Reduced Rb Phosphorylation Research Articles

CTRP13-Mediated Effects on Endothelial Cell Function and Their Potential Role in Obesity.

Obesity, a major component of cardiometabolic syndrome, contributes to the imbalance between pro- and anti-atherosclerotic factors via dysregulation of adipocytokine secretion. Among these adipocytokines, the C1q/TNF-related proteins (CTRPs) play a role in the modulation of atherosclerosis development and progression. Here, we investigated the vascular effects of CTRP13. CTRP13 is not only expressed in adipose tissue but also in vessels/endothelial cells (ECs) of mice, rats, and humans. Obese individuals (mice, rats, and humans) showed higher vascular CTRP13 expression. Human Umbilical Vein Endothelial Cells (HUVECs), cultured in the presence of serum from obese mice, mimicked this obesity-associated effect on CTRP13 protein expression. Similarly, high glucose conditions and TNF-alpha, but not insulin, resulted in a strong increase in CTRP13 in these cells. Recombinant CTRP13 induced a reduction in EC proliferation via AMPK. In addition, CTRP13 reduced cell cycle progression and increased p53 phosphorylation and p21 protein expression, but reduced Rb phosphorylation, with the effects largely depending on alpha-2 AMPK as suggested by adenoviral overexpression of dominant-negative (DN) or wild-type (WT) alpha 1/alpha 2 AMPK. The present study demonstrates that CTRP13 expression is induced in ECs under diabetic conditions and that CTRP13 possesses significant vaso-modulatory properties which may have an impact on vascular disease progression in patients.

Regulation of Cell Cycle Progression through RB Phosphorylation by Nilotinib and AT-9283 in Human Melanoma A375P Cells.

BCR-ABL tyrosine kinase inhibitors are commonly employed for the treatment of chronic myeloid leukemia, yet their impact on human malignant melanoma remains uncertain. In this study, we delved into the underlying mechanisms of specific BCR-ABL tyrosine kinase inhibitors (imatinib, nilotinib, ZM-306416, and AT-9283) in human melanoma A375P cells. We first evaluated the influence of these inhibitors on cell growth using cell proliferation and wound-healing assays. Subsequently, we scrutinized cell cycle regulation in drug-treated A375P cells using flow cytometry and Western blot assays. Notably, imatinib, nilotinib, ZM-306416, and AT-9283 significantly reduced cell proliferation and migration in A375P cells. In particular, nilotinib and AT-9283 impeded the G1/S transition of the cell cycle by down-regulating cell cycle-associated proteins, including cyclin E, cyclin A, and CDK2. Moreover, these inhibitors reduced RB phosphorylation, subsequently inhibiting E2F transcriptional activity. Consequently, the expression of the E2F target genes (CCNA2, CCNE1, POLA1, and TK-1) was markedly suppressed in nilotinib and AT9283-treated A375P cells. In summary, our findings suggest that BCR-ABL tyrosine kinase inhibitors may regulate the G1-to-S transition in human melanoma A375P cells by modulating the RB-E2F complex.

Discovery of (4-Pyrazolyl)-2-aminopyrimidines as Potent and Selective Inhibitors of Cyclin-Dependent Kinase 2.

CDK2 is a critical regulator of the cell cycle. For a variety of human cancers, the dysregulation of CDK2/cyclin E1 can lead to tumor growth and proliferation. Historically, early efforts to develop CDK2 inhibitors with clinical applications proved unsuccessful due to challenges in achieving selectivity over off-target CDK isoforms with associated toxicity. In this report, we describe the discovery of (4-pyrazolyl)-2-aminopyrimidines as a potent class of CDK2 inhibitors that display selectivity over CDKs 1, 4, 6, 7, and 9. SAR studies led to the identification of compound 17, a kinase selective and highly potent CDK2 inhibitor (IC50 = 0.29 nM). The evaluation of 17 in CCNE1-amplified mouse models shows the pharmacodynamic inhibition of CDK2, measured by reduced Rb phosphorylation, and antitumor activity.

