Search

Summary Background Current rheumatoid arthritis therapies target immune inflammation and are subject to ceiling effects. Seliciclib is an orally available cyclin-dependent kinase inhibitor that suppresses proliferation of synovial fibroblasts—cells not yet targeted in rheumatoid arthritis. Part 1 of this phase 1b/2a trial aimed to establish the maximum tolerated dose of seliciclib in patients with active rheumatoid arthritis despite ongoing treatment with TNF inhibitors, and to evaluate safety and pharmacokinetics. Methods Phase 1b of the TRAFIC study was a non-randomised, open-label, dose-finding trial done in rheumatology departments in five UK National Health Service hospitals. Eligible patients (aged ≥18 years) fulfilled the 1987 American College of Rheumatology (ACR) or the 2010 ACR–European League Against Rheumatism classification criteria for rheumatoid arthritis and had moderate to severe disease activity (a Disease Activity Score for 28 joints [DAS28] of ≥3·2) despite stable treatment with anti-TNF therapy for at least 3 months before enrolment. Participants were recruited sequentially to a maximum of seven cohorts of three participants each, designated to receive seliciclib 200 mg, 400 mg, 600 mg, 800 mg, or 1000 mg administered in 200 mg oral capsules. Sequential cohorts received doses determined by a restricted, one-stage Bayesian continual reassessment model, which determined the maximum tolerated dose (the primary outcome) based on a target dose-limiting toxicity rate of 35%. Seliciclib maximum concentration (Cmax) and area under the plasma concentration time curve 0–6 h (AUC0–6) were measured. This study is registered with ISRCTN, ISRCTN36667085. Findings Between Oct 8, 2015, and Aug 15, 2017, 37 patients were screened and 15 were enrolled to five cohorts and received seliciclib, after which the trial steering committee and the data monitoring committee determined that the maximum tolerated dose could be defined. In addition to a TNF inhibitor, ten (67%) enrolled patients were taking conventional synthetic disease modifying antirheumatic drugs. The maximum tolerated dose of seliciclib was 400 mg, with an estimated dose-limiting toxicity probability of 0·35 (90% posterior probability interval 0·18–0·52). Two serious adverse events occurred (one acute kidney injury in a patient receiving the 600 mg dose and one drug-induced liver injury in a patient receiving the 400 mg dose), both considered to be related to seliciclib and consistent with its known safety profile. 65 non-serious adverse events occurred during the trial, 50 of which were considered to be treatment related. Most treatment-related adverse events were mild; 20 of the treatment-related non-serious adverse events contributed to dose-limiting toxicities. There were no deaths. Average Cmax and AUC0–6 were two-times higher in participants developing dose-limiting toxicities. Interpretation The maximum tolerated dose of seliciclib has been defined for rheumatoid arthritis refractory to TNF blockade. No unexpected safety concerns were identified to preclude ongoing clinical evaluation in a formal efficacy trial. Funding UK Medical Research Council, Cyclacel, Research into Inflammatory Arthritis Centre (Versus Arthritis), and the National Institute of Health Research Newcastle and Birmingham Biomedical Research Centres and Clinical Research Facilities.

Over the last 50 years, there has been a steady improvement in the treatment outcome of acute myeloid leukemia (AML). However, median survival in the elderly is still poor due to intolerance to intensive chemotherapy and higher numbers of patients with adverse cytogenetics. Fadraciclib (CYC065), a novel cyclin-dependent kinase (CDK) 2/9 inhibitor, has preclinical efficacy in AML. In AML cell lines, myeloid cell leukemia 1 (MCL-1) was downregulated following treatment with fadraciclib, resulting in a rapid induction of apoptosis. In addition, RNA polymerase II (RNAPII)-driven transcription was suppressed, rendering a global gene suppression. Rapid induction of apoptosis was observed in primary AML cells after treatment with fadraciclib for 6–8 h. Twenty-four hours continuous treatment further increased efficacy of fadraciclib. Although preliminary results showed that AML cell lines harboring KMT2A rearrangement (KMT2A-r) are more sensitive to fadraciclib, we found that the drug can induce apoptosis and decrease MCL-1 expression in primary AML cells, regardless of KMT2A status. Importantly, the diversity of genetic mutations observed in primary AML patient samples was associated with variable response to fadraciclib, confirming the need for patient stratification to enable a more effective and personalized treatment approach. Synergistic activity was demonstrated when fadraciclib was combined with the BCL-2 inhibitor venetoclax, or the conventional chemotherapy agents, cytarabine, or azacitidine, with the combination of fadraciclib and azacitidine having the most favorable therapeutic window. In summary, these results highlight the potential of fadraciclib as a novel therapeutic approach for AML.

Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546).

Introduction: Targeted biologic therapies demonstrate similar efficacies in rheumatoid arthritis despite distinct mechanisms of action. They also exhibit a ceiling effect, with 10% to 20% of patients achieving remission in clinical trials. None of these therapies target synovial fibroblasts, which drive and maintain synovitis. Seliciclib (R-roscovitine) is an orally available cyclin-dependent kinase inhibitor that suppresses fibroblast proliferation, and is efficacious in preclinical arthritis models. We aim to determine the toxicity and preliminary efficacy of seliciclib in combination with biologic therapies, to inform its potential as an adjunctive therapy in rheumatoid arthritis. Methods and analysis: TRAFIC is a non-commercial, multi-center, rolling phase Ib/IIa trial investigating the safety, tolerability, and efficacy of seliciclib in patients with moderate to severe rheumatoid arthritis receiving biologic therapies. All participants receive seliciclib with no control arm. The primary objective of part 1 (phase Ib) is to determine the maximum tolerated dose and safety of seliciclib over 4 weeks of dosing. Part 1 uses a restricted 1-stage Bayesian continual reassessment method based on a target dose-limiting toxicity probability of 35%. Part 2 (phase IIa) assesses the potential efficacy of seliciclib, and is designed as a single arm, single stage early phase trial based on a Fleming-A’Hern design using the maximum tolerated dose recommended from part 1. The primary response outcome after 12 weeks of therapy is a composite of clinical, histological and magnetic resonance imaging scores. Secondary outcomes include adverse events, pharmacodynamic and pharmacokinetic parameters, autoantibodies, and fatigue. Ethics and dissemination: The study has been reviewed and approved by the North East - Tyne & Wear South Research Ethics Committee (reference 14/NE/1075) and the Medicines and Healthcare Products Regulatory Agency (MHRA), United Kingdom. Results will be disseminated through publication in relevant peer-reviewed journals and presentation at national and international conferences. Trials Registration: ISRCTN, ISRCTN36667085. Registered on September 26, 2014; http://www.isrctn.com/ISRCTN36667085 Current protocol version: Protocol version 11.0 (March 21, 2019)

