Resistant Breast Cancer Cells Research Articles

Adenovirus-mediated expression of MOAP-1, Bax and RASSF1A antagonizes chemo-drug resistance of human breast cancer cells expressing cancer stem cell markers

Cancer is one of the major leading causes of mortality globally and chemo-drug-resistant cancers pose significant challenges to cancer treatment by reducing patient survival rates and increasing treatment costs. Although the mechanisms of chemoresistance vary among different types of cancer, cancer cells are known to share several hallmarks, such as their resistance to apoptosis as well as the ability of cancer stem cells to produce metastatic daughter cells that are resistant to chemotherapy. To address the issue of chemo-drug resistance in cancer cells, a tetracistronic expression construct, Ad-MBR-GFP, encoding adenovirus-mediated expression of MOAP-1, Bax, RASSSF1A, and GFP, was generated to investigate its potential activity in reducing or inhibiting the chemo-drug resistant activity of the human breast cancer cells, MCF-7-CR and MDA-MB-231. When infected by Ad-MBR-GFP, the cancer cells exhibited round cell morphology and nuclei condensation with positive staining for annexin-V. Furthermore, our results showed that both MCF-7-CR and MDA-MB-231 cells stained positively for CD 44 and negatively for CD 24 (CD44+/CD24-) with high levels of endogenous ALDH activity whereas SNU-1581 breast cancer cells were identified as CD 44-/CD 24- cells with relatively low levels of endogenous ALDH activity and high sensitivity toward chemo-drugs, suggesting that both CD 44 and ALDH activity contribute to chemo-drug resistance. Moreover, both MCF-7-CR and MDA-MB-231 cells showed strong chemo-drug sensitivity to cisplatin when the cells were infected by Ad-MBR-GFP, leading to 9-fold and 2-fold reduction in the IC 50 values when compared to cisplatin treatment alone, respectively. The data were further supported by 3D (soft agar) and spheroid cell models of MCF-7-CR and MDA-MB-231 cells which showed a 2-fold reduction of a number of cell colonies and spheroid size when treated with both Ad-MBR-GFP and cisplatin, and compared to control. Other than chemo-sensitivity, Ad-MBR-GFP-infected cancer cells retarded cell migration. Flow cytometry analysis showed that the mechanism of action of Ad-MBR-GFP involved cell cycle arrest at the G1 phase and inhibition of cellular DNA synthesis. Taken together, our investigation showed that Ad-MBR-GFP mediated chemo-drug sensitization in the infected cancer cells involved the activation of apoptosis signaling, cell cycle arrest, and inhibition of DNA synthesis, suggesting that Ad-MBR-GFP is potentially efficacious for the treatment of chemo-drug resistant cancers.

Cholesteryl ester transfer protein knock-down in conjunction with a cholesterol-depleting agent decreases tamoxifen resistance in breast cancer cells.

The cholesterogenic phenotype, encompassing de novo biosynthesis and accumulation of cholesterol, aids cancer cell proliferation and survival. Previously, the role of cholesteryl ester (CE) transfer protein (CETP) has been implicated in breast cancer aggressiveness, but the molecular basis of this observation is not clearly understood, which this study aims to elucidate. CETP knock-down resulted in a >50% decrease in cell proliferation in both 'estrogen receptor-positive' (ER+; Michigan Cancer Foundation-7 (MCF7) breast cancer cells) and 'triple-negative' breast cancer (TNBC; MDA-MB-231) cell lines. Intriguingly, the abrogation of CETP together with the combination treatment of tamoxifen (5 μM) and acetyl plumbagin (a cholesterol-depleting agent) (5 μM) resulted in twofold to threefold increase in apoptosis in both cell lines. CETP knockdown also showed decreased intracellular CE levels, lipid raft and lipid droplets in both cell lines. In addition, RT2 Profiler PCR array (Qiagen, Germany)-based gene expression analysis revealed an overall downregulation of genes associated in cholesterol biosynthesis, lipid signalling and drug resistance in MCF7 cells post-CETP knock-down. On the contrary, resistance in MDA-MB-231 cells was reduced through increased expression in cholesterol efflux genes and the expression of targetable surface receptors by endocrine therapy. The pilot xenograft mice study substantiated CETP's role as a cancer survival gene as knock-down of CETP stunted the growth of TNBC tumour by 86%. The principal findings of this study potentiate CETP as a driver in breast cancer growth and aggressiveness and thus targeting CETP could limit drug resistance via the reduction in cholesterol accumulation in breast cancer cells, thereby reducing cancer aggressiveness.

Abstract PO1-23-11: Differential m6A modification identified by direct RNA sequencing in endocrine- resistant and sensitive breast cancer cells

