Survival In Acute Myeloid Leukemia Research Articles

P2X7 promotes the progression of MLL-AF9 induced acute myeloid leukemia by upregulation of Pbx3.

Nucleotides mediate intercellular communication by activating purinergic receptors and take part in various physiological and pathological processes. Abnormal purinergic signaling plays important roles in malignant progression. P2X7, which belongs to the P2X family of purinergic receptors, is abnormally expressed in various types of malignancies including leukemia. However, its role and molecular mechanism of action in leukemia have not been elucidated. Here, we analyzed the correlation between P2X7 expression and clinical outcome in acute myeloid leukemia (AML); we explored the role and mechanism of P2X7 in AML progression by using mouse AML, nude mouse xenograft and patientderived xenograft models. High levels of P2X7 expression were correlated with worse survival in AML. P2X7 was highly expressed in MLLrearranged AML and accelerated the progression of this type of AML both by promoting cell proliferation and by increasing leukemia stem cells. Furthermore, P2X7 caused upregulation of Pbx3 which might account for its pro-leukemic effects. The P2X7-Pbx3 pathway might also contribute to the progression of other types of leukemia as well as solid tumors with high levels of P2X7 expression. Our study provides new insights into the progression of malignancy caused by abnormal purinergic signaling.

Dysregulation of miRNA expression and their prognostic significance in paediatric cytogenetically normal acute myeloid leukaemia.

Dysregulation of miRNA expression and their prognostic significance in paediatric cytogenetically normal acute myeloid leukaemia.

Single-Cell Gene Expression Analyses Reveal Distinct Self-Renewing and Proliferating Subsets in the Leukemia Stem Cell Compartment in Acute Myeloid Leukemia.

Standard chemotherapy for acute myeloid leukemia (AML) targets proliferative cells and efficiently induces complete remission; however, many patients relapse and die of their disease. Relapse is caused by leukemia stem cells (LSC), the cells with self-renewal capacity. Self-renewal and proliferation are separate functions in normal hematopoietic stem cells (HSC) in steady-state conditions. If these functions are also separate functions in LSCs, then antiproliferative therapies may fail to target self-renewal, allowing for relapse. We investigated whether proliferation and self-renewal are separate functions in LSCs as they often are in HSCs. Distinct transcriptional profiles within LSCs of Mll-AF9/NRASG12V murine AML were identified using single-cell RNA sequencing. Single-cell qPCR revealed that these genes were also differentially expressed in primary human LSCs and normal human HSPCs. A smaller subset of these genes was upregulated in LSCs relative to HSPCs; this subset of genes constitutes "LSC-specific" genes in human AML. To assess the differences between these profiles, we identified cell surface markers, CD69 and CD36, whose genes were differentially expressed between these profiles. In vivo mouse reconstitution assays resealed that only CD69High LSCs were capable of self-renewal and were poorly proliferative. In contrast, CD36High LSCs were unable to transplant leukemia but were highly proliferative. These data demonstrate that the transcriptional foundations of self-renewal and proliferation are distinct in LSCs as they often are in normal stem cells and suggest that therapeutic strategies that target self-renewal, in addition to proliferation, are critical to prevent relapse and improve survival in AML. SIGNIFICANCE: These findings define and functionally validate a self-renewal gene profile of leukemia stem cells at the single-cell level and demonstrate that self-renewal and proliferation are distinct in AML. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/3/458/F1.large.jpg.

Prognostic Impact of Pre-Transplant Chromosomal Aberrations Detected By SNP-Array in Patients Undergoing Unrelated Donor Hematopoietic Cell Transplant for Acute Myeloid Leukemia

<h3>Background</h3> High-resolution genome-wide SNP-arrays detect large chromosomal aberrations including copy-neutral loss of heterozygosity (CNLOH), which is not captured in conventional cytogenetics. <h3>Methods</h3> We used SNP-array genotyping data generated by the DISCOVeRY-BMT study to detect chromosomal aberrations in pre-HCT blood samples from 1,974 acute myeloid leukemia (AML) patients. Clinical data and blood samples were available through the Center for International Blood and Marrow Transplant Research. We used Cox proportional hazard model for statistical analyses. <h3>Results</h3> AML patients in this study received unrelated donor HCT between 2000-2011 at a median age of 48.2 (range=0.6-78.0) years. About 53% of the patients were males, 7.4% had therapy-related disease, and 73% were in complete remission. Chromosomal aberrations were detected in 14.4% of the patients (Figure 1). Most common aberrations in patients with advanced disease at HCT (n=536) were copy losses in chr7 (5.8%), chr5 (4.5%), CNLOH in the following: chr13 (4.7%), chr11 (3.9%), or chr17 (3.5%), and copy gain in chr8 (3.5%). Common aberrations in patients in complete remission (n=1438) were copy-loss in chr7 (1.0%), chr5 (0.7%), and CNLOH in chr9 (0.9%) or in chr17 (0.8%) (Figure 2). Among patients with normal cytogenetics at diagnosis (n=572), 27.5% received HCT in advanced disease (of them 8.3% had chr13-CNLOH). In multivariable models including commonly detected aberrations and adjusted for important factors (footnote Figure 3), chr13-CNLOH was associated with an approximately 3-fold increased risk of leukemia relapse and post-HCT mortality in patients with advanced disease (relapse: HR=2.67, 95% CI=1.67-4.26; mortality: HR=2.79, 95% CI=1.81-4.28, p<0.0001). In contrast, chr17-CNLOH was associated with a statistically significant risk of post-HCT mortality in patients with remission (HR=2.85, 95% CI=1.51-5.37, p=0.001); the association with relapse was not statistically significant (HR=1.84, p=0.21). Having aberrations in ≥3 chromosomes was associated with a statistically significant excess risk of mortality in patients with advanced disease (HR=1.59, 95% CI=1.07-2.36, p=0.02) (Figure 3). In patients with normal cytogenetics at diagnosis, chr13-CNLOH pre-HCT refined risk stratification of patients with advanced disease; compared to patients in remission, the HR for OS in patients with advanced disease in the presence or absence of chr13-CNLOH= 5.7 vs. 1.7, respectively, p<0.0001 for both; HR=3.6, p=0.0004 when comparing presence or absence of chr13-CNLOH in patients with advanced disease. <h3>Conclusion</h3> Pre-HCT CNLOH in chr13 or chr17 are associated with inferior post-HCT survival in AML; possibly as a consequence of high risk of post-HCT leukemia relapse. CNLOH in chr13 or chr17 may provide new genomic prognostic markers that guide patient risk stratification and possibly therapeutic options.

High serum extracellular vesicle miR-10b expression predicts poor prognosis in patients with acutemyeloid leukemia.