Down-regulation of lncRNA MEG3 promotes chronic low dose cadmium exposure-induced cell transformation and cancer stem cell-like property

Cadmium (Cd) is a toxic heavy metal and one of carcinogens that cause lung cancer. However, the exact mechanism of Cd carcinogenesis remains unclear. To investigate the mechanism of Cd carcinogenesis, we exposed human bronchial epithelial cells (BEAS-2B) to a low dose of Cd (2.5 μM, CdCl2) for 9 months, which caused cell malignant transformation and generated cancer stem cell (CSC)-like cells. The goal of this study is to investigate the underlying mechanism. The long non-coding RNA (lncRNA) microarray analysis showed that the expression level of a tumor suppressive lncRNA maternally expressed 3 (MEG3) is significantly down-regulated in Cd-transformed cells, which is confirmed by further q-PCR analysis. Mechanistically, it was found that chronic Cd exposure up-regulates the levels of DNA methyltransferases (DNMTs), which increases the methylation of the differentially methylated region (DMR) 1.5 kb upstream of MEG3 transcription start site to reduce MEG3 expression. Functional studies showed that stably overexpressing MEG3 in Cd-transformed cells significantly reduces their transformed phenotypes. Moreover, stably overexpressing MEG3 in parental non-transformed human bronchial epithelial cells significantly impaired the capability of chronic Cd exposure to induce cell transformation and CSC-like property. Further mechanistic studies revealed that the cell cycle inhibitor p21 level is reduced and retinoblastoma protein (Rb) phosphorylation is increased in Cd-transformed cells to promote cell cycle progression. In addition, Cd-transformed cells also expressed higher levels of Bcl-xL and displayed apoptosis resistance. In contrast, stably overexpressing MEG3 increased p21 levels and reduced Rb phosphorylation and Bcl-xL levels in Cd-exposed cells and reduced their cell cycle progression and apoptosis resistance. Together, these findings suggest that MEG3 down-regulation may play important roles in Cd-induced cell transformation and CSC-like property by promoting cell cycle progression and apoptosis resistance.

SHetA2 Increases the Activity of Palbociclib in Cervical Cancer in vitro and in vivo

Introduction Cervical cancer is caused by high-risk human papillomavirus (HPV) and treated with conventional chemotherapy combined with surgery and/or radiation. HPV oncoproteins increase retinoblastoma (Rb) phosphorylation by cyclin D1/cyclin dependent kinase (CDK)4/6 complexes and cellular proliferation. We hypothesized that cyclin D1 degradation by the SHetA2 drug in combination with palbociclib inhibition of CDK4/6 activity synergistically reduces Rb phosphorylation and inhibits cervical cancer growth. Methods The effects of these drugs, alone, and in combination, were evaluated in cervical cancer cell lines using cell culture, western blots and ELISA, and in a SiHa xenograft model. Endpoints were compared by isobolograms, ANOVA, and Chi-Square. Results SHetA2 and palbociclib acted synergistically in three cervical cell lines (CI <0.5) in association with reduced Rb phosphorylation. Both drugs significantly reduced Rb phosphorylation and growth of SiHa xenograft tumors as single agents and acted additively when combined, with no evidence of toxicity. SHetA2, but not palbociclib, reduced cyclin D1 in cell cultures and tumors. Dilated CD31-negative blood vessels adjacent to, or within, areas of necrosis and apoptosis were observed in all drug-treated tumors. Conclusions The drug combination was synergistic in vitro and significantly effective in vivo. The mechanism of synergy appears to involve inhibition of Rb phosphorylation.

Anti-cell growth and anti-cancer stem cell activity of the CDK4/6 inhibitor palbociclib in breast cancer cells.

A cyclin-dependent kinase (CDK) 4/6 inhibitor, palbociclib, has been used to treat patients with estrogen receptor (ER)-positive (+) and human epidermal growth factor receptor (HER) 2-negative (-) advanced breast cancer. To investigate the mechanisms underlying the antitumor activity of palbociclib, we conducted a preclinical study on the anti-cell growth and anti-cancer stem cell (CSC) activity of palbociclib in breast cancer cells. The effects of palbociclib on Rb phosphorylation, cell growth, cell cycle progression, apoptosis, cell senescence and the proportion of CSCs were investigated in five human breast cancer cell lines of different subtypes. To investigate the mechanisms of the anti-CSC activity of palbociclib, small-interfering RNAs for CDK4 and/or CDK6 were used. Palbociclib dose-dependently reduced Rb phosphorylation and cell growth in association with G1-S cell cycle blockade and the induction of cell senescence, but without increased apoptosis, in all breast cancer cell lines. The anti-cell growth activity of palbociclib widely differed among the cell lines. Palbociclib also dose-dependently reduced the CSC proportion measured by three different assays in four of five cell lines. The inhibition of CDK4 expression, but not CDK6 expression, reduced the increased proportion of putative CSCs induced by estradiol in ER (+)/HER2 (-) cell lines. These results suggest that palbociclib exhibits significant anti-cell growth and anti-CSC activity in not only ER (+) breast cancer cell lines but also ER (-) cell lines. CDK4 inhibition induced by palbociclib may be responsible for its anti-CSC activity.

