Inhibitor Palbociclib Research Articles

LB932 Dermatomyositis following treatment with cyclin-dependent kinases inhibitors: A single-center retrospective cohort study

The cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) palbociclib, ribociclib, and abemaciclib have been reported to show an association with new-onset subacute lupus erythematosus (SCLE) but CDK4/6i’s role in new-onset dermatomyositis (DM) is unexplored. We evaluated the clinical characteristics of patients presenting with new-onset DM following CDK4/6i. In this retrospective cohort study we queried data registries of Mass General Brigham and Dana-Farber Cancer Institute from October 1, 2014 - November 30, 2021, excluding patients with rash pre-initiation or post-termination of CDK4/6i.

CDK4/CDK6 Inhibitors Synergize with Midostaurin, Avapritinib, and Nintedanib in Inducing Growth Inhibition in KIT D816V+ Neoplastic Mast Cells.

Simple SummaryAdvanced systemic mastocytosis (AdvSM) is a rare malignant disease with a poor prognosis due to the drug resistance of neoplastic mast cells. We found that drugs targeting the cell cycle regulators CDK4 and CDK6 profoundly suppress the growth and survival of neoplastic mast cells. Furthermore, these drugs can overcome resistance against KIT D816V-targeting drugs, including midostaurin, in neoplastic mast cells. Finally, the CDK4/CDK6 inhibitors applied induced apoptosis in CD34+/CD38− stem cells in AdvSM. Based on these results, we believe that CDK4/CDK6 inhibition may be a new and interesting therapeutic approach with curative potential for AdvSM. Whether combinations of KIT D816-targeting drugs and CDK4/CDK6 inhibitors can induce long-term remission in patients with AdvSM remains to be determined in clinical trials.In most patients with advanced systemic mastocytosis (AdvSM), neoplastic mast cells (MC) express KIT D816V. However, despite their disease-modifying potential, KIT D816V-targeting drugs, including midostaurin and avapritinib, may not produce long-term remissions in all patients. Cyclin-dependent kinase (CDK) 4 and CDK6 are promising targets in oncology. We found that shRNA-mediated knockdown of CDK4 and CDK6 results in growth arrest in the KIT D816V+ MC line HMC-1.2. The CDK4/CDK6 inhibitors palbociclib, ribociclib, and abemaciclib suppressed the proliferation in primary neoplastic MC as well as in all HMC-1 and ROSA cell subclones that were examined. Abemaciclib was also found to block growth in the drug-resistant MC line MCPV-1, whereas no effects were seen with palbociclib and ribociclib. Anti-proliferative drug effects on MC were accompanied by cell cycle arrest. Furthermore, CDK4/CDK6 inhibitors were found to synergize with the KIT-targeting drugs midostaurin, avapritinib, and nintedanib in inducing growth inhibition and apoptosis in neoplastic MCs. Finally, we found that CDK4/CDK6 inhibitors induce apoptosis in CD34+/CD38− stem cells in AdvSM. Together, CDK4/CDK6 inhibition is a potent approach to suppress the growth of neoplastic cells in AdvSM. Whether CDK4/CDK6 inhibitors can improve clinical outcomes in patients with AdvSM remains to be determined in clinical trials.

Abstract 1046: Combination of CDK 4/6 inhibitor Palbociclib with Rapamycin synergistically inhibits the growth of hepatocellular carcinoma cells

Abstract Introduction: Aberrant cell-cycle regulation is common in cancer, making it an attractive therapeutic target. The cell cycle inhibitor Palbociclib has been shown to have a cytostatic effect on HCC cells via the induction of quiescence and senescence, though it has limited cytocidal effect. We hypothesized that arrested cells upregulate mTOR signaling to stay metabolically active and investigated the effect of combining Palbociclib with the mTOR inhibitor Rapamycin on HCC cell lines in-vitro. Methods: We employed single sample gene set enrichment analysis (ssGSEA) of 10 paired RNA-seq and 15 paired proteomic data sets as well as 371 HCC tumors from TCGA to identify commonly altered molecular markers in HCC, including cell cycle related genes. Subsequently, we studied the effect of Palbociclib and Rapamycin on two HCC cell lines, SNU398 and HuH7. We treated the cell lines with increasing doses of Palbociclib and used cell counting to assess growth inhibition, as well as β-galactosidase activity assay to assess induction of senescence. We then treated both cell lines with Palbociclib for ten days followed by Palbociclib plus Rapamycin for 4 days and performed an MTT assay to assess relative viable cell number. We also did a fixed ratio treatment with Palbociclib and Rapamycin, followed by calculation of combination index to determine the nature of their interaction. Finally, we performed Western Blot analysis for various proteins of the cell cycle and mTOR pathways to elucidate the effects of combination treatment. Results: ssGSEA revealed significant positive enrichment of several cell cycle and mitosis related gene sets. Palbociclib treatment showed a dose-dependent inhibition of growth as well as increase in senescence associated β-galactosidase activity for both SNU398 and HuH7 cell lines. Addition of Rapamycin demonstrated a dose-dependent potentiation of Palbociclib induced growth inhibition, and fixed ratio treatment with the two drugs established a synergistic effect as indicated by a combination index of less than 1. Western blot analysis showed increased levels of intrinsic CDK inhibitors p16, p15, p21 and p27 with Palbociclib treatment. Western blot analysis of proteins of mTOR pathway revealed that Palbociclib treatment caused an increase in levels of p-mTOR as well as its downstream signaling proteins p-4eBP1, p-p70 and p-s6, and addition of Rapamycin suppressed their levels. Conclusion: HCC cells over-express genes and proteins that drive cell cycle progression. Palbociclib induces senescence in hepatocellular carcinoma cell lines along with an associated upregulation in mTOR signaling, and combination with Rapamycin has a synergistic effect on growth inhibition in-vitro possibly by eliminating senescent cells. Citation Format: Ankur Tiwari, Araceli Huerta, Hakim Bouamar, Debodipta Das, Francisco G. Cigarroa, LuZhe Sun. Combination of CDK 4/6 inhibitor Palbociclib with Rapamycin synergistically inhibits the growth of hepatocellular carcinoma 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 1046.

