Inhibitor Palbociclib Research Articles

PRECYCLE: multicenter, randomized phase IV intergroup trial to evaluate the impact of eHealth-based patient-reported outcome (PRO) assessment on quality of life in patients with hormone receptor positive, HER2 negative locally advanced or metastatic breast cancer treated with palbociclib and an aromatase inhibitor or palbociclib and fulvestrant

BackgroundEfficacy and quality of life (QoL) are key criteria for therapy selection in metastatic breast cancer (MBC). In hormone receptor positive (HR +) human epidermal growth factor receptor 2 negative (HER2 −) MBC, addition of targeted oral agents such as everolimus or a cycline-dependent kinase 4/6 (CDK 4/6) inhibitor (e.g., palbociclib, ribociclib, abemaciclib) to endocrine therapy substantially prolongs progression-free survival and in the case of a CDK 4/6i also overall survival. However, the prerequisite is adherence to therapy over the entire course of treatment. However, particularly with new oral drugs, adherence presents a challenge to disease management. In this context, factors influencing adherence include maintaining patients’ satisfaction and early detection/management of side effects. New strategies for continuous support of oncological patients are needed. An eHealth-based platform can help to support therapy management and physician–patient interaction.MethodsPreCycle is a multicenter, randomized, phase IV trial in HR + HER2 − MBC. All patients (n = 960) receive the CDK 4/6 inhibitor palbociclib either in first (62.5%) or later line (37.5%) together with endocrine therapy (AI, fulvestrant) according to national guidelines. PreCycle evaluates and compares the time to deterioration (TTD) of QoL in patients supported by eHealth systems with substantially different functionality: CANKADO active vs. inform. CANKADO active is the fully functional CANKADO-based eHealth treatment support system. CANKADO inform is a CANKADO-based eHealth service with a personal login, documentation of daily drug intake, but no further functions. To evaluate QoL, the FACT-B questionnaire is completed at every visit. As little is known about relationships between behavior (e.g., adherence), genetic background, and drug efficacy, the trial includes both patient-reported outcome and biomarker screening for discovery of forecast models for adherence, symptoms, QoL, progression free survival (PFS), and overall survival (OS).DiscussionThe primary objective of PreCycle is to test the hypothesis of superiority for time to deterioration (TTD) in terms of DQoL = “Deterioration of quality of life” (FACT-G scale) in patients supported by an eHealth therapy management system (CANKADO active) versus in patients merely receiving eHealth-based information (CANKADO inform).EudraCT Number: 2016–004191-22

Resistance to anti-HER2 therapy associated with the TSC2 nonsynonymous variant c.4349 C > G (p.Pro1450Arg) is reversed by CDK4/6 inhibitor in HER2-positive breast cancer

HER2-positive breast cancer patients carrying the germline TSC2 nonsynonymous variant c.4349 C > G (p.Pro1450Arg) are resistant to anti-HER2 therapy. Multi-predictor in silico analysis reveals that this variant is deleterious. We explore the potential mechanism of this TSC2 variant and investigate methods for overcoming anti-HER2 resistance. TSC2 c.4349 C > G reverses the inhibitory effect on mTOR and downstream signaling by increasing TSC2 phosphorylation at Thr1462 and confers significant lapatinib resistance in vitro and in vivo. The combination of lapatinib and the CDK4/6 inhibitor palbociclib inhibits cyclin D1/CDK4/Rb alternative pathway and TSC2 phosphorylation, thereby partially attenuating mTOR activity and inducing TSC2-mutant cell blockage at G1/G0. In in vitro and xenograft models, palbociclib+lapatinib shows higher anti-tumor activity than monotherapy and overcomes the resistance of the TSC2 c.4349 C > G-related variant to anti-HER2 therapy. We reveal a new mechanism of resistance to anti-HER2 therapy and provide a strategy to increase the efficiency of anti-HER2 therapy in HER2-positive breast cancer.

Identification of the KIF and MCM protein families as novel targets for combination therapy with CDK4/6 inhibitors in bladder cancer.

CDK4/6 inhibitors have proven their potency for the treatment of cancer but only in combination with hormone or targeted therapies. The aim of this study was the identification of molecules that are involved in response mechanisms to CDK4/6 inhibitors and the development of novel combination therapies with corresponding inhibitors in bladder cancer. Genes of response to therapy and genes that confer resistance to the CDK4/6 inhibitor palbociclib were identified by performing an analysis of published literature and own published data using a CRISPR-dCas9 genome wide gain of function screen. Genes that were down-regulated upon treatment were compared with genes that confer resistance when up-regulated. Two of the top 5 genes were validated by quantitative PCR and western blotting upon treatment with palbociclib in the bladder cancer cell lines T24, RT112 and UMUC3. As inhibitors for combination therapy, we used ciprofloxacin, paprotrain, ispinesib and SR31527. Analysis of synergy was done using the "zero interaction potency" model. Cell growth was examined using sulforhodamine B staining. A list of genes that met the requirements for inclusion in the study was generated from 7 publications. Of the 5 most relevant genes, MCM6 and KIFC1 were chosen and their down-regulation upon treatment with palbociclib was confirmed by qPCR and immunoblotting. The combination of inhibitors against both, KIFC1 and MCM6 with PD resulted in a synergistic inhibition of cell growth. We have identified 2 molecular targets whose inhibition has promising potential for effective combination therapies with the CDK4/6 inhibitor palbociclib.

