Sproat, Lisa

Abstract Background The prognosis for AML or MDS patients (pts) with monosomal karyotype (MK) is dismal. Although a benefit for allogeneic hematopoietic cell transplantation (allo-HCT) compared to chemotherapy or autologous HCT has been demonstrated in young (≤ 60) pts (Cornelissen, 2012), the curative potential of allo-HCT in older (≥ 60) pts with monosomal karyotype AML/MDS is not known. Patients and methods In this retrospective review, we analyzed outcomes for 69 AML (n = 49) or MDS (n = 20) pts ≥ 60 who underwent allo-HCT at Mayo Clinic Arizona between 2005 and 2013, based on cytogenetic grouping as follows: cytogenetically normal (CN, n = 23), cytogenetically abnormal but not MK (CA, n = 32), and MK (n = 14). Monosomal karyotype was defined (Breems, 2008) as a karyotype with at least two autosomal monosomies or a single autosomal monosomy in combination with at least one structural abnormality. Univariate and multivariate statistical analyses were performed to evaluate the impact of these 3 cytogenetic groupings on outcome (primary endpoint progression-free survival). The median age for all pts was 66 (60-76), and median follow-up for surviving pts was 22 months (range 70 days – 7.1 years). Thirty-seven pts had high-risk disease (MDS > 5% blasts or AML not in CR), while 32 were considered standard risk (MDS <5% blasts or AML in CR). The majority of pts had good performance status (KPS ≥ 90% in 66), but 34 pts (49%) had an HCT-CI score of ≥ 3. Conditioning was myeloablative (Bu-Cy) in 4, reduced toxicity FBM (Fludarabine, BCNU, Melphalan) in 36, and RIC/NMA in 29. Donors were matched related in 18, matched unrelated in 36, and mismatched unrelated in 15. GVHD prophylaxis included tacrolimus in all pts combined with mycophenolate mofetil (37) or methotrexate (32); 49 pts received in-vivo T-cell depletion with rabbit anti-thymocyte globulin (rATG, Thymoglobulin, Genzyme, Cambridge, MA). Results The Kaplan-Meier estimate of progression-free survival (PFS) at 2 yrs was 59% (CN), 39% (CA), and 1.5% (MK); P = 0.004 (Fig. 1). The cumulative incidence of relapse at 2 yrs (with death without relapse as a competing risk) was 34% (CN), 25% (CA), and 94% (MK); P= 0.009). In a Cox proportional hazards analysis adjusted for age (≤ 66 vs. > 66), risk status (standard vs. high), conditioning (myeloablative [Bu-Cy + FBM] vs. RIC/NMA), use of ATG (yes/no), donor type (unrelated vs. related), and HCT-CI (0-2 vs. ≥ 3), the only factor predictive for inferior PFS was cytogenetic status (MK vs. CN, HR 4.64 [95% CI, 1.7 – 13]; P = 0.003); (MK vs. CA, HR 2.4 [95% CI 0.97 – 5.9]; P = .057). Conclusions Standard allo-HCT in AML or MDS pts ≥ 60 with a monosomal karyotype appears to have limited curative potential. Although larger studies will be required to confirm our results, novel transplant approaches or post-transplant strategies to prevent relapse should be a focus of future studies in this incurable pt population. Disclosures: Reeder: Novartis: Research Funding; Celgene: Research Funding; Millennium: Research Funding.

Introduction Allogeneic hematopoietic stem cell transplant (HCT) patients are at risk for having a low Vitamin D (VD) level. VD level has been correlated with cancer incidence, pulmonary disease and infection. VD also has immunomodulatory properties and reports suggest that a low VD level increases the incidence of chronic GVHD and may impact mortality in HCT. HCT morbidity and mortality is attributed to infection, organ system toxicity, GVHD and recurrent disease. Identifying a correctable factor that would reduce the occurrence or severity of these complications would be beneficial. It is possible that VD may have significant effect on outcome after HCT due to its described properties. Normal VD level may infer better outcome for HCT patients by improving response in or preventing infection, protecting organ function, decreasing risk of relapse and/or decrease incidence or severity of GVHD. We hypothesize that there is a relationship between VD level and morbidity and mortality after HCT. We therefore studied VD levels pre- and post-HCT to determine if there was an impact of VD level on these outcomes. Patients and Methods Two hundred and fifty patients underwent myeloblative or non-myeloablative HCT between 3/12/2009 and 10/29/2012 at our institution. Baseline demographic data, disease characteristics, transplant variables and outcomes data were obtained from the transplant database. These data were supplemented by retrospective chart review for VD levels prior to transplant, at day 100 and 1 year post-HCT. Data were collected through day 100 for occurrence of infection and admissions to the ICU. Categories for VD level included normal (≥30 ng/ml) or abnormal (<30 ng/ml). Patient