Abstract 5378: PMD-026, a first in class oral RSK inhibitor, demonstrates activity against hormone receptor positive breast cancer with acquired CDK4/6 inhibitor resistance

Abstract PMD-026 is a first in class, reversible, oral small molecule inhibitor of p90 ribosomal S6 kinase (RSK), a kinase family activated by the MAPK, PDK-1 and PI3K pathways, which regulate substrates involved in cancer cell proliferation and drug resistance. Specifically, both MAPK and PI3K pathways are implicated in resistance to standard of care (SOC) CDK4/6 inhibitors in hormone receptor positive (HR+) breast cancer (BC). RSK2 is expressed in 80% of HR+ BC, therefore we hypothesized that disrupting these critical pathways with PMD-026 could overcome resistance to CDK4/6 inhibitors through cell growth inhibition, apoptosis induction and downstream target inhibition alone and in combination with SOC SERD fulvestrant. In preclinical studies and a Phase I/Ib clinical trial in metastatic BC, PMD-026 demonstrated a good safety profile, making it an ideal candidate for the CDK4/6 inhibitor resistant HR+ BC population. Here in we demonstrate that PMD-026 inhibits growth of HR+ BC cell lines MCF-7 and T47D in vitro at IC50 values ranging from 3.7 to 10.1 µM, resulting in dephosphorylation of the RSK downstream effector YB-1 and induction of apoptosis through PARP cleavage. Comparatively, the MAPK pathway remains active in the presence of fulvestrant, as indicated by the expression of pYB-1. We determined that PMD-026 synergizes with fulvestrant in the MCF-7 model in vitro with a combined drug index (CDI) of 0.73 to 0.92. The next step was to determine if this combination is effective in cells resistant to CDK4/6 inhibitors. For that purpose, MCF-7 and T47D cells were treated with increasing concentrations of the CDK4/6 inhibitor palbociclib (Ibrance) up to 2 µM for several months. The generated Ibrance resistant cell lines (MCF-7-IBR, T47D-IBR) are insensitive to Ibrance up to 2 µM, but their parental counterparts have IC50 values ranging from 0.05 to 0.15 µM. In addition, cross-resistance to an additional CDK4/6 inhibitor, abemaciclib (Verzenio), was observed in the resistant cell lines, but the parental lines remained sensitive at IC50 values ranging from 0.15 to 0.25 µM. Most importantly, both parental and resistant cell lines remained sensitive to PMD-026 at similar IC50 values, indicating that CDK4/6 inhibitor resistance does not affect PMD-026 sensitivity. PMD-026 was then tested in combination with fulvestrant in anchorage independent conditions in the MCF-7-IBR cells. It was determined that this combination was highly synergistic with CDI ranging from 0.55 to 0.79. Together, these data support the application of PMD-026 and fulvestrant as a novel method to stop the growth of HR+ BC with acquired resistance to CDK4/6 inhibitors. Additionally, given that the MAPK pathway is upregulated in response to PI3K inhibition, the PMD-026 and fulvestrant combination is a potential alternative treatment for HR+ BC patients. Citation Format: Aarthi Jayanthan, Lambert Yue, My-my Huynh, Gerrit Los, Sandra E. Dunn. PMD-026, a first in class oral RSK inhibitor, demonstrates activity against hormone receptor positive breast cancer with acquired CDK4/6 inhibitor resistance [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 5378.

Abstract 2978: E2F1 regulates cell competition, increase proliferation and inhibits chemotherapy resistance in cancer cells

