Palbociclib-resistant Cells Research Articles

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.

Abstract 1774: Circulating IL-6, an early biomarker in HR-positive, HER2-negative metastatic breast cancer patients progressing on CDK4/6 inhibitors

Abstract Background: CDK4/6 inhibitors (CDK4/6i, e.g. palbociclib) in combination with endocrine therapy (ET) have proven successful in delaying progression in hormone receptor-positive (HR+), HER2-negative (HER2-) metastatic breast cancer patients; however, 60-70% will progress within 1-2 years of treatment. Thus, understanding the mechanisms of resistance and identifying novel treatment strategies are needed to improve survival. Our previously published data shows that palbociclib resistance is marked by significant upregulation of the IL6-STAT3 signaling pathway in HR+, HER2- breast cancer cells and treatment with TTI-101, a small-molecule STAT3 inhibitor completing Phase I studies in solid tumor patients, significantly decreased cell viability. Additionally, matched biopsies from advanced HR+, HER2- breast cancer patients who progressed on palbociclib plus ET had an upregulation in activated STAT3 (STAT3 phosphorylated on Y705, pY-STAT3) as compared to their pre-treatment biopsy. Collectively, these data suggest that targeting the IL-6/STAT3 pathway is a viable therapeutic strategy. Our ongoing studies are directed at delineating the role of IL-6 and pY-STAT3 as predictive biomarkers to provide preclinical rationale for targeting STAT3 in patients who develop CDK4/6i resistance. Methods: We downregulated IL-6 using shRNA in MCF7 and T47D palbociclib sensitive and resistant cells. We tested the efficacy of TTI-101 in patient-derived xenograft (PDX) models from patients with metastatic HR+, HER2- breast cancer who progressed on treatment with palbociclib + ET. We also collected plasma samples from patients with metastatic HR+, HER2- breast cancer just prior to CDK4/6i treatment and at disease progression. Results: Downregulation of IL-6 in palbociclib resistant cells reduced pY-STAT3 levels and re-sensitized cells to ET and palbociclib. Induction of IL-6 is an early event by treating sensitive cells with 1uM palbociclib for 21 days during which the cells adapted and eventually developed resistance. Suppression of IL-6 in sensitive cells revealed that it is needed for cells to overcome growth inhibition by palbociclib. Targeting STAT3 in palbociclib resistant PDX mouse models using TTI-101 (50mg/kg twice daily by oral gavage), resulted in significantly reduced tumor volume; a synergistic effect was observed when TTI-101 was combined with palbociclib. Lastly, IL-6 levels in plasma samples from metastatic HR+, HER2- breast cancer patients were significantly higher at disease progression compared to pre-treatment with CDK4/6i. Conclusion: Collectively, these studies suggest that IL-6 signaling through STAT3 is a key mediator of resistance to palbociclib. We provide the rationale for using circulating IL-6 as a biomarker of disease progression and tumor pY-STAT3 as a selection criterion for treatment with TTI-101 to prevent or reverse resistance. Citation Format: Nicole M. Kettner, Tuyen N. Bui, Juliana Navarro-Yepes, T Kris Eckols, Akshara S. Raghavendra, David J. Tweardy, Kelly K. Hunt, Debu Tripathy, Khandan Keyomarsi. Circulating IL-6, an early biomarker in HR-positive, HER2-negative metastatic breast cancer patients progressing on CDK4/6 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1774.

Abstract 5802: Isotopic tracing reveals distinct metabolic changes with palbociclib or abemaciclib treatment & resistance in breast cancer cell lines

Abstract Background: CDK4/6 inhibitors (CDK4/6i) palbociclib (palbo) and abemaciclib (abema) have significantly improved outcomes in patients with hormonal receptor positive (HR+)/human epidermal growth factor receptor negative (HER2-) breast cancer. Yet almost 20% of patients never respond to treatment and all patients eventually develop acquired resistance. In vitro palbo and abema have different affinities for CDK4/6 and other cellular kinases, and in vivo they have different toxicity profiles. At present there are no clinically validated biomarkers to determine CDK4/6i sensitivity or resistance. Further it is not clear if and how to choose between the CDK4/6i for optimal outcomes. In this study, using 13C-isotopically enriched glucose we sought to identify metabolic changes associated with palbo and abema treatment in sensitive and resistant cell lines. Methods: MCF-7 cells (wild-type), palbociclib resistant cells (PDR) and abemaciclib resistant cells (ABR) treated with DMSO, palbo or abema were incubated for 24hrs in DMEM supplemented with 13C-glucose. After the incubation, cells were harvested, metabolites were extracted and analyzed via 1D and 2D NMR spectroscopy. Biological triplicates were prepared for each condition. Results: In wild-type MCF-7 cells treated with palbo or abema significant metabolite changes were observed compared to DMSO. In both palbo and abema treatment a marked decrease in nucleotide metabolites was observed, which is consistent with the ability of CDK4/6i to prevent cell cycle progression. In abema treated cells there was also significant decrease in metabolites involved in the serine-glycine pathway, which was not observed in the palbo treated cells. Palbo and abema treated cells were easily distinguishable via hierarchical clustering. PDR and ABR also had distinct metabolic profiles when they were maintained with palbo and abema, respectively or if the drugs were removed. Notably the serine-glycine pathway was increased in ABR cells but decreased in PDR cells. In PDR cells we observed an increase in glucose-1-phosphate and shift towards galactose/glycogen metabolism. Conclusions: Using cell line models we were able to demonstrate that glucose metabolism is altered in palbo and abema treatment and that resistance to each drug may activate different metabolic pathways. To the best of our knowledge this is the first direct evidence of palbo-specific and abema-specific metabolite signatures. We are assessing whether the results of these in vitro studies translate to clinical samples. If so, the altered metabolites could lead to biomarkers that correlate with response and resistance for specific CDK4/6i. Further, we are exploring if by targeting the pathways associated with resistance, we can restore CDK4/6i sensitivity. This could lead to novel therapeutic targets or treatment regimens that improve patient outcomes. Citation Format: Chandrashekhar Honrao, Kanchan Sonkar, Cristina Guarducci, Agostina Nardone, Wen Ma, Srihari Raghavendra Rao, Chen Dong, Leo Rodrigues, Rinath Jeselsohn, Elizabeth O'Day. Isotopic tracing reveals distinct metabolic changes with palbociclib or abemaciclib treatment & resistance in breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5802.

