB at three dose levels of CB-839: 7 pts at 400 mg BID, 5 at 600 mg BID and 3 at 800 mg BID with the latter dose level not completed. 40% of enrolled patients have received >5 prior lines of systemic therapy for adv/met disease, and 10 pts have received prior taxane therapy including 5 in the adv/met setting. The Pac-CB combination has been well tolerated with one DLT during dose escalation (G4 neutropenia at 400 mg BID) and a low rate of dose reductions (2 for Pac and 1 for CB-839). Of 15 pts, the best overall response rate (BORR, see Table) has been PR in 20% (3 pts), SD in 47% (7 pts) and PD in 33% (5 pts) with 5 patients remaining on study. At doses ≥600 mg BID (n=8) the BORR is 38% (3 pts), and disease control rate (CR + PR + SD) is 88% (7 pts). All 3 pts with PRs have received prior Pac, including 2 pts with disease that was refractory to Pac in the advanced/metastatic setting. Conclusions: The Pac-CB combination has been well tolerated and has demonstrated clinical activity in heavily pre-treated pts with TNBC. At doses ≥600 mg BID, BORR has been 38% and DCR 88%. Notably, PRs have occurred in pts with prior Pac therapy, including 2 pts with Pac-refractory disease in the adv/met setting. Updated data on the escalation and expansion cohorts will be presented. Dose LevelTotal400 mg BID600 mg BID800 mg BIDRECIST Response Evaluable (N)15753PR3 (20%)02 (40%)1 (33%)SD7 (47%)3 (43%)2 (40%)2 (67%)DCR (CR+PR+SD)10 (67%)3 (43%)4 (80%)3 (100%)PD5 (33%)4 (57%)1 (20%)0 Citation Format: DeMichele AM, Harding JJ, Telli ML, Münster P, McKay RR, Iliopoulos O, Whiting S, Orford KW, Bennett MK, Mier JW, Owonikoko TK, Patel MR, Kalinsky K, Carvajal RD, Infante JR, Merit-Bernstam F. Phase 1 study of CB-839, a small molecule inhibitor of glutaminase (GLS), in combination with paclitaxel (Pac) in patients (its) with triple negative breast cancer (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 P6-11-05.

Abstract OT1-01-06: PIQHASSO: Open label, non-randomized, multicenter phase 1/2b study investigating safety and efficacy of PQR309 and eribulin combination in patients (pts) with locally advanced (LA) or metastatic HER2 (-) and triple-negative breast cancer (TNBC) (study PQR309-007)

Abstract BACKGROUND: The PI3K/AKT/mTOR (PAT) pathway alteration has been strongly implicated in breast cancer and may contribute to resistance to available therapy. PQR309 is an oral pan-PI3K and mTOR inhibitor that penetrates the blood-brain barrier. Experiments of eribulin in combination with PI3K inhibitors in luminal and TNBC pre-clinical models enhanced antitumor activity. TRIAL OBJECTIVES: The primary objectives of the study are: to identify the maximum tolerated dose (MTD), evaluate the efficacy, safety and tolerability as well as the pharmacokinetics (PK) of PQR309 in combination with eribulin. In addition, exploratory objectives include assessments of: PAT pathway alterations prior to treatment, pharmacodynamics (PD) activity of PQR309 in combination with eribulin and correlation of PAT pathway alterations and PD activity with PQR309 and eribulin PK. TRIAL DESIGN: This is an open label, non-randomized, multicenter phase 1/2b clinical trial (dose escalation followed by expansion part) of PQR309 p.o. in combination with the standard dose of eribulin mesylate (1.4 mg/m2) in patients with LAMBC until progression or unacceptable adverse events (AE). The dose escalation part of the study will first investigate PQR309 administered in a continuous daily (q.d.) and two intermittent treatment schedules in combination with standard administration of eribulin mesylate in patients with HER2 negative LAMBC following the “modified” 3 by 3 design. MTD is defined as the highest dose level at which ≤1 of 6 pts experiences dose-limiting toxicity (DLT) during the 1st cycle. After the MTD of PQR309 in combination with eribulin has been defined in all the three treatment schedules, one schedule will be selected, based on the overall evaluation of clinical data from the dose escalation part of the study, for further evaluation of efficacy in the expansion part of the study. The expansion part of the trial applies Simon's MiniMax two-stage design. At the first stage, > 3 pts with TNBC with clinical benefit (CB) among 14 pts will be necessary to continue to the second stage. At the study end, > 9 pts with CB out of 28 pts are required to reject the null hypothesis. With this design, there is an 80% probability of a positive finding if the true clinical benefit rate (CBR) is ≥ 43% and a 5% probability of a positive finding if the true CBR is ≤ 21%. ELIGIBILITY: Women with HER2- LAMBC with two to 5 prior chemotherapy regimens in advanced disease. Adequate organ function and performance status. Phase II specific selection criteria are: triple negative LAMBC and RECIST v1.1 evaluable disease. ACCRUAL: Approximately 60 patients will be enrolled in approximately 10 sites Recruitment opened in March 2016. TRIAL REGISTRATION: NCT02723877. Date of registration: 21/12/2015. First patient included: 04/04/2016. Citation Format: López-Miranda E, Gávila J, Pernas S, Saura C, Oliveira M, Serra V, Schmid P, Lord S, Paez D, Perez J, Llombart A, Petrovic K, Dimitrijevic S, Cortes J. PIQHASSO: Open label, non-randomized, multicenter phase 1/2b study investigating safety and efficacy of PQR309 and eribulin combination in patients (pts) with locally advanced (LA) or metastatic HER2 (-) and triple-negative breast cancer (TNBC) (study PQR309-007) [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 OT1-01-06.