Abstract A major limitation of current adjuvant treatments for the ~ 70% of breast cancer patients with estrogen receptor α–expressing (ER+) primary breast tumors is the development of acquired and adaptive endocrine therapy (ET) resistance and metastatic spread. Chemical modifications of transcribed RNAs alters their cellular location, stability, and, for mRNAs, translation into proteins. N-6 methyladenosine (m6A) is the most common modification of mRNA. We tested the hypothesis that m6A epitranscriptomic changes alters the transcriptome resulting in changes in targets in pathways contributing to the metastatic cascade in ET-resistant breast cancer cells. We used an unbiased approach of direct mRNA sequencing using the Oxford Nanopore MinION platform on polyA-selected mRNA from MCF-7 luminal A breast cancer cells and ER+, ET-resistant LCC9 breast cancer cells treated with vehicle control or 100 nM 4-hydroxytamoxifen (4-OHT) for 24 h. In addition, MCF-7 cells were treated with the METTL3 (the catalytic methyltransferase that adds the methyl group from S-adenosylmethionine (SAM) to the adenosine on the N6 position) inhibitor STM2457 to identify m6A positions and transcripts directly regulated by METTL3 activity. Computational analysis directly identified m6A locations in transcripts. We examined the differential modification rate (DMR) between MCF-7 cells +/STM2457 treatment, between MCF-7 and LCC9 cells with vehicle control treatment or with 4-OHT treatment and within each cell line with 4-OHT treatment. We identified 183 and 3,097 genes with m6A DMRs between vehicle-treated and 4-OHT-treated MCF-7 and LCC9 cells, respectively. For 4-OHT-treated cells, we identified 2,401 and 2,638 genes with m6A DMRs in MCF-7 and LCC9 cells respectively with 1,760 genes in common. We identified many genes with multiple m6A modifications, e.g., AMIGO2 appeared to have 14 m6A sites at DRACH motifs in MCF-7 cells, but none were m6A modified in LCC9 cells. Enrichment by Pathway Maps, Process Networks, and GO Processes were performed. Overall, our data suggest a role for altered m6A positions in transcripts and pathways involved in ET-resistance in breast cancer. Citation Format: Belinda Petri, Bailey Valdes, Kellianne Piell, Eric Rouchka, Carolyn Klinge. Differential m6A modification identified by direct RNA sequencing in endocrine- resistant and sensitive breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-23-11.

Abstract PO2-27-04: Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer

Abstract Fibroblast Growth Factor Receptor 1 (FGFR1) has emerged as a critical player in various cancer types, including breast cancer. FGFR1 alterations, including gene amplification, overexpression, and activating mutations, have been identified in subsets of breast cancers, contributing to aberrant signaling pathways that promote tumor growth, angiogenesis, and metastasis. Our current research endeavors are directed towards unravelling the intricate mechanistic underpinnings of FGFR1's dual impact on breast tumor progression, specifically its contribution to endocrine resistance and its regulatory role in innate immune sensing and subsequent induction of Type 1 interferons (IFNs) Our investigative efforts have previously established a direct association between FGFR1 and endocrine resistance in hormone receptor positive T47D breast cancer cells. Moreover, a remarkable and distinct inverse correlation linking FGFR1 expression to the Type 1 IFN response was observed. We noticed that in vitro stimulation of T47D cells with Type 1 interferons induces the expression of FGFR1 and ESR1, thereby suppressing the interferon-stimulated genes. Interestingly, we observed that the FGFR1-mediated suppression of the Type 1 interferon response is associated with intracellular nucleic acid sensors, specifically toll-like receptors TLR7 and TLR9. Furthermore, we found that the inhibition of FGFR kinase enhances the expression of IFN-alpha, IFN-beta, TLR7, and TLR9 in T47D cells when stimulated with the TLR7 agonist loxoribine. These results strongly suggest that FGFR1 functions as an innate checkpoint and may be exploited by tumor cells to dampen Type 1 interferon responses, thereby inhibiting antitumor immunity in hormone receptor-positive breast cancer. Citation Format: Sushil Dhakal, Torbjørn Amundsen Lien, Jens Henrik Norum, Helga Bergholtz, Helene Midtun Flatekvål, Therese Sørlie. Unravelling the role of FGFR1 in innate immune sensing and endocrine resistance in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-27-04.

Abstract PO5-23-08: Adenylosuccinate lyase is essential for proliferation and mitochondrial function of endocrine therapy resistant breast cancer cells

Abstract Eighty percent of breast cancers express estrogen receptor alpha (ERα, ESR1) at the time of diagnosis. Endocrine therapy (ET) is the standard treatment that either block estrogen mediated ER activation (such as tamoxifen, fulvestrant) or suppress estrogen synthesis (such as exemestane, letrozole, or anastrozole). While ET is initially effective, emergence of ET-resistance is common and possess a major clinical challenge. Hence, there is an ongoing need to develop new treatments for ET-resistant-ERα positive breast cancer. In this study we investigated the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway as a potential target of ET-resistant breast cancer. The protein level of ADSL in two independent ET-resistant cell line model LCC9 and T47D-4HT cells were significantly higher in comparison to its parental ET-sensitive cell lines, MCF7 and T47D cells, respectively. Transient knockdown of ADSL using two independent siRNA in both LCC9 and T47D-4HT cells results in a significant decrease of cell growth, colony, and spheroid formation. Notably, reduced ADSL levels in LCC9 cells prevented the cells from progressing from G1 to S phase of cell cycle with concurrent elevation of cyclin D1/D3, CDK2/4, and cyclin E. On the other hand, in T47D-4HT cells, ADSL reduction caused cells to accumulate in S-phase of cell cycle and showed lower levels of total cyclin D1 protein. Mechanistically, in LCC9 cells (but not in T47D-4HT cells), ADSL depletion induced DNA replication stress which activates (phosphorylate) ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its major downstream effector checkpoint kinase 1 (Chk1) that in turn failed to de-phosphorylate the inhibitory-phospho-groups of cyclin-dependent kinase 2 (CDK2). In addition, ADSL depletion perturbed the mitochondrial function in both LCC9 and T47D-4HT cells. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was lower in both the cells along with the mitochondrial membrane potential. Pertinently, lower concentration of AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside), a product of ADSL enzyme function, partially rescued the cell growth and restored mitochondrial membrane potential in ADSL-depleted LCC9 cells. To further understand the global effects of ADSL depletion we performed RNA-sequencing of LCC9 and T47D-4HT cells after ADSL depletion and compared with respective control siRNA transfected cells followed by gene set enrichment analysis. Intriguingly, diverse effect was observed in LCC9 and T47D-4HT cells as very few genes were found to be commonly regulated between these two cell lines. Analysis of clinical data sets revealed high level of ADSL transcript was associated with adverse progression free survival and overall survival in breast cancer patients treated with endocrine therapies. Overall, our findings demonstrate that ADSL-mediated de novo purine synthesis is critical for cellular growth, proliferation, and mitochondrial function of endocrine therapy-resistant breast cancer cells. Therefore, targeting ADSL is a novel potential therapeutic approach for ERα positive ET- resistant breast cancer. Citation Format: Anil Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Adenylosuccinate lyase is essential for proliferation and mitochondrial function of endocrine therapy resistant breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-23-08.

