Cdc7 Inhibitors Research Articles

Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation

BackgroundMutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson’s disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.Methodology/Principal FindingsUsing Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.Conclusions/SignificanceWe developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.

Arylstibonic acids are potent and isoform-selective inhibitors of Cdc25a and Cdc25b phosphatases

Arylstibonates structurally resemble phosphotyrosine side chains in proteins and here we addressed the ability of such compounds to act as inhibitors of a panel of mammalian tyrosine and dual-specificity phosphatases. Two arylstibonates both possessing a carboxylate side chain were identified as potent inhibitors of the protein tyrosine phosphatase PTP-ß. In addition, they inhibited the dual-specificity, cell cycle regulatory phosphatases Cdc25a and Cdc25b with sub-micromolar potency. However, the Cdc25c phosphatase was not affected demonstrating that arylstibonates may be viable leads from which to develop isoform specific Cdc25 inhibitors.

Discovery of XL413, a potent and selective CDC7 inhibitor

CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines, with inhibition of CDC7 resulting in cell cycle arrest. In this paper, we disclose the discovery of a potent and selective CDC7 inhibitor, XL413 (14), which was advanced into Phase 1 clinical trials. Starting from advanced lead 3, described in a preceding communication, we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model.

Preclinical Assessment of a Novel CDC7 Inhibitor: Genomewide RNAi Screening Identifies Unique Synergetic and Resistance Genes,

Abstract 3570Despite extensive drug discovery efforts, drug-candidate failure and patients relapsing in the clinic remain as persistent problems. While insufficient drug-gene engagement leads to drug failure, de novo escape mutations give rise to patients relapsing, calling the need for systemic studies on how genes influence drug responsiveness. Towards this end, we have explored a functional short hairpin RNA (shRNA) based genomic screening platform aimed at interrogating drug-gene engagement and assessing its consequences on signaling pathways. We propose this concept as a novel way to evaluate drug candidates prior to clinical trials enabling liability assessment and predicting clinical outcome. We took advantage of the arrayed shRNA library produced in lentiviral particles and characterized by several obvious advantageous features including shRNA targeting one hairpin at a time and on the fly high content whole well microscopy imaging analysis. We carried out three parallel genomewide shRNA screens in the absence or presence of the novel CDC7 kinase inhibitor (MSK-777) at its IC20 and IC50 and have identified several gene candidates that influence MSK-777 sensitivity and resistance. These include synergizers that enhance MSK-777 sensitivity and rescuers that confer MSK-777 resistance. IPA analysis mapped clusters of these hits to multiple major pathways among them were the NF-kB pathway, the ubiquitin-proteasome pathway, DNA replication, and several epigenetic regulatory genes. We will present and discuss this concept together with the emerging pathways as a means to identify both key therapeutic targets and biomarkers of sensitivity and resistance. Thus, allowing for not only a broader applicability of assessing candidate genes that modulate specific drug agents, but also for the identification of a tailored and more efficacious therapeutic regimen to treat cancer. Disclosures:No relevant conflicts of interest to declare.

Mechanisms of Action of a Dual Cdc7/Cdk9 Kinase Inhibitor against Quiescent and Proliferating CLL Cells

