Triple-negative Breast Cancer Xenograft Research Articles

Folate Receptor-Alpha Targeted 7x19 CAR-γδT Suppressed Triple-Negative Breast Cancer Xenograft Model in Mice.

Background Triple-negative breast cancer (TNBC) is the worst prognosis subtype of breast cancer due to lack of specific targets. Recent studies have shown that immunotherapy may solve that problem by targeting folate receptor-alpha (FRα). Methods Gene modified γδ T cells were manufactured to express FRa specific chimeric antigen receptor (FRa CAR) and secrete interleukin-7 (IL-7) and chemokine C–C motif ligand 19 (CCL19). CAR-γδT cells that secrete IL-7 and CCL19 (7 × 19 CAR-γδT) were evaluated for their antitumor activity both in vitro and in vivo. Results 7 × 19 CAR-γδT showed remarkable antitumor activity in vitro. Combined with PBMC, 7 × 19 CAR-γδT inhibited TNBC xenograft model growth superiorly compared with single-application or conventional CAR-γδT cells. Histopathological analyses showed increased DC or T cells infiltration to tumor tissues. Conclusion Taken together, our results showed that 7 × 19 CAR-γδT have remarkable anti-TNBC tumor activity and showed a broad application prospect in the treatment of incurable TNBC patients.

Abstract P5-07-01: Proteogenomic analysis of differential chemotherapy responses in patient-derived xenografts of triple-negative breast cancer

Abstract Background: Chemotherapy is essential for the management of patients with triple-negative breast cancer (TNBC). Identification of biomarkers that may indicate treatment efficacy will be critical to improve patient stratification prior to treatment. To elucidate molecular determinants underlying chemotherapy response, we conducted a proteogenomic study using TNBC patient-derived xenografts (PDXs) treated with chemotherapy. Methods: Orthotopic TNBC PDXs were treated with four weekly cycles of docetaxel, carboplatin, or the combination. Changes in tumor volume after 4 weeks of treatment were evaluated. Genomic, transcriptomic, and mass-spectrometry-based proteomic profiling were performed on baseline tumors prior to treatments to identify associations with chemotherapy response (https://pdxportal.research.bcm.edu). Fisher’s exact test was used to identify significant associations between mutation and copy number events and dichotomized treatment response. For gene level analyses, Spearman’s correlation was calculated between mRNA or protein abundance and log2 fold change in tumor volumes after treatment. Signed -log10 p-values from Spearman’s correlation analysis were used as input for Gene Set Enrichment Analysis. Validation in external datasets was performed using METABRIC (PMID: 22522925) and BrighTNess clinical trial (PMID: 29501363) human datasets, an independent PDX dataset (PMID: 32546838), and shRNA screen data from DepMap (PMID: 30389920). Results: Combination carboplatin and docetaxel was largely ineffective at generating enhanced responses over the best single agent, suggesting de-escalation of chemotherapy may be possible. Genomic aberrations in BRCA2 and BCL9 were enriched in carboplatin-responsive PDXs while aberrations in RAF1 were enriched in docetaxel-resistant PDXs. Genes with gene-drug response correlations supported by both mRNA and protein measurements, but not mRNA or protein alone, for both carboplatin and docetaxel treatment in PDXs were associated with prognosis from basal human breast tumors receiving any chemotherapy from the METABRIC dataset. These data suggest that the combination of mRNA and protein data provided increased accuracy in identifying genes associated with clinical outcome in TNBC. Some of the top genes with genomic aberrations and/or overexpression at both mRNA and protein levels in chemoresistant PDXs, many of which have not been evaluated for their ability to augment response to taxane- or platinum-based chemotherapies, were validated in independent datasets with PDXs and TNBC patients receiving carboplatin and taxane combination. Further, some were found to be dependencies in TNBC cell lines from a publicly available genetic perturbation dataset. At the pathway level, both mRNA and protein data associated models resistant to both agents with enhanced oxidative phosphorylation and proteostatic pathways including proteosome degradation and the unfolded protein response (UPR) pathway related to endoplasmic reticulum stress. Pharmacological targeting the UPR pathway in combination with docetaxel showed activity in PDX models resistant to single-agent docetaxel. These results suggest targeting the UPR pathway as a novel therapeutic strategy to overcome chemotherapy resistance in TNBC. Conclusion: Proteogenomic analysis of PDX tumors identified diverse genes and pathways associated with chemotherapy resistance and response and further suggests potential therapeutic opportunities in TNBC. Citation Format: Jonathan T Lei, Chen Huang, Ramakrishnan R Srinivasan, Suhas Vasaikar, Lacey E Dobrolecki, Alaina N Lewis, Christina Sallas, Susan G Hilsenbeck, C. Kent Osborne, Mothaffar F Rimawi, Matthew J Ellis, Varduhi Petrosyan, Alexander B Saltzman, Anna Malovannaya, Gerburg Wulf, Daniel C Kraushaar, Tao Wang, Xi Chen, Gloria V Echeverria, Meenakshi Anurag, Bing Zhang, Michael T Lewis. Proteogenomic analysis of differential chemotherapy responses in patient-derived xenografts of triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-07-01.