Background:Current rheumatoid arthritis (RA) therapeutics target immune inflammation and are subject to ceiling effects, with non-response observed in a third of recipients together with low remission rates. Synovial fibroblasts (SFs) are stromal cells not yet targeted in RA, whose hyperplastic and proliferative properties drive inflammation and tissue destruction. Seliciclib (R-roscovitine) is an orally available cyclin-dependent kinase (CDK) inhibitor that suppresses SF proliferation and ameliorates inflammatory arthritis in rodents.Objectives:To determine the maximum tolerated dose (MTD) of seliciclib in patients with active RA despite anti-TNF, with or without background conventional disease modifying anti-rheumatic drugs (cDMARDs). Safety and pharmacokinetics (PK) were also evaluated.Methods:A restricted, one-stage Bayesian continual reassessment method (CRM) determined MTD based on a target dose-limiting toxicity (DLT) probability of 35%. RA patients (DAS28 ≥3.2) were recruited sequentially to cohorts of 3 subjects each. Cohort 1 received 400mg seliciclib daily for 4 consecutive days each week for 4 weeks, added to existing therapy. Each subsequent cohort received a dose determined by the toxicity-based CRM algorithm, calculated upon conclusion of the previous cohort. Safety was assessed through adverse event (AE) monitoring. Associations with relevant PK parameters were sought.Results:15 anti-TNF recipients were enrolled, 10 of whom were also taking cDMARDs (median DAS28 4.9). Application of the CRM algorithm prompted one dose increment during the study (to 600mg for cohort 2), but reversion to 400mg for subsequent cohorts (Figure 1A). After treatment of 5 cohorts, 400mg was determined the MTD, with a DLT probability of 0.35 (CI 0.18-0.52; Figure 1B). 6 patients experienced DLTs, of which two were classified as serious AEs (SAEs) in keeping with the safety profile of seliciclib; these are summarised in Table 1. Of 43/65 total AEs reported at any dose that didnotcontribute to a DLT, 26 were possibly, probably or definitely related to seliciclib; 19 of these 26 were mild, 7 moderate and none severe. The most frequent AE was mild nausea. No relationship of safety and/or tolerability with concomitant cDMARD use or PK was seen.Table 1.Characteristics of patients who developed HZ at initiation of baricitinibDLTSeliciclib dose (mg)Doses receivedContributing AEsContributing SAEsDescriptionOutcomeA1400830Constipation, N+V, liver injury; fatigue.Resolved2600430Constipation, N+V.Resolved3600101BFever, N+V, renal injury.Resolved4400831BConstipation, N+V, jaundice, liver injury.Resolved5400840Fever, dizziness, liver injury.Resolved6400890Dizziness, N+V, liver injury, bilirubin rise.Persistent AST riseConclusion:The MTD of seliciclib has been defined for RA. No unexpected safety concerns were identified to preclude ongoing evaluation in patients, which focuses on clinical, radiological and biological indicators of efficacy.Disclosure of Interests:Arthur Pratt Grant/research support from: Pfizer, GlaxoSmithKlein, Stefan Siebert Grant/research support from: BMS, Boehringer Ingelheim, Celgene, GlaxoSmithKline, Janssen, Novartis, Pfizer, UCB, Consultant of: AbbVie, Boehringer Ingelheim, Janssen, Novartis, Pfizer, UCB, Speakers bureau: AbbVie, Celgene, Janssen, Novartis, Michael Cole: None declared, Deborah Stocken: None declared, Stephen Kelly: None declared, Muddassir Shaikh: None declared, Amy Cranston: None declared, Miranda Morton: None declared, Jennifer Walker: None declared, Sheelagh Frame Employee of: Cyclacel Ltd., Wan-fai Ng: None declared, Chris Buckley Consultant of: Janssen, Pfizer, GSK, Galapagos, Gillead, Iain McInnes Grant/research support from: Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Janssen, and UCB, Consultant of: AbbVie, Bristol-Myers Squibb, Celgene, Eli Lilly and Company, Gilead, Janssen, Novartis, Pfizer, and UCB, Andrew Filer: None declared, John D Isaacs Consultant of: AbbVie, Bristol-Myers Squibb, Eli Lilly, Gilead, Janssen, Merck, Pfizer, Roche

Aim Phase I study of seliciclib (CYC202, R-roscovitine), an inhibitor of cyclin-dependent kinases 2, 7 and 9, causing cell cycle changes and apoptosis in cancer cells. Patients and methods This phase I trial aimed at defining the toxicity profile, the maximum tolerated dose (MTD), the recommended phase II dose (RD) and the main pharmacokinetic and pharmacodynamic parameters of oral seliciclib. Three schedules were evaluated: seliciclib given twice daily for 5 consecutive days every 3 weeks (schedule A), for 10 consecutive days followed by 2 weeks off (schedule B) and for 3 d every 2 weeks (schedule C). Results Fifty-six patients received a total of 218 cycles of seliciclib. Dose-Limiting Toxicities (DLT) consisting of nausea, vomiting, asthenia and hypokalaemia occurred at 1600 mg bid for schedule A and in schedule C, DLT of hypokalaemia and asthenia occurred at 1800 mg bid. The evaluation of longer treatment duration in schedule B was discontinued because of unacceptable toxicity at lower doses. Other adverse events included transient serum creatinine increases and liver dysfunctions. Pharmacokinetic data showed that exposure to seliciclib and its carboxylate metabolite increased with increasing dose. Soluble cytokeratin 18 fragments allowed monitoring of seliciclib-induced cell death in the blood of patients treated with seliciclib at doses above 800 mg/d. One partial response in a patient with hepatocellular carcinoma and sustained tumour stabilisations were observed. Conclusions The MTD and RD for seliciclib are 1250 mg bid for 5 d every 3 weeks and 1600 mg bid for 3 d every 2 weeks, respectively.

CYC065 is a cyclin-dependent kinase (Cdk) inhibitor that is highly selective towards Cdk2 and Cdk9. In chronic lymphocytic leukemia (CLL), a disease that is addicted to the over-expression of anti-apoptotic proteins for survival, inhibition of Cdk9 by CYC065 reduced phosphorylation of the C-terminal domain of RNA polymerase II and blocked transcription. These actions depleted the intrinsically short-lived anti-apoptotic protein Mcl-1, but not Bcl-2, and induced apoptosis in CLL cells in vitro. The IC50 for CYC065-induced CLL cell death after a 24-hr incubation was 0.8 μM, a concentration that is achievable in the clinic at tolerated doses. CYC065 killed the CLL cells equally efficiently in the presence or absence of the human stromal cell line, StromaNKtert, and with or without a stimulation condition that mimics the lymphoid tissue microenvironment (anti-IgM, anti-CD-40, IL-4). Venetoclax, which specifically inhibits Bcl-2 function, is approved for treatment of CLL with del(17p); however upregulation of Mcl-1 is associated with resistance to venetoclax in the lymph nodes. Therefore, we hypothesized that the combination of CYC065 with venetoclax would target the parallel mechanisms that promote the survival control in CLL cells, and induce synergistic cell death by apoptosis. A time course study of the single agents showed that under conditions that mimic the lymph node microenvironment, cell death induction by venetoclax required 6-8 hr to reach the plateau of cell killing and maximal killing by CYC065 occurred after 24 hr, consistent with the different mechanisms of action of the two compounds. Following the removal of CYC065 or venetoclax after 4, 8, 12, or 24 hr incubations, there was no evidence for additional cell death after an additional 48 hr in drug-free medium regardless of the duration of drug incubation. Immunoblots showed recovery of RNA pol II phosphorylation, and restored Mcl-1 expression upon washout of CYC065. The reversible action of these compounds has potential implications for clinical scheduling combining these compounds. Median effect analysis indicated that CYC065 and venetoclax combined synergistically in CLL samples with or without 17p deletion. A dose reduction analysis confirmed mutual potentiation of each other when combined. Combination of IC50 concentrations of CYC065 and venetoclax for 24 hr was sufficient to decrease the viability of CLL cells by over 90% in the lymph node mimicking microenvironment. Thus, these data provided rationale for clinical combination of CYC065 and venetoclax in CLL. CYC065 is currently in a Phase I clinical trial in patients with advanced solid tumors (NCT02552953) using an intermittent dosing regimen which causes at least 24 hr Mcl-1 downregulation in patient PBMCs at well tolerated dose levels. Citation Format: Rong Chen, Yuling Chen, Sheelagh Frame, David Blake, William G. Wierda, Daniella Zheleva, William Plunkett. Strategic combination of the cyclin-dependent kinase inhibitor CYC065 with venetoclax to target anti-apoptotic proteins in chronic lymphocytic leukemia [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 3905.