In chronic lymphocytic leukemia (CLL) the proliferation rate and resistance to drug-induced apoptosis are recognized as important factors in the outcome of treatment. In this study, we assess the activity and the mechanism of action of the prototype cell division cycle kinase 7 (Cdc7) inhibitor, PHA-767491, which inhibits the initiation of DNA replication but also has cyclin-dependent kinase 9 (Cdk9) inhibitory activity. We have studied the effects of this dual Cdc7/Cdk9 inhibitor in both quiescent CLL cells and CLL cells that have been induced to proliferate using a cellular coculture system that mimics the lymph node microenvironment. We find that this compound, originally developed as a DNA replication inhibitor, is particularly active in promoting mitochondrial dependent apoptosis in quiescent CLL cells purified from peripheral blood of patients regardless of recognized risk factors. In this setting, apoptosis is preceded by a decrease in the levels of Mcl-1 protein and transcript possibly due to inhibition of Cdk9. Following stimulation by CD154 and interleukin-4, CLL cells become highly chemoresistant, reenter into the cell cycle, reexpress Cdc7 kinase, a key molecular switch for the initiation of DNA replication, replicate their DNA, and undergo cell division. In this context, treatment with PHA-767491 abolished DNA synthesis by inhibiting Cdc7 but is less effective in triggering cell death, although Mcl-1 protein is no longer detectable. Thus, dual Cdc7/Cdk9 inhibition has the potential to target both the quiescent and actively proliferating CLL populations through two distinct mechanisms and may be a new therapeutic strategy in CLL.

Abstract LB-273: Preclinical assesment of a novel CDC7 inhibitor: Genomewide RNAi screening identifies unique synergetic and resistance genes

Abstract Despite extensive drug discovery efforts, drug-candidate failure and patients relapsing in the clinic remain as persistent problems. While insufficient drug-gene engagement leads to drug failure, de novo escape mutations give rise to patients relapsing, calling the need for systemic studies on how genes influence drug responsiveness. Towards this end, we have explored a functional short hairpin RNA (shRNA) based genomic screening platform aimed at interrogating drug-gene engagement and assessing its consequences on signaling pathways. We propose this concept as a novel way to evaluate drug candidates prior to clinical trials enabling liability assessment and predicting clinical outcome. We took advantage of the arrayed shRNA library produced in lentiviral particles and characterized by several obvious advantageous features including shRNA targeting one hairpin at a time and on the fly high content whole well microscopy imaging analysis. We carried out three parallel genomewide shRNA screens in the absence or presence of the novel CDC7 kinase inhibitor (MSK-777) at its IC20 and IC50 and have identified several gene candidates that influence MSK-777 sensitivity and resistance. These include synergizers that enhance MSK-777 sensitivity and rescuers that confer MSK-777 resistance. IPA analysis mapped clusters of these hits to multiple major pathways among them were the NF-kB pathway, the ubiquitin-proteasome pathway, DNA replication, and several epigenetic regulatory genes. We will present and discuss this concept together with the emerging pathways as a means to identify both key therapeutic targets and biomarkers of sensitivity and resistance. Thus, allowing for not only a broader applicability of assessing candidate genes that modulate specific drug agents, but also for the identification of a tailored and more efficacious therapeutic regimen to treat cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-273. doi:10.1158/1538-7445.AM2011-LB-273

Abstract 4125: Identification of candidate predictive biomarkers for the cdc7 kinase inhibitor BMS-863233/XL413

