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BackgroundMyelodysplastic Syndrome (MDS) is characterized by ineffective clonal hematopoiesis with peripheral blood cytopenias, leading to death from infection or bleeding. Azacitidine (AZA), a hypomethylating agent (HMA) is the standard of care for treatment of MDS patients (pts) with higher-risk MDS [Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017]. Responses to AZA occur in 50% of pts with significant effects on hematopoiesis ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence [Silverman LR, et al. Leukemia, 1993]. AZA treatment is associated with global DNA hypomethylation, including human endogenous retroviruses (HEV) which further activates innate immune signaling [Chiappinelli KB, et al.Cell, 2015]. The exact mechanism by which AZA improves hematopoiesis is unknown. AZA improves overall survival of pts, yet despite this, 100% of pts ultimately fail treatment with worsening cytopenias or transformation to leukemia [Silverman LR, et al. B. J Clin Oncol, 2002; Cancer, 2011]. Thus, understanding the mechanism of resistance and identification of targets which can reverse HMA failure and improve hematopoiesis in MDS pts is critical. Our clinical data demonstrate that AZA combined with Rigosertib (RIGO), a novel Ras mimetic that inhibits Ras/Raf signaling [Athuluri-Divakar SK, et al.Cell, 2016], yields a response rate of 54% of pts who were HMA failures [Navada SC, et al. EHA 2019]. The response was associated with significant improvement in hematopoiesis and represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The precise mechanism that leads to reversal of the resistance phenotype is poorly understood.

Background:Myelodysplastic syndrome (MDS) is a clinically heterogenous disease of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis, uni/multi-lineage dysplasia and a high tendency to transform into acute myeloid leukemia. Aberrant chromosomal and genetic lesions contribute to MDS pathogenesis which has been associated with chronic activation of the innate immune response and a hyperinflammatory microenvironment (Barryero L, et al.Blood, 2018). Dysfunction of Toll like receptors (TLR) and downstream effectors has been associated with the loss of progenitor function and differentiation of bone marrow (BM) cells in MDS patients. Azacitidine (AZA), a hypomethylating agent (HMA), is the mainstay of therapy for patients with higher-risk MDS (Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017) and carries an overall response rate (ORR) of 50% in patients with significant effects on hematopoiesis, ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence with survival benefits (Silverman LR, et al., Leukemia, 1993). The response to AZA is not durable and all patients relapse with worsening bone marrow failure.

We have shown that human high molecular weight kininogen is proangiogenic due to release of bradykinin. We now determined the ability of a murine monoclonal antibody to the light chain of high molecular weight kininogen, C11C1, to inhibit tumor growth compared to isotype-matched murine IgG. Monoclonal antibody C11C1 efficiently blocks binding of high molecular weight kininogen to endothelial cells in a concentration-dependent manner. The antibody significantly inhibited growth of human colon carcinoma cells in a nude mouse xenograft assay and was accompanied by a significant reduction in the mean microvascular density compared to the IgG control group. We also showed that a hybridoma producing monoclonal antibody C11C1 injected intramuscularly exhibited markedly smaller tumor mass in a syngeneic host compared to a hybridoma producing a monoclonal antibody to the high molecular weight kininogen heavy chain or to an unrelated plasma protein. In addition, tumor inhibition by purified monoclonal antibody C11C1 was not due to direct antitumor effect because there was no decrease of tumor cell growth in vitro in contrast to the in vivo inhibition. Our results indicate that monoclonal antibody C11C1 inhibits angiogenesis and human tumor cell growth in vivo and has therapeutic potential for treatment of human cancer. (Blood. 2004;104:2065-2072)

