Small-molecule Inhibitor Of Cyclin-dependent Kinases Research Articles

Synthetic Approaches and Clinical Application of Representative Small-Molecule Inhibitors of Cyclin-Dependent Kinase for Cancer Therapy.

The regulation of the cancer cell cycle heavily relies on cyclin-dependent kinases (CDKs). Targeting CDKs has been identified as a promising approach for effective cancer therapy. In recent years, there has been significant attention paid towards developing small-molecule CDK inhibitors in the field of drug discovery. Notably, five such inhibitors have already received regulatory approval for the treatment of different cancers, including breast tumors, lung malignancies, and hematological malignancies. This review provides an overview of the synthetic routes used to produce 17 representative small-molecule CDK inhibitors that have obtained regulatory approval or are currently being evaluated through clinical trials. It also discusses their clinical applications for treating CDK-related diseases and explores the challenges and limitations associated with their use in a clinical setting, which will stimulate the further development of novel CDK inhibitors. By integrating therapeutic applications, synthetic methodologies, and mechanisms of action observed in various clinical trials involving these CDK inhibitors, this review facilitates a comprehensive understanding of the versatile roles and therapeutic potential offered by interventions targeting CDKs.

Discovery of Potent Inhibitors of Cyclin-Dependent Kinases 7 and 9: Design, Synthesis, Structure-Activity Relationship Analysis and Biological Evaluation.

Cyclin-dependent kinases (CDKs) 7 and 9 are deregulated in various types of human cancer and are thus viewed as therapeutic targets. Accordingly, small-molecule inhibitors of both CDKs are highly sought-after. Capitalising on our previous discovery of CDKI-73, a potent CDK9 inhibitor, medicinal chemistry optimisation was pursued. A number of N-pyridinylpyrimidin-2-amines were rationally designed, chemically synthesised and biologically assessed. Among them, N-(6-(4-cyclopentylpiperazin-1-yl)pyridin-3-yl)-4-(imidazo[1,2-a]pyrimidin-3-yl)pyrimidin-2-amine was found to be one of the most potent inhibitors of CDKs 7 and 9 as well as the most effective anti-proliferative agent towards multiple human cancer cell lines. The cellular mode of action of this compound was investigated in MV4-11 acute myeloid leukaemia cells, revealing that the compound dampened the kinase activity of cellular CDKs 7 and 9, arrested the cell cycle at sub-G1 phase and induced apoptosis.

Effectiveness and Safety of Palbociclib plus Endocrine Therapy in Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer: Real-World Results.

Real-world data are critical to demonstrate the reproducibility of evidence and the external generalizability of randomized clinical trials. Palbociclib is an oral small-molecule inhibitor of cyclin-dependent kinases 4/6 that has been shown to improve progression-free survival when combined with letrozole or fulvestrant in phase 3 clinical trials. To evaluate real-world outcomes in patients with metastatic breast cancer who received palbociclib in combination with endocrine therapy in routine clinical practice. In this retrospective observational multicentre study, data were evaluated for all women with metastatic breast cancer who were treated with palbociclib from April 2017 to September 2019. Treatment response was assessed through progression-free survival according to the Response Evaluation Criteria in Solid Tumors, version 1.1. Fifty-three patients were included in the study, with median age 57 years (range 31-87 years). For all patients treated with palbociclib, median progression-free survival by the end of the study period was 14.4 months (95% confidence interval [CI] 6.2-22.2 months). Twenty-three women who received palbociclib as a first-line treatment did not experience progression-free survival; for these patients, the median treatment duration was 12.1 months (95% CI 1.4-28.0 months). For the 23 patients who received palbociclib as second-line therapy for metastatic breast cancer, median progression-free survival was 13.3 months (95% CI 4.1-22.4 months). Among the 7 women who received palbociclib as third-line therapy, median progression-free survival was 6.0 months (95% CI 0.9-11.1 months). The most common adverse events were hematologic, with grade 3 or 4 neutropenia occurring in 20 (38%) of the 53 patients. This study provides data from a real-world setting that match the results of previous studies in terms of effectiveness (i.e., progression-free survival) when palbociclib plus endocrine therapy was used as second- or third-line treatment. Palbociclib had appropriate tolerability and a profile of easily manageable adverse effects, with none of the patients suspending their treatment because of toxic effects.

Diisothiocyanate-Derived Mercapturic Acids Are a Promising Partner for Combination Therapies in Glioblastoma.