Pradimicin-IRD exhibits antineoplastic effects by inducing DNA damage in colon cancer cells

DNA-damaging agents are widely used in cancer therapy; however, their use is limited by dose-related toxicities, as well as the development of drug resistance. Drug discovery is essential to overcome these limitations and offer novel therapeutic options. In a previous study by our research group, pradimicin-IRD—a new polycyclic antibiotic produced by the actinobacteria Amycolatopsis sp.—displayed antimicrobial and potential anticancer activities. In the present study, cytotoxic activity was further confirmed in a panel of five colon cancer, including those with mutation in TP53 and KRAS, the most common ones observed in cancer colon patients. While all tested colon cancer cells were sensitive to pradimicin-IRD treatment with IC50 in micromolar range, non-tumor fibroblasts were significantly less sensitive (p < 0.05). The cellular and molecular mechanism of action of pradimicin-IRD was then investigated in the colorectal cancer cell line HCT 116. Pradimicin-IRD presented antitumor effects occurring after at least 6 h of exposure. Pradimicin-IRD induced statistically significant DNA damage (γH2AX and p21), apoptosis (PARP1 and caspase 3 cleavage) and cell cycle arrest (reduced Rb phosphorylation, cyclin A and cyclin B expression) markers. In accordance with these results, pradimicin-IRD increased cell populations in the subG1 and G0/G1 phases of the cell cycle. Additionally, mass spectrometry analysis indicated that pradimicin-IRD interacted with the DNA double strand. In summary, pradimicin-IRD exhibits multiple antineoplastic activities—including DNA damage, cell cycle arrest, reduction of clonal growth and apoptosis—in the HCT 116 cell line. Furthermore, pradimicin-IRD displays a TP53-independent regulation of p21 expression in HCT 116 TP53−/−, HT-29, SW480, and Caco-2 cells. This exploratory study identified novel targets for pradimicin-IRD and provided insights for its potential anticancer activity as a DNA-damaging agent.

IFITM3 knockdown reduces the expression of CCND1 and CDK4 and suppresses the growth of oral squamous cell carcinoma cells.

Oral squamous cell carcinoma (OSCC) is a challenging disease to treat. Up to 50% of OSCC patients with advanced disease develop recurrences. Elucidation of key molecular mechanisms underlying OSCC development may provide opportunities to target specific genes and, thus, to improve patient survival. In this study, we examined the expression and functional role of interferon transmembrane protein 3 (IFITM3) in OSCC development. The expression of IFITM3 in OSCC and normal oral mucosal tissues was assessed by qRT-PCR and immunohistochemistry. The role of IFITM3 in driving OSCC cell proliferation and survival was examined using siRNA-mediated gene knockdown, and the role of IFITM3 in driving cell cycle regulators was examined using Western blotting. We found that IFITM3 is overexpressed in more than 79% of primary OSCCs. We also found that IFITM3 knockdown led to impaired OSCC cell growth through inhibition of cell proliferation, induction of cell cycle arrest, senescence and apoptosis. In addition, we found that IFITM3 knockdown led to reduced expressions of CCND1 and CDK4 and reduced RB phosphorylation, leading to inhibition of OSCC cell growth. This information may be instrumental for the design of novel targeted therapeutic strategies. From our data we conclude that IFITM3 is overexpressed in OSCC and may regulate the CCND1-CDK4/6-pRB axis to mediate OSCC cell growth.

YAP1-Mediated CDK6 Activation Confers Radiation Resistance in Esophageal Cancer - Rationale for the Combination of YAP1 and CDK4/6 Inhibitors in Esophageal Cancer.

