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Covalent Bruton tyrosine kinase (BTK) inhibitors, such as ibrutinib, have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop as the result of a mutation in cysteine 481 of BTK (C481S), which prevents irreversible binding of the drugs. In the present study we performed preclinical characterization of vecabrutinib, a next-generation noncovalent BTK inhibitor that has ITK-inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wild-type BTK. In the murine Eμ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, whereas the naive populations were increased. Of importance, vecabrutinib treatment significantly reduced the frequency of regulatory CD4+ T cells in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on the activation and proliferation of isolated T cells. Lastly, combination treatment with vecabrutinib and venetoclax augmented treatment efficacy, significantly improved survival, and led to favorable reprogramming of the microenvironment in the murine Eμ-TCL1 model. Thus, noncovalent BTK/ITK inhibitors, such as vecabrutinib, may be efficacious in C481S BTK mutant CLL while preserving the T-cell immunomodulatory function of ibrutinib.

Introduction. Chronic graft-versus-host disease (cGVHD) can manifest as a complication in patients following allogeneic hematopoietic stem cell transplant resulting in morbidity and mortality. Effective treatment strategies for cGVHD are currently lacking. Ibrutinib, an irreversible BTK inhibitor with activity against several other Tec-family kinases, has been clinically developed for cGVHD treatment due to regulation of pathogenetic B cells and T-cell subsets. Vecabrutinib is a more selective, reversible inhibitor of BTK with a distinct kinase domain interaction, which could result in differentiated safety and activity profiles compared to ibrutinib. Vecabrutinib has the capacity to overcome the C481S mutation that mediates resistance to ibrutinib. Vecabrutinib also also demonstrates activity against ITK, which is expressed in T cells. In this study, we investigated the activity and immune modulation of vecabrutinib treatment in a murine model of sclerodermatous cGVHD. Ibrutinib was utilized for comparison. Methods. A murine model of sclerodermatous cGVHD was initiated by adoptive transfer of T-cell depleted bone marrow plus whole splenocytes from B10.D2 donors into BALB/c recipients that were previously subjected to sub-lethal irradiation. Total bone marrow and irradiation only groups were included as controls. Mice with established cGVHD characterized by weight loss and skin irritation symptoms were treated with vecabrutinib once daily at 50mg/kg by oral gavage or ibrutinib at 30mg/kg in drinking water 5 days per week for approximately 3 weeks beginning on day 27 post-adoptive transfer and ending on day 45 (n=10 mice per group). Body weight and clinical symptoms (appearance, activity, skin symptoms, diarrhea, conjunctivitis) were measured throughout the study. Immunophenotyping for B cells and T cells was performed on spleens collected from euthanized mice by flow cytometry at two timepoints (day 40 during treatment and on day 76 post-treatment). Levels of circulating immunoglobulins were measured by multiplex cytokine assay at both timepoints. Results. Clinical symptoms, including appearance, activity, skin irritation, redness, alopecia and diarrhea were significantly reduced in vecabrutinib- and ibrutinib-treated groups. Furthermore, there was a trend toward a more favorable clinical score overall for the vecabrutinib-treated group, however no statistical difference was observed compared to ibrutinib possibly due to small sample size. Weight loss was slightly elevated during vecabrutinib and ibrutinib treatment compared to vehicle (trend), however mice recovered body weight post-treatment and continued to maintain benefit. On day 40 (during treatment) and day 76 (post-treatment) groups of mice were euthanized for immunophenotyping analysis utilizing a 22-color flow cytometry panel. During treatment, both vecabrutinib and ibrutinib-treated mice retained total B cell numbers but exhibited reduced B-cell activation, proliferation, and number of B220+ CD138+ plasma cells. In addition, B cells secreted less IL-10, and IL-4/5. Expression of antigen-presentation molecules CD80 and CD86 on B cells were unchanged. Total CD3+ T cells, activated and proliferating CD4+ and CD8+ T cells, and cytotoxic granzyme-B+ CD8+ T cells were reduced in treated mice. Interestingly, CD4+ CD25+ FoxP3+ Treg number, expression of PD-1 on Tregs and CD4+ CXCR5+ PD-1+ T follicular helper cells were also reduced in both treatment groups. In addition, there were globally reduced numbers of cytokine-secreting CD4+ cells but no differences in Th1/Th2 or Th17/Treg ratios were observed. Post-treatment, proliferation and cytokine secretion of B cells and T cells were still lowered but less impaired than during treatment. Tregs and PD-1 expression were still reduced post-treatment. Finally, circulating levels of IgA were reduced during and post treatment in vecabrutinib-treated mice compared to vehicle, while IgG1, IgG2b were reduced in both treated groups. No changes in IgM levels were observed in either treatment group. In conclusion, vecabrutinib treatment demonstrated efficacy and beneficially regulated B cell and T cell immune subsets in a preclinical murine model of sclerodermatous cGVHD. Studies to further evaluate differences between vecabrutinib and ibrutinib treatment are ongoing. Figure 1 Figure 1. Disclosures Fox: Sunesis Pharmaceuticals: Current Employment. Taverna: Sunesis Pharmaceuticals: Current Employment. Pinilla Ibarz: Sellas: Other: ), patents/royalties/other intellectual property; MEI, Sunesis: Research Funding; AbbVie, Janssen, AstraZeneca, Takeda: Speakers Bureau; AbbVie, Janssen, AstraZeneca, Novartis, TG Therapeutics, Takeda: Consultancy, Other: Advisory.