C-Myc-Targeting PROTAC Based on a TNA-DNA Bivalent Binder for Combination Therapy of Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is highly aggressive with a poor clinical prognosis and no targeted therapy. The c-Myc protein is a master transcription factor and a potential therapeutic target for TNBC. In this study, we develop a PROTAC (PROteolysis TArgeting Chimera) based on TNA (threose nucleic acid) and DNA that effectively targets and degrades c-Myc. The TNA aptamer is selected in vitro to bind the c-Myc/Max heterodimer and appended to the E-box DNA sequence to create a high-affinity, biologically stable bivalent binder. The TNA-E box-pomalidomide (TEP) conjugate specifically degrades endogenous c-Myc/Max, inhibits TNBC cell proliferation, and sensitizes TNBC cells to the cyclin-dependent kinase inhibitor palbociclib in vitro. In a mouse TNBC model, combination therapy with TEP and palbociclib potently suppresses tumor growth. This study offers a promising nucleic acid-based PROTAC modality for both chemical biology studies and therapeutic interventions of TNBC.

Abstract LB159: Deregulation of CTC-associated RPL/RPS protein expression inhibits melanoma brain metastasis

Abstract Melanoma brain metastasis (MBM) is associated with poor prognosis and low survival rates. MBM is diagnosed clinically in up to 60% of melanoma patients and in up to 80% of patients at autopsy. Circulating Tumor Cells (CTCs) are “seeds” of fatal metastasis and the smallest functional units of cancer. Our previous studies have employed a multilevel approach to identify a common CTC signature for ribosomal protein large/small subunits (RPL/RPS) which is linked to MBM development and progression1. Here, we hypothesized that targeting ribosomal biogenesis prevents MBM onset in CTC-derived xenografts. First, we treated CTC-injected MBM mice with RPL15 inhibitor omacetaxine to monitor metastatic progression. Second, parallel cohorts of mice were treated with CDK4/CDK6 inhibitor palbociclib with or without omacetaxine to assess effects of cell proliferation. Total flux of MBM signal was measured by IVIS and found to be higher in untreated mice. Mouse necropsies revealed decreased levels of MBM, along with metastatic dissemination to other organs. Third, collected mouse blood was analyzed by the FDA-approved ParsortixTM platform to enumerate CTC/CTC clusters (IHC), and evaluate their gene expression (RNA-seq). Lastly, metabolic profiling of inhibitor-treated versus untreated MBM CTC-derived clones was performed in vitro. Collectively, our findings indicate that the deregulation of ribosomal proteins inhibits MBM progression. This implicates a specific vulnerability of aggressive CTC subsets with high RPL/RPS content in MBM patients. RPL/RPS genetic screening could be a prognostic indicator of MBM severity and clinical outcomes. Our work promotes the concept that therapies targeting RPL/RPS proteins merit exploration as potential suppressors of metastatic progression in general, MBM in particular. 1. Bowley T, Lagutina I, Francis C, Sivakumar S, Selwyn R, Taylor E, et al. The RPL/RPS gene signature of melanoma updates CTCs associates with brain metastasis. Cancer Research Communications 2022;2: 1445-57. Citation Format: Tetiana Yuriyivna Bowley, Seth Merkley, Irina Lagutina, Dario Marchetti. Deregulation of CTC-associated RPL/RPS protein expression inhibits melanoma brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB159.

Abstract LB134: Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth

Abstract Background: In-frame chimeric proteins that are encoded by ESR1 exons 1 to 6 (ESR1-e6) fused to C-terminal gene partners can induce hormone-independent growth and endocrine therapy (ET) resistance of estrogen receptor-α (ERα) positive breast cancer by driving a unique transcriptional signature (PMID: 34711608). However, the mechanism how active ESR1 fusions reprogram transcription is not understood. Moreover, the lack of targeted treatment due to the loss of ligand binding domain (LBD) in ESR1 gene fusions necessitates the need to develop new therapies targeting active ESR1 fusions that promote tumorigenesis. Methods: An estrogen response element (ERE) DNA pull down assay was performed to identify coactivators recruited by active ESR1 fusions. An alamar blue assay was utilized to assess cell growth after treatment with pharmacological inhibitors to the coactivators. Single cells isolated from ERα+ patient-derived xenografts (PDX) were used to form organoids (PDxOs). A CellTiter-Glo 3D assay was conducted to measure the growth of PDxOs after treatment with coactivator inhibitors. Results: Using ERE DNA pull down assays, we identified bromodomain-containing proteins (BRDs) and steroid receptor coactivators (SRCs/p160 family members) as key regulators recruited by active ESR1 fusions from nuclear extracts of breast cancer cells. A pan-BET family inhibitor (BETi), birabresib, and pan-SRC inhibitors (SRCi), SI-2 and SI-12, inhibited the growth of ERα positive breast cancer cells and PDxOs expressing an active ESR1 fusion or LBD point mutation. Importantly, the combination of birabresib and a CDK4/6 kinase inhibitor (palbociclib) conferred the largest growth reduction of PDxOs expressing an ESR1-YAP1 fusion. RNA sequencing showed BETi and SRCi led to downregulation of ERα target gene expression and cell cycle pathways, with upregulation of apoptosis pathways. Here, we propose a model whereby BRD and SRC protein families are major regulators of active ESR1 fusion-driven gene transcription and growth and positioned the potential therapeutic significance of BETi and SRCi to be tailored to treat active ESR1-translocated breast tumors. Conclusions: Active ESR1 fusions cannot be treated with standard-of-care ET due to the lack of the LBD of ERα. Therefore, it is critical to investigate how active, in-frame ESR1 fusion proteins induce gene transcription and to develop molecular mechanism-based targeted therapies. Here, we revealed two groups of coactivators (BRDs and SRCs) as critical transcriptional regulators of active ESR1 fusion-driven tumor biology. Our study demonstrated the efficacy of a BETi (birabresib) and SRCi (SI-2/SI-12) in inhibiting the viability of active ESR1 fusion/LBD mutation expressing breast cancer cells and 3D grown PDxOs. Importantly, birabresib combined with a standard-of-care CDK4/6 inhibitor conferred the best PDxO growth reduction. Citation Format: Xuxu Gou, Craig T. Vollert, Yue Chen, Diana Fandino, Matthew V. Holt, Beom-Jun Kim, Qin Feng, Jin Wang, David M. Lonard, Bert W. O'Malley, Matthew J. Ellis, Charles E. Foulds. Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB134.