Abstract Introduction: Tumor heterogeneity is a key element during the development of chemo-resistance and tumor relapse. It remains unknown how resistant tumor cells influence sensitive cells during conditions of cohabitation. The goal of our study was to determine driving factors mediating interactions between resistant and sensitive cancer cells and the effects of cohabitation on both phenotypes. Materials and methods: We used two isogenic ovarian cancer cell lines pairs, sensitive and resistant to platinum: OVCAR5 (IC50 = 2.05 uM) vs. OVCAR5-CisR (IC50 = 7.26 uM) and PEO1 (IC50 = 5.92 uM) vs. PE04 (IC50 = 14.45 uM) labeled with either GFP or RFP. The cell pairs were seeded together in long direct term culture, separated by fluorescence activated cell sorting (FACS), and used for RNAseq analysis, proliferation assays, Cisplatin IC50 determinations, and cell cycle analysis. Results: Long Term direct co-culture (14 Days) of platinum-sensitive and -resistant cells induced increased proliferation (p < 0.001) and a greater proportions of cells in the S and G2 phases of the cell cycle in sensitive PEO1 and OVCAR5 cells compared with cells cultured by themselves. Resistance to platinum of the sensitive cell lines decreased further by co-culture (IC50 change from 5.92 uM to 2.79 uM in PE01, and from 2.05 uM to 1.51 uM in OVCAR5). No changes in cell proliferation, or resistance to platinum occurred in the platinum resistant cells. Use of conditioned media of resistance cells did not change proliferation, or response to platinum of sensitive cells. RNAseq detected 3840 differentially expressed genes; 1774 upregulated and 2066 downregulated. IPA analysis showed enrichment of the cell cycle pathways (Cell Cycle Control, Cyclins and Cell Cycle Regulation, among others) and upregulation of cell proliferation genes including the E2F1 transcription factor. Western blot and qRT-PCR confirmed upregulation of E2F1 in co-cultured platinum sensitive cells vs cells in monoculture. Furthermore, western blot measurements showed increased levels of other proteins involved in cell cycle regulation such as CDK4 and CDK6. Cell cycle analysis indicated an increase of cells in S and G2 phase in the co-cultured platinum sensitive cells (18.8% up G2 phase; p = 0.031). Treatment with the CDK4/6 inhibitor Palbociclib or with the E2F1 inhibitor HLM006474 reverted the increase in proliferation rate. Conclusion: Long term cohabitation of platinum-sensitive and -resistant cancer cells drives sensitive cells to a higher proliferative state, which is more responsive to platinum. Direct cell-cell contact modulates cell cycle progression in sensitive cells through a mechanism regulated by E2F1. Our data reveal unexpected effects caused by interactions between cancer cells with different proliferative rates and levels of platinum resistance, modelling competition between cells in heterogeneous tumors. Citation Format: Andres Felipe Valdivia, Matthew Cowan, Guangyuan Zhao, Horacio Cardenas, Daniela Matei. E2F1 regulates cell competition, increase proliferation and inhibits chemotherapy resistance in 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 2978.

Abstract 2563: FAK inhibitor APG-2449 and CDK4/6 inhibitor palbociclib synergistically suppress mesothelioma tumor growth via autophagy induction

Abstract Malignant mesothelioma is a rare but highly lethal malignancy, with a 5-year survival rate of less than 10%. Current treatment, which combines surgery, chemotherapy, radiation, and immunotherapy, yields marginal benefit and ultimately results in drug resistance or recurrence. Therefore, developing more effective treatments is an urgent unmet need. Among the most common genomic abnormalities in mesothelioma are alterations of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) and neurofibromin 2 (NF2). Combined CDKN2A deletions and NF2 loss is a negative prognostic factor for both progression-free and overall survival. Mesothelioma cells lacking expression of CDKN2A/B or NF2 are reported to be sensitive to CDK4/6 or focal adhesion kinase (FAK) inhibition, respectively. This study explored the potential utility of combining CDK4/6 inhibitor palbociclib and APG-2449, an investigational, clinical-stage ALK/ROS1/FAK inhibitor, in preclinical models of mesothelioma. Compared to controls and either single agent, the combination of palbociclib and APG-2449 enhanced inhibition of growth of cultured mesothelioma cells. This binary combination also synergistically induced durable antitumor activity in multiple murine mesothelioma models, including both cell line- and patient-derived xenografts. Mechanistically, treatment of mesothelioma cells with this combination likely downregulated FAK phosphorylation and enhanced cellular senescence and autophagy, as evidenced by an increase in the percentage of β-galactosidase-positive cells and upregulation in protein expression levels of autophagy markers P62 and LC3B. The combination also downregulated DNA damage-relevant proteins high mobility group box protein 1 (HMGB1) and γ-H2A histone family member X (ϒ-H2AX). Taken together, the data implicate that an interplay between autophagy and DNA damage repair contributes to the synergistic antitumor effects. In summary, our results unveil a novel potential therapeutic strategy of targeting both FAK and CDK4/6 in order to synergistically inhibit tumor growth in mesothelioma. The synergistic effects are evidently mediated by induced autophagy and enhanced cellular senescence. This preclinical study lays a foundation for future clinical development of APG-2449 combined with CDK4/6 inhibitors for mesothelioma. Citation Format: Ran Tao, Douglas D. Fang, Yuanbao Li, Guoqin Zhai, Feng Zhou, Qixin Wang, Jing Deng, Dajun Yang, Yifan Zhai. FAK inhibitor APG-2449 and CDK4/6 inhibitor palbociclib synergistically suppress mesothelioma tumor growth via autophagy induction [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 2563.

Abstract 1789: Chromatin modifier alterations confer resistance to endocrine deprivation and CDK4/6 inhibitors in ER+ breast cancer and drive convergent evolution in patient autopsy lesions