characteristics were compared using the Chi-square test or Wilcoxon rank sum test. The logrank test was used to compare the survival curves between groups, and the Cox proportional hazard model was used to measure the association between outcomes (mortality, relapse, acute or chronic GVHD) and VD level. Results Of the 250 patients undergoing HCT VD level was performed on 180 (72%) patients pre-HCT, 149 (60%) patients at day 100 post-HCT and 81 (32%) patients at 1 year post-HCT. The rate of acute or chronic GVHD was not significantly associated with VD level pre or post-HCT. Overall infection risk was not significant pre or post-HCT but when sorted by type of infection bacterial infection (p=0.01), radiologic evidence of pulmonary nodules/consolidation (p=0.03), and ICU admissions (p=0.0313) within the first 100 days post-transplant were significantly increased in patients who had a low VD level at day 100 post -HCT. Pre-HCT and 1 year post-HCT VD level did not impact mortality or relapse but there was a significant increase in one year mortality (p=0.02, HR = 5.99) and relapse (p = 0.03, HR=9.47) in patients who had a low VD level at day 100 post-HCT. Discussion This study shows that VD may play a significant role in bacterial infection, pulmonary infection, ICU admission, mortality and relapse after HCT. Those patients with a low VD level at day 100 post-HCT had significantly worse outcomes for relapse and mortality. Those patients with a low VD level at day 100 post-HCT had a higher incidence of lung infection, bacterial infection and admission to the ICU. In contrast to previous studies, we found no correlation between VD level and incidence of acute or chronic GVHD. This study is one of the largest reported and the first to our knowledge that reports decreased risk of certain types of infection (bacterial and lung), mortality and relapse in those with an adequate VD level receiving HCT. It is possible that supplementation of VD to keep levels ≥30 would reduce these complications of HCT. Future prospective study of VD is warranted. Finding a low cost and easy to administer therapy (such as VD replacement) that would reduce morbidity, mortality and relapse in the HCT population is needed. Disclosures: Reeder: Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

Abstract Background: Relapse after ATRA-based induction therapy is relatively uncommon in patients with acute promyelocytic leukemia (APL). Long term follow up studies have estimated a 15-25% relapse rate. Autologous and allogeneic hematopoietic stem cell transplantation (HCT) has been used as an effective salvage strategy. We present a single institution study of the outcomes of patients with relapsed APL that underwent HCT. Methods: After due IRB approval, the Mayo Clinic Transplant Database was retrospectively reviewed to identify all patients across three Mayo Clinic sites with relapsed APL that underwent HCT from 1995-2013. Data was abstracted at diagnosis and at relapse. The aims of this study were to report the descriptive characteristics of patients, with a focus on HCT outcomes. Results: Baseline Patient Characteristics A total of 15 patients (9 (60%) males) with relapsed APL that underwent HCT were identified. Median age was 36 years (range: 19-63) and median follow up from diagnosis was 8.8 years (range: 1.7-17). 2 (13%) deaths (1 from relapse) were recorded. 14 (93%) received ATRA and anthracycline-based induction therapy and 1 received anthracycline-based induction without ATRA. All relapsed patients achieved a complete remission (CR) after induction. Median time to relapse was 1.6 years (range: 0.6-3.9). 10 (67%) patients had a medullary relapse (hematologic-7(70%), cytogenetic -2 (20%) and molecular-1(10%)), 3 (20%) had additional extramedullary (EM) disease (CNS-2, myeloid sarcoma-1), while 2 (13%) presented with EM disease only (CNS-2). Salvage regimens included ATRA in 5 (33%) patients and arsenic trioxide (ATO) in 8 (53%) patients. 12 (80%) patients were transplanted in CR2 and 1 in CR3 (7%), while 2 (13%) had persistent disease (PD) at time of HCT. Allogeneic HCT 4 (27%) patients with a median age of 37 years (range: 33-49) underwent allogeneic HCT. 2 (50%) patients received myeloablative (MA) conditioning and 2 received reduced-intensity conditioning (RIC). 2 patients who underwent MA HCT had PD at time of transplant, while 1 patient in CR3 received a RIC. 2 (50%) patients developed acute GVHD while 2 (50%) had extensive stage chronic GVHD. At a median follow up of 2.6 years (range: 0.3-10.9), 3 (75%) patients remain alive and disease-free. 1 patient died of infectious complications within 100 days of transplant. Autologous Transplantation 11 (73%) patients with a median age of 40 years (range: 23-68) underwent autologous HCT with MA conditioning. 