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.

Preclinical Evaluation of Fatty Acid Synthase and EGFR Inhibition in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks an approved targeted therapy. Despite initial good response to chemotherapy, 30% of the patients relapse within 5 years after treatment. EGFR overexpression is a common marker in TNBC, and its expression has been correlated with poor outcome. Inhibition of fatty acid synthase (FASN) activity leads to apoptosis of human carcinoma cells overexpressing FASN. We tested the hypothesis that blocking FASN in combination with anti-EGFR signaling agents would be an effective antitumor strategy in sensitive and chemoresistant TNBC. Several TNBC cell lines and 29 primary tumors were included to determine whether FASN is a potential target in TNBC. Doxorubicin-resistant TNBC cell lines (231DXR and HCCDXR) have been developed and characterized in our laboratory. Cellular and molecular interactions of anti-FASN compounds (EGCG and C75) with cetuximab were analyzed. In vivo tumor growth inhibition was evaluated after cetuximab, EGCG, or the combination in TNBC orthoxenograft models. TNBC cell lines showed overexpression of FASN enzyme and its inhibition correlated to FASN levels. FASN staining was observed in all of the 29 TNBC tumor samples. In vitro, EGCG and C75 plus cetuximab showed strong synergism in sensitive and chemoresistant cells. In vivo, the combination of EGCG with cetuximab displayed strong antitumor activity against the sensitive and chemoresistant TNBC orthoxenografts, without signs of toxicity. Our results show that the simultaneous blockade of FASN and EGFR is effective in preclinical models of sensitive and chemoresistant TNBC. Clin Cancer Res; 22(18); 4687-97. ©2016 AACR.

Abstract A39: Patient-derived xenografts to test emerging therapies for triple negative breast cancer

Abstract Introduction: Patients with triple negative breast cancer (TNBC) that do not respond to cytotoxic neoadjuvant therapy have a poor prognosis, thus there is a pressing need to develop pre-clinical models of chemoresistant TNBC and identify optimal therapeutic targets. Patient-derived xenografts (PDX) are a simple, expandable approach to model TNBC in vivo. We have generated a collection of triple negative PDXs that have undergone molecular characterization at the DNA, RNA and protein levels and have initiated characterization of their therapeutic sensitivity to standard chemotherapeutics regimens as well as targeted therapies. Methods: Tumors from patients and matching PDXs have been characterized by cancer-specific targeted exome sequencing, RNA sequencing, and Reverse Phase Protein Arrays. We have established single agent sensitivities for a set of PDXs to standard TNBC therapies (paclitaxel, carboplatin, doxorubicin, eribulin, gemcitabine) and the following targeted therapies: talazoparib (PARP), MLN0128 (mTOR), buparlisib (PI3K), and trametinib (MEK1/2). Results: We have generated 25 hormone receptor-low/HER2- PDXs from 24 patients (1 primary-local recurrence pair) with an overall engraftment rate of 49% for this patient population. Exome sequencing found that the PDXs mostly maintain the somatic mutation profile of the originating tumor and exhibit alterations common to TNBC: 16 (73%) with TP53 mutations, 2 (9%) with PI3KCA mutations and 4 (18%) with PTEN deletions. RNA sequencing demonstrated that the PDXs maintain gene expression profiles similar to the originating tumor and that our panel is comprised of PDXs representative of five TNBC molecular subtypes. Treatment with standard therapies established differential sensitivity to paclitaxel and carboplatin between PDXs. The two models with PI3KCA alterations were responsive to mTOR/PI3K inhibition and unresponsive to trametinib and talazoparib, supporting heightened reliance on mTOR/PI3K signaling in PI3KCA mutant TNBC. Talazoparib was the only targeted agent tested that caused regression as a single agent in any model. All three models that regressed with talazoparib treatment had genetic alterations linked to DNA damage pathways (BRCA1 germline alteration, ATM deletion; PTEN deletion); these models also had low BRCA1 mRNA expression and were the only PDXs to regress when treated with carboplatin. Conclusions: PDXs of all TNBC subtypes can be generated from TNBC patients that are resistant to neoadjuvant chemotherapy. These PDXs maintain molecular alterations of TNBC, retain patient-specific gene expression, and display differential response to therapies enabling identification of associations between responses and molecular traits. Citation Format: Kurt W. Evans, Erkan Yuca, Stephen M. Scott, Natalia Arango Paez, Xiaofeng Zheng, Ken Chen, Laura Gonzales, Coya Tapia, Emily Tarco, Dalliah M. Black, Gordon B. Mills, Funda Meric-Bernstam. Patient-derived xenografts to test emerging therapies for triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A39.