Abstract CT120: The randomized phase 1/2 LITESPARK-024 study of belzutifan with or without palbociclib in patients with advanced renal cell carcinoma (RCC)

Abstract Background: First-line treatment with immunotherapy alone or in combination with antiangiogenic agents is a standard of care for advanced RCC. Many patients (pts) develop resistance to first-line treatment and effective second-line and beyond options are needed. The von Hippel-Lindau (VHL) gene is inactivated in approximately 90% of RCC cases, which results in the constitutive activation of hypoxia-inducible factor 2α (HIF-2α), a key oncogenic driver in RCC. The first-in-class HIF-2α inhibitor belzutifan has demonstrated promising antitumor activity with manageable safety in previously treated pts with advanced RCC. The cyclin-dependent kinase (CDK) pathway has also been implicated in RCC and is associated with poor clinical outcomes. In RCC cell lines, the CDK 4/6 inhibitor palbociclib inhibited cell growth. The addition of CDK 4/6 inhibition had synergistic antiproliferative effects with HIF-2α inhibition in HIF-2α-dependent VHL -/- RCC cell lines. Palbociclib could therefore potentially enhance the efficacy of belzutifan as combination therapy for previously treated pts with advanced RCC. Methods: The two-part, open-label, multicenter, phase 1/2 randomized LITESPARK-024 study (NCT05468697) is intended to establish the recommended phase 2 dose (RP2D) of belzutifan + palbociclib in combination with a modified toxicity probability interval design (Part 1), followed by a direct comparison of belzutifan monotherapy to the combination with respect to safety and efficacy in pts with advanced RCC (Part 2). Pts with histologically confirmed unresectable stage IV RCC with a clear cell component, whose disease progressed on or after having received at least 2 systemic treatments (both an anti-PD-1/PD-L1 monoclonal antibody and a VEGF receptor-targeted tyrosine kinase inhibitor, in sequence or in combination), have measurable disease per RECIST v1.1 by blinded independent central review, and have KPS score of ≥70% will be enrolled. Up to 30 pts will be enrolled into 3 dose groups in Part 1 and will receive belzutifan 120 mg once daily + palbociclib (75, 100, or 125 mg) daily for 21 consecutive days followed by 7 days off. In part 2, approximately 150 pts will be randomly assigned 2:1 to receive belzutifan 120 mg once daily + palbociclib RP2D (21 consecutive days/7 days off) or belzutifan 120 mg once daily. Pts will be stratified by IMDC risk (0 vs 1-2 vs 3-6) and sarcomatoid histology (yes vs no) at randomization in part 2. The primary end point for part 1 is to assess dose-limiting toxicities and adverse events to determine the RP2D of belzutifan + palbociclib. The primary end point for part 2 is ORR per RECIST v1.1 by investigator assessment. Secondary end points for part 2 are CBR, DOR, PFS, OS, and safety and tolerability. This abstract was accepted and previously presented at the 2023 ASCO Genitourinary Cancers Symposium. All rights reserved. Citation Format: David F. McDermott, Avivit Peer, Neeraj Agarwal, Michael B. Atkins, Jerry Cornell, Rodolfo F. Perini, Kenneth F. Grossmann, Howard Gurney. The randomized phase 1/2 LITESPARK-024 study of belzutifan with or without palbociclib in patients with advanced renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT120.

A phase I study of the combination of palbociclib and dexamethasone for the treatment of relapsed or refractory B-cell acute lymphoblastic leukemia

PurposeDespite advances in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), outcomes for relapsed/refractory (R/R) disease remain poor. Preclinical studies suggest that the combination of the CDK4/6 inhibitor palbociclib and dexamethasone may be effective in targeting leukemic cell growth. We conducted a phase I study of escalating doses of palbociclib in combination with dexamethasone in adults with R/R B-ALL. MethodsCycle 1 consisted of single agent palbociclib given for 7 days and continued for 28 additional days in combination with dexamethasone 20 mg daily. Palbociclib dosing began at 100 mg daily. Patients with a response were eligible for maintenance consisting of 1 week of palbociclib plus dexamethasone (20 mg daily × 2 days, 16 mg daily × 2 days, 12 mg daily × 2 days, 6 mg daily × 1 day), followed by 3 weeks of palbociclib alone. Safety, efficacy, and the expression of phospho-RB and c-MYB/BCL-2 were measured. ConclusionsSeven patients were treated on study before it was closed early due to slow accrual. No dose limiting toxicities were identified. One patient had a complete response with incomplete hematologic recovery, suggesting possible efficacy of the treatment. Reduction in CD34+ cells, p-RB, c-MYB, and BCL-2 expression also suggested on-target therapy effects.

