Palbociclib-resistant Cells Research Articles

Abstract PO3-28-12: Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance

Abstract Standard of care for breast cancer (BC) treatment including Selective-Estrogen Receptor (ER)-modulators and/or degraders (SERM; tamoxifen, SERD;Fulvestrant) or cyclin-dependent kinases CDK4 and CDK6 (palbociclib) can be effective therapeutic strategies. However, drug toxicity and/or drug resistance severely limit these approaches and patients succumb to the disease. Therefore, there is an urge to develop new therapeutic strategies against ER+-BC. Ion channels are general pharmacological targets however, little is known about the role of this class of proteins in cancer biology. We discovered that expression of the KCa3.1 potassium ion channel is downregulated in ER+-BC. Also, patients expressing high level of KCa3.1 present a better overall survival (OS) and relapse free survival when compared with patients with low expression. Thus, these data indicate that KCa3.1 is a prognostic factor in ER+-BC and that high activity of this channel plays a fundamental role in an oncosuppressor signature. Our central hypothesis is that pharmacological targeting KCa3.1 with activator molecules arrests growth of ER+-BC. We discovered that ER+-BC patient-derived organoids (PDO) and cell lines treated with KCa3.1 agonists including the FDA approved Chlorzoxazone, (CZX) arrests significantly inhibit tumor growth. Activation of KCa3.1 arrested the cell cycle in the G0/G1 phase by activating a senescent-like phenotype. Furthermore, our studies on the biochemical signaling that underlines this event demonstrated that increased expression level of KCa3.1 protein associated with lower expression level of ERa in a cell cycle phase-dependent manner. Furthermore, use of KCa3.1activator produced a proteasomal-dependent ERa degradation. To better characterize the therapeutic benefit of targeting the KCa3.1, we assessed the effects of CZX on developed several tamoxifen and/or palbociclib resistant cell lines and PDO models. We found that the combination of CZX strongly potentiated the lethal effects of both tamoxifen and palbociclib in the relative resistant models. We conclude that pharmacological targeting KCa3.1 potassium channel in ER+-BC can be considered a novel, safe and efficacious therapeutic strategy. Citation Format: Davide Delisi, Metin Cetin, Ozge Saatci, Ozgur Sahin, Saverio Gentile. Targeting the KCa3.1 potassium channel arrests ER+ breast cancer growth and re-sensitizes to drug resistance [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 PO3-28-12.

A deep learning model of tumor cell architecture elucidates response and resistance to CDK4/6 inhibitors

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6is) have revolutionized breast cancer therapy. However, <50% of patients have an objective response, and nearly all patients develop resistance during therapy. To elucidate the underlying mechanisms, we constructed an interpretable deep learning model of the response to palbociclib, a CDK4/6i, based on a reference map of multiprotein assemblies in cancer. The model identifies eight core assemblies that integrate rare and common alterations across 90 genes to stratify palbociclib-sensitive versus palbociclib-resistant cell lines. Predictions translate to patients and patient-derived xenografts, whereas single-gene biomarkers do not. Most predictive assemblies can be shown by CRISPR–Cas9 genetic disruption to regulate the CDK4/6i response. Validated assemblies relate to cell-cycle control, growth factor signaling and a histone regulatory complex that we show promotes S-phase entry through the activation of the histone modifiers KAT6A and TBL1XR1 and the transcription factor RUNX1. This study enables an integrated assessment of how a tumor’s genetic profile modulates CDK4/6i resistance.

Quercetin inhibits the epithelial-mesenchymal transition and reverses CDK4/6 inhibitor resistance in breast cancer by regulating circHIAT1/miR-19a-3p/CADM2 axis.

Breast cancer (BC) cells have a high risk of metastasis due to epithelial-mesenchymal transition (EMT). Palbociclib (CDK4/6 inhibitor) is an approved drug for BC treatment. However, the drug resistance and metastasis can impair the treatment outcome of Palbociclib. Understanding the mechanisms of EMT and Palbociclib drug resistance in BC is conducive to the formulation of novel therapeutic strategy. Here, we investigated the role of circHIAT1/miR-19a-3p/CADM2 axis in modulating EMT and Palbociclib resistance in BC. circHIAT1 and CADM2 were down-regulated in BC tissues and cell lines, and miR-19a-3p showed an up-regulation. circHIAT1 could interact with miR-19a-3p and suppress its activity, while miR-19a-3p functioned to negatively regulate CADM2. Forced over-expression of circHIAT1 could impaired the EMT status and migratory ability of BC cells, and this effect was inhibited by miR-19a-3p mimic. In addition, we also generated Palbociclib resistant BC cells, and showed that circHIAT1 and CADM2 were down-regulated in the resistant BC cells while miR-19a-3p showed an up-regulation. Forced circHIAT1 over-expression re-sensitized BC cells to Palbociclib treatment. Quercetin, a bioactive flavonoid, could suppressed the migration and invasion of BC cells, and re-sensitized BC cells to Palbociclib. The anti-cancer effect of quercetin could be attributed to its regulatory effect on circHIAT1/miR-19a-3p/CADM2 axis. In vivo tumorigenesis experiment further revealed that quercetin administration enhanced the anti-cancer effect of Palbociclib, an effect was dependent on the up-regulation of circHIAT1 by quercetin. In summary, this study identified quercetin as a potential anti-cancer compound to reverse Palbociclib resistance and impair EMT in BC cells by targeting circHIAT1/miR-19a-3p/CADM2 axis.

Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer

BackgroundBreast cancer is the global leading cancer burden in women and the hormone receptor-positive (HR+) subtype is a major part of breast cancer. Though cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are highly effective therapy for HR+ subtype, acquired resistance is inevitable in most cases. Herein, we investigated the paternally expressed gene 10 (PEG10)-associated mechanism of acquired resistance to CDK4/6 inhibitors.MethodsPalbociclib-resistant cells were generated by exposing human HR+ breast cancer cell lines to palbociclib for 7–9 months. In vitro mechanistic study and in vivo xenograft assay were performed. For clinical relevance, public mRNA microarray data sets of early breast cancer were analyzed and PEG10 immunohistochemical staining was performed using pre-CDK4/6 inhibitor tumor samples.ResultsWe observed that PEG10 was significantly upregulated in palbociclib-resistant cells. Ectopic overexpression of PEG10 in parental cells caused CDK4/6 inhibitor resistance and enhanced epithelial–mesenchymal transition (EMT). On the contrary, PEG10-targeting siRNA or antisense oligonucleotides (ASOs) combined with palbociclib synergistically inhibited proliferation of palbociclib-resistant cells and growth of palbociclib-resistant xenograft in mice and suppressed EMT as well. The mechanistic study confirmed that high PEG10 expression suppressed p21, a natural CDK inhibitor, and SIAH1, a post-translational degrader of ZEB1, augmenting CDK4/6 inhibitor resistance. Then PEG10 siRNA combined with palbociclib suppressed cell cycle progression and EMT via activating p21 and SIAH1, respectively. Consequently, combined PEG10 inhibition and palbociclib overcame CDK4/6 inhibitor resistance. Furthermore, high PEG10 expression was significantly associated with a shorter recurrence-free survival (RFS) based on public mRNA expression data. In pre-CDK4/6 inhibitor treatment tissues, PEG10 positivity by IHC also showed a trend toward a shorter progression-free survival (PFS) with CDK4/6 inhibitor. These results support clinical relevance of PEG10 as a therapeutic target.ConclusionsWe demonstrated a novel PEG10-associated mechanism of CDK4/6 inhibitor resistance. We propose PEG10 as a promising therapeutic target for overcoming PEG10-associated resistance to CDK4/6 inhibitors.

Combining the AKT inhibitor capivasertib and SERD fulvestrant is effective in palbociclib-resistant ER+ breast cancer preclinical models

Combining the selective AKT inhibitor, capivasertib, and SERD, fulvestrant improved PFS in a Phase III clinical trial (CAPItello-291), treating HR+ breast cancer patients following aromatase inhibitors, with or without CDK4/6 inhibitors. However, clinical data suggests CDK4/6 treatment may reduce response to subsequent monotherapy endocrine treatment. To support understanding of trials such as CAPItello-291 and gain insight into this emerging population of patients, we explored how CDK4/6 inhibitor treatment influences ER+ breast tumour cell function and response to fulvestrant and capivasertib after CDK4/6 inhibitor treatment. In RB+, RB− T47D and MCF7 palbociclib-resistant cells ER pathway ER and Greb-1 expression were reduced versus naïve cells. PI3K-AKT pathway activation was also modified in RB+ cells, with capivasertib less effective at reducing pS6 in RB+ cells compared to parental cells. Expression profiling of parental versus palbociclib-resistant cells confirmed capivasertib, fulvestrant and the combination differentially impacted gene expression modulation in resistant cells, with different responses seen in T47D and MCF7 cells. Fulvestrant inhibition of ER-dependent genes was reduced. In resistant cells, the combination was less effective at reducing cell cycle genes, but a consistent reduction in cell fraction in S-phase was observed in naïve and resistant cells. Despite modified signalling responses, both RB+ and RB− resistant cells responded to combination treatment despite some reduction in relative efficacy and was effective in vivo in palbociclib-resistant PDX models. Collectively these findings demonstrate that simultaneous inhibition of AKT and ER signalling can be effective in models representing palbociclib resistance despite changes in pathway dependency.

