Wild-type Triple Negative Breast Research Articles

Abstract 1943: Radiosensitization of PIK3CA wild type triple negative breast cancers with Bcl-family inhibition

Abstract Purpose: Compared to other breast cancer subtypes, triple negative breast cancers (TNBC) derive the least benefit from adjuvant radiation (RT) which contributes to higher rates of locoregional recurrence. Thus, there is a critical need to identify clinical strategies to increase the effectiveness of RT therapy in TNBC. Methods: Alamar blue proliferation assays were used to calculate half maximal inhibitory concentration (IC50) values for each Bcl-2 family inhibitor 72 hours after drug treatment. Clonogenic survival assays were used to evaluate radiosensitivity and to calculate the radiation enhancement ratio (rER) after combination treatment. Apoptosis was assessed through formation of cleaved PARP and annexin V/PI-based flow cytometry. Xenograft models with MDA-MB-231 cells and TNBC patient-derived xenografts (PDX4664) were used to assess radiosensitization in vivo. Results: A novel radiosensitizer screen identified Bcl-2 family inhibition as a potentially effective treatment strategy in radioresistant breast cancer models. Single-agent response to pan Bcl-2 family inhibition (ABT-263) or Bcl-xL inhibition (WEHI-539, A-1331852) was more effective in PIK3CA wild type (wt) TNBC (IC50 < 1µM) compared to PIK3CA mutant TNBC. Inhibition of apoptosis with ABT-263 led to radiosensitization of PIK3CA/PTEN wild-type TNBC cell lines (rER: 1.09-1.74), but had no effect on PIK3CA/PTEN mutant TNBC (rER: 0.87-1.18). Radiosensitization was observed to be Bcl-xL-dependent, with Bcl-xL inhibitor-specific radiosensitization (rER: 1.12-2.38) but a lack of Bcl-2 inhibitor (ABT-199, rER: 0.94 - 1.21) or MCL-1 inhibitor-mediated radiosensitization (S63845, rER: 0.91 - 1.06). In PIK3CA wt TNBC, combination treatment of Bcl-2 family inhibition and RT significantly increased the percent of apoptotic cells (p < 0.001) and led to increased formation of cleaved PARP 48 hours after RT. Sensitivity to RT was dependent on expression of MCL-1, an anti-apoptotic protein that is overexpressed in PIK3CA/PTEN mutant TNBC. Overexpression of MCL-1 in PIK3CA/PTEN wild type TNBC rescued radioresistance (rER: 0.99-1.09), whereas co-inhibition of MCL-1 and Bcl-xL in PIK3CA/PTEN mutant TNBC was sufficient to overcome radioresistance (rER: 2.32 - 2.35). In vivo, nonspecific Bcl-2 family inhibition or specific Bcl-xL inhibition in combination with RT decreased tumor growth and increased time to tumor tripling (p < 0.0001) in PIK3CA wt models of TNBC. Conclusions: In this study, we demonstrated that inhibition of Bcl-2 family proteins in combination with RT led to increased levels of apoptosis and cell death in PIK3CA/PTEN wt - but not PIK3CA/PTEN mutant - TNBC and we identified MCL-1 as a critical mediator of this radiosensitIvity. Together, these results indicate that Bcl-xL inhibition may be a feasible clinical strategy for the radiosensitization of PIK3CA/PTEN wild-type TNBC. Citation Format: Andrea M. Pesch, Benjamin C. Chandler, Anna R. Michmerhuizen, Nicole Hirsh, Kari Wilder-Romans, Meilan Liu, Tanner Ward, Dana Messinger, Charles Nino, Cassandra Ritter, James M. Rae, Corey W. Speers. Radiosensitization of PIK3CA wild type triple negative breast cancers with Bcl-family inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1943.

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

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

Polysaccharide hydrogel based 3D printed tumor models for chemotherapeutic drug screening

A series of stable and ready-to-use bioinks have been developed based on the xeno-free and tunable hydrogel (VitroGel) system. Cell laden scaffold fabrication with optimized polysaccharide-based inks demonstrated that Ink H4 and RGD modified Ink H4-RGD had excellent rheological properties. Both bioinks were printable with 25–40 kPa extrusion pressure, showed 90% cell viability, shear-thinning and rapid shear recovery properties making them feasible for extrusion bioprinting without UV curing or temperature adjustment. Ink H4-RGD showed printability between 20 and 37 °C and the scaffolds remained stable for 15 days at temperature of 37 °C. 3D printed non-small-cell lung cancer (NSCLC) patient derived xenograft cells (PDCs) showed rapid spheroid growth of size around 500 µm in diameter and tumor microenvironment formation within 7 days. IC50 values demonstrated higher resistance of 3D spheroids to docetaxel (DTX), doxorubicin (DOX) and erlotinib compared to 2D monolayers of NSCLC-PDX, wild type triple negative breast cancer (MDA-MB-231 WT) and lung adenocarcinoma (HCC-827) cells. Results of flow property, shape fidelity, scaffold stability and biocompatibility of H4-RGD suggest that this hydrogel could be considered for 3D cell bioprinting and also for in-vitro tumor microenvironment development for high throughput screening of various anti-cancer drugs.

