ALBERT

Description: Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II. The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. Many leukemic cell lines, which are dependent upon the expression of short half-life transcripts such as Mcl-1 for survival, undergo apoptosis following transcriptional inhibition and the cell cycle effects of such inhibitors are masked. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 in leukemia cell lines (HL60 and MOLT-4). AT7519 causes rapid induction of apoptosis (within 6 hours of initial exposure) in the absence of cell cycle arrest. This induction of apoptosis occurs in parallel with a reduction in the levels of anti-apoptotic proteins such as Mcl-1. In HL60 xenograft models anti-tumour efficacy is observed following 2 cycles of daily dosing of 15 or 10mg/kg for 5 days followed by 2 days off treatment. We observe a 50% cure rate (4/8 mice) at the 15mg/kg dose and a 30% cure rate at the 10mg/kg dose level 40 days following dosing. Pharmacodynamic biomarker studies demonstrate that a greater extent and duration of Mcl-1 knockdown and apoptosis induction are associated with efficacious doses. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. Therefore, we also characterised the transcriptional effects of AT7519 on CLL cells isolated from patients. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–300nM. These effects were correlated with the cytogenetic background of individual patients and the data supports further clinical investigation of AT7519 in B-Cell lymphoproliferative disorders where survival proteins play a pivotal role.

Description: Introduction: The prognosis of patients (pts) with relapsed or refractory (rel/ref) Chronic Lymphocytic Leukemia (CLL) is poor. AT7519M is a small molecule inhibitor of cyclin-dependent kinases (CDKs) 1, 2, 4, 5 and 9 with lower potency against CDKs 3, 6 and 7. AT7519 has potent anti-proliferative activity against peripheral blood mononuclear cells isolated from CLL patients. Exposure of CLL cells to AT7519 results in cell cycle arrest and, ultimately, cell death by apoptosis. Based on the results of a NCIC CTG phase I study in advanced malignancies, the recommended phase II dose (RP2D) was 27 mg/m2 given as a 1 hour infusion twice weekly for 2 out of every 3 weeks. Using this schedule, Tumor Lysis Syndrome (TLS) and QTc prolongation were not observed. Methods: In a phase II clinical trial, we evaluated the clinical and pharmacodynamic effects of AT7519 using the RP2D schedule in pts with rel/ref CLL. Eligible patients were those with documented CLL with at least one prior systemic treatment regimen and either lymphocyte count 〉 10 x 109/L or at least one measurable lymph node 〉 2 cm x 2 cm. The primary objective was complete or partial remission (CR/PR) as defined by the 2008 International Working Group (IWG) Guidelines. We used a Fleming Phase II design, aiming for a total of 30 subjects, assuming an HA of 〉0.20. Cycle 1, dose 1 was administered as an in-patient with prophylaxis and monitoring for TLS. Results: Seven pts were accrued over 16 months. As a result, the trial was prematurely closed. Reasons for slow accrual included concerns about the risk of TLS, the need for in-patient care during cycle 1, and the relative stringency of eligibility criteria regarding baseline hematopoietic and renal function. All seven pts were male, with median age 70 years (range 47-81). Median number of previous regimens was 3 (range 1-4). Two pts had 11q- and one had 17p-. A total of 21 cycles were administered (median 4 cycles) with 71% of patients receiving 〉 90% of the planned dose intensity. Two pts required a dose reduction due to thrombocytopenia and one of these patients came off study early due to hypoxia and fever. The most common non-hematologic adverse events that were at least possibly related were grade 1 and 3 fatigue, grade 1 nausea (57%), grade 1 diarrhea and grade1/2 anorexia (43%) and grade1/2 fever (29%). Hematologic toxicities were primarily grade 1 and 2. One pt developed grade 4 neutropenia and grade 3 thrombocytopenia on cycle 1 day 2; a second patient developed grade 3 thrombocytopenia on C1D2. Both required dose reductions and counts recovered. Biochemical toxicity was minimal; all grade 1 except one pt with transient grade 2 bilirubin and 2 pts with grade 2 hypophosphatemia. According to the protocol defined 2008 IWG response assessment criteria, there were no responses observed. Four pts had stable disease (SD), two pts had progressive disease (PD) and one pt was inevaluable (came off due to toxicity after 2 doses). However, updated response criteria for CLL recommend that lymphocytosis alone should not be considered evidence of progressive disease in clinical trials testing novel agents that affect cellular migration and adhesion. Based on these new criteria, 1 of 7 patients developed PD, 3 demonstrated tumor shrinkage at time of coming off protocol therapy including 2 with corresponding improvement in blood counts although none achieved objective partial response (see Table). Conclusion: The CDK inhibitor, AT7519M was safely administered to pts with rel/ref CLL. While some patients had tumor shrinkage, there were no objective responses over the course of this study. Unfortunately, the small sample size of this trial precluded any other definitive conclusions. Table: Responses Graded By Traditional and Revised CLL Response Criteria Table:. Responses Graded By Traditional and Revised CLL Response Criteria Disclosures Toguchi: Astex Pharmaceuticals, Ltd: Employment. Lyons:Astex Pharmaceuticals: Employment.