Glioblastoma represents the most aggressive tumor of the central nervous system. Due to invasion of glioblastoma stem cells into the healthy tissue, chemoresistance, and recurrence of the tumor, it is difficult to successfully treat glioblastoma patients, which is demonstrated by the low life expectancy of patients after standard therapy treatment. Recently, we found that diisothiocyanate-derived mercapturic acids, which are isothiocyanate derivatives from plants of the Cruciferae family, provoked a decrease in glioblastoma cell viability. These findings were extended by combining diisothiocyanate-derived mercapturic acids with dinaciclib (a small-molecule inhibitor of cyclin-dependent kinases with anti-proliferative capacity) or temozolomide (TMZ, standard chemotherapeutic agent) to test whether the components have a cytotoxic effect on glioblastoma cells when the dosage is low. Here, we demonstrate that the combination of diisothiocyanate-derived mercapturic acids with dinaciclib or TMZ had an additive or even synergistic effect in the restriction of cell growth dependent on the combination of the components and the glioblastoma cell source. This strategy could be applied to inhibit glioblastoma cell growth as a therapeutic interference of glioblastoma.

CDK9 activity is critical for maintaining MDM4 overexpression in tumor cells

The identification of the essential role of cyclin-dependent kinases (CDKs) in the control of cell division has prompted the development of small-molecule CDK inhibitors as anticancer drugs. For many of these compounds, the precise mechanism of action in individual tumor types remains unclear as they simultaneously target different classes of CDKs – enzymes controlling the cell cycle progression as well as CDKs involved in the regulation of transcription. CDK inhibitors are also capable of activating p53 tumor suppressor in tumor cells retaining wild-type p53 gene by modulating MDM2 levels and activity. In the current study, we link, for the first time, CDK activity to the overexpression of the MDM4 (MDMX) oncogene in cancer cells. Small-molecule drugs targeting the CDK9 kinase, dinaciclib, flavopiridol, roscovitine, AT-7519, SNS-032, and DRB, diminished MDM4 levels and activated p53 in A375 melanoma and MCF7 breast carcinoma cells with only a limited effect on MDM2. These results suggest that MDM4, rather than MDM2, could be the primary transcriptional target of pharmacological CDK inhibitors in the p53 pathway. CDK9 inhibitor atuveciclib downregulated MDM4 and enhanced p53 activity induced by nutlin-3a, an inhibitor of p53-MDM2 interaction, and synergized with nutlin-3a in killing A375 melanoma cells. Furthermore, we found that human pluripotent stem cell lines express significant levels of MDM4, which are also maintained by CDK9 activity. In summary, we show that CDK9 activity is essential for the maintenance of high levels of MDM4 in human cells, and drugs targeting CDK9 might restore p53 tumor suppressor function in malignancies overexpressing MDM4.

Small Molecule CDK Inhibitors for the Therapeutic Management of Cancer.

Cyclin-dependent kinases (CDKs) are a group of multifunctional enzymes consisting of catalytic and regulatory subunits. The regulatory subunit, cyclin, remains dissociated under normal circumstances, and complexation of cyclin with the catalytic subunit of CDK leads to its activation for phosphorylation of protein substrates. The primary role of CDKs is in the regulation of the cell cycle. Retinoblastoma protein (Rb) is one of the widely investigated tumor suppressor protein substrates of CDK, which prevents cells from entering into cell-cycle under normal conditions. Phosphorylation of Rb by CDKs causes its inactivation and ultimately allows cells to enter a new cell cycle. Many cancers are associated with hyperactivation of CDKs as a result of mutation of the CDK genes or CDK inhibitor genes. Therefore, CDK modulators are of great interest to explore as novel therapeutic agents against cancer and led to the discovery of several CDK inhibitors to clinics. This review focuses on the current progress and development of anti-cancer CDK inhibitors from preclinical to clinical and synthetic to natural small molecules.

Tolerance of concurrent CDK inhibitor and radiation therapy in metastatic breast cancer patients.

e12598 Background: Palbociclib, a small-molecule inhibitor of cyclin-dependent kinases (CDK4 and CDK6), combined with letrozole increases progression-free survival among patients with previously untreated ER-positive, HER2 negative advanced breast cancer. The purpose of our study was to retrospectively evaluate the tolerance of the concomitant association of Palbociclib and radiation therapy (RT) at Curie Institute. Methods: Between April 2017 and August 2019, 30 women with ER-positive, HER2 negative metastatic breast cancer received locoregional (LR) and/or symptomatic irradiation at a metastatic site concurrently with Palbociclib at a daily dose of 125 mg, from d1 to d21 every 28 days. Palbociclib was always associated with endocrine therapy: letrozole (with or without an LHRH analogue) or fulvestrant. Thirty-five sites were irradiated: nine patients received post-operative locoregional RT, including the chest wall or breast and lymph node areas, and 26 sites of metastases were irradiated: 17 at the spine, 7 peripheral skeletal lesions, 1 brain lesion and 1 choroidal lesion. The dose prescribed for locoregional mammary radiotherapy was 50 Gy in 25 fractions and varied for the treatment of metastatic sites: 20 Gy in 5 fractions (n = 13), 30 Gy in 10 fractions (n = 10) and 8 Gy in 1 fraction (n = 2). The brain metastasis was stereotactically treated (1 fraction of 18 Gy). The primary endpoint was toxicity scored according to the common terminology criteria for NCI adverse events, version v5.0. Results: Mean number of days of Palbociclib during RT was 8.8 days (range, 1 to 24 days). The most common acute toxicities were dermatitis (12/35, including 2 grade 2) and neutropenia (12/35, including 9 grade 2). Palbociclib had to be stopped during the RT of two patients (2/30): one patient treated locoregionally (bilateral breast and lymph nodes irradiation) developed a grade 3 dermatitis and febrile neutropenia, another treated locoregionally developed grade 2 dysphagia. After a median follow-up since the end of RT of 17 months (6 – 31 months, SD 8), none of the patients have so far exhibited late toxicity. Conclusions: Concomitant administration of palbociclib with RT was reasonably well tolerated in our series of 30 patients. Given this experience, palbociclib should not be discontinued during radiation therapy. Nevertheless, our findings should be confirmed in prospective registration studies collecting larger number of patients.

