Cyclin D-CDK4 Research Articles

18F-FDG PET/CT Metabolic Response With Hormone Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer Treated With Cyclin-Dependent 4/6 Kinase Inhibitors.

Dysregulation of the cyclin D-CDK4/6-INK4-RB pathway, which leads to uncontrolled cell proliferation, is frequently observed in breast cancer. Recently, 3 CDK4/6 inhibitors have been FDA approved as first-line treatment for patients with hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer. Despite promising clinical results, the metabolic response to treatment with these new drugs has not been elaborately demonstrated yet. Herein, we presented a patient with hormone receptor-positive, human epidermal growth factor receptor 2-negative breast cancer who demonstrated a complete metabolic response on 18F-FDG PET/CT to treatment with a CDK4/6 inhibitor (ribociclib).

Non-phosphorylatable cyclin D1 mutant potentiates endometrial hyperplasia and drives carcinoma with Pten loss.

Cyclin D1 is a regulatory subunit of -Cyclin Dependent Kinases 4 and 6 (CDK4/6) and regulates progression from G1 to S phase of the cell cycle. Dysregulated cyclin D1-CDK4/6 contributes to abnormal cell proliferation and tumor development. Phosphorylation of threonine 286 of cyclin D1 is necessary for ubiquitin-dependent degradation. Non-phosphorylatable cyclin D1 mutants are stabilized and concentrated in the nucleus, contributing to genomic instability and tumor development. Studies investigating the tumor-promoting functions of cyclin D1 mutants have focused on the use of artificial promoters to drive the expression which unfortunately may not accurately reflect tumorigenic functions of mutant cyclin D1 in cancer development. We have generated a conditional knock-in mouse model where cyclin D1T286A is expressed under the control of its endogenous promoter following Cre-dependent excision of a lox-stop-lox sequence. Acute expression of cyclin D1T286A following tamoxifen-inducible Cre recombinase triggers inflammation, lymphocyte abnormality and ultimately mesenteric tumors in the intestine. Tissue-specific expression of cyclin D1T286A in the uterus and endometrium cooperates with Pten loss to drive endometrial hyperplasia and cancer. Mechanistically, cyclin D1T286A mutant activates NF-κB signaling, augments inflammation, and contributes to tumor development. These results indicate that mutation of cyclin D1 at threonine 286 has a critical role in regulating inflammation and tumor development.

DHA Suppresses Hepatic Lipid Accumulation via Cyclin D1 in Zebrafish.

With the widespread use of high-fat diets (HFDs) in aquaculture, fatty livers are frequently observed in many fish species. The aim of this study was to investigate if docosahexaenoic acid (DHA) could be used to reduce the fatty liver in zebrafish generated by a 16% soybean oil-HFD over 2 weeks of feeding. The DHA was added to iso-lipidic HFD at 0.5, 1.0, and 2.0% of diet. Supplementation of DHA reduced growth and feed efficiency in a dose dependent manner being lowest in the HFDHA2.0 group. Hepatic triglyceride (TG) in zebrafish fed 0.5% DHA-supplemented HFD (HFDHA0.5) was significantly lower than in the HFD control. Transcriptional analyses of hepatic genes showed that lipid synthesis was reduced, while fatty acid β-oxidation was increased in the HFDHA0.5 group. Furthermore, the expression of Cyclin D1 in liver of zebrafish fed HFDHA0.5 was significantly reduced compared to that in fish fed HFD. In zebrafish liver cells, Cyclin D1 knockdown and blocking of Cyclin D1-CDK4 signal led to inhibited lipid biosynthesis and elevated lipid β-oxidation. Besides, DHA-supplemented diet resulted in a rich of Proteobacteria and Actinobacteriota in gut microbiota, which promoted lipid β-oxidation but did not alter the expression of Cyclin D1 in germ-free zebrafish model. In conclusion, DHA not only inhibits hepatic lipid synthesis and promotes lipid β-oxidation via Cyclin D1 inhibition, but also facilitates lipid β-oxidation via gut microbiota. This study reveals the lipid-lowering effects of DHA and highlights the importance of fatty acid composition when formulating fish HFD.