Abstract 1772: EGFR signaling as a mechanism of resistance to CDK4/6 inhibitors in Palbociclib-resistant ER+ breast cancer cells

Abstract Breast cancer driven by estrogen receptor (ER) is responsible for approximately 60-70% of cases among women. Selective inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) and ER signaling is now standard-of-care therapy for ER positive (ER+) metastatic breast cancer. CDK4/6 inhibitors, including palbociclib (PD), used in combination with endocrine therapy have shown improvement in progression-free survival compared to endocrine therapy alone in the metastatic setting. However, the inevitable development of acquired resistance significantly limits the efficacy of this targeted therapy. In ER+ breast cancer resistant cells, rewired signaling driven by different oncogenes, including the epidermal growth factor receptor (EGFR), overcomes the inhibition of CDK4/6 by PD, which, in turn, allows cell cycle progression contributing to PD-resistance. We investigated the dependency of ER+ breast cancer cell lines sensitive and resistant to PD on EGFR signaling in vitro. We evaluated the expression and activity of EGFR in two different matching pairs of ER+ palbociclib-sensitive (pS) and palbociclib-resistant (pR) breast cancer cell lines (MCF7 and T47D) and observed elevated EGFR expression in MCF7/pR cells. Increased EGFR expression in MCF7/pR cells correlated with elevated ER alpha phosphorylation at S118 and S167 sites, suggesting a direct cross-talk between EGFR and ER signaling in PD-resistant cells. Serum starvation significantly decreased EGFR activity, cdk2 and cyclin D1 expression in both MCF7 and T47D PD-resistant cells compared to the parental cell lines, confirming their dependency on the presence of growth factors. Notably, stimulation with EGF promoted ER phosphorylation and expression of cell cycle related targets: cdk2, cyclin E and wee1 in both MCF7 and T47D cell lines sensitive and resistant to PD. Furthermore, stimulation with estrogen promoted EGFR activation in MCF7/pS and MCF7/pR cells, indicating a direct cross-regulation between these molecular targets. Further, exposure to the EGFR inhibitor erlotinib dramatically inhibited the proliferation of MCF7/pR cells compared to the parental cells, suggesting that EGFR could be an important bypass signaling regulator contributing to resistance to PD. We found that dual treatment with erlotinib and PD significantly inhibited Rb phosphorylation in MCF7/pR cells improving the effect of PD on cell cycle arrest. Moreover, dual treatment of MCF7/pR cells attenuated ER expression suggesting the higher dependency of ER signaling on EGFR activity in resistant cells. Taken together, our findings suggest that EGFR signaling is a mechanism of resistance in ER+ cell lines with acquired resistance to PD in vitro and that EGFR targeting can be a promising approach to improve palbociclib efficacy in breast cancer patients. Citation Format: Nadiia Lypova, Lilibeth Lanceta, Susan M. Dougherty, Jason A. Chesney, Yoannis Imbert-Fernandez. EGFR signaling as a mechanism of resistance to CDK4/6 inhibitors in Palbociclib-resistant ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1772.

Abstract 2307: Sequential treatment with abemaciclib plus endocrine therapy inhibits cell proliferation and triggers apoptosis in cell lines resistant to CDK4/6i

Abstract CDK4/6 inhibitors combined with endocrine therapy (ET) have shown clinical benefit in HR+, HER2- breast cancer. However, development of resistance highlights the need for treatment strategies to improve outcomes. To study resistance mechanisms, breast cancer cell lines were treated with CDK4/6i (abemaciclib or palbociclib) in combination with 4-OH-tamoxifen (tamoxifen) for 120-144h and sorted for resistant cells defined as geminin positive (GEM+), a marker of S/G2/M cell cycle accumulation. To confirm the resistant phenotype, cell lines were treated with tamoxifen plus the CDK4/6 inhibitor used to drive resistance. To understand if sequential CDK4/6i treatment is effective in controlling cell proliferation, resistant cells generated through treatment with palbociclib + tamoxifen or abemaciclib + tamoxifen were treated with abemaciclib + ET (fulvestrant or tamoxifen) or palbociclib + ET, respectively. Geminin/Ki67, annexin V and colony formation assays were performed to evaluate cell proliferation and viability. Molecular characterization by western blot and RNAseq analysis were utilized to provide insights into mechanisms of resistance and the effects of sequential treatment with CDK4/6i + ET. Cell lines resistant to palbociclib + tamoxifen subsequently treated with abemaciclib + ET showed decreased %GEM+ and colony formation ability, decreased Ki67 levels, and increased apoptosis. These effects were not observed in cell lines resistant to abemaciclib + tamoxifen following subsequent treatment with palbociclib + ET. Western blot analysis showed that palbociclib and abemaciclib-resistant cells had increased CDK6 and pERK levels, compared to control. Importantly, treatment of palbociclib-resistant cells with abemaciclib + ET decreased FOXM1, a key regulator of senescence and apoptosis. Cyclin A, a marker of mitosis, was also decreased, consistent with decreased %GEM+ subpopulation in palbociclib + ET resistant cells. These effects were not observed in abemaciclib-resistant cells treated with palbociclib + ET.In summary, our in vitro data provides mechanistic insights into resistance to combination CDK4/6i + tamoxifen and suggests the potential benefit of sequential treatment with abemaciclib + ET to overcome resistance in breast cancer. Citation Format: Elisabet Zapatero-Solana, Maria P. Ganado, Maria J. Ortiz-Ruiz, Cecilia Mur, Lacey Litchfield, Farhana Merzoug, Oscar Puig, Maria Jsoe Lallena. Sequential treatment with abemaciclib plus endocrine therapy inhibits cell proliferation and triggers apoptosis in cell lines resistant to CDK4/6i [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2307.