7 (64%) patients were in molecular remission at time of HCT, 3 (27%) patients transplanted before 2004 were in cytogenetic remission and information about type of remission was unavailable in 1 patient. At a median follow up of 6.8 years (range: 0.5-16) since HCT, 1 (9%) patient had a documented relapse at 175 days after HCT and died of disease-related complications. 10 (91%) are alive and in remission. Conclusions: Our single-institution study reaffirms the efficacy of HCT for APL patients with medullary and EM relapse. Autologous HCT has excellent long-term results in selected patients in CR2. Allogeneic HCT was mainly reserved for patients with high risk disease (CR3) and persistent disease at time of HCT. Table 1: Baseline Patient Characteristics N (%) Number of patients 15 Follow up (years) (median, range) 8.8 (1.7-17) Age at diagnosis (years) (median, range) 36 (19-53) Gender Male 9 (60%) Female 6 (40%) Sanz risk score High 4 (29%) Intermediate 8 (57%) Low 2 (14%) Induction Regimen ATRA + anthracycline 14 (93%) Anthracycline-based 1 (7%) Response to induction therapy CR 15 (100%) Time from diagnosis to relapse (years) (median, range) 1.6 (0.6 - 3.9) Type of relapse Hematologic 7 (47%) Cytogenetic 2 (13%) Molecular 1 (7%) Hematologic and EM 3 (20%) CNS only 2 (13%) Salvage regimen ATRA-based 5 (33%) ATO-based 8 (53%) Chemotherapy only 2 (13%) Table 2: Outcomes at HCT N (%) Age at HCT (years) (median, range) 38 (23-68) Disease status at HCT CR2 12 (80%) CR3 1 (7%) PD 2 (13%) Type of HCT Allogeneic 4 (27%) Autologous 11 (73%) Conditioning regimen Myeloablative 13 (87%) Non-myeloablative 2 (13%) Follow up since HCT (years) (median, range) 4.5 (0.3 - 16.3) Disease status at last follow up CR 13 (86%) Relapsed disease 1 (7%) Unable to assess 1 (7%) Alive at last follow up 13 (87%) Disclosures No relevant conflicts of interest to declare.

Abstract Introduction Although allogeneic hematopoietic cell transplantation (HCT) is an expensive modality of treatment for hematological disorders, very little is known about the financial burden for the patients themselves. We analyzed factors associated with financial burden and its impact on health behaviors of allogeneic HCT recipients. Patients and Methods A questionnaire was mailed to adult patients who were alive after allogeneic HCT performed between 1/06 to 6/12 at Mayo Clinic to collect information regarding financial concerns, household income, employment status, insurance, out-of-pocket expenses and health and functional status. A multivariable logistic regression analysis was performed to determine socio-demographic and clinical factors associated with financial hardship. Results Of 482 patients, 268 responded (response rate 56%). Respondents were more likely to be older and Non-Hispanic Whites. No significant differences were seen in disease or transplant characteristics between the two groups except that respondents were more likely to have received reduced intensity conditioning. Median follow-up after HCT was 2.3 years (range 0.7-6.7 years). All the patients for whom the insurance information was available (missing n=13) were insured. Median physical and mental component score, as derived from the SF-12, were 36.8 [Interquartile range (IQR) 29.3-49.0] and 53.1 (IQR 42.1-57.1) respectively, where the general population score is 50, and lower scores indicate worse functioning. The proportion of patients that reported being on medical disability increased from 8% prior to HCT to 29% after HCT. 73% of the respondents reported that being sick had hurt them financially. 54% experienced adverse financial consequences such as decrease in household income by &gt;50% due to the HCT, need to sell/mortgage home, withdraw money from retirement accounts or having to pay more for medical care than they could afford. 3% declared bankruptcy. 12% had out-of pocket costs for the past 3 months that were&gt; $5000. Median monthly medication copayments were $100 (range $ 0-3000). 36% patients felt that their insurance coverage was poor which was strongly correlated with adverse financial consequences (p=0.0013). Other risk factors for financial burden were poor physical functioning reflected by the physical component score &lt;40 (OR 3.2; p=0.005), lower household income (OR 3.08; p=0.006) and lack of commercial/private insurance (OR 4.2; p&lt;0.001) while adjusting for patient and transplant related characteristics. Time from transplant was a significant predictor (OR 1.2; p=0.03) in the univariate analysis, but lost its significance in the multivariate analysis. 