Abstract We studied resistance mechanisms to hormone therapy and CDK4/6 cell cycle inhibitors in ER+ breast cancer by analyzing whole-exome, whole-genome, single-cell, and bulk transcriptomes in 120 autopsy samples from 13 patients obtained by the Massachusetts General Hospital Rapid Autopsy program. For each patient, we inferred the clonal structure of the samples and tracked the metastatic spread of different clones throughout the body. For 7 patients, we also analyzed serial cfDNA samples to identify clones that were selected for during treatment. We identified significantly recurrent and convergent (arising independently in distinct clones) acquired mutations in ESR1, KRAS, and chromatin modifier genes, in particular, mutations in KMT2C, which may represent mechanisms of drug resistance in this clinical setting. To experimentally study the role of KMT2C mutations, we used CRISPR/Cas9 to knock out KMT2C in the ER+ CAMA1 breast cancer cell line that is sensitive to both ER and CDK4/6 inhibition. KMT2C knock-out cells demonstrated significantly increased viability under treatment with fulvestrant (ERi), palbociclib (CDK4/6i), or their combination compared to the control cell lines. We show that this increased drug resistance is driven by downregulation of the ESR1 pathway, suggesting a decreased dependency on ER signaling for cell cycle progression. In addition to the early survival benefit, KMT2C knock-out resulted in a dramatic outgrowth of cells under long-term fulvestrant treatment. The KMT2C KO fulvestrant-resistant outgrown cells were highly resistant to the CDK4/6 inhibitors palbociclib, ribociclib, and abemaciclib compared to control cells, as well as to novel ERalpha inhibitors and a range of targeted therapies currently in clinical trials. By testing a panel of compounds on KMT2C KO and control cell lines, we propose potential novel therapeutic strategies that may help overcome the development of resistance in KMT2C-mutant cells. These findings suggest that KMT2C mutations may be a mechanism of acquired resistance to CDK4/6 inhibitor combinations, and subsequent treatment with therapies directed towards ER or CDK4/6 pathways may be ineffective and other treatment avenues need to be developed. Citation Format: Elizaveta Leshchiner, Ignaty Leshchiner, Elizabeth E. Martin, Christopher T. Chen, Thomas Zhang, Christopher Pinto, Kahn Rhrissorrakrai, Filippo Utro, Chaya Levovitz, Raquel A. Jacobs, Brian P. Danysh, Kara Slowik, Maida Broudo, Laxmi Parida, Dejan Juric, Gad Getz. Chromatin modifier alterations confer resistance to endocrine deprivation and CDK4/6 inhibitors in ER+ breast cancer and drive convergent evolution in patient autopsy lesions [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 1789.

Abstract LB196: SPR965, a PI3K/mTORC1/C2 inhibitor for treatment of chordoma

Abstract Introduction: Chordoma is a rare tumor with a median survival of 7 years in US. At present, standard treatment involves surgery and/or radiation. Complete surgical resection, however, can be challenging as the tumor is located on the skull base or spine. Moreover, there is currently no approved systemic therapy for advanced chordoma. These challenges have resulted in significant morbidity and a dismal cure rate of approximately 30%, highlighting the need for development of new therapeutic strategies for this orphan disease. Emerging evidence suggests a key role for the PI3K/mTOR pathway in chordoma and numerous reports point to an enrichment of chordoma cases in individuals with tuberous sclerosis syndrome, which is driven by dysregulated mTOR activity. Another study of chordoma tumors from 111 patients demonstrated that the mTOR pathway was activated in 46% of the tumors, and sequencing studies have revealed oncogenic alterations to the PI3K/mTOR pathway in approximately 15-20% of chordomas. We surmised that dual inhibition of both the mTOR and the PI3K pathways may represent a desirable profile as it will not only have the potential to address the effects of activation of both these targets, but also prevent feedback reactivation of the pathway resulting from the inhibition of one or the other. Previously, SPR965 was shown as a potent, selective and orally bioavailable PI3K and mTOR C1/C2 inhibitor with an excellent PK/ADME profile. Methods: SPR965 and comparator compounds were evaluated in vitro in the chordoma cell lines JHC7, U-CH1, and U-CH2 by (Resarufin assay). SPR965 was evaluated in vivo in multiple chordoma PDX models. Results: SPR965 in vitro showed significant effect on cell proliferation with EC50 of 50-90nM. SPR965 was more active at 1uM than PI3K/mTOR inhibitors VS-5584, Bimiralisib, PF-04691502, WYE-687, Dactolisib, and Voxtalisib in the Kinase Chemogenomic Set. SPR965 in vivo monotherapy in SF8894 PDX at 3mg/kg had similar tumor growth inhibition (TGI) to CDK4/6 inhibitor Palbociclib at 50mg/kg. SPR965 combination with Palbocicilib had similar TGI to PI3K inhibitor Buparlisib at 35mg/kg. In CF459 PDX, SPR965 potently inhibited tumor growth at 10mg/kg. TGI% was statistically significant with SPR965 versus untreated controls in both models. Conclusion: In SF8894 PDX model SPR965 was more efficacious as it produced similar tumor growth inhibition at 12- and 17-fold lower dose than Buparlisib (PI3K) and Palbociclib (CDK4/6) respectively, both these compounds are in phase 2 clinical trials. In CF459 PDX model SPR965 exhibited dose-dependent tumor growth inhibition, and at 10mg/kg profoundly slowed tumor growth compared to Palbociclib. Studies are underway to elucidate potential mechanisms of SPR965 sensitivity. SPR965 represents a significant opportunity for development of a targeted therapeutic for chordoma patients. Citation Format: Sundeep Dugar, Somdutta Sen, Paul Maffuid, Dan Freed, Josh Sommer, Edward Anderson, Anthony Hickey. SPR965, a PI3K/mTORC1/C2 inhibitor for treatment of chordoma [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 LB196.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [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 SY24-03.

