Mutant Triple Negative Breast Cancer Research Articles

Abstract 6027: 89Zr-matuzumab and 225Ac-matuzumab as a theranostic for epidermal growth factor receptor-positive KRAS wild-type colorectal and breast cancer xenografts

Abstract Background: About 80% of colorectal cancer (CRC) and 50 % of Triple-Negative breast cancer (TNBC) patients overexpress epidermal growth factor receptor (EGFR). Mutations in the KRAS oncogene (present in 40% of mCRC) lead to constitutive over-activation of EGFR and drive de novo resistance to anti-EGFR drugs. Here, we propose to target KRAS-mutant/BRAF mutant EGFR positive mCRC and TNBC using 225Ac-labeled matuzumab, a humanized anti-EGFR monoclonal antibody. Methods: p- -SCN-macropa was used to conjugate matuzumab for radiolabeling with 225Ac and SCN-deferoxamine was used to conjugate matuzumab for labelling with 89Zr. The radioimmunoconjugates were characterized by flow cytometry, HPLC and iTLC. in vitro cytotoxicity was evaluated in EGFR-positive mCRC, TNBC cell lines and 3D spheroids with different levels of EGFR density. Tumor growth was monitored using digital caliper. Mice were treated with either 10 MBq of 89Zr-matuzumab for imaging or three doses of 13 KBq/dose administered of 225Ac-matuzumab 10 days apart. In vivo study endpoint was tumor volume greater than or equal to 1500 mm3. Results: Flow cytometry showed about 95% binding to the cells in all EGFR-positive colorectal cell lines (DLD-1, SW620, SNU-C2B, HT-29) and breast cancer cell lines (MDA-MB-468, MDA-MB-231). in vitro studies showed enhanced cytotoxicity of 225Ac-matuzumab compared with matuzumab. IC50 in the MDA-MB-468 cell line for 225Ac-matuzumab (1.86 ± nM) was 35 times more effective than matuzumab (65.25± 7 nM). Similar trends were observed in the other KRAS-mutant mCRC cell lines and breast cancer cell lines. In 3D spheroid models, the IC50 of 225Ac-matuzumab (3.0 ± 2 nM) in MDA-MB-468, was 20.79 times more effective than matuzumab (62.3 ± 1.38 nM). Similar trends were observed for all the other spheroid models as well. High uptake of 89Zr-matuzumab was observed in both mCRC and TNBC xenografts. 225Ac-matuzumab slowed tumor growth rate in MDA-MB-468 and MDA-MB-231 tumors models compared with control antibody or non-treated controls for 30 days. This study is still ongoing and will last for 2 more months. Conclusion: 225Ac-matuzumab shows very promising outcomes in KRAS-mutant mCRC models and breast cancer models and warrants further investigation. Citation Format: Florence Anjong Tikum, Humphrey Fonge, Fabrice Njotu, Hanan Babeker, Jessica K. Pougoue, Nikita Henning, Alireza Doroudi. 89Zr-matuzumab and 225Ac-matuzumab as a theranostic for epidermal growth factor receptor-positive KRAS wild-type colorectal and breast cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6027.

Abstract 522: TP53mutations promote death of breast cancer cells following Kif11 inhibition

Abstract Background: Triple-Negative Breast Cancer (TNBC) comprises 15-20% of breast cancer diagnoses. These aggressive breast cancers are characterized by their lack of expression of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and progesterone receptor (PR), resulting in few targeted therapy options for TNBC patients. While 85-90% of TNBCs contain mutations in TP53, mutant p53 is challenging to target directly. Instead, we aim to identify other survival pathways critical for the growth and survival of TP53 mutant cells. Through a combined in vitro and in silico drug screen, we identified the Kif11 inhibitor SB-743921 as more effective in TP53 mutant compared to wild-type breast cancer cells. Hypothesis: We hypothesized that Kif11 inhibition causes TP53 mutant cells to undergo cell death due to failure of activation of p53-mediated cell cycle checkpoints. Methods: Clinical data from the TCGA and METABRIC cohorts was obtained from cBioPortal. Expression and survival analyses were performed using GraphPad Prism. Cell growth assays were conducted by seeding cells, treating under designated conditions, then staining with Hoechst 33342 for cell counting on the ImageXpress Pico. Cell death was determined by staining with Annexin V and PI or DAPI Staining followed by flow cytometry analysis. Cell cycle analysis was conducted following synchronization with Lovastatin and Mevalonate release followed by imaging analysis of cell lines expressing the Fast FUCCI reporter or PI staining and flow cytometry analysis. Immunofluorescence imaging was conducted by staining with anti-alpha-tubulin-AlexaFluor488 and DAPI and imaging on the Nikon TI2 or ImageXPress PICO. In vitro expression experiments were carried out through qPCR and western blotting. Results: KIF11 is most highly expressed in TP53 mutant and triple-negative tumors and high expression of KIF11 is associated with poorer overall survival. In breast cancer cell lines, Kif11 inhibition leads to a cell cycle block in both TP53 mutant and wild-type cells, but TP53 mutant cells then die following this cell cycle block. Furthermore, Kif11 inhibition causes mitotic dysfunction, including monopolar spindle formation and multinucleated cells in TP53 mutants. Introduction of a TP53 mutation into TP53 wild-type cells also induces cell death following Kif11 inhibition. Conclusions: KIF11 is highly expressed in aggressive breast cancers and is associated with poorer prognosis. Using the small molecule inhibitor SB-74321 that has been tested in phase I/II clinical trials with favorable tolerability, we found that Kif11 inhibition results in mitotic dysfunction and death of TP53 mutant breast cancer cells due to mitotic catastrophe. These results suggest that Kif11 is a promising target for the treatment of TP53 mutant TNBCs. Citation Format: Amanda L. Lanier, William Tahaney, Jing Qian, Cassandra Moyer, Yanxia Ma, Banu Arun, Abhijit Mazumdar, Powel H. Brown. TP53mutations promote death of breast cancer cells following Kif11 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 522.