18 to 27% patients reported deleterious health behaviors due to financial constraints such as not taking prescribed medications, not using a recommended medical service or not making physician appointments. Conclusions A significant proportion of allogeneic HCT survivors experience adverse financial consequences with a few reporting severe outcomes like bankruptcy, despite having health insurance coverage. Future research should investigate potential interventions such as early financial counseling and connecting with sources of financial support to help at-risk patients and prevent long-term adverse financial outcomes after this life-saving procedure. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Treatment options for elderly acute myeloid leukemia (AML) patients, despite various novel therapies, are still limited. Relapse is common after chemotherapy, and progression on lower intensity treatment with hypomethylating agents is inevitable in all patients even after initial response. Lysine deacetylase inhibitors (KDIs or HDAC inhibitors) have shown limited single-agent clinical activity in AML. Therefore, to develop potential novel treatment options for AML, we aimed to identify synergistic KDI-based drug combinations to build upon the single-agent activity and clinical-availability of KDIs. We performed RNA-interference (RNAi) drug modifier screens of the kinome and phosphatome with the KDI vorinostat in AML cells in order to identify targets to inhibit in combination with KDIs. Based on pathway analysis of RNAi screen results that highlighted an unexpected role for p38/JNK/SAPK signaling in vorinostat activity in AML, we explored the putative p38 substrate-selective MK2 inhibitor CMPD1 combined with KDIs, and demonstrated potent synergistic activity in AML regardless of molecular, cytogenetic or lineage background. As published evidence suggests that CMPD1 can also act as a microtubule-destabilizing/targeting agent (MTA), and that MK2 can associate with microtubules and cytoskeleton-associated proteins, we examined several MTAs in combination with KDIs in AML, including vinca alkaloid microtubule-destabilizing agents vinblastine/vincristine, and taxol as a microtubule-stabilizing agent. Results: Vinblastine and vincristine dose-dependently synergized with vorinostat similarly to CMPD1 in all AML cell lines tested, with vinblastine being more potently synergistic than vincristine. Taxol only potentiated KDI activity in approximately half of AML cell lines. Importantly, preliminary data suggest preferential, dose-dependent synergy of KDIs and vinca alkaloid MTAs in the CD34+ progenitor AML cell fraction, as compared to the paired CD34-depleted fraction from the same primary AML sample in ex vivo culture. This preferential CD34+ activity was similar to that observed with CMPD1 combined with KDIs in ex vivo models. Consistent with synergistic activity of MTAs and KDIs, multiple RNAi screen hits also converged on microtubule dynamics. Subsequently, we identified the LKB1 signaling network, a critical integrator of cellular stress metabolism and hematopoietic stem cell quiescence/homeostasis, as a determinant of vorinostat activity in AML. Of 14 known kinases directly regulated by LKB1, 4 belong to microtubule-associated protein (MAP) or microtubule-affinity regulating kinases (MARK) families (e.g. screen hit MARK4). RNAi screen hits also converged on regulation of microtubule proteins, or directly on microtubule proteins themselves, (e.g. atypical MAPK4 and MAPK6 which phosphorylate microtubule-associated protein 2, or TAOK2 which has a kinase-independent role in stabilizing microtubules). Additionally, several RNAi hits such as PACSIN1 and -2, and MAST2 and -4 link the actin cytoskeleton to microtubule dynamics. Discussion: Herein we show that several types of clinically-used MTAs (i.e. vinca alkaloids or taxanes) potently synergize with KDIs in AML, and that this synergy can occur preferentially in primary CD34+ cells, as compared to the CD34-negative AML compartment. Although at high in vitro doses microtubule-destabilizing agents such as vinca alkaloids, and microtubule-stabilizing agents such as taxanes, can both induce apoptosis via mitotic perturbation, many publications show that low therapeutic MTA doses can induce apoptosis in tumor cells in vivo independent of mitosis. Therefore, we hypothesize that clinically-available KDIs (i.e. vorinostat, panobinostat) and MTAs (i.e. vinca alkaloids or taxanes) represent novel combinations with the potential to target the AML progenitor pool even in a non-proliferate state, and thus have the potential to eradicate minimal residual disease. Disclosures Mesa: Celgene: Research Funding; Incyte Corporation: Research Funding; Ariad: Consultancy; Galena: Consultancy; Novartis: Consultancy; Promedior: Research Funding; CTI: Research Funding; Gilead: Research Funding. Tibes:Italfarmaco S.p.A.: Consultancy, Honoraria.

