ALBERT

Description: The cytotoxic nucleoside cytarabine (Ara-C) is a cornerstone of AML induction and consolidation therapies, but drug resistance contributes to disease relapse. Among clinically relevant mechanisms of Ara-C resistance is the over-expression of cytidine deaminase (CDA). CDA degrades Ara-C through deamination into inactive metabolites and its increased expression in AML cells results in drug resistance. Therefore, nucleoside analogues that are not degraded by CDA may be new therapeutic agents for this disease. Through our efforts to develop novel nucleoside analogues that overcome mechanisms of resistance to Ara-C, we identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside. First, we tested whether 5FTRX was a substrate of CDA by evaluating its cytotoxicity in HEK-293 cells over-expressing CDA. Compared to wild type cells, Ara-C was 6-fold less active in cells over-expressing CDA (IC50 wild type HEK293: 3.7µM (95% CI: 3.2-4.2uM) vs IC50 CDA over-expression: 25.6uM (95% CI 21.6-30.3uM), consistent with degradation of Ara-C by CDA. In contrast, cells over-expressing CDA were more sensitive to 5FTRX, compared to wild type cells (IC50 wild type HEK293: 5.5uM (95% CI 4.7-6.3uM) vs IC50 CDA over-expression: 0.4uM (95% CI 0.4-0.5uM), potentially due to depletion of endogenous nucleotide pools by increased CDA. Thus, 5FTRX is not a substrate of CDA. We then treated 6 AML cell lines for 72 hours with increasing concentrations of 5FTRX and then measured cell growth and viability using the MTS assay. 5FTRX reduced the growth of 5 of 6 tested AML cell lines, with mean IC50 values (n= 〉 3) of 92nM (TEX), 130nM (KG1a), 150nM (MV4-11), 410nM (NB4), and 250nM (OCI-AML2). In contrast, K562 cells that have mutant p53 were resistant with an IC50 〉50,000nM. The K562 cells were also resistant to Ara-C. 5FTRX reduced the clonogenic growth of primary AML samples (n=2) with a 〉90% reduction in growth at 50nM, demonstrating that 5FTRX targets leukemia initiating cells. To test whether 5FTRX induced DNA damage in AML cells, we measured changes in phosphorylated H2AX (pH2AX) after 5FTRX treatment. In the tested cell lines (OCI-AML2, TEX, NB4 and MV4-11 cells), 5FTRX increased pH2AX at concentrations associated with loss of viability. Finally, we evaluated the efficacy and toxicity of 5FTRX in mouse models of AML. 5FTRX displayed robust and dose-dependent inhibition of MV4-11 and OCI-AML2 tumors in mouse xenograft models, with complete tumor regressions and long-lasting tumor growth delays (〉20 days at 100mg/kg in MV4-11 and 〉30 days in OCI-AML2) after a single cycle of 5 days of once-daily drug treatment, with no changes in body weight or behavior. The efficacy of 5FTRX was superior to Ara-C dosed for daily for 10 days at its MTD, 60 mg/kg (35%TGI, no regressions). MV4-11 tumors treated with 5FTRX displayed induction of pH2AX, reduction in proliferation (BrdU incorporation) and induction of necrosis, consistent with the mode of action and dramatic tumor regressions. The anti-leukemic effects of 5FTRX were further evaluated in mice engrafted intrafemorally with primary AML cells. 5FTRX (100 mg/kg i.p, x 5 days) reduced primary AML engraftment 〉95% compared to controls without toxicity (Fig 1). In summary, 5FTRX was identified as a potent inhibitor of AML cell proliferation in vitro and in vivo. In contrast to Ara-C, 5FTRX was not a substrate for CDA. Further development of analogues of 5FTRX is ongoing using protide prodrugs of the 5FTRX monophosphate to further increase potency and evade additional resistance mechanisms. Taken together, our findings support the further development of 5FTRX-based therapies for the treatment of AML, including AML patients with reduced sensitivity to Ara-C through high CDA expression. #AB and TPS contributed equally to this work. #ADS and MA contributed equally to this work. Disclosures Rizoska: Medivir AB: Employment, Equity Ownership. Rydergård:Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Eneroth:Medivir AB: Employment, Equity Ownership. Pinho:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership. Schimmer:Jazz Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Otsuka Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership.

