Target Cyclin-dependent Kinase Research Articles

Protein phosphatase 5 and the tumor suppressor p53 down-regulate each other's activities in mice

Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53+/-pp5+/- or p53+/-pp5-/- mice revealed that complete loss of PP5 reduces tumorigenesis in the p53+/- mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.

The Emerging Role of Cyclin-Dependent Kinases (CDKs) in Pancreatic Ductal Adenocarcinoma.

The family of cyclin-dependent kinases (CDKs) has critical functions in cell cycle regulation and controlling of transcriptional elongation. Moreover, dysregulated CDKs have been linked to cancer initiation and progression. Pharmacological CDK inhibition has recently emerged as a novel and promising approach in cancer therapy. This idea is of particular interest to combat pancreatic ductal adenocarcinoma (PDAC), a cancer entity with a dismal prognosis which is owed mainly to PDAC’s resistance to conventional therapies. Here, we review the current knowledge of CDK biology, its role in cancer and the therapeutic potential to target CDKs as a novel treatment strategy for PDAC.

How Selective Are Pharmacological Inhibitors of Cell-Cycle-Regulating Cyclin-Dependent Kinases?

Cyclin-dependent kinases (CDKs) are an important and emerging class of drug targets for which many small-molecule inhibitors have been developed. However, there is often insufficient data available on the selectivity of CDK inhibitors (CDKi) to attribute the effects on the presumed target CDK to these inhibitors. Here, we highlight discrepancies between the kinase selectivity of CDKi and the phenotype exhibited; we evaluated 31 CDKi (claimed to target CDK1-4) for activity toward CDKs 1, 2, 4, 5, 7, 9 and for effects on the cell cycle. Our results suggest that most CDKi should be reclassified as pan-selective and should not be used as a tool. In addition, some compounds did not even inhibit CDKs as their primary cellular targets; for example, NU6140 showed potent inhibition of Aurora kinases. We also established an online database of commercially available CDKi for critical evaluation of their utility as molecular probes. Our results should help researchers select the most relevant chemical tools for their specific applications.

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 4912: Targeting cyclin-dependent kinase 7 in gastric cancer

Abstract CDK7 is a member of the cyclin-dependent kinase family, which forms the CDK activating kinase (CAK) complex with Cyclin H and RING finger protein Mat1 to control cell cycle progression and transcription by phosphorylating other CDKs and RNA polymerase II. Upregulation of CDK7 frequently occurs in human gastric cancer (GC). In this study, we demonstrated that inhibition of CDK7 was able to induce cell growth inhibition, cell cycle arrest at G2/M phase and apoptosis with the increasing intracellular reactive oxidative species (ROS) levels. Pretreatment with ROS scavenger N-acety-L-cysteine partly reversed GC cell apoptosis induced by CDK7 inhibition. In the nude mice, inhibition of CDK7 also suppressed the growth of xenograft tumors of GC. Overall, our data showed that inhibition of CDK7 in GC presented outstanding anticancer effect in vitro and in vivo, suggesting that CDK7 is a potential therapeutic target for GC patients. Citation Format: Jia-Rong Huang, Wen-Ji Zhang, Qi-Wei Jiang, Yang Yang, Meng-Ning Wei, Zhi Shi. Targeting cyclin-dependent kinase 7 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4912.

Comment on: MiR-877-5p suppresses cell growth, migration and invasion by targeting cyclin dependent kinase 14 and predicts prognosis in hepatocellular carcinoma.

Comment on: MiR-877-5p suppresses cell growth, migration and invasion by targeting cyclin dependent kinase 14 and predicts prognosis in hepatocellular carcinoma.

MiR-877-5p suppresses cell growth, migration and invasion by targeting cyclin dependent kinase 14 and predicts prognosis in hepatocellular carcinoma.