Abstract P1-06-04: Small-molecule screening nominates diverse combination therapies that sensitize BRCA mutant and wild-type triple negative breast cancer to PARP inhibition

Abstract Background: Triple negative breast cancer (TNBC) remains a heterogeneous clinical phenotype with few, known therapeutic targets. PARP inhibitors (PARPi) are the first approved, targeted therapy in TNBC, limited to germline BRCA mutant (BRCAm) cancers that lack homologous recombination repair capacity. Even in this context, resistance quickly emerges via secondary mutations that restore DNA repair ability. While DNA damage repair is an intriguing target in BRCA wild type (BRCAwt) TNBC due to inherent, genomic instability, PARPi alone have been ineffective in unselected populations. Systematic approaches to define novel drugs that sensitize BRCAwt and BRCAm TNBC to PARPi would greatly improve therapeutic efficacy and durability. Methods: BRCAwt (HCC1806) and BRCAm (SUM149PT) cell lines were screened in duplicate using a 2,100-compound small molecule library. Cell lines were plated in media containing DMSO or sub-lethal doses of the PARPi, olaparib, onto Selleck Bioactive drug plates. Cell viability was assessed after 72 hours, then normalized to vehicle control. Hit cut-offs were predefined as log2 drug/DMSO of ≤ -0.7 with a viability difference greater than 20% -where stringent scoring thresholds were chosen to exceed the full range of scores observed in 816 empty control wells. Hits were sorted by target and pathway to provide mechanistic insight into the synergy of combinations. Drug combinations with the highest potential for near term translation were validated using GI50 viability assays in 9 BRCAwt and BRCAm TNBC cell lines. The most promising combination was further validated via immunoblotting, colony formation, and apoptosis assays. Results: Several drug classes affecting well-known oncogenic signaling pathways conferred sensitivity to PARPi, with more hits in the BRCAm cell line. Relevant druggable targets sensitizing cells to olaparib in BRCAm TNBC that met the predefined cut-point were inhibitors of PI3K (pan-PI3K, PI3Kα and PI3Kβ specific), VEGFR, MEK, EGFR, NF-kB, aurora kinase and several DNA damaging agents. Aurora kinase, EGFR, and NF-kB inhibition sensitized cells to olaparib, yet upon further validation, synergy was mild. The screen identified ATM inhibitors, KU-55933 and KU-60019, as sensitizers of BRCAm cells to olaparib. The potent ATM inhibitor, AZD0156, and olaparib were a highly synergistic combination validated in all 9 BRCAm and BRCAwt TNBC cell lines via cell viability, annexin V, and colony formation assays. Immunoblotting of relevant DNA damage repair proteins showed that olaparib caused upregulation of p-ATM in BRCAm and BRCAwt cells. p-ATM expression decreased in response to combination ATM and PARP inhibition. Attenuated levels of p-ATM resulted in increased levels of p- and T-γH2AX, indicating an accumulation of double stranded DNA breaks. Conclusion: In vitro, inhibition of several relevant, oncogenic pathways yielded sensitivity to PARPi in TNBC. We identified the ATM inhibitor, AZD0156, and olaparib as a potent combination regardless of BRCA status, a finding currently being evaluated in patient-derived in vivo models. Combination ATM plus PARP inhibitor therapy is a promising and feasible approach for near term translation in metastatic TNBC. Citation Format: Sammons S, Yip C, Anderson G, Force J, Marcom K, Westbrook K, Anders CK, Blackwell K, Wood K. Small-molecule screening nominates diverse combination therapies that sensitize BRCA mutant and wild-type triple negative breast cancer to PARP inhibition [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-06-04.