Abstract P4-12-05: A retrospective study of 27 patients with ER-positive, HER2 negative metastatic breast cancer treated with concomitant palbociclib and radiation therapy

Abstract Background: Palbociclib, a small-molecule inhibitor of cyclin-dependent kinases (CDK4 and CDK6), combined with letrozole increases progression-free survival among patients with previously untreated ER-positive, HER2 negative advanced breast cancer. However, the data on the safety of the concomitant association between Palbociclib and radiation therapy are scarce. The purpose of our study was to retrospectively evaluate the tolerance of the first patients treated with this concomitant combination at Institut Curie. Materials and methods: Between April 2017 and July 2019, 27 women with ER-positive, HER2 negative metastatic breast cancer received locoregional and/or symptomatic irradiation at a metastatic site concomitantly with Palbociclib at a daily dose of 125 mg, from d1 to d21 every 28 days. Palbociclib was always associated with an endocrine treatment: Letrozole (17 patients /27, with or without an LHRH analogue) or Fulvestrant (10 patients /27). Median age at the time of diagnosis of metastases was 60 years (range, 35-85 years). Thirty-two sites were irradiated: 9 patients received post-operative locoregional radiation therapy, including the chest wall or breast and lymph node areas, and 23 sites of metastases were irradiated: 15 at the spine (3 cervical, 6 thoracic, 4 lumbar, 2 sacral), 2 pelvic lesions, 4 peripheral skeletal lesions, 1 brain lesion and 1 choroidal lesion (two patients received locoregional treatment and irradiation in one metastatic site and two patients were treated in two different metastatic sites). The dose prescribed for locoregional mammary radiotherapy was 50 Gy in 25 fractions and varied for the treatment of metastatic sites: 20 Gy in 5 fractions (n=14), 30 Gy in 10 fractions (n=7) and 8 Gy in 1 fraction (n=1). The brain metastasis was stereotactically treated (1 fraction of 18 Gy). The primary endpoint was toxicity scored according to the common terminology criteria for NCI adverse events, version v4.0. Results: Median follow-up was 22 months (range, 4 to 54). Mean number of days of Palbociclib during radiation therapy was 8.7 days (range, 1 to 23 days). Most common acute toxicities were radiodermatitis (12/27, including 1 grade 3, 2 grade 2 and 9 grade 1) and neutropenia (13/27, including 1 grade 4, 7 grade 3, 5 grade 2). Palbociclib had to be stopped during the radiation therapy of three patients (3/27): one patient treated locoregionally (bilateral breast and lymph nodes irradiation) developed a grade 3 radiodermatitis and febrile neutropenia, another treated locoregionally developed grade 2 dysphagia (loss of 4 kg in 1 month) and one patient treated for sacral metastases (20 Gy in 4 fractions) developed grade 3 proctitis. Of the 19/27 patients with a follow-up of more than 6 months none developed late toxicities. Conclusion: Concomitant administration of Palbociclib with radiation therapy was reasonably well tolerated in our series of 27 patients. Caution should be exercised when a large volume is irradiated, and when the dose per fraction is high. It remains unsure whether the interruption-causing side effects of Palbociclib was based on synergistic toxicity with radiation therapy. We suggest that our findings are to be confirmed in prospective registration studies collecting larger number of patients. Citation Format: Arnaud Beddok, Alexandre Arsene-Henry, Baptiste Porte, Kim Cao, Louis Bazire, Nathaniel Scher, Alain Labib, Ilan Darmon, Joëlle Otz, Mathieu Minsat, Hamid Mammar, François Clément Bidar, Alain Fourquet, Paul Cottu, Phlip Poortmans, Youlia Kirova. A retrospective study of 27 patients with ER-positive, HER2 negative metastatic breast cancer treated with concomitant palbociclib and radiation therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-12-05.

