Wild-type Triple-negative Breast Cancer Research Articles

Characterization and Targeting Cancer Stem‐like Cells in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem‐like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore, CSC‐targeted therapies are keenly sought. There is strong evidence for the involvement of canonical‐Notch signaling in TNBC, however the role of non‐canonical Notch signaling in TNBC remains unknown. Notch1 is highly expressed in Basal‐like 1 (BL1) and especially Mesenchymal (M) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch activation by Jagged1‐expressing stromal cells enhances transcription of the anti‐apoptotic gene cIAP‐2 (BIRC3), a known NF‐κB target. This event is dependent on recruitment to the cIAP‐2 promoter of NF‐κB subunits, IKKα and Notch1. Short term exposure of MDA‐MB‐231 cells (M, PTEN wild‐type), but not MDA‐MB‐468 cells (BL1, PTEN‐null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by AKT inhibitors, IKK inhibitors, and dual mTORC1/2 inhibitors but not an mTORC1‐selective inhibitor. These observations support a model where canonical and non‐canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF‐κB activation in PTEN wild type TNBC cells. Importantly, we find co‐localization of Notch1 with Mitochondria in MDA‐MB‐231 cells by confocal microscopy and Western blot of isolated mitochondrial fractions. We demonstrate that recombinant Jagged1 increases the metabolism of TNBC cells. Knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. CSCs derived from MDA‐MB‐231 cells have increased Notch1, p‐AKT, and oxidative metabolism compared to non‐stem cells. AKT inhibition or IKK inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF‐03084014) in combination with AKT inhibitor (MK‐2206) or IKK inhibitor (Bay11‐7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. Notably, we find similar results in TNBC patient derived xenograft (PDX) models. However, Notch inhibition may have adverse effects on tumor immunity, due to the role of Notch in CD4 and CD8 T‐cell activation. Thus, we explored an immunocompetent tumor mouse model to study the effects of these pharmacological interventions in that setting. We developed an immunocompetent TNBC mouse model by injecting TNBC mouse cells (C0321) into mammary fat pads of isogenic FVB female mice. Combination treatments with GSI (PF‐03084014) and AKT inhibitor (MK‐2206) significantly delayed tumor growth as well as decreased CSCs number in that model. These data suggest that combination treatments affecting the intersection of Notch and AKT pathways have potential therapeutic applications in targeting CSCs in TNBC cases with high Notch1 expression. The possible immunological effects of such combination treatments require additional investigation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P2-10-05: The impact of TP53 mutation and pathological response to neoadjuvant chemotherapy on triple negative breast cancer outcomes