Purpose: Seliciclib is a small-molecule cyclin-dependent kinase inhibitor, which has been reported to induce apoptosis and cell cycle arrest in EBV-negative nasopharyngeal carcinoma cell lines. Because most nasopharyngeal carcinoma patients harbor EBV, we proceeded to evaluate the cytotoxic effects of seliciclib in EBV-positive nasopharyngeal carcinoma models. Experimental Design: Cytotoxicity of seliciclib was investigated in the EBV-positive cell line C666-1 and the C666-1 and C15 xenograft models. Caspase activities and cell cycle analyses were measured by flow cytometry. Efficacy of combined treatment of seliciclib with radiation therapy was also evaluated. Results: Seliciclib caused significant cytotoxicity in the C666-1 cells in a time- and dose-dependent manner, with accumulation of cells in both sub-G1 and G2-M phases, indicative of apoptosis and cell cycle arrest, respectively. Caspase-2, -3, -8, and -9 activities were all increased, with caspase-3 being the most significantly activated at 48 h after treatment. These cells also showed a reduction of Mcl-1 mRNA and protein levels. Combined treatment of seliciclib with radiation therapy showed a synergistic interaction with enhanced cytotoxicity in C666-1 cells and delayed repair of double-strand DNA breaks. For in vivo models, significant delays in tumor growth were observed for both C666-1 and C15 tumors, which were associated with enhanced apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and immunohistochemistry analyses. Conclusions: Seliciclib enhanced the antitumor efficacy of radiation therapy in EBV-positive nasopharyngeal carcinoma, characterized by G2-M arrest, and apoptosis, associated with an induction in caspase activity. This process is mediated by reduction in Mcl-1 expression and by attenuation of double-strand DNA break repair.

FRAT1, like its Xenopus homolog glycogen synthase kinase-3 (GSK-3)-binding protein, is known to inhibit GSK-3-mediated phosphorylation of beta-catenin. It is currently unknown how FRAT-GSK-3-binding protein activity toward GSK-3 is regulated. FRAT1 has recently been shown to be a phosphoprotein in vivo; however, the responsible kinase(s) have not been determined. In this study, we identified Ser188 as a phosphorylated residue in FRAT1. The identity of the kinase that catalyzes Ser188 phosphorylation and the significance of this phosphorylation to FRAT1 function were investigated. Protein kinase A (PKA) was found to phosphorylate Ser188 in vitro as well as in intact cells. Importantly, activation of endogenous cAMP-coupled beta-adrenergic receptors with norepinephrine stimulated the phosphorylation of FRAT1 at Ser188. GSK-3 was also able to phosphorylate FRAT1 at Ser188 and other residues in vitro or when overexpressed in intact cells. In contrast, endogenous GSK-3 did not lead to significant FRAT1 phosphorylation in cells, suggesting that GSK-3 is not a major FRAT1 kinase in vivo. Phosphorylation of Ser188 by PKA inhibited the ability of FRAT1 to activate beta-catenin-dependent transcription. In conclusion, PKA phosphorylates FRAT1 in vitro as well as in intact cells and may play a role in regulating the inhibitory activity of FRAT1 toward GSK-3.

Introduction: CYC140 is a selective and potent ATP-competitive inhibitor of PLK1, which has completed IND-enabling studies. The aim of this translational project was to inform the clinical development path of CYC140. Esophageal cancer was investigated as a potential target indication based on unmet medical need and the observation that PLK1 is frequently overexpressed in esophageal tumors and carries a poor prognosis. Experimental procedures: The anticancer activity of CYC140 was examined across multiple tumor types to identify sensitive target indications or tumor subsets. 6 h pulse exposure to CYC140 was used to determine sensitivity in a panel of over 250 cancer cell lines, including 15 esophageal cancer cell lines, using CellTiter Glo and resazurin-based assays. Candidate pharmacodynamic markers were examined in malignant and non-malignant cells. Drug combination testing was undertaken in several esophageal cell lines using approved and targeted agents. Solid tumor and leukemia xenograft models were performed to assess CYC140 dosing schedules and efficacy. Summary: Inhibition of PLK1 by CYC140 perturbs the entry into and exit from mitosis. In malignant cells, CYC140 treatment leads to the appearance of mitotic cells with monopolar spindles and a persistent increase in the proportion of cells in G2 and M phase, resulting in complete growth inhibition and induction of cell death. In non-malignant cells, the growth arrest is transient, and cells resume cycling once compound is removed. Short (6 h) pulse treatments of CYC140 maximise the difference in cellular response between malignant esophageal cell lines and cells of a non-malignant origin. In the esophageal cell line panel, CYC140 cellular IC50 correlates with induction of apoptosis. The effect of CYC140 on pharmacodynamic markers of PLK-1 activity such as phospho-nucleophosmin and phospho-histone H3 was characterized in malignant and non-malignant cells. Several promising combinations of CYC140 with targeted agents were identified, including EGFR inhibitors, HDAC inhibitors and PI3K pathway inhibitors, and CYC140 can also be combined with cytotoxic agents approved for use in esophageal cancer, such as cisplatin or irinotecan. CYC140 anti-tumor efficacy was demonstrated in solid tumor and leukemic xenograft models with responses including tumor regression and tumor-free cures. Conclusions: CYC140 is a promising anti-cancer agent with potent anti-proliferative activity and therapeutic potential in a variety of cancers, including esophageal cancer and acute leukemia. The mode of action of CYC140 is consistent with PLK1 inhibition and cell death is preferentially triggered in sensitive malignant cells. Suitable pharmacodynamic markers and several promising combinations have been identified that could assist clinical development of CYC140. Citation Format: Sylvie Moureau, Craig MacKay, Chiara Saladino, Elizabeth Pohler, Karin Kroboth, Jonathan Hollick, Daniella Zheleva, Sheelagh Frame, David Blake. The novel PLK1 inhibitor, CYC140: Identification of pharmacodynamic markers, sensitive target indications and potential combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4178. doi:10.1158/1538-7445.AM2017-4178

