Inhibitor PD0332991 Research Articles

CDK4/6 inhibitor PD-0332991 suppresses hepatocarcinogenesis by inducing senescence of hepatic tumor-initiating cells

IntroductionOwing to the limited treatment options for hepatocellular carcinoma (HCC), interventions targeting pre-HCC stages have attracted increasing attention. In the pre-HCC stage, hepatic tumor-initiating cells (hTICs) proliferate abnormally and contribute to hepatocarcinogenesis. Numerous studies have investigated targeted senescence induction as an HCC intervention. However, it remains to be clarified whether senescence induction of hTICs could serve as a pre-HCC intervention. ObjectivesThis study was designed to investigate whether senescence induction of hTICs in the precancerous stage inhibit HCC initiation. Methods and ResultsHCC models developed from chronic liver injury (CLI) were established by using Fah-/- mice and N-Ras + AKT mice. PD-0332991, a selective CDK4/6 inhibitor that blocks the G1/S transition in proliferating cells, was used to induce senescence during the pre-HCC stage. Upon administration of PD-0332991, we observed a significant reduction in HCC incidence following selective senescence induction in hTICs, and an alleviation liver injury in the CLI-HCC models. PD-0332991 also induced senescence in vitro in cultured hTICs isolated from CLI-HCC models. Moreover, RNA sequencing (RNA-seq) analysis delineated that the “Cyclin D-CDK4/6-INK4-Rb” pathway was activated in both mouse and human liver samples during the pre-HCC stage, while PD-0332991 exhibited substantial inhibition of this pathway, thereby inducing cellular senescence in hTICs. Regarding the immune microenvironment, we demonstrated that senescent hTICs secrete key senescence-associated secretory phenotypic (SASP) factors, CXCL10 and CCL2, to activate and recruit macrophages, and contribute to immune surveillance. ConclusionWe found that hTICs can be targeted and induced into a senescent state during the pre-HCC stage. The SASP factors released by senescent hTICs further activate the immune response, facilitating the clearance of hTICs, and consequently suppressing HCC occurrence. We highlight the importance of pre-HCC interventions and propose that senescence-inducing drugs hold promise for preventing HCC initiation under CLI.

CDK4/6 inhibitor-induced bone marrow micronuclei might be caused by cell cycle arrest during erythropoiesis

BackgroundA micronucleus test is generally used to evaluate the genotoxic potential of chemicals. Exaggerated erythropoiesis, as occurs following bleeding, may induce an unexpected increase in micronucleus frequency. This false positive result would be typical in a genotoxicity study due to the enhanced progression of the cell cycle that restores decreased blood cells. The cyclin-dependent kinase (CDK) family is known to play an essential role in preventing genomic instability. Conversely, a selective CDK4/6 inhibitor PD0332991, clinically named Palbociclib, is reported to have genotoxic potential, shown by positive results in both in vitro and in vivo micronucleus studies. To clarify the mechanism by which cell cycle arrest induced by a CDK4/6 inhibitor increases micronucleus frequency, we investigated the positive results of the bone marrow micronucleus test conducted with PD0332991.ResultsRats treated with PD0332991 exhibited increased micronucleus frequency in an in vivo bone marrow micronucleus test whereas it was not increased by treatment in human lymphoblastoid TK6 cells. In addition, all other genotoxicity tests including the Ames test and the comet assay showed negative results with PD0332991. Interestingly, PD0332991 treatment led to an increase in erythrocyte size in rats and affected the size distribution of erythrocytes, including the micronucleus. The mean corpuscular volume of reticulocytes (MCVr) in the PD0332991 treatment group was significantly increased compared to that of the vehicle control (83.8 fL in the PD0332991, and 71.6 fL in the vehicle control.). Further, the average micronucleated erythrocytes (MNE) size of the PD0332991 group and vehicle control was 8.2 and 7.3 µm, respectively. In the histogram, the vehicle control showed a monomodal distribution with a peak near 7.3 µm. In contrast, the PD0332991 group showed a bimodal distribution with peaks around 7.5 and 8.5 µm. Micronucleated erythrocytes in the PD0332991 group were significantly larger than those in the vehicle control.These results suggest that the increase in micronucleus frequency induced by the CDK4/6 inhibitor is not due to genotoxicity, but is attributable to disturbance of the cell cycle, differentiation, and enucleation of erythroblasts.ConclusionsIt was suggested that the positive outcome of the in vivo bone marrow micronucleus test resulting from treatment with PD0332991 could not be attributed to its genotoxicity. Further studies to clarify the mechanism of action can contribute to the development of drug candidate compounds lacking intrinsic genotoxic effects.