Abstract PO3-27-02: A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells

Abstract Background. PTK6/Brk, a non-receptor tyrosine kinase, is an oncogenic driver for several tumor types, including breast, prostate, liver, and pancreatic cancers. PTK6 is expressed in all breast cancer subtypes and higher levels of expression are associated with worse patient outcomes. The optimal approach to targeting PTK6 for therapeutic benefit has not been established. Although several inhibitors of PTK6 kinase activity have been developed, none have thus far been successfully translated to clinical trials. This may be in part due to kinase activity-independent functions of PTK6, for which there is growing evidence, that are not effectively targeted by traditional small molecule kinase inhibitors. Downregulation of PTK6 using shRNA inhibits growth, invasion/migration, survival and metastasis of breast cancer cells, including those that are resistant to standard therapies. PTK6 kinase inhibitor treatment phenocopies effects of PTK6 downregulation with respect to some, but not all PTK6-driven oncogenic activities. Moreover, growth of PTK6 CRISPR-targeted cells can be rescued either with wild type or catalytically inactive PTK6, supporting kinase activity-independent activities of PTK6 in regulating growth and viability of breast cancer cells. Therefore, PROTAC degraders may be a better way to chemically target and downregulate PTK6 expression in cancer, thereby phenocopying the effects of PTK6 shRNA. Methods. We developed a first-in-class PTK6 degrader using PROTAC (proteolysis-targeting chimeric) technology to target PTK6 expression in breast cancer cells of diverse subtypes. Degrader controls were also synthesized to assess specificity of MS105’s effects. Results. PTK6 degrader, but not degrader controls, potently downregulates PTK6 expression in multiple breast cancer cell types. Proteomic and kinome profiling confirm the specificity of our PTK6 degrader. PTK6 PROTAC degrader inhibits viability and induces apoptosis of PTK6-expressing breast cancer cells, whereas treatment with parent kinase inhibitor or degrader controls that do not engage PTK6 or E3 ligase have minimal effect. Treatment with PTK6 degrader, but not kinase inhibitor or degrader controls, induces expression of pro-apoptotic Bim, phenocopying the effects observed with PTK6 shRNA. In contrast, PTK6 degrader and kinase inhibitor are comparably effective in inhibiting cell migration, supporting the differential kinase dependency of PTK6-dependent oncogenic functions. Therefore, our PTK6 PROTAC degrader effectively inhibits both kinase-dependent and independent oncogenic activities of PTK6. Our studies support the development of PTK6 PROTAC degraders as a preferred approach to clinically targeting PTK6 in cancer. Citation Format: Hanna Irie, Criseyda Martinez, Yan Xiong, Alison Bartkowski, Jessica Byerly, Jian Jin. A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-27-02.

Abstract PO4-24-09: Grape seed extract inhibits EGFR signaling pathway as a main factor of tamoxifen resistant in hormone receptor positive breast cancer

Abstract Purpose Because more than 60% of breast cancers are hormone-receptor (HR) positive, endocrine therapy plays an important role. Tamoxifen is an antagonist of estrogen receptor-α and main treatment drug for HR-positive breast cancers. However, efficacy is not satisfactory because of the development of resistance. EGFR expression was associated with poor prognosis in HR-positive breast cancers. Loss of ER by EGFR activation induced tamoxifen resistance. And extracellular signal-regulated kinase (ERK) is another important factor for tamoxifen resistance. Activation of ERK signaling in breast cancer is not only associated with augmented tumor growth and metastasis but also with tamoxifen resistance. Therefore, the inhibition of ERK/EGFR signaling associated with tamoxifen resistance could improve the survival rate. Grape seed extract (GSE) is which contains a high content of flavonoid polyphenolic compounds. GSE is known to have various pharmacological effects, including antioxidant, anti-inflammatory, antitumor activity. In this study, we explored the activity of GSE in tamoxifen resistant breast cancer cells by inhibiting EGFR signaling pathway. Method We examined the suppressive effect of GSE on various signaling pathways. To confirm the pharmacological activity of GSE, we treated EGFR positive breast cancer cells with GSE. Levels of mRNA and protein expression were analyzed by real-time PCR and western blot. And cell proliferation and invasiveness were analyzed by the colony-forming assay and the Boyden chamber assay. Results 1. GSE down regulates EGFR and ERK in EGFR positive breast cancer cell line. The cells were treated with the indicated concentrations GSE for 48 hrs. The levels of p- and t-ERK and t-EGFR were measured through Western blotting. The levels of phosphorylated EGFR and ERK were decreased by GSE at the indicated doses for 48hrs. 2. EGF, a ligand of EGFR, upregulates the MMP-9 and GSE suppresses MMP-9. Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) are upregulated in most cancers and play crucial roles in modulating invasion and metastasis. EGFR with its ligand epidermal growth factor (EGF) regulates Matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). After treating EGFR positive breast cancer cells with EGF and TGF, MMP-9 levels were increased. However, MMP-9 level was decreased with GSE as indicated doses for 48hrs. Conclusion Taken together, GSE inhibited the ERK/EGFR and EGF/MMP-9 signaling pathway in EGFR positive breast cancer cells. Therefore, we propose the possibility of GSE as an effective adjuvant for tamoxifen resistant breast cancer. Citation Format: Seung Pil Jung, Eun-Shin Lee, Ji Young You, Jung Hwa Chun, Hyunyou Kim. Grape seed extract inhibits EGFR signaling pathway as a main factor of tamoxifen resistant in hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-24-09.