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 2606: ZN-c3, a potent and selective Wee-1 inhibitor demonstrates anti-tumor activities in combination with other targeted therapies and overcomes PARP inhibitor resistance

Abstract Inhibition of the DNA damage response (DDR) pathway is an attractive approach to target cancers cells. Unlike normal cells which relay on two cell cycle checkpoints to repair DNA damage, most tumor cells only depend on the G2 checkpoint because their G1 checkpoint is defective. Wee1 regulates the G2/M checkpoint and abrogating the G2 checkpoint by inhibiting Wee-1 in cancer cells with DNA damage can preferentially sensitize cancer cells to undergo mitotic catastrophe. We have developed and characterized ZN-c3, a potent and selective Wee-1 inhibitor with a differentiated kinase selectivity and pharmacokinetics profile. ZN-c3 was evaluated in ER-positive, triple negative, triple negative PARP inhibitor resistant, and Her-2 positive trastuzumab resistant breast cancers. In the ER-positive MCF-7 efficacy model, ZN-c3 demonstrated anti-tumor activity as a single agent as well as in combinations with a CDK4/6 inhibitor (Palbociclib) or ZN-c5, a selective estrogen receptor degrader (SERD). In the MDA-MB-436, a triple negative breast cancer tumor xenograft model, ZN-c3 demonstrated efficacy as a single agent and in combination with the PARP inhibitors Olaparib or Niraparib. We extended these combination studies by evaluating the efficacy of Niraparib in combination with ZN-c3 in patient-derived xenograft (PDX) triple negative breast tumors. ZN-c3 was efficacious as a single agent and the efficacy was enhanced in the combination with Niraparib. PARP inhibitors are effective and have been approved for treatment of some cancer types. However, most patients develop resistance to these agents. We sought to investigate whether ZN-c3 can overcome the acquired resistance to PARP inhibitors (Olaparib and Niraparib). We generated PARP inhibitor resistant breast tumor xenograft models. A significant tumor growth inhibition was observed in these resistant tumor xenograft models when treated with ZN-c3. These data suggest that ZN-c3 as a single agent can overcome the acquired PARP inhibitor resistance. Finally, we also evaluated ZN-c3 activity in a HER-2 positive trastuzumab resistant breast tumor xenograft model (JIMT-1) and ZN-c3 showed anti-tumor activity alone and in combination with trastuzumab. In summary, these results demonstrate that ZN-c3 is effective as a single agent and in combination with other targeted therapies in breast cancer models. Moreover, ZN-c3 can overcome the acquired resistance to PARP inhibitors and trastuzumab. ZN-c3 is currently in clinical trials for several indications and has demonstrated promising clinical activity and good tolerability. Citation Format: Jiali Li, Jianhui Ma, Petrus R. de Jong, Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Brant Boren, Sayee Hegde Hegde, Fernando Doñate, Ahmed A. Samatar. ZN-c3, a potent and selective Wee-1 inhibitor demonstrates anti-tumor activities in combination with other targeted therapies and overcomes PARP inhibitor resistance [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 2606.

Abstract 2573: PI3K/AKT/mTOR pathway and CDK4/6 inhibitors efficiently inhibit cell growth of HPV-positive and HPV-negative head and neck squamous cell carcinoma in vitro

Abstract Introduction: Both HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) often show activation of the PI3K/AKT/mTOR pathway, due to, amongst others, PI3KCA mutations, PTEN loss, or receptor tyrosine kinase activation. In HPV-negative tumors, CDKN2A (p16) inactivation or CCND1 (cyclin D1) amplification frequently occurs resulting in sustained cyclin dependent kinase (CDK) 4/6 activation. The aim of our study was to investigate the efficacy of PI3K/AKT/mTOR pathway inhibitors (PI3Ki) (alpelisib, buparlisib and gedatolisib) and CDK4/6 inhibitors (CDKi) (palbociclib and ribociclib) in HPV-positive and -negative HNSCC cell lines. Methods: The efficacy of the inhibitors was assessed using MTT assays and changes in PI3K and Cyclin D1/CDK pathway protein expression were determined by Western blotting. Cell cycle analysis was performed with flow cytometry and apoptosis was assessed by Annexin-V staining. Changes in cell metabolism were assessed by Seahorse XF assays. Results: Both HPV-positive and -negative HNSCC cell lines were highly sensitive to the PI3Ki (Gedatolisib, IC50 5-30 nM > Buparlisib, IC50 0.6-3.6 µM > Alpelisib, IC50 3-23 µM). In general, PI3Ki decreased pathway activity, resulted in moderate cell cycle arrest and apoptosis, and decreased oxidative and glycolytic metabolism. CDKi were particularly effective in blocking HPV-negative cell line growth (Palbociclib, IC50 0.5-2 µM > Ribociclib, IC50 4-7 µM). CDK inhibition showed decreased pRb expression and G1 cell cycle arrest, whereas apoptosis was not induced. Conclusion: PI3Ki and CDKi efficiently inhibit their respective pathways and HNSCC cell growth in vitro, the latter only in HPV-negative cell lines. Whereas PI3Ki especially show an effect on oxidative and glycolytic metabolism, CDKi particularly lead to cell cycle arrest. Further research should elucidate whether these inhibitors may be effective therapeutic agents in HNSCC patients. Citation Format: Imke Demers, Femke Verhees, Dion Leegemaate, Robin Jacobs, Ann Hoeben, Frank Hoebers, Bernd Kremer, Ernst-Jan Speel. PI3K/AKT/mTOR pathway and CDK4/6 inhibitors efficiently inhibit cell growth of HPV-positive and HPV-negative head and neck squamous cell carcinoma in vitro [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 2573.