Anti-proliferative effects of a small molecule inhibitor of CDK AT7519 on chronic myeloid leukemia (CML) cells through halting the transition of cells from G2/M phase of the cell cycle

Pathogenesis of chronic myeloid leukemia (CML) has mostly been studied with regard to the oncogenic role of BCR/ABL fusion; however, recent disclosures have declared that the challenges with the treatment of CML patients would not be resolved until the role of other aberrancies is ignored. Given the involvement of cyclin-dependent kinases (CDKs) in the pathogenesis of CML, the present study aimed to investigate the effects of a multi-CDK inhibitor AT7519 on BCR/ABL-harboring CML-derived K562 cells. Our results showed that AT7519 effectively reduced the survival of K562 and induced its anti-proliferative effect through the induction of G2/M arrest due to elevated p21 and p27. The resulting data also revealed that either direct or indirect suppression of c-Myc using specific c-Myc inhibitor 10058-F4 and selective PI3K inhibitor CAL-101 resulted in a superior cytotoxicity, suggesting that the activation of PI3K pathway could attenuate antileukemic effects of the inhibitor, at least partly, through a c-Mycdependent mechanism. To the best of our knowledge, to date, no study has addressed the effect of autophagy on CML cell response to AT7519, and, herein, we proposed for the first time that the suppression of autophagy boosted AT7519 cytotoxicity against K562. Overall, we suggested that selective CDK inhibitor AT7519 exerted antileukemic effect against CML cells and propose a novel therapeutic application for the inhibitor either as a single agent or in combination with c-Myc and/or PI3K inhibitors.

A new route for the synthesis of Palbociclib

In this paper, a novel synthetic method for Palbociclib was reported. It was synthesized in eight steps from 2-(methylthio) pyrimidin-4-(3H)-one with approximately 10% overall yield. This protocol started material 2-(methylthio) pyrimidin-4-(3H)-one, involved nucleophilic substitution by thionyl chloride, bromination, nucleophilic substitution by cyclopentylamine, a one pot-two step method (Heck reaction, ring close sequence), oxidation and bromination, cross-coupling reaction, Heck reaction, aqueous workup to afford Palbociclib. This synthetic route used inexpensive raw material and reagents, involved readily controllable reaction conditions and reduced environmental hazards. Synthesis of Palbociclib, a small molecule CDK inhibitor, starting from 2-(methylthio) pyrimidin-4-(3H)-one by 8 steps reaction. This method afforded the Palbociclib in 10% yield.

Real-world clinical outcomes and toxicity in metastatic breast cancer patients treated with palbociclib and endocrine therapy

Real-world data are critical to demonstrate the reproducibility of evidence and external generalizability of randomized clinical trials. Palbociclib is an oral small-molecule inhibitor of cyclin-dependent kinases 4 and 6 that has been shown to improve progression-free survival (PFS) when combined with letrozole or fulvestrant in phase 3 clinical trials. We evaluated real-world outcomes in metastatic breast cancer patients who received palbociclib in combination with endocrine therapy in routine clinical practice. Records of patients with advanced hormone receptor (HR)-positive breast cancer treated with palbociclib at the Cleveland Clinic health system from February, 2015 to December, 2017 were retrospectively reviewed. The primary end point was PFS. In this cohort, 411 women were included. The median age and follow-up times were 53.5years and 10.2 months, respectively. The median PFS for palbociclib plus letrozole was 15.1months for patients treated in first line, 10.5months in second line, and 4.2months in third line and beyond. For patients who received fulvestrant plus palbociclib, the median PFS in first, second, and third line and beyond were 11.6, 12.3, and 6.4months, respectively. The most common adverse events were hematologic, with grade 3-4 neutropenia occurring in 58% of patients. Thirty-one (8%) patients permanently discontinued palbociclib due to adverse events. Among patients with HR-positive advanced breast cancer, the estimated PFS in patients treated with fulvestrant and palbociclib was comparable to a previously reported phase 3 trial.However, the median PFS with letrozole and palbociclib was shorter than previously reported data from phase 2 and 3 trials. Palbociclib toxicity was very manageable, with a low drug discontinuation rate.