Abstract OT2-28-01: A phase 2 study of sitravatinib in metastatic, pre-treated, triple negative breast cancer, NCT # 04123704

Abstract Background:Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer, disproportionally affecting African American and Hispanic patients, and germline BRCA1/2 mutation carriers. Prognosis of relapsed, metastatic disease is extremely poor and effective well-tolerated therapies are urgently needed. Our collaborators have discovered that TNBCs often lose feedback control of receptor tyrosine kinases (RTKs) including PDGFR and MET, locking these receptors in a chronically active state. These receptors share a common negative regulator called Protein Tyrosine Phosphatase, Non-Receptor Type 12 (PTPN12). Baylor investigators have shown that PTPN12- deficient TNBCs may be responsive to combined RTK inhibition. Sitravatinib is a spectrum selective RTK inhibitor that has shown strong antitumor activity in patient derived xenografts (PDXs) of TNBC with low levels of PTPN12. We have therefore designed a phase II, multi-institution, two cohort trials to evaluate the efficacy of sitravatinib in patients with metastatic TNBC (mTNBC). Methods: Patients with diagnosis of mTNBC who have received at least one line of chemotherapy +/- atezolizumab for advanced disease would be considered for enrollment. Patients would need to have tissue from metastatic site available for evaluation or agree to undergo biopsy if banked tumor tissue is not available. Sitravatinib at 100 mg daily, the recommended phase 2 dose for sitravatinib monotherapy, will be started at the initiation of the study and continued until progression or unacceptable toxicity. The study will employ a modified optimal Simon’s two stage design. Patients will be recruited into two cohorts: PTPN12 low or PTPN12 high/normal cohorts simultaneously and independently. Seven patients will be enrolled to each arm during the first stage. After the first stage, depending on the observed number of responses, the study will proceed to the 2nd stage. The primary endpoint is progression-free survival status at 24 weeks (PFS24). Current Trial status: The trial is funded by Mirati Therapeutics and opened to enrollment at Dan L Duncan Comprehensive Cancer Center (DLDCCC) clinics as of 6/8/21. Trial is due to open at UT Southwestern Dallas by 11/2021. The correlative science is funded by an institutional SPORE grant. Conclusion: This trial aims to show that sitravatinib is safe and to investigate whether it could be a treatment option in mTNBC. Citation Format: Maryam Nemati Shafaee, Kristen Otte, Nicholas J Neill, Kent C Osborne, Thomas F Westbrook, Susan Hilseneck, Matthew J Ellis. A phase 2 study of sitravatinib in metastatic, pre-treated, triple negative breast cancer, NCT # 04123704 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-28-01.

Abstract OT2-01-01: Phase 1 study of ST101, a first-in-class peptide antagonist of CCAAT/enhancer-binding protein β (C/EBPβ), in patients with advanced solid tumors, with a phase 2 expansion in patients with hormone receptor positive breast cancer (HR+ BC)

Abstract Background: CCAAT/Enhancer Binding Protein Beta (C/EBPβ) is a transcription factor that is active during embryofetal development but held in an inactive state in most mature cells. C/EBPβ is upregulated or overactivated in multiple cancers, where it inversely correlates with disease prognosis. In breast cancer, C/EBPβ drives the expression of factors that promote tumor survival, proliferation and inhibit differentiation. ST101 is a cell-penetrating all D amino acid peptide antagonist of C/EBPβ. ST101 exposure inhibits C/EBPβ target gene expression, leading to selective tumor cell death in multiple human cancer cell lines, including hormone receptor positive breast cancer (HR+ BC) and triple negative breast cancer (TNBC), without impacting normal cell viability. In vivo, ST101 displays rapid uptake into multiple organs, the ability to cross the blood-brain barrier, and a long plasma half-life due to its resistance to proteolytic degradation. Potent ST101 anti-tumor activity, demonstrated by dose-dependent inhibition of tumor growth in subcutaneous HR+ and orthotopic TNBC xenograft models in vivo, supported advancing ST101 into clinical development.Trial design: This phase 1-2 study uses a standard 3+3 design with dose doubling for the first 4 dose levels then 50% escalations thereafter. The recommended phase 2 dose will be used in 4 expansion cohorts in specific tumor types, including HR+ BC. Patients receive intravenous ST101 once weekly.Eligibility criteria: The dose-escalation phase is enrolling patients ≥18 years of age with advanced, unresectable metastatic solid tumors refractory to or intolerant of other therapeutic options. In expansion, patients with HR+ BC must have progressed after 1-3 prior hormone-based therapies. Previous treatment with CDK 4/6 inhibitor, mTOR inhibitor, or chemotherapy is allowed as monotherapy or in combination. Specific aims: The primary objective of phase 1 is to evaluate safety and tolerability of ST101. Secondary objectives include the recommendation of a dose and regimen of ST101 for further evaluation, analysis of pharmacokinetics, assessment of several pharmacodynamic measures, and to assess preliminary efficacy. Statistical design: The recommended phase 2 dose will be used in a 15-30 patient HR+ BC expansion cohort, with a Simon 2-stage design, which requires one response to expand the cohort to 30 patients. Up to 120 patients are planned in a total of four expansion cohorts, which should be enrolling by Q3 2021.Accrual: We began recruitment in August 2020. Enrollment is ongoing, and by July 2021, 18 patients were recruited in five dose-escalation cohorts up to 6 mg/kg; a 6th cohort (9 mg/kg) is ongoing. Dose escalation should be complete by Sept 2021, and the phase 2 portion in the HR+ BC cohort will be underway (n=15-30). Please contact rob.michel@bexonclinical.com if you have a specific interest in this trial. Citation Format: Alice S Bexon, Hendrik-Tobias Arkenau, Jeff Evans, Gerald S Falchook, Stefan N Symeonides, Meredith A McKean, Elisa Fontana, Manojkumar Bupath, Alistair McLaren, Sreenivasa Chandana, Tze-en Ding, Emerson A Lim, Jim Rotolo, Gina Capiaux, Rob Michel, Stephen Kaesshaefer, Nehal J Lakhani. Phase 1 study of ST101, a first-in-class peptide antagonist of CCAAT/enhancer-binding protein β (C/EBPβ), in patients with advanced solid tumors, with a phase 2 expansion in patients with hormone receptor positive breast cancer (HR+ BC) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-01-01.