Increasing evidence have demonstrated that serum extracellular vesicle microRNAs (EV-miRNAs) are promising noninvasive biomarkers for various cancer types. In this study, we aimed to investigate and evaluate the potential clinical significance of serum EV-miR-10b for acute myeloid leukemia (AML). Blood samples were collected from a cohort of 95 de novo AML patients and 80 healthy individuals. Of all AML patients, 51 patients were considered as cytogenetic normal AML (CN-AML). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to measure the expression levels of serum EV-miR-10b. The extracellular vesicles we extracted from the serum samples were positive for EV/exosome markers including TSG101, CD63, Flotillin-1 and CD9. The expression levels of serum EV-miR-10b were significantly higher in AML/CN-AML patients than healthy controls. In addition, serum EV-miR-10b expression was strongly correlated with aggressive clinical characteristics. Moreover, receiver operating characteristic analysis showed serum EV-miR-10b yielded an area under the curve of 0.875, with 77.89% specificity and 82.50% sensitivity in identifying AML patients from normal controls. Furthermore, AML patients with higher serum EV-miR-10b expression had significantly shorter survival and serum EV-miR-10b was demonstrated to be an independent prognostic marker for overall survival in AML. Taken together, serum EV-miR-10b might serve as a promising biomarker for predicting the prognosis of AML.

Acute Myeloid Leukemia in Mexico: The Specific Challenges of a Developing Country. Results From a Multicenter National Registry

BackgroundIn the past decades, long-term survival outcomes for younger patients with acute myeloid leukemia (AML) have improved. Nonetheless, developing nations might be lagging behind, highlighting the need to assess real-world outcomes in such regions. MethodsWe performed a multicenter retrospective study, which included patients with AML diagnosed between January 2013 and December 2017 from 13 centers in Mexico. ResultsA total of 525 patients with AML met the inclusion criteria and were included in the study. Median age for the entire cohort was 47 years. The patients were classified according to cytogenetic risk: favorable 16.0%, intermediate 55.6%, and unfavorable 28.4%. Most patients received intensive chemotherapy (80.2%), and among these 74.1% underwent a 7 + 3 induction regimen. A complete remission was achieved in 71.3% of patients. Induction-related mortality occurred in 17.8% and we identify the following as independent risk factors: >60 years (odds ratio [OR] 2.09 [1.09-4.02]), Eastern Cooperative Oncology Group >2 (OR 4.82 [2.46-9.43]), prior solid tumor (OR 3.8 [1.24-11.59]) and active infection (OR 1.82 [1.06-3.12]). Further, allogeneic hematopoietic stem-cell transplantation (AlloHSCT) was performed in 8.2% in CR1. The 3-year overall survival (OS) was 34.8%. In a multivariate analysis, several factors were independently associated with a worse OS, including secondary AML (hazard ratio [HR] 2.14 [1.15-4.01]) and unfavorable cytogenetic risk (HR 1.81 [1.16-2.82]), whereas maintenance therapy (HR 0.53 [0.32-0.86]) and AlloHSCT (HR 0.40 [0.17-0.94]) were associated with better OS. ConclusionsThis is the first multicenter report analyzing AML survival in Mexico. Challenges in this setting include a high induction-related mortality and low AlloHSCT rate, which should be addressed to improve outcomes.

Cell adhesion to collagen promotes leukemia resistance to doxorubicin by reducing DNA damage through the inhibition of Rac1 activation

Chemoresistance is a major hurdle in anti-cancer therapy. Growing evidence indicates that integrin-mediated cell adhesion to extracellular matrix plays a major role in chemoresistance. However, the underlying mechanisms are not fully understood. We have previously shown that the collagen-binding integrin α2β1 promoted doxorubicin resistance in acute T cell lymphoblastic leukemia (T-ALL). In this study, we found that acute myeloid leukemia (AML) cell lines also express α2β1 integrin and collagen promoted their chemoresistance as well. Furthermore, we found that high levels of α2 integrin correlate with worse overall survival in AML. Our results showed that doxorubicin-induced apoptosis in leukemic cells is associated with activation of Ras-related C3 botulinum toxin substrate 1 (Rac1) and that collagen inhibited this pathway. The protective effect of collagen is associated with the inhibition of Rac1-induced DNA damage as evaluated by the comet assay and the phosphorylated levels of histone H2AX (γ-H2AX). Together these results show that by inhibiting pro-apoptotic Rac1, α2β1 integrin can be a major pathway protecting leukemic cells from genotoxic agents and may thus represent an important therapeutic target in anti-cancer treatment.

Durable Responses and Survival in High Risk AML and MDS Patients Treated with an Allogeneic Leukemia-Derived Dendritic Cell Vaccine

Background. Maintenance of induced responses remains a significant unmet medical need in patients with acute myeloid leukemia (AML) or high risk myelodysplastic syndrome (MDS) not eligible for allogeneic hematopoietic stem cell transplantation (HSCT). The allogeneic leukemia-derived dendritic cell vaccine, DCP-001, has shown to generate both humoral and cellular immune responses and is safe for clinical practice (van de Loosdrecht et al., Cancer Immunol. Immunother. 2018). In this study we show additional data on long-term follow up and patient-related risk factors from the phase 1 study. Methods. AML- or MDS-patients (n=12), ineligible for HSCT, were included in a post-hoc clinical analysis of the DCP-001 phase I trial, receiving DCP-001 vaccination at the Amsterdam University Medical Center, VU University Medical Center, Amsterdam, the Netherlands (NCT01373515). All clinical data, including baseline characteristics as cytogenetics, pre-and post-vaccination treatment and morphologic blast counts in follow up, were retrospectively collected from corresponding patient files. Targeted next generation sequencing (NGS) gene panel data and cytogenetics were utilized to determine (cyto-)genetic risks following the European Leukemia Net (ELN) 2017 criteria. We defined 'response' as decreased or stable morphologic blast counts in bone marrow at day 126 (last day of official study evaluation) compared to study entry. Results. Seven out of twelve patients responded to treatment, the other patients showed progressive disease (data are summarized in table 1). The median relapse free survival (RFS) for the responding patients was 420 days (range 90-1849) and overall survival (OS) was 1090 days (90-2160); two patients died early in complete remission (CR) due to infection (respectively at 90 and 184 days). Notably, three of these patients were intermediate risk and four were poor risk patients based on the ELN risk classification for AML or Revised International Prognostic Scoring System (IPSS-R) for MDS. The median OS for the non-responders was 144 days (range 59-209). These five patients had relapsed or refractory disease at start of vaccination with detectable circulating peripheral blasts; four of them received additional azacitidine (AZA) therapy prior to vaccination. Moreover, they were vaccinated after induction and consolidation therapy with a median time between diagnosis and first vaccination of 611 days (219-2310) compared to 240 days (105-1398) for the seven responding patients. Of the latter, all were in complete remission (CR) (n=5) or had morphologic marrow blast counts &amp;lt;10% (n=2) at the start of vaccination. Two responders that progressed and two that relapsed after vaccination were treated with AZA, resulting in one complete remission and two partial responses (one non-evaluable due to sample failure), see figure 1. Conclusion/discussion. Clinical data of the phase 1 trial show that DCP-001 may prolong duration of remission or smoldering disease in intermediate and high risk AML or MDS patients. Response was associated with treatment by vaccination shortly after achieving CR. This should be taken into account in selection criteria of future vaccination studies. Hence, an international multi-center phase 2 study (ADVANCE II; NCT03697707) is currently being conducted to determine the effect of DCP-001 on measurable residual disease (MRD) in AML patients, aiming to convert them to a MRD negative state. Furthermore, we demonstrated that AZA can be applied as rescue therapy upon progression after vaccination. Disclosures Rovers: DCprime: Employment. Cloos:Daiicchi Sankyo: Speakers Bureau; Glycomimetics: Research Funding; Takeda: Research Funding; Novartis: Other: MRD assessments of clinical trials, Research Funding; Merus: Other: MRD assessment of a clinical trial , Research Funding; Genentech: Consultancy, Research Funding; Helsinn: Other: MRD assessment of a clinical trial; DCPrime: Other: MRD assessment of a clinical trial. de Gruijl:DCprime: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Treatment Outcome and a Practical 10-Point Prognostic Index in Adult Patients with Non-M3 Acute Myeloid Leukemia: A Retrospective, Multicenter Study in Malaysia