Monoclonal antibodies to activated CDK4: use to investigate normal and cancerous cell cycle regulation and involvement of phosphorylations of p21 and p27

ABSTRACT Cyclin-dependent kinase 4 (CDK4) is a master integrator that couples mitogenic/oncogenic signaling with the cell division cycle. It is deregulated in most cancers and inhibitors of CDK4 have become standard of care drugs for metastatic estrogen-receptor positive breast cancers and are being evaluated in a variety of other cancers. We previously characterized the T-loop phosphorylation at T172 of CDK4 as the highly regulated step that determines the activity of cyclin D-CDK4 complexes. Moreover we demonstrated that the highly variable detection of T172-phosphorylated CDK4 signals the presence or absence of the active CDK4 targeted by the CDK4/6 inhibitory drugs, which predicts the tumor cell sensitivity to these drugs including palbociclib. To date, the phosphorylation of CDK4 has been very poorly studied because only few biochemical techniques and reagents are available for it. In addition, the available ones including 2D-IEF separation of CDK4 modified forms are considered too tedious. The present report describes the generation, selection and characterization of the first monoclonal antibodies that specifically recognize the active CDK4 phosphorylated on its T172 residue. One key to this success was the immunization with a long phosphopeptide corresponding to the complete activation segment of CDK4. These monoclonal antibodies specifically recognize T172-phosphorylated CDK4 in a variety of assays, including western blotting, immunoprecipitation and, as a capture antibody, a sensitive ELISA from cell lysates. The specific immunoprecipitation of T172-phosphorylated CDK4 allowed to clarify the involvement of phosphorylations of co-immunoprecipitated p21 and p27, showing a privileged interaction of T172-phosphorylated CDK4 with S130-phosphorylated p21 and S10-phosphorylated p27. Abbreviations:2D: two-dimensional; CAK: CDK-activating kinase; CDK: cyclin-dependent kinase; HAT: Hypoxanthine-Aminopterin-Thymidine; FBS: fetal bovine serum; IP: immunoprecipitation; ID: immunodetection; mAb: monoclonal antibody; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate buffer saline; pRb: retinoblastoma susceptibility protein; SDS: sodium dodecyl sulfate; DTT: dithiotreitol; TET: tetracyclin repressor; Avi: Avi tag; TEV: tobacco etch virus cleavage site; EGFP: enhanced green fluorescent protein; BirA: bifunctional protein biotin ligase BirA; IRES: internal ribosome entry site; HIS: poly-HIS purification tag; DELFIA: dissociation-enhanced lanthanide fluorescent immunoassay; 3-MBPP1: 1-(1,1-dimethylethyl)-3[(3-methylphenyl) methyl]-1H-pyrazolo[3,4-d] pyrimidin-4-amine; BSA: bovine serum albumin; ECL: Enhanced chemiluminescence

Chemical composition, anticancer activities and related mechanisms of the essential oil from Alpinia coriandriodora rhizome

Alpinia coriandriodora is widely cultivated as a medicinal and edible Zingiberaceae plant in Guangxi, China. Since essential oils from Zingiberaceae plants can be used as a potential source of anticancer drugs, this study was designed to identify the chemical composition of A. coriandriodora rhizome essential oil (EO) and firstly elucidate its anticancer activities and related mechanisms. The GC-FID/MS results indicated that the most predominant component of EO was (E)-2-decenal (56.3%). Based on the MTT results, EO exhibited comparable cytotoxicity to A549 cells and lower cytotoxicity to non-cancerous cells compared with the positive control (elemene injection). For related mechanism studies, EO inhibited the A549 cells proliferation, resulting from inducing cell cycle arrest at G1 phase via downregulation of cyclin E2-CDK2 and cyclin D3-CDK4/CDK6 complexes and upregulation of p21. Meanwhile, it induced the apoptosis through mitochondrial pathway with the successive enhancement in the Bax/Bcl-2 ratio, Δψm decline, cyt c release, activation of caspase-9 and caspase-3, and PARP cleavage. Moreover, it inhibited the migration and invasion ability of A549 cells via downregulation of MMP-2 and N-cadherin, as well as upregulation of E-cadherin. Hence, EO has outstanding anticancer properties in vitro, and this fact encourages further studies using in vivo models.

Licorice extract inhibits growth of non-small cell lung cancer by down-regulating CDK4-Cyclin D1 complex and increasing CD8+ T cell infiltration

BackgroundTargeting tumor microenvironment (TME) may provide therapeutic activity and selectivity in treating cancers. Therefore, an improved understanding of the mechanism by which drug targeting TME would enable more informed and effective treatment measures. Glycyrrhiza uralensis Fisch (GUF, licorice), a widely used herb medicine, has shown promising immunomodulatory activity and anti-tumor activity. However, the molecular mechanism of this biological activity has not been fully elaborated.MethodsHere, potential active compounds and specific targets of licorice that trigger the antitumor immunity were predicted with a systems pharmacology strategy. Flow cytometry technique was used to detect cell cycle profile and CD8+ T cell infiltration of licorice treatment. And anti-tumor activity of licorice was evaluated in the C57BL/6 mice.ResultsWe reported the G0/G1 growth phase cycle arrest of tumor cells induced by licorice is related to the down-regulation of CDK4-Cyclin D1 complex, which subsequently led to an increased protein abundance of PD-L1. Further, in vivo studies demonstrated that mitigating the outgrowth of NSCLC tumor induced by licorice was reliant on increased antigen presentation and improved CD8+ T cell infiltration.ConclusionsBriefly, our findings improved the understanding of the anti-tumor effects of licorice with the systems pharmacology strategy, thereby promoting the development of natural products in prevention or treatment of cancers.