Esophageal cancer is a lethal disease that is often resistant to therapy. Alterations of YAP1 and CDK6 are frequent in esophageal cancer. Deregulation of both molecules may be responsible for therapy resistance. Expressions of YAP1 and CDK6 were examined in esophageal cancer cells and tissues using immunoblotting and immunohistochemistry. YAP1 expression was induced in esophageal cancer cells to examine YAP1-mediated CDK6 activation and its association with radiation resistance. Pharmacologic and genetic inhibitions of YAP1 and CDK6 were performed to dissect the mechanisms and assess the antitumor effects in vitro and in vivo. YAP1 expression was positively associated with CDK6 expression in resistant esophageal cancer tissues and cell lines. YAP1 overexpression upregulated CDK6 expression and transcription, and promoted radiation resistance, whereas treatment with the YAP1 inhibitor, CA3, strongly suppressed YAP1 and CDK6 overexpression, reduced Rb phosphorylation, as well as sensitized radiation-resistant/YAP1high esophageal cancer cells to irradiation. CDK4/6 inhibitor, LEE011, and knock down of CDK6 dramatically inhibited expression of YAP1 and sensitized resistant esophageal cancer cells to irradiation indicating a positive feed-forward regulation of YAP1 by CDK6. In addition, suppression of both the YAP1 and CDK6 pathways by the combination of CA3 and LEE011 significantly reduced esophageal cancer cell growth and cancer stem cell population (ALDH1 + and CD133 + ), sensitized cells to irradiation, and showed a strong antitumor effect in vivo against radiation-resistant esophageal cancer cells. Our results document that a positive crosstalk between the YAP1 and CDK6 pathways plays an important role in conferring radiation resistance to esophageal cancer cells. Targeting both YAP1 and CDK6 pathways could be a novel therapeutic strategy to overcome resistance in esophageal cancer.

Abstract P3-11-04: Phase I/II trial of palbociclib in combination with bicalutamide for the treatment of androgen receptor (AR)+ metastatic breast cancer (MBC)

Abstract Background: The androgen-signaling pathway plays a role in breast cancer (BC) pathogenesis and emerging evidence suggests the androgen receptor (AR) is a therapeutic target. TBCRC011 established safety and efficacy of inhibiting AR with bicalutamide (B) in patients (pts) with androgen AR+/ER/PgR- metastatic BC (MBC). Treatment with B at 150 mg oral daily demonstrated a clinical benefit rate (CBR) of 19% in this population. The rationale for combining palbociclib (P) with AR blockade stems from prior work in ER+ MBC. P is an oral, selective inhibitor of CDK4/6 activity which significantly improved median progression-free survival (PFS) in combination with letrozole compared to letrozole monotherapy for the treatment of postmenopausal pts with ER+ MBC in the first-line setting. Consistent with preclinical data, P has been shown to reduce growth of AR+ ER/PgR- MDA-MB-453 breast cancer cells via reduced Rb phosphorylation. It has been shown that AR+ TNBC expresses a luminal profile and has intact Rb protein, the target of palbociclib activity. Therefore, we hypothesize that P will increase the efficacy of B in pts with metastatic AR+ TNBC. NCT02605486. Methods: Pts with AR+ (IHC ≥ 1%)/ER any/HER2(-) MBC on central review at MSK were eligible if met following criteria: ECOG ≤2, postmenopausal, no limit to prior regimens. Pts with ER+ BC must have had 1 prior endocrine therapy. Treatment: B orally daily and P orally daily 3 weeks on 1 week off. DLT period = 28 days. Pts are evaluated for toxicity every 2-4 weeks and for response every 8 weeks. Phase I standard 3+3 design with 3 dose escalations. The primary objective of the phase I portion of the study is to determine the recommended phase II dose of P in combination with B. Plasma for pharmacokinetics (PK) was collected throughout the study. Results: As of 5/11/17, 15 pts with AR+ MBC were enrolled to the phase I portion. Target accrual has been met for the phase I and has since closed. No DLTs were reported in any of the dose escalation cohorts. The maximum tolerated dose was determined to be B 150 mg daily and P 125 mg daily for 21 days in a 28 day cycle. Treatment has been well tolerated with no related Grade 4 or 5 adverse events (AEs). The most common grade 3 significant AEs were hematologic including neutropenia n =5, leukopenia n= 4, and lymphocytopenia n=3 and thought to be related to P. One Grade 4 hypercalcemia led to hospitalization and was deemed related to disease progression. Median time on study was 8 weeks (2-47 weeks). One patient from the phase I remains on study. PK analysis is ongoing and will be presented, including drug-drug interaction data. Conclusions: The combination of B+P has been well tolerated with no unexpected toxicity observed. Updated safety, response, and PK data will be presented. Enrollment on phase II is ongoing. Citation Format: Gucalp A, Edelweiss M, Patil S, Gounder MM, Feigin KN, Corben A, Arumov A, Traina TA. Phase I/II trial of palbociclib in combination with bicalutamide for the treatment of androgen receptor (AR)+ metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-11-04.