Description: Background:Although outcomes for patients with multiple myeloma (MM) have improved, it remains an incurable malignancy for which new therapies are needed. Aurora kinases are overexpressed in MM. Inhibition of Aurora A kinase, a key component of the centrosome, in MM cell lines induces apoptotic cell death. Inhibition of Aurora B, a chromosome passenger protein, has therapeutic effects against myeloma lines, primary bone marrow plasma cells and murine xenografts. Histone H3 is a direct downstream substrate of Aurora B. The Aurora A and B kinase inhibitor AT9283 has shown anti-myeloma activity in pre-clinical studies. Based on an NCIC CTG phase I trial in patients with advanced malignancy the recommended phase 2 dose (RP2D) was 40 mg/m2 on days 1 and 8 of a 21 day cycle. This single arm phase II trial was designed to explore the efficacy of AT9283 in patients with relapsed and refractory multiple myeloma. Methods: Eligibility criteria included age ≥ 18, neutrophils ≥ 1 and platelets ≥ 70 x109/L. There was no upper limit on the number of prior regimens. The primary endpoint was response rate; secondary endpoints included toxicity and evaluation of potential biomarkers. According to the 2-stage design at least 1 response was required in the first 15 patients to proceed to a full sample size of 30, assuming an HA of ≥ 0.20. Initially AT9283 was administered by 24 hour continuous infusion at the RP2D. Hematological toxicity in the first 2 patients met protocol specified criteria for reduction of the starting dose to 30 mg/m2 for subsequent patients. Adverse events were graded according to CTCAE V4.0. Phosphorylated Histone H3 (Ser10), a marker of Aurora B activity, and total Histone H3 protein expression was measured by western blot analysis in baseline and day 21 bone marrow samples. Results:Over 18 months, eight eligible patients (3 male, 5 female) with MM (4 IgG, 2 IgA, 2 light chain) were accrued. All were evaluable for toxicity and 7 evaluable for response. Median age was 60 (range 48 – 75). The median number of prior regimens was 3 (range 1 - 5). Patients received between 1 – 4 cycles of AT9283 (median 1) with 25% of patients receiving ≥ 90% of the planned dose intensity. No responses were observed. The trial was closed due to slow accrual and toxicity, in particular myelosuppression. Transient neutropenia occurred in all patients (63% grade 3, 37% grade 4). Recovery from nadir to normal range was rapid in the majority (range 1 – 8 days, median 5) but prolonged in 2 patients who were neutropenic at baseline (11 and 13 weeks). Grade 3/4 thrombocytopenia occurred in 50% of patients. There were 3 infections ≥ grade 3 including 1 death. Grade 3 skin ulceration thought possibly related to AT9283 by the investigator occurred in 1 patient. The most common non-hematologic adverse events reported as possibly, probably or definitely related to AT9283 were grade 1-3 nausea (50%), grade 1 and 3 vomiting (38%), grade 4 febrile neutropenia (25%), grade 1/2 anorexia (25%), grade 1/2 diarrhea (25%) and grade 1/2 fatigue (25%). Paired baseline and day 21 bone marrow samples were provided from 4 patients; sufficient protein was extracted from both samples for western blot analysis from 3 patients. Of these, 2 showed a reduction in Histone 3 phosphorylation (H3-P) after the first cycle of treatment consistent with inhibition of Aurora B. Both of these patients had a best response of stable disease (SD). Abstract 5734. Table Patient Dose (mg/m2) Number of cycles Best response Non-hematological toxicity ≥ Grade 3* Reason off study H3-P/H3Baseline Day 21 1 40 1 PD Gr 3 skin ulceration Toxicity 0.3 0.5 2 40 1 SD Gr 3 lung infection Intercurrent illness 1.6 0.5 3 30 3 SD None PD 0.6 0.5 4 30 1 PD Gr 4 febrile neutropenia & gr 4 left ventricular systolic dysfunction Toxicity NE NE 5 30 1 NE Gr 4 febrile neutropenia & gr 5 sepsis** Death NE NE 6 30 4 PD Gr 3 nausea & vomiting PD NE NE 7 30 3 SD None PD NE NE 8 30 1 PD None Death NE NE * Reported as possibly, probably or definitely related to AT9283 by investigator ** Neutrophil count recovered to normal range prior to death PD progressive disease, NE not evaluable, Gr grade Conclusions: This study failed to reach full accrual. No objective responses were observed. Administration of AT9283 to patients with previously treated myeloma at this dose and schedule is associated with toxicity, particularly myelosuppression. Disclosures Sandhu: Janssen: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Reece:Otsuka: Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Millenium: Honoraria, Research Funding; Merck: Research Funding; Bristol-Myers Squibb: Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria. Lyons:Astex: Employment. Seymour:Astex: Research Funding.