Abstract P457: Systems Analysis To Understand The Impact Of The CDK Module On Cardiomyocyte Proliferation

Rationale: Heart failure is caused by the inability of adult mammalian hearts to overcome the loss of cardiomyocytes (CMs). This is due partly to the limited proliferative capacity of CMs, which exit the cell cycle and do not undergo cell division. Current knowledge in cardiac regeneration lacks an understanding of the molecular regulatory networks that determine whether CMs will progress through the cell cycle to proliferate. Our goal is to use computational modeling to understand the expression and activation levels of the core cell cycle network, specifically cyclins and cyclin-cyclin-dependent kinase (CDK) complexes. Methods: A model of core cell cycle dynamics was curated using previously published studies of CM proliferation regulators. This model incorporates those regulators known to stimulate G1/S and G2/M transitions through the core CDKs. The activity of each of the 22 network nodes (22 reactions) was predicted using a logic-based differential equation approach. The CDK model was then coupled with a minimal ODE model of cell cycle phase distributions and validated based on descriptions and experimental data from the literature. To prioritize key nodes for experimental validation, we performed a sensitivity analysis by stimulating individual knockdown for every node in the network, measuring the fractional activity of all nodes. Results: Our model confirmed that the knockdown of p21 and Rb protein and the overexpression of E2F transcription factor and cyclinD-cdk4 showed an increase in cells going through DNA synthesis and entering mitosis. A combined knockdown of p21 and p27 showed an increase of cells entering mitosis. Cyclin D-cdk4 and p21 overexpression showed a decrease and increase of Rb expression, respectively. Of the 14 model predictions, 12 agreed with experimental data in the literature. A comprehensive knockdown of the model nodes suggests that E2F (a key transcription factor driving DNA synthesis) is positively regulated by cyclin D while negatively regulated by GSK3B, SMAD3, and pRB. Conclusion: This model enables us to predict how cardiomyocytes respond to stimuli in the CDK network and identify potential therapeutic regulators that induce cardiomyocyte proliferation.

NP-ALT, a Liposomal:Peptide Drug, Blocks p27Kip1 Phosphorylation to Induce Oxidative Stress, Necroptosis, and Regression in Therapy-Resistant Breast Cancer Cells.

Resistance to cyclin D-CDK4/6 inhibitors (CDK4/6i) represents an unmet clinical need and is frequently caused by compensatory CDK2 activity. Here we describe a novel strategy to prevent CDK4i resistance by using a therapeutic liposomal:peptide formulation, NP-ALT, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. We find that NP-ALT blocks proliferation in HR+ breast cancer cells, as well as CDK4i-resistant cell types, including triple negative breast cancer (TNBC). The peptide ALT is not as stable in primary mammary epithelium, suggesting that NP-ALT has little effect in nontumor tissues. In HR+ breast cancer cells specifically, NP-ALT treatment induces ROS and RIPK1-dependent necroptosis. Estrogen signaling and ERα appear required. Significantly, NP-ALT induces necroptosis in MCF7 ESRY537S cells, which contain an ER gain of function mutation frequently detected in metastatic patients, which renders them resistant to endocrine therapy. Here we show that NP-ALT causes necroptosis and tumor regression in treatment naïve, palbociclib-resistant, and endocrine-resistant BC cells and xenograft models, demonstrating that p27 is a viable therapeutic target to combat drug resistance. IMPLICATIONS: This study reveals that blocking p27 tyrosine phosphorylation inhibits CDK4 and CDK2 activity and induces ROS-dependent necroptosis, suggesting a novel therapeutic option for endocrine and CDK4 inhibitor-resistant HR+ tumors.

CDK4: A Novel Therapeutic Target for Extramammary Paget's Disease.

BackgroundThe outcome of extramammary Paget’s disease (EMPD) is poor when it progresses to metastasis because of the lack of effective systemic therapies. Recently, CDK4-targeted therapy has attracted attention as a potential therapeutic target for some cancers. The aim of this study was to analyze the impact of CDK4 expression on the survival of patients with EMPD.MethodsWe retrospectively reviewed 110 patients with EMPD. We conducted immunohistochemical analysis of CDK4 and cyclin D1 expression, and assessed the association between their expression and survival.ResultsMost EMPD lesions (108/110, 98.2%) were positive for CDK4 staining and there was a positive correlation between CDK4 expression and cyclin D1 expression (r = 0.54, p < 0.001). Tumor thickness (p = 0.0003) and the presence of regional lymph node metastasis (p = 0.015) were significantly associated with high CDK4 expression. Regarding invasive EMPD, the multivariate analysis did not show the correlation between the expression of CDK4/cyclin D1 and survival outcomes (HR: 3.14, p = 0.14).ConclusionThe overexpression of CDK4 was identified as a major risk factor for disease progression. CDK4-targeted therapy could thus be a novel treatment option for unresectable EMPD.