Abstract Background: Triple Negative Breast Cancer (TNBC) accounts for 15-20% of breast cancers, and has a poor prognosis compared with hormone receptor-positive or HER2-amplified disease. There is currently no targeted therapy to salvage chemotherapy resistant TNBC confirming a need for predictive biomarkers of sensitivity to therapy. This study aimed to evaluate the impact of TP53 mutations, common in TNBC, on sensitivity to therapy and patient outcomes. Methods: TNBC samples (pretreatment biopsies and surgical specimens, n=106) had annotated response to standardized anthracycline/taxane based neoadjuvant chemotherapy (NACT) and 3-year survival data, by which time most recurrences occur in TNBC. RNAseq was performed using Illumina HiSeq with mRNA library preparation protocol to identify TP53 mutations, which were then scored using the Evolutionary Action Score (EAS), a novel computational model shown to be successful at identifying deleterious mutations. This work was supported by an ASCO Conquer Cancer Foundation Young Investigator Award grant. Results: 106 TNBC patients were enrolled on this study, 14 had stage I, 65 stage II, and 24 had stage III disease. 64 patient samples have been analyzed to date, of which 39 (61%) harbored a TP53 mutation, and 24 (38%) were TP53 wildtype. Pathologic complete response (pCR) and survival Of 47 patients who received NACT, 14 (30%) achieved a pCR, and 33 (70%) had residual disease (RD). Patients who achieved a pCR had a significantly better 3-year recurrence free survival (RFS) (100% vs 59%, p=0.011) and overall survival (OS) (100% vs 62%, p-0.01) compared to patients who had residual disease. TP53 status and pCR The rate of pCR was significantly higher in the TP53 mutant group (11/29, 38%) compared to TP53 wildtype (2/17, 12%) TNBC (OR = 4.6, (0.9, 24), p = 0.046). Patients with wild-type and mutant TP53 TNBC who achieved a pCR had excellent 3-year RFS and OS at 100%. Among patients with RD, TP53 wild-type TNBC exhibited a non-significant trend towards higher 3-year RFS (72% vs 49%,HR = 2.5 (0.8, 8.0) p = 0.12) and OS (79% vs 48%,HR = 2.6 (0.8, 8.4) p = 0.11) compared to TP53 mutant TNBC. TP53 status and survival The 3-year OS for TP53 mutant TNBC was 72%, compared to 86% for TP53 wildtype TNBC (HR = 1.5 (0.5, 4.3), p = 0.48). There was no difference in 3-year RFS between the two groups. TP53 EAS and survival Among 39 non-silent TP53 mutant cases, EAS ranged from 42.3 to 100 with a median score of 87.6. The 3-year OS for patients with EAS ≥86.7 was 73%, compared to 74% for patients with EAS <86.7 (HR 0.8 (0.2, 3.0), p=0.78). When separated into tertiles, the 3-year OS rates were 75%, 68%, and 77% for the lower, middle, and upper thirds respectively. The 3-year OS rates for TNBC with EAS = 0 (TP53 wildtype or silent TP53 mutation) was 87%. Conclusion: TP53 mutations were associated with a higher chance of achieving a pCR; NACT Patients who achieved a pCR to NACT had excellent outcomes, regardless of TP53 mutation status. In patients with RD following NACT, TP53 mutant TNBC had a trend towards worse outcomes compared to TP53 wild-type TNBC. Larger cohorts are required to further evaluate the impact of TP53 mutations and TP53 EAS on survival outcomes in TNBC. Citation Format: Mitri Z, Lin C-H, Hess KR, Thompson A, Lichtarge O, Hubbard R, Fu C, Symmans WF, Moulder-Thompson SL. The impact of TP53 mutation and pathological response to neoadjuvant chemotherapy on triple negative breast cancer outcomes [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 P2-10-05.

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.

H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

Non‐cannonical notch signaling pathways regulate breast cancer stem‐like cells function in triple negative breast cancer

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive form of breast cancers, characterized by lack of expression of estrogen receptor α (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2).TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem‐like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC‐targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal‐like 1 (BL1) and especially Mesenchymal‐Stem‐Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch1 promotes cell survival in MDA‐MB‐231 cells, representative of MSL TNBC, in part by activating NF‐κB. Notch activation by Jagged1‐expressing stromal cells enhances transcription of the anti‐apoptotic gene cIAP‐2 (BIRC3), a known NF‐κB target. This event is rely on Notch dependent recruitment of NF‐κB subunits, IKKα to the cIAP‐2 promoter. Short term exposure of MDA‐MB‐231 cells [MSL, phosphatase and tensin homolog (PTEN) wild‐type], but not MDA‐MB‐468 cells (BL1, PTEN‐null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by gamma secretase inhibitor (GSI), dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not mTORC1‐selective inhibitor. These observations support a model where canonical and non‐canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF‐κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF‐κB activation. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1‐activated Notch pathway. We demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA‐MB‐231 cells have increased Notch1, p‐AKT, p‐IKKα, and oxidative metabolism. AKT inhibition or IKKα inhibition alone or in combination with GSI (PF‐03084014) decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by GSI (PF‐03084014) in combination with AKT inhibitor (MK‐2206) or NF‐κB inhibitor (Bay11‐7082) blocks CD90hi or CD44+CD24low (CSCs marker) sorted secondary mammospheres formation. These data suggest that blocking of non‐canonical Notch‐ NF‐κB ‐AKT pathways have potential therapeutic importance in targeting CSCs of TNBC. Our data indicate that therapeutic regimens targeting this pathway deserve further investigation in TNBCs.Support or Funding InformationFunding: NIH 3P01CA166009‐04S1