Increased phosphorylation of RB and protein expression of Cyclin E confers resistance to CDK4/6 inhibitors in dedifferentiated liposarcoma.

e23525 Background: Dedifferentiated liposarcomas (DDLPS) are malignant adipocytic cancers characterized by an amplification of the cell cycle regulatory gene, CDK4. Palbociclib, a CDK4/6-inhibitor, is a standard of care treatment; however, innate and acquired resistance to the drug limits efficacy in patients with DDLPS. Molecular mechanisms and biomarkers of palbociclib resistance have been posited in non-mesenchymal tumors but are poorly understood in DDLPS. To this end, we developed palbociclib-resistant DDLPS cell lines derived from several DDLPS patient samples to better characterize these phenotypic and genomic differences. Methods: Three human CDK4 amplified DDLPS cell lines (224A, 246, 863) and one control liposarcoma control cell line without CDK4 amplification and RB loss (LiSa2) were brought into culture. Each cell line was intermittently treated with palbociclib at their respective IC25 for 6 months to develop resistance in long-term treated cell lines. Surviving cells were allowed to recover for at least 2 weeks before retreatment. Parental lines were grown as controls in tandem with the developing resistant lines to compare molecular changes in response to palbociclib. Cell viability was measured by XTT, cell cycle analysis was measured with Flow cytometry using a PI stain, and protein expression was measured using whole cell lysates for Western blotting. Results: The four cell lines had IC50s to palbociclib ranging from 15.0, 14.8, 12.0, and 11.4 µM for LiSa-2, 224A, 246, and 863 cell lines respectively. Long-term treated strains demonstrated consistent reductions in the measured IC50s (8.99, 7.32, 8.27, and 7.08 µM respectively). After treatment with 10 µM of palbociclib for 48 hours, two parental cell lines showed a greater increase in cells in the G1 fraction than long-term treated cells: LiSa-2 (-2.4% increase in parental vs -16.5% increase in long-term treated) and 863 (16.2% vs 11.7%). 224A and 246 demonstrated the opposite where parental cell lines had fewer cells in G1 after treatment compared to long-term treated cell: 224A (12.6% vs. 20.5%) and 246 (12.6% vs. 21.6%). Western blots indicated that the initial response to palbociclib in parental cell lines decreased pRb protein levels and increased Cyclin E and CDK6. After 6 months of treatment for all cell lines, a significant increase in pRb and Cyclin E were observed in the long-term treated cell lines and no changes were seen in CDK4, Cyclin D, nor p16. Conclusions: Here we present the development of two novel, palbociclib resistant, DDLPS cell lines and two long-term treated cell lines. Our resistant cell lines indicated that the ability to bypass the G1/S-checkpoint in the presence of CDK4/6-inhibitors may result from increased phosphorylation of Rb and increased total of Cyclin E. Next generation sequencing of RNA expression in parental and resistant lines are underway.

Abstract P2-04-01: 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

Abstract CDK4/6 inhibitors are now the standard treatment in advanced HR+/HER2- breast cancer patients. Nevertheless, the resistance to CDK4/6 inhibitors is inevitable, limiting its use and efficacy. The mechanisms and the strategies to overcome resistance are of great interest. Here, we show that the palbociclib-resistant breast cancer cells express significantly higher levels of Cyclin D1 and CDK4 proteins because of upregulated protein synthesis. Silencing Cyclin D1 or CDK4 directly led to cell cycle arrest while silencing Cyclin E1 or CDK2 restored the sensitivity to palbociclib in palbociclib-resistant cells. 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 in resistant cells both in vitro and in vivo. The tumors from breast cancer patients who progressed after palbociclib treatment had significantly higher protein expression of Cyclin D1, CDK4, p-AKT and p-4E-BP1 than the paired samples before 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. Citation Format: Qiang Liu, Zijie Cai, Jingru Wang, Yudong Li, Lok Lam Wong. 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 [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-04-01.

P27kip1 expression and phosphorylation dictate Palbociclib sensitivity in KRAS-mutated colorectal cancer

In colorectal cancer, mutation of KRAS (RASMUT) reduces therapeutic options, negatively affecting prognosis of the patients. In this setting, administration of CDK4/6-inhibitors, alone or in combination with other drugs, is being tested as promising therapeutic strategy. Identifying sensitive patients and overcoming intrinsic and acquired resistance to CDK4/6 inhibition represent still open challenges, to obtain better clinical responses. Here, we investigated the role of the CDK inhibitor p27kip1 in the response to the selective CDK4/6-inhibitor Palbociclib, in colorectal cancer. Our results show that p27kip1 expression inversely correlated with Palbociclib response, both in vitro and in vivo. Generating a model of Palbociclib-resistant RASMUT colorectal cancer cells, we observed an increased expression of p27kip1, cyclin D, CDK4 and CDK6, coupled with an increased association between p27kip1 and CDK4. Furthermore, Palbociclib-resistant cells showed increased Src-mediated phosphorylation of p27kip1 on tyrosine residues and low doses of Src inhibitors re-sensitized resistant cells to Palbociclib. Since p27kip1 showed variable expression in RASMUT colorectal cancer samples, our study supports the possibility that p27kip1 could serve as biomarker to stratify patients who might benefit from CDK4/6 inhibition, alone or in combination with Src inhibitors.