Abstract Cdc7 is a serine-threonine kinase that plays a critical role in the licensing of DNA replication origins and has been shown to play a role in DNA damage-induced checkpoint responses. Inhibition of cdc7 activity can block DNA replication, arrest cell cycle progression and can also result in apoptosis. Because of its proximal role in DNA replication, cdc7 kinase represents a novel therapeutic target for cancer treatment that has the potential to provide better control and fewer side effects than traditional chemotherapeutic agents that function distally during chain elongation. To complement the clinical development of BMS-863233/XL413, an ATP-competitive reversible cdc7 kinase inhibitor, we used cell line models to identify gene expression markers that are potentially predictive of a clinical response to BMS-863233/XL413. The in vitro sensitivity for BMS-863233/XL413 was established in a panel of 64 cell lines of mixed histotypes. Neither mutational status (as defined by the Sanger Institute COSMIC database), doubling time nor cdc7, ask/dbf4 or mcm2 basal mRNA expression were significantly associated with intrinsic sensitivity. Using gene expression profiling, we identified an intrinsic sensitivity gene signature consisting of 63 candidate genes whose basal expression was significantly associated with intrinsic sensitivity to BMS-863233/XL413. Evaluation of these markers using the Oncomine™ platform demonstrated enrichment in the ER/PR/HER2-negative breast cancer subtype. Dose titration studies revealed that 49 of these candidate genes were responsive to BMS-863233/XL413 treatment; 17 had IC50 values < 50 uM. A cell line model of acquired resistance to BMS-863233/XL413 was developed and was found to display an overlapping gene expression signature similar to the intrinsic sensitivity signature. Pathway analysis of differentially expressed genes in the acquired resistance model showed enrichment for genes involved in cell cycle and checkpoint functions. Among nine genes associated with intrinsic sensitivity, responsive to BMS-863233/XL413 treatment and that were differentially expressed in the acquired resistance model, seven (RBMS1, CD55, UGCG, NOL3, DUSP5, FOSL2 and PLK2) are biologically plausible, playing roles in cell cycle regulation, cellular proliferation and/or apoptosis. In conclusion, we identified candidate gene expression markers which are potentially predictive of a clinical response to the cdc7 inhibitor BMS-863233/XL413. These markers appear to be enriched in triple negative breast cancer–a patient population with poor prognosis and few effective treatment options–suggesting the potential for clinical efficacy following cdc7 inhibition in this population. Some of these markers are likely to play a role in the biology of intrinsic and acquired resistance to cdc7 inhibition and may consequently serve as potential surrogate pharmacodynamic endpoints. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4125. doi:10.1158/1538-7445.AM2011-4125

136 Activity of the Cdc7 inhibitor NMS-1116354 as single agent and in combination in breast cancer models

In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC50 = 0.70 nM) with high selectivity (Cdk2/Cdc7 ≥ 14,000, ROCK1/Cdc7 = 200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2.

385 A first in human phase 1 study of the safety and pharmacokinetics of a novel Cdc7 inhibitor NMS-1116354, administered orally to patients with solid tumors

Tripartite Motif Containing 11 (TRIM11), a member of TRIM proteins is overexpressed in gliomas and lung cancer. However, the role of TRIM11 in hepatocellular carcinoma (HCC) is unknown.Herein, we aimed to investigate the expression and clinical significance role of TRIM11 in HCC.In this study, our data showed significant higher TRIM11 in HCC tissues (n = 117) than in the matched non-tumor liver (NTL) tissues (P < 0.01). In consistent with above data, we also found TRIM11 protein expression was significantly increased compared with the matched NTL (P < 0.01) by immunohistochemistry analysis. Additionally, our results showed that TRIM11 protein expression in HCC tissues was significantly associated with pathological grade (P < 0.01), tumor postoperative metastasis (P = 0.031), recurrence (P = 0.022), and serum a-fetoprotein (AFP) (P < 0.01). Moreover, patients’ survival was negatively correlated with TRIM11 protein expression. Furthermore, we found that TRIM11 protein was an independent prognostic factor for disease-free (P < 0.01) and overall survival (P < 0.01) in HCC patients.Our data showed that TRIM11 expression was significantly elevated in HCC tissues. The overexpression of TRIM11 is closely associated with HCC progression and poor survival of the patients, indicating TRIM11 is a potential therapeutic target for HCC patients.

Targeting Cell Division Cycle 7 Kinase: A New Approach for Cancer Therapy

The cell division cycle 7 (Cdc7) is a serine-threonine kinase, originally discovered in budding yeast, required to initiate DNA replication. Human Cdc7 phosphorylates the minichromosome maintenance protein 2 (Mcm2), a component of the DNA replicative helicase needed for genome duplication. Inhibition of Cdc7 in cancer cells impairs progression through S phase, inducing a p53-independent apoptotic cell death, whereas in normal cells, it does not affect cell viability. Small molecule compounds able to interfere with Cdc7 activity have been identified and shown to be effective in controlling tumor growth in animal models. Two Cdc7 inhibitors are currently in phase I clinical development. Inhibition of Cdc7 kinase activity in cancer cells restricts DNA replication and induces apoptotic cell death by an unprecedented molecular mechanism of action.