Abstract P5-16-02: Pathological complete response is associated with excellent outcomes in BRCA mutation associated triple negative breast cancer

Abstract Introduction: Pathological complete response (pCR) in unselected triple negative breast cancer (TNBC) is associated with excellent long-term survival. However, controversy remains as to whether pCR in BRCA mutation associated (BRCA[+]) TNBC is predictive of improved long-term outcome. A recent study suggests that pCR was not a surrogate for outcomes in BRCA1 associated TNBC. All of the patients in this study harbored an Ashkenazi Jewish founder BRCA1 mutation and the majority of mutation carriers underwent lumpectomy. Impact of pCR as it relates to BRCA status in a larger, heterogeneous TNBC cohort treated in a contemporary time frame is not known. Aim: Evaluate and compare the prognostic impact of pCR as it relates to the BRCA mutation status in patients enrolled in a prospective multisite TNBC registry. Methods: 453 patients with stage I-III TNBC were enrolled within a multisite registry between 2011- 2015, out of which 173 received neoadjuvant chemotherapy (NAC) and also underwent germline BRCA testing. pCR in the breast and axilla was evaluated and patients were followed for reoccurrence and survival. Recurrence free survival (RFS) was estimated according to the Kaplan-Meier method and compared among groups with log-rank statistic. Results: For the 173 eligible patients the median age was 49 years; African-American:14%; median tumor size:3 cm; 42%:Lymph node positive; and 18% (32/173) demonstrated BRCA mutation (BRCA1=28, BRCA2=4). All patients received anthracycline and/or taxane based NAC. pCR rates for BRCA[+] and wild type (BRCA[-]) patients was 72% and 46% respectively (p=0.01). 97% of BRCA[+] and 42% of BRCA[-] patients underwent bilateral mastectomy (p=0.001). The three year RFS was 92% and 81% in BRCA[+] and BRCA[-] patients, respectively (p=0.18). Attainment of pCR was associated with excellent 3 year RFS of 95% and 97% in BRCA[+] and BRCA[-] patients, respectively (p=0.85). Among BRCA[-] patients lack of pCR was associated with significantly worse 3 year RFS (70% RFS in patients without pCR, compared to 97% in patients with pCR; p=0.001). Among BRCA[+] patients lack of pCR was associated with numerically lower but not statistically significant worse 3 year RFS (83% RFS in patients without pCR, compared to 95% in patients with pCR; p=0.41). On multivariable Cox regression analysis, only stage III disease was associated with higher risk of relapse (p<0.001). Conclusions: Our observation of higher pCR in BRCA-carriers compared to wild-type TNBC patients is consistent with previously published literature. In this contemporary cohort of TNBC patients for whom the majority of BRCA[+] patients underwent bilateral mastectomy, attainment of pCR carried an excellent prognosis in both BRCA[+] and BRCA[-] patients. On the other hand, BRCA[+] patients who do not attain pCR may have better outcomes compared to BRCA[-] patients without pCR. Further research to explore the underlying biological mechanisms involved in tumor response and relapse in BRCA[+] and BRCA[-] TNBC patients is needed. Furthermore, given these observations, germline BRCA mutation status should be used as a stratification variable in studies evaluating pCR and long term outcomes with investigational therapies in TNBC. Citation Format: Prochaska LH, Godwin AK, Kimler BF, Lehn C, Klemp JR, O'Dea A, Elia M, Hoffmann MS, Crane G, McKittrick R, Sheehan M, Graff SL, Madhusudhana S, Khan QJ, Jensen RA, Sharma P. Pathological complete response is associated with excellent outcomes in BRCA mutation associated 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 P5-16-02.

Efficacy of Neoadjuvant Carboplatin plus Docetaxel in Triple-Negative Breast Cancer: Combined Analysis of Two Cohorts.