Introduction: Double-hit lymphomas (DHL), defined by concurrent MYC and BCL2 rearrangements, have poor prognosis compared to standard-risk diffuse large B cell lymphomas (DLBCL). Current treatment regimens involve multiple chemotherapies, but do not specifically exploit these molecular features of the disease. Several studies have established that: (i) DLBCL show frequent overexpression of Mcl-1, an anti-apoptotic member of the Bcl-2 family (30% and 50% in the ABC and GCB subtypes, respectively), (ii) MYC-driven lymphomas are highly sensitive to depletion of Mcl-1, (iii) MYC overexpression and inhibition of CDK activity are synthetically lethal and (iv) resistance to BH3 mimetics targeting Bcl-2 can be conferred by upregulation of Mcl-1. CYC065 is a specific and potent CDK2/9 inhibitor, currently in a Phase 1 trial in patients with advanced cancer (NCT02552953). The mechanism of action of CYC065 involves rapid reduction of the levels of both Mcl-1 and MYC, suggesting a therapeutic rationale for investigating this agent in DLBCL. This preclinical study explored the single agent activity of CYC065 in B cell lymphoma, and its potential to combine with other molecularly targeted agents of interest, including venetoclax (ABT-199, a Bcl-2 inhibitor), and BET inhibitors. Methods: Single agent activity of CYC065 was explored using short pulse treatments (6-8 h) in B cell lymphoma cell lines. Viability and total cell number were assessed 24, 48 or 72 h following treatment. Levels of MYC, Bcl-2 and Mcl-1 were determined by Western blotting at baseline and following treatment with CYC065. Cell fate was examined by flow cytometry. CYC065 was combined with venetoclax or BET inhibitors ((+)-JQ-1, GSK525762 and OTX-015), which were administered concomitantly for a 6 h pulse or up to 72 h. Combination data were analyzed by the Chou & Talalay method. Results: The median IC50 for CYC065 in 13 B cell lymphoma cell lines was 0.43 μM. No obvious difference was observed between ABC and GCB subtypes of DLBCL, and DHL DLBCL lines had similar IC50 values to non-DHL DLBCL lines (median 0.47 μM vs 0.29 μM; p = 0.95). As expected from the target inhibitory profile, CYC065 caused a rapid decrease in the phosphorylation of S2 of the CTD of RNA polymerase II followed by downregulation of Mcl-1 and MYC, and rapid induction of apoptosis in sensitive cell lines. CYC065 had no impact on Bcl-2 levels. Combining CYC065 with venetoclax was highly synergistic in DHL lines (median CI values range from 0.1-0.8), and resulted in >90% cell death. The combination of CYC065 and BET inhibitors was also highly synergistic. Conclusions: CYC065 targets key oncogenic and survival pathways in DLBCL. CYC065 is a potent and effective inducer of cell death and combines synergistically with Bcl-2 or BET inhibitors in B cell lymphoma cell lines, including DHLs, which represent an unmet clinical need. Citation Format: Sheelagh M. Frame, Elizabeth Pohler, Craig MacKay, Daniella Zheleva, David Blake. CYC065, a novel CDK2/9 inhibitor, is an effective inducer of cell death and synergizes with BCL2 and BET inhibitors in B-cell lymphoma, including double-hit lymphomas. [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 1309.

CYC065 is a novel CDK inhibitor, which inhibits CDK2 and 9 with IC50 values of 5 and 26 nM, respectively. Following completion of IND-enabling studies, CYC065 has been cleared by FDA for first-in-human Phase 1 clinical trials. Triple-negative breast cancers (TNBC), particularly the basal subtype, often exhibit aggressive characteristics. Despite good initial responses to chemotherapy, patients experience early relapse and diminished 5 year survival. Molecular features of basal-like TNBC include amplification or overexpression of cyclin E and MYC, suggesting potential utility for a CDK2/9 inhibitor such as CYC065. CYC065 is effective in cyclin E-overexpressing tumors, such as uterine serous carcinoma1 and trastuzumab-resistant Her2+ breast cancer2. Moreover, CDK inhibition has also been reported to be synthetic lethal with overexpressed MYC3. This led us to assess the potency and mechanism of action of CYC065 in basal-like TNBC models to evaluate the potential for CYC065 development in this indication. In vitro cell-based experiments support twice weekly pulse dosing using submicromolar concentrations of CYC065 to achieve maximum impact on cell growth in the majority of breast cancer cell lines tested. Preclinical toxicology data indicate that such levels and durations of exposure are achievable and well tolerated. As a single agent, CYC065 treatment in breast cancer cells resulted in inhibition of RNA-Pol II phosphorylation, down-regulation of Mcl-1, up-regulation of p53 and rapid induction of apoptosis. The impact of CYC065 on CDK2 targets, cyclin E and MYC was also explored. Interestingly immortalized cell lines obtained from non-malignant tissue displayed similar effects on RNA Pol II, Mcl-1, and p53 but did not undergo apoptosis and consequently exhibited relative resistance to CYC065, indicative of a potential therapeutic window. Cell cycle analysis demonstrated that CYC065 treatment induced an increase in G1 population with no significant induction of cell death in non-malignant derived cell lines, compared to cancer cell lines, in which there was significant induction of cell death. CDKs have a role in DNA repair which can be exploited to enhance the effectiveness of DNA damaging agents. Seliciclib, an oral, first generation CDK2/9 inhibitor, can be effectively combined with DNA damaging agents, such as the oral nucleoside analogue sapacitabine, or its active metabolite CNDAC. A Phase 1 clinical trial is currently underway to evaluate this combination (NCT00999401). Similarly to seliciclib, we demonstrate that CYC065 is synergistic in combination with CNDAC when given sequentially across multiple breast cancer cell lines. CNDAC-induced double strand breaks persisted for longer when cells were subsequently treated with CYC065, supporting the conclusion that these CDK inhibitors suppress DNA double-strand break repair capacity, which may contribute to the observed synergy. Taken together the data establish CYC065 as a promising anti-cancer agent in basal-like TNBC, with the potential to be combined effectively in this indication with DNA damaging agents. 1. Cocco, E. et al. AACR, 2015, Abstract 3103. 2. Scaltriti, M. et al. Proc Natl Acad Sci USA. 201, 108, 3761-6. 3. Horiuchi, D. et al. J Exp Med. 2012, 209, 679-96. Citation Format: Blake DG, MacKay C, Frame S, Zheleva D. CYC065, a novel CDK2/9 inhibitor: Molecular basis for clinical development in basal-like triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-10.

Purpose: The aims of this study were to investigate whether the cyclin-dependent kinase inhibitor seliciclib could synergize with agents that target ErbB receptors and to elucidate the molecular mechanism of the observed synergy. Experimental Design: Synergy between seliciclib and ErbB receptor targeted agents was investigated in various cell lines using the Calcusyn median effect model. The molecular mechanism of the observed synergy was studied in cultured cells, and the combination of seliciclib and the epidermal growth factor receptor (EGFR) inhibitor erlotinib was evaluated in an H358 xenograft model. Results: Seliciclib synergized with the anti-HER2 antibody trastuzumab in a breast cancer cell line, which overexpresses the HER2 receptor, and with the erlotinib analogue AG1478 in non–small cell lung cancer cell lines. In the H358 non–small cell lung cancer cell line, synergy involved decreased signaling from the EGFR, with AG1478 directly inhibiting kinase activity while seliciclib decreased the levels of key components of the receptor signaling pathway, resulting in enhanced loss of phosphorylated extracellular signal-regulated kinase and cyclin D1. The combination of seliciclib and erlotinib was evaluated further in an H358 xenograft and shown to be significantly more active than either agent alone. An enhanced loss of cyclin D1 was also seen in vivo. Conclusions: This is the first report that investigates combining seliciclib with an EGFR inhibitor. The combination decreased signaling from the EGFR in vitro and in vivo and was effective in cell lines containing either wild-type or mutant EGFR, suggesting that it may expand the range of tumors that respond to erlotinib, and therefore, such combinations are worth exploring in the clinic.