Abstract 2679: Combination of CDK 4/6 inhibitor palbociclib with rapamycin synergistically inhibits growth of hepatocellular carcinoma in preclinical models

Abstract Introduction: The cell cycle inhibitor palbociclib has been shown to have a cytostatic effect on hepatocellular carcinoma (HCC) cells via the induction of quiescence and senescence, though it has limited cytocidal effect. We hypothesized that arrested HCC cells upregulate mTOR signaling to stay metabolically active and investigated the effect of combining palbociclib with the mTOR inhibitor rapamycin on preclinical models of HCC. Methods: We identified commonly altered molecular markers in HCC, including cell cycle related genes, using single sample gene set enrichment analysis (ssGSEA) of 13 paired (tumor and adjacent non-tumor) RNA-seq and 15 paired proteomic data sets as well as 50 paired RNA-seq data sets from TCGA. Subsequently, we studied the in-vitro effects of palbociclib and rapamycin by evaluating induction of senescence, inhibition of cell growth with fixed ratio combination treatment as well as Western Blotting to establish mechanistic effects. We tested the in-vivo anti-tumor efficacy of palbociclib plus rapamycin in athymic nude mice with growing xenograft tumors. We then generated immunocompetent murine HCC models using hydrodynamic transfection of oncogenes ΔN90-β-catenin and c-Met, and measured plasma AFP levels as a marker of HCC to assess the efficacy of treatment with palbociclib and/or rapamycin. Results: ssGSEA revealed significant positive enrichment of several cell cycle related gene sets in all our HCC cases. Palbociclib treatment in vitro showed an increase in senescence associated β-galactosidase staining as well as increased levels of intrinsic CDK inhibitors p16, p15, p21 and p27, indicating senescence. Fixed ratio treatment with palbociclib and rapamycin established a synergistic interaction between the drugs, as indicated by a combination index of less than 1. We also observed an increase in levels of proteins of the mTOR pathway (p-mTOR, p-4eBP1, p-p70 and p-s6) after palbociclib treatment, followed by suppression of the levels on addition of rapamycin. In the xenograft HCC model, combination treatment with palbociclib and rapamycin resulted in slower tumor growth and significantly increased time to critical tumor volume (2000 mm3) (log rank, p <0.001). In the hydrodynamic transfection HCC model, we observed a significantly lower mean AFP level in the combination treatment group, along with lower liver weights which were closer to the liver weights of normal mice. Conclusion: HCC cells overexpress genes and proteins that drive cell cycle progression. Palbociclib induces senescence in HCC cells along with an associated upregulation in mTOR signaling, and combination with rapamycin has a synergistic effect on inhibition of tumor growth in vitro as well as in vivo, possibly by abrogating both actively growing and quiescent/senescent HCC cells. The combination of the two drugs may have potential as a novel therapy for HCC. Citation Format: Ankur Tiwari, Araceli Huerta, Hakim Bouamar, Debodipta Das, Francisco Cigarroa, LuZhe Sun. Combination of CDK 4/6 inhibitor palbociclib with rapamycin synergistically inhibits growth of hepatocellular carcinoma in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2679.

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

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

Abstract 1746: Broad-spectrum kinome profiling identifies CDK6 upregulation as a driver of lenvatinib resistance in hepatocellular carcinoma

Abstract Lenvatinib is the FDA-approved targeted drug for advanced hepatocellular carcinoma (HCC), but the efficacy is modest due to drug resistance. Tumor kinome re-wiring governs drug resistance in resistant cancer cells, which is an obstacle for efficient cancer therapy. Therefore, identification of the kinases critical for this rewiring process in HCC is crucial. Upon mass spectrometry analysis using a lysine-targeted sulfonyl fluoride probe named XO44, CDK6 was identified to be the most upregulated kinase in lenvatinib-resistant HCC cell lines. Lentiviral-based overexpression and knockdown approaches were performed to characterize functional roles of CDK6 in regulation of cancer stemness and lenvatinib resistance. The upregulation of CDK6 in lenvatinib-resistant HCC cells is found to be mediated by the ERK/YAP signaling cascade. CDK6 is crucial in the regulation of acquired lenvatinib resistance via augmentation of cancer stemness and the overexpression of CDK6 led to poorer patient survival. We identified a novel noncanonical pathway of CDK6 in which it binds and regulates the activity of GSK3β, leading to activation of the Wnt/β-catenin signaling pathway. CDK6 inhibition by CDK4/6 inhibitor palbociclib or CDK6 degradation by proteolysis targeting chimeras (PROTACs) is highly synergistic with lenvatinib in vitro. Interestingly, palbociclib not only exerts the maximal growth suppressive effect with lenvatinib in lenvatinib-resistant HCC xenografts and Trp53KO/MYCOE-induced HCC models but also reshapes the tumor immune microenvironment. CDK6 blockade impedes the lenvatinib-induced activation of the ERK/YAP/CDK6/β-catenin signaling cascade. Targeting CDK6 with palbociclib in combination with lenvatinib may be a novel therapeutic strategy against HCC. Citation Format: Carmen Oi Leung, Yang Yang, Karl Kam So, Qian Zhao, Terence Kin Lee. Broad-spectrum kinome profiling identifies CDK6 upregulation as a driver of lenvatinib resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1746.