Background: AMV564 is a novel bivalent, bispecific (2x2) CD33/CD3 targeted immunotherapy that binds both CD33 and the invariant CD3ε on T-cell receptors with strong avidity, thus creating an immune synapse between CD33-expressing cells and T cells, initiating T-cell directed lysis of CD33 expressing cells, and inducing expansion, differentiation and proliferation of T cells. AMV564 is being evaluated in clinical trials for patients with acute myeloid leukemia (AML, NCT03144245). Previously, we demonstrated that the parent molecule, T564, had specific, T-cell mediated cytotoxic activity against a KG-1 CD33+ cell line in vitro and eliminated blasts in an autologous AML patient-derived xenograft mouse model (Eissenberg, et al. 2015 ASCO). Here we investigated factors that may contribute to antileukemic activity of AMV564 in another, more aggressive, preclinical model of AML. Methods: Disseminated activity studies in NOD scid gamma mice (NSG) were performed by injecting MOLM13 AML cells transduced with click beetle red luciferase and green fluorescent protein (MOLM13-CG) in the tail vein. In a series of experiments, AMV564-mediated clearance of MOLM13-CG cells and overall mouse survival was determined. Tumor cells (either 3.3 x 103 or 1 x 105) were engrafted in non-conditioned NSG mice for 3-7 days. Variables included dose of AMV564 (0.5 to 25 mcg, i.v.), number of AMV564 cycles (1 or 2) and cycle length (4-5 days), number of T cell injections (1 or 2), and total number of human T cells administered (0.2 to 1.2 x 107). Tumor burden was serially measured by bioluminescence (BLI) and survival measured for 51 days. Results: Mouse survival was greatest when AMV564 was injected along with a total number of T cells ≥ 8 x 106 regardless of the number of T cell injections or their timing relative to tumor injection. AMV564 mediated up to a 3-fold increase in survival time compared to untreated mice. In the experiment shown in Figure 1, untreated mice died at day 28, yet about half of the treated mice were still alive when the experiment was terminated on day 51. In the absence of AMV564 (i.e., T cell treatment only), animal survival and tumor burden were similar to that of untreated animals. In the same experiment we administered, AMV564 daily from days 7-11 after MOLM13-CG infusion along with one injection of 8 x 106 human T cells on day 7, then measured tumor burden by BLI signal (Figure 2). By day 14 we observed a median 4.0 log or 4.8 log reduction in tumor using 5 or 25 mcg AMV564. In fact by day 14, 4 of 10 mice treated with 25 mcg AMV564 and 2 of 10 treated with 5 mcg had no detectable signal above background. No rebound in tumor burden occurred in these mice throughout the 51-day study. Conclusions: AMV564 is a potent and effective immunotherapy against an aggressive human AML cell line in NSG mice. AMV564 very effectively prolonged survival and dramatically reduced tumor in the bone marrow and peripheral blood. These results are consistent with our previous preclinical data with primary AML and support the testing of this reagent in patients with relapsed and refractory AML in the clinic. Disclosures Eissenberg: Amphivena Therapeutics: Research Funding; Novimmune: Research Funding. Rettig:Novimmune: Research Funding; Amphivena Therapeutics: Research Funding. Fox:Sunesis Pharmaceuticals: Employment; Amphivena Therapeutics: Employment. Guenot:Amphivena Therapeutics, Inc: Employment.

B cell receptor signaling (BCR) in chronic lymphocytic leukemia (CLL) drives tumor cell proliferation and survival. Inhibition of Bruton's tyrosine kinase (BTK), a key enzyme in the BCR pathway, has proved to be efficacious even in poor-risk and chemo-refractory patients. However resistance to the BTK inhibitor ibrutinib has been shown to emerge in a subset of CLL patients. Of importance, the C481S BTK mutation conferred resistance by preventing the covalent binding of ibrutinib to its target cysteine 481 in BTK. Vecabrutinib (formerly known as SNS-062, a succinate salt) is a novel, highly potent, next generation noncovalent BTK inhibitor which demonstrated biochemical and cellular activity against C481S BTK mutant in vitro. However, the efficacy of vecabrutinib and its impact on the T-cell microenvironment has not been studied in in vivo preclinical CLL models. In the present study, the efficacy of vecabrutinib was investigated using the Eµ-TCL1 adoptive transfer model. Mice were randomized to treatment with either 40mg/kg vecabrutinib succinate, twice daily by oral gavage (n=6) or vehicle control (n=6). The mice were sacrificed after 2 weeks of treatment and changes in tumor burden as well as alterations in T-cell microenvironment were analysed in detail. Treatment with vecabrutinib decreased tumor burden as observed by a significant decrease in WBC count (36.5 vs. 17.1 giga/L; P=0.002), spleen weight (median 0.56g vs. 0.31g; P=0.005) and liver weight (median 1.5g vs. 1.2g; P=0.005) compared to vehicle treatment. Correspondingly, the CD5+ CD19+ tumor cells were significantly decreased in blood (P=0.002) and spleen (P=0.002) while no significant difference was observed in bone marrow (P=0.818) upon treatment with vecabrutinib. Since BTK inhibition is known to reshape the tumor microenvironment, we studied the impact of vecabrutinib specifically on T-cell subsets. Firstly, no difference in the proportions of CD4 or CD8 expressing T-cells was observed in mice treated with vehicle or vecabrutinib. However, of interest, the percentage of CD4+ CD25+ FoxP3+ regulatory T cells (Tregs) were significantly decreased upon treatment with vecabrutinib in peripheral blood (P=0.026) and spleen (P=0.009). The decrease in Tregs was due to reduced proliferation of these cells upon exposure to the drug as measured by Ki-67 staining. Also, the Tregs expressing the maturation and activation markers such as CD103 and GITR were significantly decreased in blood and spleen upon drug treatment. Further, we analysed the changes in CD8 T-cell subsets following treatment with vecabrutinib. Treatment with the drug resulted in expansion of the CD127+ CD44- naïve CD8 T-cells in blood, bone marrow and spleen (all P values 0.002) while the CD127+ CD44+ memory CD8 T-cells were significantly decreased