Description: The cytotoxic nucleoside cytarabine forms the backbone of AML induction and consolidation therapies, but is associated with severe toxicities that preclude its use in patients unable to tolerate aggressive chemotherapy. Options for patients that do not respond to cytarabine, or relapse post-treatment, are limited. Elderly patients and those with relapsed/refractory AML would particularly benefit from the availability of new agents to develop treatment regimens that provide increased efficacy and tolerability compared to cytarabine, and that have a decreased susceptibility to mechanisms of cytarabine resistance, such as decreased deoxycytidine kinase (dCK) and/or upregulation of cytidine deaminase (CDA). Our preclinical evaluation of potential new anti-proliferative chemotherapeutics identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside, as having promising anti-proliferative activity against AML cell lines, and resistance to degradation by CDA. To understand potential mechanisms of resistance to 5FTRX, we selected a population of THP1 (THP1-R) cells resistant to 5FTRX. THP1-R cells were 66-fold resistant to 5FTRX and cross resistant to cytarabine (35-fold) with CC50 values for both nucleosides 〉50 μM. We discovered that THP1-R cells had decreased levels of dCK (〉95% by Western blot), the kinase responsible for the phosphorylation of cytidine and cytidine analogues such as troxacitabine and cytarabine to their corresponding monophosphates. Confirming the importance of dCK in the activation of 5FTRX and cytarabine, chemical inhibition of dCK also rendered THP1 cells 〉90-fold resistant to 5FTRX and cytarabine. To develop molecules that overcome resistance to both high CDA and low dCK, we used protide technology to construct nucleotide monophosphate prodrugs of 5FTRX, including one potent example, MV806. MV806 was not dependent upon dCK as it maintained similar efficacy in THP1-R cells with low dCK and against THP1 cells treated with the selective dCK inhibitor. We tested MV806 and 5FTRX in a panel of AML cell lines (n=7). MV806 was more potent than 5FTRX with CC50 values ranging from 0.0020-0.19 μM, compared to 0.057-1.2 μM for 5FTRX. MV806 also demonstrated CC50s 100), demonstrating future opportunities for clinical combinations. Finally, we showed that MV806 had DMPK profiles suitable for preclinical and clinical development. Leading protides were highly soluble, had a predicted half-life of 〉6h in human blood and demonstrated IC50 values 〉1 μM against major CYP isoforms (2A6, 2C9, 2D6, 3A4) with no evidence of time-dependent inhibition at 1 μM. To conclude, we used protide technology to directly deliver the active monophosphate species of 5FTRX intracellularly and thereby overcome resistance to cytarabine due to down-regulation of dCK and increased CDA expression. Taken together, our findings support the further development of protides of 5FTRX for the treatment of AML, including AML patients with reduced sensitivity to cytarabine through high CDA expression and/or low dCK expression. Disclosures Pinho: Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Rydergård:Medivir AB: Employment, Equity Ownership. Rizoska:Medivir AB: Employment, Equity Ownership. Eneroth:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Schimmer:Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership.