Retracted: Expression of CDK6 in Stomach Cancer and the Effect of CDK4/6 Inhibitor PD-0332991 on the Function of Stomach Cancer Cells.

[This retracts the article DOI: 10.1155/2022/2402567.].

Expression of CDK6 in Stomach Cancer and the Effect of CDK4/6 Inhibitor PD-0332991 on the Function of Stomach Cancer Cells.

Objective To study the expression and prognostic value of CDK6 in stomach cancer and the function of CDK4/6 inhibitor PD-0332991 on the proliferation of stomach cancer cells. Methods Immunohistochemistry was used to detect the expression of CDK6 in stomach cancer tissues and adjacent normal tissues and to analyze the effect of CDK6 on clinicopathological parameters of stomach cancer patients. Kaplan-Meier plotter was employed to study the relationship between CDK6 and overall survival in stomach cancer. Western blot and RT-PCR were used to detect protein and gene expression of CDK6 in different cells. The effects of CDK4/6 inhibitor PD-0332991 on apoptosis and aging of stomach cancer cells were detected by flow cytometry and β-galactosidase aging staining assay. The effects of CDK4/6 inhibitor PD-0332991 on the invasion and migration of stomach cancer cells were explored by the wound healing experiment and the Transwell experiment. The supernatant of stomach cancer cells was collected, and the effect of CDK4/6 inhibitor PD-0332991 on tumor markers of stomach cancer cells was detected by biochemical immunoassay. Results (1) CDK6 was highly expressed in stomach cancer tissues and cells. (2) Abnormally elevated CDK6 expression results in shorter survival in stomach cancer patients. (3) CDK4/6 inhibitor PD-0332991 could block the proliferation of stomach cancer cells, but not stomach epithelial proliferation. PD-0332991 could inhibit the secretion of pro-GRP by MGC 823. (4) PD-0332991 could advance the development of the apoptosis and senescence of stomach cancer cells and suppressed the invasion and migration of stomach cancer cells. Conclusion CDK6 expression is elevated in gastric cancer, and the CDK4/6 inhibitor PD-0332991 can remarkably promote apoptosis and senescence of stomach cancer cells and effectively inhibit the migration and invasion of stomach cancer cells.

Abstract B06: Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses Kras-mutated non-small cell lung cancer

Abstract Activating KRAS mutation is the most common driver mutation in the initiation and promotion of non-small cell lung cancer (NSCLC). Direct inhibition of the enzymatic activity of mutated Kras has been proved unsuccessful. Deregulation of PI3K-Akt-mTOR signaling, one of the most important downstream effectors of Kras, plays important roles in tumorigenesis and drug resistance in NSCLC. In this study, we found that a novel PI3Kα-specific inhibitor, CYH33, possessed variable activity against a panel of Kras-mutated NSCLC cell lines. Though CYH33 blocked Akt phosphorylation in all tested cells, phosphorylated Rb decreased in CYH33-sensitive but not resistant cells, which was consistent with G1 phase arrest in CYH33-sensitive cells. Consequently, combination of a CDK4/6 inhibitor PD0332991 with CYH33 displayed synergistic activity against the proliferation of both CYH33-sensitive and -resistant cells, which was accompanied with enhanced cell population in G1 phase compared to single-agent treatment. Moreover, downregulation of cyclin D1 with siRNA sensitized A549 and H23 cells to CYH33. Reciprocally, CYH33 abrogated PD0332991-induced upregulation of phosphorylated Akt and cyclin D1 in A549 cells. Cotreatment of these two drugs demonstrated synergistic activity against A549 and H23 xenografts with enhanced inhibition on the phosphorylation of Akt and Rb compared to administration of CYH33 or PD03332991 alone. Thus, simultaneous inhibition of PI3Kα and CDK4/6 displayed synergistic activity against Kras-mutated NSCLC and provided a mechanistic rationale for a combination approach using PI3Kα inhibitor and CDK4/6 inhibitor CYH33 for the therapy of Kras-mutated NSCLC. Citation Format: Yu-xiang Wang, Xian Li, Xue-ling Liu, Bo-bo Wang, Yi Chen, Chun-hao Yang, Jian Ding, Ling-hua Meng. Simultaneous inhibition of PI3Kα and CDK4/6 synergistically suppresses Kras-mutated non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B06.