We report here that Janus kinase 3 (Jak3) is a primary response gene for interleukin-6 (IL-6) in macrophage differentiation, and ectopic overexpression of Jak3 accelerates monocytic differentiation of normal mouse bone marrow cells stimulated with cytokines. Furthermore, we show that incubation of normal mouse bone marrow cells with a JAK3-specific inhibitor results in profound inhibition of myeloid colony formation in response to granulocyte-macrophage colony-stimulating factor or the combination of stem cell factor, IL-3, and IL-6. In addition, mutagenesis of the Jak3 promoter has revealed that Sp1 binding sites within a -67 to -85 element and a signal transducer and activator of transcription (Stat) binding site at position -44 to -53 are critical for activation of Jak3 transcription in murine M1 myeloid leukemia cells stimulated with IL-6. Electrophoretic mobility shift assay (EMSA) analysis has demonstrated that Sp1 can bind to the -67 to -85 element and Stat3 can bind to the -44 to -53 STAT site in IL-6-stimulated M1 cells. Additionally, ectopic overexpression of Stat3 enhanced Jak3 promoter activity in M1 cells. This mechanism of activation of the murine Jak3 promoter in myeloid cells is distinct from a recently reported mechanism of activation of the human JAK3 promoter in activated T cells.

We report here that Janus kinase 3 (Jak3)is a primary response gene for interleukin-6 (IL-6) in macrophage differentiation, and ectopic overexpression of Jak3 accelerates monocytic differentiation of normal mouse bone marrow cells stimulated with cytokines. Furthermore, we show that incubation of normal mouse bone marrow cells with a JAK3-specific inhibitor results in profound inhibition of myeloid colony formation in response to granulocyte-macrophage colony-stimulating factor or the combination of stem cell factor, IL-3, and IL-6. In addition, mutagenesis of the Jak3promoter has revealed that Sp1 binding sites within a -67 to -85 element and a signal transducer and activator of transcription (Stat) binding site at position -44 to -53 are critical for activation of Jak3transcription in murine M1 myeloid leukemia cells stimulated with IL-6. Electrophoretic mobility shift assay (EMSA) analysis has demonstrated that Sp1 can bind to the -67 to -85 element and Stat3 can bind to the -44 to -53 STAT site in IL-6-stimulated M1 cells. Additionally, ectopic overexpression of Stat3 enhanced Jak3promoter activity in M1 cells. This mechanism of activation of the murine Jak3promoter in myeloid cells is distinct from a recently reported mechanism of activation of the human JAK3promoter in activated T cells.

Jak3, a member of the Janus kinase family of cytoplasmic tyrosine kinases, is expressed at low levels in immature hematopoietic cells and its expression is dramatically up-regulated during the terminal differentiation of these cells. To better understand the role of Jak3 in myeloid cell development, we have investigated the role of Jak3 in myeloid cell differentiation using the 32Dcl3 cell system. Our studies show that Jak3 is a primary response gene for granulocyte colony-stimulating factor (G-CSF) and the accumulation of tyrosine phosphorylated Jak3 correlated with cell growth inhibition and terminal granulocytic differentiation in response to G-CSF. Ectopic overexpression of Jak3 in 32Dcl3 cells resulted in an acceleration of the G-CSF–induced differentiation program that was preceded by G1 cell cycle arrest, which was associated with the up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 and down-regulation of Cdk2, Cdk4, Cdk6, and Cyclin E. In addition, ectopic overexpression of Jak3 appears to result in the inactivation of PKB/Akt and Stat3-mediated proliferative pathways in the presence of G-CSF. Similarly, overexpression of Jak3 in primary bone marrow cells resulted in an acceleration of granulocytic differentiation in the presence of granulocyte-macrophage colony-stimulating factor, which was associated with their growth arrest in the G1 phase of the cell cycle. Taken together, these results indicate that Jak3-mediated signals play an important role in myeloid cell differentiation.

Navada: Onconova Therapeutics Inc: Research Funding. Reddy:Onconova Therapeutics Inc: Equity Ownership, Research Funding. Silverman:Medimmune: Research Funding; Onconova Therapeutics Inc: Patents & Royalties, Research Funding; Celgene: Research Funding.

Navada: Onconova: Research Funding. Silverman:Mount Sinai School of Medicine: Employment; Onconova Therapeutics Inc.: Patents & Royalties, Research Funding; Celgene: Research Funding; Medimmune: Research Funding; Johnson and Johnson: Research Funding; Bayer: Research Funding.