Abstract 3506: Carfilzomib demonstrates antiproliferative and proapoptotic activities in preclinical triple-negative breast cancer models

Abstract BACKGROUND: The proteosome is clinically validated as a target for cancer therapeutics. Carfilzomib (CFZ) is a proteasome inhibitor that selectively and irreversibly binds to its target and has been approved in the US for treatment of relapsed and refractory multiple myeloma. Phase 1/2 studies of CFZ reported signals of clinical activity in solid tumors. The aim of this study was to investigate the activity of CFZ in triple negative breast cancer models. METHODS: A diverse panel of human triple negative breast cancer cell lines was used to investigate the anti-tumor activity of CFZ. Cell viability following CFZ treatment was assessed using MTT assay. Proliferation was monitored both with IncuCyte Zoom real-time imaging system and 3D-ON-TOP clonogenic assay. Annexin-V flow cytometry was used to measure apoptosis. Apoptotic protein levels were measured with Western blot and proteome profiler array. MCL1 and Ki67 expression were also assessed using immunocytochemistry. RESULTS: CFZ had marked anti-proliferative activity in MDA-MD231, MDA-MB468, BT20, and SUM149 TNBC cell lines, with IC50 values following 96-hour exposure from 8 nM to 25 nM by MTT assay. The anti-proliferative activity of CFZ was observed by 3D-ON-TOP clonogenic assay. The anti-proliferative activity of CFZ was also demonstrated with IncuCyte proliferation assay. Proliferation was inhibited by low nanomolar concentrations in both MDA-MB468 and BT20 cells. Western blot analysis of CFZ-treated BT20, MDA-MB231 and MDA-MB468 cells showed cleavage of poly ADP ribose polymerase (PARP) and CASPASE-3, indicative of apoptosis, and induction of microtubule-associated protein-1 light chain-3B (LC3B), indicative of autophagy. However, expression level of anti-apoptotic protein MCL1 was increased in RAS-RAF mutated cells (MDA-MB231) but not in PTEN-null MDA-MD468 cells following the treatment of CFZ. Double immune-fluorescence staining of MDA-MB231 cells following the treatment of CFZ showed higher expression of MCL1 but no change in Ki67 expression. Interestingly, a combination of CFZ and TOR kinase inhibitor (MLN0128) enhanced cleaved CASPASE3 and BIM expression with a concomitant increased induction of apoptosis (annexinV expression) in MDA-MB231 cells. CONCLUSIONS: CFZ demonstrated anti-proliferative activity in TNBC cell lines in vitro. Flow cytometric analysis of annexin V-positive cells and Western blot expression of cleaved CASPASE 3, cleaved PARP and BIM indicated that CRZ -induced TNBC cell death through an apoptosis-dependent manner. Citation Format: Mathew Larson, Jennifer H. Carlson, Yuliang Sun, Tonia Bucholz, Casey Williams, Nandini Dey, Brian Leyland-Jones, Pradip De. Carfilzomib demonstrates antiproliferative and proapoptotic activities in preclinical triple-negative 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 3506.

Abstract 4995: anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer

Abstract Background: Effective treatment of metastatic, triple-negative breast cancer (TNBC) remains a highly unmet medical need. We have developed ES425, a bispecific ADAPTIR™ (modular protein technology) molecule that redirects T-cell cytotoxicity to tumor cells expressing ROR1 (receptor tyrosine kinase-like orphan receptor 1), an oncofetal antigen expressed on TNBC and other malignancies. Results are presented for studies run to examine in vitro and in vivo activity of ES425 in preclinical models of TNBC. Materials and Methods: Target-dependent cytotoxic activity was examined in vitro by treating ROR1(+) cell lines and ROR1(−) cell lines with ES425 in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined using chromium release assays. T cells were assessed for activation and proliferation using multi-color flow cytometry. Pharmacokinetics of ES425 in NOD/SCID gamma (NSG) mice was determined using single intravenous dose of approximately 10 mg/kg. Serum concentrations at time points ranging from 15 minutes to 504 hours were used to calculate the terminal elimination half-life of ES425. To assess activity in vivo, NOD/SCID mice were implanted subcutaneously with the ROR1(+) TNBC tumor cell line MDA-MB-231 and purified human T cells and treated with ES425. This model was run twice with T cells from different human donors. Tumor growth was assessed by measuring tumor volume. Results: ES425 efficiently redirected T cell cytotoxicity against ROR1(+) cell lines at low picomolar concentrations in vitro. Cytotoxic activity was dependent on expression of ROR1 by the target cells. T cells were activated and proliferated in response to ES425 in the presence of ROR1(+) target cells; proliferation was not observed in response to ROR1(−) cells. In vivo, pharmacokinetic analysis showed a serum half-life of approximately 7 days in NSG mice, and ES425 inhibited growth of MDA-MB-231 tumors in mouse xenografts. Repeat experiments showed similar inhibition of tumor growth and an improvement in overall survival. Conclusions: These studies show that ES425 may be an efficient agent for redirecting T cell cytotoxicity in preclinical TNBC models and merits investigation as a potential therapeutic in TNBC and other malignancies. Citation Format: John W. Blankenship, Lynda Misher, Danielle Mitchell, Nicole Zhang, Philip Tan, Gabriela H. Hoyos, Padma Ravikumar, Robert Bader, Catherine J. McMahan, Robert E. Miller, Jeannette Bannink, Hang Fang, Lara Parr, Maria Dasovich, David Bienvenue, Megan Aguilar, Carina Xu, Mollie Daugherty, Brian Woodruff, Jane A. Gross. anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer. [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 4995.