Recent studies demonstrate that addition of neoadjuvant (NA) carboplatin to anthracycline/taxane chemotherapy improves pathologic complete response (pCR) in triple-negative breast cancer (TNBC). Effectiveness of anthracycline-free platinum combinations in TNBC is not well known. Here, we report efficacy of NA carboplatin + docetaxel (CbD) in TNBC. The study population includes 190 patients with stage I-III TNBC treated uniformly on two independent prospective cohorts. All patients were prescribed NA chemotherapy regimen of carboplatin (AUC 6) + docetaxel (75 mg/m2) given every 21 days × 6 cycles. pCR (no evidence of invasive tumor in the breast and axilla) and residual cancer burden (RCB) were evaluated. Among 190 patients, median tumor size was 35 mm, 52% were lymph node positive, and 16% had germline BRCA1/2 mutation. The overall pCR and RCB 0 + 1 rates were 55% and 68%, respectively. pCRs in patients with BRCA-associated and wild-type TNBC were 59% and 56%, respectively (P = 0.83). On multivariable analysis, stage III disease was the only factor associated with a lower likelihood of achieving a pCR. Twenty-one percent and 7% of patients, respectively, experienced at least one grade 3 or 4 adverse event. The CbD regimen was well tolerated and yielded high pCR rates in both BRCA-associated and wild-type TNBC. These results are comparable with pCR achieved with the addition of carboplatin to anthracycline-taxane chemotherapy. Our study adds to the existing data on the efficacy of platinum agents in TNBC and supports further exploration of the CbD regimen in randomized studies. Clin Cancer Res; 23(3); 649-57. ©2016 AACR.

The autophagy inhibitor chloroquine targets cancer stem cells in triple negative breast cancer by inducing mitochondrial damage and impairing DNA break repair

Triple negative breast cancer (TNBC), characterized by an abundance of treatment-resistant breast cancer stem cells (CSCs), has a poorer prognosis than other types of breast cancers. Despite its aggressiveness, no effective targeted therapy exists for TNBC. Here, we demonstrate that CQ effectively targets CSCs via autophagy inhibition, mitochondrial structural damage, and impairment of double-stranded DNA break repair. Electron microscopy demonstrates CQ-induced mitochondrial cristae damage, which leads to mitochondrial membrane depolarization with a significant reduction in the activity of cytochrome c oxidase and accumulation of superoxide and double-stranded DNA breaks. CQ effectively diminishes the TNBC cells' ability to metastasize in vitro and in a TNBC xenograft model. When administered in combination with carboplatin, CQ effectively inhibits carboplatin-induced autophagy. This combination treatment significantly diminishes the expression of DNA repair proteins in CSC subpopulations, resulting in tumor growth reduction in carboplatin-resistant BRCA1 wild-type TNBC orthotopic xenografts. As TNBC's high treatment failure rate has been attributed to enrichment of CSCs, CQ, an autophagy inhibitor with anti-CSC effects, may be an effective adjunct to current TNBC chemotherapy regimens with carboplatin.

Abstract OT2-05-04: The Talazoparib Beyond BRCA (TBB) trial: A phase II clinical trial of talazoparib (BMN 673) in BRCA1 and BRCA2 wild-type patients with (i) advanced triple-negative breast cancer (TNBC) and homologous recombination deficiency (HRD) as assessed by myriad genetics HRD assay, and (ii) advanced HER2-negative breast cancer (BC) with either a germline or somatic mutation in homologous recombination (HR)