Oxidative phosphorylation is a metabolic vulnerability of endocrine therapy and palbociclib resistant metastatic breast cancers

Resistance to endocrine treatments and CDK4/6 inhibitors is considered a near-inevitability in most patients with estrogen receptor positive breast cancers (ER + BC). By genomic and metabolomics analyses of patients’ tumours, metastasis-derived patient-derived xenografts (PDX) and isogenic cell lines we demonstrate that a fraction of metastatic ER + BC is highly reliant on oxidative phosphorylation (OXPHOS). Treatment by the OXPHOS inhibitor IACS-010759 strongly inhibits tumour growth in multiple endocrine and palbociclib resistant PDX. Mutations in the PIK3CA/AKT1 genes are significantly associated with response to IACS-010759. At the metabolic level, in vivo response to IACS-010759 is associated with decreased levels of metabolites of the glutathione, glycogen and pentose phosphate pathways in treated tumours. In vitro, endocrine and palbociclib resistant cells show increased OXPHOS dependency and increased ROS levels upon IACS-010759 treatment. Finally, in ER + BC patients, high expression of OXPHOS associated genes predict poor prognosis. In conclusion, these results identify OXPHOS as a promising target for treatment resistant ER + BC patients.

Co-targeting RANK pathway treats and prevents acquired resistance to CDK4/6 inhibitors in luminal breast cancer

The combination of endocrine therapy (ET) and cyclin-dependent kinase 4/6 (CDK4/6) inhibitors (CDK4/6i) was a hallmark in metastatic luminal breast cancer (BC). However, intrinsic and acquired resistance affects long-term efficacy. Here, we study the role of the receptor activator of nuclear factor-κB (RANK) pathway in CDK4/6i resistance. We find that RANK overexpression in luminal BC is associated with intrinsic resistance to CDK4/6i, both invitro and in mouse xenografts, and decreased proliferation rate and chronic interferon (IFN) γ response are highlighted as resistance drivers. Gene expression data from the NeoPalAna CDK4/6i clinical trial, and studies with palbociclib-resistant cell lines, show that RANK is upregulated after treatment with CDK4/6i, supporting a role in acquired resistance. Our study shows that RANK ligand (RANKL) inhibitors can restore sensitivity to CDK4/6i and prevent acquired resistance. On the basis of these findings, we conclude that pharmacological inhibition of the RANK pathway through RANKL blocking could represent an add-on to ET+ CDK4/6i, warranting further clinical studies.

Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer.

e13077 Background: The past decade has seen significant advancement in increasing survival estrogen receptor alpha (ERα) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) and cyclin-dependent kinase inhibitors aided to extend overall survival of breast cancer patients. However, resistance to endocrine therapy is a common occurrence and patients will eventually succumb to their disease. Fatty acid synthase (FASN), a key enzyme in lipid biosynthesis, is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease (≥16 weeks). Methods: We generated therapy-resistant cells by long term exposure to tamoxifen (MCF7/TamR cells), and fulvestrant (MCF7/FR cells). Palbociclib-resistant (MCF7/RB1Crispr and ZR75/RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor in these cells on proliferation, viability, cell cycle, apoptosis, ERα expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate gene expression. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blot after treatment with TVB with or without the addition of palmitate or BAPTA. Results: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERα positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress. Conclusions: Our preclinical data provide evidence that FASN inhibition presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.

Abstract 5989: Targeting RRM2 &amp; cell cycle for breast cancer treatment

Abstract Endocrine treatment is an effective first-line therapy for targeting ER positive, HER2 negative breast cancer. But success is limited by development of acquired resistance due to long-term therapy. Cyclin D1 and cyclin dependent kinase 4/6 (CDK4/6) complex causes phosphorylation and subsequent inactivation of retinoblastoma (Rb) tumor suppressor protein which promotes progression of the cell cycle from G1 to S phase. This observation led the development of the first CDK4/6 inhibitor palbociclib (Ibrance; Pfizer) which induces cell cycle arrest at G1 phase in cancer cells. Owing to intrinsic and acquired drug resistance development, success is limited despite promising clinical outcomes. This situation necessitates the development of potential combination strategies to overcome drug resistance. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. Didox inhibits ribonucleotide reductase subunit 2 (RRM2) which ultimately blocks DNA synthesis. Combination of didox with palbociclib is a potential strategy to target ER positive and ER negative/triple negative breast cancer. Previously, we have shown that didox (DDX) can significantly halt malignant breast cancer cell division in combination with the anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. Recently, we confirmed that didox in combination with palbociclib significantly lowers ER positive and ER negative breast cancer and their palbociclib resistant counterparts’ growth compared to no treatment or palbociclib treatment. We also confirmed that ER positive MCF7 and ER negative MDA-MB-468 parental breast cancer cells IC50 of palbociclib drug is lower than their palbociclib resistant counterparts. Here, we are reporting that didox decreases cell cycle proteins alone or in combination with palbociclib in ER positive MCF7 and ER negative MDA-MB-468 parental and palbociclib resistant breast cancer cells. This finding opens a novel approach for targeting both ER positive as well as ER negative breast cancer treatment. We are also reporting that didox treatment modulates cyclin D1 (CCND1) and RRM2 expression in MCF7 and MDA-MB-468 breast cancer along with their palbociclib resistant counterparts. Additionally, we observed that didox alone or in combination with palbociclib alters the cell cycle of MCF7 and MDA-MB-468 parental and palbociclib resistant breast cancer cells. Our data presents a novel and promising approach for the treatment of ER positive and ER negative breast cancer that involves inhibition of RRM2 and cell cycle that merits further clinical investigation in animal and human models. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 &amp; cell cycle for breast cancer treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5989.