Recent studies reveal that hepatocellular carcinoma (HCC) express aberrant microRNAs. Dysregulation of miR-877-5p has been observed in HCC. The objective of the present study was to explore the clinical significance, function and underlying mechanism of miR-877-5p in HCC. Quantitative reverse transcription PCR (qRT-PCR) was used to measure the levels of miR-877-5p in HCC specimens and HCC cell lines. Correlations between miR-877-5p expression and the clinicopathological features and prognosis of HCC patients were then evaluated. MTT assays, colony formation assays, scratch test, transwell assays were used to explore the biological function of miR-877-5p in HCC. A luciferase reporter assay and Western blot were conducted to confirm the target gene of miR-877-5p, and the results were validated in HCC cell lines. We found that the expression of miR-877-5p was downregulated in HCC tissues or cell lines. Clinicopathologic analysis revealed that low miR-877-5p expression correlated with histologic grade (p = 0.008) and TNM stage (p = 0.018). The Kaplan-Meier method indicated that low miR-877-5p levels in HCC were associated with shorter overall survival (p = 0.0041) and disease-free survival (p = 0.0005). Multivariate analysis demonstrated that miR-877-5p expression was an independent poor prognostic factor for HCC patients. Functional assay revealed that upregulation of miR-877-5p could inhibit proliferation, migration, and invasion of HCC cells in vitro. We further identified cyclin-dependent kinase 14 (CDK14) as a direct target of miR-877-5p in HCC cells. Ectopic expression of CDK14 reversed the inhibitory effects of miR-877-5p. Low miR-877-5p expression was a poor prognostic factor for HCC patients, and miR-877-5p functioned as a tumor suppressor in HCC cells via targeting CDK14.

Discovery of N1-(4-((7-Cyclopentyl-6-(dimethylcarbamoyl)-7 H-pyrrolo[2,3- d]pyrimidin-2-yl)amino)phenyl)- N8-hydroxyoctanediamide as a Novel Inhibitor Targeting Cyclin-dependent Kinase 4/9 (CDK4/9) and Histone Deacetlyase1 (HDAC1) against Malignant Cancer.

A series of novel, highly potent, selective inhibitors targeting both CDK4/9 and HDAC1 have been designed and synthesized. N1-(4-((7-Cyclopentyl-6-(dimethylcarbamoyl)-7 H-pyrrolo[2,3- d] pyrimidin-2-yl)amino)phenyl)- N8-hydroxyoctanediamide (6e) was discovered. The lead compound 6e with excellent CDK4/9 and HDAC1 inhibitory activity of IC50 = 8.8, 12, and 2.2 nM, respectively, can effectively induce apoptosis of cancer cell lines. The kinase profiling of compound 6e showed excellent selectivity and specificity. Compound 6e induces G2/M arrest in high concentration and G0/G1 arrest in low concentration to prevent the proliferation and differentiation of cancer cells. Mice bared-breast cancer treated with 6e showed significant antitumor efficacy. The insight into mechanisms of 6e indicated that it could induce cancer cell death via cell apoptosis based on CDK4/9 and HDAC1 repression and phosphorylation of p53. Our data demonstrated the novel compound 6e could be a promising drug candidate for cancer therapy.

Targeting cyclin-dependent kinase 9 enhances sensitization to ionizing radiation in esophageal adenocarcinoma and identifies AXL as a novel downstream target

Targeting cyclin-dependent kinase 9 enhances sensitization to ionizing radiation in esophageal adenocarcinoma and identifies AXL as a novel downstream target

MiR-1202 suppresses hepatocellular carcinoma cells migration and invasion by targeting cyclin dependent kinase 14