Phase 1 safety, pharmacokinetic and pharmacodynamic study of the cyclin-dependent kinase inhibitor dinaciclib administered every three weeks in patients with advanced malignancies

Background:Dinaciclib is a potent inhibitor of cell cycle and transcriptional cyclin-dependent kinases. This Phase 1 study evaluated the safety, tolerability and pharmacokinetics of various dosing schedules of dinaciclib in advanced solid tumour patients and assessed pharmacodynamic and preliminary anti-tumour activity.Methods:In part 1, patients were enrolled in escalating cohorts of 2-h infusions administered once every 3 weeks, utilising an accelerated titration design until a recommended phase 2 dose (RP2D) was defined. In part 2, 8- and 24-h infusions were evaluated. Pharmacokinetic parameters were determined for all schedules. Pharmacodynamic effects were assessed with an ex vivo stimulated lymphocyte proliferation assay performed in whole blood.Effects of dinaciclib on retinoblastoma (Rb) phosphorylation and other CDK targets were evaluated in skin and tumour biopsies. In addition to tumour size, metabolic response was evaluated by 18F-fluorodeoxyglucose-positron emission tomography.Results:Sixty-one patients were enrolled to parts 1 and 2. The RP2Ds were 50, 7.4 and 10.4 mg m−2 as 2- 8- and 24-hour infusions, respectively. Dose-limiting toxicities included pancytopenia, neutropenic fever, elevated transaminases, hyperuricemia and hypotension. Pharmacokinetics demonstrated rapid distribution and a short plasma half-life. Dinaciclib suppressed proliferation of stimulated lymphocytes. In skin and tumour biopsies, dinaciclib reduced Rb phosphorylation at CDK2 phospho-sites and modulated expression of cyclin D1 and p53, suggestive of CDK9 inhibition. Although there were no RECIST responses, eight patients had prolonged stable disease and received between 6 and 30 cycles. Early metabolic responses occurred.Conclusions:Dinaciclib is tolerable at doses demonstrating target engagement in surrogate and tumour tissue.

Phase I/II trial of palbociclib in combination with bicalutamide for the treatment of androgen receptor (AR)+ metastatic breast cancer (MBC).

TPS1103 Background: Patients (pts) with triple negative breast cancer (TNBC) have limited effective therapeutic options and generally experience a more aggressive clinical course. Emerging evidence demonstrates that the androgen-signaling pathway plays a role in BC pathogenesis and may be a valuable therapeutic target. In TBCRC011, we tested the efficacy of the AR antagonist, bicalutamide (B) in pts with AR+/ER/PgR- MBC. B was well-tolerated and demonstrated a clinical benefit rate (CBR) of 19% in this population (Gucalp et al. CCR 2013). Palbociclib (P) is a competitive inhibitor of CDK4/6 and has been shown to reduce growth of AR+/ER/PgR- MDA-MB-453 BC cells via reduced Rb phosphorylation. In postmenopausal pts with luminal ER+ MBC the addition of P to letrozole significantly improved median progression-free survival (PFS) in comparison to letrozole monotherapy. Given the luminal signature of AR+ TNBC and the presence of intact Rb in this ER(-) subtype, we are testing the efficacy of B+P in pts with metastatic AR+ TNBC. NCT02605486. Methods: Pts with MBC consent to central AR testing at MSK. Key eligibility criteria include: Ph I AR+ (IHC ≥ 1%)/ER any/HER2(-) MBC/Ph II AR+ TN MBC, ECOG ≤ 2, postmenopausal (LHRH agonist allowed), measurable/non-measurable disease. Any number of prior regimens is permitted; pts with ER+ BC must have progression on 1 prior endocrine therapy. Treatment consists of B 100 mg orally daily and P 100 mg orally daily 3 weeks on 1 week off for the 1st dose-escalation cohort (cycle = 28 days). Pts are evaluated for toxicity every 2-4 weeks and for response every 8 weeks. Primary endpoint: Ph 1: determine recommended Ph II dose; Ph II: 6 month (mo) PFS. Secondary endpoints: CBR, toxicity, correlative studies to better characterize AR+TNBC. Ph I standard 3+3 design with 3 dose escalations; Ph II Simon two-stage minimax design, if 5/18 pts are alive and progression free at 6 mo then accrual would continue to a total 33 pts in stage 2. If ≥ 11/33 pts alive and progression free at 6 mo then B+P will warrant further study. As of 2/1/16, 3 pts with AR+ MBC are enrolled in the 1st dose-escalation cohort of Ph I. Clinical trial information: NCT02605486.