The CDK4/6 inhibitor PD0332991 stabilizes FBP1 by repressing MAGED1 expression in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly solid tumors in the world. Aerobic glycolysis is among the characteristic features of pancreatic cancer. However, the regulatory process of aerobic glycolysis in pancreatic cancer is too complicated, and the underlying mechanism remains unexplained. Reportedly, CDK4/6 inhibitors repress breast cancer cell proliferation by modulating glucose metabolism. Here, we reveal that the CDK4/6 inhibitor, PD0332991 stabilized FBP1 to hinder aerobic glycolysis in pancreatic cancer. We also show that the CDK4/6-E2 F1 signaling pathway mediated an increase in MAGED1 expression, promoting FBP1 degradation in pancreatic cancer. We, therefore, might have identified a novel mechanism by which the CDK4/6 inhibitor, PD0332991 blocks the Warburg effect of pancreatic cancer by stabilizing FBP1.

Differentiation of PTH-Expressing Cells From Human Pluripotent Stem Cells.

Differentiation of pluripotent stem cells into functional parathyroid-like cells would accelerate development of important therapeutic options for subjects with parathyroid-related disorders, from the design and screening of novel pharmaceutical agents to the development of durable cellular therapies. We have established a highly reproducible directed differentiation approach leading to PTH-expressing cells from human embryonic stem cells and induced pluripotent stem cells. We accomplished this through the comparison of multiple different basal media, the inclusion of the CDK inhibitor PD0332991 in both definitive endoderm and anterior foregut endoderm stages, and a 2-stage pharyngeal endoderm series. This is the first protocol to reproducibly establish PTH-expressing cells from human pluripotent stem cells and represents a first step toward the development of functional parathyroid cells with broad applicability for medicinal and scientific investigation.

Palbociclib, a selective CDK4/6 inhibitor, restricts cell survival and epithelial-mesenchymal transition in Panc-1 and MiaPaCa-2 pancreatic cancer cells.

The mortality rate of pancreatic cancer has close parallels to its incidence rate because of limited therapeutics and lack of effective prognosis. Despite various novel chemotherapeutics combinations, the 5-year survival rate is still under 5%. In the current study, we aimed to modulate the aberrantly activated PI3K/AKT pathway and epithelial-mesenchymal transition (EMT) signaling with the treatment of CDK4/6 inhibitor PD-0332991 (palbociclib) in Panc-1 and MiaPaCa-2 pancreatic cancer cells. It was found that PD-0332991 effectively reduced cell viability and proliferation dose-dependently within 24 hours. In addition, PD-0332991 induced cell cycle arrest at the G1 phase by downregulation of aberrant expression of CDK4/6 through the dephosphorylation of Rb in each cell lines. Although PD-0332991 treatment increased epithelial markers and decreased mesenchymal markers, the nuclear translocation of β-catenin was not prevented by PD-0332991 treatment, especially in MiaPaCa-2 cells. Effects of PD-0332991 on the regulation of PI3K/AKT signaling and its downstream targets such as GSK-3 were cell type-dependent. Although the activity of AKT was inhibited in both cell lines, the phosphorylation of GSK-3β at Ser9 increased only in Panc-1. In conclusion, PD-0332991 induced cell cycle arrest and reduced the cell viability of Panc-1 and MiaPaCa-2 cells. However, PD-0332991 differentially affects the regulation of the PI3K/AKT pathway and EMT process in cells due to its distinct influence on Rb and GSK-3/β-catenin signaling. Understanding the effect of PD-0332991 on the aberrantly activated signaling axis may put forward a new therapeutic strategy to reduce the cell viability and metastatic process of pancreatic cancer.