MRCK as a Potential Target for Claudin-Low Subtype of Breast Cancer.

To find new molecular targets for triple negative breast cancer (TNBC), we analyzed a large-scale drug screening dataset based on breast cancer subtypes. We discovered that BDP-9066, a specific MRCK inhibitor (MRCKi), may be an effective drug against TNBC. After confirming the efficacy and specificity of BDP-9066 against TNBC in vitro and in vivo, we further analyzed the underlying mechanism of specific activity of BDP-9066 against TNBC. Comparing the transcriptome of BDP-9066-sensitive and -resistant cells, the activation of the focal adhesion and YAP/TAZ pathway were found to play an important role in the sensitive cells. Furthermore, YAP/TAZ is indeed repressed by BDP-9066 in the sensitive cells, and active form of YAP suppresses the effects of BDP-9066. YAP/TAZ expression and activity are high in TNBC, especially the Claudin-low subtype, consistent with the expression of focal adhesion-related genes. Interestingly, NF-κB functions downstream of YAP/TAZ in TNBC cells and is suppressed by BDP-9066. Furthermore, the PI3 kinase pathway adversely affected the effects of BDP-9066 and that alpelisib, a PI3 kinase inhibitor, synergistically increased the effects of BDP-9066, in PIK3CA mutant TNBC cells. Taken together, we have shown for the first time that MRCKi can be new drugs against TNBC, particularly the Claudin-low subtype.

Estrogen Receptor β4 Regulates Chemotherapy Resistance and Induces Cancer Stem Cells in Triple Negative Breast Cancer.

Triple Negative Breast Cancer (TNBC) has the worst prognosis among all breast cancers, and survival in patients with recurrence is rarely beyond 12 months due to acquired resistance to chemotherapy, which is the standard of care for these patients. Our hypothesis is that Estrogen Receptor β1 (ERβ1) increases response to chemotherapy but is opposed by ERβ4, which it preferentially dimerizes with. The role of ERβ1 and ERβ4 in influencing chemotherapy sensitivity has never been studied before. CRISPR/CAS9 was used to truncate ERβ1 Ligand Binding Domain (LBD) and knock down the exon unique to ERβ4. We show that the truncated ERβ1 LBD in a variety of mutant p53 TNBC cell lines, where ERβ1 ligand dependent function was inactivated, had increased resistance to Paclitaxel, whereas the ERβ4 knockdown cell line was sensitized to Paclitaxel. We further show that ERβ1 LBD truncation, as well as treatment with ERβ1 antagonist 2-phenyl-3-(4-hydroxyphenyl)-5,7-bis(trifluoromethyl)-pyrazolo[1,5-a] pyrimidine (PHTPP), leads to increase in the drug efflux transporters. Hypoxia Inducible Factors (HIFs) activate factors involved in pluripotency and regulate the stem cell phenotype, both in normal and cancer cells. Here we show that the ERβ1 and ERβ4 regulate these stem cell markers like SOX2, OCT4, and Nanog in an opposing manner; and we further show that this regulation is mediated by HIFs. We show the increase of cancer cell stemness due to ERβ1 LBD truncation is attenuated when HIF1/2α is knocked down by siRNA. Finally, we show an increase in the breast cancer stem cell population due to ERβ1 antagonist using both ALDEFLUORTM and SOX2/OCT4 response element (SORE6) reporters in SUM159 and MDA-MB-231 cell lines. Since most TNBC cancers are ERβ4 positive, while only a small proportion of TNBC patients are ERβ1 positive, we believe that simultaneous activation of ERβ1 with agonists and inactivation of ERβ4, in combination with paclitaxel, can be more efficacious and yield better outcome for chemotherapy resistant TNBC patients.

Abstract P2-16-01: The Kinesin-like protein Kif11 is essential for the Survival of TP53-mutant Triple Negative Breast Cancer Cells

Abstract Background: Breast cancer is the most commonly diagnosed non-cutaneous malignancy in American women and one of the leading causes of cancer deaths. Breast cancer can be divided into several subtypes, the most aggressive of which is Triple-Negative Breast Cancer (TNBC), a disease with few targeted therapies. TP53 mutations are found in 80% or more of TNBCs. However, direct targeting of mutant TP53 has been difficult. To identify drugs that can specifically induce the death of TP53-mutant breast cancers, we conducted a drug screen in TP53-mutant and TP53-wild type breast cancer cells. Through this combined in silico and in vitro drug screen, we discovered that TP53 mutant TNBC cells have an increased sensitivity to KIF11 inhibition as compared to TP53 wild-type cells. Hypothesis: We hypothesize that TP53 mutational status confers sensitivity of triple-negative breast cancer cells to KIF11 inhibition. Methods: We obtained data on TP53 mutational status, KIF11 mRNA expression levels, and clinical characteristics from publicly-available TCGA, METABRIC, and CCLE datasets. To demonstrate the effect of KIF11 inhibition on cell growth, we treated TP53 mutant and wild-type breast cancer cell lines with the small molecule KIF11 inhibitor SB-743921 and KIF11 siRNAs. Cell counts were obtained through staining with DAPI or Hoechst nuclear stains and imaging on the MetaXPress PICO instrument. To determine protein expression of p53 and Kif11 across various cell lines, western blotting was performed. We then investigated the biological mechanisms of growth inhibition by DRAQ7 staining and flow cytometry analysis following Annexin V-PI staining. Results: Using cell growth assays we demonstrated that TP53 mutant cells are more sensitive to KIF11 inhibition. We next utilized cell lines in which TP53 mutations had been introduced into TP53 wild-type cells to show that overexpression of a TP53 mutant gene can sensitize cells to KIF11 inhibition. Using assays of proliferation and apoptosis, we showed that TP53 mutant breast cancer cells treated with a KIF11 inhibitor undergo cell death. Using publicly-available datasets of breast cancers, we showed that KIF11 is upregulated in TNBCs and TP53 mutant cancers, and that high expression of KIF11 in breast cancer is correlated with poorer clinical prognosis. Conclusions: Our results show that the Kif11 protein is essential for the survival of TP53 mutant TNBC cells. Inhibitors of Kif11 induce death of TP53 mutant TNBCs and thus, this kinesin-like protein involved in cell spindle mechanics is a potential target for the treatment of these aggressive cancers. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers TL1TR003169 and UL1TR003167. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the John Charles Cain Endowment. Citation Format: Amanda Lanier, Abhijit Mazumdar, William Tahaney, Powel Brown. The Kinesin-like protein Kif11 is essential for the Survival of TP53-mutant Triple Negative Breast Cancer Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-01.