Combinatorial PX-866 and Raloxifene Decrease Rb Phosphorylation, Cyclin E2 Transcription, and Proliferation of MCF-7 Breast Cancer Cells.

As a potential means to reduce proliferation of breast cancer cells, a multiple-pathway approach with no effect on control cells was explored. The human interactome being constructed by the Center for Cancer Systems Biology will prove indispensable to understanding composite effects of multiple pathways, but its discovered protein-protein interactions require characterization. Accordingly, we explored the effects of regulators of one protein on downstream targets of the other protein. MCF-7 estrogen receptor-positive (ER+) breast cancer cells were treated with raloxifene to upregulate the TGF-β pathway and PX-866 to down-regulate the PI3K/Akt pathway. This resulted in highly significant downstream reduction of cell cycle proliferation in breast cancer cells with no significant proliferation reduction following similar treatment of noncancerous MCF10A breast epithelial cells. Reduced phosphorylation of p107 and substantial reduction of Rb phosphorylation were observed in response. The effects of reduced Rb and p107 phosphorylation were reflected in significant decline in E2F-1 transcriptional activity, which is dependent on pocket protein phosphorylation status. The reduced proliferation was related to decreased expression of cyclins, including E2F-1-regulated Cyclin E2, which was also in response to raloxifene and PX-866. All combinations of raloxifene and PX-866 produced significant or highly significant results for reduced MCF-7 cell proliferation, reduced Cyclin E2 transcription, and reduced Rb phosphorylation. These studies demonstrated that uncontrolled proliferation of ER+ breast cancer cells can be significantly reduced by combinational targeting of two relevant pathways. J. Cell. Biochem. 117: 1688-1696, 2016. © 2015 Wiley Periodicals, Inc.

CDK4 Amplification Reduces Sensitivity to CDK4/6 Inhibition in Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes a PAX3- or PAX7-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in an aggressive subset of fusion-positive RMS. CDK4/6 inhibitors have antiproliferative activity in CDK4-amplified liposarcoma and neuroblastoma, suggesting CDK4/6 inhibition as a potential therapeutic strategy in fusion-positive RMS. We examined the biologic consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition by LEE011 in fusion-positive RMS cell lines and xenografts. Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and nonamplified fusion-positive RMS cells via G1-phase cell-cycle arrest. This arrest was mediated by reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, cell-cycle distribution, proliferation, or transformation were not observed in RMS cells overexpressing CDK4. Treatment with LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell-cycle arrest. Although all fusion-positive cell lines showed sensitivity to CDK4/6 inhibition, there was diminished sensitivity associated with CDK4 amplification and overexpression. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and nonamplified fusion-positive RMS. Our data demonstrate that CDK4 is necessary but overexpression is not sufficient for RB-E2F-mediated G1-phase cell-cycle progression, proliferation, and transformation in fusion-positive RMS. Our studies indicate that LEE011 is active in the setting of fusion-positive RMS and suggest that low CDK4-expressing fusion-positive tumors may be particularly susceptible to CDK4/6 inhibition.