MiR-770-5p-induced cellular switch to sensitize trastuzumab resistant breast cancer cells targeting HER2/EGFR/IGF1R bidirectional crosstalk

miR-770-5p-induced cellular switch to sensitize trastuzumab resistant breast cancer cells targeting HER2/EGFR/IGF1R bidirectional crosstalk

SiRNA treatment targeting integrin α11 overexpressed via EZH2-driven axis inhibits drug-resistant breast cancer progression

BackgroundBreast cancer, the most prevalent cancer in women worldwide, faces treatment challenges due to drug resistance, posing a serious threat to patient survival. The present study aimed to identify the key molecules that drive drug resistance and aggressiveness in breast cancer cells and validate them as therapeutic targets.MethodsTranscriptome microarray and analysis using PANTHER pathway and StemChecker were performed to identify the most significantly expressed genes in tamoxifen-resistant and adriamycin-resistant MCF-7 breast cancer cells. Clinical relevance of the key genes was determined using Kaplan-Meier survival analyses on The Cancer Genome Atlas dataset of breast cancer patients. Gene overexpression/knockdown, spheroid formation, flow cytometric analysis, chromatin immunoprecipitation, immunocytochemistry, wound healing/transwell migration assays, and cancer stem cell transcription factor activation profiling array were used to elucidate the regulatory mechanism of integrin α11 expression. Tumour-bearing xenograft models were used to demonstrate integrin α11 is a potential therapeutic target.ResultsIntegrin α11 was consistently upregulated in drug-resistant breast cancer cells, and its silencing inhibited cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT) while restoring sensitivity to anticancer drugs. HIF1α, GLI-1, and EZH2 contributed the most to the regulation of integrin α11 and EZH2 expression, with EZH2 being more necessary for EZH2 autoinduction than HIF1α and GLI-1. Additionally, unlike HIF1α or EZH2, GLI-1 was the sole transcription factor activated by integrin-linked focal adhesion kinase, indicating GLI-1 as a key driver of the EZH2-integrin α11 axis operating for cancer stem cell survival and EMT. Kaplan-Meier survival analysis using The Cancer Genome Atlas (TCGA) dataset also revealed both EZH2 and integrin α11 could be strong prognostic factors of relapse-free and overall survival in breast cancer patients. However, the superior efficacy of integrin α11 siRNA therapy over EZH2 siRNA treatment was demonstrated by enhanced inhibition of tumour growth and prolonged survival in murine models bearing tumours.ConclusionOur findings elucidate that integrin α11 is upregulated by EZH2, forming a positive feedback circuit involving FAK-GLI-1 and contributing to drug resistance, cancer stem cell survival and EMT. Taken together, the results suggest integrin α11 as a promising prognostic marker and a powerful therapeutic target for drug-resistant breast cancer.

Ganoderic acid D attenuates gemcitabine resistance of triple-negative breast cancer cells by inhibiting glycolysis via HIF-1α destabilization

BackgroundGemcitabine (GEM) resistance is the primary reason why combination chemotherapy is limited in triple-negative breast cancer (TNBC). Ganoderic acid D (GAD), a natural triterpenoid compound obtained from Ganoderma lucidum, has been shown to have antitumor activities. However, whether GAD can reverse GEM resistance in TNBC requires further investigation. PurposeThis study investigated whether and how GAD could reverse GEM resistance in TNBC as an antitumor adjuvant. MethodsThe effects of GAD on cell proliferation, cell cycle, and glycolysis were studied in vitro using a GEM-resistant (GEM-R) TNBC cell model. We enriched key pathways affected by GAD using proteomics techniques. Western blotting and qPCR were used to detect the expression of glycolysis-related genes after GAD treatment. A mouse resistance model was established using GEM-R TNBC cells, and hematoxylin-eosin staining and immunohistochemistry were used to assess the role of GAD in reversing resistance in vivo. ResultsCellular functional assays showed that GAD significantly inhibited proliferation and glucose uptake in GEM-R TNBC cells. GAD reduces HIF-1α accumulation in TNBC cells under hypoxic conditions through the ubiquitinated protease degradation pathway. Mechanistically, GAD activates the p53/MDM2 pathway, promoting HIF-1α ubiquitination and proteasomal degradation and downregulating HIF-1α-dependent glycolysis genes like GLUT1, HK2, and PKM2. Notably, GAD combined with gemcitabine significantly reduced the growth of GEM-R TNBC cells in a subcutaneous tumor model. ConclusionsThis study reveals a novel antitumor function of GAD, which inhibits glycolysis by promoting HIF-1α degradation in GEM-R TNBC cells, offering a promising therapeutic strategy for TNBC patients with GEM resistance.

