Wild-type Triple-negative Breast Cancer Research Articles

Discovery of dual-targeted molecules based on Olaparib and Rigosertib for triple-negative breast cancer with wild-type BRCA

PARP inhibitors (PARPis) demonstrate significant potential efficacy in the clinical treatment of BRCA-mutated triple-negative breast cancer (TNBC). However, a majority of patients with TNBC do not possess BRCA mutations, and therefore cannot benefit from PARPis. Previous studies on multi-targeted molecules derived from PARPis or disruptors of RAF-RAF pathway have offered an alternative approach to develop novel anti-TNBC agents. Hence, to broaden the application of PARP inhibitors for TNBC patients with wild-type BRCA, a series of dual-targeted molecules were constructed via integrating the key pharmacophores of Olaparib (Ola) and Rigosertib into a single entity. Subsequent studies exhibited that the resulting compounds 13a ∼ 14c obtained potential anti-proliferative activity against BRCA-defected or wild-type TNBC cells. Among them, an optimal compound 13b showed good inhibitory activity toward PARP-1, displayed approximately 34-fold higher inhibitory activity than that of Ola in MDA-MB-231 cells, and exerted multi-functional mechanisms to induce apoptosis. Moreover, 13b displayed superior antitumor efficacy (TGI, 61.3 %) than the single administration of Ola (TGI, 38.5 %), 11b (TGI, 51.8 %) or even their combined administration (TGI, 56.7 %), but did not show significant systematic toxicity. These findings suggest that 13b may serve as a potential condidate for BRCA wild-type TNBC.

Abstract PO4-28-01: Molecular profiling revealed activation of cytokine signaling and suppression of lipid metabolism as the hallmarks of doxorubicin-resistant TNBC

Abstract Background: Chemotherapeutic resistance observed in triple-negative breast cancer (TNBC) poses a substantial clinical hurdle, emphasizing the imperative to enhance our comprehension of the underlying mechanisms with the aim of potentially mitigating or reversing this phenomenon. Methods: Doxorubicin-resistant (DoxR) TNBC models (MDA-MB-231 and BT-549) were established through continuous exposure to increasing concentrations of doxorubicin. RNA-Seq was conducted using the Illumina platform, while bioinformatics analyses were performed using CLC genomics workbench 20.2 and iDEP.91. Colony forming unit (CFU), flow cytometry and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. Western blotting was used to confirm the expression and phosphorylation of protein targets. Ingenuity pathway analysis (IPA) and STRING database v 11.5 were used for network and pathway analyses, while Kaplan-Meier Plotter was used for survival analyses. Results: Herein, we provide a comprehensive functional and transcriptomic characterization of DoxR TNBC models, revealing multiple affected signaling networks and functional categories. The defense and immune response, response to stress, response to cytokine and external stimulus and cytokine-mediated signaling pathways were among the top activated, while suppression of cholesterol, sterol and lipid biosynthesis and metabolism were the hallmarks of DoxR cells. Upstream regulator analysis revealed IL-1B cytokine network activation in DoxR cells. Concordantly, disease and function analysis on the downregulated genes in DoxR TNBC cells revealed the most pronounced enrichment in pathways associated with cell invasion, colony formation, as well as lipid and carbohydrate metabolism. A number of upregulated genes were validated by qRT-PCR, which correlated with unfavorable overall survival (OS). Functionally, DoxR cells exhibited remarkable suppression of cell proliferation, as evidenced by diminished CFU potential, sphere formation and growth under 3D organoid culture, and cell migration. Although DoxR TNBC cells exhibited a slow cell cycling under normal culture conditions, wild-type TNBC cells exhibited remarkable arrest in the G2M phase when exposed to doxorubicin, which was less evident in DoxR TNBC models, suggesting DoxR cells undergo a quiescent state in response to chemotherapeutic challenge. In agreement with those data, increased phosphorylation of CHK2 and p53 proteins was remarkable in DoxR cells, thus promoting cellular dormancy. Conclusion: Our data revealed cellular dormancy and suppression of lipid metabolism as the hallmark associated with doxorubicin-resistant TNBC cells. On the other hand, cytokine signaling and stress response were the most enriched functional categories in DoxR TNBC cells. Therapeutic targeting to reverse the quiescent state or suppression of cytokine signaling networks could enhance the efficacy of doxorubicin-based therapeutics. Citation Format: Vishnubalaji Radhakrishnan, Nehad Alajez. Molecular profiling revealed activation of cytokine signaling and suppression of lipid metabolism as the hallmarks of doxorubicin-resistant TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-01.