CYC065 is a novel CDK2/9 inhibitor. Following completion of IND-enabling studies CYC065 has been cleared by the FDA for first-in-human clinical trials. This study aims to establish the mechanistic rationale and dosing schedule for targeting tumors that are either dependent on sustained expression of certain CDK9 transcriptional targets including Mcl-1 and MYC, or on activation of CDK2, e.g. by overexpression of cyclin E. CYC065 is efficacious in ALL and AML preclinical models, including MLL rearranged (MLLr) leukemia1. Mcl-1 is critical for survival of AML2, and MLLr AML are dependent on CDK9-driven expression of MLL target genes3. CYC065 is effective in cyclin E-overexpressing tumor models, such as uterine serous carcinoma4 and trastuzumab-resistant Her2+ breast cancer5. Pharmacologic CDK inhibition or CDK knockdown is synthetically lethal with overexpressed MYC in triple negative breast cancer (TNBC)6. A common feature of the basal-like subtype of TNBC is amplification or overexpression of cyclin E and MYC, suggesting potential utility for a CDK2/9 inhibitor. The efficacy and mechanism of action of CYC065 was analyzed in AML and basal-like TNBC cell panels by measuring the effects on cell proliferation, levels of key CDK9-dependent proteins, cell cycle distribution and apoptosis induction. We show that submicromolar CYC065 inhibits CDK9-dependent RNA Polymerase II phosphorylation, resulting in downregulation of target proteins and rapid induction of apoptosis. Sensitive tumor subtypes undergo rapid apoptosis after short pulse treatments at doses and durations predicted to be clinically achievable and well tolerated. However, significant apoptosis induction was not observed in non-malignant cells despite showing similar upstream effects. The data predicts a therapeutic window between cancer and normal proliferating cells and indicates the potential for stratification markers to enrich for sensitive patients. Combinability of anti-cancer agents is an important consideration for clinical development. In preclinical models Cyclacel's first generation CDK inhibitor seliciclib combines effectively with the novel nucleoside analogue sapacitabine (or its active metabolite CNDAC). This combination is currently being evaluated in a phase 1 clinical trial (NCT00999401)7. We show that CYC065 combines effectively with CNDAC in a TNBC panel. Increased expression and activity of other anti-apoptotic Bcl-2-family members can counteract the pro-apoptotic effects of Mcl-1 downregulation by CYC065. This can be overcome by combined treatment with CYC065 and Bcl-2 inhibitors such as ABT-199, resulting in a highly synergistic combination. CYC065 has the potential to be efficacious in cancers that require sustained expression of CDK9-dependent transcripts or activation of CDK2. These include Mcl-1-dependent and MLLr leukemia, MYC-driven lymphoma, and MYC- or cyclin E-dependent breast or gynecological cancers. CYC065 can act synergistically with selected DNA damaging or targeted agents. Together this data suggests that CYC065 may be a promising novel anti-cancer agent. 1) Saladino et al. AACR 2015, Abs 1650 2) Glaser et al. Genes Dev. 2012, 26, 120-5 3) Dou & Hess. Int J Haematol. 2008, 87, 10-8 4) Cocco et al. AACR, 2015, Abs 3103 5) Scaltriti et al. Proc Natl Acad Sci USA. 201, 108, 3761-6 6) Horiuchi et al. J Exp Med. 2012, 209, 679-96 7) Shapiro et al. AACR, 2013, Abs LB-202 Citation Format: Craig MacKay, Sheelagh Frame, Chiara Saladino, Elizabeth Pohler, Daniella Zheleva, David G. Blake. Molecular basis for clinical development of the novel CDK2/9 inhibitor CYC065 in oncology. [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 B182.

Background: CYC065 is a novel, second generation aminopurine inhibitor highly selective for CDK2, CDK5 and CDK9 over other CDKs and non-CDK enzymes. CYC065 has completed IND-directed drug development and is ready for FIH studies. The unique target profile of CYC065 can be exploited in cyclin E-overexpressing tumors, including a trastuzumab-resistant Her2+ breast cancer model (Scaltriti et al, PNAS 2011; 9:3761). The aim of this study was to gather key dosing information for this compound in advance of FIH studies, and provide understanding of the compound mechanism of action in additional key target indications. CDK9 is a key regulator of mixed lineage leukemia (MLL)-driven leukemogenesis and Mcl-1-dependent survival and therefore represents a potential therapeutic target for AML and MLL leukaemia. Methods: A panel of acute leukemia cell lines with different MLL status (WT, MLL translocations and MLL PTD) was used in this study. Resazurin and flow cytometry-based annexin V assays were used to determine cell viability and apoptosis induction. PI staining was used to examine cell cycle distribution. Protein levels were quantified by Western blotting and quantitative imaging. Combinations were studied using the Chou and Talalay method. The in vivo activity of CYC065 was studied in murine xenograft models. Results: CYC065 rapidly decreased phosphorylation of Ser2 -RNAP II and expression of proteins involved in cell cycle control, survival and leukemogenesis (such as Mcl-1, Cyclin E, PNUTS, Hoxa9 and Meis 1), leading to robust and early induction of apoptosis (within 4 h). Time- and dose-dependent studies indicated that 6 h pulse treatment at 0.5-1 μM CYC065 was sufficient to cause ≥ 90% cell death in sensitive cell lines. Cell lines with MLL rearrangements were highly sensitive to CYC065, whilst sensitivity of AML cell lines with WT MLL correlated with the level of Bcl-2 family proteins. Cell lines with reduced sensitivity to CYC065 could be effectively targeted by exposure to longer, 10 h pulse treatments with CYC065, or in combination with short pulses of Bcl-2 inhibitors. In vivo, CYC065 achieved >90% TGI at well tolerated dose levels in HL60 (WT MLL) and EOL-1 (MLL PTD) xenograft models. Conclusion: CYC065 has therapeutic potential for AML and MLL leukemia. MLL status and the levels of Bcl-2 family proteins might be useful patient stratification markers for predicting sensitivity/resistance. Citation Format: Chiara Saladino, Sheelagh Frame, Susan Davis, David Blake, Daniella Zheleva. CYC065, a novel CDK2/5/9 inhibitor: detailed mechanistic studies, determinants of sensitivity and synergistic combinations. [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 1650. doi:10.1158/1538-7445.AM2015-1650