Rigosertib (RIG) which functions as a RAS-mimetic, binds to the RAS-binding domain (RBD) impacting many pathways that involve RBD-mediated interactions, including the RAS-Raf and AKT/PI3-kinase pathways (Divakar et al., Cell 2016). Recently, we demonstrated that RIG also functions as an epigenetic modifier in MDS. RIG alone or in combination with Azacitidine (AZA) exerted epigenetic effects on the global histone post-translational modifications (PTMs), chromatin remodelers, HDACs, and DNMTs and caused enhanced apoptosis in MDS (MDS-L) and AML (BW-90) cell lines as well as in bone marrow cells (BM) derived from pts with MDS (Chaurasia et al ASCO 2016). In clinical trials, the combination of RIG and AZA demonstrated an overall response rate of 76% in all MDS patients; 62% response in pts who had failed prior hypomethyating agent (HMA) (Navada et al ASH 2016) and 85% in HMA naive pts. The growing evidence suggests that combined therapy may be more effective than either agent alone. To further study these effects, we explored activity of RIG in combination with Vorinostat (VOR), a histone deacetylase inhibitor, on the cell lines. Combined treatment in sequential order VOR/RIG and RIG/VOR potentiates far greater effects on the global PTMs, HDACs, DNMTs, chromatin remodelers, cell cycle checkpoint proteins and PI3/AKT pathway than RIG/AZA or AZA/RIG.

Reddy: Onconova Therapeutics Inc: Research Funding. Divakar:Onconova Therapeutics Inc: Research Funding. Vasquez-Del Carpio:Onconova Therapeutics Inc: Research Funding. Dutta:Onconova Therapeutics Inc: Research Funding. Baker:Onconova Therapeautics Inc: Consultancy. Reddy:Onconova Therapeutics Inc: Consultancy. Aggarwal:Onconova Therapeutics Inc: Research Funding.

Chari: Array BioPharma: Scientific Advisory Board Other; Celgene Corporation: Scientific Advisory Board, Scientific Advisory Board Other; Millennium Pharmaceuticals, Inc: Scientific Advisory Board, Scientific Advisory Board Other; Onyx Pharmaceuticals, Inc.: Scientific Advisory Board, Scientific Advisory Board Other. Reddy:Onconova Therapeutics, Inc.: Consultancy, Patents & Royalties. Reddy:Onconova Therapeutics, Inc.: Consultancy, Patents & Royalties. Dudley:Ecoeos, Inc: Consultancy, Equity Ownership; GNS Healthcare: Consultancy; GlaxoSmithKline: Consultancy; Ayasdi, Inc: Equity Ownership; NuMedii, Inc: Equity Ownership; Ubalo, Inc.: Equity Ownership. Jagannath:Onyx Pharmaceuticals, Inc.: Consultancy; Celgene Corporation: Scientific Advisory Board, Scientific Advisory Board Other; Millennium Pharmaceuticals, Inc. : Scientific Advisory Board, Scientific Advisory Board Other.