Abstract CT203: Multi-omic analysis of serial biopsies to inform biomarkers of sensitivity to olaparib and durvalumab in patients with metastatic BRCA-wildtype triple negative breast cancer (mTNBC)

Abstract Background: The phase 2 Adaptive Multi-Drug Treatment of Evolving Cancers (AMTEC) trial (NCT03801369) evaluated the efficacy of the combination of the PARP inhibitor Olaparib (Ola) and the PD-L1 inhibitor Durvalumab (Durva) in participants with BRCA-wildtype mTNBC. The combination was found to be effective with a median progression free survival (mPFS) of 5.5 months (AACR 2022). Here we report on updated biomarker analyses from paired biopsies (Bx) pre- and on-Ola therapy from 14 AMTEC participants. Methods: AMTEC participants undergo a pre-Ola Bx (Bx1), then one (28-day) cycle of Ola monotherapy with a repeat on-Ola Bx (Bx2) before adding Durva to Ola. Multi-omic profiling of DNA, RNA, and protein signals in Bx1 and Bx2 using Deep candidate gene sequencing, RNAseq, Nanostring Digital Spatial Profiling (DSP), and multiplex immunohistochemistry (mIHC) was correlated with clinical outcomes to identify predictors of Ola + Durva sensitivity, and adaptive resistance to PARPi therapy. Results: We identified over 30 biomarkers, with the optimal predictive value arising from Bx2. Key potential biomarkers were confirmed in an independent test set. Markers of good prognosis: • Basal Immune Activated (BLIA)/Basal Immune Suppressed (BLIS subtype) - A BLIA subtype on Bx2 was associated with a mPFS of 7.36 months compared to 1.71 months for BLIS/Luminal androgen receptor subtypes (p=0.001) • Multi-omic immune composite - A positive immune consensus signature (RNA, DSP, mIHC) on Bx2 was associated with a mPFS of 7.36 months compared to 2.29 months (p=0.005) • B cell activation signature - We developed a novel B-cell RNA activation signature that was highly predictive of response in Bx2, with mPFS of 8.54 and 1.04 months in signature-positive and signature-negative tumors, respectively (p=0.0004) Markers of poor prognosis: • AKT/MAPK pathway - AKT pathway gene mutations in Bx2 with concomitant protein phosphorylation of pathway members correlated with a shorter mPFS of 2.4 months compared to 7.13 months for no pathway mutation/activation (p=0.03). MAPK pathway protein phosphorylation in Bx2 was a negative prognostic factor, with mPFS of 2.17 months (p=0.004) • Angiogenesis - An upregulated Hallmark of Cancer angiogenesis RNA signature on Bx2 was associated with a shorter mPFS of 1.94 months compared to 7.95 months with no angiogenesis upregulation (p=0.0009) The following markers did not show significant association with survival outcomes on AMTEC: • PD-L1 positivity by IHC (defined as >1% Tumor Proportion Score, 22C3 antibody) • Tumor mutational burden • HRD, as measured by Rad51 foci or by mutations in HR pathway members Conclusions: Findings highlight the value of paired Bxs to identify predictive biomarkers of PARPi + immune checkpoint inhibitor sensitivity. The striking predictive value of the BLIA/BLIS signature warrants evaluation in a larger trial. Emerging resistance mechanisms justify AMTEC trial expansion to include PARPi + MEKi or PARPi + AKTi in biomarker selected patients, which is now in progress. Citation Format: Zahi I. Mitri, Allison L. Creason, Jayne M. Stommel, Daniel Bottomly, Jeong Youn Lim, SMMART Clinical Trials Program, Christopher L. Corless, Shannon McWeeney, Gordon B. Mills. Multi-omic analysis of serial biopsies to inform biomarkers of sensitivity to olaparib and durvalumab in patients with metastatic BRCA-wildtype triple negative breast cancer (mTNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT203.

Combined PARP and WEE1 inhibition triggers anti-tumor immune response in BRCA1/2 wildtype triple-negative breast cancer

Novel therapeutic strategies that can effectively combine with immunotherapies are needed in the treatment of triple-negative breast cancer (TNBC). We demonstrate that combined PARP and WEE1 inhibition are synergistic in controlling tumour growth in BRCA1/2 wild-type TNBC preclinical models. The PARP inhibitor (PARPi) olaparib combined with the WEE1 inhibitor (WEE1i) adavosertib triggered increases in anti-tumour immune responses, including STING pathway activation. Combinations with a STING agonist resulted in further improved durable tumour regression and significant improvements in survival outcomes in murine tumour models of BRCA1/2 wild-type TNBC. In addition, we have identified baseline tumour-infiltrating lymphocyte (TIL) levels as a potential predictive biomarker of response to PARPi, WEE1i and immunotherapies in BRCA1/2 wild-type TNBC.