Phase 1 study of CB-839, a small molecule inhibitor of glutaminase (GLS) in combination with paclitaxel (Pac) in patients (pts) with triple negative breast cancer (TNBC).

1011Background: CB-839 is a first-in-class highly selective inhibitor of GLS, a key enzyme in the utilization of glutamine by many cancer cells. TNBC is very glutaminolytic, with high GLS expression and a high glutamate:glutamine ratio, and highly dependent on glutamine for growth. CB-839 has selective activity in most in vitro and in vivo TNBC preclinical models tested. CX-839-001 is an ongoing Phase 1 basket trial of CB-839 as monotherapy and in combination with approved agents. BID dosing of CB-839 with meals provided optimal PK and tolerability and achieved robust inhibition of GLS in blood and tumors. After achieving strong GLS inhibition with acceptable toxicity and observing a TNBC patient (pt) with a 23% reduction in tumor burden on study for >18 months, a combination arm testing CB-839 with Pac (Pac-CB) for TNBC was opened. Methods: Patients with metastatic TNBC received escalating doses of CB-839 (400-800 BID) in combination with a fixed weekly Pac dose (80 mg/kg on Days 1, 8, 15 of 28 day cycle...

MTOR Inhibitors Suppress Homologous Recombination Repair and Synergize with PARP Inhibitors via Regulating SUV39H1 in BRCA-Proficient Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease and has the worst outcome among all subtypes of breast cancers. Although PARP inhibitors represent a promising treatment in TNBC with BRCA1/BRCA2 mutations, there is great interest in identifying drug combinations that can extend the use of PARP inhibitors to a majority of TNBC patients with wild-type BRCA1/BRCA2 Here we explored whether mTOR inhibitors, through modulating homologous recombination (HR) repair, would provide therapeutic benefit in combination with PARP inhibitors in preclinical models of BRCA-proficient TNBC. We have studied the effects of mTOR inhibitors on HR repair following DNA double-strand breaks (DSB). We further demonstrated the in vitro and in vivo activities of combined treatment of mTOR inhibitors with PARP inhibitors in BRCA-proficient TNBC. Moreover, microarray analysis and rescue experiments were used to investigate the molecular mechanisms of action. We found that mTOR inhibitors significantly suppressed HR repair in two BRCA-proficient TNBC cell lines. mTOR inhibitors and PARP inhibitors in combination exhibited strong synergism against these TNBC cell lines. In TNBC xenografts, we observed enhanced efficacy of everolimus in combination with talazoparib (BMN673) compared with either drug alone. We further identified through microarray analysis and by rescue assays that mTOR inhibitors suppressed HR repair and synergized with PARP inhibitors through regulating the expression of SUV39H1 in BRCA-proficient TNBCs. Collectively, these findings strongly suggest that combining mTOR inhibitors and PARP inhibitors would be an effective therapeutic approach to treat BRCA-proficient TNBC patients.

Cabozantinib (XL184) Inhibits Growth and Invasion of Preclinical TNBC Models.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is associated with poor clinical outcome. There is a vital need for effective targeted therapeutics for TNBC patients, yet treatment strategies are challenged by the significant intertumoral heterogeneity within the TNBC subtype and its surrounding microenvironment. Receptor tyrosine kinases (RTK) are highly expressed in several TNBC subtypes and are promising therapeutic targets. In this study, we targeted the MET receptor, which is highly expressed across several TNBC subtypes. Using the small-molecule inhibitor cabozantinib (XL184), we examined the efficacy of MET inhibition in preclinical models that recapitulate human TNBC and its microenvironment. To analyze the dynamic interactions between TNBC cells and fibroblasts over time, we utilized a 3D model referred to as MAME (Mammary Architecture and Microenvironment Engineering) with quantitative image analysis. To investigate cabozantinib inhibition in vivo, we used a novel xenograft model that expresses human HGF and supports paracrine MET signaling. XL184 treatment of MAME cultures of MDA-MB-231 and HCC70 cells (± HGF-expressing fibroblasts) was cytotoxic and significantly reduced multicellular invasive outgrowths, even in cultures with HGF-expressing fibroblasts. Treatment with XL184 had no significant effects on MET(neg) breast cancer cell growth. In vivo assays demonstrated that cabozantinib treatment significantly inhibited TNBC growth and metastasis. Using preclinical TNBC models that recapitulate the breast tumor microenvironment, we demonstrate that cabozantinib inhibition is an effective therapeutic strategy in several TNBC subtypes.

IO-125: A novel supramolecular platinum chemotherapy for triple negative breast cancer.