in bone marrow and spleen (all P values 0.009). Also, the CD127low CD44int-hi effector CD8 T-cells were decreased in blood (P=0.004), bone marrow (P=0.004) and spleen (P=0.002) upon vecabrutinib treatment. Therefore, vecabrutinib treatment did not alter the percentage of CD4+ and CD8+ T cells in mice however, significant changes in the subset composition of the CD4 and CD8 T cells were observed. Lastly, to analyse the impact of vecabrutinib on survival, a cohort of mice (n=12) were transplanted with 5 million splenic tumor cells isolated from Eµ-TCL1 transgenic mice. After allowing for engraftment, the mice were randomized to treatment with the drug (n=6) or vehicle (n=6). Of note, the mice treated with the drug showed a significant increase in survival (median 35 days from transplant; P In summary, vecabrutinib was efficacious in vivo in a preclinical CLL adoptive transfer model, decreasing tumor burden in different organs and significantly improving survival. Treatment with the drug altered the T-cell architecture in vivo. Of interest, the immunosuppressive Tregs, which protect the tumor from immune surveillance were decreased in various tissue compartments; however, a decrease in the effector CD8 T cells might impact anti-tumor immunity if there is a consistent effect upon drug treatment. Vecabrutinib antitumor activity and effects on T-cell populations in vivo in this preclinical CLL model are intriguing, merits further investigation and supports the ongoing phase 1b/2 study in patients with previously treated B-lymphoid malignancies (NCT03037645). Disclosures Tausch: AbbVie: Consultancy, Other: Travel grants; Celgene: Consultancy, Other: Travel grants; Gilead: Consultancy, Other: Travel grants. Fox:Sunesis Pharmaceuticals: Employment; Amphivena Therapeutics: Employment. Taverna:Sunesis Pharmaceuticals: Employment. Stilgenbauer:Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Hoffmann La-Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmcyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Background: SNS-062 is a potent, noncovalent (reversible) BTK inhibitor in development for B-cell malignancies and other cancers. SNS-062 has the potential for activity in patients whose cancers are sensitive to BTK inhibition, as well as those that are resistant to ibrutinib through acquisition of a BTK Cys481Ser mutation. In vitro studies have demonstrated that SNS-062 antitumor activity in cells with the mutation is unaffected (Binnerts et al, EORTC 2015, Abstract C186), in contrast to the substantially reduced activity seen with ibrutinib and acalabrutinib. This study was designed to evaluate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of SNS-062 in healthy subjects. Methods: This was a phase 1a, first-in-human, randomized, double-blind, placebo-controlled, sequential-group, single-dose study conducted in 3 stages. In stage 1, four sequential cohorts of 8 subjects were randomly assigned to receive ascending SNS-062 dose levels (50, 100, 200, and 300 mg; n=6, 3 male, 3 female) or placebo (n=2, 1 male, 1 female) as a single dose administered orally. The primary endpoint was safety, assessed by adverse events (AEs), laboratory parameters, and cardiac monitoring. Secondary endpoints included PK parameters and PD parameters (inhibition of phosphorylation BTK [pBTK] as determined by ELISA in whole blood lysates). Stages 2 and 3 were designed to evaluate the effects of food and CYP3A4 inhibition, respectively, on the PK of SNS-062. Results: In stage 1 (n=32), the median age was 55 years (range: 22-64) among those who received SNS-062 (n=24) and 42.5 years (range: 29-65) among those who received placebo (n=8). Treatment-emergent AEs (TEAEs) were reported for 8 (33%) subjects who received SNS-062 and for 3 (38%) subjects who received placebo. TEAEs reported for subjects who received SNS-062 included headache (n=5) and nausea, constipation, bronchitis, fatigue, orthostatic hypotension, and supraventricular tachycardia (n=1 each) without obvious evidence of dose dependency. AEs in the placebo group included headache (n=2), nausea (n=2), and diarrhea (n=1). AEs were all reported as Grade 1 except for 1 subject (who received 300 mg SNS-062) who experienced Grade 2 headache and fatigue. No Grade 3 or higher AEs and no serious AEs were reported. SNS-062 was rapidly absorbed (median Tmax: 1 hour [range: 0.5-3.0 hours]). SNS-062 concentrations declined in a multiphasic manner. Exposure increased approximately proportional to dose. Mean PK parameters for each cohort are shown in the Table. SNS-062 demonstrated rapid and near complete inhibition of pBTK at all dose levels. Stages 2 and 3 are in progress and results of the completed study will be reported at the meeting. Conclusions: The observed safety, PK, and PD profiles of SNS-062 in this phase 1a study in healthy subjects support further clinical investigation. This study continues to evaluate the effects of food and CYP3A4 inhibition on SNS-062 PK. Mean SNS-062 exposure at 50 mg, the lowest dose level studied, exceeded those reported for ibrutinib (Imbruvica [package insert]. Sunnyvale, CA: Pharmacyclics, LLC; 2016) and acalabrutinib (Byrd et al, N Engl J Med 2016;374:323:32) when those drugs are administered at recommended dose levels. The extent of SNS-062 exposure and duration of pBTK inhibition are encouraging and support twice-daily dosing in a planned phase 1b/2 study in patients with advanced B-cell malignancies with and without the BTK Cys481-Ser mutation. This study was sponsored by Sunesis Pharmaceuticals. Disclosures Neuman: Puma Biotechnology: Employment; Sunesis Pharmaceuticals: Employment. Ward:Sunesis Pharmaceuticals: Consultancy, Employment. Arnold:Sunesis Pharmaceuticals: Consultancy. Combs:Sunesis Pharmaceuticals: Consultancy. Gruver:Sunesis Pharmaceuticals: Employment. Hill:Sunesis Pharmaceuticals: Employment. Miller:Sunesis Pharmaceuticals: Consultancy. Fox:Amphivena Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: Patent #9212225; Bispecific CD33 and CD3 Binding Proteins; Sunesis Pharmaceuticals: Consultancy, Equity Ownership, Patents & Royalties: Patent Application #20150202189; Methods of Using SNS-595 for Treatment of Cancer Subjects with Reduced BRCA2 Activity.