Abstract P5-10-01: Leukemia inhibitory factor receptor inhibition reduces obesity driven progression of triple negative breast cancer

Abstract Background: The obesity epidemic is rapidly increasing in the USA and obese women are at a higher likelihood of developing triple negative breast cancer (TNBC). Several studies implicated the importance of the breast microenvironment on the aggressive cancer biology especially obese microenvironment. However, the underlying mechanism(s) by which obesity contributes to the progression of TNBC remains unclear. The objective of this study is to test a novel concept that obesity upregulates leukemia inhibitory factor receptor (LIFR) oncogenic signaling in TNBC and test whether LIFR inhibition blocks TNBC progression. Methods: Established TNBC cell lines were co-cultured with human primary adipocytes or incubated with adipocyte conditioned medium or with high glucose (HG) followed by treatment with LIFR inhibitor EC359. The effect of adiposity on TNBC cells was determined using cell viability, colony formation, and invasion assays. Mechanistic studies were performed using CRISPR/Cas9 KO of LIFR, Western blotting, RT-qPCR, and reporter gene assays. Utility of LIFR inhibitor EC359 was tested using xenografts, and patient derived organoid (PDO) models. Results: Treatment of TNBC cells with adipose conditions or HG increased the proliferation and invasion of TNBC cells. Western blot and RT-qPCR analyses confirmed that increased expression of LIFR correlated with enhanced downstream LIFR signaling such as STAT3 and subsequent activation of STAT3 target genes. CRISPR KO of LIFR or treatment of TNBC cells with EC359 significantly reduced the cell viability, colony formation and invasion under adipose conditions. Western blotting results showed that co-culture with adipocytes significantly enhanced LIFR downstream signaling in TNBC model cells and is effectively blocked by LIFR KO or EC359 treatment. Further, EC359 treatment blocked the adipose environment mediated growth of organoids. Importantly, co-implantation of adipocytes significantly enhanced TNBC xenograft tumor growth, however treatment with EC359 significantly attenuated adipocyte induced TNBC progression. Conclusions: Collectively, these results suggest that adiposity contributes to increased TNBC cell growth via upregulation of the LIF/LIFR pathway. The LIF/LIFR axis represents a potential therapeutic target for adiposity driven TNBC and the LIFR inhibitor EC359 could be used as a new therapeutic agent to treat obesity associated TNBC. Citation Format: Suryavathi Viswanadhapalli, Uday P Pratap, Behnam Ebrahimi, Logan Blankenship, Jaitri Joshi, Zexuan Liu, Kristin A Altwegg, Xiaonan Li, Gangadhara R Sareddy, Bindu Santhamma, Swapna Konda, Manjeet Rao, Edward Kost, Rajeshwar R Tekmal, Hareesh B Nair, Ratna K Vadlamudi. Leukemia inhibitory factor receptor inhibition reduces obesity driven progression of triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-01.

Abstract PD3-07: Combined inhibition of CDK4/6 and AKT is effective in Rb-intact triple-negative breast cancer of the luminal androgen receptor (LAR) subtype

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease group with variable clinico-pathologic features. Based on gene expression profiles, TNBCs are grouped into 6 major subtypes. The Luminal androgen receptor (LAR) subtype is enriched for potentially targetable biomarkers, including high androgen receptor (AR) expression, high rates of PIK3CA mutations, and intact Rb. The purpose of this study was to investigate the most effective combinations of CDK4/6, AR, and PI3K-AKT inhibitors in pre-clinical models of LAR TNBC for future clinical trial design. Methods: MDA-MB-453 and MFM-223 (both Rb-intact/PTEN-intact/PIK3CA-mutant) and CAL-148 (Rb-null/PTEN-null/PIK3CA-mutant) LAR TNBC cell lines were treated with the CDK4/6 inhibitor palbociclib, the PI3Kα inhibitor alpelisib, the AKT inhibitor capivasertib, and the AR antagonist enzalutamide, each alone or in different combinations. Drug sensitivity was determined by coulter counter cell counts in 2D, colony formation, and the CellTiterGlo cell viability assay. The combination index (CI) which defines synergism (CI < 1), additive effect (CI = 1) and antagonism (CI > 1), calculated by the CompuSyn method, was used to evaluate the synergistic effects of drug combinations. Expression of cell cycle and PI3K-AKT downstream signaling molecules was measured by western blot analysis. An androgen response element (ARE) luciferase-based reporter assay was used to evaluate AR transcriptional activity. Results: Rb-intact LAR TNBC cell lines were sensitive to single-agent palbociclib, alpelisib or capivasertib (IC50, ~500 nM). Enzalutamide had minimal growth inhibitory activity (IC50, 15-25 μM). Palbociclib combined with either alpelisib or capivasertib synergistically inhibited proliferation of LAR TNBC cells (CI values, 0.07-0.86). Treatment of Rb-intact LAR TNBC cells with palbociclib monotherapy suppressed Rb phosphorylation and resulted in adaptive phosphorylation/activation of S473 AKT and AKT substrates GSKβ and PRAS40 at 24h. These responses were not observed in Rb-null CAL-148 cells. Palbociclib-induced phosphorylation of AKT substrates as well as induction P-S6 and P-4EBP1 were better suppressed by capivasertib than by alpelisib over a dose range. Addition of the PI3Kβ/δ inhibitor AZD8186 to alpelisib markedly enhanced the inhibition of P-AKT, P-PRAS40 and P-Sin, suggesting inhibition of PI3Kα is inadequate to block the adaptive response to palbociclib in these cells. Mean CI values showed that the combination of palbociclib/capivasertib was more synergistic against LAR TNBC cells compared to palbociclib/alpelisib (mean CI, 0.29 vs. 0.78). ARE reporter activity did not change upon inhibition of PI3K or AKT with alpelisib or capivasertib, respectively. Conclusions: Our results suggest that addition of an AKT inhibitor to palbociclib suppresses the rebound activation of AKT following treatment with the CDK4/6i and is effective in LAR TNBC with wild type Rb. In vivo studies are underway to investigate the antitumor activity of the combination of palbociclib and capivasertib in LAR TNBC xenografts. Citation Format: Gun Min Kim, Kyung-min Lee, Dhivya Sudhan, Albert Lin, Arnaldo Marin, Sumanta Chatterjee, Dan Ye, Vishal Kandagatla, Saurabh Mendiratta, Ariella Hanker, Carlos Arteaga. Combined inhibition of CDK4/6 and AKT is effective in Rb-intact triple-negative breast cancer of the luminal androgen receptor (LAR) subtype [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-07.