RUVBL1 ubiquitination by DTL promotes RUVBL1/2-β-catenin-mediated transcriptional regulation of NHEJ pathway and enhances radiation resistance in breast cancer

Radiotherapy effectiveness in breast cancer is limited by radioresistance. Nevertheless, the mechanisms behind radioresistance are not yet fully understood. RUVBL1 and RUVBL2, referred to as RUVBL1/2, are crucial AAA+ ATPases that act as co-chaperones and are connected to cancer. Our research revealed that RUVBL1, also known as pontin/TIP49, is excessively expressed in MMTV-PyMT mouse models undergoing radiotherapy, which is considered a murine spontaneous breast-tumor model. Our findings suggest that RUVBL1 enhances DNA damage repair and radioresistance in breast cancer cells both in vitro and in vivo. Mechanistically, we discovered that DTL, also known as CDT2 or DCAF2, which is a substrate adapter protein of CRL4, promotes the ubiquitination of RUVBL1 and facilitates its binding to RUVBL2 and transcription cofactor β-catenin. This interaction, in turn, attenuates its binding to acetyltransferase Tat-interacting protein 60 (TIP60), a comodulator of nuclear receptors. Subsequently, ubiquitinated RUVBL1 promotes the transcriptional regulation of RUVBL1/2-β-catenin on genes associated with the non-homologous end-joining (NHEJ) repair pathway. This process also attenuates TIP60-mediated H4K16 acetylation and the homologous recombination (HR) repair process. Expanding upon the prior study’s discoveries, we exhibited that the ubiquitination of RUVBL1 by DTL advances the interosculation of RUVBL1/2-β-catenin. And, it then regulates the transcription of NHEJ repair pathway protein. Resulting in an elevated resistance of breast cancer cells to radiation therapy. From the aforementioned, it is evident that targeting DTL-RUVBL1/2-β-catenin provides a potential radiosensitization approach when treating breast cancer.

Abstract ND06: First disclosure of AZD8421, a highly selective CDK2 inhibitor to address resistance to CDK4/6 inhibitors in breast and CCNE1-high cancers

Abstract Cyclin-dependent kinases (CDKs) are an attractive class of cancer targets due to their role in cell-cycle regulation. CDK2 is a Ser/Thr kinase activated via interaction with its cyclin partners CCNE and CCNA, driving G1/S progression of the cell cycle. CDK2 inhibition has the potential to address multiple resistance mechanisms to CDK4/6 inhibitors in breast cancer. In addition, CCNE1 is frequently amplified and over-expressed in cancers including ovarian, with pre-clinical data linking CDK2 inhibition sensitivity to high CCNE1. First-generation inhibitors targeting CDK2 suffered from poor tolerability in the clinic likely due to off-target activities across both the CDK family and across the protein kinome. As well as achieving CDK family selectivity in cells versus key off-targets (CDK1, CDK4/6, CDK9), it’s noteworthy that AZD8421 had no significant kinase inhibition outside the CDK family. X-ray co-crystal structures of AZD8421 with CDK2 provided the basis for the observed selectivity among the CDK-family. For example, hydrogen bonding interaction of AZD8421 with Lys89 (a CDK2-specific residue) near the solvent region of the ATP-binding pocket was the key requisite for achieving >300-fold selectivity over CDK9. In nanoBRET assays measuring cellular target engagement, AZD8421 had an IC50 against CDK2 of 9nM with selectivity over CDK1, CDK4 and CDK6. Further kinetic characterization of AZD8421 binding revealed a long residence time at CDK2 (2 hours) but not CDK1 (7 minutes). These kinetic studies showed that AZD8421 has a low Kd,app for CDK2 and thus even higher selectivity vs CDK1 than was measured by single timepoint assays. In a CCNE1 amplified cell line, AZD8421 potently inhibited cell proliferation (69nM, OVCAR3), correlated with inhibition of pRB, arrest in G1/S phase of the cell cycle and induction of senescence. In vitro combination assays in CDK4/6 inhibitor resistant breast cancer cell lines showed combination benefit with AZD8421 plus approved CDK4/6 inhibitors. In vivo, AZD8421 potently suppressed phosphorylation of Rb, and demonstrated robust monotherapy and CDK4/6i combination activity in breast and ovarian in vivo models. In vivo PD marker suppression and efficacy was demonstrated in CDK4/6 inhibitor resistant breast PDXs in combination with palbociclib (CDK4/6i). AZD8421 showed robust monotherapy activity in a CCNE1 amplified ovarian model OVCAR3 with regressions seen with monotherapy and in combination with palbociclib. These data, along with suitable physical and pharmacokinetic properties, supported progression of AZD8421 into clinical studies [NCT06188520]. Citation Format: Christopher R. Denz, Michael Grondine, Jun Fan, Jessie Hao-Ru Hsu, Anne Jackson, James Robinson, Grace Guo, Wen Li, Yanjun Wang, Maryann San Martin, Laura Prickett, Jeffrey Johannes, Avipsa Ghosh, Kun Song, Dhivya Sudhan, Christina Vasalou, Ryan Richards, Kevin Beaumont, Matthew Peters, Lisa Drew, Stephen Fawell, Frederick W. Goldberg. First disclosure of AZD8421, a highly selective CDK2 inhibitor to address resistance to CDK4/6 inhibitors in breast and CCNE1-high cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND06.

Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.