Preclinical study of PD-0332991 on EBV-positive NPC cell lines and xenografts.

e17506 Background: Originated from nasopharynx, nasopharyngeal carcinoma is a subtype of head and neck cancer, which is consistently associated with EBV infection. It is prevalent exclusively in south of China, Malaysia and southeast Asia. Previously, there was only one EBV positive cell line, c666-1, for NPC study, the new EBV positive cell lines and xenografts established by our lab recently represent better models for preclinical drug test. The objective of this preclinical study is to evaluate the therapeutic effect of specific CDK4/6 inhibitor PD-0332991 in our new established EBV positive NPC cell lines and Xenografts. Methods: Cell lines were cultured in both 2D and 3D condition, cytotoxic test was conduct by Resazurin in 2D and by cell titer-glo in 3D. Cell cycle was analyzed by Western blot and flow cytometry. In the animal study, PD-0332991 was first used as single drug administrated to xenograft mice daily by oral gavage. Secondly, SAHA as a HDAC inhibitor was used to combine with PD-0332991 in this study, which was i.p. injected into animal every other day. Tumor size, body weight well as micro-pets scan data was recorded. The data from each experiment was analyzed by ANOVA and unpaired t-test for significant drug responses. Results: PD-0332991 was first identified to be effective during in vitro study. Beside single drug effect, when combination with SAHA, a synergistic effect was observed both in vitro and in vivo. In single drug study, PD-0332991 can significantly inhibit tumor growth in 3 of our xenografts (XENO76: P < 0.0001, XENO32: P < 0.0001, XENO23: P = 0.0019). In combination study, through tumor size and micro-pets scan, combination treatment induced significant decrease in tumor size and tumor metabolism states in animal models compare to vehicle, PD-0332991 and SAHA treatment. Conclusions: PD-0332991 is identified to be effective in our preclinical test by using our new established EBV positive cell lines and xenografts, this could be a promising treatment for NPC patients after clinical trial.

The interplay of CDK4 and CDK6 in melanoma.

The cyclin-dependent kinases CDK4 and CDK6 promote progression through the cell cycle, where their functions are considered to be redundant. Recent studies have identified an additional role for CDK6 in the transcriptional regulation of cancer-relevant genes such as VEGF-A and EGR1 in hematopoietic malignancies. We show that the CDK4/6 inhibitor PD0332991 causes a significant decrease in tumor growth in a xenotransplantation mouse model of human melanoma. shRNA knockdown of either CDK4 or CDK6 significantly reduces cell proliferation and impedes their migratory capacity in vitro, which translates into a strong inhibition of tumor growth in xenotransplantation experiments. CDK4/6 inhibition results not only in the pronounced reduction of cell proliferation but also in an impaired tumor angiogenesis. CDK6 knockdown in melanoma cell lines impairs VEGF-A expression and reduces the potential stimulation of endothelial cell growth. The knockdown of CDK4 ends in similar results. The effect is caused by changes of CDK6 localization, less CDK6 is detected on the VEGF-A promoter. Bioinformatic analysis of human melanoma patient data verifies the key role of CDK6 in tumor angiogenesis in melanoma. The results highlight the importance of the delicate balance between CDK4 and CDK6 in regulating the cell cycle and transcription.