Abstract 5011: Targeting kinome reprogramming in ESR1 fusion-driven breast cancer

Abstract Background: Transcriptionally active ESR1 gene fusions (ESR1-TAF) are a potent cause of estrogen receptor alpha-positive (ERα+) breast cancer endocrine therapy (ET) resistance. These ESR1-TAF are gain-of-function mutations, exhibiting estrogen-independent cell growth, motility and ET resistance. They are not directly druggable because the ERα C-terminal ligand binding domain (LBD) encoding sequence is replaced with a translocated in-frame partner gene sequence. Herein we utilized proteomic approaches to develop novel targeted therapies against ESR1-TAF driven tumorigenesis. Methods: ESR1 fusion cDNA constructs were expressed in ERα+ breast cancer cell lines (T47D and MCF7). Cell growth was assayed by an Alamar blue assay. A mass spectrometry (MS)-based Kinase Inhibitor Pulldown Assay (KIPA) was employed to identify druggable kinases that are commonly upregulated by diverse ESR1-TAFs. A panel of 22 ERα+ patient-derived xenograft (PDX) models were profiled using proteomics and phosphoproteomics to identify models with sensitivity to RET kinase inhibition. Results: KIPA detected an increased abundance of a receptor tyrosine kinase, RET, in T47D cells expressing ESR1-TAFs in an estrogen-independent manner, compared to stable cell lines expressing transcriptionally inactive fusions as well as wild-type ERα protein. Interestingly, RET was also increased when constitutive activating ERα LBD point mutants, Y537S and D538G, were expressed in breast cancer cells. Inhibition of the RET kinase in vitro by repurposing pralsetinib, an FDA-approved RET inhibitor for advanced thyroid and non-small-cell lung cancers, demonstrated a significant reduction in the growth of cells expressing ESR1-TAFs and ERα LBD mutants. These data nominate RET kinase as a common therapeutic vulnerability for ESR1-TAF expressing breast cancers. Proteomic profiling of 22 biologically heterogenous ERα+ PDX tumors defined targetable pathways and predicted tumor subsets that were responsive to RET inhibition therapy. Organoids and xenografts from the pan-ET resistant WHIM18 PDX (that expresses the ESR1-YAP1 TAF) were inhibited by pralsetinib to a similar extent as the CDK4/6 inhibitor palbociclib. These data provide key preclinical rationale for the consideration of RET inhibition for the treatment of ESR1-TAF-driven ET-resistant breast cancer. Interestingly, the growth of WHIM37 PDX (that expresses ERα D538G) that had low level of RET and high level of GFRα-1, the co-receptor of RET, was also suppressed by pralsetinib. This data suggests that either RET or GFRα-1 is a predictive biomarker for RET inhibitor efficacy. Conclusions: Kinome analysis of ESR1 translocated breast tumors using KIPA followed by drug sensitivity studies nominated RET as a new therapeutic target for ET-resistant ERα+ breast cancer. Citation Format: Xuxu Gou, Beom-Jun Kim, Meenakshi Anurag, Jonathan T. Lei, Meggie N. Young, Matthew V. Holt, Diana Fandino, Craig T. Vollert, Purba Singh, Mohammad A. Alzubi, Anna Malovannaya, Lacey E. Dobrolecki, Michael T. Lewis, Shunqiang Li, Matthew J. Ellis, Charles E. Foulds. Targeting kinome reprogramming in ESR1 fusion-driven breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5011.

Abstract 5986: The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study

Abstract INTRODUCTION: CDKN2A, coding for p16INK4a, a CDK4/6 inhibitory protein, is among the most commonly lost tumor suppressors in oral cavity squamous cell carcinoma (OCSCC). Drugs like palbociclib are CDK4/6 inhibitors that phenomenally also induce senescence. Senescence can be pro-tumorigenic. Studies suggests prosurvival BCL-2 family support tumor cell survival in senescent state. Agents targeting BCL-2 family e.g. navitoclax, are “senolytics” The current study examines combining palbociclib (P) with the senolytic agent navitoclax (N) as a treatment strategy. We demonstrate efficacy of combining P+N in OCSCC, and examine senescence and apoptosis processes as related mechanistic response of combination. EXPERIMENTAL PROCEDURE: We examined cell viability after sequential treatment: 72 h P followed by 72 h N (P+N) in 2-dimensional (2D) model of immortalized cell line HN5 and 3D model of a human OCSCC patient-derived conditional reprogrammed cell line (CR18). Flow cytometry measured apoptosis with Annexin V assay in 2D. Confocal imaging in 3D spheroids corroborated pro-survival protein levels. An in vivo xenograft study was performed with HN5 in 8 weeks old male nude mice to examine tumor responses to sequential P+N treatment. 3 million cells were injected in right flank. Tumors were treated at ~300 mm3 (P-100 mg/kg o.p; N -17 mg/kg i.p). After treatment termination, Bcl-2 family proteins (BcLxl); apoptosis markers (caspase 3, Cleaved PARP) and β-galactosidase (β -gal) for senescence were evaluated with western blot. QPCR measured expression of CDK4/6 target (FOXM1) to examine senescence and pro survival genes. RESULTS: Sequential treatment of P+N (1µM each) demonstrated consistent synergy in both 2D and 3D viability assays. Bright field images of P+N revealed senescence with P followed with apoptosis on N application. β -gal levels increased to 1.5 fold with P and Caspase 3 showed 2-fold increase with P+N. Flow cytometry data revealed 2 folds increase in the apoptotic population with P+N. Confocal imaging in organoids demonstrated increased levels of BcLxL with P, and repression of BCL-xL following N administration. Sequential P+N significantly reduced tumor growth in vivo. P+N demonstrated a robust 5-fold decrease in tumor volume (TV) with a complete cure (TV<20 mm3) in 60% population in comparison to either of the cohorts post 30 days of treatment (control: 2000 mm3, P- 1200 mm3; N- 1500 mm3, p<0.05). Compared to control sequential P+N treatment in vivo i) reduced anti-apoptotic BcLxL mRNA expression to 2.5 fold ii) Cleaved PARP increased 10 folds and iii) BcLxL levels decreased 2 fold. CONCLUSION: Our results suggest that CDK4/6 inhibition makes OCSCC undergo senescence and make it more susceptible to apoptosis when combined with the BCL2-family inhibitor, navitoclax. We propose a sequential P+N as a rational and potentially effective treatment strategy in OCSCC. Citation Format: Nitisha Shrivastava, Nicole Kawachi, Michael B. Prystowsky, Evripidis Gavathiotis, Chandan Guha, Thomas J. Ow. The CDK4/6 inhibitor palbociclib sensitizes oral cavity squamous cell carcinoma to navitoclax-induced apoptosis-An in vitro and in vivo study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5986.