Abstract P5-08-14: Maternal embryonic leucine zipper kinase is associated with metastasis in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) has high rates of relapse and metastasis. It has a high proportion of cancer stem-like cells (CSCs), which possess self-renewal and tumor initiation capacity. MELK (maternal embryonic leucine zipper kinase), a protein kinase of the Snf1/AMPK kinase family, plays a critical role in promoting CSC maintenance, cell proliferation, and malignant transformation. Here, we assessed the role of MELK in TNBC aggressiveness. Methods: The clinical relevance of MELK in TNBC was analyzed using the World IBC Consortium dataset (n = 314). The effects of MELK knockdown (siRNA), knockout (CRISPR-Cas9), and MELK inhibition with a novel selective small molecule inhibitor MELK-In-7 on migration and invasion were determined using the migration and invasion assays, respectively. MELK’s role in regulating CSC phenotypes was assessed using the mammosphere assay and flow cytometry. The impact of MELK knockdown on epithelial-to-mesenchymal transition (EMT) was determined using Western blotting. MELK’s protumorigenic role was determined in vitro using soft agar assay and in vivo using TNBC xenograft mouse models. Results: MELK mRNA expression level was higher in TNBC tumors than in tumors expressing hormone receptor, HER2, or both (P < 0.0001). In univariate analysis, breast cancer patients with high-MELK-expressing tumors had worse overall survival (OS, P < 0.001) and distant metastasis-free survival (DMFS, P < 0.01) than patients with low-MELK-expressing tumors, suggesting a prognostic impact of MELK in breast cancer. MELK knockdown using siRNA or MELK inhibition using MELK-In-7 significantly reduced migration, invasion, mammosphere formation, CD24-/CD44+ subpopulations, and ALDH activity, and reversed EMT in TNBC cells. This result suggests a role for MELK in regulating CSCs and EMT, which play crucial roles in tumor invasion and metastasis. Nude mice injected with MELK-knockout MDA-MB-231 cells exhibited suppression of lung metastasis and significantly improved OS compared with the mice injected with parental MDA-MB-231 cells (P < 0.05). Further, MELK-selective inhibitor (MELK-In-7) suppressed the growth of 4T1 mouse TNBC tumors in a dose-dependent manner in syngeneic BALB/c mice (P < 0.001). These results indicate an essential role for MELK in promoting TNBC tumor growth and metastasis. To uncover the underlying molecular mechanism of MELK’s regulation of TNBC metastasis, we performed microarray analysis and identified SERPINE1, a serine protease inhibitor known to regulate metastasis, as a downstream target of MELK. We validated that mRNA levels of SERPINE1 were reduced in MELK-knockout MDA-MB-231 cells and MELK-In-7-treated MDA-MB-231 cells. Moreover, SERPINE1 knockdown using siRNA reduced migration and invasion of TNBC cells. These results suggest that MELK regulates TNBC cell motility through SERPINE1. Conclusion: This work confirms MELK as a driver of aggressiveness and metastasis in TNBC. The mechanisms underlying MELK’s regulation of TNBC metastasis via SERPINE1 require further studies. Citation Format: Xuemei Xie, Gaurav B. Chauhan, Ramakrishna Edupuganti, Takahiro Kogawa, Jihyun Park, Moises T Tacam, Fnu Vidhu, Diane D. Liu, Juliana M. Taliaferro, Mary Kathryn Pitner, Yu Shen, Naoto T. Ueno, Savitri Krishnamurthy, Gabriel N. Hortobagyi, Debu Tripathy, Steven J. Van Laere, Geoffrey Bartholomeusz, Kevin Dalby, Chandra Bartholomeusz. Maternal embryonic leucine zipper kinase is associated with metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-14.

Abstract GS1-09: Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by lack of ER, PR, and Her2. Up to 85% of TNBCs have a p53 mutation as their oncogenic driver. TP53 is a tumor suppressor known as the “guardian of the genome” for its roles in regulating growth and death after genomic insult. Due to this high frequency of p53 mutations in TNBCs, targeting p53 mutants in a clinical setting is highly attractive. However, there are currently no FDA-approved drugs that can directly target p53 mutant TNBCs. We propose pathways exist that, when inhibited, will induce the specific death of p53-mutant breast cancer cells, but not p53-wild type breast cells. To investigate this hypothesis, we performed high-throughput drug screening to identify drugs that induce death in p53-mutant TNBCs. We then characterized the identified drugs for mechanism of death induction and in vivo drug effect. Methods: In vitro and in silico drug screens were conducted with small-molecule libraries of drugs with known protein targets, and screens were integrated and common drugs identified. P53-wild type and mutant cells were treated with identified drugs and stained with DAPI and DRAQ7 to identify growth-suppressive and death-inductive effects. One of these drugs, the GPX4 inhibitor ML-162, was selected for further study. Mechanism of death induction by ML-162 was determined with AnnexinV/PI, caspase cleavage, and ferroptosis assays. To confirm these results, GPX4 was knocked out and replicated the effect of ML-162. To determine the reliance of this phenotype on p53 mutational status, a series of inducible p53-mutant cell lines were created in ER+ and TNBC cell lines and then treated with small molecule inhibitors. ML-162 in vivo effect was demonstrated by treating p53-mutant TNBC xenografts in nude mice. Results: Integration of in vitro and in silico screens identified 6 common drugs, representing cell cycle inhibitors, cell division inhibitors, a proteasome inhibitor, and a peroxidase inhibitor. Identified drugs were demonstrated to reduce growth or induce death of p53-mutant TNBC cell lines. The GPX4 inhibitor ML-162 was further characterized, and found to induce death through ferroptosis, and not apoptosis or necroptosis. GPX4 knockout in p53-mutant TNBC cell lines induced ferroptosis and can be blocked with an anti-ferroptosis drug. Inducible p53-mutations in ER+ and TNBC cell lines were treated with ML-162. P53 mutations in TNBC, and not ER+, cell lines showed increased sensitivity to ML-162. To demonstrate in vivo effect of ML-162, TNBC cell line xenografts were grown in nude mice and treated with ML-162. This treatment significantly reduced tumor volume and also induced lipid peroxidation, a hallmark of ferroptosis. Conclusion: Our high-throughput screening demonstrated several of the identified drugs suppress growth or induce death preferentially in p53-mutant breast cancers. One of these drugs, ML-162, induces death of p53-mutant triple-negative breast cancer cells through induction of ferroptosis, both in vitro and in vivo. These studies provide the basic science foundation to further develop ferroptosis inducers for the targeted treatment of p53-mutant breast cancers. This work was funded by the John Charles Cain Endowment. Citation Format: William M Tahaney, Jing Qian, Reid Powell, Cassandra L Moyer, Yanxia Ma, Nghi Nguyen, Jamal Hill, Clifford Stephan, Abhijit Mazumdar, Peter JA Davies, Powel H Brown. Inhibition of GPX4 induces preferential death of p53-mutant triple-negative breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS1-09.