Rigosertib is a small molecule anti-cancer agent which inhibits the PI-3K and PLK pathways, promotes G2/M arrest, and selectively induces apoptosis in cancer cells. We have previously reported results of a phase I/II study of rigosertib in patients (pts) with MDS and AML who had relapsed or were refractory to hypomethylating agents (HMA), a population for which there are currently no approved second line therapies. Rigosertib appeared to be well tolerated in this pt population and to have biologic activity with reduction or stabilization of bone marrow (BM) blasts and improvement in the peripheral blood (PB) counts in few treated pts. Reduction in BM blasts by rigosertib was associated with increased survival. In the current analysis, we evaluate pt characteristics that may predict for response.We analyzed the results of a phase I/II study of Rigosertib that was conducted in pts with MDS and AML. In the phase I component, pts were entered in cohorts of escalating doses in a classic 3+3 design ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) administered for 72 to 144 hours. A MTD of 1375 mg/m2 was identified for the phase II component, and subsequent pts were treated with this dose as a CIV for 72 hours. BMs were performed at baseline, week 4, 8, and then q3 months.Twenty-two pts with MDS or AML refractory or relapsing to a HMA have been treated with rigosertib. The study cohort comprised pts with a diagnosis of int-2 MDS (2), high risk MDS (6), CMMOL (1), and AML (13 pts all with antecedent MDS). Responses according to IWG 2006 criteria were observed in the BM and PB: marrow CR (4), marrow PR (2). Two pts also had hematologic improvement of the erythroid (1) and platelet (1) lineages. Four pts had stable disease (SD) after treatment but their courses were complicated by infections requiring hospitalization and removal from study. Three pts were deemed to be inevaluable because they received < 2 cycles of treatment or did not have a follow-up BM evaluation. Thus, 10/19 evaluable pts (53%) demonstrated either a BM/PB response (6) or SD (4).The median overall survival (OS) of pts with marrow CR+PR (n=6) was 12 months versus 1.8 months for those without a BM response (n=9) (p=0.0159, log-rank test). Age was not a predictor of response. 1 out of 6 responders had a major elimination in the size of the clonal population. Non-responders did not have a change in the magnitude of the clone. Prior response to HMA was not a predictor for response to rigosertib. Those who did have a marrow CR or PR responded early with median time to response of 2-4 cycles. Those with higher blast counts were less likely to respond. At study entry, the median blast percentage of responders was 16% versus 44% for non-responders. Less than 20% blasts at study entry was a positive predictor of response (p=0.047). Of those pts who did not respond or were inevaluable, the majority (75%) had AML, many with a proliferative course. Nine of 19 pts developed cystitis manifested by dysuria and/or hematuria as a side-effect of therapy. Among responding patients, 5 of 6 had cystitis [grade (GR) 1 (2); GR 2 (1); GR 3 (2)] compared with 3 of 9 non-responders [GR 1(1); GR 2 (1); GR 3(1)] (p=0.08). In one responding pt with grade 3 cystitis, a cystoscopy was performed which revealed polypoid inflammatory changes of the mucosa with hemorrhage. Biopsy showed neutrophilic inflammation without malignant cells. Upon resolution of symptoms, treatment was restarted at 50% of the original dose without complications. Pts who developed symptomatic cystitis were treated with sodium bicarbonate with improvement. The relationship between cystitis and response is being investigated.Rigosertib appears to have biologic activity with reduction in BM blasts associated with increased survival and improvement in the PB counts in a subset of treated pts. Pts with <20% blasts at study entry had a greater likelihood of response. Pts with proliferative disease with rapidly rising or high wbc did not respond. Age, cytogenetic profiles, and response to prior therapy were not predictors of response. Cystitis may be a response related biomarker and requires further analysis. A phase III multicenter randomized trial is underway to compare rigosertib to best supportive care with a primary endpoint of OS in pts with higher risk MDS who have failed, progressed, or relapsed after treatment with HMA, and can be used to validate the observations reported here in a larger study.Reddy: Onconova: Equity Ownership, Research Funding. Holland:Onconova: Research Funding. Wilhelm:Onconova Therapeutics: Employment, Equity Ownership. Silverman:Onconova: Research Funding.