Abstract 5993: Dissecting the role of CDK4 in liposarcoma

Abstract Background: Liposarcoma (LPS) encompass one of most common soft tissue sarcoma (STS) histological subtypes accounting for 15% of all cases. They can be divided into four entities including atypical lipomatous tumor or well-differentiated LPS (ALT/WDLPS), dedifferentiated LPS (DDLPS), myxoid LPS (MLPS) and pleomorphic LPS (PLS). Their heterogeneity is reflected in their morphology, molecular landscape, prognosis and clinical behavior. Current treatment options for localized disease include surgery, (neo)adjuvant radiotherapy and chemotherapy; for the metastatic setting chemotherapy represents the cornerstone but its role needs to be elucidated. The unavailability of predictive biomarkers makes the management of these mesenchymal diseases even more challenging. Recent evidences have shed light on the key role of cyclin-dependent kinase 4 (CDK4) in cancer cells proliferation. Methods: This study involved 21 adult patients affected by liposarcoma (n= 5 ALT/WDLPS and n= 16 DDLPS). IHC analyses of CDK4 and MDM2 were performed on FFPE surgically resected specimens by experienced sarcoma pathologists. Moreover, a patient-derived DDLPS cell line was established and pharmacological profiling was performed. Next, standard and innovative drugs currently used for LPS management were assessed in both 2D and 3D culture systems. Finally, in silico analyses based on public repositories on STS were carried out including 260 sarcoma patients. Results: IHC results highlighted an higher expression of CDK4 and MDM2 biomarkers in DDLPS compared to ALT/WDLSP. Moreover, although CDK4 and MDM2 are codified by the same genomic region, their expression did not correlate (e.g. 100% of CDK4 and 2% of MDM2 in the same patient). Therefore we hypothesized that even exhibiting a low MDM2 expression, a patient could benefit from CDK4 inhibition. Furthermore, our data showed that in DDLPS cases CDK4 expression ranged from 90% to 100% in spermatic cord, skin and abdomen while it ranged from 5% to 50% in the retroperitoneum. The above observations prompted us to hypothesize a correlation of CDK4 expression with specific anatomical sites. Moreover, pharmacological profiling showed a synergistic effect exerted by sequential treatment with palbociclib and some chemotherapeutics including trabectedin, dacarbazine, eribulin and lenvatinib. Indeed we observed a 10% increase in cell proliferation inhibition compared to standard treatment. In addition, in silico analyses revealed the role of CDK4 as negative prognostic biomarker for PFS and OS in STS. Conclusions: Our study highlighted the promising role of CDK4 in the management of LPS patients. In particular, we pointed out encouraging results of sequential treatment combining CDK4 inhibitor palbociclib and chemotherapy. Furthermore, preliminary analyses highlighted the involvement of this biomarker as a potential prognostic tool for STS. Citation Format: Silvia Vanni, Graziana Gallo, Valentina Fausti, Giacomo Miserocchi, Chiara Liverani, Chiara Spadazzi, Claudia Cocchi, Chiara Calabrese, Giovanni De Luca, Massimo Bassi, Manlio Gessaroli, Angelo Campobassi, Federica Pieri, Giorgio Ercolani, Davide Cavaliere, Lorena Gurrieri, Nada Riva, Giovanni Martinelli, Laura Mercatali, Alessandro De Vita. Dissecting the role of CDK4 in liposarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5993.

Abstract 5974: Synergistic activity of the CDK4/6 antagonist narazaciclib (ON123300) with irreversible BTK inhibition in ibrutinib-resistant mantle cell lymphoma