RB expression confers sensitivity to CDK4/6 inhibitor-mediated radiosensitization across breast cancer subtypes.

Standard radiation therapy (RT) does not reliably provide locoregional control for women with multinode-positive breast cancer and triple-negative breast cancer (TNBC). We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor–positive (ER+) cells, but also of TNBC that expresses retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB WT TNBC (n = 4, radiation enhancement ratio [rER]: 1.49–2.22) but failed to radiosensitize RB-null TNBC (n = 3, rER: 0.84–1.00). RB expression predicted response to CDK4/6i + RT (R2 = 0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88–1.13) after RB1 knockdown in isogenic and nonisogenic models. CDK4/6i suppressed homologous recombination (HR) in RB WT cells but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB reexpression. Radiosensitization was independent of nonhomologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult-to-control RB-intact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.

MAPK4 promotes triple negative breast cancer growth and reduces tumor sensitivity to PI3K blockade

About 15–20% of breast cancer (BCa) is triple-negative BCa (TNBC), a devastating disease with limited therapeutic options. Aberrations in the PI3K/PTEN signaling pathway are common in TNBC. However, the therapeutic impact of PI3K inhibitors in TNBC has been limited and the mechanism(s) underlying this lack of efficacy remain elusive. Here, we demonstrate that a large subset of TNBC expresses significant levels of MAPK4, and this expression is critical for driving AKT activation independent of PI3K and promoting TNBC cell and xenograft growth. The ability of MAPK4 to bypass PI3K for AKT activation potentially provides a direct mechanism regulating tumor sensitivity to PI3K inhibition. Accordingly, repressing MAPK4 greatly sensitizes TNBC cells and xenografts to PI3K blockade. Altogether, we conclude that high MAPK4 expression defines a large subset or subtype of TNBC responsive to MAPK4 blockage. Targeting MAPK4 in this subset/subtype of TNBC both represses growth and sensitizes tumors to PI3K blockade.

Synthesis and Evaluation of Radioiodine-Labeled pH (Low) Insertion Peptide Variant 7-Like Peptide as a Noninvasive Tumor Microenvironment Imaging Agent in a Mouse MDA-MB-231 Triple-Negative Breast Cancer Model.

The pH (low) insertion peptide (pHLIP) family can target the tumor microenvironment (TME). If pHLIP can be labeled with radioiodine, the imaging and treatment of tumors can be considered. However, tyrosine and tryptophan can bind with iodine in the insertion region of pHLIP, and radioiodine labeling may affect the formation of α-helix structures in acidic environments; therefore, it is necessary to adjust the structure of pHLIP. This study aims to develop an 125I-labeled pH (low) insertion peptide variant 7-like peptide (pHLIP (Var7) LP) for imaging the TME in MDA-MB-231 triple-negative breast cancer (TNBC) xenograft tumor models. Based on pHLIP (Var7), a new peptide sequence, pHLIP (Var7) LP, was obtained by the sequence modification method and then characterized. The binding of pHLIP (Var7) LP to MDA-MB-231 cells was analyzed. pHLIP (Var7) LP was labeled with 125I by the iodogen iodination method. Serial biodistribution studies and small-animal single photon emission computed tomography (SPECT)/computed tomography (CT) imaging in subcutaneous MDA-MB-231 TNBC-bearing mice were performed using [125I] I-pHLIP (Var7) LP. A novel peptide, pHLIP (Var7) LP, has the characteristics of an α-helix structure, electronegativity, and amphiphilicity. Circular dichroism (CD) spectroscopy showed that the peptide presented a typical pH-dependent transition from an unstructured conformation to an α-helix structure when the pH was reduced from 8.0 to 4.0. The relative fluorescence intensities of 5-carboxytetramethylrhodamine (5-TAMRA)-pHLIP(var7) LP at pH = 6.0, 6.6, and 7.4 were 100.00 ± 5.98%, 72.10 ± 4.65%, and 13.72 ± 1.41%, respectively. The distribution of [125I] I-pHLIP (Var7) LP in tumors reached the highest level (8.7 ± 1.6% ID/g) at 2h after injection, and the tumor-to-muscle ratios and tumor-to-blood ratios increased with time. Of the measured off-target organs, the stomach, kidney, and bladder showed higher uptake levels. SPECT imaging revealed rapid and sustained tumor uptake of [125I] I-pHLIP (Var7) LP in breast cancer-bearing mice. This study showed that [125I]I-pHLIP (Var7)LP had rapid and sustained tumor uptake in MDA-MB-231 TNBC and provided a new method for TNBC imaging and further treatment.