Abstract 5979: TY-0540, a highly potent CDK2/4/6 inhibitor, attenuates acquired resistance against CDK4/6 inhibition

Abstract The introduction of cyclin-dependent kinase (CDK) 4 and 6 dual inhibitors significantly improves the progression-free survival of patients with ER+/HER2- advanced or metastatic breast cancer. A large cohort of patients, however, eventually relapse to CDK4/6 therapy. To further empower the CDK therapy, simultaneous targeting of CDK2, CDK4, and CDK6 has been proposed as a new strategy based on the finding that abnormal activation of CDK2/CyclinE1 due to CCNE1 gene amplification is determined the key resistant mechanism to CDK4/6 inhibition. Here we present preclinical data of TY-0540, a novel CDK2/4/6 inhibitor for the treatment of breast cancer that is resistant to CDK4/6 inhibition. TY-0540 demonstrates high selectivity against CDK2, CDK4, and CDK6 compared to that of CDK1, CDK7, and CDK9 in a CDK panel screening. In vitro cell proliferation data shows that tumor cell lines OVCAR3 and HCC1806, both bearing CCNE1 amplification, are highly sensitive to TY-0540 treatment. To test the effectiveness of the clinical candidate compound on CDK4/6i resistance models, two Palbociclib resistant cell populations (T47D-R, HCC1428-R) were in-house generated via gradient exposure of the cells to Palbociclib. As expected, TY-0540 potently inhibits T47D-R and HCC1428-R cell proliferation whereas Palbociclib only confers mild interruption to cell proliferation. TY-0540 abolishes Rb phosphorylation at all its three phosphorylation sites and down-regulates E2F1, FOXM1, and c-Myc expression levels in the model cell line OVCAR3. Meanwhile, cell cycle analysis suggests the occurrence of strong G1 arrest at 24 hours after TY-0540 treatment. Consistent with the in vitro results, TY-0540 treatment confers extraordinary in vivo efficacy with a spectrum of tumor CDX mouse models and PDX models in mice. To examine the in vivo effectiveness of TY-0540 over resistance models to CDK4/6 inhibition, we developed Palbociclib-resistant MCF7 tumor model (Palbociclib-R-MCF7) through a combination of in vitro and in vivo evolution of the cells under the selection pressure of Palbociclib. In agreement with its mode of action, TY-0540 is able to suppress Palbociclib-R-MCF7 tumor growth and maintain tumor size at stable disease status. Taken together, we have identified a potent CDK2/4/6 inhibitor which may grant new therapeutic opportunities for cancer patients who relapse or refractory to CDK4/6 signaling blockage therapy. #Meihua Li and Chengshan Niu contributed equally to this work. *Jun Li, Meihua Li and Chengshan Niu are the correspondent authors. Citation Format: Meihua Li, Chengshan Niu, Mingtao Chen, Kaige Ji, Hui Xu, Shengli Dong, Yan Zhang, Qinguo Meng, Yuge Dou, Yijun Wang, Rui Wu, Yian Tu, Chao Zhou, Apeng Liang, Huan Wang, Rongzhen Ni, Aishen Gong, Hui Su, Mingyu Jiang, Feng Xing, Shaoqing Chen, Xiugui Chen, Jun Li, Yusheng Wu. TY-0540, a highly potent CDK2/4/6 inhibitor, attenuates acquired resistance against CDK4/6 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5979.