Currently, hepatocellular carcinoma (HCC) patients still have poor survival outcomes mainly due to the powerful mobility of HCC cells. Increasing evidences hint that abnormally expressed miRNAs are capable to modulate HCC cells invasion and migration. MiR-1202 has been proposed as a ponderable molecular tumor marker in a variety of tumors. Here, results from real-time PCR indicated the decreased expression of miR-1202 in HCC. Clinically, statistical analysis showed that miR-1202 under-expression was closely associated with metastasis-related clinicopathologic characteristics. In addition, 5-year overall survival (OS) and disease-free survival (DFS) rates of HCC patients with high miR-1202 expression were much better than that in low miR-1202 group. Functionally, gain- and loss-of -function studies were performed to investigate the roles of miR-1202. Intriguingly, Would healing assay and Transwell assays indicated that elevated miR-1202 weakened HCC cells migration and invasion abilities, while miR-1202 knockdown presented the contrary effects. Furthermore, cyclin dependent kinase 14 (CDK14) was identified as a downstream target of miR-1202 by bioinformatics analysis, Dual luciferase reporter assay, detection of CDK14 expression and Pearson correlation analysis. More importantly, rescue experiments demonstrated that CDK14 mediated miR-1202 to exert its anti-tumor effects, which further confirmed the above finding. Taken together, miR-1202 may act as a new biomarker and potential therapeutic target for HCC.

Targeting cyclin-dependent kinase 9 enhances sensitization to ionizing radiation in esophageal adenocarcinoma and identifies AXL as a novel downstream target

Targeting cyclin-dependent kinase 9 enhances sensitization to ionizing radiation in esophageal adenocarcinoma and identifies AXL as a novel downstream target

ISY15-2 - Targeting CDK 4/6 in ER+ Breast cancer

Significant improvements in outcomes have been achieved in hormone receptor positive (HR+) breast cancer with the use of anti-hormonal agents including tamoxifen, aromatase inhibitors (AIs), and fulvestrant. Still, patients with will have either de novo or develop acquired resistance to these approaches. Several alternative signalling pathways have been pursued clinically to overcome resistance including receptor tyrosine kinase signalling, angiogenesis, and intracelullar targets. Except for the mTOR inhibitor everolimus, none of these approaches have improved outcomes. There has been an effort to target the cellular machinery that controls progression through the cell cycle to control cancer growth. These strategies have been hindered by non-selective compounds and the lack of a targeted population for trial selection. Cyclin-dependent kinases (CDKs) are a large family of serine-threonine kinases that play a pivotal role in cell cycle control. Pre-lcinical work identified a role for the first-in-class CDK 4/6 inhibitor palbociclib in HR+ breast cancer and demonstrated its ability to work synergistically with anti-estrogens and reverse endocrine resistance. This lead to the randomized Phase 2 PALOMA1 study that demonstrated a 10 mos improvement in PFS with palbociclib and letrozole vs letrozole alone. These data supported the accelerated approval of this combination in advanced ER+ breast cancer by the US FDA in 2015. Since this time, these findings have been validated in several phase 3 studies with not only palbociclib, but now with other CDK 4/6 inhibitors including ribociclib and abemaciclib. In addiition, these findings are consistent with combinations with other AIs and fulvestrant. CDK 4/6 inhibitors have changed the landscape of ER+ breast cancer and ongoing studies will define their role in early breast cancer. In the lecture we will discuss there development, clinical efficacy and side effects, and future opportunities.