Seliciclib (CYC202, R-roscovitine) is a cyclin-dependent kinase (CDK) inhibitor that competes for the ATP binding site on the kinase. It has greatest activity against CDK2/cyclin E, CDK7/cyclin H, and CDK9/cyclin T. Seliciclib induces apoptosis from all phases of the cell cycle in tumor cell lines, reduces tumor growth in xenografts in nude mice and is currently in phase II clinical trials. This study investigated the mechanism of cell death in multiple myeloma cells treated with seliciclib. In myeloma cells treated in vitro, seliciclib induced rapid dephosphorylation of the carboxyl-terminal domain of the large subunit of RNA polymerase II. Phosphorylation at these sites is crucial for RNA polymerase II–dependent transcription. Inhibition of transcription would be predicted to exert its greatest effect on gene products where both mRNA and protein have short half-lives, resulting in rapid decline of the protein levels. One such gene product is the antiapoptotic factor Mcl-1, crucial for the survival of a range of cell types including multiple myeloma. As hypothesized, following the inhibition of RNA polymerase II phosphorylation, seliciclib caused rapid Mcl-1 down-regulation, which preceded the induction of apoptosis. The importance of Mcl-1 was confirmed by short interfering RNA, demonstrating that reducing Mcl-1 levels alone was sufficient to induce apoptosis. These results suggest that seliciclib causes myeloma cell death by disrupting the balance between cell survival and apoptosis through the inhibition of transcription and down-regulation of Mcl-1. This study provides the scientific rationale for the clinical development of seliciclib for the treatment of multiple myeloma.

Phosphorylation of the endogenous GSK3alpha (glycogen synthase kinase-3alpha) at Tyr279 and GSK3beta at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3beta transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3beta expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216 in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3beta was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3beta lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.

Publisher Summary The chapter provides a short summary of the structure, substrate specificity, functions, and regulation of glycogen synthase kinase 3 protein. Glycogen synthase kinase 3 (GSK3) is a protein that phosphorylates and inhibits glycogen synthase, the enzyme that catalyzes the transfer of glucose from UDPG to glycogen. GSK3 is a key player in two distinct signal transduction pathways: the phosphatidylinositol-3–kinase-dependent pathway that is triggered by insulin and growth factors, and the Wnt signaling pathway that is required for embryonic development. Insulin induces the activation of glycogen synthase, mainly by stimulating the dephosphorylation of the serine residues in glycogen synthase that are targeted by GSK3, and stimulates the activation of eIF2B by promoting the dephosphorylation of Ser535. GSK3 appears to play a role in the insulin-regulated transcription of the genes. Inhibitors of GSK3 may lower the levels of blood glucose in vivo by suppressing the production of glucose as well as by enhancing the conversion of glucose to glycogen. There is considerable evidence that the inhibition of GSK3 triggered by growth factors contributes to the anti-apoptotic effects of these signals. It is a therapeutic target for the design of inhibitory drugs to treat diseases such as head trauma, stroke, epilepsy, and motor neuron disease. GSK3 plays a key role in embryonic development as a central player in the Wnt signaling pathway. It mimics the Wnt signaling pathway and stimulates the accumulation of β-catenin.

Glycogen synthase kinase 3 (GSK3) was initially described as a key enzyme involved in glycogen metabolism, but is now known to regulate a diverse array of cell functions. The study of the substrate specificity and regulation of GSK3 activity has been important in the quest for therapeutic intervention.

The inhibition of GSK3 is required for the stimulation of glycogen and protein synthesis by insulin and the specification of cell fate during development. Here, we demonstrate that the insulin-induced inhibition of GSK3 and its unique substrate specificity are explained by the existence of a phosphate binding site in which Arg-96 is critical. Thus, mutation of Arg-96 abolishes the phosphorylation of "primed" glycogen synthase as well as inhibition by PKB-mediated phosphorylation of Ser-9. Hence, the phosphorylated N terminus acts as a pseudosubstrate, occupying the same phosphate binding site used by primed substrates. Significantly, this mutation does not affect phosphorylation of "nonprimed" substrates in the Wnt-signaling pathway (Axin and β-catenin), suggesting new approaches to design more selective GSK3 inhibitors for the treatment of diabetes.

Background: Sapacitabine is an oral nucleoside analogue that is metabolised in-vivo to CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine). It has a novel mechanism of action by causing single-strand breaks and replication-associated double-strand breaks (DSBs) after its incorporation into DNA. DSB, if unrepaired, are cytotoxic and are predominantly repaired by homologous recombination (HR). Over 50% of high-grade serous ovarian cancers are estimated to be defective in HR, only a small proportion of which are accounted for by germline BRCA mutations. HR-defective ovarian cancers have been shown to be sensitive to PARP inhibitors ex-vivo and platinum in-vivo and we therefore hypothesised that HR defective ovarian cancers would be sensitive to CNDAC and aimed to explore the potential use of CNDAC in ovarian cancer stratified for HR function. Methods: Primary cultures (PCO) were generated from ascitic fluid collected from patients undergoing ovarian cancer surgery, characterised and confirmed to be ovarian and epithelial in origin based upon morphology, expression of immunofluorescent-labelled markers and cross-referencing with formal examination of FFPE tissue and cytology of ascites. The functional status of HR was determined for each cell line and PCO culture by quantification of nuclear Rad51 focus formation following induction of DNA DSB. SRB assays were used to calculate the GI50 for cisplatin and CNDAC, (Cyclacel, UK), in cell lines nad PCO cultures. Results: As hypothesised, HR defective cell lines were highly sensitive to CNDAC (VC8 BRCA2 mutant: GI50=366 nM) in comparison to their matched HR competent cell lines (VC8-BRCA2 reconstructed: GI50=18 μM; VC8-PARPi-resistant BRCA revertant: GI50=3 μM). Sensitivity to CNDAC in patient PCO cultures (n=39) was heterogeneous with a mean GI50 of 297 nM (95% CI 136-646 nM). Stratifying PCO cultures according to their HR status demonstrated greater sensitivity in HR defective cultures with a mean GI50 of 135 nM (46-394; n=12) in defective cultures, compared to 477 nM in HR competent cultures (164-1386; n=27). Of the 34 cisplatin resistant PCO cultures (GI50 >5 μM), 20 (59%) remained sensitive to CNDAC with a mean GI50 of 426 nM (148-1223; n=13) in HR competent cultures and a GI50 of 230 nM (70-759; n=7) in HR defective cultures. CNDAC sensitivity was evaluated in base excision repair (BER) defective EM9 cell line (GI50=374 nM) compared with parental wild type AA8 cells (GI50=6.9 μM), p Conclusions: Cell lines defective in HR or BER are highly sensitive to CNDAC suggesting a possible use of HR or BER status as biomarkers for the stratified use of CNDAC in the treatment of ovarian cancer. When applied to ex-vivo studies HR status as a single marker cannot reliably predict sensitivity and a panel of pathways may therefore be needed to enable reliable treatment stratification. Nevertheless, stratification based on these markers is likely to permit enrichment of patients more likely to respond to CNDAC. Additionally CNDAC may have a role in the treatment of platinum resistant disease as approximately 60% of patients with resistant disease may benefit. Further work is required to explore the possibility of CNDAC as a chemosensitiser in platinum sensitive disease and as a second line agent in platinum resistant disease. Citation Format: Rachel L. O'Donnell, James CC Murray, Adriana GB Buskin, Sheelagh Frame, David G. Blake, Michelle Dixon, Aiste McCormick, Angelika Kaufmann, E Ruth Plummer, Richard J. Edmondson, Nicola J. Curtin. Therapeutic potential of sapacitabine in ovarian cancers defective in homologous recombination. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A33.