Rigosertib is a small molecule anti-cancer agent with a multi-targeted mechanism of action. It is a multi-kinase/PI3 kinase inhibitor that promotes G2/M arrest and selectively induces apoptosis in cancer cells. Leukemic cells exhibit significantly higher levels of sensitivity to rigosertib compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Chen Proc AACR 2008). Azacitidine is first-line therapy for patients (pts) with higher-risk MDS and produces a response rate of 50%. Pts relapsed or refractory to hypomethylating agents have a short life expectancy of approximately 4 to 6 months (Jabbour 2010, Prebet 2011). There are no approved second line therapies for this patient population.A phase I/II study of Rigosertib is being conducted in pts with MDS and AML. Pts with higher-risk disease had to have failed a hypomethylating agent. In the phase I component, pts were entered in cohorts of escalating doses in a classic 3+3 design ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) for durations from 72 hours to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments were administered every 3 to 4 weeks. A maximum tolerated dose of 1375 mg/m2 was identified for the phase II component, and subsequent pts were treated with this dose as a CIV for 72 hours. A CBC was performed weekly and a bone marrow (BM) was performed at baseline and week 4, 8, and then q3 months afterwards.Twenty-one patients with MDS or AML refractory/relapsed to a hypomethylating agent have been treated with rigosertib. The study cohort comprised pts with a diagnosis of intermediate-2 MDS (2 pts), high risk MDS (5 pts), chronic myelomonocytic leukemia (1 pt), and AML (13 pts) (all AML had an antecedent MDS). The median age was 79 years. 86% of pts were male. Patients received between 1–19 cycles of treatment. Their cytogenetic profiles were diverse with the most recurrent abnormalities including a complex karyotype (5 pts), normal (5 pts), monosomy 7 (4 pts), and trisomy 8 (2 pts). Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: marrow CR (4), hematologic improvement (HI) (2); erythroid (1) platelet (1). Time to response was 2–4 cycles. An additional 2 pts had a >50% BM blast decrease from baseline but not to < 5%. Three pts had stable disease after treatment but their courses were complicated by infections requiring hospitalization and removal from the study. Three pts were deemed to be inevaluable because they received less than 2 cycles of treatment or did not have a follow-up bone marrow evaluation. Thus, 9/18 evaluable pts (50%) demonstrated either a bone marrow/peripheral blood response (6) or stable disease (3). The median overall survival of those with marrow CR+PR was 10.1 months versus 2 months for those without a bone marrow response (p=0.0011, log-rank test). Of those pts who did not respond or were inevaluable, the majority (83%) had AML, many with a proliferative course. The most frequent grade 1–2 side-effects included dysuria, hematuria, fatigue, anorexia, nausea, and diarrhea. Possibly related grade 3 side-effects included fatigue, hematuria, and dyspnea, each in one pt. Six of 21 pts developed cystitis manifested by dysuria and/or hematuria. Among responding or stable patients, 5 of 9 had cystitis compared with 1 of 12 non-responders. Patients who developed symptomatic cystitis were treated with sodium bicarbonate with improvement. The relationship between dysuria and/or cystitis and response is being investigated.Rigosertib appears to be safe and well tolerated in patients with refractory or relapsed MDS and AML. It has biologic activity with reduction in BM blasts and improvement in the peripheral blood counts in a subset of treated pts, and these effects are associated with increased survival. Dysuria/cystitis may be a response related biomarker and requires further analysis. Data regarding pharmacokinetics and pharmacodynamics will be presented and correlated to response. Given promising initial results, a phase III multicenter randomized trial is underway to compare rigosertib to best supportive care with a primary endpoint of overall survival in patients with higher risk MDS who have failed, progressed, or relapsed after treatment with hypomethylating agents.Reddy: Onconova: Research Funding. Holland:Onconova: Research Funding. Wilhelm:Onconova: Employment, Equity Ownership. Silverman:Onconova: Research Funding.

No relevant conflicts of interest to declare.