Abstract Background: Poly-ADP-ribose polymerase (PARP) inhibition induces synthetic lethality in tumor cells bearing deleterious mutations in the genes BRCA1/2. Talazoparib (BMN 673) is a novel, dual-mechanism PARP inhibitor that potently inhibits the PARP enzyme and effectively traps PARP on DNA. Talazoparib has shown promising single-agent anti-tumor efficacy in several BRCA1/2 mutation-associated advanced cancers. The efficacy of PARP inhibition in BRCA1/2 wild-type TNBC with HR defects and in breast tumors with mutations in other non-BRCA1/2 HR pathway genes is currently unknown. Trial Design & Eligibility: This Phase 2 trial (TBB) explores the activity of single agent talazoparib in BRCA1/2 wild-type BC patients using an optimal Simon two-stage design. Eligible subjects will be assigned to one of two parallel cohorts: 1) Cohort A: Subjects (n=29) with advanced TNBC with underlying HR defects as assessed by the HRD assay and, 2) Cohort B: Subjects (n=29) with advanced HER2-negative BC with a somatic or germline deleterious mutation in a non-BRCA1/2 HR pathway gene. Gene mutations of interest are: PTEN, PALB2, CHEK2, ATM, NBN, BARD1, BRIP1, RAD50, RAD51C, RAD51D, MRE11, ATR, Fanconi anemia complementation group of genes (FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FANCL). Other key eligibility criteria include metastatic disease treated with at least one prior line of chemotherapy, no deleterious BRCA1/2 mutation, and no prior platinum exposure. In cohort A, TNBC patients must have a HRD score of ≥ 42. Eligible patients will receive oral talazoparib (1.0 mg/day, 28-day cycles) until disease progression or unacceptable toxicity. Endpoints & Statistical Plan: The primary endpoint is objective response rate (ORR). Secondary endpoints include clinical benefit rate (CBR), progression free survival (PFS), and safety. In this study, we have set a null hypothesis of ≤ 5% objective response rate and alternative response rate of ≥ 20% based on RECIST 1.1. Interim analysis will be performed after accrual of 10 patients in each cohort. If at least one out of the 10 patients responds, then we will accrue 19 additional patients for a total of 29 patients in each cohort. At least four patients have to respond out of the 29 patients in each cohort to declare statistical significance at a one-sided 5% level with 80% power or better. This trial is enrolling patients at Stanford University in California (NCT 02401347). Citation Format: Afghahi A, Chang P-J, Ford JM, Telli ML. The Talazoparib Beyond BRCA (TBB) trial: A phase II clinical trial of talazoparib (BMN 673) in BRCA1 and BRCA2 wild-type patients with (i) advanced triple-negative breast cancer (TNBC) and homologous recombination deficiency (HRD) as assessed by myriad genetics HRD assay, and (ii) advanced HER2-negative breast cancer (BC) with either a germline or somatic mutation in homologous recombination (HR) pathway genes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT2-05-04.

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.

Efficacy of Carboplatin Alone and in Combination with ABT888 in Intracranial Murine Models of BRCA-Mutated and BRCA-Wild-Type Triple-Negative Breast Cancer.

Patients with breast cancer brain metastases have extremely limited survival and no approved systemic therapeutics. Triple-negative breast cancer (TNBC) commonly metastasizes to the brain and predicts poor prognosis. TNBC frequently harbors BRCA mutations translating to platinum sensitivity potentially augmented by additional suppression of DNA repair mechanisms through PARP inhibition. We evaluated brain penetrance and efficacy of carboplatin ± the PARP inhibitor ABT888, and investigated gene-expression changes in murine intracranial TNBC models stratified by BRCA and molecular subtype status. Athymic mice were inoculated intracerebrally with BRCA-mutant: SUM149 (basal), MDA-MB-436 (claudin-low); or BRCA-wild-type (wt): MDA-MB-468 (basal), MDA-MB-231BR (claudin-low). TNBC cells were treated with PBS control [intraperitoneal (IP), weekly], carboplatin (50 mg/kg/wk, IP), ABT888 (25 mg/kg/d, oral gavage), or their combination. DNA damage (γ-H2AX), apoptosis (cleaved caspase-3, cC3), and gene expression were measured in intracranial tumors. Carboplatin ± ABT888 significantly improved survival in BRCA-mutant intracranial models compared with control, but did not improve survival in BRCA-wt intracranial models. Carboplatin + ABT888 revealed a modest survival advantage versus carboplatin in BRCA-mutant models. ABT888 yielded a marginal survival benefit in the MDA-MB-436, but not in the SUM149 model. BRCA-mutant SUM149 expression of γ-H2AX and cC3 proteins was elevated in all treatment groups compared with control, whereas BRCA-wt MDA-MB-468 cC3 expression did not increase with treatment. Carboplatin treatment induced common gene-expression changes in BRCA-mutant models. Carboplatin ± ABT888 penetrates the brain and improves survival in BRCA-mutant intracranial TNBC models with corresponding DNA damage and gene-expression changes. Combination therapy represents a potential promising treatment strategy for patients with TNBC brain metastases warranting further clinical investigation.