Background: Sapacitabine is an orally administered nucleoside analogue; the active metabolite, CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine), generates single-strand DNA breaks that are converted to double-strand DNA breaks (DSBs) during subsequent replication, resulting in cell death if unrepaired. Repair of CNDAC-induced DSBs is dependent on the homologous recombination (HR) repair pathway. Depletion or inhibition of components of the HR pathway (including ATM, BRCA1/2, Rad 51, and XRCC3) greatly sensitizes tumor cell lines to CNDAC-induced cell death in vitro. Seliciclib is an orally bioavailable inhibitor of cyclin-dependent kinases (CDKs) 2, 7 and 9. CDK2 has been shown to participate in DNA repair and to be a therapeutic target in BRCA-deficient cancers. Seliciclib inhibits DSB repair, and also reduces BRCA1 and BRCA2 mRNA levels in cancer cell lines, sensitizing tumor cells to CNDAC. This phase I study evaluates sequential sapacitabine and seliciclib. Methods: Dose escalation was conducted in patients with incurable solid tumors and adequate organ function with sapacitabine bid x 7 consecutive days (d1-7), seliciclib bid x 3 consecutive days (d8-10) followed by 11 days of rest. At least 3 patients were evaluated per dose level. MTD was the highest dose level at which less than one-third of at least 6 patients experienced cycle 1 DLT. Skin biopsies were obtained to assess DNA damage following sapacitabine (d8 vs pre-treatment) and further augmentation of DNA damage after seliciclib (d11 vs d8). Results: 38 patients were treated. The MTD is sapacitabine 50 mg bid/seliciclib 1200 mg bid. DLTs were reversible transaminase elevations and neutropenia. The most frequent adverse events (all cycles, regardless of causality) included, fatigue, abdominal pain, diarrhea, constipation, decreased appetite, nausea, vomiting, anemia, neutropenia, pyrexia, AST elevation, alkaline phosphatase elevation, creatinine elevation, hyperglycemia, hypophosphatemia, cough, and alopecia, the majority mild to moderate in intensity. Skin biopsies showed a 2.3-fold increase in γ-H2AX staining post-sapacitabine (n=16; p=0.007) and a further 0.58-fold increase post-seliciclib (n=12; p=0.069). Four confirmed PRs occurred in patients with pancreatic, breast (2 pts) and ovarian cancer, all BRCA mutation carriers, lasting 21, 78+, 36+ and 42+ weeks, respectively. SD as best response ≥= 12 weeks was observed in 8 additional patients, including two BRCA mutation carriers with ovarian and breast cancer, lasting 64 and 21 weeks, respectively. Conclusions: Sequential sapacitabine and seliciclib is safe with preliminary antitumor activity. BRCA mutation carrier status may be a potential biomarker for response across multiple tumor types. An alternative schedule with concomitant administration of sapacitabine and seliciclib is currently under evaluation. Citation Format: Geoffrey I. Shapiro, John Hilton, James M. Cleary, Sara M. Tolaney, Leena Ghandi, Eunice L. Kwak, Jeffrey W. Clark, Andrew Wolanski, Tracy Bell, John Schulz, Sheelagh Frame, Chiara Saladino, Morag Hogben, Scott J. Rodig, Judy H. Chiao, David Blake. Responses to sequential sapacitabine and seliciclib in patients with BRCA-deficient solid tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-202. doi:10.1158/1538-7445.AM2013-LB-202

3053 Background: Sapacitabine is an orally administered nucleoside analogue; the active metabolite CNDAC generates ss DNA breaks that are converted to ds DNA breaks (DSB) during subsequent replication, resulting in cell death if unrepaired. CNDAC-induced DSB repair is dependent on homologous recombination (HR). Seliciclib is a potent orally bioavailable inhibitor of CDK2, 7 and 9, and sensitizes cells to CNDAC by decreasing DSB repair via compromise of HR protein activation and transcriptional inhibition of HR components. This phase I study evaluates sequential sapacitabine and seliciclib. Methods: Dose escalation was conducted in patients with incurable solid tumors and adequate organ function with sapacitabine b.i.d. x 7 consecutive days (d1-7), seliciclib b.i.d. x 3 consecutive days (d8-10) followed by 11 days of rest. At least 3 patients were evaluated per dose level. MTD was the highest dose level at which less than one-third of at least 6 patients experienced cycle 1 DLT. Skin biopsies were obtained to assess DNA damage following sapacitabine (d8 vs pre-treatment) and further augmentation of DNA damage after seliciclib (d11 vs d8). Results: 27 patients were treated. The MTD and RP2D is sapacitabine 50 mg b.i.d./seliciclib 1200 mg b.i.d. DLTs were reversible transaminase elevations and neutropenia. The most frequent adverse events (all cycles, regardless of causality) included anorexia, fatigue, abdominal pain, dizziness, nausea, anemia, neutropenia, creatinine elevation, hyperglycemia, hyperbilirubinemia, hypophosphatemia, hypokalemia and hypomagnesemia, the majority mild to moderate in intensity. Skin biopsies showed a 2.3-fold increase in H2AX staining post-sapacitabine (n=16; p=0.007) and a further 0.58-fold increase post-seliciclib (n=12; p=0.069). Two confirmed PRs occurred in patients with pancreatic and breast cancer, both BRCA mutation carriers. SD as best response >/= 12 weeks was observed in 6 additional patients, including one BRCA mutation carrier with ovarian cancer (ongoing at 24 weeks). Conclusions: Sequential sapacitabine and seliciclib is safe with preliminary antitumor activity. BRCA mutation carrier status may be a potential biomarker for response across multiple tumor types.

Polo-like kinase 1 (Plk1) is one of the emerging new generation of anti-mitotic targets. Plks are a family of serine-threonine kinases involved in the regulation of mitosis and maintenance of DNA integrity. Plk1 is required for almost every step of mitosis: promotes mitotic entry, contributes to centrosome maturation, regulates kinetochore assembly, contributes to spindle assemble checkpoint and initiates mitotic exit. Besides promoting proliferation, Plk1 overexpression contributes to oncogenesis by promoting chromosome instability and aneuploidy. Overexpression of Plk1 is associated with tumor development and many cancers have elevated Plk1 levels compared to surrounding normal tissues. Numerous studies have shown that Plk1 expression levels correlate with disease progression, invasiveness and poor patient prognosis. Furthermore, pre-clinical studies have demonstrated that cancer cell proliferation is blocked in vitro and in vivo via inhibition of Plk1 using siRNA or small molecule inhibitors. Collectively, these observations support the selection of Plk1 as an attractive target for cell cycle-directed cancer therapy and several small molecule inhibitors are currently being investigated in clinical trials. Using a de novo ligand design approach we have identified pyrimidodiazepinone small molecules, which are ATP-competitive, highly selective and potent Plk1 inhibitors. These molecules have good drug-like properties, oral bioavailability and efficacy in mouse xenograft models. To characterize the cell sensitivity toward the Plk1 inhibitors we have used washout and outgrowth assays in a panel of oesophageal, lung and colon cancer cell lines. The anti-proliferative activity of Plk1 inhibitors was correlated with the Plk1 expression level, p53 and Ras mutational status. Generally, we found that cell lines harbouring p53 mutations are more sensitive towards these inhibitors. Time-lapse microscopy and flow cytometry studies of RKO cell line (WT p53) treated with Plk1 inhibitors demonstrated that a proportion of cells arrested just before the G2-/-M transition and were able to resume normal proliferation upon compound removal. These results suggest that Plk1 inhibitors should have better therapeutic window when used for treatment of tumors with mutant p53, p53 mutational status could be used as a patient stratification marker, and tumor types with high frequency of p53 mutations, such as oesophageal cancer, could be suitable therapeutic indications for Plk1 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2814. doi:1538-7445.AM2012-2814