Inhibitors of cyclin-dependent kinases (Cdks) have been reported to have activities in chronic lymphocytic leukemia cells by inhibiting Cdk7 and Cdk9, which control transcription. Here we studied the novel Cdk inhibitor SNS-032, which exhibits potent and selective inhibitory activity against Cdk2, Cdk7, and Cdk9. We hypothesized that transient inhibition of transcription by SNS-032 would decrease antiapoptotic proteins, resulting in cell death. SNS-032 effectively killed chronic lymphocytic leukemia cells in vitro regardless of prognostic indicators and treatment history. This was associated with inhibition of phosphorylation of RNA polymerase II and inhibition of RNA synthesis. Consistent with the intrinsic turnover rates of their transcripts and proteins, antiapoptotic proteins, such as Mcl-1 and X-linked inhibitor of apoptosis protein (XIAP), were rapidly reduced on exposure to SNS-032, whereas Bcl-2 protein was not affected. The initial decrease of Mcl-1 protein was the result of transcriptional inhibition rather than cleavage by caspase. Compared with flavopiridol and roscovitine, SNS-032 was more potent, both in inhibition of RNA synthesis and at induction of apoptosis. SNS-032 activity was readily reversible; removal of SNS-032 reactivated RNA polymerase II, which led to resynthesis of Mcl-1 and cell survival. Thus, these data support the clinical development of SNS-032 in diseases that require short-lived oncoproteins for survival.

Introduction: Despite 40 years of intense clinical research, there remain no new approved treatments or standard of care for patients with relapsed or refractory (R/R) acute myeloid leukemia (AML). New safe and effective salvage treatments are urgently needed. Vosaroxin is a first-in-class anticancer quinolone derivative that is active in AML. Vosaroxin is minimally metabolized, evades P glycoprotein receptor–mediated efflux and has activity independent of p53 status. VALOR is a rigorously designed and conducted phase 3, adaptive design, randomized, double-blind, placebo-controlled trial evaluating vosaroxin plus cytarabine (vos/cyt) vs placebo plus cytarabine (pla/cyt) in patients with R/R AML (NCT01191801). Methods: Patients were randomized 1:1 to receive cytarabine (1 g/m2 IV over 2 hr, d 1-5) plus either vosaroxin (90 mg/m2 IV over 10 min d 1 and 4; 70 mg/m2 in subsequent cycles) or placebo. Up to 2 induction and 2 consolidation cycles were administered. Eligible patients had refractory disease (persistent disease after induction, or first complete remission [CR1] < 90 d) or were in first relapse (early relapse: CR1 of 90 d to 12 mo; late relapse: CR1 of 12 mo to 24 mo). Patients had received 1-2 cycles of prior induction chemotherapy including at least 1 cycle of anthracycline (or anthracenedione) and cytarabine. Randomization was stratified by disease status (refractory, early relapse, late relapse), age (< 60, ≥ 60 years), and geographic location (US, non-US). Primary efficacy and safety endpoints were overall survival (OS) and 30- and 60-day mortality; secondary endpoints were complete remission (CR) rate and incidence of adverse events (AEs). Results: Between Dec 2010 and Sept 2013, 711 patients were randomized to receive vos/cyt (n = 356) or pla/cyt (n = 355) at 124 sites; per the adaptive design, a prespecified 1-time sample size increase of 225 patients was implemented after the interim analysis. At the final analysis, median OS was 7.5 mo (95% CI: 6.4-8.5) with vos/cyt vs 6.1 mo (95% CI: 5.2-7.1) with pla/cyt (HR = 0.866 [95% CI: 0.73-1.02]; 2-sided unstratified log-rank P = 0.06) (Figure). The OS difference was statistically significant in a preplanned analysis accounting for the stratification factors at randomization (2-sided stratified log-rank P = 0.02). Overall, 29.5% of patients underwent allogeneic stem cell transplant (ASCT), including 45.8% of patients < 60 years and 20.2% of patients ≥ 60 years. Transplant rates were comparable between the 2 treatment arms (30.1% with vos/cyt and 29.0% with pla/cyt). In a predefined analysis censoring for subsequent ASCT, median OS was improved with vos/cyt (6.7 mo vs 5.3 mo with pla/cyt; HR = 0.81 [95% CI: 0.67-0.97]; P = 0.02; stratified P = 0.03) (Figure). In predefined subgroup analyses, OS benefit was greatest in patients aged ≥ 60 years (7.1 mo with vos/cyt vs 5.0 mo with pla/cyt; HR = 0.75; P = 0.003) (Figure) and those with early relapse (6.7 mo vs 5.2 mo; HR = 0.77; P = 0.04). OS with vos/cyt vs pla/cyt was 9.1 mo vs 7.9 mo in patients < 60 years (HR = 1.08; P = 0.60); 6.7 mo vs 5.0 mo in patients with refractory disease (HR = 0.87; P = 0.23); and 14.1 mo vs 12.3 mo in patients with late relapse (HR = 0.98; P = 0.96), respectively. A CR was achieved in 30.1% of patients treated with vos/cyt vs 16.3% treated with pla/cyt (P = 0.00001). Thirty-day and 60-day all-cause mortality was similar in the 2 arms (30-day: 7.9% vs 6.6%; 60-day: 19.7% vs 19.4% with vos/cyt vs pla/cyt, respectively). Most common serious AEs were febrile neutropenia (11.3% with vos/cyt vs 7.4% with pla/cyt), sepsis (8.7% vs 4.3%), pneumonia (7.6% vs 4.9%), bacteremia (8.5% vs 2.9%), and stomatitis (3.4% vs 1.4%). Serious and non-serious cardiac, renal, neurologic, and hepatic AEs were comparable between treatment groups. Conclusion: Vos/cyt demonstrated improved OS and higher CR rates in patients with R/R AML without increased early mortality. In the primary OS analysis, the overall clinical benefit associated with vosaroxin may be underestimated, particularly in younger patients, due to the confounding effect of high transplant rates, a methodological limitation of AML trials. Vosaroxin-containing therapy had acceptable tolerability. VALOR results represent one of the largest datasets available in this setting, and the OS benefit was confirmed by a robust sensitivity analysis. These data