Abstract 2333

ON 01910.Na a novel benzyl styryl sulfone derivative is under clinical development in hematologic malignancies. It is a multi-kinase/PI3 kinase inhibitor that promotes G2/M arrest and selectively induces apoptosis in cancer cells. Leukemic cells exhibit significantly higher levels of sensitivity to ON 01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). Azacitidine (AzaC), is first line therapy for patients (pts) with higher-risk MDS and produces a response rate of 50%. Pts relapsed or refractory to hypomethylating based therapies have a poor prognosis and there are no accepted effective second line treatments, thus a need for new agents.A phase I/II study of ON 01910.Na is being conducted in pts with hematological malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CIV) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks. A CBC is performed weekly and a bone marrow (BM) is performed at baseline and week 4, 8, and then q3 months thereafter. Pts with higher-risk disease had to have failed a hypomethylating agent.Ten pts with MDS or AML relapsed/refractory to a hypomethylating agent have been treated with ON 01910.Na thus far (table 1). The study cohort comprised pts with a diagnosis (Dx) RAEB-2 (4 pts), RAEB-T (1 pt), and AML (5 pts) (median age of 75 years). Their cytogenetic profile included 1 pt with normal, 2 with intermediate (+8), and 7 pts with poor risk cytogenetics (monosomy 7 and/or complex). Patients were treated between 5 and 70 weeks. Responses according to IWG 2006 criteria were observed in the BM and peripheral blood: Marrow CR (3), hematologic improvement (HI-P) (2); erythroid (1) platelet (1). An additional 2 pts had a >50% BM blast decrease from baseline but not to <5%. Thus, 5/10 (50%) demonstrate a bone marrow response. Survival of these pts was 7.3, 15.7, and 16.4 months; one patient remains on study 5+ months. Four of the five responders had MDS at the initiation of treatment: RAEB-2 (3), CMMoL (1), AML (1). Responders had monosomy 7 (2), trisomy 8 (1) and complex cytogenetics (2). One pt had an elimination of the MDS clone and the others had persistence of the abnormal karyotype throughout their treatment course. Five pts had SD without HI at 4 weeks, 2 pts progressed to AML. All 5 non-responders had AML; 4 with a proliferative course. These latter received only 2 (2) or 3 (3) cycles before succumbing to disease related infectious complications. Survival for these patients ranged from 1.3 – 2 months with a median duration on study of 42 days. The most frequent side effects grade2 2 for all pts included fatigue, anorexia, nausea, and dysuria in patients receiving extended duration infusions. One pt had a grade 3 urinary frequency. No hematologic toxicities occurred and no bone marrow toxicity or hypoplasia was noted. Pharmacokinetic studies are ongoing, data to date demonstrate no evidence of drug accumulation in patients who are treated repeatedly.ON 01910.Na appears to be safe and well tolerated in patients with refractory or relapsed MDS and AML. ON 01910.Na has biologic activity with reduction in BM blasts, eradication of the MDS clone and improvement in the peripheral blood counts in some pts. These effects are associated with increased survival albeit in limited numbers of pts treated thus far. Further study of ON 01910.Na is warranted to better define biologic activity, appropriate target populations and to define mechanism of action.Silverman: Onconova Therapeutics Inc: Research Funding, Research support of Clinical Trial. Wilhelm:Onconova Therapeutics Inc: Employment, Equity Ownership.

No relevant conflicts of interest to declare.