Abstract 3043: Targeting CDK4/6 inhibitor resistance in relapsed osteosarcoma via PI3 Kinase inhibition

Abstract Osteosarcoma (OS) is the most common primary bone malignancy in children as well as in adolescents and young adults (AYA). Approximately 35% of OS patients develop metastases and relapse after first-line treatment emphasizing the need for new therapies. Our objective was to use patient -omics analyses to prioritize models of relapsed pediatric and AYA OS to investigate an unexplored combination therapy. Genomic data from pediatric and AYA patients enrolled in the Precision Genomics program at Riley Hospital for Children, Indiana University Health indicate that dysregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is one of the top actionable signatures. The cyclin D-CDK4/6 complex regulates retinoblastoma protein (RB)-E2F transcription factor interactions. Upon cyclin D-CDK4/6-mediated RB phosphorylation, the RB-E2F complex dissociates, and cell cycle progression ensues. CDK4/6 inhibitors (CDK4/6i) typically induce cell cycle arrest rather than cell death and can activate compensatory pathways such as PI3K. Moreover, aberrant PI3K activation has been reported in OS. Our hypothesis is that inhibition of CDK4/6 and PI3K pathways will be efficacious and well-tolerated in RB1-proficient (RB+) OS models exhibiting hyperactivation of the cyclin D-CDK4/6 complex. Cell growth response to CDK4/6i (Palbociclib or Abemaciclib) and a PI3K/mTOR inhibitor (PI3K/mTORi, Voxtalisib) was evaluated in RB+ OS cell lines and an RB+ patient-derived xenograft (PDX)-derived xenoline (TT2-77). Combination index and Bliss independence analyses indicated that CDK4/6i+PI3K/mTORi resulted in additive to synergistic inhibition of growth in RB+ OS cell lines at clinically relevant concentrations. In the TT2-77 PDX, whole genome sequencing indicated that the original OS biopsy and the TT2-77 PDX generated from a resection sample harbor signatures associated with CDK4/6 pathway up-regulation. Global and phosphoproteome analysis were conducted on TT2 PDX tumors from NOD/SCIDγnull mice treated with vehicle vs. Palbociclib for 5 days. Modulation of 6226 phosphopeptides and 3870 total proteins were observed and included downregulation of phosphopeptides and total protein levels of CDK1 as well as DNA replication proteins in Palbociclib-treated TT2-77 PDX mice. Furthermore, TT2-77 PDX mice were treated with Palbociclib daily (10-120 mg/kg) for 3 weeks. TT2-77 PDX growth was completely blocked by high dose Palbociclib. While all mice survived, clinical observation criteria indicated that this dose was not optimal. In TT2-77 PDX mice treated with 50 mg/kg Palbociclib, tumor growth significantly decreased compared to vehicle (p&amp;lt;0.01) and was well tolerated. However, tumors progressed over time while still on treatment; evaluation of PI3K pathway activation is in progress. These data provide an opportunity to evaluate efficacy of targeting CDK4/6i-induced compensatory pathways in relapsed pediatric and AYA OS. Citation Format: Farinaz Barghi, Pankita H. Pandya, M. Reza Saadatzadeh, Khadijeh Bijangi-Vishehsaraei, Barbara J. Bailey, Erika A. Dobrota, Courtney Young, Melissa A. Trowbridge, Kathy Coy, Anthony L. Sinn, Harlan E. Shannon, Jamie L. Renbarger, Karen E. Pollok. Targeting CDK4/6 inhibitor resistance in relapsed osteosarcoma via PI3 Kinase inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3043.

Abstract 1415: Triple combination targeting ER, CDK4/6, and PI3K inhibits tumor growth in ER+ breast cancer resistant to combined fulvestrant and CDK4/6 or PI3K inhibitor