Background: Literature on Acute Myeloid Leukemia (AML) survival and prognostic factors were often derived from strict trial studies from developed country. A simple yet practical prognosis index has not been developed and tested in resource limited setting such as Malaysia. We described the treatment outcome and designed a 10 point prognostic index to predict survival of adult AML (non-M3) in real clinical practice in Malaysia. Methods: Data were retrospectively collected and analyzed from all adults with AML diagnosed and treated from 2007 to 2017 in three main hematology centers in Malaysia, Ampang Hospital, Sarawak General Hospital and Miri General Hospital. Treatment pattern and survival outcome were described. Multivariable analysis using Cox regression statistics were performed to identify significant prognostic variables affecting overall survival. Each variable were assigned points based on hazard ratios. A sum of the points led to a maximum score of 10. Patients were then categorized into low (0 point), intermediate (1 to 3 points) or high-risk group (4 points or above). Results: Demographics and treatment outcome of patients are shown in Table 1 &amp; 2. There were 1277 adult patients, diagnosed with AML where 86.5% (n= 1106) of them were non M3 AML. Out of these, 908 patients (82.2%) received intensive chemotherapy treatment. Median age of diagnosis was 45 years. The remission post induction rate was 64.3% with induction death, refractory and relapse rate of 8.8%, 20.0% and 27.7% respectively. Median overall survival (OS) and Event Free Survival (EFS) time was 15 months and 12 months. The 3-year OS and EFS was 32.9% and 28.5% respectively. At the time of analysis, 66.1% of patients were dead (n=600) with disease progression being the main cause of death (n=416, 45.8%). Three year overall OS for patients who underwent allogeneic stem cell transplant (n=301, 33.1%) versus patients without transplantation were 53.7 % versus 22.0 % (HR 2.597, p &lt;0.001). Cumulative incidence of relapsed and non-relapse mortality for transplant patients, shown in Figure 1 were 27.5% and 22.1%. Multivariate analysis in Table 3 showed that age 60 years old and above, male gender, white cell count more than 100 x 109 /L ,relapsed less than 12 months of treatment, refractory state after induction and high risk genetic group (based on EuropeanLeukemiaNet/Medical Research Council risk stratification by genetics) are prognostic factors associated with worse OS and EFS. The information was used to develop a 10 point prognostic index based on calculation described in Table 3. Overall survival decreased with each additional index point. When stratified according to risk group, the 3 year OS for low risk, intermediate risk and high risk group was 53.3%, 34.3% and 4.9% respectively. This is shown in Table 4 &amp; Figure 2. Relapse rate was also lower in the low-risk group (8.8%), compared to intermediate-risk group (19.2%) and high-risk group (35.2%). Comparing transplant and non transplant cohort shown in Figure 3, there was no survival benefit in the low-risk group (58.6% vs 49.2%, p=0.122) but significant survival benefit in both intermediate-risk group (56.6% vs 23%, p&lt;0.001) and adverse-risk group (13% vs 7%, p=0.002). Discussion/Conclusion: This is one of few survival studies that involved patients of different ethic groups in Asia (Malay, Chinese, Indian and native Borneo Sarawakians). Our results are comparable to data from large population based database such as US SEER and EURO CARE. This is the first prognostic index incorporating genetics, baseline characteristics and dynamic response, eg. refractory and/or relapsed post induction in non M3 AML. The results reaffirmed the importance of these factors in determining the clinical outcome and prognosis of patients with AML. When stratified using our 10 point prognostic index, our cohort of patients who is in low risk group has lower relapse rate and did not have significant survival benefit from allogeneic transplant compare to stratification using only the ELN/MRC genetic classification.(Table 5 &amp; 6). In resource limited setting, measurable residual disease (MRD) monitoring and advanced genetic testing are difficult financially. This prognostic scoring index is an economical and practical alternative to guide physicians on treatment after induction therapy. However, it still needs to be validated by a larger cohort of patients in a prospective study. Disclosures No relevant conflicts of interest to declare.

Low-Dose Azacitidine, Pioglitazone and All-Trans Retinoic Acid Versus Standard-Dose Azacitidine in Patients ≥ 60 Years with Acute Myeloid Leukemia Refractory to Standard Induction Chemotherapy (AMLSG 26-16/AML-ViVA): Results of the Safety Run-in Phase I