Identification and evaluation of a novel PARP1 inhibitor for the treatment of triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a particularly invasive subtype of breast cancer and usually has a poor prognosis due to the lack of effective therapeutic targets. Approximately 25% of TNBC patients carry a breast cancer susceptibility gene1/2 (BRCA1/2) mutation. Clinically, PARP1 inhibitors have been approved for the treatment of patients with BRCA1/2-mutated breast cancer through the mechanism of synthetic lethality. In this study, we identified compound 6 {systematic name: 2-[2-(4-Hydroxy-phenyl)-vinyl]-3H-quinazolin-4-one} as a novel PARP1 inhibitor from established virtual screening methods. Compound 6 exerted stronger PARP1 inhibitory activity and anti-cancer activity as compared to olaparib in BRCA1-mutated TNBC cells and TNBC patient-derived organoids. Unexpectedly, we found that compound 6 also significantly inhibited cell viability, proliferation, and induced cell apoptosis in BRCA wild-type TNBC cells. To further elucidate the underlying molecular mechanism, we found that tankyrase (TNKS), a vital promoter of homologous-recombination repair, was a potential target of compound 6 by cheminformatics analysis. Compound 6 not only decreased the expression of PAR, but also down-regulated the expression of TNKS, thus resulting in significant DNA single-strand and double-strand breaks in BRCA wild-type TNBC cells. In addition, we demonstrated that compound 6 enhanced the sensitivity of BRCA1-mutated and wild-type TNBC cells to chemotherapy including paclitaxel and cisplatin. Collectively, our study identified a novel PARP1 inhibitor, providing a therapeutic candidate for the treatment of TNBC.

A randomized phase II clinical trial of talazoparib maintenance therapy in patients with triple-negative breast cancer who showed platinum-sensitivity on first- or second-line platinum-based chemotherapy: KCSG BR21-10.

TPS1132 Background: A potent PARP inhibitor, talazoparib, demonstrated superior clinical activity compared to standard chemotherapy in germline BRCA1/2-mutant advanced breast cancer patients. However, the role of talazoparib treatment in BRCA1/2 wild-type triple-negative breast cancer (TNBC) patients remains undefined, although high incidence of homologous recombination deficiency (HRD) is one of the major characteristics of the TNBC. Previous studies indicated clinical responsiveness to platinum agents is a useful surrogate for HRD that may predict favorable PARP inhibitor response. Here, we present a phase II study to test talazoparib maintenance therapy in germline BRCA1/2 mutation unselected advanced TNBC patients after platinum-based chemotherapy. Methods: The KCSG BR21-10 study (NCT04755868) is a randomized double-blind placebo-controlled phase II clinical trial of talazoparib maintenance therapy in metastatic TNBC patients whose tumor showed platinum-sensitivity to first- or second-line platinum-based chemotherapy. The criteria for platinum-sensitivity were set as follows: remaining complete response (CR), partial response (PR), or stable disease (SD) after 6 to 9 tri-weekly doses or 18 to 27 weekly doses of platinum-based chemotherapy. The eligible TNBC patients are enrolled to the trial after completion of platinum-based therapy and 1:1 randomized to receive talazoparib (once daily 1.0 mg) or placebo maintenance therapy. The patient with any germline BRCA1/2 mutation status is eligible, and randomization stratification factors include line of platinum-based chemotherapy, response to platinum-based chemotherapy, and germline BRCA1/2 mutation status. The talazoparib/placebo maintenance treatment is initiated within 8 weeks after the last platinum chemotherapy. At the time of progression of disease, unblinding can be performed and patients in placebo arm may be eligible for crossover to talazoparib single treatment. The primary endpoint is progression-free survival (PFS) after randomization by RECIST 1.1, and the key secondary endpoints are overall survival, PFS2, objective response rate, adverse events by CTCAE 5.0 criteria, and quality of life. We also planned exploratory study to find predictive biomarker by tumor tissue and blood analysis. The median PFS from the randomization is expected to be 3 months in the placebo maintenance arm and we expect that talazoparib maintenance will improve PFS with hazard ratio of 0.65. It is predicted that a total of 206 patients (103 patients in each arm) are required with the α of 0.05 and power of 0.8 (1-β) by two-sided test, considering monthly 3% dropout rate. Currently, 23 of planned 206 patients have been enrolled. Clinical trial information: NCT04755868 .

Bcl-xL inhibition radiosensitizes PIK3CA/PTEN wild-type triple negative breast cancers with low Mcl-1 expression.