Abstract 5399: Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype. Despite the early response to chemotherapy, high incidence of recurrence leads to short overall survival and poor prognosis. Native hyaluronic acid (HA) interacts with CD44 and receptors for hyaluronan mediated motility (RHAMM) overexpressed in TNBC mediating chemo-resistance and metastasis. In contrast, oligomeric HA (oHA) can disrupt HA-CD44/RHAMM interactions and attenuate oncogenic and pro-metastatic pathways, such as the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway, reducing downstream DNA repair and drug resistance markers. Our previous work demonstrated that oHA can exert synergistic anti-tumor and anti-metastatic effects when combined with doxorubicin (DOX) and co-loaded in an integrin-targeted iRGD-modified nanoparticle system (iRGD-DOX-oHA-PLNs). This study aims to investigate the inhibitory effect of iRGD-DOX-oHA-PLNs on both DNA single-strand break (SSB) and double-strand break (DSB) repair proteins and drug efflux pumps responsible for multidrug resistance to enhance DOX efficacy in breast cancer gene 1 (BRCA1) mutant and non-mutant TNBC. Methods: The cytotoxicity of DOX, oHA and their combinations in free solution or in nanoparticles was evaluated by clonogenic assay in human MDA-MB-231-luc-D3H2LN and MDA-MB-436 (BRCA1 mutant) TNBC cells. The in vitro expression of a DNA DSB marker, DNA repair markers, and drug efflux pump P-glycoprotein (P-gp) were measured by Western blot. The in vivo expression level of BRCA1 and Rad51 in an orthotopic TNBC mouse model was determined by immunohistochemical staining. Results: The combination of DOX-oHA showed synergism against both BRCA1 mutant and non-mutant TNBC cells. The iRGD-DOX-oHA-PLNs induced great increases in DNA DSBs demonstrated by the highest γH2AX level compared to other treatment groups. These nanoparticles also showed inhibitory effects on the expression of both DNA SSB repair protein (poly (ADP-ribose) polymerase) and DNA DSB repair proteins (Rad50 and Rad51), contributing to the enhanced efficacy of chemotherapy. The immunohistochemical staining of tumor tissues indicated lower levels of BRCA1 and Rad51 after iRGD-DOX-oHA-PLN treatment than the formulation without oHA, attributable to the effect of intracellularly delivered oHA on limiting the MAPK signaling. Additionally, iRGD-DOX-oHA-PLNs reduced the expression of the drug efflux pump P-gp as compared to DOX treatment groups without oHA. Conclusion: The co-delivery of oHA and DOX in the iRGD-DOX-oHA-PLNs efficiently blocked DNA damage repair and down-regulated the drug efflux pump P-gp, thus improving the efficacy of DOX. Collectively, this nanoparticle system could be a promising option for metastatic TNBC treatment. Citation Format: Pei Zhi, Ibrahim Alradwan, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Michael Rauth, Xiao Yu Wu. Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5399.

Abstract 5990: Targeted nanoparticles reduce tumor progression and lung metastasis by limiting DNA damage repair and immune suppression in metastatic triple-negative breast cancer