Highlights of This Issue

Treatment options for osteosarcoma remain anchored to surgery and conventional chemotherapy, and are in need of additional and less toxic approaches. Ratti and colleagues developed osteosarcoma models in immunocompetent mice and showed the therapeutic efficacy of trabectedin, a clinically approved marine-derived drug, in halting osteosarcoma growth and metastases. The action of trabectedin was dual: direct on neoplastic cells, inducing their differentiation, and indirect on the immune microenvironment, increasing the infiltration of T cells. However, the latter highly expressed the inhibitory immune checkpoint PD-1. Consistently, combination of trabectedin and anti PD-1 antibody resulted in the best therapeutic effect.The authors investigate whether epigenetic changes are involved in the response to tyrosine kinase inhibitor (TKI) treatment of epidermal growth factor receptor (EGFR) mutated lung cancer. To investigate methylation changes at the whole-genome level, Niu and colleagues applied methyl-sensitive cut counting sequencing (MSCC) in EGFR 19 deletion lung adenocarcinoma tissues before and after erlotinib treatment. A differential methylated region (DMR) of the GABBR2 gene was identified and validated based on the results of whole methylome profiling, and upregulation of GABBR2 rescued erlotinib-induced apoptosis in vitro. This research shows promise in the epigenetic study of EGFR-mutated lung adenocarcinoma treated with TKI.Conditioning strategies constitute a relatively unexplored and exciting opportunity to shape tumor fate by targeting the tumor microenvironment. In this study, Jaworski and colleagues adopted a strategy to remodel the stroma using hemin, a well-known inducer of the enzyme Heme Oxygenase-1, to counteract tumor development. Their results showcase a novel function of an already human-used drug as a means of boosting the endogenous response against prostate cancer, a major health care problem worldwide. In particular, hemin conditioning limits tumor development by simultaneously targeting both tumor vascularization and the cytotoxic T-cell responses.Hormone receptor positive (HR+)/human epidermal growth factor receptor 2-negative (HER2−) metastatic breast cancer (mBC) patients have limited treatment options. Targeting cyclin-dependent kinases (CDK), key components in the cell cycle that are dysregulated in many cancers, has emerged as a viable therapy strategy. In a phase II study of heavily pretreated refractory HR+/HER2− mBC patients, abemaciclib, a selective oral inhibitor of CDK4 and CDK6, exhibited promising single-agent activity and a safety profile that allowed for dosing on a continuous schedule. Abemaciclib may represent a possible therapy advancement for HR+/HER2− mBC patients.

MicroRNA let - 7c inhibits proliferation of hepatocellular carcinoma cells by targeting cyclin dependent kinase 6

Objective To explore let-7c’s influence on the proliferation of human hepatocellular carcinoma cells and to clarify its target gene. Methods The let-7c expression in human hepatocellular carcinoma cells (MHCC-97L, HepG2, HCCLM3, SMMC-7721) and human hepatic cell L02 were detected by real-time quantitative polymerase chain reaction (Real-time PCR). MicroRNAs (miRNAs) were transfected into HCCLM3 cells via Lipofectamine™2000. The cells were divided into three groups: let-7c group transfected with let-7c, negative control group transfected with negative control miRNA, blank control group transfected with no miRNAs. The proliferation of cells was assessed via cell counting kit-8 (CCK-8), cell cycle of each group was detected by flow cytometry, Western blotting was used to evaluate the protein expression of cyclin dependent kinase 6 (CDK6) after transfection. The 3’untranslated region (3’-UTR) of CDK6 containing the putative binding sites of let-7c was inserted into a firefly luciferase reporter vector named pMIR-REPORT. MiRNAs, pMIR-REPORT and renilla luciferase control plasmid were transfected into 293T cells together, the Dual-Luciferase Reporter Assay System was used to assay the luciferase activity. Results The expression level of let-7c in MHCC-97L, HepG2, HCCLM3, SMMC-7721 were (3.22±0.08)×10-4, (2.34±0.11)×10-4, (1.85±0.03)×10-4 and (3.03±0.11)×10-4 respectively, lower than that in L02 [(4.37±0.09)×10-4,P 0.05). The percentage of G1 stage in let-7c group was (56.95±2.40)% at 72 h after transfection, higher than that of the other two groups (P 0.05). The protein level of CDK6 in let-7c group was lower than that of the other two groups at 48 h after transfection (P 0.05). The Dual-Luciferase Reporter Assay System revealed that let-7c could suppress the luciferase’s activity compared with negative control miRNA (P<0.05). Conclusion The expression level of let-7c is lower in the hepatocellular carcinoma cells compared with normal hepatic cell. let-7c can inhibit the proliferation of hepatocellular carcinoma cells and cause a delay in G1-S transition, CDK6 is its target gene. Key words: Carcinoma, hepatocellular; MicroRNAs; Cyclin-dependent kinase 6; Let-7c

O3-5-3 - Phase I study of roniciclib (BAY1000394), an oral CDK inhibitor in Japanese patients with advanced solid tumors