Abstract P3-11-07: Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer

Abstract INTRODUCTION: The past decade has seen significant advancement in increasing survival in estrogen receptor alpha (ERα) positive breast cancer. The use of selective estrogen receptor down-regulators and modulators (SERMs) (e.g. fulvestrant and tamoxifen), mTOR inhibitors (e.g. everolimus), aromatase inhibitors (AIs), and cyclin-dependent kinase inhibitors (e.g. palbociclib, ribociclib, and abemaciclib) have helped to extend overall survival of breast cancer patients. Unfortunately, resistance to endocrine therapy is a common occurrence and all patients will eventually succumb to their disease. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and is overexpressed in more aggressive and therapy-resistant tumors, including breast cancers. FASN inhibitor, TVB-2640, has been evaluated in multiple tumor cell lines and in a phase 1 clinical study, and showed partial responses in 5 patients and multiple patients with prolonged stable disease (≥16 weeks). METHODS: We generated tamoxifen- and fulvestrant-resistant MCF7 cells by long term exposure to tamoxifen (MCF7/TamR cells) and fulvestrant (MCF7/FR cells), and palbociclib-resistant (MCF7/RB1Crispr and ZR75/RB1Crispr) cells were generated through CRISPR/Cas9 knockout of the retinoblastoma (RB) gene. We assessed the impact of TVB-3166 inhibitor (and analog of TVB-2640 with slightly lower molecular weight for in vitro use) on proliferation, viability, cell cycle, and apoptosis in these cells. We evaluated the impact of TVB on proliferation and ERα expression in patient derived explants, and tumor growth in xenografts. RNA sequencing of tamoxifen- and fulvestrant-resistant cells was performed to investigate alterations in gene expression. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB with or without the addition of palmitate or BAPTA. RESULTS: TVB treatment leads to a marked inhibition of proliferation in tamoxifen-, fulvestrant- and palbociclib-resistant cells compared to the parental cells. RNA sequencing of explants of patients with ERα positive disease showed down regulation of ESR1 related genes and genes involved in invasiveness. RNA sequencing of fulvestrant-resistant cells showed that treatment with TVB results in down regulation of EMT and E2F target genes, cholesterol homeostasis genes and mTORC1 signaling. Additionally, TVB significantly inhibited tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB treatment is mediated through the induction of endoplasmic reticulum stress in tamoxifen-resistant cells. CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered. Citation Format: Henriette Balinda. Inhibition of FASN as a potential treatment of advanced endocrine therapy-resistant breast cancer [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 P3-11-07.

Abstract P1-13-01: Targeting RRM2 for the treatment of palbociclib resistant breast cancer

Abstract Hormone receptor (HR) positive breast cancer (BC) is a prevalent disease accounting for almost 2 million new cases globally. Almost 70-80% of breast cancer patients are women with a positive score for the estrogen receptor (ER). HR positive BCs of all stages are selectively treated with endocrine therapy targeting ER activity. Intrinsic and acquired resistance are common phenomena, impacting patient outcomes negatively. Palbociclib is an FDA approved checkpoint inhibitor for the treatment of HR+/HER2- breast cancer in combination with aromatase inhibitors or selective estrogen receptor degraders. However, patients responding well to the therapy in the beginning, lose sensitivity to palbociclib with time. Ribonucleotide reductase (RR) is a rate limiting enzyme in DNA synthesis consisting of two subunits RRM1 and RRM2. In our previous study, we have shown that RRM2 is upregulated in ER positive tamoxifen resistant BC. Previously, we have also reported that didox (DDX) which is a unique RRM2 enzyme inhibitor can significantly halt malignant breast cancer cell division in combination with an anthracycline drug doxorubicin by targeting RRM2, mutant p53 and NFkB regulatory proteins. In this study, we target palbociclib resistant BC with DDX to circumvent palbociclib resistance. For this purpose, we have developed palbociclib resistant ER positive MCF7 and ER negative MDA-MB-468 breast cancer cell lines. Here, we report morphological changes in parental as compared to palbociclib resistant cells after treatment with palbociclib alone, DDX alone or with palbociclib as compared to non-treated cells. Cell proliferation studies confirm the synergistic combinatorial effect of palbociclib and DDX compared to palbociclib or DDX alone. We also report that DDX alone or in combination with palbociclib downregulates cell cycle and NFkB proteins in palbociclib resistant ER positive MCF7 and ER negative MBA-MB-468 breast cancer cell lines. Citation Format: Nahid Sultana, Howard L. Elford, Jesika S. Faridi. Targeting RRM2 for the treatment of palbociclib resistant breast cancer [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 P1-13-01.

Abstract P1-13-21: Loss of CDKN2B expression as a potential marker of resistance to CDK4/6 inhibitor in Luminal Breast Cancer cells