Introduction: Patients (pts) with acute myeloid leukemia (AML) who are refractory to intensive frontline treatment have a dismal outcome. In case of ineligibility for allogeneic stem cell transplantation (HSCT), the median survival of chemo-refractory AML is about 2 months and less than 5% of these pts are alive after 1-year (retrospective analysis from the AMLSG database). To date, there is no universally accepted standard approach for the treatment of chemo-refractory AML in older pts. Several retrospective studies have assessed the role of hypomethylating agents in this patient group, but complete remission (CR) rates were disappointingly low (≤10%) when compared to first line treatment. The presented study represents a novel approach focusing on hematopoietic tissue reprogramming (i.e. anakoinosis) (ClinicalTrials.gov Identifier: NCT02942758). Methods: The initial dose-finding phase I of the study evaluated the combination of azacitidine (AZA) 75 mg/d s.c. for 7 days, repeated every 28-days, pioglitazone 45 mg/d p.o. continuously from day 1 and all-trans retinoic acid (ATRA). A modified 3+3 design has been used to establish the maximum-tolerated dose of ATRA. Patients have been enrolled at an ATRA dose of 45 mg/m²/d from day 1 to day 28 and 15 mg/m²/d continuously thereafter if no dose limiting toxicity (DLT) occurred until start of next cycle on day 29. The safety DLTs were defined as toxicities attributable to ATRA, expected or unexpected, except if these are likely associated with another cause. Eligible patients had confirmed diagnosis of AML refractory to induction therapy and were not eligible for further intensive induction therapy or were not immediate candidates for allogeneic HSCT. The severity of adverse events was graded using the Common Terminology Criteria for Adverse Events (CTCAE) V. 4.03. The response to treatment was evaluated using standard criteria defined by the expert panel on behalf of the European LeukemiaNet and international working group (IWG) response. Results: Ten pts were enrolled in the safety-run-in phase I (one pt withdrew informed consent on day 9 of cycle 1). Among all treated pts, the median age was 67 years (range, 62-76 years), and the majority of pts (70%) had an ECOG PS of 1 (see table 1). Two pts had secondary AML; another two pts had therapy-related AML (t-AML). Eight pts had a complex karyotype. Concerning safety, hematological adverse events (AEs) were the most common toxicities observed. Because pts with baseline cytopenia were included (leukopenia n=8; 80%; thrombocytopenia n=9; 90%), occurrences of many hematological AEs began before study drug initiation and were attributed to underlying hematologic disease. Common 3°/4° AEs included neutropenia (50%), anemia (50%), thrombocytopenia (30%), and infections (40%). 50% of pts experienced a serious AE; one 5° AE (gastric hemorrhage) occurred. No DLTs were observed. Five pts discontinued the study, with progressive disease (PD) or relapse being the most common reason for discontinuation. Concerning efficacy, 3 pts (30%) achieved a CR and one pt a long-lasting stable disease (14 months). Morphologic review showed signs of differentiation of blasts in responding pts, which has already been shown in in-vitro analysis. In line with this observation, one pt demonstrated resolution of fungal pneumonia during the study. Conclusions: In summary, the low-intensity, biomodulatory regimen of low-dose AZA, pioglitazone, and ATRA demonstrated a tolerable safety profile and encouraging signals for efficacy in pts with AML refractory to standard induction chemotherapy warranting further investigation. S.T. and A.R. contributed equally to this abstract as senior co-authors. Disclosures Paschka: Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau; Abbvie: Other: Travel expenses; Amgen: Other: Travel expenses; Otsuka: Membership on an entity's Board of Directors or advisory committees; BMS: Other: Travel expenses, Speakers Bureau; Astex: Membership on an entity's Board of Directors or advisory committees, Travel expenses; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Other: Travel expenses; Takeda: Other: Travel expenses; Sunesis: Membership on an entity's Board of Directors or advisory committees. Döhner:Celgene, Novartis, Sunesis: Honoraria, Research Funding; AROG, Bristol Myers Squibb, Pfizer: Research Funding; AbbVie, Agios, Amgen, Astellas, Astex, Celator, Janssen, Jazz, Seattle Genetics: Consultancy, Honoraria. Thomas:Celgene: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Medigene AG: Consultancy, Other: Travel support; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Medac: Other: Travel support; Janssen: Other: Travel support.

Increased Toxicity Among Adolescents and Young Adults Treated for Acute Myeloid Leukemia at US Children's Hospitals

Introduction Adolescents and Young Adults (AYAs) with cancer are known to experience disparities in access to care along with delays in diagnosis and treatment. Acute Myeloid leukemia (AML) represents 15-20% of childhood leukemias and 33% of adolescent leukemias. Although improvements in the survival of pediatric AML is encouraging, older age has historically been an adverse prognostic factor, and survival does not appear to be improving to the same extent in AYAs as in children. AYA patients treated on pediatric trials had better outcomes than those treated on adult trials. As a result, AYAs are increasingly being treated in children's hospitals on pediatric protocols, which generally consist of intensified regimens compared with adult protocols. However, minimal data exists regarding how well AYAs tolerate the intensity of chemotherapy at doses and regimens designed for children. Our study aims to compare toxicities between pediatric and AYA patients treated for AML in children's hospitals in the United States. Methods With IRB approval, the Pediatric Health Information Systems (PHIS) database was queried to analyze healthcare outcomes in pediatric and AYA patients with AML admitted between January 2004 and December 2018. We chose to utilize the PHIS database, as these AYA patients with AML were more likely to have been treated on pediatric protocols. AYA was defined as patients between the ages of 15 and 39 per the National Cancer Institute (NCI) definition. We extracted relevant ICD-9 and -10 diagnoses, procedure codes and medications for each patient related to toxicities as outlined by the NCI. For the descriptive statistics, demographics and clinical characteristics of patients were described using median and interquartile ranges for continuous variables, and frequency and percentages for categorical variables. Categorical variables were analyzed with the chi-square test. Continuous variables were assessed by the Mann Whitney U test. Multiple logistic regression was performed with 'discharge mortality' as the primary outcome. The age was categorized into two levels (0-14 y.o and ≥ 15 y.o). P-value of less than 0.05 or absence of 1 in the 95% confidence intervals were considered as statistically significant for the analysis. All statistical analyses were performed using SAS software, version 9.4 (SAS institute, Cary, NC). Results A total of 31,868 admissions met inclusion criteria, representing a total of 7,229 AML patients. Of these, 8,606 admissions (27%) were in the AYA group. 64% of children and 67% of AYA patients were Caucasians. AYA patients had a significantly higher incidence of ICU stay [20.1% vs 15.7%, p&amp;lt;0.0001] and overall mortality [23.4% vs 15.5%, p&amp;lt;0.0001]. Out of a total of 7,229 AML patients, 1277 (18%) passed away. Table 1 describes our findings and show statistically significant differences among the two groups, with AYA patients showing markedly increased toxicity, and requiring more supportive medications and procedural interventions. No difference in terms of need for blood and platelets products was observed among the two groups. 24.4% of children and 31.6% of AYA patients underwent stem cell transplant. We identified no significant differences in mortality between these two groups. After adjusting for other factors, diagnosis prior to 2014, public insurance, African American ethnicity in ≥ 15 y.o, ICU stay and use of antifungals other than fluconazole, were associated with inferior outcome. ICU stay remained the strongest predictor of mortality (OR 10.5, 95% CI = 8.85 - 12.47 for pediatric patients, and OR 13.3, 95% CI = 10.39 - 17.03 for AYA patients). Conclusion To our knowledge, this is the largest multicenter database study evaluating toxicity and mortality in AML patients. Compared to children, AYA patients developed disproportionately higher toxicities from chemotherapy and required more supportive medications and procedures. We found overall increased mortality in AYA patients, however the negative impact of older age lost its significance in an additional analysis focusing on transplant patients. Despite improvements in treatments and outcomes over time, there is still need for more effective strategies for preventing toxicities and mortality in AYA patients. Prospective studies are needed to assess whether dose modifications for certain chemotherapeutics may improve the toxicity profile of AYA patients with AML treated in Children's hospitals. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia Export Mitochondria in Extracellular Vesicles Which Induces Pro-Tumoral Changes in Bone Marrow Macrophages