ON 01910.Na, a novel benzyl styryl sulfone derivative is currently under clinical development as an anticancer agent in solid tumors and hematologic malignancies. ON 01910.Na selectively targets tumor cells at the mitotic phase of the cell cycle, arrests leukemia cells at the G2/M stage and induces apoptosis in a time and dose dependent manner. ON 01910.Na affects multiple cellular pathways, has multikinase activity and has been shown to target multiple genetic pathways, including those that are regulated by cyclin dependent kinases. Our studies indicate that leukemic cells exhibit significantly higher levels of sensitivity of ON01910.Na compared to normal marrow progenitors and increasing cytotoxicity upon prolonged and repetitive exposure (Skidan Proc AACR 2006; Chen Proc AACR 2008). AzaC alters the natural history of higher risk MDS with effects on marrow function, reduction in the risk of leukemic transformation and increased overall survival (Silverman JCO 2002, 2006; Fenaux Lancet 2009). However, 50% of patients (pts) do not respond to AzaC and upon progression after treatment their prognosis is poor, thus, a need for alternative agents.A phase I/II study of ON 01910.Na is being conducted in pts with heme malignancies. In the phase I component pts are entered in cohorts of escalating doses in a classic 3+3 design in doses ranging from 650 up to 1700 mg/m2/d continuous IV infusion (CI) for durations from 72 hours up to 144 hours every 2 weeks (1 cycle) for 4 cycles of treatment during the induction phase. Subsequent treatments are administered every 3 to 4 weeks.This report details two pts with progressive disease following treatment for MDS with AzaC at a dose of 75mg/m2/d x 7 days SC every 4 weeks. Upon progression they were treated with ON 01910.Na at 1050 mg/m2/d CI x 72h to 144 hours. Patient 002 presented with MDS int-2 with myelofibrosis and mild splenomegaly, 8% marrow blasts, normal karyotype, and RBC transfusion dependence. AzaC therapy was initiated in 9/2004. A hematologic improvement (HI) was achieved according to modified IWG criteria with marrow myeloblasts < 5%, RBC transfusion independence and a persistence of mild splenomegaly. A stable HI was maintained for 4 years on monthly AzaC therapy until relapse. At progression (10/2008), marrow myeloblasts increased to 11%, RBC transfusion requirement resumed, platelets fell to 20 × 109/L, the spleen enlarged and a new cytogenetically abnormal clone emerged, 46,XY,del(20)(q11q13),i(21)(q10),der(21)t(1;21)(q12;p11) resulting in trisomy for 1q. Administration of ON 01910.Na 1050 mg/m2/d x 72 hours CI was initiated 3 months after the last dose of AzaC. After 2 cycles of treatment evaluation demonstrated a reduction in: marrow myeloblast to 1%, spleen size by 50% and the MDS clone by 74% by standard cytogenetic analysis. Following the 4th and 7th cycles of treatment with ON 01910.Na, marrow myeloblasts ranged from 1 to 3%, palpable splenomegaly resolved, RBC transfusions decreased by 33% and platelets rose to 80 × 109/L. Although the conventional karyotype normalized after the 4th cycle, all 3 abnormalites were detected by FISH in 30% of cells following the 4th and 7th cycles suggesting a loss of proliferative capacity of the MDS clone. Patient 004 presented with high risk MDS, 19% marrow blasts, pancytopenia and a complex karyotype. AzaC treatment was initiated in 9/2002 leading to a stable HI, with marrow myeloblasts < 5%, RBC transfusion independence, and persistence of the MDS clone with no change in the complex karyotype, including deletion of the MLL locus. The HI was maintained for 6.5 years with monthly AzaC therapy until progression. At relapse marrow myeloblasts increased to 19%, and the complex karyotype persisted unchanged from initial diagnosis. ON 01910.Na treatment started (3/2009) and evaluations at the 2nd, 4th and 7th cycles revealed: a) reduction in % marrow myeloblasts to 12%, 8% and 7% respectively; b) persistence of abnormal karyotype in over 90% of metaphase and interphase cells, irrespective of the decreased % blasts; c) increased platelets from 20 to 40 × 109/L. Patient 002 and 004 remain on treatment with ON 01910.Na at 8+ and 5+ months, respectively.Preliminary data suggest that ON 01910.Na has an anti-proliferative modulatory effect on the MDS clone in pts progressing after AzaC therapy and should be explored in a broader MDS population.Silverman: Onconova Therapeutics: Research Funding. Off Label Use: ON01910Na is investigational and not FDA approved. Wilhelm:Onconova: Employment. Reddy:Onconova Therapeutics Inc.: Consultancy, Equity Ownership, Grantee, Membership on an entity's Board of Directors or advisory committees. Holland:Onconova Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