Abstract LB078: Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity

Abstract Introduction: Small molecule therapeutics of estrogen receptor-positive/HER2-negative breast cancer remains an area of active investigation where novel agents are greatly needed for treatment of hormone therapy resistant metastatic disease. The biguanide hexyl-benzyl-biguanide (HBB) is a potent inhibitor of CYP3A4 arachidonic acid (AA) epoxygenase activity and inhibits breast cancer cell proliferation and MCF-7 breast cancer tumor growth in nude mice. To explore the impact of bioisosteric substitution of the benzyl moiety of HBB with a cubane moiety, we synthesized hexyl-(cuban-1-yl-methyl)-biguanide (HCB) and tested its potency for the inhibition of the cognate CYP3A4 target AA epoxygenase activity as well as breast cancer cell proliferation of hormone therapy sensitive and resistant cell lines. Results: HCB selectively inhibited CYP3A4-mediated biosynthesis of (±)-14,15-EET with an IC50 of 4.7±0.2 uM vs. 64.8±6.5 uM for 8,9-EET and 26.5±1.9 uM for 11,12-EET. At 24 hours, HCB inhibited proliferation of MCF-7 (ER+HER2-), BT474 (ER+HER2+) and MDA-MB-231 (ER-HER2-) cells at IC50 of 8.4±1.2, 11±1.3 and 15±0.9 uM, respectively. At 48 hours, HCB inhibited proliferation of aromatase inhibitor and fulvestrant resistant (LR,FR), and cyclin dependent kinase inhibitor (CDKi) palbociclib resistant (LR,FR,PR) MCF-7 cell lines; LR,FR MCF-7AC1 (IC50 =1.34±0.1 uM) and LR,FR,PR MCF-7AC1 (IC50 =1.64±0.2 uM). Addition of 14,15-EET (1 uM) partially rescues MCF-7 cells from HCB-mediated inhibition of proliferation. OXPHOS is promoted, in part, by EETs. HCB is a potent OXPHOS inhibitor and rapidly inhibits O2 consumption of the MCF-7 and ZR75 (ER+HER2-) cells in a dose-dependent fashion (P<0.05). HCB treatment (10 uM) reduces mitochondrial membrane potential to 57.4±15.3% (P<0.001) of vehicle control in MCF-7 cells. Treatment with HCB at 20 uM for 0.5 hour also causes mitochondrial swelling in MCF-7 cells. HCB (10 uM) activates AMPK within 0.5 hour and increases the level of phosphorylation from 2.4±0.3 to 25.1±6.0 folds in a time dependent fashion in MCF-7 cells from 0.5-24 hours. Conclusion: These results show that HCB inhibits proliferation of ER+HER2- breast cancer cells, in part through inhibition of OXPHOS and suppression of the CYP product 14,15-EET. This inhibition is highly active in hormonal therapy and CDKi resistant ER+HER2- breast cancer cells. These results suggest that HCB is a novel and potent biguanide that has potential to be developed for inhibition of hormone therapy resistant and CDKi resistant breast cancer. Citation Format: Zhijun Guo, Jianxun Lei, Kwon Ho Hong, Beverly Norris, Craig M. Flory, Swaathi Jayaraman, Connor McDermott, Elizabeth Ambrose, Irina Sevrioukova, Tom Poulos, Ilia Denisov, Stephen Sliga, Robert J. Schumacher, Gunda I. Georg, John R. Hawse, Matthew P. Goetz, David A. Potter. Hexyl-(cuban-1-yl-methyl)-biguanide (HCB) inhibits hormone therapy resistant breast cancer cells, in part by Inhibiting CYP3A4 arachidonic acid epoxygenase activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB078.

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.

Deregulated Immune Pathway Associated with Palbociclib Resistance in Preclinical Breast Cancer Models: Integrative Genomics and Transcriptomics.

Recently, cyclin-dependent kinase (CDK) 4/6 inhibitors have been widely used to treat advanced hormone receptor-positive breast cancer. Despite promising clinical outcomes, almost all patients eventually acquire resistance to CDK4/6 inhibitors. Here, we screened genes associated with palbociclib resistance through genomics and transcriptomics in preclinical breast cancer models. Palbociclib-resistant cells were generated by exposing hormone receptor-positive breast cancer cell lines to palbociclib. Whole-exome sequencing (WES) and a mRNA microarray were performed to compare the genomic and transcriptomic landscape between both palbociclib-sensitive and resistant cells. Microarray analysis revealed 651 differentially expressed genes (DEGs), while WES revealed 107 clinically significant mutated genes. Furthermore, pathway analysis of both DEGs and mutated genes revealed immune pathway deregulation in palbociclib-resistant cells. Notably, DEG annotation revealed activation of type I interferon pathway, activation of immune checkpoint inhibitory pathway, and suppression of immune checkpoint stimulatory pathway in palbociclib-resistant cells. Moreover, mutations in NCOR1, MUC4, and MUC16 genes found in palbociclib-resistant cells were annotated to be related to the immune pathway. In conclusion, our genomics and transcriptomics analysis using preclinical model, revealed that deregulated immune pathway is an additional mechanism of CDK4/6 inhibitor resistance besides the activation of cyclin E-CDK2 pathway and loss of RB, etc. Further studies are warranted to evaluate whether immune pathways may be a therapeutic target to overcome CDK4/6 inhibitor resistance.

Combined CDK2 and CDK4/6 Inhibition Overcomes Palbociclib Resistance in Breast Cancer by Enhancing Senescence.