Abstract 650: Reparixin, CXCR1/2 inhibitor in combination with CDK4/6 inhibitors attenuates endocrine resistant breast cancer growth and metastasis

Abstract Various endocrine therapies have been developed such as selective estrogen receptor modulation (SERM), selective estrogen receptor downregulator (SERD) and ligand deprivation using aromatase inhibitors (AI). Tamoxifen (TAM), a SERM and AIs are the most commonly used adjuvant treatment for postmenopausal women with early-stage estrogen receptor positive breast cancer. Despite the relative safety and significant anti-neoplastic and chemopreventive activities of tamoxifen and AIs, many initially responsive breast tumors develop resistance and ultimately recur. Many underlying molecular events that confer resistance are known, but a unifying theme is yet to be revealed. While the tumor microenvironment is being increasingly recognized as a key factor in multiple stages of disease progression, particularly local resistance, immune-escaping, and distant metastasis, our previous work shows that the critical molecular and cellular players secreted from a crosstalk between breast cancer cells and stromal cells can regulate the tumor growth and process of metastasis in breast cancer. In this report, using established endocrine resistant breast cancer (ERBC) cells and ESR1 mutant cells (Y537S and D538G), we screened the secreted factors in crosstalk between cancer cells and fibroblasts using cytokine antibody arrays and found that CXCL1 is highly upregulated in fibroblasts. To investigate a potential impact given the current clinical landscape of endocrine resistant breast cancer, we performed a high throughput cell-based screen to identify effective combinations of drugs that will be tested in vivo using reparixin, CXCR1/2 inhibitor, tamoxifen (SERM), fulvestrant (SERD), and CDK4/6 inhibitors (Palbociclib and Ribociclib) in co-culture with ERBC cells and fibroblast. Significantly, reparixin decreased ERBC cell viability compared to vehicle while reparixin in combination with tamoxifen and fulvestrant, enhanced the inhibitor effects in ERBC cell growth. We found that the combination treatment of reparixin and Palbociclib and ribociclib significantly inhibited ERBC cell growth and migration compared the single treatment. The inhibitory effect of reparixin was higher in combination with CDK4/6 inhibitors that the combination with tamoxifen and fulvestrant. Furthermore, reparixin in combination with CDK4/6 inhibitors decreased ERBC cell migration and promotes apoptosis.These findings implicate CXCL1 signaling as a critical event in ERBC tumor growth and metastasis via crosstalk between cancer cells and stromal components. Further, these studies suggest that CXCL1 act as key regulators orchestrating ERBC. Therefore, we have provided evidence that supports the hypothesis that functional inhibition of the CXCL1 and CDK4/6 signaling pathway has the potential to circumvent ERBC growth and metastasis. Citation Format: Sneha Pandithar, Olivia Reff, Kideok Jin. Reparixin, CXCR1/2 inhibitor in combination with CDK4/6 inhibitors attenuates endocrine resistant breast cancer growth and metastasis [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 650.

Abstract 2304: Comprehensive characterization of CDK inhibitors using a complete panel of all 20 human cyclin-dependent kinases

Abstract The family of human Cyclin dependent kinases (CDKs) comprises 20 different CDKs that play critical roles in the regulation of cell cycle progression, gene transcription and neuronal function. Deregulation of different CDKs is frequently observed in human cancer. Enzymatic kinase activity of CDKs is dependent on the binding of a member of the Cyclin protein family. So far more than 15 Cyclins have been described most of them can bind and activate different CDKs. Current data suggest the physiological relevance of at least 50 different CDK/Cyclin complexes. Since the 20 CDKs share significant structural homology and regulate different function in cell growth and development, selectivity of compounds within the CDK family is of critical importance. Approval of a first CDK inhibitor (Palbociclib) targeting CDK4/6 for the treatment of ER+/HER+- breast cancer served as a clinical proof that targeting specific members of the CDK protein kinase family is a versatile approach to treat cancer. The approval of the first CDK inhibitor sparked the research and development of other inhibitors targeting different members of the CDK-family. Currently, four additional CDK4/6 inhibitors have been approved and more than 15 CDK inhibitors with limited selectivity are in different preclinical or clinical development phases. However, the critical importance of selectivity within the CDK family is underlined by the fact that the clinical development of four CDK9 inhibitors has been stopped due to the lack of selectivity and high toxicity. Different approaches (including inhibition by covalent binding) have resulted in more selective inhibitors, especially against CDKs like CDK7, CDK9 and CDK12. Although several biochemical activity assays for different CDKs have been set up and used for selectivity testing, so far no panel covering all 20 human CDKs using one assay technology has been described. We report here the setup of a biochemical in-vitro activity assay panel of 32 CDK/Cyclins complexes generated recombinantly in insect cells covering the complete set of all 20 human CDKs. For all 32 complexes a radiometric biochemical activity assay has successfully been established allowing to characterize inhibitors with respect to their biochemical selectivity applying the same assay technology. Using the comprehensive CDK panel we determined the IC50 values of more than 15 CDK inhibitors that have been either approved or are in different preclinical and clinical development phases. Results will be presented showing the selectivity of these inhibitors not only for all 20 CDKs but also for specific CDKs forming active complexes with two or more different cyclins. This CDK screening panel allows the generation of comparative data on compound selectivity early in development, thereby helping to reduce the risk of designing compounds with suboptimal target selectivity. Citation Format: Daniel Müller, Frank Totzke, Thomas Weber, Andreas Gericke, Diane Krämer, Carolin Heidemann-Dinger, Constance Ketterer, Michael H. Kubbutat. Comprehensive characterization of CDK inhibitors using a complete panel of all 20 human cyclin-dependent kinases [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 2304.