Patients with radioresistant breast cancers, including a large percentage of women with triple negative breast cancer (TNBC), demonstrate limited response to radiation (RT) and increased locoregional recurrence; thus, strategies to increase the efficacy of RT in TNBC are critically needed. We demonstrate that pan Bcl-2 family inhibition (ABT-263, rER: 1.52-1.56) or Bcl-xL specific inhibition (WEHI-539, A-1331852; rER: 1.31-2.00) radiosensitized wild-type PIK3CA/PTEN TNBC (MDA-MB-231, CAL-120) but failed to radiosensitize mutant PIK3CA/PTEN TNBC (rER: 0.90 - 1.07; MDA-MB-468, CAL-51, SUM-159). Specific inhibition of Bcl-2 or Mcl-1 did not induce radiosensitization, regardless of PIK3CA/PTEN status (rER: 0.95 - 1.07). In wild-type PIK3CA/PTEN TNBC, pan Bcl-2 family inhibition or Bcl-xL specific inhibition with RT led to increased levels of apoptosis (p < 0.001) and an increase in cleaved PARP and cleaved caspase 3. CRISPR-mediated PTEN knockout in wild-type PIK3CA/PTEN MDA-MB-231 and CAL-120 cells induced expression of pAKT/Akt and Mcl-1 and abolished Bcl-xL inhibitor-mediated radiosensitization (rER: 0.94 - 1.07). Similarly, Mcl-1 overexpression abolished radiosensitization in MDA-MB-231 and CAL-120 cells (rER: 1.02 - 1.04) but transient MCL1 knockdown in CAL-51 cells promoted Bcl-xL-inhibitor mediated radiosensitization (rER 2.35 ± 0.05). In vivo, ABT-263 or A-1331852 in combination with RT decreased tumor growth and increased tumor tripling time (p < 0.0001) in PIK3CA/PTEN wild-type TNBC cell line and patient-derived xenografts. Collectively, this study provides the preclinical rationale for early phase clinical trials testing the safety, tolerability, and efficacy of Bcl-xL inhibition and RT in women with wild-type PIK3CA/PTEN wild-type TNBC at high risk for recurrence.

Selective degradation of PARP2 by PROTACs via recruiting DCAF16 for triple-negative breast cancer

Triple negative breast cancer (TNBC) is a complex and heterogeneous neoplasm, and till now no effective therapies are available. PARP inhibitors, which target DNA repair, are lethal to those cells that have impaired homologous recombination (HR) pathway. So, PARP inhibitors might exert promising results in the treatment of BRCA-mutated TNBC, but show compromised effect to those wild-type TNBC. Herein, we describe a novel PROTACs C8, which was obtained by conjugating PARP1/2 inhibitor Olaparib to KB02, can induce potent and specific degradation of PARP2 by recruiting DCAF16 E3 ligase for treatment of wild-type TNBC. Moreover, C8 exhibits therapeutic potential in TNBC cell lines MDA-MB-231 both in vitro and in vivo. These studies demonstrated that the DCAF16 E3 ligases can be used in PARP2 PROTACs design, and C8, as a novel PARP2 selective DCAF16 based PROTACs, might be a promising lead compound for the treatment of BRCA-wild-type TNBC.

ATR Inhibitor AZD6738 (Ceralasertib) Exerts Antitumor Activity as a Monotherapy and in Combination with Chemotherapy and the PARP Inhibitor Olaparib.

This detailed preclinical investigation, including pharmacokinetics/pharmacodynamics and dose-schedule optimizations, of AZD6738/ceralasertib alone and in combination with chemotherapy or PARP inhibitors can inform ongoing clinical efforts to treat cancer with ATR inhibitors.

Abstract PO-131: RAD51 is a biomarker for aggressive disease and racial disparities in triple-negative breast cancer