Abstract Background Cyclin-Dependent Kinase (CDK) 4/6 inhibitors have significantly improved progression-free survival of Hormone Receptor positive (HR+), Human Epidermal Growth Factor Receptor type 2 negative (HER2-) luminal breast cancers (LBC). Several studies demonstrated that the addition of CDK4/6 inhibitors to endocrine therapy results in a significant prolongation of progression-free survival in patients with endocrine-sensitive or endocrine-resistant LBCs. However, the percentage of patients unresponsive or refractory to these therapies is as high as 40%, and no reliable and reproducible biomarkers able to select a priori responder or resistant patients have been validated till now. The main cause of resistance is the selection of mutant clones in the target oncoprotein. Other mechanisms, like oncogene amplification/overexpression or mutations in other pathways, have been described in several models. Here, we focused on palbociclib, a selective inhibitor of CDK4/6. Methods: We generated and characterized human luminal breast cancer MCF-7 and T47D derived cell lines, able to survive and proliferate at different palbociclib concentrations, which also shows cross-resistance to abemaciclib. The resistant MCF7 cell line was characterized by RNA sequencing. Results: To confirm resistance, we performed cell viability assays in MCF-7 and T47D palbociclib sensitive cells (MCF-7pS and T47pS) versus MCF-7 and T47D palbociclib resistant cells (MCF-7pR5), showing a 10-fold increase of IC50 in MCF-7pR5 compared to parental MCF-7pS cells (16.7 vs 1.8 µM) and a 3-fold increase of IC50 in T47DpR5 vs parental T47DpS. We also confirmed a significant cross resistance using abemaciclib in MCF-7pR5 with an IC50 equal to 6.8 vs 0.35 µM and in T47DpR with an IC50 of 10.72 vs 0.5 µM. RNA sequencing, qRT-qPCR and Western blot results demonstrated a dramatic downregulation of the CDK4 inhibitor CDKN2B in both cell lines and we found upregulation of an miR-31, a putative regulator of CDKN2B. This finding was further validated in a biopsy from a patient progressing on CDK4/6 inhibitor therapy. Conclusions: This study provides new relevant information regarding the mechanism of resistance to CDK4/6 inhibitors and suggests potential new markers to follow up patients during the treatment. Citation Format: Nicoletta Cordani, Luca Mologni, Rocco Piazza, Viola Cogliati, Francesca Pepe, Serena Capici, Camillo Di Bella, Marta Jaconi, Maria Grazia Cerrito, Matteo Villa, Pietro Tettamanti, Guido Cavaletti, Marialuisa Lavitrano, Marina Elena Cazzaniga. Loss of CDKN2B expression as a potential marker of resistance to CDK4/6 inhibitor in Luminal 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 P1-13-21.

Abstract PD10-04: PD10-04 Combination of the next generation oral SERD camizestrant (AZD9833) with CDK4/6 and mTOR/AKT inhibitors delivers robust efficacy in a broad range of ER+ breast tumors

Abstract Next-generation oral selective estrogen receptor degraders (ngSERDs) are aiming to become the backbone endocrine therapy (ET) for patients with estrogen receptor positive (ER+) breast cancer (BC) by achieving greater ER signaling blockade than current therapies and tackling key mechanisms of resistance. Camizestrant (AZD9833) is an ngSERD for the treatment of ER+ BC which has demonstrated selective ERα degradation, pure ER antagonism and significant anti-tumor activity in ESR1 wild-type (ESR1wt) and mutant (ESR1m) tumors, as well as encouraging clinical activity in early phase trials. ER+ BC is responsive to therapies targeting ER and CDK4/6 signaling in the adjuvant and metastatic settings. To explore camizestrant combination potential as a backbone ET, camizestrant was partnered with either palbociclib or abemaciclib in CDK4/6 inhibitor (CDK4/6i) naïve and resistant in vitro and in vivo models. Combination benefit was observed in vitro in 3 parental ER+ BC cell lines, moreover camizestrant plus abemaciclib showed activity in palbociclib-resistant cell lines including lines harboring CCNE1amp and RB1 loss. In vivo, combination of camizestrant and either CDK4/6i was well tolerated with the combinations promoting improved efficacy in ESR1wt and ESR1m PDX tumor models compared to monotherapy arms. ER+ BC has a high prevalence of alterations in the PI3K/AKT/PTEN pathway which provides opportunities for treatment with PI3K/AKT pathway inhibitors. Camizestrant delivered enhanced efficacy when combined with mTORC1 inhibitor everolimus and AKT inhibitor capivasertib in CTC-174, an ESR1m and PI3KCAm tumor model. Additionally, combination with capivasertib was more efficacious than single treatments, at clinically relevant dose and schedules, in both PI3Kwt and mutated pathway in PDX models. These data demonstrate the interplay between PI3K/AKT/PTEN pathway inhibition and camizestrant mechanism of action. Finally, we explored a potential triple combination of camizestrant, capivasertib and palbociclib in Palbociclib-resistant models representative of PI3KCA/AKT/PTEN wt and altered tumors. This strategy delivered robust efficacy across these models comparing to single treatments and double combinations. The triplet led to durable regressions at clinically achievable doses of all compounds, irrespective of genetic background and aligned to biomarker modulation of the three signaling axis. These preclinical data demonstrate the potential of camizestrant to become the backbone ET, with high combinability in vivo with inhibitors of CDK4/6, mTOR and AKT. These combinations demonstrate the opportunity to impact care of patients with early and metastatic ER+BC, delivering benefit to broad patient populations including those with ESR1wt or ESR1m tumors, independent of PI3K/AKT/PTEN pathway mutation status, and in patients with both CDK4/6i sensitive and resistant tumors. Citation Format: Larissa Carnevalli, Susana Ros, Jelena Urosevic, Natalie Cureton, Sophie Darcy, Mandy Lawson, Stuart Williamson, Pablo Morentin Gutierrez, Azadeh Bashi, Jennifer Moss, Christopher Morrow, Barry Simon, Teresa Klinowska. PD10-04 Combination of the next generation oral SERD camizestrant (AZD9833) with CDK4/6 and mTOR/AKT inhibitors delivers robust efficacy in a broad range of ER+ breast tumors. [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 PD10-04.

