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Abstract Abstract 4479 Background: In vivo T-cell depletion with antithymocyte globulin (ATG) is known to decrease the incidence of acute and chronic graft vs. host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). However, the detailed patterns of GVHD (incidence, severity, timing, and quality) after ATG-based conditioning have not been examined in large patient cohorts, and it is unknown whether they differ from those seen in patients who do not receive ATG during conditioning therapy. Patients and Methods: We analyzed the incidence and characteristics of acute and chronic GVHD, requirements for immunosuppressive therapy (IST) and survival in a cohort of 174 patients who underwent a first HCT for hematologic malignancy with ATG as a part of their conditioning regimen. The median age was 54 years (range 19–76); all but 5 pts received PBSC, and median follow-up of survivors was 16.9 months (range 3–70 months). Donors were matched related in 18% (n=32), matched unrelated in 44% (n=77), and mismatched unrelated in 37% (n=65). Conditioning regimens were myeloablative in 33% (n=57) and reduced intensity in 67% (n=117). Additional GVHD prophylaxis included tacrolimus in all patients combined with either methotrexate (42%) or MMF (58%). Results: The cumulative incidence of grade II-IV and III-IV acute GVHD at 100 days was 34% and 7%, respectively, with the median time of onset at 43 days (range 11–98 days) after transplant. Eleven patients (6.3%) required additional immunosuppressive treatment due to steroid refractory GVHD. Late/persistent acute GVHD without any evidence of chronic occurred in 25% of patients. NIH chronic GVHD developed in 25 patients, with a cumulative incidence of 24.4% at 2 years. Forty four percent of these patients were classified as classic chronic, and 56% as overlap. The onset of chronic GVHD was quiescent in 20 (80%), progressive in 3 (12%), and de-novo in 2 (8%) patients. The global severity was mild in 9 (36%), moderate in 11 (44%) and severe in 5 (20%) cases. The median time of onset for chronic GVHD was 185 days (range 99–763). In a multivariate analysis of factors predictive for development of chronic GVHD, the only factor associated with development of chronic GVHD was prior grade II-IV acute GVHD (HR 2.5, p =.03). The most common diagnostic organ was mouth (n=16), followed by skin (n=8) and eye (n=1). The median number of sites involved during the course of chronic GVHD was 4 (range 1–7), and the median number of systemic immunosuppressive agents for treatment was 2 (range 0–4). Among the 25 chronic GVHD patients, 5 have discontinued immunosuppression at a median time of 13.1 months (range 6–26) since the diagnosis of chronic GVHD. The cumulative incidence of discontinuation of IST was 23% at one year and 50% at two years. Three deaths in the overall cohort were attributed to complications related to acute (n=2) or chronic GVHD (n=1). At 2 years, the overall survival among all 174 pts was 62.4%, cumulative incidence of relapse was 23.1%, and non-relapse mortality was 22.7%. Conclusion: These data from a large, uniformly treated and graded, predominantly peripheral blood stem cell transplant recipient population, confirm that ATG decreases both the incidence and severity of acute and chronic GVHD. In particular, the rate of moderate to severe chronic GVHD is extremely low, resulting in minimal need for tertiary treatment and decreased duration of immunosupression. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction - Allogenic hematopoietic cell transplantation (HCT) remains potential curative treatment for hematologic malignancies. Anti-thymocyte globulin (ATG) is an immunoglobulin directed to human T lymphocytes, and may decrease the risk of chronic graft versus host disease (cGVHD). Use of ATG has been associated with higher incidence of disease relapse and infection. While the optimal dosing of ATG has not been well established, higher doses (≥ 7.5 mg/kg) have a stronger association with infection and disease relapse. Our institution utilizes low dose rabbit ATG at 2.5 mg/kg for in-vivo T-cell depletion. In this retrospective study we evaluated outcomes in patients receiving low dose ATG to those not undergoing in-vivo T-cell depletion with the hypothesis that low dose ATG protects against cGVHD without impacting infectious complications or relapse. Patients and Methods - We evaluated all patients with 8/8 HLA Matched unrelated Donors (MUD) undergoing HCT between 2007 and 2016. Patients were divided in to two cohorts, those receiving low dose ATG to those that did not. Both cohorts received standard GVHD prophylaxis with calcineurin inhibition plus methotrexate or mycophenolate. The primary outcome was overall survival (OS). Secondary outcomes included relapse free survival (RFS), EBV reactivation, CMV reactivation, CMV disease, invasive fungal infection, incidence of acute GVHD (aGVHD) grades III-IV, and moderate to severe cGVHD. A subset analysis was performed to assess relapse free survival (RFS) in patients with myeloid malignancies. Descriptive statistics were utilized for baseline characteristics; and a series of univariate and multivariant Cox proportional hazard regression models were conducted for outcomes of interest. A Kaplan-Meier (K-M) survival curve was used to depict probability of survival by ATG cohort. Results - We identified 209 patients, with 129 (62%) in ATG group and 80 (38%) in non-ATG group. At baseline, there were no differences in the age at transplant, sex, ABO incompatibility, or performance status. The ATG cohort had fewer low disease risk index (DRI) patients and more patients with intermediate or high DRI (2.4%, 64.6%, 28.3% vs 18.4%, 51.3%, 23.7% respectively), and Fisher's Exact test showed that this difference between ATG groups was statistically significant, p = <0.001. The ATG cohort also had significantly different rates of myeloablative (MA) conditioning regimens from the non-ATG cohort with fewer receiving MA (31% vs 53% p < 0.01). No statistically significant differences in OS were observed between the two ATG cohorts at up to 5 years post-transplant (p=0.35; Fig. 1). The primary predictor of OS was DRI very high vs low [HR 6.0 (2-18) p < 0.01]. The ATG exposed cohort had significantly lower risk of cGVHD [HR 0.17 (0.08-0.37) p = <0.001]. There was no difference in risk of death from relapse, incidence of EBV or CMV reactivation, CMV disease, invasive fungal infection, or grade III-IV acute GVHD. Low dose ATG was not associated with inferior RFS, including in subset analysis of patients with myeloid malignancies [HR 0.85 (0.5-1.4) p = 0.549]. Conclusions - Our study shows no significant difference in survival or death from relapse for patients who receive ATG. Importantly, patients receiving ATG had a lower rate of cGVHD with no increased rates of infectious complications. Low dose ATG did not appear to increase the risk of relapse, even when considering only myeloid malignancies. These findings suggest that low dose ATG can improve cGVHD with no impact on survival, relapse, or infectious complications. Additional prospective trials using low dose ATG compared to no ATG are warranted. Disclosures Palmer: Novartis: Research Funding.