support the use of this combination as a new option for salvage therapy in patients with R/R AML. Figure 1 Figure 1. Disclosures Ravandi: Sunesis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Sayar:Sunesis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Strickland:Sunesis: Membership on an entity's Board of Directors or advisory committees. Schiller:Sunesis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Erba:Sunesis: Consultancy; Seattle Genetics: Consultancy; Novartis: Consultancy; Incyte: Consultancy; Celgene: Consultancy; Amgen: Consultancy. Pigneux:Sunesis: Consultancy. Horst:Sunesis: Research Funding. Recher:Sunesis: Consultancy; Celgene: Consultancy, Research Funding; Chugai: Research Funding. Klimek:Sunesis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Craig:Sunesis: Equity Ownership. Fox:Sunesis: Consultancy, Equity Ownership. Ward:Sunesis: Employment, Equity Ownership. Smith:Sunesis: Employment, Equity Ownership. Acton:Sunesis: Consultancy. Mehta:Sunesis: Consultancy. Stuart:Sunesis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 635 Background: Voreloxin is a first-in-class anticancer quinolone derivative (AQD) that intercalates DNA and inhibits topoisomerase II, inducing apoptosis. Nonclinical studies showed synergistic activity when combined with cytarabine (ara-C). We report preliminary results of a Phase (Ph) 1b/2 study of combination voreloxin plus ara-C in relapsed/refractory AML patients. Objectives: 1) safety, tolerability and MTD of escalating doses of voreloxin in combination with ara-C; 2) voreloxin PK; 3) clinical activity by IWG; 4) pharmacodynamic (PD) markers; 5) ex vivo voreloxin resistance in bone marrow aspirates (BMA). Methods: Ph 1b/2 study with dose escalation of voreloxin given on Days 1 and 4 in combination with 2 schedules (sch) of ara-C: Sch A (A-CIV): 400 mg/m2/d continuous IV infusion ara-C X 5 days; Sch B (B-Bolus): 1 g/m2/d 2 hr IV infusion ara-C X 5 days. Voreloxin Ph 1b doses: 10 - 90 mg/m2 (A-CIV); 70 - 90 mg/m2(B-Bolus). At MTD, pts are enrolled into Ph 2 arms (dose expansion) to assess response and safety in a first relapse (1st REL) population (both sch), and a primary refractory (1oREF) population (B-Bolus only). Pre- and post-dose PBMC were probed for mechanism-based PD responses by western blot probing for DNA damage markers. Pt BMA taken prior to dosing were analyzed ex vivo for resistance to voreloxin and ara-C by a proliferation assay. Clinical response was determined by IWG criteria. Results: To date, 94 patients have been treated. Preliminary safety and overall response rate (ORR = CR + CRp) are available for Ph 1b for both cytarabine schedules and Ph 2 1st REL for A-CIV. Ph 2 B-Bolus 1st REL has completed enrollment (N=15); enrollment continues for Ph 2 B-Bolus (1oREF). Grade 3 or higher non-hematologic AEs ≥ 10% incidence were markedly reduced in Ph 1b B-bolus vs Ph1b A-CIV, notably for infection related AEs and mucositis and 30-day all-cause mortality. Activity: Tabulated results show activity with both ara-C schedules; CR+CRp seen in difficult-to-treat pt populations. In Ph 1b at doses ≥ 80 mg/m2, 1oREF pts ORR was 36% (5 of 14); all but one had failed multiple cycles of induction therapy. A high number of pts have been bridged to transplant (BMT): 8 BMT of 20 CR and 1 PR. CR + CRp have been noted in both B-Bolus Ph 2 expansions, but outcome and safety data are immature. PK/PD: Voreloxin PK were dose proportional from 10–90 mg/m2. DNA damage PD response markers, increased pDNA-PKcs and/or pCHK2, were seen in 12 of 23 patients analyzed who received ≥ 34 mg/m2 voreloxin. Ex vivo resistance assays of pts' BMA to voreloxin and ara-C suggest that voreloxin is a major contributor to the observed CRs and that the combination has enhanced activity. Conclusions: Voreloxin given in combination with continuous infusion ara-C is well-tolerated, with an encouraging number of pt successfully bridged to BMT. An ORR of 33% was observed in 1st REL pts txd with ≥ 80 mg/m2 voreloxin (all remissions were in pts txd at MTD, 80 mg/m2 voreloxin). An ORR rate of 36% was observed in 1oREF pts, a population with an historical remission rate of 10-15%. CR + CRp have been noted in both B-Bolus Ph 2 expansions, but outcome and safety data are in process. Enrollment in Ph 2 B-Bolus 1oREF patients is ongoing.DemographicsPhase1b A-CIV1b B-Bolus2 A-CIV2 B-Bolus2 B-BolusN391816156 of 25Gender71%M72%M69%MECOG 0-198%89%94%Disease StatusFrontline3%0%1st REL23%11%100%100%2nd REL15%11%1oREF33%55%100%REF REL21%22%Not available5%0%CytogeneticsPendingFavorable3%11%0%Intermediate64%28%50%Unfavorable31%22%31%Unknown3%6%6%Not available033%13%Safety–Grade 3 or Higher Adverse Events ≥ 10%Phase1b A-CIV1b B-Bolus2 A-CIVN391816Febrile N54%40%38%Pneumonia15%0%13%Sepsis/bact.38%27%56%Infections15%7%19%Upper GI13%7%6%Lower GI13%0%13%Hypokalaemia28%7%31%Hyperglycaemia13%7%13%Hypophosphataemia10%0%19%Hypoxia3%0%13%Pulmonary Hemorrhage0%0%13%Hypotension10%0%0%DLTN=2 of 7 pts at 90 mg/m2 Grade 3 diarrhea and bowel obstruction Grade 3 oral mucositis lasting ≥ 7 daysN=1 of 6 pts at 80 mg/m2and 90 mg/m2 Odynophagia Grade 3 oral mucositis lasting ≥ 7 daysMTD/Recommended Ph 2 Dose80 mg/m290 mg/m280 mg/m2Outcome1b A-CIV1b B-Bolus2 A-CIV2 B-Bolus2 B-BolusPopulationREL or REFREL or REF1st REL1st REL1oREFN391816156 of 25Median OS days (95% CI)209 (74-258)NANANANACR6363 CR, 1 CRp of 10 evaluable1 CR of 2 evaluableCRp311%CR+CRp22%23%44%TETETETE30-day all-cause mortality21%0%6%0 of 10 evaluable0 of 4 evaluable Disclosures: Lancet: Sunesis Pharmaceuticals: Study Steering Committee. Roboz:Sunesis Pharmaceuticals: Study Steering Committee. Cripe:Sunesis Pharmaceuticals: Research Funding. List:Sunesis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Fox:Sunesis: Employment. Michelson:Sunesis: Employment. Karp:Sunesis Pharmaceuticals: Research Funding.