PRMT1 Regulates EGFR and Wnt Signaling Pathways and Is a Promising Target for Combinatorial Treatment of Breast Cancer.

Simple SummaryPatients with triple-negative breast cancer (TNBC) respond well to chemotherapy initially but are prone to relapse. Searching for new therapeutic targets, we found that PRMT1 is highly expressed in TNBC tumor samples and is essential for breast cancer cell survival. Furthermore, this study proposes that targeting PRMT1 in combination with chemotherapies could improve the survival outcome of TNBC patients.Identifying new therapeutic strategies for triple-negative breast cancer (TNBC) patients is a priority as these patients are highly prone to relapse after chemotherapy. Here, we found that protein arginine methyltransferase 1 (PRMT1) is highly expressed in all breast cancer subtypes. PRMT1 depletion decreases cell survival by inducing DNA damage and apoptosis in various breast cancer cell lines. Transcriptomic analysis and chromatin immunoprecipitation revealed that PRMT1 regulates the epidermal growth factor receptor (EGFR) and the Wnt signaling pathways, reported to be activated in TNBC. PRMT1 enzymatic activity is also required to stimulate the canonical Wnt pathway. Type I PRMT inhibitors decrease breast cancer cell proliferation and show anti-tumor activity in a TNBC xenograft model. These inhibitors display synergistic interactions with some chemotherapies used to treat TNBC patients as well as erlotinib, an EGFR inhibitor. Therefore, targeting PRMT1 in combination with these chemotherapies may improve existing treatments for TNBC patients.

TOP1 inhibition induces bifurcated JNK/MYC signaling that dictates cancer cell sensitivity.

Rationale: Triple-negative breast cancer (TNBC) does not respond to anti-estrogen and anti-HER2 therapies and is commonly treated by chemotherapy. TNBC has a high recurrence rate, particularly within the first 3 years. Thus, there is an urgent clinical need to develop more effective therapies for TNBC. Topoisomerase I (TOP1) inhibitors cause DNA damage, making these drugs desirable for TNBC treatment since DNA repair machinery is defective in this subtype of breast cancer. Among the main molecular subtypes of breast cancer, the TNBC cell lines exhibited the highest TOP1 inhibition sensitivity. However, clinically used TOP1 inhibitors, such as topotecan and irinotecan, have shown limited clinical applications and the reasons remain unclear. Understanding the mechanism of differential responses to TOP1 blockade and identifying the predictive markers for cancer cell sensitivity will help further TOP1-targeted therapy for TNBC treatment and improve the clinical use of TOP1 inhibitors.Methods: Viability assays were used to evaluate breast cancer cell sensitivity to topotecan and other TOP1 inhibitors as well as TOP2 inhibitors. An in vitro-derived topotecan-resistant TNBC cell model and TNBC xenograft models were employed to confirm cancer cell response to TOP1 blockade. RNA-seq was used to identify potential predictive markers for TNBC cell response to TOP1 blockade. Western blotting and qRT-PCR were performed to measure the protein levels and RNA expression. ATAC-seq and luciferase reporter assays were used to examine MYC transcriptional regulations. The effects of MYC and JNK in cancer cell response to TOP1 inhibition were validated via loss-of-function and gain-of-function experiments.Results: We observed two distinct and diverging cancer cell responses - sensitive versus resistant to TOP1 inhibition, which was confirmed by TNBC xenograft mouse models treated by topotecan. TNBC cells exhibited bifurcated temporal patterns of ATR pathway activation upon TOP1 inhibitor treatment. The sensitive TNBC cells showed an “up then down” dynamic pattern of ATR/Chk1 signaling, while the resistant TNBC cells exhibited a “persistently up” profile. On the contrary, opposite temporal patterns of induced expression of MYC, a key regulator and effector of DNA damage, were found in TNBC cells treated by TOP1 inhibitors. Mechanistically, we showed that TOP1-induced JNK signaling upregulated MYC expression. Furthermore, pharmacological inhibition of ATR reversed TNBC cell resistance to topotecan, whereas MYC knockdown and JNK inhibition reduced cancer cell sensitivity.Conclusions: Dynamic temporal profiles of induced ATR/Chk1 and JNK activation as well as MYC expression, may predict cancer cell response to TOP1 inhibitors. JNK activation-mediated constitutive elevation of MYC expression may represent a novel mechanism governing cancer cell sensitivity to TOP1-targeting therapy. Our results may provide implications for identifying TNBC patients who might benefit from the treatment with TOP1 inhibitors.

The Curcumin Analog EF24 Inhibits Proliferation and Invasion of Triple-Negative Breast Cancer Cells by Targeting the Long Noncoding RNA HCG11/Sp1 Axis.

EF24, a curcumin analog, exerts a potent antitumor effect on various cancers. However, whether EF24 retards the progression of triple-negative breast cancer (TNBC) remains unclear. In this study, we explored the role of EF24 in TNBC and clarified the underlying mechanism. In a mouse model of TNBC xenograft, EF24 administration reduced the tumor volume, suppressed cell proliferation, promoted cell apoptosis, and downregulated long noncoding RNA human leukocyte antigen complex group 11 (HCG11) expression. In TNBC cell lines, EF24 administration reduced cell viability, suppressed cell invasion, and downregulated HCG11 expression. HCG11 overexpression reenhanced the proliferation and invasion of TNBC cell lines suppressed by EF24. The following mechanism research revealed that HCG11 overexpression elevated Sp1 transcription factor (Sp1) expression by reducing its ubiquitination, thereby enhanced Sp1-mediated cell survival and invasion in the TNBC cell line. Finally, the in vivo study showed that HCG11-overexpressed TNBC xenografts exhibited lower responsiveness in response to EF24 treatment. In conclusion, EF24 treatment reduced HCG11 expression, resulting in the degradation of Sp1 expression, thereby inhibiting the proliferation and invasion of TNBC cells.

