Event In Acute Myeloid Leukemia Research Articles

Influence of Bone Marrow Blast Enumeration and Co-Occurring Myelodysplasia Related Gene Mutations in NPM1-Mutated Myeloid Malignancies

Introduction Both World Health Organization (WHO 5th) and International Consensus Classification (ICC) guidelines recognize mutations in nucleophosmin 1 ( NPM1mut) as a class defining molecular event in acute myeloid leukemia (AML). WHO 5th omitted the blast percentage requirement and ICC lowered the blast threshold to 10%. NPM1mut AML without FLT3 ITD or adverse risk cytogenetics is considered favorable risk in European LeukemiaNet (ELN) 2022, however co-occurring adverse-risk myelodysplasia related mutations (MR mut) do not influence ELN 2022 risk stratification. This analysis addresses the prognostic impact of blast enumeration in NPM1mut myeloid neoplasms (MN) with <10% blasts and influence of co-occurring mutations in NPM1mut AML. Methods We interrogated publicly available data from adult (≥18 years) patients (pts) with chronic myeloid neoplasms included in the International Working Group for Prognosis in MDS (N= 3,323; Bernard et al. NEJMEvid 2022) and two independent AML cohorts (AMLSG [N=1540] Papaemmanuil et al. NEJM 2016; UKNCRI [N=2,113] Tazi et al. Nat. Commun. 2022). Between group differences were assessed using the Wilcoxon rank-sum test or Fisher's exact test as appropriate. Time to event endpoints were analyzed using the log-rank method. Multivariate analysis (MVA) used cox proportional hazards regression. Results NPM1 mut was identified in 17% (N=1148) of pts (AML: 30% [1109/3653], MN with <20% blasts: 1.2% [39/3,323]). Compared to pts with NPM1mut AML, pts with NPM1mut MN (MDS, CMML, aCML) with blast < 20% were older (median age 65 vs. 55 years, p < 0.001), had a lower median total WBC count (5 vs. 32 x10 9 /L, p < 0.001) and bone marrow (BM) blast percentage (8% vs. 76%, p < 0.001). No significant difference was observed between NPM1mut AML and MN with <20% blasts in median hemoglobin (9.1 vs. 9 x10 9, p: 0.44), platelets (62 vs. 66 x10 9 /L, p: 0.55), or NPM1mut variant allele frequency (VAF; 35% vs. 37%, p: 0.64). After a median follow up of 4.7 years (range: 0-12), no difference in overall survival (OS) was observed between pts with low blast (LB; <10%) NPM1mut MN (N=19) vs. ≥10% blasts (N=18) (median OS: 0.64 years [95% CI: 0.31-3.0] vs. 1.1 years [95% CI: 0.95-NR], p=0.32). Median leukemia free survival (LFS) was 0.81 years (95% CI: 0.67-1.5) and not significantly different between NPM1mut MN pts with <10 vs. ≥10% blasts (median LFS: 0.55 vs. 0.91 years, p=0.27). In pts with NPM1mut AML (N=1109), 52% (N=581) were ELN 2022 favorable-risk; 15% (N=92) had MR mut including SRSF2 (51%, N=47), STAG2 (21%, N=19), ASXL1 (9%, N=8), BCOR (9%, N=8), RUNX1 ( 8%, N=7), EZH2 (5%, N=5), SF3B1 (5%, N=5), U2AF1 (1%, N=1), and ZRSR2 (1%, N=1). Pts with MR mut were older (median age 62 vs. 53 years, p < 0.001), had higher BM blasts (80% vs. 72% p=0.019), and lower platelets (45 vs. 77 x10 9/L p < 0.001) compared to pts with NPM1mut AML without MR mut. No significant difference in NPM1mut VAF was observed (34% in both groups). After a median follow up of 3.5 years (range: 0-15), median OS was 6.2 years in pts with NPM1mut AML (95% CI: 4.2-9.7). Inferior OS was observed in ELN favorable risk NPM1mut pts with vs. without MR mut, respectively (2 vs. 8.4 years, p=0.0001). When stratified by age < 60 vs. ≥ 60 years, younger pts with MR mut had an increased risk of death (HR 1.7 95% CI: 1.1-2.8, p: 0.02). No OS difference was observed in older pts with or without MR mut (HR: 1.2, 95% CI: 0.80-1.68, p=0.44), however OS was significantly shorter in older pts with NPM1mut compared to younger pts (median 1.7 years vs. NR, p < 0.001). Patients with NPM1mut /MR mut AML had survival comparable to patients with ELN 2022 intermediate risk AML (2.0 vs. 2.1 years, p=0.35). In MVA of NPM1mutAML adjusted for MR mut, BM blast percentage, age, performance status, baseline WBC, hemoglobin, platelet count, NPM1mut VAF, and treatment setting (AMLSG or UKNCRI), the presence of an MR mut (HR 1.39 [95% CI: 1.0-1.9], p=0.04), age < 60 (HR 0.43 [95% CI: 0.32-0.56], p < 0.001), and performance status (HR 1.35 [95% CI: 1.14-1.60], p=0.0004) retained statistical significance. Conclusion Pts with low blast NPM1mut MN have outcomes similar to pts with NPM1mut AML, supporting NPM1mut as an AML defining event irrespective of BM blast enumeration. In patients with ELN favorable risk NPM1mut AML, co-occurrence of MR mut is independently associated with inferior survival, comparable to ELN intermediate risk AML. These results may inform future refinements of current consensus guidelines.