Simple SummaryCyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are widely used to treat metastatic hormone receptor-positive/human epidermal growth factor receptor 2-negative breast cancer. Despite the effectiveness of CDK4/6 inhibitors, acquired resistance occurs in almost all cases. Strategies to address this issue have not been developed yet. We investigated mechanisms of resistance to CDK4/6 inhibitor in breast cancer and potential therapeutic strategies. We found that cyclin E-CDK2 mediated phosphorylation of C-MYC is responsible for resistance to CDK4/6 inhibitor by suppressing C-MYC induced senescence. On the contrary, the synergistic anti-proliferative effect of the combined inhibition of CDK2 and CDK4/6 overcomes acquired resistance to CDK4/6 inhibitors by enhancing senescence. Our findings could pave the way for the development CDK2-specific kinase inhibitor for the treatment of breast cancers that are resistant to CDK4/6 inhibitor.Breast cancer represents the number one global cancer burden in women and the hormone receptor (HR)-positive subtype comprises approximately 70% of breast cancers. Unfortunately, acquired resistance ultimately occurs in almost all cases, even though cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are a highly effective therapy for HR-positive/human epidermal growth factor receptor 2-negative subtype. Here, we investigated mechanisms of resistance to CDK4/6 inhibitor and potential therapeutic strategies using our palbociclib-resistant preclinical model. We observed that cyclin E was significantly overexpressed in palbociclib-resistant cells, and similar association was also confirmed in pleural effusion samples collected from HR-positive breast cancer patients. After confirmation of cyclin E-CDK2 interaction by co-immunoprecipitation, we demonstrated CDK2 inhibition combined with palbociclib synergistically suppressed proliferation of palbociclib-resistant cells and growth of palbociclib-resistant xenograft in mice. We also proved that enhancing C-MYC-mediated senescence is a novel mechanism behind the synergism created by targeting both CDK2 and CDK4/6. Furthermore, the clinical relevance of cyclin E as a therapeutic target was supported by significant association between CCNE1 overexpression and poor prognosis based on large-scale public gene expression data sets in HR-positive breast cancer patients. Therefore, we propose cyclin E-CDK2 signaling as a promising therapeutic target for overcoming cyclin E-associated resistance to CDK4/6 inhibitor.

Abstract 5811: Combined inhibition of CDK2 and CDK4/6 overcomes acquired resistance to CDK4/6 inhibitors in breast cancer via RB-independent pathway

Abstract Objective: FDA-approved CDK4/6 inhibitors (palbociclib, ribociclib and abemaciclib) are widely used to treat metastatic hormone receptor-positive (HR+) breast cancer. Despite the excellent disease control with CDK4/6 inhibitors, acquired resistance emerges in almost all cases. The strategies to combat the resistance are not yet defined. We investigated therapeutic strategies as well as mechanisms of resistance to CDK4/6 inhibitors in breast cancer. Methods: Palbociclib resistant cells (MCF7-PR and T47D-PR) were generated from the parental MCF and T47D cell lines by treating them with gradually increasing concentration of palbociclib for over 9 months. Cell viability assay, cell cycle, and western blot were performed to confirm the acquired resistance. Microarray analyses were carried out to identify altered gene expression. We validated the overexpressed genes by qRT-PCR and western blot and further confirmed the significant inhibition by CDK2 siRNA in combination with palbociclib both in vitro and in vivo. Results: Palbociclib-resistant MCF7 and T47D cells had about 10 fold higher half maximal inhibitory concentration (IC50), compared with parental cells. Morphological changes and alteration in EMT markers were observed in resistant cells. Loss of RB and overexpression of cyclin E1 confirmed that the cells gained resistance. Overexpression of C-MYC was observed in resistant cells compared to sensitive counterparts. Furthermore, from analysis of pleural effusion samples from HR+ breast cancer patients, we confirmed again that loss of RB and overexpression of cyclin E are significantly correlated with resistance to CDK4/6 inhibitors. Mechanistically, we found that, cyclin E-CDK2 mediated phosphorylation of C-MYC was responsible for suppressing C-MYC induced senescence. To overcome CDK4/6 inhibitor resistance, combined inhibition of CDK2 and CDK4/6 showed a synergistic effect in palbociclib-resistant in vitro and in vivo model. Further, combination-treated group showed significantly higher β-galactosidase expression compared to no- treatment control or palbociclib alone treated group. Conclusion: The synergistic anti-proliferative effect of the combined inhibition of CDK2 and CDK4/6 overcame acquired resistance to CDK4/6 inhibitors via RB-independent pathway, that is, suppressing C-MYC. These findings could pave the way for the development of next generation CDK2 inhibitor in CDK4/6 inhibitor-resistant breast cancer, which is characterized by overexpressed cyclin E and C-MYC. Keywords: CDK4/6, hormone receptor-positive breast cancer, drug resistance Citation Format: Kamal Pandey. Combined inhibition of CDK2 and CDK4/6 overcomes acquired resistance to CDK4/6 inhibitors in breast cancer via RB-independent pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5811.

Resensitization of palbociclib-resistant MCF-7 cells by inhibiting TGF-β-induced epithelial-mesenchymal transition.

e13061 Background: Inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) is the most recent therapeutic method to treat estrogen receptor (ER)-positive breast cancer. Three CDK4/6 inhibitors have been approved by the FDA. The acquired resistance to CDK4/6 inhibitors are expected to have a negative impact on success of breast cancer therapy. It is critical to understand the molecular mechanisms underlying drug resistances to devise a better regimen or overcome drug resistance. Methods: The derivative MCF-7 (MCF-7:PR) cells which exert acquired resistance to palbociclib (PCB), one of the FDA-approved CDK4/6 inhibitors, are recently established. The EMT characteristics, major mechanisms for the EMT, and inhibition of TGF-β signaling pathways are studied in MCF-7:PR cells. Results: The mesenchymal markers are increased and the epithelial ones are decreased at both mRNA and protein levels in our cell line model. Transwell migration and would healing assays also demonstrated that MCF-7:PR cells exert EMT properties. Hyperactivation of TGF-β/Smad signaling was observed in MCF-7:PR cells. Chemical inhibition of TGF-β signaling lead to diminished cell migration and resistance to PCB. Conclusions: Resistance to PCB in MCF-7 cells resulted in significant changes in cell motility and molecular markers associated with EMT. In particular, TGF-β signaling is closely related to EMT and its inhibition reversed the EMT and cellular response to PCT. Our findings suggest that the modulation of EMT via inhibition of TGF-β signaling can be one of strategies to bypass PCB resistance in ER-positive breast cancer.

Transcriptomic Profiling Identifies Differentially Expressed Genes in Palbociclib-Resistant ER+ MCF7 Breast Cancer Cells.