Abstract LBA008: CS5001, a novel ROR1-targeting antibody drug conjugate (ADC) armed with tumor-cleavable β-glucuronide linkers and pyrrolobenzodiazepine (PBD) prodrugs for hematological and solid malignancies

Abstract ROR1 (receptor tyrosine kinase-like orphan receptor 1) is an oncofetal protein prevalently expressed in a variety of hematological and solid malignancies but largely absent in normal adult tissues, making it an attractive ADC target. CS5001/ABL202/LCB71 is an ADC composed of a human monoclonal antibody targeting ROR1, site-specifically conjugated with a proprietary cleavable β-glucuronide linker to a prodrug of PBD dimer. Both linker and prodrug are selectively cleaved by the lysosomal b-glucuronidase, which is overexpressed in many cancerous cells, to allow tumor-selective release of the DNA-crosslinking PBD dimer. In vitro and in vivo pharmacology of CS5001 were evaluated and benchmarked against CS5001-BMK1 (an MMAE-based ROR1 ADC). Binding specificity, affinity and internalization were determined by Octet, ELISA and flow cytometry. Cytotoxicity and predictive biomarkers were evaluated in 20 human cancer cell lines with various levels of ROR1. In vivo efficacy was studied in Jeko-1 mantle cell lymphoma and MDA-MB-231 triple-negative breast cancer xenografts. CS5001 bound to human ROR1, but not ROR2, with a KD value of 1.38 nM. CS5001 has cross reactivity against mouse, rat and cynomolgus ROR1 at similar affinities. Upon binding, CS5001 was rapidly internalized by ROR1-expressing cancer cells at 37°C. CS5001 demonstrated potent cytotoxicity towards ROR1 high expressing cell lines such as Jeko-1 and MDA-MB-231, with an IC50value of 0.161 and 0.900 nM, whereas the IC50 value of CS5001-BMK1 was 10.8 nM and 129 nM, respectively. The growth inhibition activity of CS5001 but not CS5001BMK was significantly correlated with ROR1 density (Pearson correlation= -0.697, P = 6.4 x 10-4). CS5001 potently induced DNA damage in MDA-MB-231 cells, and resulted in increased apoptosis and G2-M cell cycle arrest in a concentration-dependent manner, indicating a mechanism of cytotoxicity consistent with that of the PBD payload. CS5001 exhibited prominent antitumor activity in both Jeko-1 and MDA-MB-231 xenograft models in a dose-dependent manner. In Jeko-1, complete regression (CR) was observed after a single administration of CS5001 at 1mg/kg (approximate 1/5th the maximum tolerated dose (MTD) in mice), whereas no CR was achieved for CS5001-BMK1 at the highest dose (2.5mg/kg, QWx3). When both CS5001 and CS5001-BMK1 were administrated at 1/20th of their respective MTDs, CS5001 (0.25mg/kg, single dose) produced significantly superior efficacy to CS5001-BMK1 (2.5mg/kg, single dose) with tumor growth inhibition (TGI) of 60% and 38%, respectively (p=0.018). In MDA-MB-231, 106% and 65.7% of TGI were observed for CS5001 at 1mg/kg (single dose) and CS5001-BMK1 at 2.5mg/kg (QWx3) respectively. CS5001 exhibited potent and selective cytotoxicity to a variety of ROR1-expressing cell lines and showed remarkable in vivo antitumor activity. ROR1 cytometric density predicts sensitivity to CS5001 in vitro. CS5001 is a promising therapeutic candidate for ROR1-expressing hematological and solid malignancies with precision medicine potential. Citation Format: Fu Li, Yongwang Li, Lan Zhang, Clarence K. Zhang, Hui-Han A. Hu, Juan Zhang, Ying Pan, Jinwon Jung, Sang Hoon Lee, Hyun-Min Ryu, Yun-Hee Park, Haixiang Yu, Archie N. Tse. CS5001, a novel ROR1-targeting antibody drug conjugate (ADC) armed with tumor-cleavable β-glucuronide linkers and pyrrolobenzodiazepine (PBD) prodrugs for hematological and solid malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA008.

Abstract 14414: Embryonic Stem Cell Derived Exosomes Enhance Anti-Tumor Efficacy of Doxorubicin and Improve Cardiac Function in Triple-Negative Breast Cancer Xenograft Mice

Background: Doxorubicin (Dox) is a potent chemotherapeutic drug widely used for the treatment of triple negative breast cancer (TNBC). However, Dox treatment results in inadvertent cardiotoxic effects which often require supplemental therapy. We previously showed that treatment with embryonic stem cells derived exosomes (ES-Exos) enhanced chemosensitivity of Dox in breast cancer cells. Here we tested the hypothesis whether adjuvant therapy with ES-Exos in mice with orthotopic TNBC xenograft can enhance the anti-tumor effect of Dox and improve cardiac function. Methods and Results: Athymic female nude mice (6-wk old) were injected with human MDA-MB-231-Luc cell line in mammary intraductal T4 region to generate orthotopic xenografts of TNBC. After 2 weeks of tumor growth, mice (n=10/group) were treated for 4 weeks with: Saline (Con); Dox (5 mg/kg/week); ES-Exos (50 μg, twice a week); Dox+ES-Exos; and MEF (50 μg/twice a week; non-stem cell exosomes derived from mouse fibroblasts). ES-Exos were labelled with ExoGlow-Vivo near-IR dye to track the localization of exosomes in the tumor. Treatment with Dox+ES-Exos and Es-Exos showed improved survival as compared to Dox (2 deaths) and Con (3 deaths) (Fig.A) . Tumor size was significantly reduced with Dox+ES-Exos and ES-Exos treatments as compared to Con and Dox treatment (Fig.B) . Cardiac function measured by echocardiography showed improved ejection fraction in Dox+ES-Exos and ES-Exos as compared to Con group (Fig.C) . Cell proliferation rate assessed by Ki-67 score with immunofluorescence showed significantly higher rate in Con as compared to Dox and Dox+ES-Exos groups. RNA seq analysis identified upregulation of unique antitumorigenic regulators such as Semaphorin 5 (Sema5) and FOXO15 in ES-Exos compared to MEF, which may contribute to the therapeutic effect. Conclusion: Treatment with ES-Exos may be a promising novel strategy to improve chemosensitivity with attenuation of its cardiotoxic effect in TNBC.