Abstract Bruton tyrosine kinase inhibitors (BTKi) have transformed the therapeutic landscape of B-cell lymphoma including the aggressive mantle cell lymphoma (MCL) subtype, although primary and acquired resistance is frequently observed. Interestingly, preliminary results have suggested that the second-generation, orally bioavailable and clinical-stage CDK4/6 inhibitor, ON123300 (narazaciclib), may be effective in preclinical models of BTKi-resistant MCL. We compared the efficacy and safety of ON123300 vs the approved CDK inhibitors palbociclib, abemaciclib or ribociclib, in association with various BTKi, in a panel of 10 MCL cell lines with distinct sensitivity to the first-in-class BTKi, ibrutinib, or the second generation acalabrutinib. We evaluated the effects of these combinations by CellTiter-Glo proliferation assay, FACS-mediated analysis of cell cycle and apoptosis, RT-PCR and western blot. Efficacy and safety of ON123300 in vivo was evaluated in a chicken embryo chorioallantoic membrane (CAM) xenograft model of MCL. We observed that ON123300 exhibited a significant antitumor activity in MCL cell lines independently of their sensitivity to ibrutinib, with IC50 at 72h ranging from 0.7 to 7.1µM (mean: 3.61±2.1µM), while it achieved a 32% tumor growth inhibition, with no detectable toxicity, in the CAM-MCL model. In vitro, ON123300 demonstrated a superior activity than palbociclib and ribociclib, and a similar effect that abemaciclib. Interestingly, ON123300 exhibited an antitumor activity comparable to ibrutinib but was more effective than acalabrutinib. ON123300 single agent treatment repressed several positive regulators of the G2/M cell cycle phase and triggered the accumulation of the CDK inhibitors p21, p16, and phospho-p27, with the consequent loss of phospho-Histone H3 and phospho-CDK2. Accordingly, ON123300 treatment evoked a 20-35% increase in G1 cell cycle fraction at 24h, which preceded the ignition of mitochondrial apoptosis. When combined with ibrutinib, rather than with acalabrutinib, ON123300 exhibited a significant antitumor activity with synergistic combination indexes in both BTK-sensitive and BTK-resistant cell lines. This phenomenon was associated with a slight but constant (+10-15%) augmentation of G1 blockade, histone H3/CDK2 dephosphorylation and p27/p16 accumulation in cells exposed to ibrutinib-ON123300 combination, with no detectable potentiation of apoptosis. In conclusion, ON123300 is safe and effective as single agent in preclinical models of MCL, including BTKi-resistant cases. Due to its complete distinct MoA from BTKi involving the direct modulation of cell cycle, ON123300, but not abemaciclib or palbociclib, can achieve significant synergistic activity in combination with ibrutinib, and in lower extent with acalabrutinib, especially in BTKi-resistant MCL cases. Citation Format: Nuria Profitos-Peleja, Marcelo L. Ribeiro, Adar Makovski Silverstein, Stephen Cosenza, Gaël Roué. Synergistic activity of the CDK4/6 antagonist narazaciclib (ON123300) with irreversible BTK inhibition in ibrutinib-resistant mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5974.

CDK4/6 are necessary for UCP1-mediated thermogenesis of white adipose tissue

AimsIn white adipose tissue (WAT) the cell cycle regulators CDK4 and CDK6 (CDK4/6) promote adipogenesis and maintain the adipocyte mature state. Here we aimed to investigate their role in the Ucp1-mediated thermogenesis of WAT depots and in the biogenesis of beige adipocytes. Main methodsWe treated mice with the CDK4/6 inhibitor palbociclib at room temperature (RT) or cold and analyzed thermogenic markers in the epididymal (abdominal) and inguinal (subcutaneous) WAT depots. We also assessed the effect of in vivo palbociclib-treatment on the percentage of beige precursors in the stroma vascular fraction (SVF), and on its beige adipogenic potential. Finally, we treated SVFs and mature adipocytes from WAT depots with palbociclib in vitro to study the role of CDK4/6 in beige adipocytes biogenesis. Key findingsIn vivo CDK4/6 inhibition downregulated thermogenesis at RT and impaired cold-induced browning of both WAT depots. It also reduced the percentage of beige precursors and beige adipogenic potential of the SVF upon differentiation. A similar result was observed with direct CDK4/6 inhibition in the SVF of control mice in vitro. Importantly, CDK4/6 inhibition also downregulated the thermogenic program of beige differentiated- and depots-derived adipocytes. SignificanceCDK4/6 modulate Ucp1-mediated thermogenesis of WAT depots in basal and cold-stressing conditions controlling beige adipocytes biogenesis by adipogenesis and transdifferentiation. This shows a pivotal role of CDK4/6 in WAT browning that could be applied to fight obesity or browning-associated hypermetabolic conditions such as cancer cachexia.

CDK4/6 inhibitors in metastatic breast cancer, a comparison of toxicity and efficacy across agents in a real-world dataset.

CDK4/6 inhibitors (ribociclib, palbociclib and abemaciclib) are 1st line therapy in metastatic breast cancer (MBC). No comparative data exists between agents regarding toxicity or efficacy. A retrospective study was performed at our tertiary referral centre evaluating patients on a CDK4/6 inhibitor for MBC between July 2017 and December 2021. Toxicity was evaluated along with variability in full blood counts and liver function over the first 12 weeks of therapy. Two hundred and seventeen patients were treated (palbociclib 59%, abemaciclib 25% and ribociclib 16%). 86% received the agent as 1st line therapy. Most patients were white women with a median age of 61 years (32-95) and ECOG 0/1. Twelve patients were switched to an alternative CDK4/6 inhibitor due to toxicity and two did not tolerate this. Toxicity profiles of agents were consistent with published trials. However, there was greater overlap in hepatitis, diarrhoea and bone marrow suppression. Blood results indicated a minimum of four weeks treatment before development of neutropenia. Forty percent of patients went onto have subsequent lines of therapy. The progression-free survival per agent was palbociclib 27.9 months (95% CI 23-32.5), ribociclib 29 months (95% CI 21.5-37.0) and abemaciclib 20.6 months (95% CI 15.0-26.0). The overall survival was palbociclib 38.0 months (95% CI 33.5-42.5), ribociclib 33.9 months (95% CI 26.7-41.1) and abemaciclib 27.3 months (95% CI 22.5-32.1). Toxicity across CDK4/6 inhibitors overlaps. The optimal sequence of therapies post CDK4/6 inhibitors remains unknown but rechallenge with an alternative agent is possible.