Abstract Despite the improved outcome of ER+ advanced breast cancer patients treated with combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy, the disease will eventually progress. Furthermore, the recently approved combined therapy with the α-specific PI3K inhibitor (PI3Ki) alpelisib and fulvestrant in PIK3CA-mutated ER+ metastatic breast cancer patients who had previously received endocrine therapy demonstrated a clinically meaningful benefit in clinical trials, but drug resistance is inevitable. Thus, novel and more effective treatment combinations are urgently needed, as well as defining the subsequent optimal sequence of treatment following progression on combined treatment with either CDK4/6i or Pi3Ki and endocrine therapy. We developed 4 PIK3CA-mutated ER+ breast cancer cell models resistant to combined fulvestrant and CDK4/6i or PI3Ki, derived from MCF-7 (MPFR and MPiFR) and T47D (TPFR and TPiFR) cells, and 2 orthotopic cell line xenograft models (MPFR and MPiFR). We used these models to investigate whether ER+ breast cancer cells resistant to combined fulvestrant and CDK4/6i or PI3Ki would benefit from switching between these combined therapies, or whether triple combination of these agents would be required. We observed that triple inhibition of CDK4/6, PI3K and ER significantly reduced growth of breast cancer cells and xenografts models resistant to combined fulvestrant and CDK4/6i or PI3Ki. The triple combination also induced apoptosis and cell cycle arrest and reduced the expression of key proteins of the ER, PI3K/AKT/mTOR and cyclin D-CDK4/6-Rb pathways, including ER, p-PRAS40 and p-S6, in cell lines resistant to combined fulvestrant and CDK4/6i or PI3Ki. Finally, the triple inhibition significantly prevented and/or delayed the acquisition of resistance in cells resistant to combined fulvestrant and CDK4/6i or PI3Ki. Importantly, switching between combined fulvestrant and CDK4/6i to fulvestrant and PI3Ki or vice versa upon resistance did not prevent disease progression, suggesting that simultaneous blockage of the 3 pathways is required in these tumors. Finally, we found high CDK2 levels in cells resistant to combined fulvestrant and PI3Ki, and targeting ER, PI3K, and CDK2 offered an additional treatment option for ER+ breast cancer resistant to combined fulvestrant and PI3Ki. Evaluation of the efficacy of these different targeted therapy combinations in patient-derived xenografts models is ongoing. Our data supports the use of combined therapy with either PI3Ki or CDK4/6i and fulvestrant following progression on or after an endocrine-based regime. However, following disease progression on combined therapy with fulvestrant and either CDK4/6i or PI3Ki, triple combination targeting ER, PI3K and either CDK4/6 or CDK2 is needed to inhibit tumor progression. Citation Format: Leena Karimi, Carla L. Alves, Mikkel G. Terp, Martina Tuttolomondo, Henrik J. Ditzel. Triple combination targeting ER, CDK4/6, and PI3K inhibits tumor growth in ER+ breast cancer resistant to combined fulvestrant and CDK4/6 or PI3K inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1415.

Abstract LB115: Blocking p27Kip1phosphorylation with a liposomal:peptide drug induces Reactive Oxygen Species,necroptosis and tumor regression in breast cancer cells

Abstract Resistance to cyclin D-cdk4/6 inhibitors (CDK4/6i) represents an unmet clinical need and is frequently caused by compensatory CDK2 activity. Here we describe a novel strategy to prevent CDK4i resistance by using a therapeutic liposomal:peptide formulation, NP-ALT, to inhibit the tyrosine phosphorylation of p27Kip1(CDKN1B), which in turn inhibits both CDK4/6 and CDK2. We find that NP-ALT blocks proliferation in HR+ breast cancer (BC) cells, as well as CDK4i-resistant cell types, including Triple Negative (TN) BC. The peptide ALT is not as stable in primary mammary epithelium, suggesting that NP-ALT has little effect in non-tumor tissues. In HR+ BC cells specifically, NP-ALT treatment induces ROS and RIPK1-dependent necroptosis. Estrogen signaling and ERα appear required. Significantly, NP-ALT induces necroptosis in MCR7 ESRY537S cells, which contain an ER gain of function mutation frequently detected in metastatic patients, which renders them resistant to endocrine therapy. Here we show that NP-ALT causes necroptosis and tumor regression in treatment naïve, palbociclib-resistant and endocrine-resistant BC cells and xenograft models, demonstrating that p27 is a viable therapeutic target to combat drug resistance. Because the RAS/MAPK axis is the target of many therapies, NP-ALT, with its ability to target p27, can deal with the backend drug resistance seen in the presence of other inhibitors, extending the addressable market of this line of therapy. Citation Format: Stacy Wister Blain, Irina Jilishitz, Jason L. Quinones, Priyank Patel, Grace Chen, Jared Pavetsky, Allison VanInwegen VanInwegen, Scott Schoninger, Manasi P. Jogalekar, Vladislav Tsiperson, Lingyue Yan, Yun Wu, Susan R. Gottesman, Jonathan Somma. Blocking p27Kip1phosphorylation with a liposomal:peptide drug induces Reactive Oxygen Species,necroptosis and tumor regression in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB115.