Dexamethasone Accelerates the Transition of Human BFU-E to CFU-E and Enhances CFU-E Proliferation through Cell Cycle Regulation

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome that is frequently managed with glucocorticoids to increase red cell mass. Despite sustained effective clinical use for many decades in DBA and other anemias, we still do not fully understand the mechanistic basis for the regulation of human erythropoiesis by glucocorticoids. In order to improve the clinical management of patients with DBA, we studied the mechanisms of action of dexamethasone (Dex) on erythroid progenitors.To study the effects of Dex on erythroid progenitor biology, we employed a serum-free erythroid culture system to differentiate primary human CD34+ cells isolated from the peripheral blood of adults or DBA patients as well as cord blood samples. We found that Dex increases the total proliferation of CD34+ cells from adult peripheral blood but not in cord blood over 14 days in culture. Dex treatment also led to an acceleration of the BFU-E to CFU-E transition in peripheral blood with a minimal effect in cord blood. In methylcellulose colony forming assays, peripheral blood derived CFU-E treated with Dex for 24hrs produced more colonies or larger colonies than the untreated controls while a lesser effect was seen in cord blood derived CFU-E. Both assays revealed that Dex treatment enhanced self-renewal of peripheral blood derived CFU-E.We also noted that BFU-E and CFU-E differentially express the glucocorticoid receptor (GR) isoforms, GRα and GRβ. BFU-E expressed comparable levels of GRα and the dominant-negative GRβ, whereas only the GRα transcript was identified in CFU-E. We observed similar expression patterns for GR between peripheral and cord blood derived progenitors. Importantly, following Dex treatment, GRα was shown to translocate to the nucleus of erythroid progenitors derived from peripheral blood derived CD34+ cells but not in the cord blood derived progenitors.When we examined the cell cycle progression of sorted human erythroid progenitors, CFU-E and BFU-E, we observed a G0/G1 arrest of peripheral blood CFU-E treated with Dex compared to untreated CFU-E. In marked contrast, no such change was noted for either Dex treated BFU-E from peripheral blood or for CFU-E or BFU-E from cord blood. In addition, we found an increase in the expression of cell cycle regulators p57Kip2 and p27Kip1 in Dex treated peripheral blood CFU-E but no such increased expression was seen in cord blood CFU-E.When peripheral blood derived erythroid progenitors were treated with olomoucine (Olo), a small molecule CDK inhibitor, we observed an acceleration of the BFU-E to CFU-E transition similar to that induced by Dex. Upon co-treatment with Dex and Olo, there was a synergistic effect in the accelerated formation of CFU-E population from BFU-Es. To validate this finding of the dependence of CFU-E on cell cycle regulators, we transduced peripheral blood derived CD34+ cells with a lentiviral shRNA construct to knockdown p57 expression. Following successful knockdown of the expression levels of p57, the numbers of CFU-E indeed decreased implying a role for cell cycle in defective transition of BFU-E to CFU-E.Erythroid progenitors derived from cultures of primary CD34+ cells from DBA patients were also studied. Importantly, we noted increased baseline expression of p57Kip2 and p27Kip1 in erythroid progenitors from steroid resistant DBA patients and in contrast to controls, their expression did not increase following Dex treatment.Our findings provide novel mechanistic insights into the regulation of human erythropoiesis by Dex. We have shown that alterations in cell cycle regulation of erythroid progenitors plays a key role in the effects of glucocorticoids on red blood cells. Furthermore, this work has direct relevance for the development of improved therapeutic treatment strategies for patients with bone marrow failure syndromes such as DBA. DisclosuresNo relevant conflicts of interest to declare.

Abstract B43: The CDK4 and 6 inhibitor abemaciclib induces both epigenetic and immunogenic responses in preclinical models of Ewing’s sarcoma

Abstract Ewing’s sarcoma (ES) is a rare but aggressive cancer typically occurring in the bone or soft tissue of children and adolescents. Current treatment regimens composed of surgery, radiation, and high-dose chemotherapy have improved outcomes for localized disease cases; however, ES continues to present a clinical challenge in the recurrent or metastatic setting. ES is often characterized by a chromosomal translocation that results in a chimeric EWS/ETS transcription factor. This fusion protein may contribute to tumorigenesis through modulation of downstream targets leading to cell cycle dysregulation, due in part to the overexpression of Cyclin D1. In addition, ES also has a high degree of DNA methylation when compared to other tumor types, which can lead to epigenetic silencing of key tumor suppressor genes. Abemaciclib is a small-molecule inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 and 6) currently under clinical evaluation for metastatic breast cancer, non-small cell lung cancer, and other adult solid tumors. Abemaciclib shows antitumor activity in preclinical models of human adult and pediatric cancer with functional Rb and high cyclin D expression, and has been reported to elicit an immune response in addition to its cell cycle effects in breast cancer. This study was designed to assess abemaciclib-induced antitumor effects, in addition to epigenetic and immunogenic changes, in preclinical ES models. Six ES cell lines were treated with abemaciclib and the resulting changes in protein expression and methylation were evaluated by Western blot and DNA promoter methylation analysis, respectively. In addition, tumor-secreted factors from conditioned media were measured by ELISA and activation of the interferon (IFN) pathway was assessed through Western blot, qRT-PCR, and RNAseq analysis. In vivo studies of abemaciclib as a single agent or in combination with ES standard-of-care (SOC) in multiple mouse xenograft models were also performed to measure abemaciclib-mediated antitumor effects and epigenetic and immunogenic changes. Single-agent abemaciclib elicited tumor growth delay or tumor stasis, while the combination with SOC was generally superior to either monotherapy arm, often resulting in objective responses. Abemaciclib induced global DNA demethylation through the Rb-E2F-DNMT1 axis, and a ligand-independent type III IFN response. In addition, abemaciclib increased expression of the tumor cell antigen presentation protein MHC-1, and this effect was more durable than abemaciclib-dependent cell cycle effects. These data demonstrate that abemaciclib activity in ES goes beyond cell cycle arrest to include a measurable and durable immunogenic response. Therefore, the multipronged antitumor effects of abemaciclib, namely CDK4/6 inhibition, global DNA demethylation, and resulting immunogenic changes, may be a viable therapeutic option for ES patients when used in combination with appropriate chemotherapy, and additional studies to better understand this unique biology are ongoing. Citation Format: Michele Dowless, Caitlin Lowery, Matthew Renschler, Wayne Blosser, Jennifer Stephens, Robert Flack, Kelly Credille, Mia Chen, Frank Dorsey, Lillian Sams, Philip Ebert, Jonathan Olsen, Terry Shackleford, Peter Houghton, Louis Stancato. The CDK4 and 6 inhibitor abemaciclib induces both epigenetic and immunogenic responses in preclinical models of Ewing’s sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B43.