Targeted immunotherapy of triple-negative breast cancer by aptamer-engineered NK cells

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer comprised of cells that lack expression of targetable biomarkers. Nucleic acid aptamers are a group of molecular ligands that can specifically bind to their targets with high affinity. The ssDNA aptamer PDGC21-T recognizes poorly differentiated cancer cells and tumor tissues through an unidentified cell surface target(s). Because TNBC tumor cells are poorly differentiated, the aptamer PDGC21-T is a promising therapeutic candidate to target TNBC tumor cells. In vitro study revealed that synthetic aptamer probes selectively targeted TNBC cell lines. To assess aptamer immunotherapeutic targeting capability, we generated aptamer-engineered NK cells (ApEn-NK) using aptamer probes as a targeting ligand and NK cells as a therapeutic agent. Cell clustering formation assays revealed that ApEn-NK bound both suspended and adherent TNBC cells with high affinity. In a functional study, ApEn-NK treatment triggered apoptosis and death of cultured TNBC cells. Finally, systemic administration of ApEn-NK in mice harboring TNBC xenografts resulted in significant inhibition of lung metastasis relative to parental NK cell treatments. Unlike chemotherapy, ApEn-NK treatment did not affect body weight in treated mice. We demonstrate a novel approach for targeted TNBC immunotherapy.

Expression and function of SLC38A5, an amino acid-coupled Na+/H+ exchanger, in triple-negative breast cancer and its relevance to macropinocytosis.

Metabolic reprogramming in cancer necessitates increased amino acid uptake, which is accomplished by up-regulation of specific amino acid transporters. However, not all tumors rely on any single amino acid transporter for this purpose. Here, we report on the differential up-regulation of the amino acid transporter SLC38A5 in triple-negative breast cancer (TNBC). The up-regulation is evident in TNBC tumors, conventional and patient-derived xenograft TNBC cell lines, and a mouse model of spontaneous TNBC mammary tumor. The up-regulation is confirmed by functional assays. SLC38A5 is an amino acid-dependent Na+/H+ exchanger which transports Na+ and amino acids into cells coupled with H+ efflux. Since cell-surface Na+/H+ exchanger is an established inducer of macropinocytosis, an endocytic process for cellular uptake of bulk fluid and its components, we examined the impact of SLC38A5 on macropinocytosis in TNBC cells. We found that the transport function of SLC38A5 is coupled to the induction of macropinocytosis. Surprisingly, the transport function of SLC38A5 is inhibited by amilorides, the well-known inhibitors of Na+/H+ exchanger. Down-regulation of SLC38A5 in TNBC cells attenuates serine-induced macropinocytosis and reduces cell proliferation significantly as assessed by multiple methods, but does not induce cell death. The Cancer Genome Atlas database corroborates SLC38A5 up-regulation in TNBC. This represents the first report on the selective expression of SLC38A5 in TNBC and its role as an inducer of macropinocytosis, thus revealing a novel, hitherto unsuspected, function for an amino acid transporter that goes beyond amino acid delivery but is still relevant to cancer cell nutrition and proliferation.

Inhibition of Vasculogenic Mimicry and Angiogenesis by an Anti-EGFR IgG1-Human Endostatin-P125A Fusion Protein Reduces Triple Negative Breast Cancer Metastases

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. Metastasis is the major cause of TNBC mortality. Angiogenesis facilitates TNBC metastases. Many TNBCs also form vascular channels lined by tumor cells rather than endothelial cells, known as ‘vasculogenic mimicry’ (VM). VM has been linked to metastatic TNBC behavior and resistance to anti-angiogenic agents. Epidermal growth factor receptor (EGFR) is frequently expressed on TNBC, but anti-EGFR antibodies have limited efficacy. We synthesized an anti-EGFR antibody–endostatin fusion protein, αEGFR IgG1-huEndo-P125A (αEGFR-E-P125A), designed to deliver a mutant endostatin, huEndo-P125A (E-P125A), to EGFR expressing tumors, and tested its effects on angiogenesis, TNBC VM, and motility in vitro, and on the growth and metastasis of two independent human TNBC xenograft models in vivo. αEGFR-E-P125A completely inhibited the ability of human umbilical vein endothelial cells to form capillary-like structures (CLS) and of TNBC cells to engage in VM and form tubes in vitro. αEGFR-E-P125A treatment reduced endothelial and TNBC motility in vitro more effectively than E-P125A or cetuximab, delivered alone or in combination. Treatment of TNBC with αEGFR-E-P125A was associated with a reduction in cytoplasmic and nuclear β-catenin and reduced phosphorylation of vimentin. αEGFR-E-P125A treatment of TNBC xenografts in vivo inhibited angiogenesis and VM, reduced primary tumor growth and lung metastasis of orthotopically implanted MDA-MB-468 TNBC cells, and markedly decreased lung metastases following intravenous injection of MDA-MB-231-4175 lung-tropic TNBC cells. Combined inhibition of angiogenesis, VM, and TNBC motility mediated by αEGFR-E-P125A is a promising strategy for the prevention of TNBC metastases.

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.