Sapacitabine, a novel, orally available, pro-drug of CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine), is currently being evaluated in SEAMLESS, a pivotal Phase 3 trial in elderly patients with AML, and Phase 2 trials in AML, MDS, NSCLC, and CLL/SLL. CNDAC acts by a unique DNA-damaging mechanism. After CNDAC is incorporated into DNA, single-strand DNA breaks are generated via a β-elimination reaction. If unrepaired, these are subsequently converted to double-strand breaks during further replication. These DNA breaks can also activate the G2 checkpoint, leading to cell cycle arrest in G2. Parallel approaches have been utilized to identify further rational therapeutic development strategies for sapacitabine, particularly focusing on solid tumors. These approaches include synthetic lethality screening using focused siRNA libraries in CNDAC-treated cells, and screening for agents that could synergistically induce cell death when administered in combination with CNDAC. These have demonstrated that depletion or inhibition of components of the homologous recombination DNA repair (HRR) pathway (including ATM, BRCA1/2, Rad 51 and XRCC3) or disruption of the G2 checkpoint greatly sensitizes tumor cell lines to CNDAC-induced cell death. For example, cell lines in which BRCA1 or BRCA2 has been depleted through siRNA are significantly more sensitive to the compound than control cells. Moreover, inhibitors of the ATM or Chk1 kinases also sensitize cells to CNDAC, the latter through abrogation of the G2 checkpoint. These complementary approaches have underscored the potential clinical utility of sapacitabine in patients carrying homologous recombination DNA repair deficient (HRD) tumors, and have also highlighted promising combination strategies. Drug combination screening has also assessed the potential for combining sapacitabine with other agents active in HRD tumors, including platins and PARP inhibitors. Ongoing work is extending these findings to several solid tumor indications, including breast, ovarian and non-small cell lung cancers. Correlation of HRR status and CNDAC sensitivity will be reported for a panel encompassing cell lines derived from each of these target indications, in order to guide future clinical development of sapacitabine in solid tumors, and to aid development of biomarkers to identify suitable patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5666. doi:1538-7445.AM2012-5666

Chromosomal rearrangements involving the human mixed - lineage leukaemia (MLL) gene at 11q23, are associated with the development of acute leukemias. Abnormalities in the MLL gene can be detected in de novo acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) as well as in therapy-related AML, particularly after treatment with DNA topoisomerase II inhibitors. Some malignancies, such as infant ALL, are characterised by exceedingly high incidence (∼80%) of MLL gene rearrangements, which is a strong predictor of an adverse outcome. Current therapies fail in >50% of the infant MLL cases, indicating that innovative therapeutic strategies are urgently needed to improve prognosis. Re-arranged MLL interacts with the transcription complex including CDK9. The association of CDK9 results in an increase in RNApol II phosphorylation and hence overexpression of HoxA9 and Meis1, which are normally down regulated during haematopoietic differentiation, resulting in immortalisation and transformation. These suggest a key role of CDK9 activity in this transformation process and the potential of CDK9 inhibitors to stop the uncontrolled myeloblast proliferation. Additionally, the expression of MCL-1, an anti-apoptotic member of BCL-2 family, was shown to increase in infant MLL samples, which correlated with prednisone resistance and poor prognosis. Importantly, MCL-1 also plays an important role in the development of B and T lymphocytes as well as in the survival of haematopoietic stem cell. Therefore, targeting MCL-1 in infant MLL cells may also induce leukemic cell death of the CD34+CD19- chemoresistant leukemic stem cell. The ability of CDK inhibitors, such as seliciclib, flavopiridol, SNS-032, to down regulate MCL-1 and induce apoptosis in B-CLL and MM cells is well recorded. We studied the effect of a purine CDK inhibitor, Cmpd 5, on MLL cell lines in vitro and in vivo. Cmpd 5 is a structural analogue of seliciclib and a potent and selective inhibitor of CDKs 2, 5, and 9. AML cell lines with MLL re-arrangements were exquisitely sensitive to Cmpd 5. Treatment for 5 hours at concentrations Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3886.

CNDAC is the active metabolite of sapacitabine, which is currently being evaluated in Phase II clinical trials for acute myeloid leukaemia, myelodysplastic syndrome and non small cell lung cancer. CNDAC (2′-C-Cyano-2′-deoxy-β-D-arabino-pentafuranosylcytosine) was designed as a novel cytosine analogue with a unique mechanism of action. The presence of the cyano-group within the ribose moiety of the molecule causes the formation of single-stranded DNA strand breaks, following incorporation of CNDAC into an extending DNA chain. These breaks are difficult to repair and are processed into double-strand DNA breaks that activate the dsDNA damage checkpoint. As a consequence of this unique mechanism of action, CNDAC arrests cells in the G2/M phase of the cell cycle in contrast to other nucleoside agents such as cytarabine and gemcitabine which cause an S-phase arrest. As such CNDAC may have unique therapeutic applications as a single agent in certain tumour types as well as in combination with other agents compared with standard nucleoside analogues. In order to identify options for maximising that activity of CNDAC, two approaches were taken to evaluate the repair mechanisms involved for CNDAC induced DNA damage. First a small scale siRNA screen was used to identify genes that were synthetically lethal with CNDAC. Ten targets were initially selected; prioritising genes known to be involved in DNA repair and including BRCA, ATM, CHK and ERCC1. The most dramatic increase in CNDAC sensitivity was seen when BRCA2 was targeted by siRNA, indicating that the homologous recombination DNA repair pathway is involved in the repair of CNDAC induced DNA damage. The second approach involved a cytotoxicity screen evaluating synergy with commercially available agents that either target DNA repair or induce DNA damage themselves. The most promising combinations were then followed up with flow cytometry analysis to examine the induction of cell death. Using this approach the best synergy was detected between CNDAC and either the ATM inhibitor, KU55933 or the CHK inhibitor PF477736. Again these data indicate that the homologous recombination pathway is involved in the repair of CNDAC induced DNA damage. These data suggest that it would be feasible to explore the clinical use of sapacitabine in diseases where the homologous recombination DNA repair pathway is compromised, such as BRCA deficient tumours. Data from these ongoing studies will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3502.

Rho kinase is known to control smooth muscle contractility by phosphorylating the 110 kDa myosin-targetting subunit (MYPT1) of the myosin-associated form of protein phosphatase 1 (PP1M). Phosphorylation of MYPT1 at Thr695 has previously been reported to inhibit the catalytic activity of PP1. Here, we show that the phosphorylation of Thr850 by Rho kinase dissociates PP1M from myosin, providing a second mechanism by which myosin phosphatase activity is inhibited.