Background: Roniciclib (BAY1000394) is a novel, potent and orally available small molecule inhibitor targeting cyclin-dependent kinases, critical cell cycle and transcription regulatory proteins implicated in tumorigenesis and tumor maintenance. Treatment of patients (pts) with advanced solid tumors at 5 mg bid in 3 days-on/4 days-off schedule is feasible and showed signs of efficacy (Bahleda et al., AACR 2014). We report the safety, tolerability, PK and antitumor activity of roniciclib in Japanese pts with advanced solid tumors.Methods: Roniciclib was administered orally at 2.5/5 mg bid in 3d on/4d off schedule in 3-week cycles. DLT was evaluated in Cycle 1 and 3 + 3 design was employed for dose escalation. PK was evaluated on Days 1 and 10 of Cycle 1. Tumor assessment based on RECIST 1.1 was conducted every 2 cycles.Results: A total of 12 pts (7 male) were treated (2.5/5 mg:6/6 pts); Median age:65 yrs, primary tumor: NSCLC (7 pts). Mean duration of treatment was 57 days (2.5/5 mg:59/56 days). No G4/5 drug-related TEAE was reported. G3 drug-related TEAEs were reported in 3 pts (2.5/5 mg:1/2 pts). Drug-related SAEs were reported in one patient (2.5 mg); G3 ALT, AST and total bilirubin increased and G2 biliary tract infection. Two DLTs were reported:G3 AST, ALT and bilirubin increased in one patient (2.5 mg) and G3 nausea (5 mg). Commonly observed drug-related TEAEs were anorexia, nausea and vomiting. Maximum plasma roniciclib concentration (mean:2.5/5 mg: 42/76 µg/L) was observed 0.5-2 hrs after administration, and decreased with mean half-life of 10 hrs. Dose-proportional increase in the exposure was observed. One partial response in an adrenocortical carcinoma patient at 2.5 mg (at Cycle 20) and 4 stable diseases with disease control rate of 41.7% (2.5/5mg:33/50%) were observed.Conclusions: Roniciclib, at 2.5 and 5 mg bid in 3d on/4d off schedule, was tolerable and demonstrated potential antitumor activity in Japanese patients with advanced solid tumors. Background: Roniciclib (BAY1000394) is a novel, potent and orally available small molecule inhibitor targeting cyclin-dependent kinases, critical cell cycle and transcription regulatory proteins implicated in tumorigenesis and tumor maintenance. Treatment of patients (pts) with advanced solid tumors at 5 mg bid in 3 days-on/4 days-off schedule is feasible and showed signs of efficacy (Bahleda et al., AACR 2014). We report the safety, tolerability, PK and antitumor activity of roniciclib in Japanese pts with advanced solid tumors. Methods: Roniciclib was administered orally at 2.5/5 mg bid in 3d on/4d off schedule in 3-week cycles. DLT was evaluated in Cycle 1 and 3 + 3 design was employed for dose escalation. PK was evaluated on Days 1 and 10 of Cycle 1. Tumor assessment based on RECIST 1.1 was conducted every 2 cycles. Results: A total of 12 pts (7 male) were treated (2.5/5 mg:6/6 pts); Median age:65 yrs, primary tumor: NSCLC (7 pts). Mean duration of treatment was 57 days (2.5/5 mg:59/56 days). No G4/5 drug-related TEAE was reported. G3 drug-related TEAEs were reported in 3 pts (2.5/5 mg:1/2 pts). Drug-related SAEs were reported in one patient (2.5 mg); G3 ALT, AST and total bilirubin increased and G2 biliary tract infection. Two DLTs were reported:G3 AST, ALT and bilirubin increased in one patient (2.5 mg) and G3 nausea (5 mg). Commonly observed drug-related TEAEs were anorexia, nausea and vomiting. Maximum plasma roniciclib concentration (mean:2.5/5 mg: 42/76 µg/L) was observed 0.5-2 hrs after administration, and decreased with mean half-life of 10 hrs. Dose-proportional increase in the exposure was observed. One partial response in an adrenocortical carcinoma patient at 2.5 mg (at Cycle 20) and 4 stable diseases with disease control rate of 41.7% (2.5/5mg:33/50%) were observed. Conclusions: Roniciclib, at 2.5 and 5 mg bid in 3d on/4d off schedule, was tolerable and demonstrated potential antitumor activity in Japanese patients with advanced solid tumors.