153 Background: Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer patients, with a median survival of 13 months and limited treatment options. Recent studies have indicated clinical benefits in TNBC with platinum agents. We designed a novel platinum chemotherapeutic (IO-125), with platinum (II) tethered to cholesterol-based leaving group via a novel coordination environment, conferring properties unique from current platinum-based chemotherapies. In this study, we investigated the anti-tumor activity of IO-125 in pre-clinical models of TNBC. Methods: Cellular viability of IO-125 was assessed in TNBC cell lines using MTT assay, while Pt levels in cells, plasma and tumors were quantified using atomic absorption spectroscopy. Antitumor activity was assessed in MDA-MB-231 xenograft model. Results: We reportthat IO-125 shows enhanced cellular cytotoxicity and higher intracellular uptake as compared to oxaliplatin in TNBC lines. In addition to uptake by organic cation transporters known for transporting current platinum chemotherapies, IO-125 is also internalized via caveolin-mediated endocytic pathway, resulting in 10-20 fold greater uptake into cells as compared to oxaliplatin. Studies have implicated caveolin in TNBC progression and drug resistance, and harnessing this mechanism could confer enhanced activity. In animals bearing MDA-MB-231 xenografts, three cycles of IO-125 at well-tolerated doses (35 mg Pt/kg) resulted in significant tumor growth inhibition as compared to reported tumor inhibitions seen with existing platinum chemotherapies. Pharmacokinetic studies conducted in mice and rabbits indicate markedly improved profile of IO-125 as compared to oxaliplatin, showing a significantly higher area-under-the-curve (AUC), a substantial reduction in clearance and volume of distribution, with an enhanced half-life. Conclusions: Collectively, our findings demonstrate that IO-125 exhibits superior pharmacokinetics profile and biodistribution to tumors, resulting in enhanced anti-tumor activity in a TNBC xenograft model. Based on these observations, we propose that IO-125 may emerge as a potentially effective platinum-based drug for TNBC patients.

Abstract 4240: TORC inhibition enriches for a cancer stem cell-like population with FGFR-dependent Notch1 activation

Abstract Background. Cancer stem cells (CSCs) are associated with metastatic recurrences and poor prognosis in triple negative breast cancer (TNBC) patients. Genomic and proteomic data suggest that >30% of TNBC harbor PI3K/mTOR pathway aberrations. Furthermore, PI3K/mTOR inhibitors have shown antitumor activity in preclinical TNBC models. However, resistance to PI3K/mTOR inhibitors in breast and ovarian cancer has been recently shown. We hypothesized that resistance to TORC inhibition in TNBC is driven by a CSC-like population and targeting this population can improve antitumor action of TORC inhibitors. Methods and Results. Treatment of TNBC cells, SUM159 (PIK3CA-H1047L), BT549 (PTEN-del), and MDA468 (PTEN-del), with the PI3K/mTOR inhibitor BEZ235 or the TORC1/2 inhibitor MLN128 reduced growth. However, cells that survived BEZ235 and MLN128 treatment displayed CSC properties as assessed by FACS analysis for CSC markers and mammosphere formation. Using a Stem Cell-specific PCR Array and qRT-PCR, Notch1, Jagged1 and FGF1 mRNA levels were elevated in BEZ235 and MLN128-treated TNBC cells. Transient and long-term treatment with BEZ235 and MLN128 also increased expression of the active Notch intracellular domain (NICD), Notch target genes Hes1, Hey1 and c-Myc, and NICD-luciferase reporter activity. Nanostring analysis of post-chemotherapy primary breast cancers displayed increased expression of CSC markers (ALDH1A1 and CD44), Notch1, and Jagged1. Cells surviving the combination of the chemotherapeutic paclitaxel with BEZ235 or MLN128 displayed increased NICD and CSC-like properties. Similar changes were observed in OVCAR8 ovarian cancer cells treated with MLN128 ± cisplatin. Notch1 siRNA and the γ-secretase inhibitor (GSI) DAPT abrogated BEZ235- and MLN128-mediated enrichment of CSC markers and mammosphere formation. Treatment of SUM159 and MDA468 xenografts established in nude mice with MLN128 and DAPT did not significantly decrease tumor growth compared to MLN128 alone. However, MLN128 and DAPT-treated tumors displayed decreased tumorigenicity compared to MLN128-treated tumors as assessed by an in vivo limiting dilution assay. Furthermore, TNBC cells stably expressing mTOR shRNA exhibited increased NICD levels, CSC markers and tumorigenicity in vivo which was reduced with DAPT. With the increased FGF1 levels observed with BEZ235 and MLN128 treatment, we examined the effect of FRS2 siRNA and the FGFR1 kinase inhibitor lucitanib. Both approaches resulted in a reduction of the CSCs and NICD expression in MLN128-treated cells. Conclusions. These data suggest that treatment of TNBC harboring PI3K pathway aberrations with TORC1/2 inhibitors results in decreased tumor growth but may spare a drug-resistant CSC population with FGFR-Notch signaling. Thus, combination of Notch and TORC1/2 inhibitors in addition to chemotherapy may effectively decrease primary tumor growth and prevent recurrences in TNBC patients. Citation Format: Neil E. Bhola, Valerie M. Jansen, Carlos L. Arteaga. TORC inhibition enriches for a cancer stem cell-like population with FGFR-dependent Notch1 activation. [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 4240. doi:10.1158/1538-7445.AM2015-4240

Abstract P5-06-03: Combination of the PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of triple negative breast cancer