Phase Ib/II study of BCL-2 inhibitor lisaftoclax (APG-2575) safety and tolerability when administered alone or combined with a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor in patients with estrogen receptor-positive (ER+) breast cancer or advanced solid tumors.

TPS1122 Background: Various cancers evade apoptosis by overexpressing BCL-2 proteins. Investigational agent lisaftoclax (APG-2575) is a novel, potent, selective BCL-2 inhibitor, while palbociclib inhibits cyclin-dependent kinases (CDK) 4 and 6. In preclinical studies, palbociclib decreased proliferation of ER+ breast cancer cell lines by arresting them in the G1 cycle phase. Preclinical data demonstrate favorable, complementary effects of palbociclib when combined with a BCL-2 inhibitor and support this combination in patients with ER+/ human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer. Methods: This global multicenter open-label dose escalation and dose expansion study is assessing the safety of lisaftoclax monotherapy in patients with histologically or cytologically confirmed advanced solid tumors that have progressed on standard therapy or when combined with CDK4/6 inhibitor palbociclib in physiologically postmenopausal women with ER+/ HER2- metastatic breast cancer that has progressed or relapsed after treatment with a CDK4/6 inhibitor. This trial consists of 2 parts: a phase Ib dose escalation phase using a standard 3+3 design to determine the maximum tolerated dose (MTD) of lisaftoclax as a single agent in patients with solid tumors, as well as both the MTD and recommended phase 2 (RP2D) dose of lisaftoclax when combined with palbociclib in women with ER+/ HER2- metastatic breast cancer. Phase II of this study is a signal-seeking expansion of lisaftoclax at RP2D when combined with palbociclib in women with ER+/HER2- metastatic breast cancer. This phase is being conducted using Simon’s Minimax two-stage design. The primary objective for phase II is to determine clinical benefit response, and secondary efficacy endpoints include overall response rate, duration of response, time to response, and progression-free survival. Lisaftoclax is being administered orally once daily in a 28-day cycle at the assigned dose. Clinical trial information: NCT04946864.

The effect of BMI on the outcomes of CDK 4/6 inhibitor therapy in HR-positive metastatic breast cancer patients.

e13010 Background: The standard of care in patients with hormone receptor (HR) -positive metastatic breast cancer is a cyclin-dependent kinase (CDK) 4/6 inhibitor combined with endocrine therapy (ET). Preclinical studies show that metabolic processes and cell metabolism such as adipogenesis, lipid synthesis and glucose regulation is affected by cell cycle regulators such as CDK 4 and 6. There are limited data regarding the impact of body mass index (BMI) on the efficacy of cyclin-dependent kinase 4/6 inhibitors plus endocrine therapy (ET) in metastatic breast cancer. We aimed to investigate the effect of BMI on the progression-free survival (PFS) in HR-positive metastatic breast cancer who received CDK4/6 inhibitor. Methods: This study was conducted as a retrospective cohort study, and data were obtained from three institution medical records. Patients with metastatic HR-positive breast cancer receiving CDK 4/6 inhibitor (palbociclib or ribociclib) plus ET (letrozole, anastrozole, or fulvestrant) were enrolled in the study. 179 patients were enrolled in the study between the January 2018 and December 2021. The patients were divided into three groups according to BMI level as follows; group 1: 18.5-24.9 kg/m2, group 2: 25-29.9 kg/m2 and group 3: ≥ 30 kg/m2. Median follow-up was 10.94 months. Categorical variables were compared using the chi-square test or two-sided Fisher’s exact test. Comparison of PFS and BMI categories were performed through Kaplan-Meier curve and log-rank test. Results: 179 patients were included the study (42 [24%] in group 1, 65 [36%] in group 2, and 72 [40%] in group 3). The 12-month PFS was 52.9% in group 1, was 72.2% in group 2 and was 56.5% in group 3. There was no significant different between the groups (p = 0.054). However, group 2 patients tend to have a better outcome in term of PFS. When palbociclib plus ET and ribociclib plus ET were evaluated separately, the 12-month PFS rates between the BMI groups were higher in group 2 patients who received palbociclib plus ET, but there was no statistical significance (Ribociclib plus ET: 63.2% in group 1, 73.3% in group 2, 59.7% in group 3, p = 0.51. Palbociclib plus ET: 44.3% in group 1, 71.5% in group 2, 52.8% in group 3, p = 0.07). Hematological and non-hematological toxicity were similar between BMI groups (p > 0.05, for all). Adverse events of grade 3 or 4 occurred in 18 (42.9%) of 42 patients in group 1 and in 20 (30.8%) of 65 patients in group 2 and in 22 (30.6%) of 72 patients in group 3 (p = 0.34). Dose reduction for CDK4/6 inhibitors was also similar between groups (p = 0.42). Median PFS was not reached due to a limited number of events in each group. Conclusions: In this study, we demonstrated the tendency that overweight patients with metastatic breast cancer may benefit more from CDK4/6 inhibitors in term of PFS from interim analyses of our data. Similar toxicity rates and adverse events were observed in all BMI groups with CDK4/6 inhibitors plus ET.