Description: Introduction SGI-110 is a dinucleotide of decitabine and deoxyguanosine and is a potent inhibitor of DNA methylation in-vitro and in-vivo. A Phase 1 study established the biologically effective dose of SGI-110 in MDS/AML patients as 60 mg/m2 SQ daily x 5 and demonstrated clinical responses that correlated with hypomethylation induction. Here, we analyze AML patients treated at pharmacologically effective doses of SGI-110 to explore determinants of hypomethylation and response. Methods We studied patients with relapsed/refractory AML treated at therapeutic dose ranges of SGI-110 (36 mg/m2-125 mg/m2). DNA methylation pre/post treatment (for pharmacodynamics [PD]) was measured by bisulfite-pyrosequencing for the LINE-1 repetitive element and the INS6 CpG island gene promoter which is highly methylated in all somatic tissues. Gene expression at baseline and after treatment was measured by qPCR. Results We analyzed samples from 27 patients with AML. Median age was 64, (range, 29–86), 18 were Males (67%), 13 (48%) had poor risk cytogenetics at study entry and 59% had prior exposure to a hypomethylating agent. Overall, peak LINE-1 demethylation generally occurred on Day 8 and correlated strongly with INSL6 demethylation (R=0.95, p0.05). Unsupervised classification grouped the patients into four clusters: A (N=2), B (N=6), C (N=10), and D (N=9). Cluster D is characterized by high DNMT3b expression, low P15 expression, low CDA expression and reduced demethylation (demethylation average -10.9% in cluster D compared to -22.7% in clusters B and C, p=0.06). Next, we examined SGI-110 mediated induction of gene expression for the P15, P21, DNMT3B and CTCF genes. P15 was significantly induced in patients who were treated on the daily x 5 regimen (p=0.03) but not in patients receiving the weekly x 3 regimen. In this group of 14 patients with induced P15, P15 induction peaked on Day 8 and averaged a 2.4-fold increase. P15 induction was associated with a trend for increased demethylation on Day 8 (R=0.28) and on Day 29 (R=0.37), p〉0.05 for both. Of the 27 patients, 5 showed major clinical responses (2 CR, 3 CRi/CRp). LINE-1 and INSL6 demethylation averaged -21.1% and -16.4% in responders compared to -13.1% and -11.3% in non-responders. A three gene classifier score (low CDA, low P15 and high DNMT3B) was associated with low LINE-1 demethylation (R=0.43, p=0.025) as well as resistance to SGI-110 (mean score 6.2 in non-responders compared to 0.5 in responders, p=0.047). Finally, peak induction of P15 was similar in responders and non-responders, but sustained induction (at Day 29) was higher in responders (3.1 fold) than in non-responders (1.0 fold). While a genetic signature of response could not be identified among those 8 genes that were examined by exome sequencing, mutations in IDH1 or IDH2 were identified in 5 patients. One of these patients was positive for substitution R132H of IDH1 (described to induce epigenetic alterations and may predict poor clinical outcome in AML) and achieved a CR in response to SGI-110 treatment. The TP53 polymorphism NM_000546:c.C215G:p.P72R, (associated with differential response to chemotherapy in AML), was identified in 9 subjects, 3 of whom responded to SGI-110. Conclusions At therapeutic doses of SGI-110, we identified a gene expression signature (high DNMT3B, low P15, and low CDA) that differentiates responders from non-responders to SGI-110 and we find trends for associations between demethylation and response, as well as sustained P15 induction and response. These associations will be further investigated in the ongoing Phase 2 study of SGI-110 in AML. Disclosures: Chung: Astex Pharmaceuticals Inc.: Research Funding. Off Label Use: SGI-110. Taverna:Astex Pharmaceuticals Inc.: Employment. Lyons:Astex Pharmaceuticals Inc.: Employment. Hao:Astex Pharmaceuticals Inc.: Employment. Azab:Astex Pharmaceuticals Inc.: Employment. Kantarjian:Astex Pharmaceuticals Inc.: Research Funding. Kropf:Astex Pharmaceuticals Inc.: Research Funding. Issa:Astex Pharmaceuticals Inc.: Honoraria, Research Funding.