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition in estrogen receptor-positive (ER+) breast cancer remains a significant clinical challenge. Efforts to uncover the mechanisms underlying resistance are needed to establish clinically actionable targets effective against resistant tumors. In this study, we sought to identify differentially expressed genes (DEGs) associated with acquired resistance to palbociclib in ER+ breast cancer. We performed next-generation transcriptomic RNA sequencing (RNA-seq) and pathway analysis in ER+ MCF7 palbociclib-sensitive (MCF7/pS) and MCF7 palbociclib-resistant (MCF7/pR) cells. We identified 2183 up-regulated and 1548 down-regulated transcripts in MCF7/pR compared to MCF7/pS cells. Functional analysis of the DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified several pathways associated with breast cancer, including ‘cell cycle’, ‘DNA replication’, ‘DNA repair’ and ‘autophagy’. Additionally, Ingenuity Pathway Analysis (IPA) revealed that resistance to palbociclib is closely associated with deregulation of several key canonical and metabolic pathways. Further studies are needed to determine the utility of these DEGs and pathways as therapeutics targets against ER+ palbociclib-resistant breast cancer.

Abstract PD2-05: Differential mechanisms of acquired resistance to abemaciclib versus palbociclib reveal novel therapeutic strategies for CDK4/6 therapy-resistant breast cancers

Abstract Background: Hormone receptor positive (HR+)/human epidermal growth factor receptor 2 negative (Her2-) is the most prevalent subtype of breast cancer, representing 70% of the cases with metastatic disease. Endocrine therapy plus FDA-approved cyclin-dependent kinases (CDK) 4/6 inhibitors (palbociclib, ribociclib and abemaciclib) have improved the treatment of HR+/Her2- advanced breast cancer patients. However, patients develop clinical resistance (acquired) during the long-term treatment, and in some cases, they do not respond within the first 3 months of treatment (intrinsic resistance), leading to disease progression. Although these inhibitors have the same nominal targets, CDK4/6, abemaciclib can inhibit other kinases that are not inhibited by palbociclib/ribociclib, such as CDK9/7/2/1, GSK3α/β and CAMK2γ/δ. As such, abemaciclib has been shown to inhibit cell growth in Rb deficient cells, in which palbociclib/ribociclib are ineffective. Therefore, we hypothesized that 1) mechanisms driving palbociclib acquired resistance are distinct than those mediating abemaciclib resistance, 2) palbociclib resistant models may be responsive to abemaciclib, and 3) pathways underlying these mechanistic differences can be exploited as alternative therapies to overcome resistance. Methods: We generated several models to examine mechanisms of resistance to different CDK4/6 inhibitors: 1) MCF7 and T47D (ER+) palbociclib resistant breast cancer cells with increasing concentrations of palbociclib, starting at 1.2 µM (initial IC 50) up to 5 µM (plasma concentrations in patients) in a stepwise manner over a 6 month period, 2) MCF7 abemaciclib resistant cells (0.5, 1 and 1.5 µM), and 3) organoids derived from patient derived xenografts (PDXs) with a similar molecular profile to palbociclib acquired resistance models. Cell cycle changes were evaluated by western blot analysis and flow cytometry. Results: Western blot analysis revealed a dose dependent downregulation of ERα, Rb, p-Rb and p27, while levels of cyclin E and p-CDK2 increased in a stepwise fashion in palbociclib resistant cells, which were only partially cross resistant to abemaciclib and sensitive to CDK9 inhibitors LDC067 and SNS032. FACS analysis of abemaciclib resistant cells showed an increase in G2/M accompanied by a drastic reduction of p-Rb and a gradual decrease of Rb. ERα levels were slightly decreased in abemaciclib resistant cells compared to the complete loss in palbociclib resistant cells. As in the palbociclib resistant cells, a dose dependent increase in cyclin E and p-CDK2 were also observed. However, Rad51, a key mediator of DNA repair, was downregulated in a stepwise manner only in abemaciclib resistant cells, but not in palbociclib resistant cells. Therefore, we evaluated if a combination therapy targeting DNA repair pathways plus abemaciclib could render palbociclib resistant models more responsive to targets other than cell cycle inhibitors. To this end, we examined the combination of abemaciclib and niraparib in organoid cultures generated from PDX models that are transcriptomic surrogates of palbociclib resistance cells and found a significant reduction of viability, number and density of these organoids. In vivo, using the same PDX models, the combined treatment reduced the tumor growth rate and increased overall survival. Conclusion: Our results suggest that mechanisms underlaying palbociclib and abemaciclib acquired resistance exhibit differentially altered pathways, such as DNA damage/repair pathways, which can be exploited to overcome CDK4/6 inhibitors therapy resistance in breast cancer patients. Citation Format: Juliana Navarro-Yepes, Xian Chen, Tuyen Bui, Nicole M Kettner, Kelly K Hunt, Khandan Keyomarsi. Differential mechanisms of acquired resistance to abemaciclib versus palbociclib reveal novel therapeutic strategies for CDK4/6 therapy-resistant breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-05.

Abstract P6-03-09: Role of IL-6 in promoting endocrine therapy and palbociclib resistance estrogen receptor positive breast cancer cells