Abstract 2317: Palbociclib monotherapy exhibits potent activity in cervical cancer cell lines

Abstract Background: Over the last decades, little progress has been made in the systemic treatment of patients with advanced or recurrent cervical cancer. We recently performed an silico analysis to identify potential driver pathways of cervical carcinogenesis and candidate targets for treatment.1 Expression2Kinases (E2K) analysis revealed a protein-protein interaction (PPI) network of 162 nodes (including 20 druggable kinases) consisting of 5 signaling modules associated with MYC signaling (module 1), cell cycle deregulation and cyclin signaling (module 2), TGFβ-signaling (module 3), a PI3K - MAPK signaling (module 4) and chromatine modeling (module 5). Methods: The cervical cancer cell lines included in the study were CaSki, SiHa and HeLa. Cells were incubated for 24 or 72 hours with the PI3K pathway inhibitor BYL719 (Novartis), the multiple pathway inhibitor INC280 (inhibits cMET-dependent PI3K and RAS signaling, Novartis), or the CDK4/6 inhibitor PD-0332991 (palbociclib, Selleck Chemicals) in monotherapy. Sensitivity to drug treatment (0 - 10 μM) was investigated using the colorimetric sulforhodamine B (SRB) assay. IC50 values were calculated using WinNonlin Software. Results: Incubation with BYL719 or INC280 for 24 or 72 hours in concentrations up to 10 μM was not able to establish a distinct cytotoxic effect in the three cervical cancer cell lines included in the study. Incubation with 10 μM BYL719 for 72h resulted in only 20% cell kill, while 10 μM INC280 for 72h caused around 30% cell kill in all three cell lines. However, incubation with palbociclib for 72 hours clearly induced a concentration-dependent cytotoxic effect, with IC50 values of 5.32 ± 0.03 μM, 7.69 ± 0.02 μM and 5.68 ± 0.12 μM for HeLa, SiHa and CaSki cells, respectively. Conclusion: CDK4/6 inhibition seems effective in cervical cancer cell lines and should be further evaluated alone and in combination with chemotherapy for the treatment of advanced and recurrent cervical cancer.

PO-028 Effectiveness and molecular basis of CDK4/6 inhibition in combination with taxanes in pancreatic cancer

IntroductionPancreatic Ductal Adenocarcinoma (PDAC) is among the deadliest human cancers with a 5 year survival rate of less than 5% using the standard of care gemcitabine/nab-paclitaxel. The most frequently disrupted genes in PDACs are first K-RAS and second CDKN2A, which encodes the cyclin-dependent kinase (CDK)4/6 inhibitor p16. Recently, CDK4/6 inhibitors have been approved for breast cancer treatment, and preclinical assays for PDAC are giving promising results.Material and methodsPDAC Patient-Derived Xenografts (PDX) models and PDX-derived cell lines were used for in vivo and in vitro studies, respectively. Cellular studies were performed using proliferation and cell cycle assays in combination with flow cytometry, immunoblotting, fluorescence microscopy and live cell imaging techniques. Drug treatments were performed with the CDK4/6 inhibitor PD-0332991 (Palbociclib), and with Paclitaxel (Taxol) or Nab-Paclitaxel (Abraxane) for in vitro and in vivo studies, respectively.Results and discussionsTreatment of different PDX-derived cell lines with the combination of taxanes and CDK4/6 inhibitors resulted in a higher anti-proliferative effect than both drugs used as single agent. Cell cycle studies showed that inhibition of CDK4/6 prevented recovery from treatment with taxol. At the molecular level we found that the combined treatment induced a clear interruption in retinoblastoma pathway, even higher than CDK4/6 inhibition in monotherapy. Gene expression profiles comparing single versus combined treatment are currently being performed to further understand the molecular basis underlying the effectiveness of this type of treatment. Moreover, to assess the efficacy of this new combined treatment in vivo, we treated nine PDAC PDX models with PD-0332991 and nab-paclitaxel, following the same schedule. Importantly, eight of them presented an increased tumour growth inhibition in the combination with respect to the monotherapies.ConclusionAlthough the molecular mechanism underlying the effectiveness of this treatment is not completely understood yet, our data suggest a good therapeutic value for the combination of CDK4/6 inhibitors and taxanes in PDAC treatment.