Description: Introduction: SGI-110 is second generation HMA formulated as a dinucleotide of decitabine (DAC) and deoxyguanosine delivered as a small volume, pharmaceutically stable SQ injection allowing longer half-life and more extended decitabine exposure than DAC IV infusion. SGI-110 differentiated pharmacokinetic profile resulted in potent hypomethylation and clinical responses in previously treated MDS and AML patients in the phase 1 trial (Kantarjian et al. 2012). Here, we have identified novel DNA-methylation biomarker candidates that may be predictive of response to SGI-110 using Differential Methylation Hybridisation (DMH) profiling of the NCI-60 cell line panel (Fassbender A et al, Methods Mol Biol 2010). Cell lines were stratified based on SGI-110 EC50 values from Colony Forming Assay and degree of LINE-1 (Long Interspersed Nucleotide Elements) demethylation post-SGI-110 treatment. The stratification was used to classify cell lines into SGI-110 sensitive and resistant and to generate 249 genomic methylation sites as candidate biomarkers of response to SGI-110. Candidate markers from this analysis will now be compared in DNA samples derived from whole blood from treatment naïve and relapsed/refractory AML patients that were classified into responders and non-responders after treatment with SGI-110 in our phase 2 clinical study. Methods: DMH was used for unbiased profiling of methylated loci throughout the genome. Briefly, the genome is cleaved into a defined fragment library using methylation unspecific restriction enzymes, followed by PCR-adapter ligation. In a second step, the fragments are digested by methylation-sensitive restriction enzymes cutting only at unmethylated restriction sites, while methylated fragments and fragments that do not have any restriction site were protected from the restriction digestion. The methylated fragments and the control fragments are then selectively amplified by PCR and the PCR product is labeled and hybridized to Epigenomics' proprietary DMH microarray. Direct Bisulfite Sequencing (DBS) is used to assess the DNA-methylation pattern of marker candidates at CpG level. Regions with CpGs of interest are amplified to generate PCR products that are directly sequenced using a 4 dye labelled ddNTP Sanger sequencing. Quantitative DNA methylation of CpG region with a lengths of up to 550 bp are derived from the resulting trace files using the ESME software (Lewin J et al, Bioinformatics. 2004 Nov 22;20(17):3005-12). Results: Using this method, we have seen a clear correlation of high sensitivity to SGI-110 with high LINE-1 methylation level. Five (leukemia) cell lines classified as sensitive to SGI-110 by EC50 values were among the 6 cell lines with the highest LINE-1 methylation (〉 73% methylation) while resistant cell lines showed a LINE-1 methylation 〈 70%. A high degree of correlation was observed between LINE-1 methylation and genome-wide DNA-Methylation measured by DMH in over 50k genomic sites and supported the role of LINE-1 as a useful indicator for sensitivity to SGI-110 and global DNA methylation. Fifty genomic fragments that better characterized sensitive and resistant cell lines were selected for further validation. The differential DNA-methylation discriminating between SGI-110 sensitive and resistant cancer cell lines (n=18) was validated by DBS analysis at CpG level in genomic fragments. We will present data obtained from the DBS evaluation of methylation in these validated methylation marker candidates in up to 45 AML blood samples that were classified into responders and non-responders after treatment with SGI-110 in a phase 2 clinical trial. Conclusions: We identified DNA methylation patterns associated to sensitivity and resistance to the novel HMA SGI-110. The validated methylation biomarker discovery process based on DMA and DBS approaches may help to identify and characterise specific subgroups of AML patients that could preferentially respond to SGI-110. Disclosures Jueliger: Astex Pharmaceuticals: Employment. Lyons:Astex Pharmaceuticals: Employment. Lewin:Epitenomics AG: Employment. Azab:Astex Pharmaceuticals, Inc.: Employment. Taverna:Astex Pharmaceuticals, Inc.: Employment.