Abstract Background: Metastatic triple-negative breast cancer (TNBC), especially with BRCA1/2 mutations, is a deadly subtype of breast cancer with ~12% of 5-year survival. Despite their FDA approval, the therapeutic benefits of poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPi) are limited to BRCA1/2 mutated malignancies. Moreover, the PARPi olaparib has been shown to upregulate the expression of programmed death-ligand 1 (PD-L1) leading to immune suppression. The purpose of this study is to investigate if a novel nanoparticle formulation of oligomer hyaluronic acid (oHA) and doxorubicin (DOX) co-loaded in an iRGD-conjugated polymer-lipid nanocarrier (iRGD-DOX-oHA-PLN) can inhibit DNA damage repair and suppress PD-L1 in both mutant and non-mutant TNBC cells and reduce tumor progression and lung metastasis compared to the PARPi olaparib. We postulate that the co-administered oHA will block the signaling pathways of native hyaluronic acid (HA) receptors, the cluster of differentiation 44 (CD 44) and receptor for HA mediated motility (RHAMM), that regulate DNA damage repair and immunosuppression and enhance DOX efficacy. Methods: In vitro cellular uptake of various DOX formulations by TNBC MDA-MB-231-luc-D3H2LN cells and MDA-MB-436 (BRCA1 mutant) cells was examined using confocal laser scanning microscopy or spectrophotometer. The expression level of RHAMM, PD-L1, and PARP1 parylation (PAR) was evaluated after treatment with iRGD-DOX-oHA-PLN or olaparib in both cell lines using western blot or confocal laser scanning microscopy. The therapeutic efficacy of iRGD-DOX-oHA-PLN compared to olaparib was determined by monitoring the tumor progression and lung metastasis development in orthotopic breast tumor models of MDA-MB-231-luc-D3H2LN or MDA-MB-436 cell line. Results: The expression of native HA receptor RHAMM was profoundly reduced by ~5 folds in vivo and the DNA DSB was significantly increased by the iRGD-DOX-oHA-PLN treatment with 40% of γH2AX positive cells in tumor tissue sections. The iRGD-functionalized PLN enhanced DOX cellular uptake compared to DOX free drug or non-targeted NPs by ~3- and ~1.5-fold, in MDA-MB-231 and MDA-MB-436 cells, respectively. While olaparib upregulated PD-L1 and free DOX increased PAR level, the iRGD-DOX-oHA-PLN reduced the parylation and PD-L1 expression in both TNBC cell lines. The iRGD-DOX-oHA-PLN treatment (two dose of 10 mg/kg i.v. biweekly) outperformed olaparib (twenty doses of 50 mg/kg i.p 5x/week) in preventing tumor progression and lung metastasis in vivo over a 4-week period. Conclusion: The results suggest that the iRGD-DOX-oHA-PLN can effectively inhibit DNA damage repair and immunosuppression of cancer cells and could be a promising multitargeted nanomedicine for the treatment of both BRCA1-mutant and non-mutant metastatic TNBC. Citation Format: Ibrahim Alradwan, Pei Zhi, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Rauth, Xiao Yu Wu. Targeted nanoparticles reduce tumor progression and lung metastasis by limiting DNA damage repair and immune suppression in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5990.

Targeting MUC1-C Suppresses Chronic Activation of Cytosolic Nucleotide Receptors and STING in Triple-Negative Breast Cancer.

Simple SummaryTriple-negative breast cancers (TNBCs) are recalcitrant tumors with limited therapeutic options. Cytotoxic agents, including platinum-based drugs, are a standard of care for advanced TNBCs. Olaparib is also used for the treatment of germline BRCA mutant TNBC tumors in the adjuvant and recurrent disease settings. Notably, however, the effectiveness of these genotoxic agents is often limited by intrinsic and adaptive DNA damage resistance. We demonstrate in TNBC cells that the oncogenic MUC1-C protein chronically activates the type I interferon (IFN) pathway, drives the cGAS/STING axis and induces expression of the DNA damage resistance gene signature (IRDS). Targeting MUC1-C inhibits activation of this pathway in the response to carboplatin and olaparib and sensitizes TNBC cells to these agents. These findings indicate that MUC1-C is a target, which is druggable, for overcoming the obstacle of DNA damage resistance in the treatment of TNBCs.The MUC1-C apical transmembrane protein is activated in the acute response of epithelial cells to inflammation. However, chronic MUC1-C activation promotes cancer progression, emphasizing the importance of MUC1-C as a target for treatment. We report here that MUC1-C is necessary for intrinsic expression of the RIG-I, MDA5 and cGAS cytosolic nucleotide pattern recognition receptors (PRRs) and the cGAS-stimulator of IFN genes (STING) in triple-negative breast cancer (TNBC) cells. Consistent with inducing the PRR/STING axis, MUC1-C drives chronic IFN-β production and activation of the type I interferon (IFN) pathway. MUC1-C thereby induces the IFN-related DNA damage resistance gene signature (IRDS), which includes ISG15, in linking chronic inflammation with DNA damage resistance. Targeting MUC1-C in TNBC cells treated with carboplatin or the PARP inhibitor olaparib further demonstrated that MUC1-C is necessary for expression of PRRs, STING and ISG15 and for intrinsic DNA damage resistance. Of translational relevance, MUC1 significantly associates with upregulation of STING and ISG15 in TNBC tumors and is a target for treatment with CAR T cells, antibody–drug conjugates (ADCs) and direct inhibitors that are under preclinical and clinical development.

Molecular Characterization of BRCA1 c.5339T>C Missense Mutation in DNA Damage Response of Triple-Negative Breast Cancer

Simple SummaryRecent studies re-classified the c.5339T->C; p.Leu1780Pro (L1780P) BRCA1 missense mutation as “likely pathogenic” in ACMG classification, which shows a high prevalence in the Korean population. This study aims to reveal the molecular mechanisms and therapeutic relevance of BRCA1 L1780P mutation in DNA damaging response of triple-negative breast cancer (TNBC).BRCA1 L1780P BRCT domain mutation has been recognized as a pathogenic mutation in patients with breast cancer. However, the molecular significance of this mutation has not yet been studied in triple-negative breast cancer (TNBC) cells in vitro. We established MDA-MB 231, HCC1937, and HCC1395 TNBC cell lines expressing BRCA1 L1780P mutant. BRCA1 L1780P mutant TNBC cells showed increased migration and invasion capacity, as well as increased sensitivity to olaparib and carboplatin compared to BRCA1 wild-type cells. BRCA1 L1780P mutant TNBC cells showed decreased RAD51 expression and reduced nuclear RAD51 foci formation following carboplatin and olaparib treatment. The molecular interaction between p-ATM and BRCA1 was abrogated following introduction of BRCA1 L1780P mutant plasmid in TNBC cells, suggesting that the BRCA1 L1780P mutation disrupts the p-ATM-BRCA1 protein–protein interaction. We established an olaparib-resistant BRCA1 L1780P mutant TNBC cell line by chronic drug treatment. Olaparib-resistant cell lines showed upregulation of RAD51 expression upon olaparib treatment, and reduction in RAD51 expression in olaparib-resistant cells restored olaparib sensitivity. Collectively, these results suggest that the BRCA1 L1780P mutation impairs RAD51 recruitment by disrupting p-ATM-BRCA1 interaction, which is a crucial molecular factor in homologous recombination and olaparib sensitivity. Further therapeutic targeting of RAD51 in BRCA1 L1780P mutant breast cancer is warranted.