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.

Background Steroid-refractory (SR) acute graft-versus host disease (aGVHD) is a serious complication of allogeneic HCT, for which there is no established standard treatment. Both ECP and cytokine inhibitors such as Infliximab have apparent single agent activity in the therapy of SR aGVHD, but there are no published data that address the efficacy of combination anticytokine therapy and ECP in this setting. We report here results in 18 adult patients (pts) with grade III or IV SR aGVHD who were treated with concurrent Infliximab and ECP. Patients All pts underwent allogeneic HCT (first transplant, n=15, second transplant, n=3) for a hematologic malignancy at Mayo Clinic Arizona between March 2012 and May 2013. The median age was 61 (range 22-70), and the median follow-up of 6 surviving pts is 263 days (range 79-433). Donors were related in 3, matched unrelated in 8, and mismatched unrelated in 7. GVHD prophylaxis consisted of tacrolimus and mycophenolate mofetil in 13 pts and tacrolimus/methotrexate in 5; in addition, 17 pts received in vivo T-cell depletion with Thymoglobulin 2.5 – 7.5 mg/kg. All pts developed grades III-IV aGVHD at a median day 27 post-transplant (range 10-107) and failed to respond to a course of high-dose corticosteroids (methylprednisolone or equivalent 2 mg/kg daily) for at least 5 days. Treatment Regimen At diagnosis of SR aGVHD, pts were scheduled to receive weekly Infliximab 10 mg/kg (maximum 4 doses), concurrent with ECP performed twice or thrice weekly for one month, followed by a pre-specified taper schedule (anticipated total treatment duration of 6-9 months). To minimize infectious risk and morbidity, corticosteroids were tapered rapidly after the start of Infliximab/ECP therapy. Results Thirteen pts (72%) responded to the Infliximab/ECP combination; complete response (CR; resolution of all GVHD symptoms for at least 30 days and able to discontinue corticosteroids) was observed in 6 pts (33%), and partial response (PR; improvement in GVHD symptoms, partial taper of corticosteroids, and no additional agent for at least 30 days) in 7 pts (39%). At the time of this report, 6 pts are alive, and the Kaplan-Meier estimate of one-year survival is 22.5 % (95% CI, 8.8%-47%). Only pts who achieved CR had good outcomes (5/6 surviving and clinically well); among 13 pts with PR (7) or no response/progression (6) to Infliximab/ECP, 12 have died and one is day 79 post transplant and remains on ECP and low-dose prednisone. Eleven of the 12 deaths were related to GVHD or complications of its treatment (infection, multi-organ failure). Conclusions In this retrospective study of adult pts with severe (grade III/IV) SR aGVHD, treatment with 4 doses of weekly Infliximab and concurrent ECP resulted in an overall response rate of 72%, with one-third of pts achieving CR; the regimen was well-tolerated and facilitated early taper and rapid discontinuance of corticosteroids. Unfortunately, the overall survival of 22.5% at one year does not appear superior to other single agent or combination therapies reported in the literature, or to historical data from our program. Although a small percentage of pts appear to be long-term survivors, the majority of pts succumb to complications of progressive GVHD or infection. Given poor results with available therapies, and the dismal prognosis of pts with SR aGVHD, future research should focus on better preventative strategies for this devastating complication of allogeneic HCT. Disclosures: No relevant conflicts of interest to declare.

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.