Low dose dinaciclib enhances doxorubicin-induced senescence in myeloma RPMI8226 cells by transformation of the p21 and p16 pathways.

Multiple myeloma (MM) is a hematological malignancy that lacks a cure. However, novel combination therapy is a current anti-MM strategy. Doxorubicin (DOX) is a type of anthracycline which is a first-line chemotherapeutic for treating MM and induces senescence in many types of cancer. Dinaciclib is a potent, small molecule CDK inhibitor with promise for treating several types of cancer in I/II phase clinical trials. In the present study the anticancer effects and underlying mechanisms of dinaciclib combined with DOX in MM RPMI-8226 cells were investigated. Results indicated that DOX induced cell viability inhibition, cell cycle arrest and senescence. Furthermore, DOX resulted in increased alterations in DNA damage-related proteins such as p-ATM, p-Chk2, p-p53, p21 and γH2AX, but not p16. Notably, the combination of dinaciclib and DOX inhibited cell growth and promoted senescence by transforming the suppressive effects of the ATM/Chk2/p53/p21 signaling pathway and enhancing the p16 signaling pathway. Thus, low-dose dinaciclib enhanced anti-MM effects mediated by DOX via transformation of p21-p16 signaling pathways, leading to accelerated senescence, but not apoptosis. The present findings suggest this approach may be a promising therapeutic strategy for the treatment of MM.

Targeting Cyclin-Dependent Kinases for Treatment of Gynecologic Cancers.

Ovarian, uterine/endometrial, and cervical cancers are major gynecologic malignancies estimated to cause nearly 30,000 deaths in 2018 in US. Defective cell cycle regulation is the hallmark of cancers underpinning the development and progression of the disease. Normal cell cycle is driven by the coordinated and sequential rise and fall of cyclin-dependent kinases (CDK) activity. The transition of cell cycle phases is governed by the respective checkpoints that prevent the entry into the next phase until cellular or genetic defects are repaired. Checkpoint activation is achieved by p53- and ATM/ATR-mediated inactivation of CDKs in response to DNA damage. Therefore, an aberrant increase in CDK activity and/or defects in checkpoint activation lead to unrestricted cell cycle phase transition and uncontrolled proliferation that give rise to cancers and perpetuate malignant progression. Given that CDK activity is also required for homologous recombination (HR) repair, pharmacological inhibition of CDKs can be exploited as a synthetic lethal approach to augment the therapeutic efficacy of PARP inhibitors and other DNA damaging modalities for the treatment of gynecologic cancers. Here, we overview the basic of cell cycle and discuss the mechanistic studies that establish the intimate link between CDKs and HR repair. In addition, we present the perspective of preclinical and clinical development in small molecule inhibitors of CDKs and CDK-associated protein targets, as well as their potential use in combination with hormonal therapy, PARP inhibitors, chemotherapy, and radiation to improve treatment outcomes.