Description: Background: Myeloproliferative neoplasms (MPNs) are chronic myeloid malignancies with markedly heterogeneous disease course, and are associated with underlying inflammatory states that promote development of thrombotic events and acquisition of comorbidities. There is poor understanding of health care resource utilization (HRU) and cost of treatment in patients with MPNs. Objectives:To estimate and compare the HRU and cost of treatment for MPN patients (Essential Thrombocythemia, ET; Polycythemia Vera, PV; and Myelofibrosis, MF) with matched controls, and investigate the impact of patient characteristics and health service factors on the cost of treatment. Study design: Retrospective, population-based, matched-cohort study, using provincial health databases of Ontario's single payer universal health system. Study population: Cases were individuals in the Ontario Cancer Registry, diagnosed with MPN (Total n= 7130; ET, n=3481; PV, n=2618; MF, n=1031), from 2004 to 2016. Controls were individuals in the general population of Ontario, without a diagnosis of MPN. Each case was matched with four controls on age, sex, geographical location, and neighborhood income quintile. Baseline parameters including thrombosis and other comorbidities were collected during two-years prior to the date of MPN diagnosis. The baseline comorbid disease burden was measured using the Aggregated Diagnostic Group (ADG) score with a larger number of ADGs representing a greater comorbid disease burden (https://www.johnshopkinssolutions.com/wp-content/uploads/2014/04/ACG-White-Paper-Applications-Dec-2012.pdf). Main outcome measures:For each case and its controls, direct medical costs were obtained by costing all health care-related resources and expressed as mean per person year costs ((2018 Canadian Dollars, $1 CDN = $0.76 USD) to adjust for variable length of follow-up. Linear regression analysis was performed to assess the impact of baseline factors on the cost of treatment for MPN and represented as rate ratios (95% CI). Results:The mean duration of follow-up in years (cases vs controls) was 3.9 vs 4.3 for ET; 3.9 vs 4.2 for PV and 3.2 vs 4.9 for MF. The total follow-up duration was 27449 person years for all MPN cases, and 124963 person years for all controls. Comorbidities (congestive heart failure, chronic obstructive pulmonary disease, coronary artery disease, stroke, chronic renal failure, chronic liver disease, and pre-diagnosis arterial and venous thromboses were significantly higher in cases as compared to controls (p6 months after the diagnosis incurred significantly lesser cost of treatment due to less comorbidity burden as noted by the lower ADG score for patients with 〉6 months vs

Description: Ruxolitinib is the first available JAK inhibitor (JAKi) therapy for amelioration of constitutional symptoms or splenomegaly in myelofibrosis (MF). Duration of response varies, as patients discontinue ruxolitinib due to disease progression or drug toxicity. Ruxolitinib failure is a poorly defined clinical entity, but may include suboptimal or lost spleen response, worsening cytopenias, accelerated or blast phase (AP/BP) progression, or non-hematologic side effects. It is not clear what other clinical parameters should be considered indicative of JAKi failure. Leukocytosis is a known prognostic factor in MF and is included in various prognostic models. We have observed that some patients on stable doses of JAKi therapy develop progressive leukocytosis in the absence of other signs of MF progression. The significance of this event is not known, and it is not clear whether the onset of leukocytosis should prompt changes in clinical management. To assess the clinical significance of emergent leukocytosis, we evaluated leukocyte counts in our database of MF patients receiving Ruxolitnib or Momelotinib as first-line JAKi therapy. We defined emergent leukocytosis as any of:New onset of WBC ≥25 x 109/L in patients with WBC ≤12.5 x 109/L at JAKi start.Doubling of WBC from the nadir value in patients with WBC 〉12.5 x 109/L at JAKi start and nadir WBC 〉12.5 x 109/L.WBC ≥25 x 109/L in patients with WBC 〉 12.5 x 109/L at JAKi start after attaining a nadir WBC ≤12.5 x 109/L. Leukocytosis had to be sustained over consecutive blood counts at least one month apart and had to occur in the absence of infection, steroid therapy, AP/BP transformation, splenectomy or JAKi dose reduction. Exclusion criteria included concurrent hematologic diagnoses, and splenectomy or AP/BP MF preceding JAKi initiation. Of 290 patients with MF receiving JAKi therapy, 217 met study criteria. Of these 217 patients, 27 developed emergent leukocytosis while receiving JAKi. The cumulative incidence of leukocytosis was 4%, 10% and 15% at 1, 3, and 5 years from the start of JAKi therapy, respectively. Transformation to AP/BP, splenectomy, bone marrow transplant, or death from any cause were considered as competing risks in the calculation of cumulative incidence. In a multivariate analysis, clinical parameters associated with emergent leukocytosis included presence of baseline anemia (HR 4.94 [95% CI, 1.13-21.53]; p = 0.03) or leukocytosis (HR 5.01 [95% CI, 1.44-17.41]; p = 0.01) at JAKi start and female gender (HR 0.21 for male [0.08-0.06]; p=0.002). Baseline leukocytosis as WBC ≥25 x 109/L, and anemia was as a hemoglobin