PO-031 NAA induces antitumoral effects in BXPC3 pancreatic cancer cell line

IntroductionPancreatic Ductal Adenocarcinoma (PDAC) is among the deadliest human cancers with a 5 year survival rate of less than 5% using the standard of care gemcitabine/nab-paclitaxel. The most frequently disrupted genes in PDACs are first K-RAS and second CDKN2A, which encodes the cyclin-dependent kinase (CDK)4/6 inhibitor p16. Recently, CDK4/6 inhibitors have been approved for breast cancer treatment, and preclinical assays for PDAC are giving promising results.Material and methodsPDAC Patient-Derived Xenografts (PDX) models and PDX-derived cell lines were used for in vivo and in vitro studies, respectively. Cellular studies were performed using proliferation and cell cycle assays in combination with flow cytometry, immunoblotting, fluorescence microscopy and live cell imaging techniques. Drug treatments were performed with the CDK4/6 inhibitor PD-0332991 (Palbociclib), and with Paclitaxel (Taxol) or Nab-Paclitaxel (Abraxane) for in vitro and in vivo studies, respectively.Results and discussionsTreatment of different PDX-derived cell lines with the combination of taxanes and CDK4/6 inhibitors resulted in a higher anti-proliferative effect than both drugs used as single agent. Cell cycle studies showed that inhibition of CDK4/6 prevented recovery from treatment with taxol. At the molecular level we found that the combined treatment induced a clear interruption in retinoblastoma pathway, even higher than CDK4/6 inhibition in monotherapy. Gene expression profiles comparing single versus combined treatment are currently being performed to further understand the molecular basis underlying the effectiveness of this type of treatment. Moreover, to assess the efficacy of this new combined treatment in vivo, we treated nine PDAC PDX models with PD-0332991 and nab-paclitaxel, following the same schedule. Importantly, eight of them presented an increased tumour growth inhibition in the combination with respect to the monotherapies.ConclusionAlthough the molecular mechanism underlying the effectiveness of this treatment is not completely understood yet, our data suggest a good therapeutic value for the combination of CDK4/6 inhibitors and taxanes in PDAC treatment.

Id1 and Sonic Hedgehog Mediate Cell Cycle Reentry and Apoptosis Induced by Amyloid Beta-Peptide in Post-mitotic Cortical Neurons.

Amyloid beta-peptide (Aβ), the neurotoxic component of senile plaques in Alzheimer's disease (AD) brains, is known to trigger cell cycle reentry in post-mitotic neurons followed by apoptosis. However, the underlying mechanisms remain unclear. Recently, we have reported that Aβs stimulate the expression of inhibitor of differentiation-1 (Id1) to induce sonic hedgehog (SHH) (Hung et al., Mol Neurobiol 53(2):793-809, 2016), and both are mitogens capable of triggering cell cycle progression. In this work, we tested the hypothesis that Aβ-induced Id1 and SHH contribute to cell cycle reentry leading to apoptosis in neurons. We found that Aβ triggered cell cycle progression in the post-mitotic neurons, as indicated by the increased expression of two G1-phase markers including cyclin D1 and phosphorylated retinoblastoma protein (pRb), two G2-phase markers such as proliferating cell nuclear antigen (PCNA) and incorporation of 5-bromo-2'-deoxyuridine (BrdU) into newly synthesized DNA, as well as the mitotic marker histone H3 phosphorylated at Ser-10. As expected, Aβ also enhanced caspase-3 cleavage in the cortical neurons. Id1 siRNA, the neutralization antibody against SHH (SHH-Ab), and the cyclin-dependent kinase (CDK)-4/6 inhibitor PD0332991 all attenuated, in part or in full, the Aβ-induced expression of these cell cycle markers. Indeed, exogenous recombinant Id1 protein and the biologically active N-terminal fragment of SHH (SHH-N) were both sufficient to enhance the expression of cell cycle markers independent of Aβ. Taken together, our results revealed the critical roles of Id1 and SHH mediating Aβ-dependent cell cycle reentry and subsequently caspase-dependent apoptosis in the fully differentiated post-mitotic neurons, at least in vitro.

Antiproliferative effects of the CDK6 inhibitor PD0332991 and its effect on signaling networks in gastric cancer cells.