Abstract 2828: Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for breast cancer

Abstract The cyclin D:CDK-4/6:Rb axis is dysregulated in a number of different cancers and is implicated in resistance to hormonal therapy in breast cancer. Pharmacologically targeting cyclin dependent kinase 4 and 6 (CDK4 and 6) has proven to be a successful therapeutic approach in ER+ breast cancer (BC). This study aimed to identify the molecular subtypes of BC that are sensitive to the novel CDK4 and 6 inhibitor, abemaciclib, and identify the best combination strategies for the clinical development. Growth inhibition activity of abemaciclib was assessed in a panel of 46 BC cell lines molecularly characterized by genomic, transcriptomic and proteomic profiling. IC50 values were determined from direct cell counts using a Z1-particle counter. In vivo activity of abemaciclib was assessed in cell line xenograft models of ER+ and HER2+/ER+ BCs. For ER+ BC, mice were treated daily with clinically achievable doses of abemaciclib (50-75 mg/kg) as a single agent or in combination with tamoxifen or fulvestrant. Combinations with trastuzumab, docetaxel and tamoxifen were assessed in the HER2+/ER+ xenografts. Sensitivity to abemaciclib was observed predominately in multiple luminal BC cell lines and a small subset of triple negative cell lines that had intact Rb signaling. Activating mutations in PIK3CA also marked for abemaciclib sensitivity. Abemaciclib potentiated the anti-proliferative effects of cytotoxic/anti-mitotic agents when given simultaneously or 48 hours prior to treatment in vitro. Significant tumor growth inhibition (TGI) was observed with single agent abemaciclib in the ER+ BC cell line xenografts. In ZR751 xenografts, the addition of either tamoxifen or fulvestrant to abemaciclib induced complete inhibition of tumor growth for the 12 weeks of treatment. In the MCF7 model, treatment was withdrawn after five weeks, which triggered tumor regrowth in each of the single agent arms. However, complete responses were maintained in the combination arms for a further six weeks post drug withdrawal. In HER2 amplified xenografts, abemaciclib single agent treatment induced significant TGI in trastuzumab sensitive and resistant xenografts, and combination with trastuzumab further increased this anti-tumor effect. The addition of tamoxifen to this combination induced a further increment in TGI. Consistent with the in vitro findings, the combination of abemaciclib and the anti-mitotic agent docetaxel was not antagonistic in vivo, and the addition of docetaxel to the triple combination of abemaciclib, trastuzumab, and tamoxifen induced the most efficacy of any of the treatment arms tested. Combinations were well tolerated in animals. These data highlight the potential of abemaciclib to have single agent activity in addition to combined activity with anti-mitotic or targeted therapies for breast cancer. Citation Format: Neil A. O’Brien, Dylan Conklin, Tong Luo, Ondrej Kalous, Erika von Euw, Sara A. Hurvitz, Richard P. Beckmann, Colleen Mockbee, Dennis J. Slamon. Preclinical activity of abemaciclib as a single agent or in combination with anti-mitotic or targeted therapies for breast cancer. [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 2828.

Abstract 2821: Characterization of CDK inhibitors in a biochemical assay using a comprehensive panel of human CDK-cyclin complexes