Abstract 1037 Poster Board I-59 Background: Voreloxin is a first-in-class anticancer quinolone derivative (AQD) that intercalates DNA and inhibits topoisomerase II, inducing apoptosis. Interim results of REVEAL-1, a Phase 2 (Ph 2) dose regimen optimization study of 3 schedules (sch) of single agent voreloxin in newly diagnosed elderly acute myeloid leukemia (AML) patients (pts), are reported. The initial dose regimen, voreloxin 72 mg/m2 qw x 3, was established in a Ph 1 dose-escalation study in relapsed/refractory leukemia pts (Lancet J et al., Proc ASH 2007). Overall remission rate (ORR = CR+CRp) was high (41%), but this regimen was less well tolerated in the frontline elderly population. The protocol explored 2 alternative voreloxin sch: 72 mg/m2 voreloxin qw x 2 and days 1 and 4 (d 1,4). A final cohort of pts is enrolling to the d 1,4 sch at 90 mg/m2. Dose escalation was based on safety data from the 72 mg/m2 cohort and from an ongoing Ph 1b/2 study of 90 mg/m2 voreloxin d 1,4 in combination with 1g/m2/d cytarabine x 5d (Lancet J, et al, ASCO 2009). Methods: Ph 2 study of 3 voreloxin sch (30 pts/sch): A 72 mg/m2 qw x 3 or B 72 mg/m2 qw x 2 or C 72 mg/m2 on d 1,4. C at 90 mg/m2 voreloxin is now enrolling to 20 pts. Eligibility: newly diagnosed AML (de novo or secondary AML), pts age ≥ 60 with ≥ 1 additional adverse risk factor (age ≥ 70, secondary AML, intermediate or unfavorable cytogenetics, or PS 2). PK were evaluated in a pt subset in cycle 1. Patient bone marrow aspirates (BMA) taken prior to dosing were tested ex vivo for extreme drug resistance (EDR®) to voreloxin. Results: To date, 105 pts have been treated. Preliminary safety and ORR are available for A, B and C at 72 mg/m2 voreloxin. Twelve pts in C at 90 mg/m2 are too early to evaluate (TETE). Overall incidence of infections and mucositis were reduced in B and C, sch with 2 voreloxin doses, relative to A which had 3 voreloxin doses. Voreloxin PK were similar to those in an earlier Ph 1 study in relapsed/refractory AML (Lancet J, et al., Proc ASH 2007). Pts whose BMA were inhibited < 48% by 1 μM voreloxin had a greater chance of treatment failure (p = 0.043) than those whose BMA were inhibited by ≥ 48%. Conclusions: In REVEAL-1, voreloxin demonstrates clinical activity with 3 dosing sch in previously untreated elderly (age ≥ 60) AML pts who are unlikely to benefit from standard chemotherapy. ORR across 3 sch was 35%; the majority (76%) were CR. Responses were seen in each risk factor category and with multiple risk factors. Of reinduced pts, 45% achieved CR(p). Durable remissions exceeding 6 months were observed in 50% (A) and 63% (B) thus far. C d 1,4 was selected for further development based on ORR (38%), 30 and 60 day all-cause mortality (7% and 14%, respectively) and an improved safety profile with lower rates of infection compared to previous schedules. Further accrual to C 90 mg/m2voreloxin d 1,4 is ongoing. Disclosures: Ravandi: Sunesis: sunesis study steering committee. Cripe:Sunesis: Research Funding. Chen:sunesis: Employment. Mahadocon:sunesis: Employment. Fox:Sunesis: Employment. Berman:Sunesis: Employment. Michelson:sunesis: Employment. Stuart:sunesis: sunesis study steering committee.