NPM1 mutation reprograms leukemic transcription network via reshaping TAD topology

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, molecular mechanisms underlying NPM1C+-driven leukemogenesis remain elusive. Here, we report that NPM1C+ activates signature HOX genes and reprograms cell cycle regulators by altering CTCF-driven topologically associated domains (TADs). Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to disrupted regulation of the cell cycle as well as aberrant chromatin accessibility and homeotic gene expression, which results in myeloid differentiation block. Restoration of NPM1 within the nucleus re-establishes differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators that switch the oncogenic MIZ1/MYC regulatory axis in favor of interacting with coactivator NPM1/p300, and prevents NPM1C+-driven leukemogenesis. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation.

A fetal tumor suppressor axis abrogates MLL-fusion-driven acute myeloid leukemia.

MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r can be sufficient to drive transformation. However, despite the prenatal origin of MLL-r in these patients, congenital leukemia is very rare with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-expresses MLL-ENL and LIN28B, we investigate the impact of LIN28B on AML. LIN28B impedes the progression of MLL-r AML through compromised leukemia-initiating cell activity and suppression of MYB signaling. Mechanistically, LIN28B directly binds to MYBBP1A mRNA, resulting in elevated protein levels of this MYB co-repressor. Functionally, overexpression of MYBBP1A phenocopies the tumor-suppressor effects of LIN28B, while its perturbation omits it. Thereby, we propose that developmentally restricted expression of LIN28B provides a layer of protection against MYB-dependent AML.

Analysis of Patient-Level Data from 3 Cooperative Group Trials Confirms a Survival Advantage for NPM1m Patients Achieving MRD-Negative CR after Intensive Induction