Description: Background The benefits of targeting the cyclins and RNAPII transcription in multiple myeloma (MM) was previously demonstrated using the cyclin kinase inhibitor AT7519 in preclinical studies. Predicated upon this work, a phase I/II trial was undertaken in relapsed refractory MM patients Purpose To identify the optimum dose of the AT7519M both as a single agent (Part one) and in combination with Bortezomib (Part two) in patients with MM. Methods In Part one nine patients with relapsed or refractory MM were treated with AT7519M alone using a twice weekly regimen (days one, four, eight and eleven) of a 21 day cycle. The starting dose of AT7519M was 21 mg/m2 per dose during Cycle one, and provided treatment was well tolerated the dose was escalated to 27 mg/m2 per dose for Cycle two onwards. In the second part of the study the safety and tolerability of the combination of AT7519M (dose levels 14 and 21 mg/m2 per dose) with Bortezomib (dose levels 1 and 1.3 mg/m2 per dose) were explored in an escalating fashion using a “3 + 3” design. In order to be eligible patients must have measurable MM, and initially must have had progressed through at least two previous lines of therapy or following a protocol amendment, been refractory to Bortezomib, as defined by progression on a Bortezomib-containing regimen within six weeks of starting the study. Patients with significantly abnormal liver or renal function (creatinine clearance 〈 30 ml/min) or significant underlying neuropathy were excluded. Results The median number of cycles of treatment administered in the first part of the study (9 patients) was two (range two to four) and the best response to treatment was stable disease. Two patients discontinued treatment as a result of toxicity – one as a result of a persistent elevation in serum creatinine and the second as a consequence of Grade 3 fatigue. Two patients did not undergo a dose escalation to 27 mg/m2 as planned because of poor tolerability of the 21 mg/m2 dose (fatigue) or progressive disease. Pharmacokinetic analysis of the two dose levels revealed overlapping exposure and further evaluation of the 27 mg/m2 dose was not performed. In the second part of the study, three patients per cohort, 9 patients in total, were treated at escalating AT7519/Bortezomib dose levels of 14 mg/m2/1 mg/m2; 21 mg/m2/1 mg/m2 21 mg/m2/1.3 mg/m2. The median number of previous regimens was 5 (range 2 – 6). Based on the investigator assessments at least one objective response was seen at each dose level (MR x1, PR x 2, VGPR x 1). The median number of cycles received was three (range one to 9; ongoing). There were no dose-limiting toxicities. The most commonly reported adverse events to date have been haematological in nature. There have been two adverse events ≥Grade 3 in severity (neutropenia, and dyspnea) considered to be at least possibly related to treatment. Conclusions Treatment with AT7519M was well tolerated in this patient population and full doses of this agent and Bortezomib were successfully achieved in combination. The maximum tolerated dose was 21 mg/m2 AT7519M and 1.3 mg/m2 Bortezomib. Although no significant efficacy was seen following treatment with AT7519M alone in this heavily pretreated patient population, the combination of AT7519M with Bortezomib achieved significant (33% ≥ PR) responses in a proportion of patients who were either pre-treated with or refractory to treatment with Bortezomib. Disclosures: Raje: Celgene, Millenium, Onyx, Amgen: Consultancy; Acetylon, Eli Lilly: Research Funding. Richardson:Millennium: The Takeda Oncology Company: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity’s Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity’s Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity’s Board of Directors or advisory committees; Britsol-Myers Squibb: Membership on an entity’s Board of Directors or advisory committees. Lyons:Astex Pharmaceuticals Inc.: Employment. Langford:Astex: Employment. Yule:Astex: Consultancy.