Abstract Members of the family of cyclin dependent kinases (CDKs) have been recognized as pivotal regulators of cell cycle progression for more than 20 years. Concordant to their central role in the control of cell division they have been in the focus of research of proliferation associated diseases ever since, most prominently amongst these cancer. Although initial results obtained from first and second generation, low specificity CDK inhibitors (e.g. Flavopyridol, Roscovitine, Dinaciclib, AT7519, R547) have been sobering the recent approval of the first CDK-inhibitor Palbociclib for the treatment of certain forms of breast cancer clearly demonstrates the suitability of cell cycle CDKs as targets in oncology. Furthermore, in addition to cell cycle CDKs a second group of CDKs have been shown to have important roles in the regulation of gene transcription, and several of the “transcriptional” CDKs have become interesting targets in oncology. Recent results underline the notion that for being effective in the treatment of cancer, CDK inhibition requires very high specificity towards the respective target CDK(s). For example CDK1 knockdown or CDK9 inhibition have been shown to be synthetically lethal in combination with MYC overexpression. Selectivity of compounds within the family of CDKs could so far only be tested using a quite limited number of CDK-Cyclin complexes expressed in human cells. To date there are 20 CDK genes and at least 17 different Cyclin genes described, many of which give rise to different variants, e.g. there are 3 D-type cyclins, two A- and E-type cyclins etc.. Experimental data indicates that at least 50-60 different, biologically relevant CDK-Cyclin complexes may exist, but only a limited number of these are available for biochemical testing of drug candidates so far. We have recombinantly expressed and purified 28 different CDK-Cyclin complexes, covering a significant part of the CDK family, and established in-vitro kinase-activity assays for these recombinant enzymes. The resulting CDK panel represents the most comprehensive array for biochemical testing of this enzyme group currently available. We characterized the specificity of several CDK inhibitors that have been or are currently in preclinical or clinical development with this CDK collection. Results will be presented showing the specificity of these inhibitors not only for CDKs but also for CDKs complexed to different Cyclins. In several cases we could detect signifcant differences in the inhibition of the same CDK complexed to different Cyclins, e.g. a 10fold difference was seen for CDK6 complexes with Cyclin D1-3. A &amp;gt;100 fold difference was detected for CDK3 complexed to either Cyclin E1 or Cyclin C. This screening panel allows generating data on compound selectivity early in development, diminishing the risk of designing a compound with suboptimal target specificity. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Christian Beisenherz-Huss, Diane Kraemer, Carolin Heidemann-Dinger, Constance Ketterer, Chris Eckert, Michael H.G. Kubbutat. Characterization of CDK inhibitors in a biochemical assay using a comprehensive panel of human CDK-cyclin complexes. [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 2821.

Targeting Cyclin-Dependent Kinases in Synovial Sarcoma: Palbociclib as a Potential Treatment for Synovial Sarcoma Patients

BackgroundIn synovial sarcomas alterations in the cyclin D1-CDK4/6-Rb axis have been described. Also, β-catenin, a cyclin D1 regulator, is often overexpressed. Additionally, studies have shown that the t(X;18) translocation influences tumor behavior partly through cyclin D1 activation. We investigated how alterations in the cyclin D1-CDK4/6-Rb axis impact prognosis and studied effects of targeting this axis with the CDK4/6 inhibitor palbociclib.MethodsSynovial sarcoma samples (n = 43) were immunohistochemically stained for β-catenin, cyclin D1, p16, p21, p27, Rb, and phospho-Rb. Fluorescent in situ hybridization (FISH) was performed to detect CCND1 amplification or translocation. In 4 synovial sarcoma cell lines sensitivity to palbociclib was investigated using cell viability assays, and effects on the sensitive cell lines were evaluated on protein level and by cell cycle arrest.ResultsExpression of nuclear phospho-Rb and nuclear β-catenin in the patient samples was associated with poor survival. FISH showed a sporadic translocation of CCND1 in a subset of tumors. An 8-fold CCND1 amplification was found in 1 cell line, but not in the patient samples investigated. Palbociclib effectively inhibited Rb-phosphorylation in 3 cell lines, resulting in an induction of a G1 arrest and proliferation block.ConclusionsIn this series nuclear phospho-Rb and nuclear β-catenin expression were negative prognostic factors. In vitro data suggest that palbociclib may be a potential treatment for a subset of synovial sarcoma patients. Whether this effect can be enhanced by combination treatment deserves further preclinical investigations.Electronic supplementary materialThe online version of this article (doi:10.1245/s10434-016-5341-x) contains supplementary material, which is available to authorized users.