Abstract P4-01-11: UBQLN4 regulates cisplatin-resistance in triple-negative breast cancer by targeting BAT3 for proteasomal degradation

Abstract Background: Triple-negative breast cancer (TNBC) is a heterogeneous breast cancer (BC) subtype. Cisplatin is one of the broad FDA-approved drugs to treat TNBC patients with recurrent and unresectable disease. However the treatment response is limited, and patients frequently develop resistance and recur locally and distantly. The aim of this study is to find molecular mechanisms driving cisplatin-resistance in TNBC. The ubiquitin proteasomal system (UPS) is a universal and conserved mechanism that helps to maintain cellular homeostasis. The aberrant expression of certain components of the UPS have been associated with cancer onset, progression, and metastasis as we and other groups have shown. UBQLN4 is part of the UPS and play a major role in controlling ubiquitinated proteins in response to genotoxic stress. BCL2-associated athanogene-6 (BAG6), alternately known as BAT3, is a chaperone that complex with other chaperones and ubiquitin ligases to regulate protein stability and insertion of tail-anchored membrane proteins into the endoplasmic reticulum. This is a critical step that reduce mislocalized proteins and proteotoxic stress. We hypothesized that UBQLN4 upregulation determine cisplatin-resistance in BC patients by targeting BAT3 for proteasomal degradation to reduce proteotoxic stress. Methods: TCGA BRCA database was utilized to determine clinical associations and outcomes in BC patients. A BC tissue microarray (TMA) was stained by IHC and protein levels quantified to confirm associations between UBQLN4 and TNBC patients. TNBC cell lines were CRISPR-engineered to generate UBQLN4 knockout (KO), and lentivirus transduction was used to recover the KO phenotype. Functional assays and confocal imaging were performed in BRCA mutant and wildtype human TNBC cell lines. Results: Increased UBQLN4 mRNA expression was observed in the TCGA BRCA database in primary BC tissues compared to normal adjacent tissues (p<0.0001). Using PAM50 classification, the BC patients were stratified and compared for UBQLN4 expression. BC patients with basal-like tumors showed significantly higher UBQLN4 expression than normal-like subtype (p< 0.0001). In the assessment of 5-years relapse-free survival (RFS), patients with high levels of UBQLN4 (n=221) showed significantly lower RFS rates compared to those with low UBQLN4 (n=221) (HR=1.67(1.21-2.3), log-rank test p<0.0016). These results were further evaluated by IHC using a BC TMA. Patients with TNBC showed a significantly increased UBQLN4 H-scores values compared to normal adjacent breast tissues. KO of UBQLN4 significantly increased cisplatin sensitivity in TNBC cell lines (p<0.0001). UBQLN4 overexpression restored the cisplatin-resistance in UBQLN4-KO cell line. In TGCA BRCA database, BAT3 was significantly upregulated in BC patients with basal-like tumors compared to normal-like subtype. Knockdown of BAT3 significantly increased cisplatin sensitivity in TNBC cell lines (p<0.0001). In TCGA BRCA database, BAT3 and UBQLN4 mRNA levels positively correlated (r= 0.26, p<0.0001). By using co-immunoprecipitation, endogenous BAT3 and UBQLN4 proteins interact in parental TNBC cell line, but only during cisplatin-treatment. These results were further validated using reciprocal co-immunoprecipitation and confocal imaging in TNBC cell lines with UBQLN4 overexpression. Conclusions: UBQLN4 expression controls cisplatin-resistance in TNBC cell lines. UBLQN4 interacts with BAT3 under cisplatin treatment. During cisplatin treatment, UBQLN4 targets BAT3 for proteasomal degradation to reduce proteotoxic stress induced by genotoxic cisplatin treatment. The UBQLN4 protein levels may represent a prognostic biomarker to predict RFS in TNBC patients. Citation Format: Yoshiaki Shoji, Takamichi Yokoe, Peter J Bostick, Yosef Shiloh, Dave SB Hoon, Matias A Bustos. UBQLN4 regulates cisplatin-resistance in triple-negative breast cancer by targeting BAT3 for proteasomal degradation [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 P4-01-11.

Abstract P1-04-09: Essential role for MUC1-C in chronic activation of cytosolic nucleotide sensing and the type I interferon pathway in triple-negative breast cancer