PD0332991 (palbociclib/Ibrance®) is a cyclin-dependent kinase (CDK)4/6 inhibitor that has recently been approved for the treatment of estrogen receptor-positive advanced breast cancer. The present study investigated the antiproliferative effects of PD0332991 on gastric cancer (GC) cells and the underlying molecular mechanisms. The activity of PD0332991 was tested in several GC cell lines, including AGS, KATO-III, NCI-N87 and HS746T. Growth inhibitory activity of PD0332991, alone or in combination with fluorouracil (5-FU), was measured by MTT assay. The effects of PD0332991 on cell cycle progression were analyzed by flow cytometry and western blotting. Protein pathway array and Ingenuity Pathway Analysis were used to identify signaling pathways that may mediate the antiproliferative effects of PD0332991. PD0332991 inhibited proliferation in a dose-dependent manner and enhanced the activity of 5-FU in all GC cell lines tested. Cells treated with PD0332991 exhibited cell cycle arrest in G1 phase of the cell cycle, whereas the number of cells in G2/M phase was decreased. PD0332991 also inhibited CDK6-specific phosphorylation of retinoblastoma on Ser780, reduced the expression of cyclin D1, and induced expression of p53 and p27. Furthermore, 31 proteins were identified, the expression of which was significantly altered following treatment with PD0332991 in at least three cell lines. Pathway analysis indicated that the altered proteins were frequently associated with cell death, cell cycle and the molecular mechanism of cancer. The results of the present study indicated that PD0332991 may inhibit cell proliferation via modulation of the cell cycle, and may affect numerous oncogenic signaling pathways. Therefore, PD0332991 may be considered effective for the treatment of GC.

Frequent Genetic Aberrations in the CDK4 Pathway in Acral Melanoma Indicate the Potential for CDK4/6 Inhibitors in Targeted Therapy.

Purpose: Effective therapies for the majority of metastatic acral melanoma patients have not been established. Thus, we investigated genetic aberrations of CDK4 pathway in acral melanoma and evaluated the efficacy of CDK4/6 inhibitors in targeted therapy of acral melanoma.Experimental Design: A total of 514 primary acral melanoma samples were examined for the copy number variations (CNV) of CDK4 pathway-related genes, including Cdk4, Ccnd1, and P16INK4a , by QuantiGenePlex DNA Assay. The sensitivity of established acral melanoma cell lines and patient-derived xenograft (PDX) containing typical CDK4 aberrations to CDK4/6 inhibitors was evaluated.Results: Among the 514 samples, 203 cases, 137 cases, and 310 cases, respectively, showed Cdk4 gain (39.5%), Ccnd1 gain (26.7%), and P16INK4a loss (60.3%). The overall frequency of acral melanomas that contain at least one aberration in Cdk4, Ccnd1, and P16INK4a was 82.7%. The median overall survival time for acral melanoma patients with concurrent Cdk4 gain with P16INK4a loss was significantly shorter than that for patients without such aberrations (P = 0.005). The pan-CDK inhibitor AT7519 and selective CDK4/6 inhibitor PD0332991 could inhibit the cell viability of acral melanoma cells and the tumor growth of PDX with Cdk4 gain plus Ccnd1 gain, Cdk4 gain plus P16INK4a loss, and Ccnd1 gain plus P16INK4a loss.Conclusions: Genetic aberration of CDK4 pathway is a frequent event in acral melanoma. Acral melanoma cell lines and PDX containing CDK4 pathway aberrations are sensitive to CDK4/6 inhibitors. Our study provides evidence for the testing of CDK4/6 inhibitors in acral melanoma patients. Clin Cancer Res; 23(22); 6946-57. ©2017 AACR.

Tailored first-line and second-line CDK4-targeting treatment combinations in mouse models of pancreatic cancer

ObjectiveExtensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase...