Abstract Introduction: In a small neoadjuvant study in patients with triple negative breast cancer (TNBC) the combination of eribulin plus carboplatin was effective, with a pathologic complete response rate of 43% following 4 cycles of treatment. Significant numbers of sporadic TNBC tumors are deficient in DNA repair capacity and share clinical and pathological features with hereditary BRCA1 mutant disease. PARP inhibitors have demonstrated synthetic lethality in cancer cells with defective DNA repair and have therapeutic potential for TNBC. In this study we describe the combination of PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of TNBC. Methods: E7449, an orally available PARP inhibitor, was administered in combination with eribulin +/- carboplatin to 4 s.c. xenograft models of TNBC: MDA-MB-436 (BRCA1 mutant, PTEN deficient), MDA-MB-468 (BRCA wild type, PTEN deficient), HCC1806 and MDA-MB-231 (BRCA and PTEN wild type). Results and Discussion: Addition of E7449 to eribulin significantly delayed tumor progression in PTEN deficient MDA-MB-468 xenografts. In the BRCA1 mutant and PTEN deficient MDA-MB-436 xenograft model, combination of E7449 with eribulin enhanced antitumor activity versus eribulin alone. Similar potentiation was observed for carboplatin upon combination with E7449. Treatment of MDA-MB-436 xenografts with the triple combination of E7449 + eribulin + carboplatin was more efficacious than any double combination and was well tolerated at the doses examined. In contrast, no significant combination activity was observed for E7449 plus eribulin in the BRCA and PTEN wild type xenografts HCC1806 and MDA-MB-231, and similarly no potentiation of carboplatin was observed in an MDA-MB-231 xenograft. Notably, combination activity was observed in the BRCA1 mutant (MDA-MB-436) and PTEN deficient (MDA-MB-436 and MDA-MB-468) xenografts and not in the BRCA and PTEN wild-type models (HCC1806 and MDA-MB-231). Data from ongoing studies to evaluate the combination activity of E7449 + eribulin in patient-derived xenograft (PDx) models of TNBC will be presented at the meeting. Potential biomarkers of sensitivity to the combination are under investigation in both cell line xenograft and PDx models and will be described. Conclusion: The addition of E7449 to eribulin +/- carboplatin increased antitumor activity in a subset of TNBC models. Biomarker studies aimed at a better understanding of the underlying cause of sensitivity are underway. The preclinical data support assessment of E7449 + eribulin + carboplatin combination therapy in the current phase I/II clinical trial. Citation Format: Sharon McGonigle, Jiayi Wu, Donna Kolber-Simonds, Natalie C Twine, Jue-lon Shie, Noel Taylor, Sergei Agoulnik, Zoltan Dezso, Shannon McGrath, Mark Matijevic, Shanqin Xu, Galina Kuznetsov, Mary Woodall-Jappe, Kenichi Nomoto. Combination of the PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-03.

Abstract 2638: Novel synergy of radiosensitizer prodrug IPdR with Aurora kinase inhibitors in triple-negative breast cancer

Abstract Introduction and Background: Breast cancer afflicts 1 in 8 women in the United States and is the second leading cause of cancer death in women. An estimated 232,340 new cases of invasive breast cancer will be diagnosed in 2013 with 39,620 deaths. Triple-negative breast cancer (TNBC) lacks estrogen (ER), progesterone receptors (PR), and human epidermal growth factor 2 (HER2) and accounts for ∼15% of breast cancers. TNBC is associated with an overall poor prognosis due to an aggressive and early pattern of metastasis and a relative lack of therapeutic targets. Chemotherapy remains the cornerstone of treatment albeit with a dismal response rate (∼ 25%) to monotherapy. It is critical to improve therapeutics in this disease. A key to improving treatment is identification of synergism between drugs which enhances effectiveness, decreases resistance and reduces toxicities. With TNBC being highly proliferative, we sought to exploit potential utility of agents targeting specific aspects of proliferation and could exhibit synergy. Experimental Methods: We assembled a library of agents, each with unique intracellular mechanisms and targets that have subtly different effects on cell division. We constructed detailed dose response curves (DRC) for these agents in MDA-MB-231, a TNBC cell line. Subsequently, we performed a novel synergy screen testing 105 unique two-drug combinations in TNBC. Our screen was especially designed to distinguish between synergism and additivity through presence of an internal control and performed in sufficient replicates to distinguish changes in viability exceeding 5%. Results: We have discovered a promising synergistic combination of 5-iodo 2-deoxypyrimidinone 2′-deoxyribose (IPdR) with Aurora kinase inhibitors, MLN 8237 and ZM 447439. IPdR is a pro-drug of 5-iodo-2′-deoxyuridine (IUdR), a potent radiosensitizer. MLN 8237 is a selective inhibitor of Aurora A kinase which activates the spindle assembly checkpoint leading to mitotic arrest while ZM 447439 disrupts activity of Aurora B kinase and results in accelerated mitotic slippage, failed cytokinesis, and ultimate apoptotic cell death. This is the first time that synergy between IPdR and anti-mitotic agents has been demonstrated and moreover, the first discovery of cytotoxic effect of IPdR without requiring hepatic metabolism to IUdR. Conclusions and Future Directions: The discovery of synergy between IPdR and Aurora kinase inhibitors is a significant finding with potential clinical implications. We are pursuing further studies understanding the underlying biology behind this synergy and validation in pre-clinical models of TNBC. We plan to pursue these synergistic combinations in early phase clinical trials through the NCI CTEP or other mechanisms. Citation Format: Murtuza M. Rampurwala, Alka Choudhary, Mark E. Burkard. Novel synergy of radiosensitizer prodrug IPdR with Aurora kinase inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2638. doi:10.1158/1538-7445.AM2014-2638