It is envisioned that improved understanding of the dependency of acute myeloid leukemia (AML) on its tumour microenvironment within the bone marrow could be exploited to offer new treatment strategies and better patient outcomes. Previously we have shown that the bone marrow (BM) microenvironment supports AML growth and survival, specifically via functional mitochondrial transfer from stromal cells to AML blasts (Marlein 2017 Blood and Abdul Aziz 2019 Blood). The increase in mitochondria in AML theoretically presents a cell intrinsic problem; accumulation of excess and dysfunctional mitochondria can induce cell death via increased reactive oxygen species and apoptotic caspases (Kroemer 2007 Physiol Rev). As such, we examine the mechanisms by which AML blasts manage excess and dysfunctional mitochondria and the impact of this process on the supporting bone marrow microenvironment. Primary AML blasts and primary AML bone marrow stromal cells (BMSC) were isolated from the bone marrow of patients. Co-culture experiments show that primary AML blasts (cultured with BMSC) maintain constant levels of mitochondria despite continuous uptake of mitochondria from BMSC. Furthermore, a combination of proteomics, confocal microscopy, image cytometry and dynamic light scattering shows that AML actively export mitochondria in large extracellular vesicles (EVs), approximately 0.6micron-1.5micron in diameter, a process which maintains a steady state of mitochondrial content in the blasts. To track the recipient cell for large EV packaged AML derived mitochondria, patient derived AML were transduced with rLV.EF.mCherry-mito-9 lentivirus which fluorescently tags the mitochondria, and then transplanted into NSG mice. BM was extracted from engrafted mice and various cell populations were analysed for increases in mCherry fluorescence as evidence of AML derived mitochondria uptake. Murine F4/80+/GR1-/CD115intBM macrophages had increased mCherry fluorescence but not mouse CD45-/CD31-/Ter119-/CD105+/CD140a+BMSC, CD45-/Ter119-/CD31+endothelial cells or CD45+leukocytes. To determine the impact of AML derived mitochondria containing EVs on the function of the BM macrophages we first isolated the AML derived mitochondrial containing EVs using cell sorting for mCherry fluorescence and then BM derived macrophages (BMDM) were incubated with EVs containing mitochondria or EV with no mitochondria and assessed for changes in pro-inflammatory genes. IL-6, IL-1B, CXLC9 and CXCL10 were all induced in BMDM by EV containing mitochondria at 24 hours post incubation. However, repeated exposure of BMDM with EV containing mitochondria resulted in significantly lower levels of IL-6, IL-1B, CXLC9 and CXCL10 upregulation. Moreover, repeated exposure of BMDM to EV containing mitochondria resulted in reduced phagocytic potential compared to control BMDM, suggesting exhaustion or senescence. To determine if AML induced BM macrophage senescence we used a syngeneic model of AML, HOXA9/Meis1 or MN1 and lineage negative cells as control. Animals were sacrificed 35 days post AML injection and BM macrophages were isolated and senescent markers were analysed by real-time PCR. p16ink4a and p21 were both upregulated in BM macrophages from HOXA9/Meis1 or MN1 engrafted animals. Data indicates that mitochondrial mass in AML blasts is maintained in equipoise through export of mitochondria in large EVs. In addition, we show that AML derived EVs containing mitochondria function as a signal which changes the function of BM macrophages, towards a senescent phenotype which promotes AML blast survival and proliferation. Disclosures Bowles: Abbvie: Research Funding; Janssen: Research Funding. Rushworth:Abbvie: Research Funding; Janssen: Research Funding.

Patient-Derived Acute Myeloid Leukemia (AML) Ex Vivo platform for Evaluation of Novel Molecular Targeted Therapeutic Agents

Acute myelogenous leukemia (AML) is the most common acute leukemia in adults. A hematologic cancer, the disease is highly heterogeneous, with multiple subtypes. Despite advances in treatment, the long-term survival for AML remains poor and the development of novel treatments is an unmet need. Due to the highly divergent subtypes and mutation profiles in AML, the use of patient-derived models may improve drug discovery and development. To address this, we have established a short-term culture system that supports the growth of primary AML cells ex vivo to permit the evaluation and/or screening of candidate therapeutic agents. Our AML bank is comprised of patient-derived specimens across a range of subtypes (which includes M1, M2, M4, M5, NOS and others), and includes models with common mutations in FLT3 (ITD), IDH1/IDH2 and NPM. Primary AML specimens were characterized for common mutations by TruSight sequencing and for surface marker expression by flow cytometry. The ex vivo assay system was evaluated for the ability to support the survival and expansion of primary AML specimens. Among these, the majority of the models showed evidence of proliferation, a few models had no net expansion, and some failed to survive. Extension of the culture period for up to 14 days was feasible, with most models having equal or increased cell numbers by the end of the culture period (8 of 10 models evaluated). All models stably expressed CD33 throughout the assay. To verify the applicability of this system for drug testing, a standard of care agent cytarabine (Ara-C) was assessed for each of the AML models. A small cohort of models with distinct mutations were tested for sensitivity towards small molecule inhibitors venetoclax, gilteritnib, glasdegib, panobinostat and Ibrutinib. Cell growth/viability was assessed using Cell titer-Glo assay. Concentration-dependent responses to Ara-C were observed across multiple models (IC50 10 nM to 150 nM), indicating a range of relatively sensitive to resistant AML models. Heterogeneous concentration-dependent responses were observed across multiple patient-derived ex vivo models when treated with venetoclax, glasdegib, gilteritnib, ibrutinib and panobinostat, with both sensitive and resistant models identified. A cohort of models were evaluated in vivo for sensitivity to Ara-C. Systemic engraftment of the patient-derived xenograft AML models (into NOG or NOG-EXL mice) was evaluated by flow cytometry. When engrafted was confirmed, AML-bearing mice were randomized into Ara-C or vehicle control groups. Two weeks later, mice were evaluated for the presence of human CD45+CD33+AML cells. Models that were relatively sensitive to Ara C ex vivo (IC50 &lt; 30 nM) generally showed a greater in vivo response as evidenced by a significant reduction in the mean circulating AML cells versus models with IC50 values &gt;100 nM that had no response to Ara C in vivo. Over thirty distinct patient-derived primary, unpassaged models of AML have been studied both in ex vivo culture and as in vivo systemic models. The diversity in these AML models is reflective of patient diversity, enhancing their utility in the evaluation of novel therapeutic candidates. These data indicate the feasibility of utilization of these primary models, ex vivo as well as in vivo, for drug discovery for AML, from screening to preclinical efficacy modeling. Disclosures Verma: Champions Oncology: Employment. Biel:Champions Oncology: Employment. Ruggeri:Champions Oncology: Consultancy. Wesa:Champions Oncology: Employment, Other: stock options.