Abstract P2-09-06: The role of p53 family tumor suppressors in mediating response to aurora kinase inhibition 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 the estrogen and progesterone receptors and lack of HER2 over-expression. TNBC carries an increased risk of developing distant metastatic disease and cancer-related death. TNBC is heterogeneous in its underlying biology, however, mutations in p53 are found in approximately 80% of tumors. The purpose of this study was to utilize TNBC cell line models to investigate the role of p53 and p73 in mediating sensitivity to MLN8237, a selective Aurora kinase A inhibitor, in the setting of mutant or wildtype p53. Additionally, we used p53 and p73 knock-down models to determine the role of p53 and p73 in mediating induction of apoptosis and cellular senescence in response to Aurora kinase inhibition. Methods: Eighteen TNBC cell lines were exposed to MLN8237 and the effects on proliferation, apoptosis, and cell cycle distribution were evaluated. Proliferation was assessed using an SRB assay, apoptosis was analyzed using a caspase 3/7 assay and cell cycle was measured using flow cytometry. Proliferation was confirmed using a cell counting technique to control for an increase in cell size following MLN8237 exposure. The p53 wildtype TNBC cell line CAL51 was transduced with several clones of shRNA constructs targeting p53 and p73 with > 80% knockdown by RT-PCR. These clones were exposed to escalating doses of MLN8237 and the effect on proliferation was determined using an SRB assay and the effect on cellular senescence was determined using a senescence associated beta-galactosidase (SA b-gal) assay. Results: In vitro exposure to MLN8237 resulted in robust inhibition of proliferation in TNBC cell lines which was associated with dose-dependent G2/M cell cycle arrest and induction of caspase-dependent apoptosis in a subset of sensitive cell lines. Knock-down of p53 and p73 in the CAL51 cell line resulted in an increase in the absolute 50% inhibitory concentration (IC50) calculated from the SRB proliferation assay curves from 0.04 μM to > 2 μM, 1.8 μM, > 2 μM and 1.5 μM in the CAL51scr, CAL51p5310, CAL51p5312, CAL51p7326, CAL51p7355, respectively. The sensitive p53 mutated MDA-MB-468 cell line (IC50 40 nM) and the sensitive p53 wild-type CAL51 cell line (IC50 45 nM) were selected for further experimentation. Exposure to MLN8237 at concentrations of 50 nM and 100 nM for 7 days resulted in induction of cellular senescence as detected by the SA b-gal assay in the CAL51 p53 wild-type cell line, but not in the MDA-MB-468 cell line where induction of apoptosis at 48 hours was observed using the caspase 3/7 assay. In the CAL51 p73 knock-down clones, induction of cellular senescence was not observed following exposure to MLN8237. Conclusions: MLN8237 exhibited robust anticancer activity towards preclinical models of p53 mutated and p53 wild-type TNBC, supporting future clinical investigation of Aurora kinase inhibitors in TNBC. In p53 wild-type TNBC, both p53 and p73 mediate sensitivity to MLN8237 and p73 is essential for induction of cellular senescence following exposure to MLN8237. Biomarkers predictive of response to MLN8237 in p53 mutated TNBC are being developed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-06.