Abstract 1953: Q901, a selective CDK7 inhibitor, a potential new strategy for primary and CDK4/6 inhibitor resistant ER-positive breast cancer

Abstract CDK4/6 inhibitors, in combination with hormone therapy, has made a breakthrough in treatment of hormone receptor-positive, HER2-negative metastatic breast cancers. However, in clinical practice, a substantial number of patients who responded to CDK4/6 inhibitors eventually develop resistance within 2-3 years. Current knowledge of the molecular mechanisms of CDK4/6 inhibitor resistance is under investigation , with the loss of function of the retinoblastoma (RB) protein the most frequently observed change in resistance to CDK4/6 inhibitors. RB loss of function by CDK4/6 inhibitors appears an irreversible mechanism of resistance while gain of cyclin E, and overexpression of CDK2 reducing cyclin D1-CDK4/6 dependency which results in the resistant cell to re-enter into s-phase. Thus, inhibition of the cyclin E-CDK2 axis may be effective in overcoming resistance to CDK4/6 inhibitors. We report a novel treatment strategy that targets Cyclin-dependent kinase 7 (CDK7) in CDK4/6 inhibitor-resistant, ER+ breast cancer. Q901 is a novel highly-selective inhibitor against CDK7 and demonstrates good functional inhibition of CDK activating kinase (CAK) activities leading to activation of CDK4, 6 and CDK2 in vitro and in vivo. Q901, monotherapy, effectively inhibited tumor growth in ER+ breast cancer xenograft model as well as CDK4/6 inhibitor resistant PDX model. The results suggest a potential for Q901 in treatment of hormone receptor-positive, HER2-negative metastatic breast cancer, regardless of resistance status to CDK4/6 inhibitors. Citation Format: Donghoon Yu, Yeejin Jeon, Dongsik Park, Mooyoung Seo, Wongyun Ahn, Jaeseung Kim, Kiyean Nam. Q901, a selective CDK7 inhibitor, a potential new strategy for primary and CDK4/6 inhibitor resistant ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1953.

Clinical CDK4/6 inhibitors induce selective and immediate dissociation of p21 from cyclin D-CDK4 to inhibit CDK2

Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors—palbociclib, ribociclib, or abemaciclib—immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms.

Molecular Modeling Studies on Biochanin-A as a Potential Dual inhibitor for VEGFR-2 and Cyclin D1-CDK-4 Complex

Biochanin-A is a known phytoestrogen that is mainly found in red clover. It has several biological activities including anticancer, anti-inflammatory and antioxidant effects. Preclinical studies showed that Biochanin-A has anticancer properties in different cancer models. This effect was found to happen through a diversity of mechanisms inducing cell cycle arrest, apoptosis and antiangiogenic effects. Moreover, despite being a promising nature-derived anticancer agent, there is a paucity of information regarding specific target validation studies for Biochanin-A. In this study we first predicted the physicochemical properties of Biochanin-A using two different online tools (SwissADME and pkCSM), and then we performed an in silico molecular docking studies for Biochanin-A as a potential dual inhibitor for Cyclin-D1-cyclin dependent kinase (CDK) 4 complex and vascular endothelial growth factor receptor (VEGFR-2) which are key molecular targets for cancer therapy. The results suggest that Biochanin-A interacts with both Cyclin D1-CDK4 complex and VEGFR-2 with a docking affinity that is comparable to their standard inhibitors. These results open the door for further follow up investigations.

Preliminary results of the abemaciclib arm in the Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT): A phase II platform trial using Bayesian adaptive randomization.

2014 Background: The cyclin D-CDK4/6-Rb pathway is activated in most glioblastomas. Abemaciclib is a potent CDK4/6 inhibitor with good brain penetration approved for HR+/HER2- breast cancer. In order to efficiently evaluate the potential impact of abemaciclib on overall survival (OS) in newly diagnosed glioblastoma and to simultaneously develop information regarding potential genomic biomarker associations, abemaciclib was included as an arm on the Individualized Screening Trial of Innovative Glioblastoma Therapy (INSIGhT) trial. INSIGhT is a phase II platform trial using response adaptive randomization and deep genomic profiling to more efficiently test experimental agents in MGMT unmethylated glioblastoma and potentially accelerate identification of novel therapies for phase III testing. Initial randomization was equal between abemaciclib, control, and two other experimental arms but subsequent randomization was adapted based on efficacy as determined by progression-free survival (PFS). Ineffective arms were discontinued and new arms added by protocol amendment. We report preliminary results for the abemaciclib arm which has completed accrual. Methods: Patients with newly diagnosed MGMT-unmethylated glioblastoma were randomized to receive either radiotherapy with concomitant and adjuvant temozolomide at standard doses or standard radiochemotherapy followed by adjuvant abemaciclib (150-200 mg orally BID). Treatment continued until progression or development of unacceptable toxicities. The primary endpoint was OS which was assessed using the log-rank test estimated via the Kaplan Meier method using a type I error of 5%. The hazard ratio (HR) was estimated using a cox proportional hazards model. Association between abemaciclib efficacy and cyclin D-CDK4/6-Rb pathway genomic alterations was also investigated. Results: There were 142 patients (69 control; 73 treated with abemaciclib). Abemaciclib was generally well-tolerated with no new toxicity signals identified. PFS was significantly longer (HR 0.67; p = 0.03, logrank test) with abemaciclib (median 6.54 months) compared to the control arm (median 5.88 months). For patients with activation of the CDK4 pathway the PFS HR was 0.64 (p-value = 0.04). However, there was no significant improvement in overall survival (HR 0.9; p-value &gt; 0.05) between abemaciclib (median 15.5) compared to the control arm (median 15.5). Conclusions: Abemaciclib was well-tolerated and prolonged PFS but there is no evidence of an overall survival improvement compared to standard radiochemotherapy. Clinical trial information: NCT02977780.