Quantitative proteomics reveals a "poised quiescence" cellular state after triggering the DNA replication origin activation checkpoint.

An origin activation checkpoint has recently been discovered in the G1 phase of the mitotic cell cycle, which can be triggered by loss of DNA replication initiation factors such as the Cdc7 kinase. Insufficient levels of Cdc7 activate cell cycle arrest in normal cells, whereas cancer cells appear to lack this checkpoint response, do not arrest, and proceed with an abortive S phase, leading to cell death. The differential response between normal and tumor cells at this checkpoint has led to widespread interest in the development of pharmacological Cdc7 inhibitors as novel anticancer agents. We have used RNAi against Cdc7 in combination with SILAC-based high resolution MS proteomics to investigate the cellular mechanisms underlying the maintenance of the origin activation checkpoint in normal human diploid fibroblasts. Bioinformatics analysis identified clear changes in wide-ranging biological processes including altered cellular energetic flux, moderate stress response, reduced proliferative capacity, and a spatially distributed response across the mitochondria, lysosomes, and the cell surface. These results provide a quantitative overview of the processes involved in maintenance of the arrested state, show that this phenotype involves active rather than passive cellular adaptation, and highlight a diverse set of proteins responsible for cell cycle arrest and ultimately for promotion of cellular survival. We propose that the Cdc7-depleted proteome maintains cellular arrest by initiating a dynamic quiescence-like response and that the complexities of this phenotype will have important implications for the continued development of promising Cdc7-targeted cancer therapies.

Molecular architecture of the DNA replication origin activation checkpoint

Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-∣Hdm2-∣p53 → p21 pathway and mediates p15(INK4B) upregulation; p53 in turn activates expression of the Wnt/β-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors.

Targeting DNA replication before it starts: Cdc7 as a therapeutic target in p53 mutant Her2 and triple negative breast cancer

Based on protein expression profiles of core regulatory proteins involved in the G1-S and G2-M phase transitions, we have identified three distinct cell cycle phenotypes in a series of 200 breast cancers: a G0 out-of-cycle state (18% of cases); a G1 arrested/delayed state (24% cases); and accelerated S-G2-M phase progression (58% of cases). The accelerated cell cycle progression phenotype had a higher risk of relapse when compared with G0 and G1-delayed/arrested phenotypes (HR = 3.90 (95% CI = 1.81 to 8.4), P < 0.001) and was associated with Her2 and triple negative subtypes (P < 0.001). High-grade tumours with the G1-delayed/arrested phenotype showed an identical low risk of relapse compared with well-differentiated G0 tumours. In addition to its prognostic significance, the cell cycle phenotype also impacts on individualised therapeutic decisions. Only patients showing the actively cycling, aggressive cell cycle phenotype are likely to benefit from conventional chemotherapeutic S-phase-directed or M-phase-directed agents or from the new generation of targeted cell cycle inhibitors that are now entering clinical trials. The DNA replication initiation factor Cdc7 is an emerging anticancer target. Cdc7 inhibition results in an abortive S phase and potent cancer cell killing. Specificity is based on normal cells undergoing a reversible G1 arrest following Cdc7 inhibition due to activation of a novel cell cycle checkpoint that is lost or impaired in cancer cells. Our analysis of the molecular circuitry underlying this replication origin activation checkpoint reveals that G1 arrest is dependent on three nonredundant checkpoint axes coordinated through the Forkhead transcription factor FoxO3a and p53. We show that only breast cancers displaying the accelerated cell cycle phenotype express elevated Cdc7 levels and are therefore highly represented in p53 mutant Her2-subtype and triple negative tumours. Breast cancers of the luminal subtype expressing low levels of Cdc7 undergo a cytostatic G1 arrest after Cdc7 inhibition due to their p53 wild-type status, a checkpoint response mimicking untransformed cells. In contrast, Her2 and triple negative tumours show a marked response to Cdc7 inhibitors with potent cancer-cell-specific killing as a result of overexpression of the target protein and a result of impairment of the origin activation checkpoint due to p53 lesions. We have thus defined a new therapy and a means of assessing response.