Reversal of drug resistance in breast cancer cells by photodynamic action mediated by curcumin/Ko143/PLGA nanoparticles

Objective: The objective of this study is to prepare Cur/Ko143/PLGA that encapsulate the photosensitizer curcumin (Cur) and the multidrug transporter-specific inhibitor Ko143 to investigate its application in reversing drug resistance in breast cancer and evaluate its properties and therapeutic efficacy. Results: After successful preparation of nanoparticles, the encapsulation rate of Cur/Ko143/PLGA was 73.8% ± 1.8%, and the drug loading rate was 2.8% ± 0.1%. The Cur/Ko143/PLGA were spherical and regular in morphology under transmission electron microscopy. The nanoparticles were morphologically intact, were uniform in size, and did not show significant aggregation under scanning electron microscopy. The average particle size of Cur/Ko143/PLGA was 232.32 ± 10.60 nm, the polymer dispersion index was 0.01, and the zeta potential was −17.25 ± 0.65 mV, as determined by laser particle sizing. In vitro ultrasound observation showed that Cur/Ko143/PLGA were enhanced in both the B-mode and contrast modes. Intracellular Cur fluorescence detection, biocompatibility testing, cell scratch experiment, flow detection of apoptosis, live–dead staining, and actin polymerization test all prove that new nanomaterials have better photodynamic efficacy. Conclusion: Drug-carrying Cur/Ko143/PLGA nanoparticles can increase the concentration of the intracellular photosensitizer Cur and increase the photodynamic efficacy by inhibiting an efflux pump, which is expected to become a new therapeutic strategy.

Blockade of a novel MAP4K4-LATS2-SASH1-YAP1 cascade inhibits tumorigenesis and metastasis in luminal breast cancer

Novel components in the noncanonical Hippo pathway that mediate the growth, metastasis, and drug resistance of breast cancer (BC) cells need to be identified. Here, we showed that SAM and SH3 domain containing protein 1 (SASH1) expression is negatively correlated with mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) expression in a subpopulation of patients with luminal-subtype BC. Downregulated SASH1 and upregulated MAP4K4 synergistically regulated the proliferation, migration, and invasion of luminal-subtype BC cells. The expression of LATS2, SASH1 and YAP1 and the phosphorylation of YAP1 were negatively regulated by MAP4K4, and LATS2 then phosphorylated SASH1 to form a novel MAP4K4-LATS2-SASH1-YAP1 cascade. Dephosphorylation of Yes1 associated transcriptional regulator (YAP1), YAP1/TAZ nuclear translocation and downstream transcriptional regulation of YAP1 were promoted by the combined effects of ectopic MAP4K4 expression and SASH1 silencing. Targeted inhibition of MAP4K4 blocked proliferation, cell migration and ER signaling both in vitro and in vivo. Our findings reveal a novel MAP4K4-LATS2-SASH1-YAP1 phosphorylation cascade, a noncanonical Hippo pathway that mediates ER signaling, tumorigenesis and metastasis in breast cancer. Targeted intervention with this noncanonical Hippo pathway may constitute a novel alternative therapeutic approach for endocrine-resistant BC.

Exosome-Derived MicroRNA-221-3p Desensitizes Breast Cancer Cells to Adriamycin by Regulating PIK3r1-Mediated Glycose Metabolism.

Background: Cancer-derived exosomes facilitate chemoresistance by transferring RNAs, yet their role in exosomal microRNA-221-3p (miR-221-3p) regulation of Adriamycin resistance in breast cancer (BC) remains unclear. Methods: Adriamycin-resistant BC cells were developed from MCF-7 and MDA-MB-231 cells by incremental Adriamycin exposure. The miR-221-3p levels were quantified by quantitative reverse transcription-polymerase chain reaction. Subsequently, exosomes were isolated and incubated with BC cells, and exosome-mediated Adriamycin sensitivity was evaluated using Cell Counting Kit-8, colony formation, and flow cytometry assays. Sensitive cells were cocultured with miR-221-3p inhibitor-treated cells to assess Adriamycin resistance. Moreover, the interaction between miR-221-3p and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was validated using a dual luciferase reporter gene assay. Mimics and inhibitors were used to determine the effects of miR-221-3p on Adriamycin resistance. Results: Elevated levels of miR-221-3p expression were observed in Adriamycin-resistant BC cells and exosomes. Sensitive cells were cocultured with exosomes from resistant cells, resulting in increased half-maximal inhibitory concentration value and proliferation, and reduced Adriamycin-induced apoptosis. However, the effects of coculturing sensitive cells with Adriamycin-resistant cells were significantly weakened by miR-221-3p inhibitor transfection in Adriamycin-resistant cells. PIK3R1 was found to be a target of miR-221-3p, and miR-221-3p mimics enhanced Adriamycin resistance in sensitive cells. miR-221-3p inhibitors increased the expression of PIK3R1, p-AKT, c-Myc, HK2, and PKM2, decreased FOXO3 expression, and weakened the Adriamycin resistance in resistant cells. Conclusions: miR-221-3p can be transferred between BC cells through exosomes. High levels of miR-221-3p were found to target PIK3R1 and promoted Adriamycin resistance in BC cells. [Figure: see text].

Abstract 5730: Targeting the cell cycle with AZD8421, a potent and highly selective CDK2 inhibitor