The Acquired Vulnerability Caused by CDK4/6 Inhibition Promotes Drug Synergism Between Oxaliplatin and Palbociclib in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult to treat cancers, and its nature of being largely refractory to most, if not all, current treatments results in generally poor prognosis and high mortality. Efficacious alternative therapies that can be used ubiquitously are urgently needed. Using acquired vulnerability screening, we observed that CCA cells that reprofile and proliferate under CDK4/6 inhibition became vulnerable to ribosomal biogenesis stress and hypersensitive to the anti-ribosome chemotherapy oxaliplatin. CCA cells overexpress the oncogenic ribosomal protein RPL29 under CDK4/6 inhibition in a manner that correlated with CDK4/6 inhibitor resistance. Depletion of RPL29 by small interfering RNAs (siRNAs) restored the sensitivity of CCA cells to CDK4/6 inhibition. Oxaliplatin treatment suppressed the RPL29 expression in the CDK4/6 inhibitor treated CCA cells and triggered RPL5/11-MDM2-dependent p53 activation and cancer apoptosis. In addition, we found that combination treatment with oxaliplatin and the CDK4/6 inhibitor palbociclib synergistically inhibited both parental and CDK4/6 inhibitor-resistant CCA, and prevented the emergence of CDK4/6 and oxaliplatin-resistant CCA. This drug combination also exerted suppressive and apoptosis effects on CCA in the in vitro 3-dimensional culture, patient-derived organoid, and in vivo xenograft CCA models. These results suggest the combination of the CDK4/6 inhibitor palbociclib and the anti-ribosome drug oxaliplatin as a potentially promising treatment for cholangiocarcinoma.

Real world incidence and management of adverse events in patients with HR+, HER2− metastatic breast cancer receiving CDK4 and 6 inhibitors in a United States community setting

Objective To examine the real-world incidence and management of select adverse events (AEs) among female patients with hormone receptor positive (HR+), human epidermal growth factor receptor 2-negative (HER2−) metastatic breast cancer (MBC), receiving a cyclin-dependent kinase 4 and 6 (CDK4 and 6) inhibitor (palbociclib, abemaciclib, or ribociclib). Methods This retrospective study analyzed data from the US Oncology Network iKnowMed electronic health record database for 396 patients with an initial MBC diagnosis on/after 1 January 2014 and receipt of first CDK4 and 6 regimen between 1 January 2017 and 31 December 2018. In this descriptive study, the proportion of patients who experienced select AEs and associated dose modifications or discontinuations were reported. The occurrence of select healthcare resource utilization categories was also reported. Results Median follow-up time was 451, 262, and 355 days for patients in the palbociclib, abemaciclib, and ribociclib cohorts, respectively. The most common AEs were neutropenia (palbociclib, 44.8%; abemaciclib, 10.6%; ribociclib, 36.3%), diarrhea (palbociclib, 8.0%; abemaciclib, 43.0%; ribociclib, 8.8%), and fatigue (palbociclib, 12.9%; abemaciclib, 17.6%; ribociclib, 16.5%). AEs resulted in a treatment hold among 91 (23.0%), a dose reduction among 86 (21.7%), and permanent discontinuation among 48 (12.1%) patients overall. Conclusions This real-world study provides insight into the occurrence of AEs which varied by CDK4 and 6 inhibitor. Compared to clinical trials, frequencies of AEs were numerically lower but dose reductions due to AEs were numerically higher. It is possible these differences reflect proactive management of AEs on the part of clinicians to help patients remain on therapy.

194P Increased blood creatinine level due to CDK 4/6 inhibitor treatments: Single center experience

Combinations of cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6) (ribociclib, palbociclib, abemaciclib) and endocrine therapy (ET) are the first-line recommended treatment in patients with hormone receptor (HR)-positive HER2-negative metastatic breast cancer, unless there is visceral crisis due to organ metastases.We aimed to report the increase in creatinine levels in our patients who developed due to ribociclib and palbociclib use, which were not reported among the common side effects of CDK 4/6 inhibitor treatment and because there was no report in the literature.

IFN-γ and TNF Induce Senescence and a Distinct Senescence-Associated Secretory Phenotype in Melanoma.

Immune checkpoint blockade (ICB) therapy is a central pillar of melanoma treatment leading to durable response rates. Important mechanisms of action of ICB therapy include disinhibition of CD4+ and CD8+ T cells. Stimulated CD4+ T helper 1 cells secrete the effector cytokines interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF), which induce senescence in tumor cells. Besides being growth-arrested, senescent cells are metabolically active and secrete a large spectrum of factors, which are summarized as senescence-associated secretory phenotype (SASP). This secretome affects the tumor growth. Here, we compared the SASP of cytokine-induced senescent (CIS) cells with the SASP of therapy-induced senescent (TIS) cells. Therefore, we established in vitro models for CIS and TIS in melanoma. The human melanoma cell lines SK-MEL-28 and WM115 were treated with the cytokines IFN-γ and TNF as CIS, the chemotherapeutic agent doxorubicin, and the cell cycle inhibitor palbociclib as TIS. Then, we determined several senescence markers, i.e., growth arrest, p21 expression, and senescence-associated β-galactosidase (SA-β-gal) activity. For SASP analyses, we measured the regulation and secretion of several common SASP factors using qPCR arrays, protein arrays, and ELISA. Each treatment initiated a stable growth arrest, enhanced SA-β-gal activity, and—except palbociclib—increased the expression of p21. mRNA and protein analyses revealed that gene expression and secretion of SASP factors were severalfold stronger in CIS than in TIS. Finally, we showed that treatment with the conditioned media (CM) derived from cytokine- and palbociclib-treated cells induced senescence characteristics in melanoma cells. Thus, we conclude that senescence induction via cytokines may lead to self-sustaining senescence surveillance of melanoma.