The roles and therapeutic potential of cyclin-dependent kinases (CDKs) in sarcoma.

Uncontrolled proliferation and cell growth is the hallmark of many different malignant diseases, including sarcomas. Cyclin-dependent kinases (CDKs) are members of the serine/threonine protein kinase family and play crucial roles in tumor cell proliferation and growth by controlling cell cycle, transcription, and RNA splicing. In addition, several CDKs influence multiple targets and phosphorylate transcription factors involved in tumorigenesis. There are many examples linking dysregulated activation and expression of CDKs to tumors, and targeting CDKs in tumor cells has become a promising therapeutic strategy. More recently, the Food and Drug Administration (FDA) has approved the CDK4/6 inhibitor palbociclib for treating metastatic breast cancer. In sarcomas, high levels of CDK mRNA and protein expression have been found in most human sarcoma cells and patient tissues. Many studies have demonstrated consistent results in which inhibition of different CDKs decrease sarcoma cell growth and induce apoptosis. Therefore, CDKs comprise an attractive set of targets for novel anti-sarcoma drug development. In this review, we discuss the roles of different members of CDKs in various sarcomas and provide a pre-clinical overview of promising therapeutic potentials of targeting CDKs with a special emphasis on sarcoma.

Abstract LB-A21: Single-agent activity and favorable pharmaceutical properties of orally bioavailable next-generation CDK4/6 inhibitor, ON 123300

Abstract Introduction: Recent proof-of-concept for targeting cyclin dependent kinases 4 and 6 (CDK4/6) culminated in the approval for breast cancer of the first-in-class CDK4/6 inhibitor, palbociclib (Ibrance®). Palbociclib and other potent but narrowly targeted CDK4/6 inhibitors are cytostatic rather than cytotoxic (Leonard et al, 2012), and require combination therapy for optimal activity. ON 123300 is a next-generation CDK4/6 inhibitor with improved single-agent cytotoxicity. This novel compound is a potent CDK4 and CDK6 inhibitor (IC50 = 3.9 nM and 9.8 nM, respectively) that is cytotoxic against breast and other cancer cell lines (Reddy et al, 2014). We present pre-IND profiling pharmacology, physicochemical and ADME studies to support ON 123300 as a clinical candidate and next-generation CDK4/6 inhibitor. Methods and Results: Non-clinical studies were conducted in order to assess the drug-like properties of ON 123300 and its pharmaceutical salts. Physicochemical parameters were determined for the compound. ON 123300 was highly soluble below pH 4.0, with reduced solubility in more basic conditions. Predicted intestinal absorption, as measured by Caco-2 permeability, was 1.95×10-5 cm/s with a low efflux ratio. Based on these characteristics, the compound was advanced to lead profiling against 68 primary molecular targets. Radioligand binding studies revealed limited receptor inhibition (11/68 targets) with 10 μM of ON 123300. Human Ether-a-go-go (hERG) inhibition testing revealed no significant effect (IC50 &amp;gt; 25 μM). In vitro metabolism studies indicated metabolism through CYP3A4 and CYP2C8. Finally, in silico based modeling (Cloe® PK) integrating all key non-clinical parameters was carried out in order to estimate the potential for oral absorption in humans. Preliminary analysis suggests good oral bioavailability and the potential to establish an acceptable therapeutic window. Conclusions: The well differentiated biochemical, anticancer and pharmaceutical properties of ON 123300 provide the basis for pre-clinical toxicology testing, leading to an investigational new drug (IND) filing and clinical exploration of this compound across tumor types that share targets accessed by this next-generation CDK inhibitor. Citation Format: Benjamin Hoffman, Ramana Reddy, Muralidhar Mallireddigari, Daniel Fox, Chen Ren, Manoj Maniar. Single-agent activity and favorable pharmaceutical properties of orally bioavailable next-generation CDK4/6 inhibitor, ON 123300. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A21.