Abstract Cyclin-dependent Kinase 2 (CDK2) is a Ser/Thr kinase activated via interaction with its cyclin partners CCNE and CCNA, driving G1/S progression of the cell cycle. CDK2 inhibition has the potential to address multiple resistance mechanisms to CDK4/6 inhibitors in breast cancer. In addition, CCNE1 is frequently amplified and over-expressed in cancers including ovarian, uterine, breast, gastric and others, with pre-clinical data linking CDK2 inhibition sensitivity to high CCNE1. The CDK2 inhibitor field has historically suffered from high attrition in Phase I due to off-target toxicity. AZD8421 is a uniquely selective CDK2 inhibitor, that offers potential to show improved therapeutic index and combinability. As well as achieving CDK family selectivity in cells versus key off-targets (CDK1, CDK4/6, CDK9), AZD8421 had no significant kinase inhibition outside the CDK family. Only 7 distinct targets showed greater than 50% inhibition in a panel of 403 kinases when screened with 1µM of AZD8421. In nanoBRET assays measuring cellular target engagement, AZD8421 had an IC50 against CDK2 of 9nM with selectivity over CDK1, CDK4 and CDK6. In vitro cellular assays demonstrated that AZD8421 inhibits endogenous phospho-substrate at 58nM with >327-fold selectivity over CDK9 phospho-substrate pSer2 of RNAPII (>19.2 µM). In a CCNE1 amplified cell line, AZD8421 potently inhibited cell proliferation (69nM, OVCAR3), correlated with inhibition of pRB, arrest in G1/S phase of the cell cycle and induction of senescence, compared to a CCNE1 non-amplified cell line (SKOV3) with IC50 of 2.05 µM. In vitro combination assays in CDK4/6 inhibitor resistant breast cancer cell lines showed combination benefit with AZD8421 plus approved CDK4/6 inhibitors. In vivo, AZD8421 potently suppressed phosphorylation of Rb, and demonstrated robust monotherapy and CDK4/6i combination activity in breast and ovarian in vivo models. In vivo PD marker suppression and efficacy was demonstrated in CDK4/6 inhibitor resistant breast PDXs in combination with palbociclib (CDK4/6i). AZD8421 showed robust monotherapy activity in a CCNE1 amplified ovarian model OVCAR3 with regressions seen with monotherapy and in combination with palbociclib. Monotherapy activity and combination benefit in vivo with standard of care agents was also observed in ovarian PDX models with elevated levels of CCNE1. AZD8421 is a potent and highly selective CDK2 inhibitor, with double digit nM potency in cells, as well as suitable physical and pharmacokinetic properties for progression into the clinic. Citation Format: Christopher R. Denz, Michael Grondine, Jun Fan, Jessie Hao-Ru Hsu, Anne Jackson, James Robinson, Grace Guo, Wen Li, Yanjun Wang, Maryann San Martin, Laura Prickett, Jeffrey Johannes, Avipsa Ghosh, Dhivya Sudhan, Christina Vasalou, Ryan Richards, Kevin Beaumont, Matt Peters, Lisa Drew, Stephen Fawell, Frederick W. Goldberg. Targeting the cell cycle with AZD8421, a potent and highly selective CDK2 inhibitor [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 5730.

Abstract 1985: Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells

Abstract Approximately 75% of breast tumors express estrogen receptors (ER+); recurrent ER+ breast cancer is generally incurable. Endocrine therapy (ET) is the mainstay of treatment for ER+ breast cancer and works by targeting either ER activity (Tamoxifen or Fulvestrant) or availability of its ligand estrogen (Letrozole, Exemestane). Unfortunately, endocrine therapies are limited by the emergence of resistance; resistance to endocrine therapies (ET) remains a significant clinical challenge. We have explored the role of adenylosuccinate lyase (ADSL), an enzyme of the de-novo purine biosynthesis pathway, in ET-resistant breast cancer cells. Protein expression of ADSL was significantly higher in two different ET-resistant cells (LCC9 and T47D-4HT) when compared with their respective parental ET-sensitive cells (MCF-7 and T47D). siRNA mediated ablation of ADSL in LCC9 and T47D-4HT cells blocked cell proliferation and both colony and spheroid formation. Depletion of ADSL in LCC9 cells obstructed cell cycle progression from G1 to S phase and elevated the protein expression of cyclin D1/D3, CDK2/4, and cyclin E. In T47D-4HT cells, ADSL depletion led to an accumulation of cells in S-phase with reduced protein expression of cyclin D1. Notably, ADSL ablation in LCC9 cells lead to DNA replication stress, resulting in activation (phosphorylation) of ataxia-telangiectasia-mutated-and-Rad3-related kinase (ATR) and its key downstream effector checkpoint kinase 1 (Chk1), which further limited the phosphatase activity of cdc25A leading to deactivation (phosphorylation) of Cyclin-Dependent Kinase 2 (CDK2). ADSL knockdown reduced the mitochondrial membrane potential and impaired the oxygen consumption (OCR) and extracellular acidification (ECAR) rates in both LCC9 and T47D-4HT cells. Of note, addition of AICAR to ADSL-depleted LCC9 cells significantly increased cell proliferation, colony formation, and restored mitochondrial membrane potential. RNA sequencing data revealed the effect of ADSL depletion on enrichment of different pathways in LCC9 and TH47D-4HT cells. Importantly, p53 signaling was significantly enriched in LCC9 cells but not in T47D-4HT cells after ADSL knockdown (T47D cells have a mutated p53 gene). ER+ breast cancer patients treated with ET showed high ADSL expression that is associated with poor recurrence-free survival. In summary, our findings indicate that ADSL-mediated de novo purine synthesis is crucial for cell proliferation. Therefore, targeting ADSL may be a novel therapeutic approach for ER, ET-resistant breast cancer. Citation Format: ANIL KUMAR Yadav, Lu Jin, Robert Clarke, Surojeet Sengupta. Ablation of adenylosuccinate lyase retarded the cell proliferation and induces mitochondrial dysfunction in endocrine therapy resistant breast cancer cells [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 1985.