Abstract P4-09-11: Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors

Abstract Background CDK4/6 inhibitors are the latest and highly specific CDK inhibitors approved for metastatic and early estrogen receptor (ER)-positive breast cancer patients, improving the survival outcomes. Prior preclinical research has indicated various aberrations of cell cycle regulation and activated oncogenic signaling pathway as the mechanisms of resistance to CDK4/6 inhibitors. However, there is currently no available biomarker except ER and human epidermal growth factor receptor 2 (HER2) in clinical practice. The aim of our study is to elucidate diverse mechanisms of acquired resistance of ER-positive breast cancers to CDK4/6 inhibitors and develop personalized medicine in ER-positive breast cancer patients. Materials and methods To generate resistant lines, the ER-positive human breast cancer cell lines MCF7 and T47D were treated individually with increasing concentrations of the CDK4/6 inhibitors palbociclib and ribociclib until a target concentration of 3μM for six months. Cells were maintained in RPMI 1640 containing 10% fetal bovine serum, 100 U/ml penicillin/streptomycin at 37°C in a 5% CO2 incubator. Western blot analysis was performed for whole cell extract proteins from resistant cells to CDK4/6 inhibitors and parental cells. Colony formation assay was done to observe cell viability. Results Western blot analysis showed elevated FGFR2, CDK6, pS6RP, and cyclin E1 in resistant cells compared to parental cells. Specifically, cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, while FGFR2 and CDK6 in T47D palbociclib- and ribociclib-resistant cells. Notably, CDK6 was upregulated in MCF7 ribociclib-resistant cells, whereas increased cyclin E1 was detected in T47D palbociclib-resistant cells, respectively. Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in generated resistant cell lines. Conclusions The tumors harboring resistance to CDK4/6 inhibitors have heterogenous mechanisms of therapeutic resistance to these agents. The novel combination treatment of targeted molecular therapy and CDK4/6 inhibition will lead development of personalized medicine for ER-positive breast cancer based on elucidation of diverse resistant mechanisms to CDK4/6 inhibition. Western blot analysis of lysates from MCF-7 cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo) Cyclin E1 and pS6RP were increased in MCF7 palbociclib- and ribociclib-resistant cells, whereas CDK6 was upregulated especially in MCF7 ribociclib-resistant cells. Western blot analysis of lysates from T47D cells treated for 24 to 48 hours with palbociclib (Palbo) and ribociclib (Ribo) FGFR2 and CDK6 were increased in T47D palbociclib- and ribociclib-resistant cells, whereas increased cyclin E1 was detected especially in T47D palbociclib-resistant cells. Viability in MCF7 ribociclib-resistant and parental cells by colony formation assay Colony formation assay revealed acquired ability of resistance to CDK4/6 inhibitors in MCF7 resistant-cells. Citation Format: Yoshie Kobayashi, Yusuke Motoi, Mutsumi Fujimoto, Hideo Shigematsu. Development of personalized medicine for breast cancer based on elucidation of resistance mechanisms to CDK4/6 inhibitors [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 P4-09-11.

Investigation on acquired palbociclib resistance by LC-MS based multi-omics analysis.

Palbociclib is a specific CDK4/6 inhibitor that has been widely applied in multiple types of tumors. Different from cytotoxic drugs, the anticancer mechanism of palbociclib mainly depends on cell cycle inhibition. Therefore, the resistance mechanism is different. For clinical cancer patients, drug resistance is inevitable for almost all cancer therapies including palbociclib. We have trained palbociclib resistant cells in vitro to simulate the clinical situation and applied LC-MS multi-omics analysis methods including proteomic, metabolomic, and glycoproteomic techniques, to deeply understand the underly mechanism behind the resistance. As a result of proteomic analysis, the resistant cells were found to rely on altered metabolic pathways to keep proliferation. Metabolic processes related to carbohydrates, lipids, DNA, cellular proteins, glucose, and amino acids were observed to be upregulated. Most dramatically, the protein expressions of COX-1 and NDUFB8 have been detected to be significantly overexpressed by proteomic analysis. When a COX-1 inhibitor was hired to combine with palbociclib, a synergistic effect could be obtained, suggesting the altered COX-1 involved metabolic pathway is an important reason for the acquired palbociclib resistance. The KEGG pathway of N-glycan biosynthesis was identified through metabolomics analysis. N-glycoproteomic analysis was therefore included and the global glycosylation was found to be elevated in the palbociclib-resistant cells. Moreover, integration analysis of glycoproteomic data allowed us to detect a lot more proteins that have been glycosylated with low abundances, these proteins were considered to be overwhelmed by those highly abundant proteins during regular proteomic LC-MS detection. These low-abundant proteins are mainly involved in the cellular biology processes of cell migration, the regulation of chemotaxis, as well as the glycoprotein metabolic process which offered us great more details on the roles played by N-glycosylation in drug resistance. Our result also verified that N-glycosylation inhibitors could enhance the cell growth inhibition of palbociclib in resistant cells. The high efficiency of the integrated multi-omics analysis workflow in discovering drug resistance mechanisms paves a new way for drug development. With a clear understanding of the resistance mechanism, new drug targets and drug combinations could be designed to resensitize the resistant tumors.