Abstract Introduction Venetoclax (ABT-199) is an approved BCL-2 inhibitor for the treatment of patients with chronic lymphocytic leukemia (CLL). Multiple clinical trials are underway to explore its efficacy in additional indications. While venetoclax demonstrated high remission rates in combination with azacitidine in early stage clinical trials, the question of durability of responses and primary and acquired resistance remain, especially given the modest activity and rapid development of resistance as a single agent. One reported mechanism of intrinsic resistance is high expression of other BCL-2 family proteins, including MCL-1. We and others have demonstrated that the CDK9 inhibitor, alvocidib, can mediate transcriptional repression of anti-apoptotic MCL-1. It has also been shown that alvocidib can increase pro-apoptotic BIM, a dual activator and sensitizer BH3-only protein that can directly induce apoptosis and simultaneously inactivate anti-apoptotic BCL-2 family proteins such as MCL-1 and BCL-2, thus having the same effect on mitochondria-associated apoptosis as MCL-1 down-regulation, with the potential to directly induce apoptosis. An alvocidib-containing cytotoxic chemotherapy regimen demonstrated favorable remission rates in high-risk AML patients over standard therapy in a randomized Phase 2 trial indicating its potential role and safety in AML. We hypothesized that alvocidib and venetoclax would synergize against AML cells by shifting the overall balance of pro- and anti-apoptotic BCL-2 proteins in favor of apoptosis and thus represent a novel active treatment regimen in AML. Aims This study seeks to examine the efficacy of a treatment regimen containing alvocidib and venetoclax in multiple preclinical studies, including in vivo models of AML. Methods Cell viability assays interrogating alvocidib and venetoclax activity in cell lines were performed using CellTiter-Glo according to manufacturer's protocol. mRNA/miRNA expression changes were assessed using standard RT-PCR technique. Protein expression changes were assessed using standard western immunoblotting technique. To assess the efficacy of an alvocidib and venetoclax combination on tumor growth in an in vivo model, the OCI-AML3 xenograft mouse model and ex vivo studies with AML patient samples were performed. Results Herein we demonstrate that alvocidib inhibits both mRNA and protein expression of MCL-1 in a time and concentration-dependent fashion in 3 out of 4 AML cell lines analyzed, while in cells where alvocidib did not reduce MCL-1 protein levels (i.e. MOLM-13) a dose-dependent decrease in miR17-92, and concomitant increase in BIM protein was observed after 24 hours of alvocidib treatment. The alvocidib-venetoclax combination resulted in very strong synergistic reductions of cell viability (with combination indices [CI] of 0.4 to 0.7), both in venetoclax-sensitive and resistant cells. The venetoclax-sensitive lines, MV4-11 and MOLM-13, exhibited 5- to 10-fold reduction of venetoclax EC50 values in the low nM range when combined with only 80 nM alvocidib. Importantly, venetoclax-resistant lines, OCI-AML3 and THP-1, exhibited at least 20-fold reduction of venetoclax EC50 values from near 1 µM to 10-50 nM, when combined with 80 nM alvocidib.In the venetoclax-resistant OCI-AML3 xenograft model, single agent alvocidib and venetoclax achieved tumor growth inhibition (TGI) of 9.7 and 31.5%, respectively, while the combination achieved 87.9% TGI at the same dose levels of individual drugs. Conclusions Taken together, our data suggest that the combination of alvocidib with venetoclax is highly synergistic in vitro and in vivo, in both venetoclax-sensitive and -resistant AML across a heterogeneous genomic background. The particularly high level of synergy achieved in venetoclax-resistant cell lines highlights the central importance of both BCL-2 and MCL-1-mediated cell survival in AML. Importantly, the addition of alvocidib to venetoclax treatment reduced IC50s to clinically achievable concentrations. Therefore, we conclude that an alvocidib/venetoclax combination may be a novel approach for the treatment of AML and warrants further pre-clinical and clinical validation. Disclosures Whatcott: Tolero Pharmaceuticals: Employment. Kim:Tolero Pharmaceuticals: Employment. Haws:Tolero Pharmaceuticals: Employment. Mesa:Celgene: Research Funding; Galena: Consultancy; Promedior: Research Funding; Ariad: Consultancy; Novartis: Consultancy; CTI: Research Funding; Incyte: Research Funding; Gilead: Research Funding. Peterson:Tolero Pharmaceuticals: Employment. Siddiqui-Jain:Tolero Pharmaceuticals: Employment. Weitman:Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties. Warner:Tolero Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties.

Abstract Background: Large case-control studies have identified common epidemiologic exposures associated with risk of AML development, including obesity [BMI ≥30 kg/m2] associated with 2-fold increase risk. However it is not known whether individual risk factors are associated with unique AML genetic lesions. We have recently identified a significant association of some common exposures including obesity with unique disease cytogenetic subgroups in a cohort of n=295 consecutive AML patients diagnosed and treated at Mayo Clinic [Cancer Epidemiol 39:1084, 2015]. Aberrant DNA methylation is a hallmark of cancer and has been reported in AML, and we therefore performed an analysis of global DNA methylation in patients in this cohort to determine whether specific epidemiologic exposures are associated with a unique pattern of DNA methylation. Methods: Patients in the initial cohort were previously reported. An available baseline AML bone marrow cytogenetic pellet from diagnosis was identified for n=148 patients (50.2%). Following IRB approval and using previously reported techniques [Leukemia 24:1283, 2010], leukemia DNA was isolated from the remnant diagnostic cytogenetic samples and processed on the HumanMethylation450 BeadChip array (Illumina). Individual CpG dinucleotides were assigned a Beta value (i.e. methylation value) ranging from 0 (unmethylated) to 1 (methylated). We excluded those with minor methylation changes (<0.2 between any two samples), resulting in 383,524 CpG's. For the purposes of this first analysis we have focused only on CpG's with an inter-quartile range (IQR, i.e. difference between 75th and 25th percentiles) of greater than 0.25, comprising a total of 39,657 CpG's. An unsupervised cluster analysis of differential methylation was generated ("Heatmap", Figure). Univariate and multivariable analyses were performed adjusting for age as a continuous