Abstract The MUC1-C transmembrane protein, which is aberrantly expressed in triple-negative breast cancer (TNBC), evolved in mammals to provide protection of epithelia from the external environment. MUC1-C functions as an effector of epithelial cell responses to inflammation and damage. MUC1-C induces inflammatory, proliferative and remodeling signaling pathways that are associated with wound healing. However, in contrast, prolonged MUC1-C activation in settings of chronic inflammation promotes cancer progression. Recent work has demonstrated that MUC1-C drives intrinsic activation of inflammatory signaling pathways in TNBC cells that are linked to immune evasion and oncogenesis (Yamashita N, JITC, 2021). By extension, what is needed now is a better understanding of how MUC1-C specifically integrates inflammation with TNBC progression. In addressing this issue, the present work uncovers a previously unrecognized MUC1-C activated inflammatory pathway that contributes to genomic instability and DNA damage tolerance in TNBC cells. Constitutive expression of pattern-recognition receptors (PRRs), which include the retinoic acid-inducible gene-1 (RIG-I/DDX58) and melanoma differentiation-associated gene 5 (MDA5/IFIH1), are activated by the presence of cytosolic dsRNA. Accumulation of DNA damage-associated molecular patterns (DAMPs) in the cytosol is recognized by activation of the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Stimulation of these RNA and DNA PRRs induces production of the type I IFNs and induction of interferon-stimulated genes (ISGs) that confer DNA damage resistance. We report here that MUC1-C is necessary for constitutive activation of RIG-I, MDA5, cGAS and STING in BRCA wild type and mutant TNBC cells. Consistent with these findings, we show that MUC1-C is necessary for expression of the downstream STAT1, STAT2 and IRF9 effectors in activation of the type I IFN pathway and the IFN-related DNA damage resistance gene signature (IRDS). One of the MUC1-C-induced IRDS genes encodes ISG15, a ubiquitin-like protein that links chronic inflammation and DNA damage resistance. Of translational significance, studies in TNBC cells treated with carboplatin or the PARP inhibitor olaparib demonstrate that MUC1-C is necessary for expression of RIG-I, MDA5, cGAS, STING and ISG15 and that targeting MUC1-C abrogates this response. In addition, we show that MUC1 significantly associates with the upregulation of STING, STAT1, STAT2, IRF9 and ISG15 in TNBC tumors. In summary, these findings in TNBC uncover an essential role for MUC1-C in chronic activation of the PRR/STING axis, type I IFN pathway and IRDS in association with conferring resistance to replicative stress. Our findings also support MUC1-C as a target for inhibiting DNA damage tolerance and synergistically sensitizing BRCA wild type and mutant TNBC cells to platinum-based agents and olaparib. Citation Format: Nami Yamashita, Atsushi Fushimi, Yoshihiro Morimoto, Atrayee Bhattacharya, Mark Long, Song Liu, Donald Kufe. Essential role for MUC1-C in chronic activation of cytosolic nucleotide sensing and the type I interferon pathway 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 P1-04-09.

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.

Abstract P027: MUC1-C integrates chronic activation of the type I and II interferon pathways in treatment resistance of triple-negative breast cancer

Abstract Chronic inflammation is intimately linked to cancer progression and resistance to treatment. Triple-negative breast cancer (TNBC) is a recalcitrant malignancy largely unresponsive to cytotoxic, targeted and immunotherapeutic agents. The MUC1-C transmembrane protein, which is aberrantly expressed in TNBCs, evolved in mammals to provide protection of epithelia from the external environment. MUC1-C is activated in epithelial cell responses to inflammation and damage, and induces inflammatory, proliferative and remodeling signaling pathways that are associated with wound healing. However, prolonged MUC1-C activation in settings of chronic inflammation promotes cancer progression and treatment resistance. Recent studies in TNBCs have demonstrated that MUC1-C drives intrinsic activation of type II interferon (IFN) pathway that is linked to chronic inflammation and immune evasion (Yamashita N, et al. JITC 2021). In extending this work, we have now found that MUC1-C activates the type I IFN pathway, which contributes to genomic instability and DNA damage tolerance in TNBC cells. Importantly, our results further demonstrate that MUC1-C interacts with interferon regulatory factor 1 (IRF1) as a key regulatory node in integrating activation of the type I and II IFN pathways. We show that stimulation of TNBC cells with IFN-γ induces nuclear complexes of MUC1-C and IRF1 that drive expression of effectors, such as IDO1 and WARS, which promote immunosuppression in the tumor microenvironment (TME). Of translational relevance, analysis of human TNBC bulk and single cell RNA-seq datasets demonstrated that MUC1 associates with (i) IDO1 and WARS expression, and (ii) depletion and dysfunction of CD8+ T cells in the TNBC TME. Our results further demonstrate that MUC1-C and IRF1 complexes are necessary for induction of RIG-I, MDA5 and STING expression and for IFN-β production in TNBC cells. Consistent with these findings, we show that MUC1-C is necessary for activation of the downstream type I IFN pathway and IFN-related DNA damage resistance gene signature, which includes ISG15, a ubiquitin-like protein that links chronic inflammation and DNA damage resistance. Of translational relevance, studies in BRCA wild type and mutant TNBC cells treated with carboplatin or the PARP inhibitor olaparib demonstrate that MUC1-C is necessary for expression of RIG-I, MDA5, cGAS, STING and ISG15 and that targeting MUC1-C abrogates this response. As a result, MUC1-C is a target for inhibiting DNA damage tolerance and synergistically sensitizing TNBC cells to platinum-based agents and olaparib. In summary, these findings in TNBC uncover an essential role for MUC1-C and IRF1 in chronic activation of the type I and II IFN pathways that promote immune evasion and resistance to replicative stress. Our findings also support MUC1-C as a target for TNBC treatment with chimeric antigen receptor T cells, antibody-drug conjugates and a functional inhibitor that are under preclinical and clinical development. Citation Format: Nami Yamashita, Atsushi Fushimi, Yoshihiro Morimoto, Atrayee Bhattacharya, Mark Long, Song Liu, Donald Kufe. MUC1-C integrates chronic activation of the type I and II interferon pathways in treatment resistance of triple-negative breast cancer [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 P027.

Selinexor, a selective inhibitor of nuclear export, enhances the anti-tumor activity of olaparib in triple negative breast cancer regardless of BRCA1 mutation status.

Triple negative breast cancer (TNBC) is a deadly disease with limited treatment options. Selinexor is a selective inhibitor of nuclear export that binds covalently to exportin 1 thereby reactivating tumor suppressor proteins and downregulating expression of oncogenes and DNA damage repair (DDR) proteins. Olaparib is a poly (ADP-ribose) polymerase (PARP) inhibitor approved for the treatment of patients with breast cancer harboring BRCA mutations. We examined the effects of co-treatment with selinexor and olaparib in TNBC cell lines. BRCA1 wildtype (BRCA1-wt) and BRCA1 mutant (BRCA1-mut) TNBC cell lines were treated with selinexor and/or olaparib and effects on cell viability and cell cycle were evaluated. The effects of treatment were also evaluated in mouse xenograft models generated with BRCA1-wt and BRCA1-mut TNBC cell lines. Treatment with selinexor inhibited cell proliferation and survival of all TNBC cell lines tested in vitro. This effect was enhanced following treatment of the cells with the combination of selinexor and olaparib, which showed synergistic effects on tumor growth inhibition in MDA-MB-468-derived (BRCA1-wt) and MDA-MB-436-derived (BRCA1-mut) xenografts. As co-treatment with selinexor and olaparib exhibits anti-tumor activity regardless of BRCA1 mutation status, the clinical implications of the combination warrant further investigation.