Inhibition of the MNK1/2-eIF4E Axis Augments Palbociclib-Mediated Antitumor Activity in Melanoma and Breast Cancer.

Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E. When phosphorylated, eIF4E specifically engenders the translation of mRNAs that code for proteins involved in cell survival. We hypothesized that cancer cells treated with palbociclib use upregulated phosphorylated eIF4E (phospho-eIF4E) to escape the antitumor benefits of this drug. Indeed, we found that pharmacologic or genetic disruption of MNK1/2 activity, the only known kinases for eIF4E, enhanced the ability of palbociclib to decrease clonogenic outgrowth. Moreover, a quantitative proteomics analysis of melanoma cells treated with combined MNK1/2 and CDK4/6 inhibitors showed downregulation of proteins with critical roles in cell-cycle progression and mitosis, including AURKB, TPX2, and survivin. We also observed that palbociclib-resistant breast cancer cells have higher basal levels of phospho-eIF4E, and that treatment with MNK1/2 inhibitors sensitized these palbociclib-resistant cells to CDK4/6 inhibition. In vivo we demonstrate that the combination of MNK1/2 and CDK4/6 inhibition significantly increases the overall survival of mice compared with either monotherapy. Overall, our data support MNK1/2 inhibitors as promising drugs to potentiate the antineoplastic effects of palbociclib and overcome therapy-resistant disease.

Elacestrant demonstrates strong anti-estrogenic activity in PDX models of estrogen-receptor positive endocrine-resistant and fulvestrant-resistant breast cancer

The selective oestrogen receptor (ER) degrader (SERD), fulvestrant, is limited in its use for the treatment of breast cancer (BC) by its poor oral bioavailability. Comparison of the orally bioavailable investigational SERD elacestrant, versus fulvestrant, demonstrates both drugs impact tumour growth of ER+ patient-derived xenograft models harbouring several ESR1 mutations but that elacestrant is active after acquired resistance to fulvestrant. In cell line models of endocrine sensitive and resistant breast cancer both drugs impact the ER-cistrome, ER-interactome and transcription of oestrogen-regulated genes similarly, confirming the anti-oestrogenic activity of elacestrant. The addition of elacestrant to CDK4/6 inhibitors enhances the antiproliferative effect compared to monotherapy. Furthermore, elacestrant inhibits the growth of palbociclib-resistant cells. Lastly, resistance to elacestrant involves Type-I and Type-II receptor tyrosine kinases which are amenable to therapeutic targeting. Our data support the wider clinical testing of elacestrant.

Indisulam synergizes with palbociclib to induce senescence through inhibition of CDK2 kinase activity.

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition.

Overexpressed Cyclin D1 and CDK4 proteins are responsible for the resistance to CDK4/6 inhibitor in breast cancer that can be reversed by PI3K/mTOR inhibitors.

CDK4/6 inhibitors are the standard treatment in advanced HR+/HER2- breast cancer patients. Nevertheless, the resistance to CDK4/6 inhibitors is inevitable and the strategies to overcome resistance are of great interest. Here, we show that the palbociclib-resistant breast cancer cells expressed significantly higher levels of Cyclin D1 and CDK4 proteins because of upregulated protein synthesis. Silencing Cyclin D1 or CDK4 led to cell cycle arrest while silencing Cyclin E1 or CDK2 restored the sensitivity to palbociclib. Furthermore, PI3K/mTOR pathway was hyper-activated in palbociclib-resistant cells, leading to more phosphorylated 4E-BP1 and higher levels of Cyclin D1 and CDK4 translation. Targeting PI3K/mTOR pathway with a specific PI3Kα inhibitor (BYL719) or an mTOR inhibitor (everolimus) reduced the protein levels of Cyclin D1 and CDK4, and restored the sensitivity to palbociclib. The tumor samples expressed significantly higher levels of Cyclin D1, CDK4, p-AKT and p-4E-BP1 after progression on palbociclib treatment. In conclusion, our findings suggest that overexpressed Cyclin D1 and CDK4 proteins lead to the resistance to CDK4/6 inhibitor and PI3K/mTOR inhibitors are able to restore the sensitivity to CDK4/6 inhibitors, which provides the biomarker and rationale for the combinational use of CDK4/6 inhibitors and PI3K/mTOR inhibitors after CDK4/6 inhibitor resistance in breast cancer.