Abstract 1677: Preclinical activity of Vintafolide/MK-8109 monotherapy and in combination with standard of care therapy in triple-negative breast cancer models

Abstract Vintafolide (also known as EC145 or MK-8109), is a small molecule drug conjugate for the treatment of cancers expressing high affinity folate receptor (FR). Vintafolide consists of the anti-mitotic vinca alkaloid desacetylvinblastine monohydrazide (DAVLBH) chemically linked to folic acid. Binding of vintafolide to FR located on the cell surface delivers the chemotherapy payload directly to the tumor cell. Clinical investigations of vintafolide are underway in ovarian and lung cancer, indications with a high prevalence of FR expression. Triple negative breast cancer (TNBC) may represent an additional indication for vintafolide due to the prevalence of FR expression (∼30%), sensitivity to vinca alkaloids, and unmet medical need. Vintafolide and/or DAVLBH were evaluated in preclinical models of TNBC as monotherapies and in combination with taxanes (paclitaxel or docetaxel). Taxanes represent a commonly used standard of care therapy for TNBC. A panel of TNBC cell lines was generally sensitive to DAVLBH, with IC50s ranging from 4-67 nM. In the majority of cell lines tested, DAVLBH in combination with paclitaxel provided combination benefit and induced more cell death than either single agent. Vintafolide and DAVLBH were further evaluated in vivo in the FR-high MDA-MB-231 and FR-low CAL51 TNBC xenograft models. Vintafolide was dosed at its MTD of 9.6 mg/kg three times per week (TIW), and at 1.5 mg/kg TIW. DAVLBH was dosed at its MTD of 0.77 mg/kg TIW. Mice were dosed for 3 weeks followed by a 6 week follow-up. Both vintafolide doses resulted in marked MDA-MB-231 tumor regressions of 56-78% at the end of therapy (Day 21) and 75% cures (6 of 8 mice) over the 6 week follow-up period. In the CAL51 model, vintafolide produced 8% and 76% tumor growth inhibition (TGI), respectively, at the 1.5 and 9.6 mg/kg doses, and no cures. DAVLBH was less efficacious compared to vintafolide, giving 96% TGI in the MDA-MB-231 model and 46% TGI in the CAL51 model. In mechanism of action studies in the MDA-MB-231 xenograft model, a dose of vintafolide that gave tumor regressions/cures was associated with increased phospho-histone H3 staining, indicative of a mitotic block of tumor cells, and consistent with the mechanism of action of vinca alkaloids. In combination therapy experiments in vivo, docetaxel (20 mg/kg weekly) monotherapy achieved near tumor stasis (98% TGI) of MDA-MB-231 tumors over 3 weeks of therapy, but all tumors re-grew upon cessation of treatment. Since vintafolide monotherapy at 1.5 mg/kg TIW gave significant regressions and cures, a combination benefit with docetaxel could not be determined. However, DAVLBH and docetaxel combination therapy delayed tumor re-growth over single agents upon follow-up, suggesting benefit of a vinca alkaloid / taxane combination. Taken together, these preclinical data support further investigation of vintafolide monotherapy and in combination with taxane therapy in TNBC. Citation Format: Brian B. Haines, Jennifer O'Neil, Marlene C. Hinton, Christopher Ware, Tammie C. Yeh, Tianxiao Sun, Kristen L. Picard, Theresa Zhang, Emmett V. Schmidt, Isabelle Dussault. Preclinical activity of Vintafolide/MK-8109 monotherapy and in combination with standard of care therapy in triple-negative breast cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1677. doi:10.1158/1538-7445.AM2014-1677

387P - Aggravation of Er Stress By Combination of Proteasome Inhibitors and Hiv Protease Inhibitors Results in Preferencial Killing of Triple-Negative Breast Cancer Cells in Vitro

ABSTRACT Aim: Breast cancer (BC) is the most frequent cancer and the principal cause of cancer death for females worldwide. Triple-negative breast cancer (TNBC), carries extraordinarily poor prognosis due to its aggressive biology, fast development of drug resistance, and lack of molecular targets such as hormone receptors and HER2/Neu. Until now, chemotherapy remains the standard care for advanced TNBC, with a poor median overall survival. Recently, pharmacological aggravation of endoplasmic reticulum stress (ERS) has become an attractive strategy for cancer therapy. In our work we present evidence that pharmacological aggravation of ERS may lead to efficient killing of breast cancer preclinical models in vitro. Methods: We determined the cytotoxic activity of proteasome inhibitors (bortezomib, bzb; carfilzomib, cfz) alone and in combination with the two HIV protease inhibitors nelfinavir (nfv) and lopinavir (lpv) in five different breast cancer cell lines. To assess cytotoxicity of each drug and their combinations, the MTS assay was applied. Results: Both proteasome inhibitors moderately reduced cell survival in all BC cell lines at clinically relevant concentrations (125 nM) when applied alone (8% - 36%). Importantly, combinations with HIV protease inhibitors yielded significantly enhanced cytotoxicity in all cell lines compared with either proteasome (see table) or HIV protease inhibitors alone. Moreover, preliminary data shows that these combinations cause strong cytotoxic effects in TNBC cell lines. Conclusions: Drug combinations of the irreversible proteasome inhibitors and an HIV protease inhibitor resulted in substantial cytotoxicity in preclinical models of TNBC. The concept of pharmacological aggravation of ERS is promising in patients with TNBC, and a respective phase IB-II clinical study is in early planning. Cell Lines Bzb (125 nM) Cfz (125 nM) - Nfv Lpv - Nfv Lpv MDA-MB 468 27 ± 8 57 ± 0.1 80 ± 1 21 ± 7 65 ± 2 79 ± 4 MDA-MB 231 36 ± 8 56 ± 0.1 57 ± 2 41 ± 2 74 ± 4 78 ± 8 BT 549 23 ± 3 90 ± 1 56 ± 0.2 44 ± 3 91 ± 1 69 ± 4 BT 474 8 ± 6 28 ± 6 32 ± 3 33 ± 6 82 ± 6 61 ± 1 SK-BR-3 - - - 30 ± 2 68 ± 3 21 ± 4 Cytotoxic effects of mono and combination experiments in BC cell lines (% cell death +/- SD) Disclosure: All authors have declared no conflicts of interest.