Abstract 3287: Learning to target CDK4/6 inhibitor resistance via a breast cancer-specific atlas of cellular mechanisms

Abstract Development of resistance to targeted, chemotherapeutic, and immune-oncology treatments alike is a major barrier to long-term remission in advanced cancer patients. We developed an integrated computational-experimental platform to mitigate treatment resistance by identifying therapeutics that selectively target clinical resistance mechanisms. The key innovation is the computational framework that comprehensively maps tumor-specific mechanisms of response and resistance to a treatment based on clinicogenomic and/or preclinical pharmacogenomic data. It then screens for drugs or targets that are synthetically lethal with these resistance mechanisms. Here we present the identification of therapeutic options that mitigate resistance to CDK4/6 inhibitors as a case study. Leveraging a breast cancer-specific atlas of cellular architecture, our framework learned genetic mechanisms of CDK4/6 inhibitor resistance from a high-throughput phenotypic screen of ~700 cell lines treated with the clinical CDK4/6 inhibitor palbociclib. The learned mechanisms formed a parsimonious set of hierarchically linked protein complexes that coordinate G1-to-S transition (P=5.6 × 10−12). The mechanisms unified distinct resistant populations such as samples resistant due to CDK4/6-Rb checkpoint bypass and those resistant due to growth factor receptor activation. Applied to 70 ER+ metastatic breast cancer patients in a real-world dataset, the learned resistance mechanisms accurately predicted patient response to palbociclib (P=3 × 10−4, log-rank test). Computationally screening 20K clinical-grade small molecules and gene targets for therapeutic options synthetically lethal with the learned resistance mechanisms identified the known CDK4/6 resistance targets CDK2, CCNE1, and E2F. It also identified a target that, to our knowledge, has not previously been rigorously evaluated for its potential to mitigate CDK4/6 inhibitor resistance. In-vitro, monotherapy inhibition of the target by siRNA selectively decreased growth of breast cancer cell lines resistant to CDK4/6 inhibitors by 52% compared to CDK4/6i sensitive cell lines (P=0.023, Mann-Whitney U-test). As a combination therapy, inhibition of the target by siRNA selectively increased the sensitivity to palbociclib by nearly an order of magnitude in an otherwise resistant breast cancer cell line (expected vs observed IC50: 9.7 uM vs. 1.3 uM) while not having a substantial effect in a palbociclib-sensitive cell line (expected vs observed IC50: 0.57 uM vs. 0.56 uM). Finally, while monotherapy inhibition of the target and CDK4/6 induced cell cycle arrest, the combination strongly induced apoptosis in cell lines resistant to CDK4/6 inhibitors. These results highlight how computational deconvolution of tumor resistance mechanisms enable algorithmic identification of therapeutic options to target treatment-resistant cell populations. Citation Format: Adam Yaari, Eduardo Farias, Francisco Guedes, Oliver Priebe, Lee McDaniel, Tyler Earnest, Trey Ideker, Maxwell A. Sherman. Learning to target CDK4/6 inhibitor resistance via a breast cancer-specific atlas of cellular mechanisms [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 3287.

Abstract 1670: Mechanistic insight into mTOR signaling identifies RNA splicing machinery as a potential therapeutic target for cancer

Abstract Aberrant signal transduction regulating cell growth and proliferation is a hallmark of many cancers. Activation of mTOR signaling is common and is an attractive therapeutic target with mechanistically different mTOR inhibitors in clinical use. One consequence of exposure to the mTOR inhibitor rapamycin is an alteration of gene expression through a global increase in alternative exon inclusion (Cheng et al. 2022, doi.org/10.3390/ijms232012416). To examine the mechanisms underlying this effect, we utilized the TempO-Seq targeted sequencing platform to profile both the whole transcriptome and a selection of alternatively spliced RNA targets in the rapamycin sensitive and resistant breast cancer cell lines MCF-7 and MDA MB 231 following exposure to 13 mTOR inhibitors, some closely related and some from distinct chemical classes. Recent publications have demonstrated that high quality dose response data can be obtained using this sequencing platform (Harrill et al. 2021, doi: 10.1093/toxsci/kfab009; Ramaiahgari et al. 2019, doi: 10.1093/toxsci/kfz065) and TempO-Seq has been widely adopted in toxicology for deriving compound-specific expression signatures to identify mechanisms of action and long-term toxicity. Treated cells were also profiled using TempO-LINC, a novel high-throughput single-cell assay based on the same platform. We observed that certain mTOR inhibitors induced both common and cell-specific differential expression as well as widespread alternative exon inclusion, as expected. Intriguingly, transcripts with the most significant changes in splicing upon treatment were those encoding members of the SR family of splicing factors, often overexpressed or amplified in breast cancers (Park, et al. 2019, doi: 10.1016/j.celrep.2019.10.110). This is significant because SR protein expression is autoregulated by alternative inclusion of an exon containing a premature stop codon in a feedback loop leading to nonsense-mediated decay (NMD) and lowered protein levels. Indeed, NMD is known to be inhibited by rapamycin (Martinez-Nunez, et al. 2017, doi: 10.1093/nar/gkw1109), which would lead to the observed accumulation of these transcripts. Thus, monitoring a targeted selection of alternative splicing events and regulators revealed mTOR regulation of mRNA isoform abundance consistent with regulation of the NMD RNA turnover process and with overexpression of SR proteins in breast cancers. Importantly, targeted bulk and single-cell TempO-Seq assays enable efficient sequencing and simplified data analysis without the expense and bioinformatic burden of RNAseq, making them particularly well-suited for drug discovery. These results support the use of this approach for further characterization of cell-specific effects of mTOR inhibitors and their mechanisms of action. Citation Format: Gaelle Rondeau, Megan Opichka, Kevin White, Joanne M. Yeakley, Dennis J. Eastburn, Bruce Seligmann. Mechanistic insight into mTOR signaling identifies RNA splicing machinery as a potential therapeutic target for cancer [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 1670.