variable to determine the association of individual exposures with unique methylation clusters (primary endpoint), and we also evaluated the association of clinical characteristics and clinical outcomes after therapy (secondary endpoint). The median age of patients was 69 yrs (range 19-89), male 65%, and Caucasian 89%. The median BMI was 27.2 (range 16.9-59.9), and n=47 patients (32%) had BMI ≥30. Standard cytogenetic risk groups were applied [favorable (n=8), normal (n=61), intermediate abnormal (n=32) and poor (n=47)]. Results: We identified 4 distinct methylation clusters (Figure: Cluster #1, Black horizontal bar; Cluster #2, Green; Cluster #3, Blue; and Cluster #4, Red). Multivariate analysis of select variables is shown in Table. Patients in cluster #1 were significantly more likely to have a BMI ≥30 [Odds Ratio (OR) 2.49, p=0.017] and more likely to have favorable risk cytogenetics although this was not statistically significant [OR 6.71, p=0.088], while those in cluster #2 were significantly more likely to be have a history of regular NSAID use [OR 3.26, p=0.03] and less likely to have normal [OR 0.40, p=0.044] and possibly intermediate abnormal cytogenetics [OR 0.34, p=0.051]. Those in cluster #3 were significantly younger [median age 48 yrs, p<0.001] and were also more likely to have prior history MDS although this was not statistically significant [OR 3.7, p=0.082]. Cluster #4 was not correlated with any distinct exposure apart from possible decreased alcohol use [OR 0.3, p=0.079]. History of past or current tobacco use, regular acetaminophen use, documented myelotoxin exposure, family history, and secondary AML did not correlate with a unique methylation cluster. There was no independent association of methylation cluster with overall survival (p=0.46), although among patients receiving intensive therapy cluster #2 was associated with lower complete remission rate [OR 0.28 (95% CI 0.09-0.86), p=0.026]. Conclusion: This first analysis of AML molecular epidemiology and clinical outcome suggests that specific disease risk factors (esp. obesity and age) are associated with unique leukemia DNA methylation clusters. In addition, aberrant methylation is associated with some clinical features and with outcome after therapy, suggesting that epidemiologic risk factors may have a prognostic impact. This provides novel insight into leukemogenesis, and will guide prospective studies of AML epidemiology and outcome. Figure. Figure. Disclosures Foran: medscape: Honoraria; pfizer: Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; novartis: Honoraria; karyopharm: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding.

Abstract Background Myeloid neoplasms (MN) occurring secondary to treatment of non-malignant disease, especially autoimmune diseases (AID), and the overlap between them, is an increasing clinically important observation. Confounding variables for myeloid transformation include the nature of autoimmune disease as an incessant, chronically activated inflammatory cascade that has been associated with an increased risk of myeloid malignancy, and disease modifying therapies, often with cytotoxic and immunosuppressive drugs. Thus, the addition of therapeutic agents for treatment of AID theoretically amplifies the opportunity for MN to develop in genetically susceptible individuals. To our knowledge, there is no large study evaluating a comprehensive primary AID population that developed MDS or AML with detailed assessment of therapeutic interventions for AID. The presented study queries the association of cytotoxic, anti-inflammatory, and immune-modulating agents to treat AID as risk factors for developing myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). Methods In a 10-year period from 2004-2014, a total of 40,011 patients with an ICD-9 coded diagnosis of AID were seen at Mayo Clinic Arizona and Florida campuses; 311 patients from this population had a concomitant coded diagnosis of MDS and/or AML. In an IRB approved, retrospective chart review, we confirmed 86 cases meeting strict inclusion criteria for AID and confirmed MDS or AML. We performed a case-control match from the same primary autoimmune population for age, gender, and autoimmune specific diagnosis at a 2:1 ratio querying for therapeutic interventions. Results Myeloid neoplasm specifics in our case cohort of 86 patients were 55 MDS, 21 de novo AML, and 10 AML with history of MDS. Average age was 72 years with a slight male predominance (57%). Rheumatoid arthritis (26.4%), psoriasis (20.7%), systemic lupus (13.8%), was the most common autoimmune profiles. Median onset of autoimmune disease to diagnosis of myeloid neoplasm was 6 years (range 1-54 years). Up to 26% controls documented no systemic agent for treatment of autoimmune disease and similarly 12.8% of the case cohort (p=0.142). Case and control populations treated with systemic therapies had similar exposures by category for immune-modulating agents (33.7% and 37.8% respectively) and cytotoxic therapies (47.7 and 44.8% respectively). A total of 57/86 cases (66.3%) received either a cytotoxic or immune-modulating agent. In comparison, 105/172 (61.1%) controls received either agent; p=0.495. Azathioprine was the only statistically significant agent exposure observed more frequently in the study cohort than controls with an OR of 7.05 (p= < 0.001). There was no increased incidence of MDS or AML in TNF-antagonist treated patients. No significant correlation for duration of agent exposure by drug category was observed. Discussion In a large primary AID population, azathioprine exposure was associated with a 7 fold risk of MDS or AML (p=< 0.001). Notable, but not statistically significant case cohort trends among cytotoxic agents was exposure to cyclophosphamide (OR 3.58) followed by mitoxantrone (OR 2.73). Methotrexate, mercaptopurine, and mycophenolate had favorable odd ratios, but these were not statistically significant. No association was found for TNF-inhibitory agents. Finally, there was no association of drug exposure time to the incidence in development of MDS or AML. Disclosures Dueck: Bayer: Honoraria. Mesa:Gilead: Research Funding; Novartis: Consultancy; Celgene: Research Funding; Incyte Corporation: Research Funding; Galena: Consultancy; Ariad: Consultancy; CTI: Research Funding; Promedior: Research Funding. Al-Kali:Onconova Therapeutics, Inc.: Research Funding; Celgene: Research Funding. Foran:Millennium Pharmaceuticals, Inc.: Research Funding; novartis: Honoraria; karyopharm: Honoraria; medscape: Honoraria; pfizer: Honoraria; boehringer ingelheim: Research Funding; agios: Research Funding; Cellerant: Research Funding.