Abstract PD1-14: Induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer: BET inhibition as a therapeutic strategy

Abstract Background: Pharmacological induced BRCAness and synthetic lethality could represent a potential therapeutic strategy in triple negative breast cancer (TNBC). Bromodomain and Extra-Terminal proteins (BET) such as BRD2 and BRD4, are involved in controlling RNA polymerase II pause/release transition mediating transcriptional activation and gene bookmarking. BRD4 is found to be associated to enhancer and promoter regions to foster gene expression. Furthermore, BRD4 overexpression is described to be associated with worse outcome and increased metastasizing potential. Aim of this study was to explore novel therapeutic strategies through pharmacological induced epigenetic BRCAness in BRCA1 wild-type TNBC cells by means of BET inhibition. Methods: The experimental models were based on the BT-549, MDA-MB-157 and MDA-MB-231 TNBC BRCA1 wild type cells lines. Two different pan-BET inhibitors were explored (i.e. JQ1 and I-BET762). BRCA1 and RAD51 levels were evaluated by both qPCR and western blotting as a marker of BRCAness phenotype onset. Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi)-mediated BRD4 silencing were performed on TNBC cells. Synthetic lethality induced by JQ1 (0.5 µM) and the effects of co-treatment with the PARP inhibitor PJ34 (5-15 µM) or cisplatin (2-10 µM) were evaluated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Results: A dose-dependent reduction in BRCA1 and RAD51 protein levels was observed after treatment with both JQ1 and I-BET762 in all the cell lines tested. To verify the direct relationship between BRD4 and BRCA1/RAD51 expression, a dual approach was performed. First, BRCA1 and RAD51 protein levels were evaluated after RNAi-mediated BRD4 knockdown, verifying that the BRCAness phenotype (exemplified by BRCA1 and RAD51 protein downregulation) was due to the absence of BRD4 protein. Furthermore, a ChIP assay was performed to confirm the direct regulation of BRCA1 and RAD51 promoters by BRD4. To test the gain in sensitivity of BRCAness-displaying TNBC cells towards platinum salts, the effects of cisplatin alone or in combination with BET inhibitors were tested. JQ1 and cisplatin combinations induced a strong reduction in TNBC cell viability in all the three cell lines tested. Moreover, the pharmacological combination between JQ1 and PJ34 displayed a significant cell viability decrease in TNBC wild type cells, corroborating the synthetic lethality mechanism. Conclusions: A decrease in BRCA1 and RAD51 levels was observed after BET inhibition by means of the JQ1 drug, exploiting synergism with PARP inhibition and use of platinum salts. Moreover, the present results demonstrate the direct interaction between BRD4, BRCA1 and RAD51, supporting the therapeutic rationale behind this novel strategy. Citation Format: Gerratana L, Puglisi F, Damante G, Mio C. Induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer: BET inhibition as a therapeutic strategy [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 PD1-14.

Abstract P3-06-16: Thrombocytopenia is associated with pathological complete response to neoadjuvant carboplatin/docetaxel chemotherapy in BRCA wild type triple negative breast cancer

Abstract Introduction: Growing evidence demonstrates activity of neoadjuvant carboplatin in triple negative breast cancer (TNBC). Underlying germline and somatic Homologous Recombination (HR) repair deficiency may predict response to DNA damaging agents like platinum compounds in TNBC. Certain DNA repair machinery genes (Fanconi Anemia gene) are also involved in the maintenance of hematopoetic stem cell (HSC) function and impaired repair of DNA double strand breaks can lead to HSC and progenitor cell dysfunction. Thus, in presence of HR defects DNA damaging chemotherapy may lead to unique hematological toxicity. It is also possible that in presence of HR defects breast cancer response and hematological toxicity with DNA damaging agents will parallel each other. Aim: To evaluate the impact of hematological toxicity on response to neoadjuvant Carboplatin/Docetaxel chemotherapy in patients with sporadic and BRCA associated TNBC utilizing clinical and BRCA mutation data from a prospective TNBC registry. Methods: 288 patients with Stage I (T>1cm) II and III TNBC were enrolled on a multisite prospective registry between 3/2011 to 4/2014, out of which 49 patients received neoadjuvant Carboplatin AUC 6 + Docetaxel 75mg/m2 every 21 days (4-6 cycles) and have undergone breast surgery. Carboplatin was dosed using the modified Cockcroft-Gault formula. Hematologic toxicity was graded using the CTCAE version 4.03. All patients received prophylactic pegfilgrastim on day 2. Pathological complete response (pCR) was defined as absence of invasive disease in the breast and axilla. All patients underwent comprehensive BRACAnalysis®(Myriad). Results: For the 49 eligible patients median age was 50 years, median weight was 172 lbs, 18% were African American, and 37% had LN+ disease. 26% (13 /49) of patients carried deleterious BRCA mutation (9 BRCA1, 4 BRCA2). pCR of the cohort was 65%. Overall 61%, 96%, and 12 % patients demonstrated > Grade1 thrombocytopenia (Tp), anemia, or neutropenia, respectively; 4%, 6%, and 6% patients demonstrated grade 3/4 thrombocytopenia, anemia, or neutropenia, respectively. There was no association between pCR and anemia or neutropenia. Carboplatin dose reductions/delays/omission was more common in patients with Tp compared to patients without Tp (33% vs. 5%; p=0.02). Patients with Tp were more likely to achieve a pCR compared to patients without Tp (85% vs. 47%; p=0.013). 77% of BRCA carriers and 64% of BRCA wild type TNBC demonstrated Tp (NS). pCR rates in BRCA wild type patients with and without Tp were 82% and 47%, respectively (p=0.041). pCR rates in BRCA mutation carriers with and without Tp were 89% and 50%, respectively (p=0.20). On multivariable platelet count was independently associated with pCR (p=0.001)Conclusions: Tp was associated with decreased dose delivery of carboplatin but an improved pCR in BRCA wild type TNBC. Comprehensive assessment of HR defects beyond germline BRCA mutation status may be required to elucidate the biological process that explains this observation. Tp may be a harbinger of underlying HR deficiency and further correlative studies exploring this association of Tp with pathological response to carboplatin in TNBC are warranted. Citation Format: Priyanka Sharma, Benjamin C Powers, Bruce F Kimler, Claire Ward, Jennifer R Klemp, Carol S Connor, Marilee K McGinness, Joshua MV Mammen, Jamie L Wagner, Qamar J Khan, Roy A Jensen, Andrew K Godwin, Carol J Fabian. Thrombocytopenia is associated with pathological complete response to neoadjuvant carboplatin/docetaxel chemotherapy in BRCA wild type 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 P3-06-16.