Stromal Senescence following Treatment with the CDK4/6 Inhibitor Palbociclib Alters the Lung Metastatic Niche and Increases Metastasis of Drug-Resistant Mammary Cancer Cells.

CDK4/6 inhibitors (CDKi) have improved disease control in hormone-receptor-positive, HER2-negative metastatic breast cancer, but most patients develop progressive disease. We asked whether host stromal senescence after CDK4/6 inhibition affects metastatic seeding and growth of CDKi-resistant mammary cancer cells by using the p16-INK-ATTAC mouse model of inducible senolysis. Palbociclib pretreatment of naïve mice increased lung seeding of CDKi-resistant syngeneic mammary cancer cells, and this effect was reversed by depletion of host senescent cells. RNA sequencing analyses of lungs from non-tumor-bearing p16-INK-ATTAC mice identified that palbociclib downregulates immune-related gene sets and gene expression related to leukocyte migration. Concomitant senolysis reversed a portion of these effects, including pathway-level enrichment of TGF-β- and senescence-related signaling. CIBERSORTx analysis revealed that palbociclib alters intra-lung macrophage/monocyte populations. Notably, lung metastases from palbociclib-pretreated mice revealed senescent endothelial cells. Palbociclib-treated endothelial cells exhibit hallmark senescent features in vitro, upregulate genes involved with the senescence-associated secretory phenotype, leukocyte migration, and TGF-β-mediated paracrine senescence and induce tumor cell migration and monocyte trans-endothelial invasion in co-culture. These studies shed light on how stromal senescence induced by palbociclib affects lung metastasis, and they describe palbociclib-induced gene expression changes in the normal lung and endothelial cell models that correlate with changes in the tumor microenvironment in the lung metastatic niche.

Cost Effectiveness of CDK4/6 Inhibitors in the First-Line Treatment of HR+/HER2- Metastatic Breast Cancer in Postmenopausal Women in the USA.

Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors improve progression-free survival when combined with endocrine therapies in patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative metastatic breast cancer. However, the comparative cost effectiveness of utilizing three US Food and Drug Administration-approved CDK4/6 inhibitors is unknown. Therefore, we aimed to evaluate the cost effectiveness of individual CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) with letrozole versus letrozole monotherapy in the first-line treatment of hormone receptor-positive/human epidermal growth factor receptor 2-negative metastatic breast cancer in the USA. We constructed a Markov-based decision-analytic model to evaluate the cost effectiveness of CDK4/6 inhibitors plus endocrine therapies over a 40-year lifetime from a third-party payer perspective. The model incorporated health states (progression-free disease, progressive disease, and death), major adverse events (neutropenia), and cancer-specific and all-cause mortality. Using clinical efficacy and quality-of-life scores (utility) data from clinical trials, we estimated quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios using Medicare charges reported in US dollars per 2022 valuation and a discount rate of 3% applied to costs and outcomes. We performed deterministic and probabilistic sensitivity analyses to evaluate parametric and decision uncertainty. Compared to letrozole, the model estimated an increase of 5.72, 5.87, and 6.39 in QALYs and costs of $799,178, $788,168, and $741,102 in combining palbociclib, ribociclib, and abemaciclib plus letrozole, respectively. Palbociclib or ribociclib plus letrozole were dominated by abemaciclib plus letrozole. Compared with letrozole, abemaciclib plus letrozole resulted in an incremental cost-effectiveness ratio of $457,538 per QALY with an incremental cost of $553,621 and an incremental QALY gain of 1.21. The results were sensitive to the cost of abemaciclib, disease progression utility, and patients' age. At a willingness to pay of $100,000/QALY gained, our model predicts that combining CDK4/6 inhibitors plus letrozole is not cost effective with a marginal increase in QALYs at a high cost. Lowering the cost of these drugs or identifying patients who can receive maximal benefit from CDK4/6 inhibitors would improve the value of this regimen in patients.

CDK4/6 Inhibitors—Overcoming Endocrine Resistance Is the Standard in Patients with Hormone Receptor-Positive Breast Cancer

Tamoxifen and aromatase inhibitors can be considered as some of the first targeted therapies. For the past 30 years, they were the endocrine treatment standard in the advanced and early breast cancer setting. CDK4/6 inhibitors, however, are the first substances in almost two decades to broadly improve the therapeutic landscape of hormone receptor-positive breast cancer patients for the upcoming years. This review is designed to discuss the recent history, current role, future directions and opportunities of this substance class. The CDK4/6 inhibitors abemaciclib, dalpiciclib, palbociclib and ribociclib have all demonstrated a statistically significant improvement in progression-free survival in advanced disease. However, to date, abemaciclib and ribociclib are the only CDK4/6 inhibitors to have shown an improvement in overall survival in patients with metastatic breast cancer. Moreover, abemaciclib is the first CDK4/6 inhibitor to also reduce the risk of recurrence in those with early-stage disease. Further CDK inhibitors, treatment combinations with other drugs and different therapy sequences are in development. Achieving significant improvements in survival rates in the advanced and early breast cancer treatment setting, CDK4/6 inhibitors have set a new standard of care for patients with advanced breast cancer. It remains important to better understand resistance mechanisms to be able to develop novel substances and treatment sequences.