Chidamide Combined with Decitabine Synergistically Induce Apoptosis of Acute Myeloid Leukemia By up-Regulating Perp gene In Vivo and in Vitro

Background: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem cells. The long term survival of AML is not satisfactory, so new treatment should be explored. Here, we show that chidamide(CH), a histone deacetylase inhibitor, combined with decitabine(DE) induces apoptosis of AML cell lines and primary refractory/relapsed AML cells by up-regulating PERP. This may provide a new option for AML treatment. Methods and results: We first examined the half-inhibitory concentrations (IC50) of chidamide and decitabine against THP-1, MV4-11, HL60 and Kasumi-1 cell lines using MTT (Fig1 A-D). And the drug combination is performed according to the IC50. In the double-drug combination experiment, we used MTT to detect the effect of drugs on the proliferation of the four cell lines (Fig1 E-H), used calcusyn 2.0 software to calculate the synergistic effect (Fig2), flow cytometry to detect apoptosis (Fig3 A-D), and western blot to detect the pro-apoptotic protein (C-CASPASE 3 and C-CASPASE 9) and anti-apoptotic proteins (CASPASE 8, BCL-2 and BCL-XL) (Fig3 E-H). We found that chidamide combined with decitabine synergistically inhibited proliferation of AML cell lines, induced apoptosis, up-regulated pro-apoptotic protein levels and down-regulated anti-apoptotic protein levels. To investigate this combination therapeutic effect in vivo, we selected 5 refractory/relapsed AML patients, extracted primary AML cells, and used ATP chemiluminescence kit for drug sensitivity test. The results confirmed that four of the five patients with AML showed sensitivity to combinations (Fig4). To further explore the mechanism of action of CH combination with DE, we performed transcriptome sequencing (Fig5). Analysis of the sequencing results, the gene PERP, which shows the significant difference in the apoptotic pathway, was further examined. The PERP is a new member of the PMP-22/GAS3 family as an apoptosis-associated target of TP53. RT-QPCR and WB verified the role of PERP in apoptosis in DE and CH combination (Fig6). The results showed that the combination could up-regulate the PERP gene than the single drug. When we explored the role of the PERP gene in AML cell lines, we knocked down the PERP gene by lentivirus and detected cell proliferation after infection. Pretreated AML cell lines by lentivirus-infection (Fig7A-F), then we tested for proliferation (Fig7G-I) (Fig8A-C), apoptosis (Fig8D-E), and pro-apoptotic protein expression (Fig8G-I). The results showed that knocking down the PERP gene promoted the proliferation of AML cell lines and attenuated the sensitivity of AML cell lines to chemotherapeutic drugs. We also compare the mRNA level of PERP between 35 AML patients and 20 normal and found that the PERP mRNA of AML patients was significantly lower than the normal (Fig9). MV4-11 cells were exposed to CH and DE alone or in combination, and proteomic sequencing was performed to examine the effect of the drug on cellular protein. The result indicates to some extent that CH contributes more to the combined effect. And the drug causes changes in multiple pathways in the cell (Fig10). Conclusion: Our experiments revealed that CH combined with DE may have therapeutic effects on AML and, to some extent, reveal the mechanism of dual drug combination. Legends to figures Fig1. 50% inhibitory concentration (IC50) values of chidamide and decitabine alone treated AML cell lines. Fig2. Chidamide acts synergistically in AML with DE. Fig3. Chidamide in combination with decitabine significantly induced apoptosis in AML cell lines. Fig4. The sensitivity of relapsed or refractory AML primary cells to chidamide and decitabine alone or in combination. Fig5. Gene expression analysis showed an obvious difference based on treatment. Fig6. Verify transcriptome sequencing results by real-time QPCR and by western blotting with or without drug treatment. Fig7. The effect of down-regulation of PERP by Lentivirus-mediated RNAi on AML cells proliferation. Fig8. PERP knockdown causes AML cells to develop resistance to combination drugs. Fig9.The level of PERP mRNA in peripheral blood mononuclear cells of AML and normal humans. Fig10. Proteomics sequencing results show the differentially expressed protein and a cluster analysis of the functions or pathways enriched by differentially expressed proteins in GO and KEGG pathways compared to single agents. Disclosures No relevant conflicts of interest to declare.

Targeting DHODH with AG-636 Induces Apoptosis and Differentiation and Inhibits Mitochondrial Function in AML, Translating into Anti-Tumor Efficacy in Vitro and in Vivo

Targeting DHODH with AG-636 Induces Apoptosis and Differentiation and Inhibits Mitochondrial Function in AML, Translating into Anti-Tumor Efficacy in Vitro and in Vivo

Combining Clinical Features with Genetic Factors Improves Survival Prediction for Adults with Acute Myeloid Leukemia: Validation of a New Score System in 3 Cohorts