Dual blockade of EGFR and CDK4/6 delays head and neck squamous cell carcinoma progression by inducing metabolic rewiring.

Despite preclinical success, monotherapies targeting EGFR or cyclin D1-CDK4/6 in Head and Neck squamous cell carcinoma (HNSCC) have shown a limited clinical outcome. Here, we aimed to determine the combined effect of palbociclib (CDK4/6) and afatinib (panEGFR) inhibitors as an effective strategy to target HNSCC. Using TCGA-HNSCC co-expression analysis, we found that patients with high EGFR and cyclin D1 expression showed enrichment of gene clusters associated with cell-growth, glycolysis, and epithelial to mesenchymal transition processes. Phosphorylated S6 (p-S6), a downstream effector of EGFR and cyclin D1-CDK4/6 signalling, showed a progressive increase from normal oral tissues to leukoplakia and frank malignancy, and associated with poor outcome of the patients. This increased p-S6 expression was drastically reduced after combination treatment with afatinib and palbociclib in the cell lines and mouse models, suggesting its utiliy as a prognostic marker in HNSCC. Combination treatment also reduced the cell growth and induced cell senescence via increasing reactive oxygen species with concurrent ablation of glycolytic and tricarboxylic acid cycle intermediates. Finally, our findings in sub-cutaneous and genetically engineered mouse model (K14-CreERtam;LSL-KrasG12D/+;Trp53R172H/+) studies showed a significant reduction in the tumor growth and delayed tumor progression after combination treatment. This study collectively demonstrates that dual targeting may be a critical therapeutic strategy in blocking tumor progression via inducing metabolic alteration and warrants clinical evaluation.

Inhibition of CDK4/6 kinases causes production of aneuploid oocytes by inactivating the spindle assembly checkpoint and accelerating first meiotic progression

Cyclin D-CDK4/6 complex mediates the transition from the G1 to S phase in mammalian somatic cells. Meiotic oocytes pass through the G2/M transition and complete the first meiosis to reach maturation at the metaphase of meiosis II without intervening S phase, while Cyclin D-CDK4/6 complex is found to express during meiotic progression. Whether Cyclin D-CDK4/6 complex regulates meiotic cell cycle progression is not known. Here, we found its different role in oocyte meiosis: Cyclin D-CDK4/6 complex served as a regulator of spindle assembly checkpoint (SAC) to prevent aneuploidy in meiosis I. Inhibition of CDK4/6 kinases disrupted spindle assembly, chromosome alignment and kinetochore-microtubule attachments, but unexpectedly accelerated meiotic progression by inactivating SAC, consequently resulting in production of aneuploid oocytes. Further studies showed that the MPF activity decrease before first polar body extrusion was accelerated probably by inactivation of the SAC to promote ubiquitin-mediated cyclin B1 degradation. Taken together, these data reveal a novel role of Cyclin D-CDK4/6 complex in mediating control of the SAC in female meiosis I.

A cyclin D-CDK6 dimer helps to reshuffle cyclin-dependent kinase inhibitors (CKI) to overcome TGF-beta-mediated arrest and maintain CDK2 activity

ABSTRACT The cyclin D-CDK4/6 complex has two distinct functions. Its kinase-dependent role involves its ability to act as serine/threonine kinase, responsible for phosphorylation of substrates required for cell cycle transitions, while its kinase-independent function involves its ability to act as a reservoir for p27Kip1. This association sequesters p27 from cyclin E-CDK2 complexes, allowing them to remain active. The aim of this current study is two-fold: to understand the contribution of the kinase-dependent and kinase-independent functions of CDK4 and CDK6 in epithelial cells and to directly compare CDK4 and CDK6 in a simple model system, TGF-β treatment, where arrest is initiated by the expression of p15Ink4b. Cells that overexpressed a catalytically inactive, p15-insensitive CDK6 variant (p27 sequestration only mutant) were able to overcome TGF-β-mediated arrest by maintaining CDK2 activity, while cells expressing the identical mutations in CDK4 were not. This result can be partially explained by the presence of a previously unidentified cyclin D-CDK6 dimer, which serves as a sink for free p27 during TGF-β treatment, enabling CDK2 to remain inhibitor free. The use of the TGF-β model system and the characterization of CDK pool dynamics and p27 switching is relevant to the CDK4/6 specific inhibitors, such as palbociclib, whose mechanism of action may resemble that of p15.