Abstract 1011: Metformin inhibits angiogenesis, local and metastatic growth of triple-negative breast cancer by targeting two classes of adipose tissue progenitors

Abstract We have recently reported that human white adipose tissue (WAT) contains two classes of CD45-CD34+ progenitors with cooperative roles in triple negative breast cancer progression. In orthotopic murine models we found that 1) purified human WAT CD34+CD13+ mesenchymal adipose stromal cell progenitors (ASCs) were not able to migrate but promoted local tumor growth in the mammary fat pad and 2) purified human WAT CD34+CD31+ endothelial progenitors (EPCs) were able to migrate towards lymph nodes and blood and promoted breast cancer cell EMT, migration, invasion and metastatic growth (Orecchioni et al, 2013). Here we report that in several in vitro and in vivo models the biguanide Metformin, a drug commonly used in type 2 diabetes management, is able to inhibit angiogenesis, local and metastatic growth of triple negative breast cancer by targeting WAT progenitors. First, we and others (Dallaglio et al, in press) have found in collaborative studies that Metformin inhibits the formation of capillary-like networks by HUVEC cells. This effect was partially AMPK-dependent. Metformin inhibited VEGF-dependent activation of ERK1/2, angiogenesis in matrigel pellets in vivo, and decreased microvessel density in tumor-free mice. The effect of Metformin on WAT progenitors was then studied in vivo in several immune-competent and immune-deficient murine strains with and without triple negative breast cancer. In immune-competent mice, low-dose Metformin (2mg/mL drinking water) induced apoptosis and increased hypoxia of CD34+CD31+ WAT EPCs. As a result of low-dose Metformin administration, WAT and cancer vessels were quantitatively reduced, had a significant impairment of the pericyte/endothelial cell ratio and displayed a branching and dysplastic phenotype. This effect was also observed in immune-competent mice on high fat diet receiving Metformin. WAT CD34+ ASC were targeted by higher doses of Metformin, which were associated with a reduced pericyte coverage of new blood vessels. As Metformin is frequently administered along with aspirin and beta-blockers in diabetic and obese patients, we studied in vivo the association of these three drugs in murine models of triple negative breast cancer. In several immune-deficient murine models, the beta-blocker Atenolol significantly synergized with Metformin in inhibiting both local and metastatic breast cancer growth. In immune-competent murine strains, the anti-metastatic effect of Metformin and Atenolol was further enhanced by the addition of aspirin. These effects were observed in several murine models generated by both p53 mutated or p53 wild type triple negative breast cancer cells. We are currently investigating the molecular mechanisms used by Metformin to inhibit WAT progenitors and the most efficient schedules of administration of Metformin, Atenolol and Aspirin to target local and metastatic breast cancer growth. Citation Format: Stefania Orecchioni, Giuliana Gregato, Francesca Reggiani, Patrizia Mancuso, Angelica Calleri, Giovanna Talarico, Valentina Labanca, Francesco Bertolini. Metformin inhibits angiogenesis, local and metastatic growth of triple-negative breast cancer by targeting two classes of adipose tissue progenitors. [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 1011. doi:10.1158/1538-7445.AM2014-1011