Abstract Breast cancer (BC) is the second most diagnosed malignant disease in women, and one of the leading causes of cancer-related deaths. Triple-negative breast cancer (TNBC) is the most aggressive and difficult to treat subtype of BC because it is highly metastatic and lacks targeted therapies. African American (AA) women have a higher death rate from BC than women of other races and ethnicities. The higher incidences of TNBCs and their aggressive growth in young AA women contributing to higher death rates indicate a biological basis for this difference. Thus, it is imperative to understand the molecular mechanisms that contribute to aggressive tumor growth in AA women, identify biomarkers to select patients who will respond to existing therapies, and develop effective therapeutics to reduce this disparity. Our studies identified that multiple TNBC cells derived from AA women are inherently chemoresistant and exhibit aggressive growth behavior compared to TNBC cells derived from European American (EA) patients. Our preliminary screenings showed that the DNA repair protein, RAD51, is overexpressed in AA TNBC patients and correlates a poor prognosis relative to EA TNBC patients. Analysis of AA and EA TNBC tumor specimens indicated the epigenetic regulation of RAD51 by promoter methylations and microRNAs. Furthermore, AA women diagnosed with TNBC, have a considerably lower incidence of germline BRCA1 mutations than women of other racial or ethnic groups. This indicates most TNBC tumors in AA patients are DNA repair proficient and have intact cell cycle checkpoint mechanisms that protect them from chemotherapy-induced DNA damage and promote therapeutic resistance. Our drug screenings identified CHK1 inhibitor, Prexasertib caused DNA repair deficiency in BRCA wild-type TNBC cells by promoting proteasome-mediated degradation of BRCA1 and RAD51 proteins. Therefore, we designed a synthetic lethality-based drug combination of Prexasertib with PARP inhibitors (PARPi) in DNA repair proficient TNBC cells. Data from our preclinical evaluations show Prexasertib and Olaparib cause increased DNA strand breaks, mitotic catastrophe, and synergistic TNBC cell lethality compared to individual drug treatments. Additionally, computational analysis of TCGA data revealed a RAD51 upregulation in TNBC tumors compared to normal breast tissues and other subtypes of BC which renders as a poor prognostic marker for these patients. Remarkably, there was an interesting discrepancy in RAD51 expression levels between different racial groupings, with AA and Asian BC patients having higher levels of RAD51 expression than Caucasian BC patients. Consistent with these observations, AA and Asian TNBC patients showed decreased survival probability. Together, our data indicate that RAD51 and its epigenetic regulators could be biomarkers for aggressive TNBC and racial disparity in BC therapeutic outcomes and suggests a novel combination therapy involving Prexasertib and Olaparib may improve prognosis and reduce racial disparity in TNBC. Citation Format: Ganesh N. Acharya, Chinnadurai Mani, Upender Manne, Komaraiah Palle. RAD51 is a biomarker for aggressive disease and racial disparities in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-131.

Platinum and Taxane Based Adjuvant and Neoadjuvant Chemotherapy in Early Triple-Negative Breast Cancer: A Narrative Review.

Platinum (Pt) derivatives such as cisplatin and carboplatin are the class of drugs with proven activity against triple-negative breast cancer (TNBC). This is due to the ability of Pt compounds to interfere with the DNA repair mechanisms of the neoplastic cells. Taxanes have been efficacious against estrogen receptor-negative tumors and act by disruption of microtubule function. Due to their distinct mechanisms of action and routes of metabolism, the combination of the Pt agents and taxanes results in reduced systemic toxicity, which is ideal for treating TNBC. Also, the sensitivity of BRCA1-mutated cells to taxanes remains unsolved as in vitro evidence indicates resistance against taxanes due to BRCA1 mutations. Recent evidence suggests that the combination of carboplatin and paclitaxel resulted in better pathological complete response (pCR) in patients with TNBC, both in neoadjuvant and adjuvant settings. In vitro studies showed sequential dependency and optimal time scheduling of Pt- and taxane-based chemotherapy. Also, combining carboplatin with docetaxel in the NAC regimen yields an excellent pCR in patients with BRCA-associated and wild-type TNBC. TNBC is a therapeutic challenge that can be tackled by identifying new therapeutic sub-targets and specific cross-sections that can be benefitted from the addition of Pt- and taxane-based chemotherapy. This review summarizes the merits as well as the mechanism of Pt- and taxane-based adjuvant and neoadjuvant chemotherapies in early TNBC from the available and ongoing clinical studies.

CDK4/6 Inhibition and Radiation as a Treatment Strategy to Improve Local Disease Control in Breast Cancers With Poor Prognoses