Background:NPM1 mutations (NPM1m), predominantly tetra-nucleotide insertions in exon 12, are clonal driver events in acute myeloid leukemia (AML) and are found exclusively in leukemic cells, making them ideal for sensitive detection of measurable residual disease (MRD) using quantitative polymerase chain reaction (qPCR)- or next generation sequencing (NGS)-based methods. Over 50 peer-reviewed publications across multiple cooperative group trials have shown that NPM1m AML patients in complete remission (CR), CR with incomplete count recovery (CRi), or CR with partial hematologic recovery (CRh) have significantly better survival if they are MRD negative compared to MRD positive. Based on this body of evidence, the European LeukemiaNet recommends monitoring molecular MRD in NPM1m AML patients during treatment to help guide treatment decisions. Purpose: To provide further evidence for the value of NPM1 MRD as a surrogate endpoint for prospective, randomized trials in patients receiving novel agents in combination with intensive chemotherapy by evaluating the relationship between MRD negativity in patients with CR after 2 cycles of chemotherapy and event-free survival (EFS) and overall survival (OS) in a pooled analysis of patient-level data from 3 large cooperative group trials. MRD in all 3 studies was assessed using reverse transcriptase-mediated qPCR (RT-qPCR) of NPM1m transcripts normalized to ABL transcripts. Methods: Deidentified patient data for a total of 1,128 patients who achieved CR, CRi, or CRh, and who had MRD data after 2 cycles of chemotherapy, were provided by the UK National Cancer Research Institute AML Study Group (SG) for the AML17 (N = 539), the AMLSG for the AMLSG 09-09 (N = 497), and the Study Alliance Leukemia (SAL) for the AML2003 (N = 105) trials and standardized using Standard Data Tabulation Models. Data included demographic information, disease history, induction and consolidation treatment received, morphologic response, MRD data after 2 cycles of treatment, relapse, and survival. Cutoffs for MRD negativity were defined according to the definitions used in the individual studies (Ivey A, et al. N Engl J Med. 2016; Kapp-Schwoerer S, et al. Blood. 2020; Shayegi N, et al. Blood. 2013). In addition, sensitivity analyses were conducted using a range of cutoff values. CR was defined as <5% blasts in the bone marrow (BM) with count recovery, occurring within 42 days of the start of the last induction cycle; only these patients were included in the MRD analysis. EFS and OS were estimated using the Kaplan-Meier method and defined according to FDA criteria. Briefly, EFS was measured from day 1 of randomization to the date of treatment failure, hematologic relapse from CR or death from any cause; treatment failure was defined as not achieving CR after two cycles of chemotherapy. OS was measured from day 1 to the date of death from any cause. The absolute risk difference among MRD response status groups in EFS/OS at the 2-month, 1-year, 2-year, 3-year, and 5-year landmarks and their corresponding 95% confidence intervals (CI) were calculated using complementary log-log transformation. Hazard ratios (HR) were fitted using a Cox regression model with EFS/OS as the dependent variable and with MRD-response status and study as independent variables. Results: Among the 515 patients who achieved a CR within 42 days at the start of chemotherapy Cycle 2, MRD negativity confers a substantial survival advantage with EFS HR (95% CI) = 1.6 (1.1-2.3) when measured in BM and 1.8 (1.4-2.4) in peripheral blood (PB) using the study-defined cutoffs for positive and negative. This advantage is also observed using cutoff definitions of MRD negativity ranging from <10 to <1,000 NPM1m/104ABL transcripts. A survival advantage for OS was also observed with HR (95% CI) = 1.5 (1.0-2.4) and 1.7 (1.2-2.3) in BM and PB, respectively. Analysis of the predictive value of MRD negativity in the 613 patients who achieved CRi/CRh within 42 days of the start of Cycle 2 is underway. Conclusions: Analysis of pooled patient-level data confirms the survival advantage of achieving MRD-negative CR as previously reported in 3 independent, cooperative group trials using different induction regimens. The prognostic value of MRD negativity in NPM1m AML is consistent across a broad range of cutoff definitions in both BM and PB providing further evidence for the value of NPM1 MRD as a surrogate endpoint for clinical trials.

NPM1 Mutation Reprograms Leukemic Transcription Network Via Reshaping CTCF-Defined TAD Topology

C-terminal mutation of Nucleophosmin 1 (NPM1C+) was thought to be a primary driving event in acute myeloid leukemia (AML) that reprograms leukemic-associated transcription programs to transform hematopoietic stem and progenitor cells (HSPCs). However, it remains poorly understood how NPM1C+ mutation results in altered myeloid master transcription program and HOX expression profile leading to AML disease and whether NPM1 mutant associated transcription signature can be targeted to prevent pathogenesis of acute myeloid leukemia. Here, we report that NPM1C+ reprograms MIZ-1/MYC regulatory axis by altering NPM1-associated CTCF-driven topologically associated domains (TADs) that switches the balance of MIZ1 interaction with coactivator NPM1/p300 and corepressors MYC/G9A complexes to control cell cycle progression and myeloid lineage-specific PU.1/CEBPa transcription networks leading to impairment of myeloid differentiation and leukemic transformation. To test the hypothesis that mislocalization of the NPM1C+ mutant to cytoplasm may alter CTCF mediated three-dimensional (3D) TADs, we carried out Hi-C to examine whether NPM1C+ mutation altered hematopoietic TAD integrity by comparing mouse bone marrow (BM) Lin-cKit+ (LK) stem- and progenitor-enriched cells purified from WT and NPM1C+ knock-in animals, which develop a myeloproliferative neoplasm/myelodsplasia-like condition with a long latency characterized by enlarged spleens, leukocytosis, and thrombocytopenia. Hematopoietic-specific NPM1C+ knock-in alters TAD topology leading to altered chromatin accessibility and NPM1 mutant associated gene expression signature, which results in disrupted regulation of the cell cycle and myeloid master transcription factors, and eventually myeloid differentiation block. In contract, Retention of NPM1 in OCI-AML3 nucleus, a NPM1C+ mutant driven AML cells, reverses NPM1C+-driven signature TAD formation and gene transcription leading to proper cell cycle control and myeloid differentiation. Mechanistically, we demonstrated that cytoplasmic mislocalization of NPM1C+ mutant exerts two coordinated effects: (a) alteration of CTCF dependent TAD structure, and (b) MYC-MIZ-1 transcription axis. Both of these regulate target genes critical for cell cycle control and myeloid master transcription factors, Pu.1 and CEBPA, mediated myeloid differentiation. Re-activation of Pu.1 or CEBPa within the nucleus by CRISPR-dCas9 mediated endogenous promoter activation restores myeloid differentiation programs by reorganizing TADs critical for myeloid TFs and cell cycle regulators, and prevents NPM1C+-driven gene expression. FinalIy, when we transplanted NPM1OE, activated Pu.1, or activated CEBPA OCI-AML3 cell line or primary NPM1C+ AML patient cells into NSG mice along with control cells, NPM1 overexpression or re-activation of PU.1 or CEBPA in OCI-AML3 cells or NPM1C+ primary AML patient cells prolonged transplanted or PDX mice survival and mitigate AML leukemic blast in transplant or PDX mouse models. In sum, our data reveal that NPM1C+ reshapes CTCF-defined TAD topology to reprogram signature leukemic transcription programs required for cell cycle progression and leukemic transformation. Restoration of myeloid transcription program in nucleus reversed NPM1C+-driven transcription signature and promotes myeloid differentiation leading to mitigation of AML.