SNS-032, formerly BMS-387032, is a highly selective and potent inhibitor of cyclin-dependent kinases (CDK) 2, 7 and 9. CDK2 and CDK7 are involved in cell cycle regulation. CDK7, along with CDK9, regulate RNA polymerase (Pol) II-dependent transcription. Temporary inhibition of RNA Pol II-dependent transcription by SNS-032 has significant effects on short half-life transcripts and proteins, particularly survival factors, cell cycle regulatory proteins, and cytokines that are critical for the survival of malignant B-cells in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). A phase 1 dose-escalation study in patients with MM and CLL is ongoing with separate dose escalations for each indication. The study is designed to evaluate safety, pharmacokinetics (PK) and preliminary evidence of activity of a loading dose (LD) followed by a 6 hour infusion of SNS-032 given weekly for 3 consecutive weeks of each 28-day cycle. Dose and schedule aim to maintain for 6 hours threshold plasma concentrations of 115 ng/mL (in vitro IC90) and higher. The study incorporates an exploratory analysis of potential pharmacodynamic (PD) biomarkers such as decreased phosphorylation of RNA Pol II C-terminal domain to demonstrate inhibition of CDK7 and CDK9, and decreased expression of survival factors to indicate transcriptional inhibition. Methods: Previously treated patients with advanced CLL or MM, measurable disease, and ECOG status 0–1 were eligible. Increasing doses of SNS-032 given as an LD followed by a 6 hr infusion were evaluated. The total starting dose was 15 mg/m2 comprised of a LD of 5 mg/m2 followed by 10 mg/m2 over 6 hr with dose escalation by modified Fibonacci. PD studies of target modulation were performed on peripheral blood mononuclear cells (PBMC) obtained pre- and post-dose. Direct target modulation or downstream effects of target inhibition were evaluated. Results: 35 patients have been treated to date, 18 MM patients and 17 CLL patients. Median age was 61 (range 45–82), with 13 females and 24 males. Median number of prior therapies was 5 (range: 1–11). MM patients have received total doses of 15 – 75 mg/m2. No drug-related dose limiting toxicities (DLTs) or objective responses have been reported thus far in MM. CLL patients have received total doses of 15 –100 mg/m2. No drug-related DLTs were observed through the 50 mg/m2 dose cohort. At 75 mg/m2, concentrations of SNS- 032 exceeded IC90 (mean maximum concentration during the 6 hr infusion was 261 ± 45 ng/mL). Evidence of biochemical tumor lysis syndrome (TLS) was observed in all CLL patients treated at this dose level. One patient experienced a DLT of vascular leak syndrome and failure to receive all 3 cycle 1 doses. One CLL patient has been treated thus far at 100 mg/m2. This patient experienced TLS with a DLT of elevated liver function enzymes for >48 hr and received only 2 of 3 doses in cycle 1. No objective responses have been observed. PD analyses showed evidence of decreased Mcl-1 or XIAP in several patients. Conclusions: The mechanism of action of SNS-032 supports testing this agent in B-cell malignancies such as MM and CLL. A pharmacologically-derived dose regimen that sustains IC90 SNS-032 concentrations or higher for 6 hr is being studied; target levels were achieved and exceeded in cohort 5 (75 mg/m2) for both MM and CLL. No DLTs or objective responses have been observed thus far in MM. AEs and DLTs related to mild to moderate TLS were observed in CLL patients at 75 mg/m2 and higher. No objective responses have been observed. Preliminary evidence of target-specific PD modulation has been demonstrated. Enrollment in this trial is continuing.

Cyclin dependent kinases (Cdks) not only drive cell cycle progression, but also control transcription. For example, Cdk7 and Cdk9 phosphorylate specific sites on the C-terminal domain of RNA polymerase II (pol II) to promote transcription initiation and elongation. Our previous experience with flavopiridol, a non-selective pan-Cdk inhibitor, in primary chronic lymphocytic leukemia (CLL) cells demonstrated inhibition of RNA pol II-driven mRNA synthesis, reduction of the expression of short lived anti-apoptotic proteins, and induction of apoptosis in CLL cells in vitro (Blood106:2513–19, 2005). Here we studied a novel Cdk inhibitor SNS-032, a 2-aminothiazole derivative with potent and selective inhibitory activity against Cdk2, Cdk7 and Cdk9. We hypothesized that similar to flavopiridol, SNS-032 would induce apoptosis in CLL cells through transcriptional inhibition of anti-apoptotic proteins. To test this hypothesis, we first studied toxicity of SNS-032 in primary CLL samples. At the clinically relevant concentration of 0.3 μM, SNS-032 induced 61 ± 13% apoptosis after 24 hr (mean ± S.D., n=19). This occurred in a time- and concentration-dependent manner that plateaued after 10–12 hr. Additional studies demonstrated this was not due to inactivation of SNS-032. A similar amount of apoptosis was induced in samples with a deletion of p53 gene, indicating a p53-independent cell death. This is important since clinical resistance to fludarabine in CLL is associated with loss of p53 function. Peripheral blood mononuclear cells from healthy donors were less sensitive to SNS-032, suggesting SNS-032 is selective against CLL cells. In direct comparisons SNS-032 was more potent than flavopiridol; 0.1 μM SNS-032 induced a similar amount of apoptosis as did 3 μM flavopiridol. Secondly, we investigated the inhibition of transcription by SNS-032. SNS-032 strongly inhibited the phosphorylation of pol II at both the Ser2 and Ser5 sites of its C-terminal domain, which are the substrates of Cdk9 and Cdk7, respectively. Inhibition of pol II function was associated with a decrease in RNA synthesis, measured by the incorporation of [3H]uridine. After 6 hr incubation, 0.1 μM SNS-032 inhibited 80% of RNA synthesis. Again, SNS-032 was about 30-fold more potent than flavopiridol at inhibiting transcription. As the most sensitive targets of transcription inhibitors are mRNAs and proteins with short intrinsic half-lives, we measured the mRNA levels of short-lived anti-apoptotic proteins by real-time RT-PCR. SNS-032 significantly reduced the mRNA levels of Mcl-1, XIAP and Bcl-2. The protein levels of Mcl-1 and XIAP were also reduced accordingly, whereas Bcl-2 protein remained stable, consistent with a longer half-life of the protein. As CLL cells exemplify a biological context of a tumor that is dependent upon the continued expression of anti-apoptotic proteins for survival, we conclude that inhibition of their expression led to the SNS-032-induced cell death. Thus, while the inhibition of transcription induced by both SNS-032 and flavopiridol is associated with apoptosis in CLL cells, SNS-032 is about 30-fold more potent. This may be due to differences in potency against Cdks and/or differential protein binding. These in vitro results provide rationale for a phase 1 trial of SNS-032 administered to patients with advanced B-lymphoid malignancies that is currently ongoing in multiple centers.