Locoregional control remains an issue in women with multi-node positive estrogen receptor-positive (ER+) breast cancer and many women with triple-negative breast cancer (TNBC). There is growing evidence that CDK4/6 inhibition (CDK4/6i) sensitizes breast cancer cells to ionizing radiation (RT) by suppressing the DNA damage response, but the role of RB is currently unclear. We sought to understand the role of RB and the implications of RB loss in ER+ and TNBC.Clonogenic survival assays were used to calculate radiosensitivity and calculate enhancement ratios (rER). Cellular viability was quantified 72 hours after drug addition to calculate half-maximal inhibitory concentrations (IC50s). Phospho- and total RB expression levels were assessed by immunoblotting. DNA repair was assessed with γH2AX and RAD51 immunofluorescence. GFP-based plasmid reporter systems were used to assess homologous recombination (HR) and non-homologous end joining (NHEJ) competency. Isogenic RB1 knockout cells were generated with CRISPR-Cas9, and siRNA was used for transient RB1 knockdown. G1 cell cycle arrest was quantified using propidium iodide-based flow cytometry. In vivo efficacy of CDK4/6 inhibition + RT was assessed using MDA-MB-231 xenografts.Four TNBC cell lines with intact RB expression were radiosensitized (rER 1.08 - 2.22) after CDK4/6i with palbociclib, ribociclib, or abemaciclib, but two RB null TNBC models were not radiosensitized with combination treatment (rER: 0.84 - 1.00). In ER+ and TNBC cell lines, response to CDK4/6i + RT was accurately predicted by the presence or absence of RB protein, and higher RB expression led to increased radiosensitization of breast cancer cell lines at lower doses. In addition, pRB expression decreased in wild type TNBC cell lines treated with CDK4/6i + RT. HR was suppressed with CDK4/6i in RB wild type - but not mutant - cell lines. NHEJ efficiency in RB wild type TNBC was unchanged with CDK4/6i. Genetic knockdown of RB1 led to the loss of radiosensitization in ER+ and TNBC cell lines (rER: 0.84 - 1.13) as well as a decrease in sensitivity to CDK4/6i monotherapy. Parental and Cas9 control TNBC cells arrested in G1 24 hours after CDK4/6i and remained arrested at 48 hours, but RB1 CRISPR TNBC cells did not arrest after drug treatment. In a TNBC xenograft model with wild type RB expression (MDA-MB-231), CDK4/6i + RT led to significant radiosensitization in vivo and delayed time to tumor doubling.RB loss mitigates CDK4/6 inhibitor-mediated radiosensitization of both ER+ and TNBC cells. Loss of RB expression prevents CDK4/6i-mediated radiosensitization, suggesting that RB expression may be a valuable clinical biomarker to predict efficacy of CDK4/6i + RT in future clinical trials. Our data also provide the preclinical rationale for CDK4/6i with RT not just in ER+ BC, but also in women with TNBC that express RB.

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

Abstract Purpose: Cyclin-dependent kinase 4&amp;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 &amp;gt; 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 &amp;lt; 0.001) but not RB1 mutant (p &amp;gt; 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 &amp;gt; 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.

Abstract 1270: The novel PARP1-selective inhibitor, AZD5305, is efficacious as monotherapy and in combination with standard of care chemotherapy in the in vivo preclinical models

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown efficacy in homologous recombination deficient (HRD) tumours, such as those with BRCA mutations (BRCAm). In this setting PARPi treatments lead to accumulation of DNA damage and cancer cell death. PARPi currently in clinical use inhibit both PARP1 and PARP2, as well as other members of the PARP family. Here, we report for the first time in vivo profiling of AZD5305, a potent and highly selective PARP1 inhibitor and trapper, currently in Ph1 clinical trials. Dose response efficacy of AZD5305 was evaluated in the BRCA1m triple-negative breast cancer (TNBC) xenograft model MDA-MB-436. AZD5305 dosed at 0.1mg/kg QD or higher for 35 days delivered about 90% regression, compared with 83% regression caused by treatment with 100mg/kg QD olaparib. Anti-tumour effects of AZD5305 continued after cessation of treatment and complete responses were achieved which were sustained for the whole duration of the study, over 100 days after treatment withdrawal, in contrast to the olaparib-treated group where regrowth of tumours was observed from day 63 after treatment withdrawal. Investigation of the PK/PD/efficacy relationship in MDA-MB-436 showed that maximum efficacy of AZD5305 was achieved when unbound plasma concentrations were maintained above the IC95 estimated from an in vitro DLD-1 BRCA2-/- cell growth assay. Similar results were obtained in a BRCA1m patient-derived explant (PDX) model, HBCx-17. Anti-tumour efficacy of AZD5305 was also tested in the DLD-1 BRCA2-/- and wild-type (WT) isogenic xenograft models. In the DLD-1 BRCA2-/- model, AZD5305 dosed at 10mg/kg QD and 1mg/kg QD delivered 78% and 63% tumour regression, respectively. AZD5305 at 0.1mg/kg QD resulted in responses similar to those observed in the olaparib 100mg/kg QD group (40-54% tumour growth inhibition, TGI). As expected, AZD5305 and olaparib showed no anti-tumour efficacy in the DLD-1 WT tumour model. Due to improved PARP1 selectivity, AZD5305 has the potential to show improved efficacy and tolerability in combination with standard of care chemotherapy when compared to non-selective PARPi. Hence, we investigated the anti-tumour effects of AZD5305 in combination with carboplatin or paclitaxel in a BRCA1m TNBC xenograft, SUM149PT, and BRCA WT TNBC PDX model, HBCx-9. In both models, combination of AZD5305 with carboplatin was well tolerated and demonstrated clear benefit compared to each monotherapy treatment. The effects of adjusted dosing and scheduling of the combination on the anti-tumour efficacy will be presented. Citation Format: Anna D. Staniszewska, James W. Yates JWT, Andy Pike, Christine Fazenbaker, Kimberly Cook, Emily Bosco, Aaron Smith, Joanne Wilson, Elisabetta Leo. The novel PARP1-selective inhibitor, AZD5305, is efficacious as monotherapy and in combination with standard of care chemotherapy in the in vivo preclinical models [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 1270.