Abstract B165: The antitumor efficacy of eribulin is mediated via suppression of invasion and profound induction of cell death in triple negative breast cancer.

Abstract Eribulin, a microtubule-targeting antitumor drug, is FDA-approved for the treatment of certain patients with advanced or metastatic breast cancer. Some patients treated with this drug have evidence of reduced metastatic dissemination. Triple-negative breast cancer (TNBC), which accounts for approximately 15%-20% of all breast cancers, does not respond to hormone therapies so patients are dependent on chemotherapy. A subset of these patients are intrinsically resistant to chemotherapy, or subsequently relapse and develop metastatic disease, eventually leading to mortality. We explored the mechanistic basis for the antitumor efficacy of eribulin in a panel of triple negative breast cancer cell lines that histologically are classified as basal-like or claudin-low. Specifically, we determined the ability of eribulin to induce cell death versus senescence, and secondly to modulate cancer cell invasion and epithelial to mesenchymal transition (EMT) gene expression. Senescence is a clinically relevant cell fate following chemotherapy that can propel tumor progression and EMT. Eribulin was efficacious at inducing cell death in both subtypes of TNBC, and had a remarkably low propensity to induce senescence in TNBC compared to non-small cell lung cancer and colon cancer cells. A 24-hour treatment with eribulin partially prevented tumor cell invasion in the claudin-low subtype, as assayed by Boyden chamber analysis. Drug-tolerant claudin-low cells that survived high-dose eribulin treatment were also found to have a reduction in EMT markers. In summary, in vitro analysis of cell fate and metastasis indicate that eribulin has an ability to modulate invasiveness and EMT characteristics in preclinical models of TNBC. This research was funded by Eisai Inc. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B165. Citation Format: Jing Hu, Susan Band Horwitz, Hayley M. McDaid. The antitumor efficacy of eribulin is mediated via suppression of invasion and profound induction of cell death 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 B165.

Abstract A286: 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 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.

Kinome reprogramming response to MEK inhibition: A window-of-opportunity trial in triple-negative breast cancer (TNBC).

512 Background: Targeted therapy (Rx) in TNBC is challenging due to heterogeneity and cancer cell kinome “reprogramming” in response to targeted kinase inhibitors (Cell 149:307, 2012). In preclinical TNBC models specific kinases (e.g., DDR1/2, PDGFRbeta, VEGFR2, AXL) are activated in response to MEK inhibition. Our studies define unique kinome reprogramming in basal-like (BBC) versus claudin-low (CL). This study compared kinome profiles of TNBC pre- and post- MEK1/2 inhibition with GSK1120212 (trametinib, T) using multiplexed inhibitor beads coupled with mass spectrometry (MIB/MS). Methods: Eligible patients (pts) with untreated TNBC received T (initial dose 1.5mg/d raised after interim analysis of kinome effects to 2mg/d) for 7 days preceding breast surgery. Fresh tumor tissue was obtained before and after T. Kinase activation state was analyzed as post:pre(-)treatment ratio; molecular subtype was by gene expression analysis. Results: Kinome response profiling was completed in 7 of 9 pts by abstract submission. Toxicities included grade 2 (2pts) or grade 1 (1pt) rash, grade 1 diarrhea (1pt), and grade 1 nausea (1pt). Two of the 9 pts profiled did not have sufficient pre-treatment tumor for kinome analysis. Molecular subtypes included 6 BBC patients (one of which was CL after T), 2 CL patients (one of which was BBC after T), and 1 normal-like patient (which was BBC after T). MEK1/2 inhibition and kinase reprogramming was seen in 6 of the 7 tumors where both pre- and post T samples could be analyzed. The results show kinome reprogramming in response to MEK1/2 inhibition occurs in human tumors similar to preclinical modeling in TNBC cell lines and credentialed engineered mouse models; the kinase reprogramming pattern differed between BBC and CL. Conclusions: MEK1/2 inhibition upregulates and activates specific receptor tyrosine kinases in BBC that are different from CL.A subset of BBC and CL may have plasticity, changing their molecular subtype and kinome profile, and responding heterogeneously to MEK1/2 inhibition. Analysis of kinome reprogramming identifies upregulation of druggable targets for individual patients that suggest rational combinations with MEK inhibition in TNBC. Clinical trial information: NCT01467310.