Abstract B60: Omics profiling of CDK4/6 inhibitors reveals functionally important secondary targets of abemaciclib

Abstract The recent introduction of small-molecule inhibitors of cyclin-dependent kinases (CDK) 4/6 to the clinic has improved the treatment of hormone receptor-positive breast cancer, and shown promise in other malignancies. The three clinically used CDK4/6 inhibitors, palbociclib, ribociclib, and abemaciclib, are reported to be broadly similar, although recent data suggest that abemaciclib has distinct single-agent activity in patients and a unique toxicity profile. In vitro profiling of the CDK4/6 inhibitors has shown abemaciclib to be more potent on target, but less selective than either palbociclib or ribociclib. We sought to systematically investigate the functional consequences of abemaciclib’s polypharmacology. We used molecular and phenotypic profiling by mRNA sequencing, mass spectrometry-based proteomics, and GR-based dose-response assays to characterize the mechanisms of action of the three CDK4/6 inhibitors. Inhibition of CDKs other than CDK4/6 by abemaciclib resulted in an arrest in the G2 phase of the cell cycle, a prolonged inhibition of growth, and cytotoxicity, even in retinoblastoma protein (pRb)-deficient cells. Abemaciclib elicited unique molecular responses at clinically achievable concentrations that are likely to be therapeutically advantageous and prevented cross-resistance with ribociclib and palbociclib. More generally, we propose that multi-omic approaches are required to fully elucidate the spectrum of targets relevant to drug mechanisms of action in cells, and we expect such understanding to assist in stratifying patient populations and in ordering sequential therapies when resistance arises. Citation Format: Caitlin E. Mills, Marc Hafner, Kartik Subramanian, Chen Chen, Sarah A. Boswell, Robert A. Everley, Dejan Juric, Peter K. Sorger. Omics profiling of CDK4/6 inhibitors reveals functionally important secondary targets of abemaciclib [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B60.

CDK4/6 Inhibition Augments Antitumor Immunity by Enhancing T-cell Activation.

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies.Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Jenkins et al., p. 196This article is highlighted in the In This Issue feature, p. 127.

Cost-effectiveness of palbociclib in hormone receptor-positive advanced breast cancer

Palbociclib (PAL), a novel small-molecule inhibitor of cyclin-dependent kinases 4 and 6 for the treatment of advanced breast cancer, has demonstrated significant efficacy in prolonging progression-free survival when added to existing therapies. Considering the high cost of PAL, we assessed cost-effectiveness of adding PAL to usual care in treatment of advanced breast cancer. We developed a discrete event simulation model to simulate time to cancer progression and to compare life time clinical benefit and cost of alternative treatment strategies for patients with metastatic disease from societal perspective. Per approved indication, endocrine treatment naive patients were assigned to PAL plus letrozole (PAL + LET) or letrozole alone (LET). Patients with prior endocrine therapy were assigned to PAL plus fulvestrant (FUL) (PAL + FUL) or FUL alone. The model assumptions were informed based on published clinical trial data and other peer reviewed studies. We carried out one-way and probabilistic sensitivity analyses to assess the robustness of our results to the changes in model assumptions. In treatment-naive patients, the addition of PAL to LET cost an estimated $768 498 per additional quality-adjusted life-year (QALY) gained. The addition of PAL to FUL in patients with prior endocrine therapy cost an estimated $918 166 per QALY gained. Sensitivity analyses demonstrated adding PAL has a 0% chance of being cost-effectiveness in either patient groups at a willingness-to-pay threshold of $100 000 per QALY. From a societal perspective, PAL treatment of both patient groups (with and without prior endocrine therapy) is highly unlikely to be cost-effective compared with the usual care in the USA.

Incidence and risk of neutropenia with palbociclib in patients with cancer: A systematic review and meta-analysis.

e12530 Background: Palbociclib is an oral small-molecule inhibitor of cyclin-dependent kinases 4 and 6 and has improved the progression-free survival of estrogen receptor positive, metastatic breast cancer (MBC). Its application in other cancers is also undergoing clinical evaluation. Neutropenia, especially high grade (grade 3 or 4), is one of the major side-effects of palbociclib, and the incidence was reported too vary in different studies. In order to better understand the overall risk of palbociclib associated neutropenia, we conducted a systematic review and meta-analysis. Methods: We conducted a systematic literature search (including Medline, Web of Science to November, 2016 and abstracts presented at the ASCO annual meetings from 2009 to 2016). Eligible studies include prospective clinical trials of patients with cancer treated with palbociclib on a 125mg daily 3/4 week schedule and have available data on neutropenia. The incidence and relative risk (RR) of neutropenia were calculated using a random-effects or fixed-effects model, depending on the heterogeneity of the included studies. Results: Eight studies published between 2012 and 2016 included a total of 1515 patients with cancer were eligible for analysis. For patients treated with palbociclib, the overall incidence of all-grade and high grade neutropenia were 77.4% (95% CI 70.9-82.8%) and 60.9% (57.8–63.8%), respectively. Surprisingly, the incidence of neutropenia was significantly different between MBC patients and non-MBC patients (all grade: RR 1.26 [95% CI 1.01–1.56], p = 0.041; high-grade: RR 1.57 [1.23–2.00], p = 0.000) who received palbociclib. Palbociclib was associated with a significantly increased risk of all-grade neutropenia in patients with cancer with an RR of 15.03 (10.17–22.21, p < 0.001) compared with controls. Conclusions: Patients with cancer who received palbociclib have a significant risk of developing neutropenia, especially MBC patients. It is strongly recommended to monitor these patients who are treated with palbociclib to adjust dose, treatment intervals, and avoid infections.