CDK4 phosphorylation status and a linked gene expression profile predict sensitivity to palbociclib

Cyclin D‐CDK4/6 are the first CDK complexes to be activated in the G1 phase in response to oncogenic pathways. The specific CDK4/6 inhibitor PD0332991 (palbociclib) was recently approved by the FDA and EMA for treatment of advanced ER‐positive breast tumors. Unfortunately, no reliable predictive tools are available for identifying potentially responsive or insensitive tumors. We had shown that the activating T172 phosphorylation of CDK4 is the central rate‐limiting event that initiates the cell cycle decision and signals the presence of active CDK4. Here, we report that the profile of post‐translational modification including T172 phosphorylation of CDK4 differs among breast tumors and associates with their subtypes and risk. A gene expression signature faithfully predicted CDK4 modification profiles in tumors and cell lines. Moreover, in breast cancer cell lines, the CDK4 T172 phosphorylation best correlated with sensitivity to PD0332991. This gene expression signature identifies tumors that are unlikely to respond to CDK4/6 inhibitors and could help to select a subset of patients with HER2‐positive and basal‐like tumors for clinical studies on this class of drugs.

Abstract P6-07-02: CDK4 phosphorylation status and corresponding gene expression profile predict sensitivity to Palbociclib

Abstract P6-07-02: CDK4 phosphorylation status and corresponding gene expression profile predict sensitivity to Palbociclib

Abstract 2840: CDK4/6 inhibitor PD-0332991 (palbociclib) promotes cell death and synergizes with CPT-11 in colorectal cancer under hypoxia in vitro

Abstract We recently reported that a family of nucleoside analogues (sangivamycin-like molecules) can sensitize tumor cells to TRAIL through dual blockade of CDK1 and GSK3-beta (Mayes et al., Cancer Research, 2011). We further reported that CDK inhibitors can destabilize HIF1-alpha regardless of VHL or p53 status or the presence of hypoxia (Warfel et al., Cell Cycle, 2013). In order to translate this knowledge into a cancer therapeutic strategy, we investigated the effects of CDK inhibition in colorectal cancer (CRC) cell lines with or without chemotherapy. PD-0332991 (Palbociclib) is a specific inhibitor of CDK4/6 that has been tested in numerous clinical trials for breast cancer, NSCLC, GBM, lymphoma, leukemia, in combination with 5-FU and oxaliplatin in solid malignancies (NCT01522989) or with cetuximab in head and neck cancer (NCT02101034). Palbociclib was approved by the FDA in 2015 in combination with letrozole as initial endocrine therapy for post-menopausal women with ER(+)/Her2(-) breast cancer. Little is known about the effects of CDK4/6 inhibition in CRC. We investigated the therapeutic effect and anti-proliferative mechanism of CDK4/6 inhibition in CRC. We used CellTiter-Glo assays to detect CRC cell viability and determined IC50 values (50% inhibitory concentration) of single drug through nonlinear regression analysis by GraphPad Prism software. The combination index (CI) of multiple drug combinations was identified with Compusyn analysis. We found that Palbociclib promotes cell death of CRC cells under hypoxia but not under normoxia where Palbociclib inhibited cell proliferation via the pRb pathway. These results suggest that the CDK4/6 inhibitor could regulate cell fate of CRC via different molecular mechanisms under hypoxia versus normoxia. We further found that Palbociclib can upregulate ERK/MAPK signaling under hypoxia, as compared with normoxia. The IC50 values of Palbociclib were generally higher under hypoxia (Mean ± SD: 10.54 ± 3.35 μM, N = 5) versus normoxia (Mean ± SD: 6.61 ± 0.85 μM, N = 5) in CRC cell lines. Thus, hypoxia promotes resistance of CRC cells toward the cytotoxic activity of the CDK4/6 inhibitor. We found that Palbociclib synergizes with CPT-11 much better than with either 5-FU or oxaliplatin against CRC cell lines with different molecular subtypes. Based on our findings that Palbociclib can promote cell death of CRC cells under hypoxia and synergizes with CPT11, further investigation is needed to assess the novel combination therapy against CRC. Citation Format: Jun Zhang, Lanlan Zhou, Shuai Zhao, David Dicker, Wafik S. El-Deiry. CDK4/6 inhibitor PD-0332991 (palbociclib) promotes cell death and synergizes with CPT-11 in colorectal cancer under hypoxia in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2840.