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

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

Abstract PS4-39: Efficacy of combined CDK4/6 inhibitor and PARP inhibitor in the treatment of BRCA1 mutant triple negative breast cancer

Abstract Background: PARP inhibitors (PARPi) benefit only a fraction of breast cancer with BRCA mutation, and are even more limited in triple negative breast cancer (TNBC), due to clinical primary and acquired resistance. Here we found that the efficacy of PARPi in TNBC can be expanded with CDK4/6 inhibitors (CDK4/6i). Methods: We screened sensitive and resistant PARPi cell lines from existing TNBC cell lines and cultured PARPi-resistant strains by gradient concentration increase method. The effects of PARP inhibitor Olaparib and CDK4/6 inhibitor Palbociclib on TNBC cells lines were examined in vitro and in vivo. Combination index (CI) was used to evaluate the synergistic effects of drug combination. Results: We screened out the cell line MDA-MB-436, which was the most sensitive to PARPi, and the cell lines HCC1937 and SUM149, which were relatively resistant to PARPi, all of which were BRCA1 mutants. We demonstrated for the first time that the combination of PARPi and CDK4/6i has synergistic effects against some TNBCs both in vitro and in vivo, and was verified by CI<0.9. Further experiments confirmed that PARPi combined with CDK4/6i inhibited cell proliferation and migration, and increased apoptosis and DNA damage. In the PARPi sensitive BRCA-/TNBC cell (MDA-MB-436), the inhibitory effect of monotherapy PARPi was obvious; In the PARPi resistant BRCA-/TNBC cells (HCC1937 and SUM149), CDK4/6i was added to achieve significant growth inhibition. In the timing of medication, PARPi followed by CDK4/6i had better inhibitory effect.Conclusions: In some BRCA-/ TNBCs, PARPi combined with CDK4/6i had a synergistic effect. Even in PARPi-resistant cells, combined treatment could enhance the efficacy and might reverse the drug resistance to some extent. The combination index index of HCC1937 at different drug concentrations (uM)The combination index index of HCC1937 at different drug concentrations (uM)OlaparibPalbociclib25.0016.6711.117.414.943.292.191.460.980.65P-25.000.459110.668320.866540.869640.993450.989890.97841.003221.073741.00809P-20.000.537090.851131.022651.149561.231561.270271.189071.145021.14811.25795P-15.000.594121.018141.134481.056960.928140.955670.889760.842040.81060.81348P-10.000.475540.860580.77190.717480.62330.615880.561390.52960.510250.49752P-5.000.509450.762230.746560.683340.56030.499410.467390.41490.390670.37502P-2.500.573130.706410.714750.661230.519270.455960.350980.29340.319310.2756 Citation Format: Xiuzhi Zhu, Li Chen, Zhonghua Wang. Efficacy of combined CDK4/6 inhibitor and PARP inhibitor in the treatment of BRCA1 mutant triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-39.

Abstract PS16-19: Hotspot p53 mutations correlate with increased expression of stem cell markers in triple-negative breast cancer

Abstract p53 mutations occur in 80% of triple-negative breast cancer (TNBC) cases. Several types of p53 mutations have been reported, among which gain of function mutations have been associated with increased cancer stem cells, chemotherapy resistance, and disease relapse in several malignancies. Particularly, hotspot p53 mutations (mutations at amino acid location 157, 175, 248, 249, 273 predict worse overall survival in some cancer types. We have previously reported that Ganglioside GD2, a cell surface glycosphingolipid, identifies breast cancer stem cells and promotes tumorigenesis. We have also reported that GD3 synthase, a key enzyme which catalyzes the rate-limiting step of the GD2 biosynthesis pathway, is significantly upregulated in breast tumors with p53 mutations. Here, we hypothesize that mutant p53 promotes stemness in TNBC cells by regulating GD3 synthase and GD2 expression. To identify specific p53 mutations that contribute to TNBC stemness, we stratified TNBC cell lines (N=18) based on p53 mutation status including mutation type, location, and domain affected. We measured GD2 and GD3 synthase expression in the available TNBC cell lines by flow cytometry and RT-PCR, respectively. We found that GD3 synthase expression is significantly upregulated in TNBC cell lines with p53 hotspot mutations compared to cell lines with other p53 mutations (Median relative expression 0.0114 compared to 0.0003, p=0.005). Similarly, we found that GD2 expression is significantly higher in TNBC cell lines with hotspot mutations compared to non-hotspot counterparts (Median GD2+ cells 15.2% compared to 0.97%, p= 0.013) Additionally, GD2 and GD3 synthase are upregulated in basal-type TNBC cell lines and TCGA patient samples compared to other TNBC molecular subtypes. Interestingly, we also found that GD2 expression is not always directly correlated with GD3S expression, suggesting that GD2 synthase expression is also a key factor in the regulation of GD2 expression. To examine the effect of mutant p53 protein levels on TNBC stemness, we measured p53 expression in all the available TNBC cell lines by western blot, and found that p53 protein levels do not correlate with GD2 or GD3S expression. To validate these findings, we analyzed RPPA data from the MD Anderson Cancer Cell Lines Project and found similar results. These data suggest that it is the type of p53 mutation, but not the amount of p53 protein in the cells that determines GD2 and GD3 synthase expression in TNBC cells. For instance, p53 hotspot mutations involving amino acid position 248 have been shown to be strongly associated with increased cancer stemness. We found that cell lines with p53 mutations at this specific location have significantly higher GD3 synthase expression compared to other mutant p53 forms. We are currently examining the direct role of mutant p53 in the regulation of GD3S expression by stabilizing mutant p53 using MDM2 inhibitors as well as stable knockdown of p53 gene in multiple TNBC cell lines to establish a clear link between mutant p53 and stem cell marker expression in TNBC cell lines. In conclusion, stem cell markers are highly expressed in p53 mutant TNBC cell lines compared to wild type p53 counterparts. Specifically, cell lines with hotspot p53 mutations significantly correlate with increased TNBC stemness. The type of p53 mutation, rather than level of its expression correlates with stem cell marker expression in TNBC cells. Citation Format: Fouad El-Dana, Bin Yuan, Stanley Ly, Vivek Anand, Venkata Lokesh Battula. Hotspot p53 mutations correlate with increased expression of stem cell markers in triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-19.