Abstract PS10-03: Interim safety and efficacy analysis of phase IB / II clinical trial of tucatinib, palbociclib and letrozole in patients with hormone receptor and HER2-positive metastatic breast cancer

Abstract Background: In hormone receptor-positive / HER2-positive (HR+/HER2+) breast cancer, the HER2 and estrogen receptor (ER) signals merge on the cyclin D1-CDK4/6-RB1 pathway. Thus, a combined pharmacological intervention with individual drugs targeting HER2, ER and CDK4/6 is warranted. Here, we present the safety and efficacy results of the combination of tucatinib with letrozole and palbociclib in patients (pts) with HR+/HER2+ metastatic breast cancer (MBC) (NCT03054363). Methods: Pts with HR+/HER2+ MBC previously treated with at least 2 HER2-targeted agents were enrolled in this phase IB/II clinical trial. Pts with untreated asymptomatic or stable treated brain metastasis (BM) were included. Pts with treated progressing BM were enrolled after local treatment and classified as treated stable. Treatment consisted of tucatinib 300mg PO BID and letrozole 2.5mg PO daily continuously, and palbociclib 125mg PO daily 21 days on, 7 days off. Due to drug-drug interaction issues found in the middle of the trial and not related to this study, the dose of sensitive CYP3A4 substrate palbociclib was reduced to 75mg for all study participants, as it became evident that tucatinib is a strong CYP3A4 inhibitor. The primary end-points were assessment of safety using CTCAE v.4.03 criteria, and progression free survival (PFS). Secondary end-points included pharmacokinetic evaluation (PKs) and objective response rate by RECIST 1.1. BM response was evaluated using RANO-BM criteria. All pts who received at least one cycle of therapy were assessed for safety. Results: Between 11.21.2017 and 04.20.2020, we enrolled 42 pts of whom 40 were evaluable. Median age was 52.5 years (range, 22 to 82) and the median number of prior lines of therapy for MBC was 2 (range, 0 to 7); 23 pts (58%) had visceral disease and 15 (38%) had BM. All pts had prior therapy with trastuzumab and pertuzumab and 18 pt (45%) had prior T-DM1. As of 06.15.2020 data cut off, 14 patients were on active therapy while 26 were off study (22 due to progressive disease [PD], 1 due to toxicity and 3 for other reasons). Median follow up time was 6 months. The combination was well tolerated with manageable and expected adverse events (AEs). The most common grade ≥3 AEs were neutropenia (25 pts, 60%), leukopenia (10 pts, 24%), diarrhea (8 pts, 19%), fatigue (6 pts, 14%), and infections (6 pts, 14%). One pt came off study due to asymptomatic grade 4 elevated LFTs that resolved without sequelae. There were no deaths due to AEs. Among 26 pts with measurable disease at the time of data cut-off, 8 pts (31%) had partial response, 16 pts (62%) had stable disease (SD) (7 pts [27%] had SD for ≥ 6 months and 6 pts [23%] have not yet reached 6 months of follow up) and 2 pts (8%) had PD. Among 14 patients with BM and evaluable disease by RANO-BM, 1 pt had complete response in the brain, 6 pts had SD in the brain for ≥6 months, and 7 pts had SD for 2-6 months (4 pts on active therapy have not yet reached 6 months of follow up). Median PFS is 8.7 months (10.1 months for pts without BM and 6.0 months for those with BM). Updated analysis including PKs, tumor response, and PFS will be presented. Conclusion: The combination of tucatinib with letrozole and palbociclib showed a tolerable and manageable safety profile and evidence of considerable anti-tumor activity that warrant further clinical investigation in pts with HR+/HER2+ MBC. Citation Format: Elena Shagisultanova, William Gradishar, Ursa Brown-Glaberman, Pavani Chalasani, Andrew J. Brenner, Alison Stopeck, Jose Mayordomo, Jennifer R. Diamond, Peter Kabos, Virginia F. Borges. Interim safety and efficacy analysis of phase IB / II clinical trial of tucatinib, palbociclib and letrozole in patients with hormone receptor and HER2-positive metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-03.