Abstract Background: CDK4/6 inhibitors combined with endocrine therapy (ET) are mainstay to treat metastatic estrogen receptor (ER) positive patients. Yet, almost 60% develop resistance to CDK4/6 inhibition within 2 years of initial treatment. An ongoing clinical challenge has thus been identifying biomarkers of response to predict patients that will either respond or not respond to palbociclib. Further, there is an unmet need to identify actionable targets for patients that have progressed on CDK4/6 blockade regimens. Currently, the only biomarker being used to identify patients for anti-CDK4/6 therapy is estrogen receptor (ER) by IHC. The goal of our study was thus to identify the therapeutic vulnerabilities of CDK4/6 inhibitor resistance cells and identify key markers that can longitudinally correlate with development of resistance. Methods: We have developed MCF7 and T47D resistant cells by treating them with increasing doses (1.2, 2.4, 3.6, and 4.8μΜ) of palbociclib over a 6-month period. At each dose, resistant cells were harvested to observe dose dependent changes that occur as the cells acquire resistance to palbociclib. At endpoint i.e. 4.8μM, multi-omics approach was used to compare sensitive vs. resistant cells. Results: Our multi-omics data indicates that palbociclib resistant cells show i) Upregulation of the STAT3-IL6 pathway ii) Loss of ERα and iii) Loss RB protein levels. Primarily, resistant cells adapt to palbociclib treatment by a dose dependent upregulation of IL6. Further, we observe that induction of IL-6 is an early event which also correlates with senescence-associated secretory phenotype (SASP) signature in resistant cells, predicted to be induced with CDK4/6 inhibition. Importantly, time-course studies using 1μM palbociclib indicates that IL-6 is needed in overcome SASP and promote proliferation in cells adapting and eventually developing resistance to CDK4/6 inhibition. Lastly, we subjected MCF7 and T47D parental cells, which are sensitive to endocrine therapy and palbociclib, with exogenous IL-6 and IL-6 receptor (IL-6R). Treatment with IL-6/IL-6R lead to the downregulation of ER and RB protein levels. IL-6 treated parental cells are 5-fold more resistant to tamoxifen and 7-fold more resistant to fulvestrant compared to parental cells not treated with IL-6. Additionally, dose response studies with palbociclib showed parental cells treated with IL-6 are 5-fold more resistant to palbociclib treatment compared to the cells cultured in the absence of IL-6. Conclusions: Collectively, our data suggests that resistance to palbociclib results in a cascade of events initiating with induction of senescence, leading to sustained upregulation of IL-6 which leads to the promotion of an aggressive tumor growth, accompanied by resistance to endocrine therapy. Our ongoing studies are geared towards delineating the role of each step of CDK4/6 resistance and determining if IL-6 is required to maintain CDK4/6 inhibition resistance. Our long-term goal is to correlate provide the pre-clinical rationale for using IL-6 as a predictive biomarker in patients receiving CDK4/6 inhibitor based therapies. Citation Format: Nicole M Kettner, Tuyen Bui, Xian Chen, Kelly K Hunt, Debu Tripathy, Khandan Keyomarsi. Role of IL-6 in promoting endocrine therapy and palbociclib resistance estrogen receptor positive breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-09.

Abstract P6-04-12: STAT3 as a therapeutic target in estrogen receptor positive breast cancer patients refractory to CDK4/6 inhibition

Abstract Background: CDK4/6 inhibitors (i.e. palbociclib) in combination with endocrine therapy (ET) are currently standard of care for estrogen receptor (ER) positive breast cancer patients with advanced/metastatic disease. While this combination has proven successful in delaying progression, no improvement in long-term survival has been observed to date in the post-menopausal setting. Further, resistance is inevitable as 50-60% of patients’ metastatic lesions progress on CDK4/6 inhibitor therapy within 2-years of treatment. For patients experiencing resistance to CDK4/6 inhibitors, novel treatment strategies are needed to delay progression or to improve survival. Our recently published data shows that palbociclib-resistance is marked by significant upregulation of the IL6- STAT3 signaling pathway in ER-positive breast cancer cells. Knockdown of STAT3 in resistance cancer cells restored sensitivity to palbociclib. Additionally, matched biopsies from advanced ER-positive breast cancer patients who progressed on palbociclib showed upregulation in p-STAT3 as compared to their pre-treatment biopsy samples (14 out of 25 patients; p=0.042). Collectively, these data suggest that STAT3 is viable therapeutic target to overcome palbociclib resistance. Hence, the goal of this study is to translate these findings in vivo and provide pre-clinical rational to the efficacy of TTI-101 (an orally bioavailable STAT3 inhibitor) in PDX models of palbociclib resistance. Methods: To identify PDX models that recapitulate palbociclib resistance observed in cell line models, we correlated two published RNA-seq data sets of a panel of 64 PDX models from breast cancer patients to our multi-omics analysis of palbociclib resistant cell lines. Four PDXs that recapitulated the resistant-transcriptome expression pattern based on Spearman’s correlation, were narrowed down for future studies. In addition, we have also established PDX models (1st-4th passage) from two sets of patients (1) those who are intrinsically resistant to palbociclib (the patients progressed at 2-3 months while on palbociclib + ET) and (2) those who have developed resistance over time (i.e. acquired resistance-patients progress at 12-18 months while on palbociclib + ET). Results: We chose PDX model BCX94 for our preliminary studies as it showed baseline induction of p-STAT3 by immunoblot analysis. Using BCX94, we show that TTI-101 at 50mg/kg twice a day potently reduced tumor volume and improved survival of these mice. We also observed a significant reduction in tumor-derived IL-6 in circulation; as assayed by hIL-6 levels in the serum. Further, complete blood count analysis of whole blood collected at endpoint (i.e. 28 days on treatment) showed no drug toxicities. Treatment of intrinsic palbociclib resistant PDX models with TTI-101, on the other hand, showed only a brief delay in tumor progression, but no overall benefit. Conclusions: Collectively, these results indicate that TT1-101 is safely tolerated and efficacious at the dose examined in PDX models and that TTI-101 may be a suitable target for those tumors that are resistant to palbociclib. The differences in response to TTI-101 in the BCX94 versus our intrinsic PDX models, suggest that acquired vs. intrinsic resistance signatures of palbociclib may be mutually exclusive and that the IL-6-STAT3 signaling axis may be a driver in acquired palbociclib resistance, but not the intrinsic setting. Our ongoing studies are therefore geared towards testing the efficacy of IL-6-STAT3 inhibition in both settings (i.e. acquired and intrinsic) and identifying distinct therapeutic vulnerabilities of intrinsic palbociclib resistance that may be unresponsive to STAT3 inhibition. Citation Format: Nicole M Kettner, Tuyen Bui, Min Jin Ha, T Kris Eckols, David J Tweardy, Funda Meric-Bernstam, Kelly K Hunt, Debu Tripathy, Khandan Keyomarsi. STAT3 as a therapeutic target in estrogen receptor positive breast cancer patients refractory to CDK4/6 inhibition [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-12.