Abstract 3093: CDK4 amplification reduces sensitivity to CDK4/6 inhibition in fusion-positive rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and includes an aggressive, PAX3-FOXO1 fusion-positive subtype. Amplification of chromosomal region 12q13-q14, which contains the CDK4 proto-oncogene, was identified in a subset of fusion-positive RMS. Other tumor types with CDK4 amplification or overexpression, including liposarcoma and neuroblastoma, are sensitive to CDK4/6 inhibition, suggesting that CDK4/6-targeted therapies may provide a new treatment strategy in fusion-positive RMS. To evaluate the role of CDK4 in chromosomal region 12q13-14 amplification in fusion-positive RMS and the potential clinical utility of CDK4/6 inhibition in this disease setting, we examined the biological consequences of CDK4 knockdown, CDK4 overexpression, and pharmacologic CDK4/6 inhibition in fusion-positive RMS in vitro and in vivo. Knockdown of CDK4 abrogated proliferation and transformation of 12q13-14-amplified and non-amplified fusion-positive RMS cells via G1-phase cell cycle arrest. This arrest was associated with reduced RB phosphorylation and E2F-responsive gene expression. Significant differences in E2F target expression, however, were not observed in RMS cells overexpressing CDK4 or in fusion-positive tumors harboring 12q13-14 amplification relative to control cells or tumors lacking amplification, respectively. Treatment with the small molecule CDK4/6 inhibitor LEE011 phenocopied CDK4 knockdown, decreasing viability, RB phosphorylation, and E2F-responsive gene expression and inducing G1-phase cell cycle arrest. All fusion-positive RMS cell lines showed sensitivity to CDK4/6 inhibition, with evidence of differential antitumor activity resulting from an inverse relationship between CDK4 expression and inhibitor vulnerability. This variable responsiveness to LEE011 was recapitulated in xenograft models of CDK4-amplified and non-amplified fusion-positive RMS. Our findings demonstrate that CDK4 is necessary for RB-E2F-mediated G1-phase cell cycle progression, proliferation, and transformation in fusion-positive RMS regardless of CDK4 amplification status. Our studies further indicate that single-agent LEE011 is active in the setting of fusion-positive RMS and suggest that CDK4 amplification may be a marker of reduced sensitivity whereas low CDK4 expression may be associated with higher susceptibility to CDK4/6 inhibition. Collectively, our data provide preclinical evidence supporting further investigation of CDK4/6-targeted therapies in treatment regimens for fusion-positive RMS. Citation Format: Mary E. Olanich, Wenyue Sun, Stephen M. Hewitt, Zied Abdullaev, Svetlana D. Pack, Frederic G. Barr. CDK4 amplification reduces sensitivity to CDK4/6 inhibition in fusion-positive rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3093. doi:10.1158/1538-7445.AM2015-3093

Molecular Determinants for the Inactivation of the Retinoblastoma Tumor Suppressor by the Viral Cyclin-dependent Kinase UL97

The retinoblastoma (Rb) tumor suppressor restricts cell cycle progression by repressing E2F-responsive transcription. Cellular cyclin-dependent kinase (CDK)-mediated Rb inactivation through phosphorylation disrupts Rb-E2F complexes, stimulating transcription. The human cytomegalovirus (HCMV) UL97 protein is a viral CDK (v-CDK) that phosphorylates Rb. Here we show that UL97 phosphorylates 11 of the 16 consensus CDK sites in Rb. A cleft within Rb accommodates peptides with the amino acid sequence LXCXE. UL97 contains three such motifs. We determined that the first LXCXE motif (L1) of UL97 and the Rb cleft enhance UL97-mediated Rb phosphorylation. A UL97 mutant with a non-functional L1 motif (UL97-L1m) displayed significantly reduced Rb phosphorylation at multiple sites. Curiously, however, it efficiently disrupted Rb-E2F complexes but failed to relieve Rb-mediated repression of E2F reporter constructs. The HCMV immediate early 1 protein cooperated with UL97-L1m to inactivate Rb in transfection assays, likely indicating that cells infected with a UL97-L1m mutant virus show no defects in growth or E2F-responsive gene expression because of redundant viral mechanisms to inactivate Rb. Our data suggest that UL97 possesses a mechanism to elicit E2F-dependent gene expression distinct from disruption of Rb-E2F complexes and dependent upon both the L1 motif of UL97 and the cleft region of Rb.

PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis.

Preclinical and clinical studies have demonstrated the anticancer activity of PD-0332991, a selective cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, in the treatment of various types of cancer in a retinoblastoma protein (RB)-dependent manner. However, it remains unclear whether CDK4, CDK6 or both are required for RB phosphorylation in colorectal carcinoma and thus PD-0332991 can be used to target this CDK-RB axis for the cancer therapy. The aim of this study was to determine whether CDK4, CDK6 and phosphorylated RB proteins were overexpressed in colorectal carcinoma tissues as compared to matched normal colorectal tissues. The results showed that knockdown of CDK6 but not CDK4 reduced RB phosphorylation and inhibited carcinoma cell growth. Thus, CDK6 plays a critical role in RB phosphorylation and cancer growth. PD-0332991 treatment blocked RB phosphorylation and inhibited cell growth through the induction of G1 arrest of colorectal carcinoma cells. The results demonstrated that, by targeting of CDK6-RB axis, PD-0332991 may prove to be a novel therapeutic agent in treating colorectal carcinoma.