Abstract P4-22-03: Palbociclib in combination with endocrine therapy in treatment-naive and previously treated elderly women with HR+, HER2– advanced breast cancer: a pooled analysis from randomized phase 2 and 3 studies

Abstract Background: At least 40% of breast cancers are diagnosed in women ≥65 y old and most are hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-). Palbociclib (PAL) is an oral, small-molecule inhibitor of cyclin-dependent kinases 4 and 6. Randomized studies of PAL combined with endocrine therapy (ET) demonstrated significantly improved progression-free survival (PFS) in patients (pts) with treatment-naive and previously treated advanced breast cancer (ABC). Methods: We evaluated the efficacy of PAL+ET vs ET alone in pts aged ≥65-74 and ≥75 y across multiple pivotal randomized phase 2 and 3 studies. Safety and pharmacokinetics (PK) data (blood samples collected from pts in phase 1/2 [PALOMA-1] and phase 1 studies [NCT00141297 and NCT00420056]) for PAL+ET were pooled and compared across age groups. Pts who had not received treatment for ABC were randomized to receive PAL+letrozole (LET) or LET alone/with placebo (PBO; PALOMA-1, open-label/PALOMA-2, double-blind). Pts who had progressed on prior ET were randomized to receive PAL+fulvestrant (FUL) or PBO+FUL (PALOMA-3, double-blind). The primary endpoint for these studies was investigator-assessed PFS. Safety assessments and blood counts occurred at baseline and every 2 weeks for the first 2 cycles and on day 1 of subsequent cycles. Results: Among 872 pts treated with PAL+ET, 221 (25%) were aged ≥65-74 y and 83 (10%) were ≥75 y (PAL+LET: n=528, 162 and 56, respectively; PAL+FUL: n=347, 59 and 27). Median (range) treatment durations were 440 (1-1615) d, 502 (1-1615) d, and 459 (21-1404) d, respectively. Improvement in efficacy endpoints was seen with PAL+ET vs ET across all age groups (Table 1). Incidence of adverse events (AEs), serious AEs and discontinuations due to AEs were similar in the overall population (99%, 19%, 3%) and in pts aged ≥65-74 (99%, 25%, 5%) and ≥75 y (100%, 30%, 6%). Incidence of all grades and grade 3/4 neutropenia were also similar across age groups (overall: 67% and 54%; ≥65-74 y: 64% and 51%; ≥75 y: 77% and 60%). PK analysis showed no clinically relevant differences between arithmetic means, medians, and geometric means of the apparent oral clearance across age groups. Conclusions: PAL in combination with ET is an effective and well-tolerated treatment option for elderly pts with HR+/HER2- endocrine-sensitive and -resistant ABC. A dose adjustment based on age is not required. Sponsor: Pfizer Table 1. PFS in pts ?65-74 and ?75 y (ITT populations)OverallAged ≥65-74 yAged ≥75 yPALOMA-1/PALOMA-2PAL+LET vs528 vs 303162 vs 9456 vs 26LET/LET+PBO,* nHR (95% CI);0.53 (0.44-0.64);0.66 (0.45-0.97);0.31 (0.16-0.61);1-sided P value<0.00010.01620.0002Median PFS (95% CI), mo24.4 (22.0-26.2) vs27.5 (24.2-NR) vsNR (19.2-NR) vs13.6 (11.1-16.4)21.8 (16.3-31.3)10.9 (4.9-24.9)PALOMA-3PAL+FUL vs347 vs 17459 vs 3727 vs 6FUL+PBO, nHR (95% CI);0.46 (0.36-0.59);0.25 (0.14-0.45);0.87 (0.27-2.79);1-sided P value<0.0001<0.00010.4074Median PFS (95% CI), mo9.5 (9.2-11.0) vs16.1 (12.0-NR) vs13.6 (7.5-NR) vs4.6 (3.5-5.6)3.7 (1.9-5.3)7.4 (1.9-NR)HR=hazard ratio; ITT=intent to treat; NR=not reached. *Does not include 5 pts from phase 1 of PALOMA-1. Citation Format: Rugo HS, Turner NC, Finn RS, Joy AA, Verma S, Harbeck N, Moulder S, Masuda N, Im Y-H, Zhang K, Kim S, Sun W, Schnell P, Huang-Bartlett C, Slamon D. Palbociclib in combination with endocrine therapy in treatment-naive and previously treated elderly women with HR+, HER2– advanced breast cancer: a pooled analysis from randomized phase 2 and 3 studies [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-22-03.