Abstract P1-21-06: Deregulated immune pathway associated with palbociclib resistance in breast cancer preclinical models: Integrative analysis of genomics and transcriptomics

Abstract Background: Recently CDK4/6 inhibitors are being widely used to treat advanced HR-positive breast cancer. Despite promising clinical outcomes, almost all patients eventually acquire resistance to CDK4/6 inhibitors. Hence, understanding the mechanisms of acquired resistance to CDK4/6 inhibitors is crucial to develop alternate treatment strategy. Therefore, we screened genes associated with palbociclib resistance through genomics and transcriptomics in breast cancer preclinical models. Methods: We generated palbociclib-resistant cell lines, MCF7-PR and T47D-PR by exposing MCF7 and T47D cells to palbociclib for over 9 months. After confirming the acquired resistance through in vitro assays, we performed whole exome sequencing (WES) and mRNA microarray to compare genomic and transcriptomic landscape between palbociclib-sensitive and resistant cells. Real-time PCR was performed to confirm differentially expressed genes. Results: Microrray analysis revealed 651 differentially expressed genes (DEGs) (fold change ≥2) by comparing MCF7 vs. MCF7-PR cells. WES also revealed 107 mutated genes by comparing T47D vs. T47D-PR cells. Further, GO annotation of both the DEGs and mutated genes found deregulation of immune pathway commonly in MCF7-PR and T47D-PR (FDR<0.25). Representatively, activated type I interferon pathway was notable in MCF7-PR cells with annotation of the DEGs. By real-time PCR, IRF9 and SP100 were commonly increased in the resistant cells (MCF7-PR, T47D-PR) compared to each sensitive counterpart. Besides, STAT1, IFI27, IFIT2, IFIT3, and XAF1 were increased in the MCF7-PR compared to MCF7, STAT2 was also increased in the T47D-PR compared to T47D. Regarding mutations found in resistant cells, MUC4 (in MCF7-PR) and MUC16 (in T47D-PR) mutations were annotated as immune pathway including immune response activating cell surface receptor signaling or O-glycan processing, et al. Besides, RB1 mutation was detected in T47D-PR cells. Conclusions: Deregulated immune pathway was found to be associated with palbociclib resistance in preclinical breast cancer models. Further studies are warranted to evaluate whether immune pathway may be a therapeutic target to overcome CDK4/6 inhibitor resistance. Citation Format: Yong Wha Moon, Eunbyeol Lee, Sohyun Hwang, Kamal Pandey, Nahee Park, Jin Hur, Young Bin Cho, Seung Ki Kim, Seung Ah Lee, Hee-Jung An, Joohyuk Sohn. Deregulated immune pathway associated with palbociclib resistance in breast cancer preclinical models: Integrative analysis of genomics and transcriptomics [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-21-06.

Abstract 3012: Single-cell transcriptomic characterization of luminal breast cancer cell lines with acquired resistance to the CDK4/6 inhibitor palbociclib

Abstract CDK4/6 inhibitors, such as palbociclib, target the cyclin-dependent kinases 4 and 6, essential for cell cycle regulation. CDK4/6 inhibitors are established treatment options in patients with endocrine receptor positive, HER2 negative metastatic breast cancer (BC). We recently conducted a transcriptomic study of seven palbociclib resistant luminal BC cell lines, demonstrating a common transcriptional signature of resistance to palbociclib, including the over-expression of CCNE1 and under-expression of RB1. Genomic analysis based on ddPCR and whole exome sequencing (WES) demonstrated CCNE1 over-expression due to gene amplification in only two cell lines, and RB1 under-expression due to gene deletion in 3 out of 7 cell lines. This prompts the hypothesis that more complex molecular mechanisms of CCNE1 over-expression / RB1 under-expression may exist. To improve the characterization of the molecular mechanisms underlying the resistance to palbociclib, we performed single-cell transcriptomic profiling (single-cell RNA-seq, scRNA-seq) of seven BC cell line resistant to palbociclib and their sensitive counterparts. Cells were isolated using the Bio-Rad ddSEQ single cell isolator and sequenced in order to obtain about 100,000 reads per cell. A total of 10,557 cells with at least 2,000 genes read per cell were selected for downstream analysis, corresponding to 5,116 palbociclib sensitive cells and 5,441 palbociclib resistant cells. Considering the 5,000 most variable genes, dimensionality reduction analysis based on t-distributed stochastic neighbor embedding (tSNE) clearly segregated cells depending on their cell type, as expected based on previously generated bulk RNA-seq data. Using previously identified top cell-type specific differentially expressed genes between resistant and sensitive cell lines (absolute log2(fold-change) > 0.5), dimensionality reduction analysis correctly classified 98.2% of cells based on their sensitivity/resistant status, demonstrating the quality of our scRNA-seq data. Interestingly, we observed a small fraction (<1.5%) of misclassified sensitive cells showing resistant-like phenotypes. Co-expression analysis suggests modulation of key gene expression programs between sensitive and resistant cells, including E2F signaling. Further analyses to validate these data are ongoing and the results will be presented during the meeting. This study represents, to our knowledge, the first attempt to generate a single-cell transcriptomic profiling of a large panel of luminal BC cell lines resistant to palbociclib. Results from this study might lead to the identification of new biomarkers of de-novo and acquired resistance to palbociclib, which would assist to better personalize treatment of patients with endocrine receptor positive BC. Citation Format: Matteo Benelli, Giulia Boccalini, Dario Romagnoli, Martina Bonechi, Chiara Biagioni, Rachel Schiff, Carmine De Angelis, Angelo Di Leo, Ilenia Migliaccio, Luca Malorni. Single-cell transcriptomic characterization of luminal breast cancer cell lines with acquired resistance to the CDK4/6 inhibitor palbociclib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3012.