Description: Recent reports suggest that Aurora kinases (AK) A and B are overexpressed in a proportion of patients with AML and that the level of overexpression correlates with their sensitivity to AK inhibition in vitro. Inhibition of AKB results in mitotic exit in the absence of cell division resulting in polyploidy whilst AKA is responsible for the fidelity of mitotic spindle assembly and phosphorylates p53 at Ser315 leading to its ubiquitination by Mdm2 and subsequent proteolysis. Inhibition of this process increases p53 stability inducing cell-cycle arrest with 4N DNA. Clinical studies of the dual AK inhibitor AT9283 indicate that it is active in the treatment of a proportion of patients with relapsed/refractory AML. In order to explore the molecular basis of this varied sensitivity we investigated the effect of AT9283 in a panel of 10 AML cell lines from a variety of genetic backgrounds including a proportion driven by ras, Flt3 or c-kit mutations. Two phenotypes were observed; Accumulation of cells in the G2/M phase (4N) of the cell cycle followed by apoptosis, or Accumulation of cells with 〉4N DNA (polyploid) followed by apoptosis. Cell lines that exhibit the former phenotype were those driven by mutation in an oncogenic kinase such as ras, c-kit or Flt3. Further analysis of Cyclin B levels suggest that profile 1) results from a G2 block occurring as a consequence of a dominant AKA inhibitory effect in these cell lines. Profile 2) results from AKB inhibition; where cells continue to undergo rounds of DNA replication in the absence of cell division. Inhibition of both of these signaling pathways has been confirmed in AML blasts taken from patients treated with AT9283 in the ongoing clinical program. These results suggest that AT9283 triggers the mitotic checkpoint and induces apoptosis in patients harboring mutations in FLT3 or c-kit via AKA inhibition. These AML cases have been shown to be more likely to exhibit normal cytogenetics and this profile may be important in sustaining rapid peripheral blast proliferation typical of this subtype of AML. Cell lines that respond to treatment with AT9283 by becoming polyploid may be manifesting the effect of predominant AKB inhibition. The balance between these outcomes may reflect the corresponding levels of expression of AKA and AKB in individual subtypes of AML along with factors such as TP53 mutation being associated with genomic instability supporting the development of complex karyotypic abnormalities. Such preliminary findings indicate that patients with mutations in oncogenic signaling pathways may be particularly sensitive to treatment with AT9283 due to the presence of an intact mitotic checkpoint and a dominant Aurora A inhibitory phenotype. This hypothesis is currently being explored through the analysis of biological studies obtained from the ongoing clinical program.