Introduction: Risk stratification in acute myeloid leukemia (AML) is continually being refined as we learn more about the molecular pathophysiology of this heterogeneous disease. European LeukemiaNet (ELN) 2017 used widely to stratify the 'genetic' risk of AML, but there are considerable challenges in its application, particularly in centres where molecular genetic testing either not available, or not sufficiently timely for clinical decision making. Up to a third of the study subjects cannot be stratified using a full cytogenetic-molecular model in real-time, real-life setting. There are few attempts to combine clinical features and genetic factors aiming to find a scoring system to improve AML survival prediction. There is only one to our knowledge (Sorror ML et al. 2017). This study has generated an Adapted Genetic Risk (AGR) assessment, and used it in combination with clinical risk parameters to create a novel scoring system which has now been validated using two independent cohorts. Methods: A training cohort from São Paulo (FMUSP, n = 167) of intensively treated AML patients (18-65 years) was assessed using ELN2017 genetic criteria. A comparative validation with our AGR which permits missing cytogenetic or molecular data (Figure 1) split these patients into favorable-risk (FR), intermediate-risk (IR), and adverse-risk (AR). This cohort was also used for Cox Proportional-Hazard Model (CPHM) univariate and multivariate analysis to find clinical parameters that would inform a novel Survival AML Score (SAMLS). Variables which are included in SAMLS had to be either significant in both CPHM models or significant in univariate and crucial for multivariate fitness as measured for the Akaike Information Criterion (AIC). We then applied the AGR strategy and SAMLS to 2 independent test cohorts of intensively treated adult AML patients : Riberao Preto (FMRP, n=145) and Oxford (OUH, n=157). The study was approved by the institutional review boards of the 3 participating centers. Informed consent was obtained from all patients according to the Declaration of Helsinki. Results: Table 1 shows the clinical characteristics for all the 3 cohorts. The median follow-up (FUP) was 72.3, 44.4, and 70.5 months for FMUSP, FMRP, and OUH, respectively. The median Overall Survival (OS) was 12.4, 12.5, and 56.4 months and the 5-year OS were 29.6%, 29.7%, and 49.7% respectively. Both ELN2017 and AGR correlated with significant differences in OS (p-value &lt;0.001) and DFS (ELN p=0.002, AGR p=0.003) in the FMUSP cohort. The ROC c-statistic were 0.68 (ELN) and 0.66 (AGR). AGR was validated in FMRP and OUH cohorts, with statistically significant stratification of OS across the 3 risk groups in both cohorts (FMRP p=0.03, OUH p=0.003); and DFS in FMRP (p = 0.04), but not in OUH (p = 0.7)(Table 2 and Figure 2). Multivariate CPHM model for OS in FMUSP which yielded the smallest AIC were: Age, HR 1.52; Albumin, HR 0.50; WBC, HR 1.37; Haemoglobin, HR 1.48; Platelet count, HR 0.64; and AGR-IR, HR 2.23 and AGR-AR HR, 4.66 (Figure 3). Variables which met SAMLS inclusion criteria are in Table 3. SAMLS stratifies AML as low-risk (LR-AML) (SAMS &lt;=1.5) or high-risk (HR AML) (SAMLS &gt;=2) (Table 3 and Figure 4 C-D), where there is a significant difference in survival in the FMUSP training cohort (p &lt;0.001). The 5-year OS was 55% and 9% for LR and HR AML respectively. The ROC c-statistic for OS in FMUSP using SAMLS was 0.80 (21% bigger than using AGR only). SAMLS risk stratification was validated in both FMRP and OUH cohorts for OS (FMRP p-value 0.003, OUH p-value &lt; 0.001) and DFS (p-value 0.04 OUH only). Prediction accuracy was also increased since ROC c-statistic was 0.65 for OS in FMRP (a 10% increase), and 0.77 in OUH (a 22% increase) (Figure 4). Conclusion: ELN2017 genetic risk score for AML was validated in a cohort from a low-medium income country (LMIC). AGR is a proposed risk evaluation that can be used for AML patients for whom fully genetic testing is not feasible (required for ELN2017) and was as accurate as ELN2017. AGR predicts survival in 3 independent cohorts, from LMIC and developed nation settings. When other biological and clinical factors are added to AGR, we were able to show SAMLS is better to predict overall survival than AGR. Importantly, serum Albumin, a simple and routine test parameter, was an independent predictor of OS for AML in all 3 cohorts. Therefore the use of SAMLS can predict early and late mortality and can impact on treatment decisions. Disclosures Quek: Celgene: Research Funding, Speakers Bureau; Agios: Research Funding. Vyas:Astellas: Speakers Bureau; Pfizer: Speakers Bureau; Abbvie: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding; Celgene: Research Funding, Speakers Bureau.

IPO11 Is Upregulated in Relapsed AML and Supports Survival of Leukemic Stem Cells

Patients with relapsed AML (Acute Myeloid Leukemia) have a poor prognosis and few therapeutic options. Therefore, it remains critical to identify biological vulnerabilities in relapsed AML cells. To identify such vulnerabilities, we analyzed mRNA expression by RNA sequencing of 11 paired diagnosis and relapsed AML samples and identified 34 genes that were upregulated at FDR &lt;= 0.01 at relapse. In parallel, we conducted a genome-wide CRISPR screen of 91,320 barcoded gRNA in human OCI-AML2 AML cells and identified 570 genes that reduced growth and viability with a negative FDR (false discovery rate) of 0.01 or less. We then compared the upregulated genes at relapse with the genes necessary for AML growth and viability through the CRISPR screen and identified only one hit, IPO11. IPO11 is a member of the importin-β family of proteins functions that facilitate the import of protein cargo into the nucleus. To date, 10 importin-β family members have been identified, but only IPO11 was upregulated at AML relapse, although, importin β1, TNPO3 AND IPO13 were also hits in the CRISPR screen and necessary for AML growth and viability. Therefore, we focused our study on IPO11. Using independent databases of gene expression, we confirmed that IPO11 mRNA was upregulated in LSC+ (engrafting) vs. LSC- (non-engrafting) samples (rank 125, FDR &lt;=0.05, GSE76008), CD34+ vs CD34- LSC (rank 42, FDR &lt;=0.05, GSE76008) and undifferentiated cluster (M0) vs. myeloid cluster M4/M5 (rank 648, FDR &lt;=0.05, TCGA-LAML). To verify that IPO11 protein was also increased in AML stem cells, we separated 8227 low passage primary AML cells into functionally defined stem (CD34+CD38-) and bulk (CD34-CD38+) fractions. IPO11 protein was only detected in AML stem cells compared to bulk cells as measured by immunoblotting. To determine whether IPO11 is necessary for AML growth and viability, we knocked down IPO11 in OCI-AML2, TEX and NB4 leukemia cells with shRNA in lentiviral vectors. Knockdown of IPO11 induced cell cycle arrest with reduced AML growth and viability by 80-90%. In contrast, knockdown of another importin-β family member, IPO5, that was not a hit in our CRIPSR screen did not reduce AML growth and viability. In addition, we demonstrated that knockdown of IPO11 increased differentiation of AML cells as evidenced by increased CD11b expression and staining with non-specific esterase. Finally, knockdown of IPO11 reduced the clonogeneic growth of the above-mentioned AML cell lines and engraftment of TEX cells and the low passage primary AML sample 8227 cells into immune deficient mice by over 90%. To identify cargos of IPO11 whose nuclear import is necessary for AML survival, we compared IPO11 cargo identified through a Bio-ID mass spectrometry protein-protein interaction screen with hits identified from our CRISPR screen. Through this analysis, we identified 7 hits in common to both screens, including RFC5 (replication fork complex unit 5). RFC5 is an essential DNA polymerase accessory protein, involved in telomere maintenance, DNA replication and mismatch repair. Knockdown of IPO11 decreased the nuclear localization of RFC5 in AML cells. In addition, knockdown of RFC5 in AML cells decreased AML growth and decreased clonogeneic growth. Thus, in summary, comparing genes that are upregulated in paired diagnosis and relapse AML samples to our CRISPR screen we identified IPO11 as a single hit. IPO11 knockdown reduced growth and viability, promoted differentiation, reduced CFU generation of several AML cells lines. In a mouse model we show that IPO11 knockdown dramatically reduced engraftment of leukemic cells. We further identify RFC5, a protein involved in replication and DNA damage response as one of the cargos of IPO11. Our study suggests IPO11 supports LSC survival and relapse and provides a new target for novel therapeutic intervention. Disclosures Schimmer: Otsuka Pharmaceuticals: Consultancy; Medivir Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Consultancy; Novartis Pharmaceuticals: Consultancy.