Statin induces inhibition of triple negative breast cancer (TNBC) cells via PI3K pathway

Primary TNBCs are treated as if they were a single disease entity, yet it is clear they do not behave as a single entity in response to current therapies. Recently, we reported that statins might have a potential benefit for TNBCs associated with ets-1 overexpression. The aim of this study is to investigate the role of PTEN loss in the effects of statin on TNBC cells. In addition, we analyze the relationship between AKT downstream pathways and the effects of statin on TNBC cells. We investigated the effect of a statin on TNBC cells and analyzed the association of PI3K pathways using various TNBC cells in terms of PTEN loss and AKT pathways. Simvastatin treatments resulted in decreased cell viabilities in various TNBC cell lines. Compared with PTEN wild-type TNBC cells, PTEN mutant-type TNBC cells showed a decreased response to simvastatin. Expressions of phosphorylated Akt and total Akt showed an inverse relationship with PTEN expression. The TNBC cell lines, which showed increased expression of p-Akt, appeared to attenuate the expression of p-Akt by PTEN loss in simvastatin-treated TNBC cells. The Akt inhibitor, LY294002, augmented the effect of simvastatin on PTEN wild-type TNBC cells. Simvastatin induces inhibition of TNBC cells via PI3K pathway activation.

Abstract 1788: The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition.

Abstract Background: Triple Negative Breast Cancer (TNBC) comprises 15% of all breast cancers, and has a poor prognosis relative to other breast cancer subtypes. Inhibition of poly (ADP-ribose) polymerase (PARP) causes the degeneration of single-strand DNA breaks to more lethal double-strand breaks (DSBs), and also traps PARP-DNA complexes, which must be repaired or bypassed by homologous recombination (HR). BRCA1 plays a critical role in HR and its activity is in part regulated by cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation. PARP inhibition results in synthetic lethality in cells that have impaired HR, such as BRCA-deficient cells. Other CDK family members activate additional components of the HR pathway. Here, we show that HR-proficient TNBC cells can be sensitized to PARP inhibition through use of CDK inhibition to impair BRCA1 function and disrupt HR. Methods: We examined the effects of dinaciclib (CDK1, 2, 5 and 9 inhibitor), γ-irradiation and veliparib (PARP inhibitor) on a panel of BRCA1 wild type TNBC cell lines (MDA-MB-231, MDA-MB-468, and BT549). Levels of phospho-S1497 BRCA1 (pBRCA1; CDK phosphorylation site), γ-H2AX, and RAD51 were measured by western blot to assess effects on HR proteins. A direct assessment of DSB repair was measured using the U2OS-DR-GFP reporter system. Cell viability was measured using Cell-Titer Glo assays. Patient-derived TNBC xenograft models were established in immunocompromised NOD-SCID-IL2γ-/- mice, and implanted orthotopically into cohorts of mice for in vivo efficacy studies. Results: There was a significant reduction in total and pBRCA1 and RAD51 protein levels with increasing concentrations of dinaciclib in all TNBC cell lines. In response to 10 Gy γ-irradiation treatment, pretreatment with dinaciclib (20 nM) reduced the percentage of cells with greater than five BRCA1 foci from 54% to 5% (P < 0.001); and the percentage of cells with greater than 5 RAD51 foci from 26% to 4% (P = 0.001). In U2OS-DR-GFP assays, expression of GFP was detected in 3.4% of the vehicle group and in 1.9% of dinaciclib treated cells. Cell viability assays following five days of dinaciclib, veliparib or the combination revealed in vitro cytotoxic synergy in all three TNBC cell lines. In an orthotopic TNBC model derived from a primary patient sample, the relative tumor volumes over a 30-day treatment period for vehicle, dinaciclib, veliparib and combination- treated mice were 5.6-fold, 3.3-fold, 3.2-fold and 0.66-fold (P < 0.001) that of the initial tumor volume at the start of treatment. Tumor volume decreased only in the combination arm. Conclusion: CDK inhibition effectively inhibits HR, and renders BRCA-proficient TNBC cells sensitive to PARP inhibition. This combination represents an effective strategy for the treatment of TNBC. Citation Format: Shawn F. Johnson, Neil Johnson, David Chi, Benjamin Primack, Alan D. D'Andrea, Elgene Lim, Geoffrey I. Shapiro. The CDK inhibitor dinaciclib sensitizes triple-negative breast cancer cells to PARP inhibition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2013-1788