Abstract DD01-01: NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase

Abstract DD01-01: NMS-1116354: More than an inhibitor of Cdc 7 kinase in S-phase

Inhibitors of Cdc25 phosphatases as anticancer agents: a patent review

Importance of the field: The cell division cycle 25 (Cdc25) family of proteins are highly conserved dual specificity phosphatases that regulate cyclin-dependent kinases, the main gatekeepers of the eukaryotic cell division cycle. The three isoforms of Cdc25, including Cdc25A, Cdc25B and Cdc25C, appear to act on different cyclin-dependent kinase/cyclin complexes at different stages of the cell cycle. Overexpression of Cdc25A and/or Cdc25B, but not Cdc25C, has been detected in numerous cancers and is often correlated with a poor clinical prognosis. Thus, inhibition of these phosphatases may represent a promising therapeutic approach in oncology.Areas covered in this review: The main focus of the present review is to describe the development of Cdc25 inhibitors over the years. We describe different compounds according to the decade of discovery and focus attention on molecules that were published in patents.What the reader will gain: Insight into the most clinically relevant therapeutic Cdc25 analogues that have been published in over 40 patents over the past 19 years.Take home message: Some Cdc25 inhibitors have suppressed in vivo the growth of human tumor xenografts in animals; this confirmed the validity of using Cdc25 phosphatase inhibition as an anticancer strategy, but side effects and toxicity remain to be investigated.

Targeting DNA Replication before It Starts: Cdc7 as a Therapeutic Target in p53 Mutant Her2 and Triple Negative Breast Ca.

Abstract Based on protein expression profiles of core regulatory proteins involved in the G1–S and G2–M phase transitions, we have identified three distinct cell cycle phenotypes in a series of 200 breast cancers: (I) a G0 “out-of-cycle” state (18% of cases); (II) a G1 arrested/delayed state (24% cases); and (III) accelerated S-G2-M phase progression (58% of cases). The accelerated cell cycle progression phenotype had a higher risk of relapse when compared with G0 and G1-delayed/arrested phenotypes (HR=3.90 (1.81-8.4, p&amp;lt;0.001) and was associated with Her2 and triple negative subtypes (p&amp;lt;0.001). High-grade tumours with the G1 delayed/arrested phenotype showed an identical low risk of relapse compared with well-differentiated G0 tumours. In addition to its prognostic significance, the cell cycle phenotype also impacts on individualised therapeutic decisions. It is only patients showing the actively cycling, aggressive cell cycle phenotype that are likely to benefit from conventional chemotherapeutic S- or M-phase-directed agents or from the new generation of targeted cell cycle inhibitors that are now entering clinical trials.The DNA replication initiation factor Cdc7 is an emerging anti-cancer target. Cdc7 inhibition results in an abortive S phase and potent cancer cell killing. Specificity is based on normal cells undergoing a reversible G1 arrest following Cdc7 inhibition due to activation of a novel cell cycle checkpoint that is lost or impaired in cancer cells. Our analysis of the molecular circuitry underlying this replication origin activation checkpoint reveals that G1 arrest is dependent on three non-redundant checkpoint axes coordinated through the Forkhead transcription factor FoxO3a and p53. We show that only breast cancers displaying the accelerated cell cycle phenotype express elevated Cdc7 levels and are therefore highly represented in p53 mutant Her2-subtype and triple negative tumours. Breast cancers of the luminal subtype expressing low levels of Cdc7 undergo a cytostatic G1 arrest after Cdc7 inhibition due to their p53 wild type status, a checkpoint response mimicking untransformed cells. In contrast Her2 and triple negative tumours show a marked response to Cdc7 inhibitors with potent cancer-cell-specific killing as a result of (i) overexpression of the target protein and (ii) impairment of the origin activation checkpoint due to p53 lesions. Thus integrating cancer cell cycle phenotypic data with a molecular analysis of the origin activation checkpoint serves as a predictor of response to pharmacological Cdc7 inhibitors. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2148.