Novel Agents for Metastatic Triple-Negative Breast Cancer: Finding the Positive in the Negative.

Metastatic triple-negative breast cancer (TNBC) is associated with a poor prognosis, and the development of better therapeutics represents a major unmet clinical need. Although the mainstay of treatment of metastatic TNBC is chemotherapy, advances in genomics and molecular profiling have helped better define subtypes of TNBC with distinct biologic drivers to guide the therapeutic development of targeted therapies, including AKT inhibitors for PI3K/AKT-altered TNBC, checkpoint inhibitors for PD-L1-positive TNBC, and PARP inhibitors for BRCA1/2 mutant TNBC. This progress may ultimately convert TNBC from a disease traditionally defined by the absence of therapeutically actionable receptors to one that is defined by the presence of discrete molecular targets with therapeutic implications. Furthermore, antibody drug conjugates have emerged as an important therapeutic strategy to target genomically complex tumors that lack actionable oncogenes but have overexpressed actionable surface receptors such as trop-2. In this article, we discuss promising novel agents for advanced TNBC, some of which have been incorporated into current clinical practice, and others that will likely change the therapeutic landscape and redefine the TNBC terminology in the near future.

Both BRCA1-wild type and -mutant triple-negative breast cancers show sensitivity to the NAE inhibitor MLN4924 which is enhanced upon MLN4924 and cisplatin combination treatment.

Triple-negative breast cancer (TNBC) shows limited therapeutic efficacy. PARP inhibitor has been approved to treat advanced BRCA-mutant breast cancer but shows high resistance. Therefore, the development of new therapeutics that sensitize TNBC irrespective of BRCA status is urgently needed. The neddylation pathway plays a critical role in many physiological processes by regulating the degradation of proteins. MLN4924, a selective inhibitor of the key neddylation enzyme NEDD8 Activation Enzyme (NAE1), shows higher sensitivity to both BRCA1-wild type and -mutant TNBCs compared to other breast cancer subtypes. MLN4924 induced re-replication with >4N DNA content leading to robust DNA damage. Accumulation of unrepaired DNA damage resulted in S and G2/M arrest causing apoptosis and senescence, due to the stabilization of the replication initiation protein CDT1 and the accumulation of cell cycle proteins upon MLN4924 treatment. Moreover, adding MLN4924 to the standard TNBC chemotherapeutic agent cisplatin increased the DNA damage level, further enhancing the sensitivity. In vivo, MLN4924 reduced tumor growth in a NOD-SCID mouse xenograft model by inducing DNA damage which was further augmented with the MLN4924 and cisplatin cotreatment. NAE1 is overexpressed in TNBC cell lines and in patients compared to other breast cancer subtypes suggesting that NAE1 status is prognostic of MLN4924 treatment response and outcome. Taken together, we demonstrated the mechanism of TNBC sensitization by the MLN4924 and MLN4924/cisplatin treatments irrespective of BRCA1 status, provided a strong justification for using MLN4924 alone or in combination with cisplatin, and identified a genetic background in which this combination will be particularly effective.

Reversal effect of quercetin on talazoparib resistance in BRCA1 mutant triple negative breast cancer

Objectives: Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated an outstanding activity in patients with BRCA-mutated and wild-type breast cancer. However, the identification of resistance mechanisms to PARP inhibitors is a significant clinical challenge in effective treatment. Thus, new therapeutic strategies are urgently needed to overcome resistance. The aim of the current study was to explore the potential effect of quercetin on HCC1937 (BRCA1 mutant) and talazoparib (BMN 673), a PARP inhibitor, resistant HCC1937 (HCC1937-R) triple negative breast cancer cells (TNBC). Methods: We firstly generated BMN 673 resistance HCC1937 cells by continuous exposure to BMN 673 during 6 months. Then, cells were exposed to the different concentration (0-100 µM) of quercetin and the cytotoxic and apoptotic effects of quercetin on these cells were evaluated by WST-1, Annexin V and dual acridine orange-ethidiumbromide (Et-BR) staining. Results: The cell viability of HCC1937 and HCC1937-R cells reduced to 37.1% and 44.2% at a concentration of 100 μM, respectively for 48 h (p < 0.01). Apoptotic rates of HCC1937 and HCC1937-R cells treated with 100 μM quercetin were nearly 56.0% and 46.0%, respectively (p < 0.01). Additionally, theapoptotic morphologicalchanges were observed in these cells. Conclusions: In conclusion, the obtained results suggest that quercetin could potentially be used as an alternative therapeutic strategy in BRCA1 mutant TNBC to overcome acquired BMN 673-resistance.