HCD122 is a novel, fully human, IgG1 antagonistic monoclonal antibody targeting the CD40 receptor. This antibody blocks CD40-mediated signaling and is a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). Previous preclinical investigation confirmed expression of CD40 on myeloma cells in the majority of patients and reported antitumor activity of HCD122 against multiple myeloma cells ex vivo (Tai, Y et al. Cancer Res2005; 65(13): 5898–5906). This ongoing phase 1 study will determine the maximum tolerated dose of CHIR-12.12 in multiple myeloma patients (pts) who are relapsed or refractory after at least one prior therapy. Planned dose levels are 1, 3 and 10 mg/kg administered IV once weekly for 4 weeks. Each dose group will enroll 3–6 pts to evaluate safety, pharmacokinetics (PK) and clinical response. To date, 9 pts have been treated at 2 dose levels: 3 pts at 1 mg/kg and 6 pts at 3 mg/kg. Median patient age is 65 yrs (46–81 yrs); median number of prior therapies is 3 (2–12). No dose limiting toxicity (DLT) occurred at the 1mg/kg dose level. At 3 mg/kg, 1 DLT of grade 4 thrombocytopenia occurred in 1 pt. No other grade 3 and 4 lab abnormalities and adverse events have been reported. In 7 pts with available data, infusions were well tolerated, with easily managed grade 1–2 toxicities, primarily chills (5 pts), nausea (3 pts), pyrexia (2 pts), and arthralgia (2 pts) mainly reported during the first infusion. Preliminary PK analysis showed more than dose proportional - increase in Cmax and AUC at the 3 mg/kg dose level compared to the 1 mg/kg dose level. At the 3 mg/kg dose, antibody accumulation occurred week-to-week; the mean Cmax after the fourth infusion on Day 22 was 126.1 mg/mL(range 52 – 195 ug/mL) and HCD122 levels were measurable up to Day 57 and in one patient up to Day 99. One week after the last 3 mg/kg dose, trough levels ranged from 28 to 109 mg/mL. Of the 3 pts at 1 mg/kg, one showed stable disease (SD) for >23 weeks and two had progressive disease (PD) by week 5. Of the 6 pts at 3 mg/kg, one had partial response (PR) at week 9 and was confirmed at week 15, one had SD for > 5 weeks, and 4 had PD at week 5. One pt with PD terminated the study before final safety evaluation, and must be replaced before assessment of the 3mg/kg dose level is complete. Thus, in preliminary studies, HCD122 appears to be safe and well tolerated to date at doses of 1 mg/kg and 3 mg/kg weekly for 4 doses and shows promising anti-myeloma activity. Enrollment is continuing to determine MTD.

The CD40 antigen is expressed in most B-cell malignancies, including chronic lymphocytic leukemia (CLL), and represents an attractive target for antibody therapy. HCD122 is a high affinity, fully human IgG1 antagonistic anti-CD40 monoclonal antibody designed to exert antitumor activity as an inhibitor of CD40-ligand-mediated survival signals and as a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). In this phase 1 study to determine maximum tolerated dose (MTD) in CLL, eligible patients (pts) include those with relapsed or refractory disease after prior fludarabine treatment and are assigned to receive one cycle of 4 weekly infusions of HCD122 at doses ranging from 0.3 to 10 mg/kg depending upon the dose cohort. A standard phase I design is used with each dose cohort enrolling 3–6 pts for evaluation of MTD, toxicity, pharmacokinetics (PK), and pharmacodynamics (PD). To date, 14 pts have been treated at 3 dose levels: 3 pts at 0.3 mg/kg, 4 pts at 1 mg/kg, and 7 pts at 3 mg/kg. Median patient age was 65 yrs (41–74 yrs); median number of prior therapies was 3 (2–11); median WBC at enrollment was 38.8 K/μ L (3.4–284 K/μ L). No dose limiting toxicity (DLT) occurred at the 0.3 and 1 mg/kg dose levels. At 3 mg/kg, streptococcal sepsis was reported in 1 pt after 2 study infusions and was considered DLT. Transient asymptomatic grade 3 or 4 elevation of amylase and/or lipase occurred in 2 subjects. In 9 pts with available data, infusions were associated with manageable grade 1–2 toxicity, primarily chills (7 pts), nausea (4 pts), and fever (3 pts) that was most predominant with the first infusion. PK analysis showed rapid clearance of HCD122 at the 0.3 and 1mg/kg dose levels, with no detectable levels at 2 and 7 days after infusion, respectively. PK data were available from 6 of 7 patients receiving 3mg/kg. These data indicated that some accumulation of HCD122 occurred at this dose level. Complete antigen saturation data were available for 2 of these subjects. Both showed sustained 100% saturation of antigen on peripheral CD5+CD19+ cells throughout the treatment period. Peripheral CD40+ CLL cells dropped transiently with a mean of 31% during each infusion in the majority of patients but the decreases were not maintained week-to-week. There were no substantive changes in NK or T-cell populations during therapy. In summary, HCD122 was safe and well tolerated up to the 3mg/kg dose level that is currently under evaluation. PK analysis to date suggests that levels higher than 3 mg/kg will be necessary to sustain levels of HCD122 in the expected therapeutic range, and dose escalation will continue to the maximum tolerated dose.