Abstract B255: Discovery and validation of a novel class of small molecule inhibitors of the CDC7 kinase: Modulation of tumor cell growth in vitro and in vivo

Abstract High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI-7, was selected for further investigation. First, CKI-7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI-7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naive and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl_xL anti-apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase-3 activation in the absence and attenuation of the activity in the presence of Bcl_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over-expressing cell lines. We found that CKI-7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1) in addition to melanoma and non small cell lung cancer xenograft models. Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI-7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B255.

Targeting Cdc7 Kinase in Multiple Myeloma.

Abstract 3847Poster Board III-783 IntroductionMyeloma remains an incurable cancer and the development of new therapeutic approaches is of fundamental importance. A key feature of progressive myeloma is deregulation of the mechanisms that control progression through the cell cycle, in particular, the increase in the activity of S-phase promoting factors and the loss of cell cycle brakes such as the tumour suppressor protein p53. Moreover, high expression of Mcl-1, a potent anti-apoptotic Bcl2 family member, and defects in the p53 pathway are now known to be instrumental in inhibiting responsiveness to current and emerging myeloma therapeutics. The Cell division cyle 7 (Cdc7) kinase is an essential protein for the initiation of DNA replication and for cell cycle progression. Previous studies have indicated that Cdc7 inhibition can cause p53 independent apoptosis in several cancer models both in vitro and in vivo and currently Cdc7 inhibitors are entering clinical trials as experimental drugs. In this work we study the effects of the prototype Cdc7 protein kinase inhibitor, PHA-767491, in myeloma cells. In addition to targeting Cdc7, PHA-767491 also targets cyclin dependent kinase 9 (CDK9) and can downregulate Mcl-1 in a variety of cancer cell lines. Hypothesis: Given the molecular characteristics of progressive myeloma and the spectrum of activity of PHA-767491, we hypothesized that Cdc7 inhibition could be an effective treatment strategy in myeloma. Methods The Multiple Myeloma Research Consortium Validation-Core cell lines were treated with increasing doses of PHA-767491 as a single agent and in combination with drugs that currently constitute standards of care in myeloma treatment. These lines included lines resistant to conventional anti-myeloma agents (e.g. melphalan, dexamethasone, and doxorubicin) as well as novel anti-MM agents (e.g. immunomodulatory thalidomide derivatives). A number of lines were TP53 mutants. Similarly, CD138+ purified primary cells with clincal resistance to lenalidomide and bortezomib were treated with increasing doses of PHA-767491. Cell viability was measured by Cell TitreGlo 72 hours post treatment allowing IC50 calcuations. Combination indices were calculated as described by Chou et Talalay. Apoptosis was assessed by PI and Annexin V staining using flow cytometry and immunoblotting with relevant antibodies was performed for cell cycle and apoptotic proteins of interest. ResultsWe found that PHA-767491 blocks proliferation and induces apoptosis with an IC50 in the low micromolar range in six out of seven MMRC cell lines and in primary cells from patient (s) with clinical resistance to bortezomib and lenalidomide. The averaqe and median IC50 value of MMRC cell lines was 2.25μM and 2μM respectively. TP53 mutation did not alter sensitivity to PHA-767491. Following treatment of myeloma cells with PHA-767491 there was potent inhibition of phosphorylation of the replicative helicase Mcm2 at serine 40/41 (a specific biomarker of Cdc7 activity) and complete downregulation of Mcl-1 by 6 hours, with concomitant caspase 3 cleavage and apoptosis as assessed by accumulation of sub-G1 cells. Annexin V/PI staining confirmed these cells to be apoptotic. When used in combination, PHA-767491 showed synergistic activity with melphalan and doxorubicin (Combination Index IC50 – 0.3, 0.7 respectively), even in the highly doxorubicin-resistant(p170/MDR1 mediated) line RPMI-8226-Dox-40 (Combination IndexIC50 – 0.3). ConclusionWe suggest that Cdc7 inhibition with PHA-767491 or with small molecules that have similar properties could be an effective strategy in myeloma treatment and merits further evaluation. Disclosures:Montagnoli:Nerviano Medical Sciences Oncology: Employment.