Description: Cyclin Dependent Kinases (CDKs) play a central role in the eukaryotic cell cycle. The activation of these kinases is modulated by the expression and binding of their regulatory cyclin partners. Their key role in cell cycle progression, coupled to evidence that pathways leading to their activation are deregulated in a number of human cancers makes them attractive therapeutic targets. More recently the role of CDKs 7, 8 and 9 in the regulation of transcription has been explored. CDK9 has been shown to play a role in the regulation of transcription via phosphorylation of RNA polymerase II (RNA pol II). The outcome of transcriptional inhibition via CDK9 exhibits significant variation between cell lines. B-Cell lymphoproliferative disorders, including CLL, rely on the expression of transcripts with a short half-life such as Mcl-1, Bcl-2 and XIAP for survival. In vitro studies have demonstrated that compounds with transcriptional inhibitory effects are effective pro-apoptotic agents in models of this disease. AT7519 is a potent inhibitor of cyclin dependent kinases 1, 2 and 9 and is currently in early phase clinical development. These studies profile the mechanism of action of AT7519 on CLL cells isolated from patients. Primary cell samples were isolated from a total of 15 patients with CLL with various stages of disease (8 Stage 0, 0/I or II and 7 Stage IV) and who were either treatment naïve or had received a variety of prior therapies. Patient samples were characterised for cytogenetic abnormalities (11q, 17p and 13q deletion or trisomy 12) as well IgVH mutation and ZAP70 expression. AT7519 was shown to induce apoptosis (by MTS, morphology and PARP cleavage) in these samples at concentrations of 100–700nM. AT7519 appears equally effective at inhibiting the survival of CLL cells harbouring a variety of mutations including those representative of patients that fall within poorer prognosis treatment groups. The amount of AT7519 required to induce cell death in 50% of the CLL cell population increased as exposure time was decreased but significant cell death was obtained at doses approximating to 1uM following 4–6h of treatment. These doses are equivalent to exposures achieved in ongoing AT7519 clinical studies indicating that cytotoxic doses can be achieved in patients on well tolerated schedules. The mechanism of AT7519 cytotoxic effects was investigated by western blotting for a variety of cell cycle and apoptotic markers following incubation with compound. Short term treatments (4–6h) resulted in inhibition of phosphorylation of the transcriptional marker RNA pol II and the downregulation of the anti-apoptotic protein Mcl-1. Additional antiapoptotic proteins including XIAP and Bcl-2 remained unchanged. The reduction in Mcl-1 protein levels was associated with an increase in the apoptotic marker cleaved PARP. No inhibition of cell cycle markers such as phospho-retinoblastoma protein was observed in the same samples suggesting that the cytotoxic effects of AT7519 in CLL patient samples is due to its transcriptional activity alone. Together the data suggest AT7519 offers a promising treatment strategy for patients with advanced B-cell leukemia and lymphoma.

Description: Abstract 4159 Aurora kinases (AK) A and B are overexpressed in a proportion of patients with acute myeloid leukemia (AML) and the level of overexpression correlates with their sensitivity to AK inhibition in vitro. We recently reported data from a dose escalation study of AT9238, a small molecule inhibitor of AKs in patients with refractory leukemias in which eight of 24 AML patients with relapsed/refractory AML achieved a 3 33% reduction in bone marrow blasts and haematological improvement. All patients had received at least one line of previous therapy. Further analysis has revealed that of the eight patients with relapsed/refractory AML that benefited from treatment with AT9283 five had a normal karyotype and the remaining three patients showed evidence of isolated abnormalities of chromosome 7, including 7q loss. Separately, we have reported that AT9283 inhibits the proliferation and survival of AML cell lines in vitro and suggested that those cell lines with complex karyotypic abnormalities responded differently from normal diploid lines. In these experiments AML cell lines exhibit one of two phenotypes following exposure to AT9283; rapid induction of cell death at low nM concentrations (Phenotype 1) or endo-reduplication followed by cell death at a later time point (Phenotype 2). In both scenarios treatment with AT9283 results ultimately in cell death. Cell lines with a normal karyotype tended to undergo rapid apoptosis without evidence of endoreduplication at low concentrations of AT9283. These findings provide further support for the potential importance of karyotype as a determinant of outcome in the clinical study. This is the first indication that cytogenetics might be used to predict responsiveness to Aurora kinase inhibitors in the clinic. Disclosures: Lyons: astex therapeutics: Employment. Squires:Astex Therapeutics, Ldt: Employment. Goodall:astex therapeutics: Employment. Yule:Astex Therapeutics Ldt: Employment. Ravandi:BMS: Consultancy, Honoraria, Research Funding.

Description: AT9283 is a potent inhibitor of JAK2, JAK3, mutant Abl kinase (T315IAbl) and Aurora kinases A and B all of which have an IC50