Long non-coding RNA LINC-PINT attenuates paclitaxel resistance in triple-negative breast cancer cells via targeting the RNA-binding protein NONO.

The treatment of triple-negative breast cancer (TNBC) relies largely on chemotherapies. However, it is frequent that TNBC patients develop resistance to the chemotherapy drugs. Generation of drug-resistant cell lines facilitates the identification of drug resistance. Here, we established two paclitaxel (PTX)-resistant TNBC cancer cell lines using an intermittent and stepwise method and found that long non-coding RNA long intergenic non-protein-coding RNA p53-induced transcript (LINC-PINT) was significantly decreased in PTX-resistant cancer cells. Ectopic expression of LINC-PINT sensitized both PTX-resistant TNBC and wild-type TNBC to PTX. Moreover, RNA immunoprecipitation showed that LINC-PINT bound to RNA-binding protein NONO. Overexpression of LINC-PINT resulted in the degradation of NONO in a proteasome-dependent manner and vice versa. Knockdown of NONO with siRNA sensitized TNBC to PTX. We further analyzed the expression level of LINC-PINT and NONO in patient samples via online database and found that LINC-PINT and NONO may function antagonistically in all types of breast cancers. Taken together, our data illustrated a tumor suppressor role of LINC-PINT in sensitizing TNBC to chemotherapies via destabilizing NONO.

The VEGF receptor neuropilin 2 promotes homologous recombination by stimulating YAP/TAZ-mediated Rad51 expression

Vascular endothelial growth factor (VEGF) signaling in tumor cells mediated by neuropilins (NRPs) contributes to the aggressive nature of several cancers, including triple-negative breast cancer (TNBC), independently of its role in angiogenesis. Understanding the mechanisms by which VEGF-NRP signaling contributes to the phenotype of such cancers is a significant and timely problem. We report that VEGF-NRP2 promote homologous recombination (HR) in BRCA1 wild-type TNBC cells by contributing to the expression and function of Rad51, an essential enzyme in the HR pathway that mediates efficient DNA double-strand break repair. Mechanistically, we provide evidence that VEGF-NRP2 stimulates YAP/TAZ-dependent Rad51 expression and that Rad51 is a direct YAP/TAZ-TEAD transcriptional target. We also discovered that VEGF-NRP2-YAP/TAZ signaling contributes to the resistance of TNBC cells to cisplatin and that Rad51 rescues the defects in DNA repair upon inhibition of either VEGF-NRP2 or YAP/TAZ. These findings reveal roles for VEGF-NRP2 and YAP/TAZ in DNA repair, and they indicate a unified mechanism involving VEGF-NRP2, YAP/TAZ, and Rad51 that contributes to resistance to platinum chemotherapy.

A phase II trial of olaparib and durvalumab in metastatic BRCA wild type triple-negative breast cancer.

TPS1111 Background: There is an urgent need to develop novel non chemotherapy treatments for metastatic triple negative breast cancer (mTNBC) patients who otherwise have a poor prognosis. Immune checkpoint blockade (ICB) and PARP inhibitors (PARPi) have independently shown promise for the treatment of mTNBC, and the combination has shown early benefit in the MEDIOLA and TOPACIO trials. This trial looks to 1) evaluate the efficacy of the combination of the PARPi olaparib and the PD-L1 inhibitor durvalumab, and 2) perform extensive multi-omics including protein based image analytics (multiplex IHC, cyclic immunofluorescence) on serial biopsies to identify predictive biomarkers and resistance mechanisms. Methods: Trial Design: This is a single-arm phase II study to assess the efficacy of the combination of olaparib and durvalumab in BRCA-wildtype mTNBC. mTNBC participants will undergo a pre-treatment biopsy, then will start a 4 week induction treatment with olaparib (300 mg PO BID). At the end of 4 weeks of single agent therapy, participants will undergo a repeat on-treatment biopsy, following which durvalumab (1500 mg IV every 4 weeks) will be added to olaparib. Participants will also be offered an optional biopsy on progression. Endpoints: The primary endpoint of this study is overall response rate (ORR) to olaparib and durvalumab therapy. Secondary efficacy endpoints include clinical benefit rate, duration of response, progression-free, and overall survival. The incidence and severity of on-treatment adverse events will be collected per CTCAE 5.0. Statistical Methods: 28 participants are planned for enrollment to this study. A 2-stage analysis will be performed using a Simon 2-stage Minimax design. The null (ICB alone) and alternative (ICB + PARPi) hypotheses are: H0: π = 0.15 and Ha: π = 0.35. For the primary endpoint, a total sample size of 28 participants will achieve 80% power to detect the ORR difference of 0.20 with one-sided type I error =0.05. The trial will be terminated in stage I if 2 or less out of the first 15 participants respond. If the trial goes on to the stage II, a total of 28 participants will be studied. If the total number responding is less than or equal to 7, the combination is rejected. Current Enrollment: The study was activated on 1/7/2019. To date, 3 out of 15 patients have been accrued to stage I of the study Clinical trial information: NCT03801369.