Discovery and Validation of a Novel Class of Small Molecule Inhibitors of the CDC7 Kinase: Modulation of Tumor Cell Growth in Vitro and In Vivo.

Abstract 3771Poster Board III-707High throughput screening of compounds comprising the Memorial Sloan Kettering chemical library resulted in several confirmed hits against the recombinant Cdc7:Dbf4 heterodimeric kinase, a key regulator in the initiation of DNA replication and the G1 to S phase transition. Chemoinformatic analysis of the hits revealed an enrichment in one chemical cluster made up of several naturally occurring compounds, of which the most potent compound, CKI-7, was selected for further investigation. First, CKI-7 was found to be a non competitive inhibitor for ATP and prompted us to prolife it against a panel of 200 known kinases in order to assess its selectivity profile. The results were as predicted and very few kinases were specifically affected. Second, CKI-7 cytotoxic activity was assessed against a panel of well established cancer cell lines representing both hematopoietic and solid tumor malignancies as well as against a panel of primary hematopoietic cells derived from leukemia patients (both chemotherapy naïve and relapsed/refractory samples) and was found to be a very effective agent with potencies in the low nanomolar range. Subsequent studies using an isogenic pair of cell lines with one over expressing the Bcl_xL anti-apoptotic protein further confirmed the induction of the intrinsic apoptotic pathway via caspase-3 activation in the absence and attenuation of the activity in the presence of Bcl_xL. This was further demonstrated through standard cell cycle synchronization studies revealing that exposure to the Cdc7 inhibitor results in an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. This cell death is the direct result of Cdc7 kinase inhibition by CKI-7 as demonstrated using a Cdc7 substrate biomarker assay. Third, the physicochemical properties of this class of naturally occurring compounds also prompted us to investigate their effect on several multidrug resistence (MDR) over-expressing cell lines. We found that CKI-7 was not a substrate for the efflux pumps demonstrating that this novel compound can overcome a major mechanism of chemotherapy resistence in human tumor cells. Based of the above observations, in vivo dose-dependent anti-tumor activity of CKI-7 was subsequently demonstrated in a SCID-Beige mouse systemic tumor model utilizing a recently isolated Philadelphia chromosome positive acute lymphoblastic leukemia cell line (PhALL3.1). Taken together, our data confirm that Cdc7 is a new promising target for cancer therapy, and that the newly discovered inhibitor CKI-7, a naturally occurring selective small molecule inhibitor of this enzyme, is an equally promising novel cancer therapeutic agent. Disclosures:No relevant conflicts of interest to declare.

Cdc7 kinase – A new target for drug development

The cell division cycle 7 (Cdc7) is a serine threonine kinase that is of critical importance in the regulation of normal cell cycle progression. Cdc7 kinase is highly conserved during evolution and much has been learned about its biological roles in humans through the study of lower eukaryotes, particularly yeasts. Two important regulator proteins, Dbf4 and Drf1, bind to and modulate the kinase activity of human Cdc7 which phosphorylates several sites on Mcm2 (minichromosome maintenance protein 2), one of the six subunits of the replicative DNA helicase needed for duplication of the genome. Through regulation of both DNA synthesis and DNA damage response, both key functions in the survival of tumour cells, Cdc7 becomes an attractive target for pharmacological inhibition. There are much data available on the pre-clinical anti-cancer effects of Cdc7 depletion and although there are no available Cdc7 inhibitors in clinical trials as yet, several lead compounds are being optimised for this purpose. In this review, we will address the current status of Cdc7 as an important target for new drug development.