Identification of Novel Synthetic Lethal Partners of NAMPT Inhibitor By CRISPR-Cas9 Screens in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a hematopoietic neoplasm arising from the clonal expansion of myeloid progenitors resulting in bone marrow failure. Nicotinamide phosphoribosyltransferase (NAMPT) is a rate‐limiting enzyme which regulates the generation of the metabolic enzyme substrate NAD+. NAD+ is key in several survival pathways in AML such as DNA repair and metabolic pathways. AML subtypes differ vastly in their inherent DNA repair and metabolic capacities. Since NAMPT is essential in single and double strand DNA repair, NAMPT inhibitors (NAMPTis) were used to block DNA repair as single agents or in combinational therapies in solid tumors. However the clinical experience with the first generation of NAMPTis suggests that higher doses will be needed in order to achieve efficacy, which are associated with high toxicity. We hypothesize that identification of critical synthetic lethal partners for NAMPTi will increase efficacy with lower doses. To this end, we conducted genome-wide CRISPR KO (loss-of-function) screens by using two libraries, GeCKO and Brunello, and identified, along with genes reported essential for AML survival, 16 genes whose knockout displays synergism or resistance, after a NAMPTi (KPT-9274) treatment in AML cells. According to their gene ontology (GO) functions, these co-essential candidates are involved in DNA damage repair and metabolism. Genetic depletion of co-essential genes, SIRT6, HDAC8 and DCPS, sensitizes AML cell lines, MOLM13, Kasumi-1 and MV4-11, to KPT-9274 treatment. In addition, preclinical inhibitor for HDAC8 (PCI-34051), in combination with KPT-9274, synergistically decreased AML primary cell survival in a dose-dependent manner and decreased colony formation in AML patient samples across multiple genotypes with a minimal decrease in colony formation of normal CD34+ hematopoietic cells. In addition, drug combo treatment of AML cells decreased re-plating capacity, implying attenuated self-renewal capacity. Drug combo also increased apoptotic cells as measured by Annexin/PI staining and induced myeloid differentiation as detected by CD45+CD34- staining. In DNA homologous recombination (HR) pathway, SIRT6 and HDAC8 deacetylate CtIP and Rad51 to promote DNA end resection at double strand breaks. Since NAMPT inhibition blocks the generation of NAD+ thus compromising downstream PARP1 mediated base excision repair (BER) as well as the Sirtuin1 (Sirt1) mediated, KU- dependent NHEJ and atypical Rad51-independent HR repair, we expect that inhibiting these HR pathway components (like SIRT6 and HDAC8) may shut down these compensatory repair pathways and sensitize AML subtypes to NAMPT inhibition. Indeed, Western blotting shows drug combo increased acetyl-P53, acetyl-Rad51, phosphor-γH2Ax and Chk1 compared NAMPT inhibition alone. The in vivo efficacy of these combinatorial therapies has been investigated on PDX mouse models established in our laboratory. We demonstrated that the dug combo successfully eliminated engrafted AML cells and conferred survival advantages to mice. Our study provides evidence that co-essential genes, SIRT6, HDAC8 and DCPS, are synthetic lethal partners for NAMPT inhibition to target drug-resistant AML cells, with minimal toxicity towards normal cells. Disclosures Byrd: BeiGene: Research Funding; Ohio State University: Patents &amp; Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Ohio State University: Patents &amp; Royalties: OSU-2S; BeiGene: Research Funding; Ohio State University: Patents &amp; Royalties: OSU-2S; Novartis: Other: Travel Expenses, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau.

Effects of Obesity on Overall Survival of Adults With Acute Myeloid Leukemia

BackgroundThe role of obesity in prognosis of acute myeloid leukemia (AML) is debatable. Our retrospective study aimed to determine the effect of obesity on overall survival (OS) in AML. Patients and MethodsAML patients diagnosed at University of Nebraska Medical Center were divided into 3 groups according to body mass index (BMI): normal (18.5-25 kg/m2) or underweight (< 18.5 kg/m2); overweight (25-30 kg/m2); and obese (≥ 30 kg/m2). Chi-square test, Kruskal-Wallis test, and ANOVA were used to examine the association of BMI with baseline characteristics. Mann-Whitney test was used for pairwise comparisons of hematopoietic cell transplantation (HCT) comorbidity index. Bonferroni correction was used to adjust P values. OS, defined as time from diagnosis to death from any cause, was determined by the Kaplan-Meier method; comparisons of survival curves were done using log-rank test. Cox regression analysis was performed to detect the effect of BMI on OS. ResultsOf 314 patients, 38% were obese, 68% received intensive chemotherapy, and 30% underwent HCT. Patient characteristics for all BMI groups were similar except greater HCT comorbidity index in obese patients. Actual body weight was used to calculate the chemotherapy dose in 92% of obese patients. The rates of receipt of HCT in normal, overweight, and obese groups were 33%, 32%, and 25%, respectively (P = .6). One-year OS values for normal/underweight, overweight, and obese groups was 42%, 45%, and 39%, respectively (P = .31). On multivariate analysis, obesity was associated with worse OS compared to normal-weight (hazard ratio = 0.6; 95% confidence interval, 0.4-0.9; P = .03) but not overweight patients. ConclusionObesity confers worse prognosis in AML. Differences in OS were not the result of differences in chemotherapy dose or receipt of HCT.

Integrated nuclear proteomics and transcriptomics identifies S100A4 as a therapeutic target in acute myeloid leukemia

Inappropriate localization of proteins can interfere with normal cellular function and drive tumor development. To understand how this contributes to the development of acute myeloid leukemia (AML), we compared the nuclear proteome and transcriptome of AML blasts with normal human CD34+ cells. Analysis of the proteome identified networks and processes that significantly affected transcription regulation including misexpression of 11 transcription factors with seven proteins not previously implicated in AML. Transcriptome analysis identified changes in 40 transcription factors but none of these were predictive of changes at the protein level. The highest differentially expressed protein in AML nuclei compared with normal CD34+ nuclei (not previously implicated in AML) was S100A4. In an extended cohort, we found that over-expression of nuclear S100A4 was highly prevalent in AML (83%; 20/24 AML patients). Knock down of S100A4 in AML cell lines strongly impacted their survival whilst normal hemopoietic stem progenitor cells were unaffected. These data are the first analysis of the nuclear proteome in AML and have identified changes in transcription factor expression or regulation of transcription that would not have been seen at the mRNA level. These data also suggest that S100A4 is essential for AML survival and could be a therapeutic target in AML.