Abstract Cytarabine (AraC) resistance is a fundamental feature of refractory/relapsed AML. RNA interference (RNAi) screens conducted in our laboratory recently identified WEE1 kinase (WEE1) as one of the top candidate genes and target in leukemias in combination with AraC. WEE1 is a tyrosine kinase belonging to the Ser/Thr family of protein kinases and acts as a negative regulator of mitotic entry by controlling DNA damage (DDR) and cell cycle checkpoint responses. The WEE1 inhibitor MK1775 potently synergizes with AraC ex vivo and in vitro and clinical trials are in preparation. However, the mechanism of action for the anti-leukemic activity of MK1775 with AraC remains unknown. To elucidate genes mediating activity of the combination, we first performed siRNA rescue screens silencing a custom set of 44 genes involved in WEE1 regulation under combined AraC + MK1775 to identify sensitizers and markers of resistance. The MRN (MRE11, Rad51, NBS1) complex and particularly NBS1 were potent modifiers of AraC and MK1775. Focusing on NBS1 since it is proposed to centrally regulate the defense capacity of leukemic cells, we identified that NBS1 phosphorylation at Ser343 (the ATM regulation site) is significantly altered both in cell lines and primary AML samples under combined AraC+MK1775 treatment as compared to single agent MK1775. In parallel, lower phosphorylation of ATMS1981(an autophosphorylation site in response to DNA strand breaks), was observed indicating that the ATM-CHEK1 pathway is not activated under co-treatment. Further Homologous recombination (HR)-mediated repair was compromised by AraC+MK1775 shown by DR-GFP expression vector to measure intracellular HR capacity: post-transfection of the I-SceI nuclease which cleaves non-functioning GFP tandem repeats to form a functional GFP unit, the HR was reduced with the combination. Consistently other HR markers decreased as well. Delayed accumulation of Cyclin A (indicative of S-phase progression) and greater inhibition of phospho-Cdk2Y15in synchronized cells treated with AraC + MK1775 in comparison to controls was observed. In addition the cell cycle was globally dysregulated by slower S-phase kinetics (progression), a completely abrogated G2/M checkpoint/phase as well as de-regulated DNA replication origin formation and firing as evidenced by Cdt1 and Mus81. As a consequence high single and double strand breaks (ɣH2AX) were observed with an increase in phospho-histone H3 in AraC + MK1775 treated cells compared to untreated cells or MK1775 single agent, confirming faster mitotic entry. Changes were followed by massive induction of apoptosis. Since WEE1 is implicated in leukemic stem cell maintenance we examined the long term effects of the combination in colony forming assays. AraC + MK1775 treated leukemic cells obtained from patients with AML were re-plated on Methocult after drug washout and colonies counted after 14 days. While MK1775 as a single agent could reduce colony formation by 4 fold compared to controls and lower dose AraC, co-treatment with low to moderate doses of AraC and MK1775 reduced colony formation by more than 7 fold and to almost zero in some primary specimens. Taken together, these results suggest that leukemia cells co-treated with AraC + MK1775 lost their ability to activate DNA damage and repair pathways mainly by compromising the MRN complex via NBS1 with subsequently reduced HR. The combination (as opposed to single agents) almost complete dysregulated the cell cycle and its checkpoints lead to DNA damage, genomic instability and rapid exit from the cell cycle with cell death via apoptosis. Thus we have molecularly characterized the detailed mechanisms underlying the potent AraC+WEE1 inhibition in AML and describe for the first time a therapeutic combination that has the potential to abrogate the MRN and NBS1 repair capacity which is central for drug resistance in AML. A key implication of our work is to provide a clinical rationale, mechanistic understanding and suggestions for biomarkers to clinically evaluate AraC + MK1775 in patients with AML. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Cytotoxic agents are a well-established risk factor for therapy related myeloid neoplasms (t-MN). The incidence of t-MN in patients (pts) with autoimmune diseases (AID) treated with cytotoxic and immuno-modulatory agents are not well described in the literature. Most case series assess an MDS/AML population and co-occurrence with an AID. To our knowledge, there exists no large case-control studies evaluating a primary AID population that developed therapy-related MDS or AML, and the relationship to detailed therapeutic intervention for AID. Our study queries the association of cytotoxic and immune-modulating agents to treat AID as risk factor for developing MDS or AML. Methods Between 2004-2014, 23,671 pts with an ICD-9 coded AID were seen at Mayo Clinic Arizona; 148 pts from this population had a concomitant coded diagnosis of MDS/AML. In an IRB approved, retrospective chart review we confirmed 61 cases meeting strict inclusion criteria. We performed a case-control match from the same primary AID population for age, gender, and autoimmune specific diagnosis at a 2:1 ratio. AML/MDS cases were compared to controls by use of chi-square test for frequency data. Odds ratios with 95% confidence interval were constructed using conditional logistic regression. Case incidence was compared to SEER data for the general population Results Of 61 cases 33 were MDS, 20 de-novo AML, and 8 MDS transformed to AML. Psoriasis 16 (26.2%) and rheumatoid arthritis 14 (23.0%) were the most common autoimmune diagnoses, followed by systemic lupus 11 (18%), Crohn's disease 7 (11.5%), and ulcerative colitis 5 (8.2%). 39 out of 61 cases (63.9%) received either a cytotoxic or immune modulating agent for AID, compared to 64/122 (52.5%) in the control group; p=0.140. 7/61 cases (11.5%) received both a cytotoxic and immune modulating agent as compared to 16/122 (13.1%) in controls; p=0.753. Overall, the case population received more agents than controls. No cytotoxic or immune-modulating treatment was documented in 16% of cases and 36% controls. Exposures to cytotoxic and immune-modulating agents were similar in both case and control cohorts. Exposure time was not statistically significant. Azathioprine was the only statistically significant agent exposure observed more often in the case cohort than control. Per SEER data, over a 10-year period in a general age-matched population that is comparable to our AID population cohort, we would expect 54 MDS and 35 AML cases. Table 1. Exposures and Timeline Cases (n=61) Controls (n=122) P value * Number of Systemic Agents, Total None 1 2 3 4+ - 10 (16.4%) 26 (42.6%) 12 (19.7%) 7 (11.5%) 6 (9.8%) - 44 (36.1%) 32 (26.2%) 31 (25.4%) 9 (7.4%) 6 (4.9%) 0.020 Exposure to Cytotoxic agent < 1 year 2-5 years 6-10 years 11 years or more Unknown (n=26) 7 (26.9%) 6 (23.1%) 5 (19.2%) 3 (11.5%) 5 (19.2%) (n=43) 5 (11.6%) 8 (18.6%) 6 (13.9%) 12 (27.9%) 12 (27.9%) 0.276 Exposure to Immune modulating agent < 1 year 2-5 years 6-10 years 11 years or more Unknown (n=20) 6 (30%) 6 (30%) 3 (15%) 1 (5%) 4 (20%) (n=37) 6 (16.2%) 10 (27.0%) 5 (13.5%) 3 (8.1%) 13 (35.1%) 0.661 Table 2. Treatments and Odds Ratio Odds Ratio 95% CI for OR P value * Cytotoxic OVERALLAzathioprine Cyclophosphamide Methotrexate Mitoxantrone 6-MP Mycophenolate 1.39 4.16 1.28 0.74 2.73 1.00 1.21 0.72, 2.67 1.29, 13.44 0.24, 6.89 0.29, 1.88 0.23, 33.0 0.23, 4.35 0.28, 5.21 0.322 0.017 0.773 0.528 0.429 0.999 0.803 Immune modulating agent OVERALLAnti-TNF Leflunomide Plaquenil 1.19 0.73 2.00 2.73 0.52, 2.73 0.24, 2.24 0.40, 9.91 0.80, 8.33 0.672 0.585 0.396 0.077 Discussion Treatment of AID does not seem to increase the incidence of MDS or AML. Furthermore, the incidence of myeloid neoplasm occurring in association with treatment of AID is not higher than expected in the general SEER population. However, the case cohort was exposed to more agents overall and more likely to have exposure to azathioprine than controls. Limitations of our study are associated with its retrospective nature, possible associated underrepresentation of t-MN, and autoimmune population profile. Nevertheless, this is the largest case study with detailed drug exposures with timelines for pts with AID, contributing to a better understanding regarding possible risk of developing MDS/AML in pts treated for AID. Confirmation of the same study at Mayo Clinic Rochester and Florida are ongoing. Disclosures Al-Kali: Novartis: Research Funding.