ON 01910.Na, a small molecule multikinase inhibitor, promotes G2/M arrest and apoptosis. Key targets for this inhibitor include Plk1 (polo-like kinase, a cell cycle regulator), Cdk1, (cyclin dependent kinase, a mitotic regulator) and the PI-3 kinase pathway (Ramana Reddy et al. J. Med. Chem. 2008, Park et al, Oncogene, 2007, Gumireddy et al., Cancer Cell, 2005). The drug has been shown to have anti-tumor activity in in vitro and in vivo models. Phase I studies in >100 advanced cancer patients revealed that the drug is well tolerated. Further, in several ongoing Phase 1 clinical trials in patients with myelodysplastic syndromes (MDS), positive effects on hematological indicators have been noted (Sloand et al, ASH 2008). Based on these data, a Phase 2 single-arm study is in progress to assess the efficacy and safety of the drug in IPSS Intermediate-2 and High risk MDS patients. Single Cell Network Profiling (SCNP) using flow cytometry is a platform that measures multiple fluorescent parameters (up to 10) in each cell, including both surface markers and intracellular signaling proteins in response to extracellular network inputs. By simultaneously measuring the effects of drug exposure on several pathways within each cell type in a heterogeneous patient tissue sample, valuable data can be gained about drug interactions with specific cellular pathways and cell type selectivity. This information has potential implications for dose/schedule optimization and development of patient stratification biomarkers.Studies were designed to evaluate the in vitro effects of ON 01910.Na, at clinically relevant concentrations, on intracellular pathways in the human GM-CSF-dependent erythroblastic TF-1 cell line using SCNP in order to monitor transitional changes in the cell cycle, with a focus on the G2-M phase and to perform dose-dependent titrations of drug using these cell cycle readouts.The reagents chosen to measure cell cycle readouts were fluorochrome-conjugated antibodies that recognize cyclin B1, p-histone H3(S28) and p-Cdk1(Y15) and 4'6'-diamino-2-phenylindole (DAPI), a fluorescent dye that binds strongly to DNA. The phosphorylation status of p-histone H3(S28) and p-Cdk1(Y15), and the level of cyclin B1 expression are all determinants of the G2-M and/or M phase of the cell cycle. Dose dependent titrations of ON 01910.Na and its inactive analog ON 01911 were performed over a dose range starting at 10-5 M and decreasing to 10-10 M (dose range which includes pharmacologically achievable concentrations in humans) with 3-fold serial dilutions for eleven points after an exposure to the drug for either 24 or 48 hrs. Cells were processed for multiparameter flow cytometry by fixation, permeabilization and incubation with fluorchrome-conjugated antibodies.The data showed that at 24 hours after ON 01910.Na exposure there was a simultaneous increase in phosphorylation of histone H3(S28), a decrease in phosphorylation of Cdk-1(Y15), and accumulation of cyclin B1. These data suggest that ON 01910 exposure disrupted the G2/M cell cycle transition leading to mitotic arrest with subsequent apoptosis. TF-1 cell DNA content measured by DAPI verified this to be the case as increases in G2/M and sub-G1 (a measure of apoptotic cell death) were simultaneously observed. No significant effects on G2/M targets were observed when TF-1 cells were exposed to ON 01911, indicating the effects of ON 01910.Na on the cell cycle were specific to the drug. Maximal effects of ON-01910.Na on cell cycle signaling molecules were observed at a drug concentration of 0.37 mM and no further changes were seen at higher concentrations. These effects were also observed at 48 hours, although with more cell death.These data indicate that intracellular phosphorylation changes of histone H3(S28) and Cdk-1(Y15), in addition to accumulation of cyclin B1 with subsequent apoptosis, reflect possible mechanisms of action of ON 01910.Na. The assay will be used in ongoing clinical trials to measure the pharmacodynamic activity of the drug in MDS patient samples pre- and post-treatment.Soper: Nodality Inc.: Employment, Equity Ownership. Huang:Nodality Inc.: Employment, Equity Ownership. Wilhelm:Onconova Therapeutics Inc: Employment. Cosenza:Onconova Therapeutics Inc.: Consultancy. Reddy:Onconova Therapeutics Inc.: Consultancy, Equity Ownership, Grantee, Membership on an entity's Board of Directors or advisory committees. Cesano:Nodality Inc.: Employment, Equity Ownership. Greenberg:Nodality Inc.: Research Funding; Onconova Therapeutics Inc.: Research Funding. Fantl:Nodality, Inc.: Employment, Equity Ownership.

Signature abnormalities in the cell cycle and apoptotic pathway have been identified in mantle cell lymphoma (MCL), affording the opportunity to develop targeted therapies. Here, we report on a novel class of kinase inhibitors, styryl sulfones, that differ from prior cell cycle inhibitors in that they are not related to purines or pyrimidines. We observed that two closely related compounds, ON013100 and ON01370, altered the growth and cell cycle status of MCL lines and potently inhibited the expression of several important molecules, including CDK4, p53, MDM2, cyclin D, and cyclin B. Using both TUNEL and PARP assays, we found that these compounds caused apoptosis in MCL cells. In addition, using molecular analyses, we observed the modulation of caspase-3 activity but not the expression of Bcl family molecules in these cells. Next, we investigated the cytotoxicity of the MCL lines upon treatment with styryl sulfone compounds in combination with other currently used chemotherapeutic agents, such as doxorubicin (DOX) or vincristine (VCR). We found that the combination of DOX plus styryl sulfone or VCR plus styryl sulfone increased cytotoxicity by one log scale, compared with the single styryl sulfone compound. Thus styryl sulfones alone, or in combination with chemotherapeutic agents, present attractive opportunities for new drug development in MCL.