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.

BET proteins regulate homologous recombination-mediated DNA repair: BRCAness and implications for cancer therapy.

Bromodomain and Extra-Terminal (BET) proteins are historically involved in regulating gene expression and BRD4 was recently found to be involved in DNA damage regulation. Aims of our study were to assess BRD4 regulation in homologous recombination-mediated DNA repair and to explore novel clinical strategies through the combinations of the pharmacological induction of epigenetic BRCAness in BRCA1 wild-type triple negative breast cancer (TNBC) cells by means of BET inhibitors and compounds already available in clinic. Performing a dual approach (chromatin immunoprecipitation and RNA interference), the direct relationship between BRD4 and BRCA1/RAD51 expression was confirmed in TNBC cells. Moreover, BRD4 pharmacological inhibition using two BET inhibitors (JQ1 and GSK525762A) induced a dose-dependent reduction in BRCA1 and RAD51 levels and is able to hinder homologous recombination-mediated DNA damage repair, generating a BRCAness phenotype in TNBC cells. Furthermore, BET inhibition impaired the ability of TNBC cells to overcome the increase in DNA damage after platinum salts (i.e., CDDP) exposure, leading to massive cell death, and triggered synthetic lethality when combined with PARP inhibitors (i.e., AZD2281). Altogether, the present study confirms that BET proteins directly regulate the homologous recombination pathway and their inhibition induced a BRCAness phenotype in BRCA1 wild-type TNBC cells. Noteworthy, being this strategy based on drugs already available for human use, it is rapidly transferable and could potentially enable clinicians to exploit platinum salts and PARP inhibitors-based treatments in a wider population of TNBC patients and not just in a specific subgroup, after validating clinical trials.

Notch Signaling Regulates Mitochondrial Metabolism and NF-κB Activity in Triple-Negative Breast Cancer Cells via IKKα-Dependent Non-canonical Pathways.

Triple negative breast cancer (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. Notch signaling is a key pathway regulating TNBC CSC survival. Treatment of TNBC with PI3K or mTORC1/2 inhibitors results in drug-resistant, Notch-dependent CSC. However, downstream mechanisms and potentially druggable Notch effectors in TNBC CSCs are largely unknown. We studied the role of the AKT pathway and mitochondrial metabolism downstream of Notch signaling in TNBC CSC from cell lines representative of different TNBC molecular subtypes as well as a novel patient-derived model. We demonstrate that exposure of TNBC cells to recombinant Notch ligand Jagged1 leads to rapid AKT phosphorylation in a Notch1-dependent but RBP-Jκ independent fashion. This requires mTOR and IKKα. Jagged1 also stimulates mitochondrial respiration and fermentation in an AKT- and IKK-dependent fashion. Notch1 co-localizes with mitochondria in TNBC cells. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor PF-03084014 in combination with AKT inhibitor MK-2206 or IKK-targeted NF-κB inhibitor Bay11-7082 blocks secondary mammosphere formation from sorted CD90hi or CD44+CD24low (CSCs) cells. A TNBC patient-derived model gave comparable results. Besides mitochondrial oxidative metabolism, Jagged1 also triggers nuclear, NF-κB-dependent transcription of anti-apoptotic gene cIAP-2. This requires recruitment of Notch1, IKKα and NF-κB to the cIAP-2 promoter. Our observations support a model where Jagged1 triggers IKKα-dependent, mitochondrial and nuclear Notch1 signals that stimulate AKT phosphorylation, oxidative metabolism and transcription of survival genes in PTEN wild-type TNBC cells. These data suggest that combination treatments targeting the intersection of the Notch, AKT and NF-κB pathways have potential therapeutic applications against CSCs in TNBC cases with Notch1 and wild-type PTEN expression.