Selective Disruption of Rb–Raf-1 Kinase Interaction Inhibits Pancreatic Adenocarcinoma Growth Irrespective of Gemcitabine Sensitivity

Inactivation of the retinoblastoma (Rb) tumor suppressor protein is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb-Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n = 95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (P = 0.0272), which correlated with reduced overall patient survival (P = 0.0186). To define the antitumor effects of RRD-251 (50 μmol/L), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were conducted on gemcitabine-sensitive and -resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (P < 0.01). Drug efficacy on subcutaneous and orthotopic xenograft models was tested by intraperitoneal injections of RRD-251 (50 mg/kg) alone or in combination with gemcitabine (250 mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (P < 0.01). Combination of RRD-251 with gemcitabine showed cooperative effect on tumor growth (P < 0.01). In conclusion, disruption of the Rb-Raf-1 interaction significantly reduces the malignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb-Raf-1 interaction might be a promising strategy targeting pancreatic cancer.

TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin

Members of the transforming growth factor beta (TGF-β) superfamily are multifunctional cytokines that regulate several cellular processes, including cell cycle arrest, differentiation, morphogenesis, and apoptosis. TGF-β promotes extracellular matrix production and morphological change. Morphogenetic responses to TGF-β include cell migration and epithelial–mesenchymal transition (EMT), which are critical during embryogenesis, development of fibrotic diseases, and the spreading of advanced carcinomas. The purpose of this study was to clarify how TGF-β regulates the fate of retinal pigment epithelial (RPE) cells. TGF-β1 promoted cell cycle progression and phosphorylation of retinoblastoma protein (Rb) in ARPE-19 cells. TGF-β1 induced survivin expression, which in turn stabilized tubulin and Aurora B. RT-PCR and western blot analysis revealed that survivin expression increased in ARPE-19 cells following TGF-β1 treatment. When survivin was depleted, TGF-β1 induced cell cycle arrest and apoptosis and also reduced Rb phosphorylation. In conclusion, the present study shows that induction of EMT in human RPE cells upregulates survivin, leading to survivin-dependent inhibition of cell cycle arrest and apoptosis. Whether cells undergo EMT or apoptosis in response to TGF-β1 is dependent on their cell cycle state, and TGF-β1 regulates the cell cycle via survivin.

Lopinavir inhibits meningioma cell proliferation by Akt independent mechanism

Recent studies suggest that HIV-1 protease inhibitors may have anti-neoplastic effects on some malignancies. The anti-neoplastic effects of lopinavir have not been established or studied in brain tumors. Primary cultures of three fetal leptomeninges and 18 meningiomas were treated with lopinavir alone or with PDGF-BB. DNA synthesis was assessed by CyQUANT. Lopinavir effects on basal and PDGF-stimulated phosphorylation of the Akt-mTOR, MEK1/2-MAPK and STAT3 pathways, phosphorylation of Rb, Caspase 3 activation and reductions in survivin were assessed by Western blots. Lopinavir produced a significant reduction in PDGF-BB stimulation of DNA synthesis in a leptomeningeal culture (P = 0.0013) and 1 of 6 WHO grade I and 1 of 4 grade II meningiomas at 24 h and in 3 of 6 WHO grade I, 4 of 4 grade II and 1 of 1 grade III cell cultures (P = 0.0001) at 72 h. Lopinavir reduced PDGF-BB stimulation of phosphorylation/activation of MAPK in the 22 week fetal leptomeningeal cell cultures and in cells from 1 grade I meningioma at 24 h, but in none of 4 grade I and 5 grade II meningiomas at 6 h. Lopinavir had no notable effect on basal or PDGF-stimulated p-mTOR, p-MEK1/2, or p-STAT3, activation of Caspase 3 or survivin levels. Lopinavir treatment for 24 h had no effect on basal Rb phosphorylation but reduced Rb phosphorylation in all four meningioma cultures. These studies suggest that lopinavir may inhibit meningioma growth, and does so in part by cell cycle arrest. Additional evaluation of lopinavir as a potential adjunct chemotherapy is warranted.