AML-226 Impairment of Primary Hemostasis as an Additional Risk Factor of Hemorrhage in Patients With Newly Diagnosed Acute Myeloid Leukemia: Ukrainian Prospective Study

Bleeding events in acute myeloid leukemia (AML) are frequent and life threatening, whereas widely used treatment approaches are not always able to satisfy expected outcomes. The primary endpoints were the qualitative and quantitative functions of plasma von Willebrand factor (vWF), which include measurements of vWF activity (vWF:ristocetin cofactor [vWF:RCo]), vWF level (vWF:antigen [vWF:Ag]), factor VIII coagulation activity regarding vWF (vWF:FVIII), and FVIII activity (FVIII) in AML patients. Forty-two patients with newly diagnosed AML (excluding acute promyelocytic leukemia) were included in the study group. The control group included 20 age-matched and sex-matched healthy individuals. The grade (G) of bleeding events was assessed according to the Modified WHO Bleeding Scale (WHO BS). Hemorrhage (G1-4) occurred in 53.3% of patients in the study group during induction therapy. vWF:Ag below normal ranges had been observed in 19% of patients with AML and was associated with the occurrence of severe hemorrhage compared with the patients who had normal vWF:Ag levels and no bleeding episodes (P=0.012). It is important to mention that vWF:Ag levels (median 134, mean 163±18.25) were elevated in most cases (56%) in the main group and are probably associated with a compensatory mechanism for stopping bleeding compared with the control group. In 23.8% of patients with AML, we observed levels of vWF:RCo below normal ranges. Among them, the most frequent bleeding events were G2 (5/10, 50%) and G4 (3/10, 30%); three of them died during the first course of chemotherapy due to gastrointestinal and CNS bleeding. Therefore, we found a statistically significant association between decreased levels of vWF:RCo below normal ranges and the occurrence of severe bleeding episodes (G2-G4) in patients with AML compared with the group that had normal ranges of vWF:RCo and no bleeding events (G0) (P=0.0001). Furthermore, low vWF:FVIII (P=0.033) and FVIII (P=0.037) levels in patients with AML and bleeding episodes G2-G4 had statistical differences compared to non-hemorrhage patients (G0). According to our study, vWF:RCo, vWF:Ag, and vWF:FVIII were associated with hemorrhage in patients with newly diagnosed AML and could be considered predictive factors of bleeding.

Clinical implications and genetical insights of SOX6 expression in acute myeloid leukemia.

Transcription factor SOX6 belongs to Sry-related high-mobility-group box (SOX) family, has been reported to be downregulated and acts as a tumor-suppressor gene in various solid tumors, but in acute myeloid leukemia (AML) is incompletely understood. The SOX6 expression was analyzed between AML patients and normal controls from public data and our research cohort. Correlations between SOX6 expression and clinical, genetic features together with survival were further analyzed. In both public and our present datasets, we demonstrated that SOX6 expression is notably downregulated in AML patients compared with normal controls. Moreover, the expression level of SOX6 was dynamic, along withthediseasestatus. SOX6 was significantly decreased in relapsed/refractory AML compared with complete remission AML. Clinically, SOX6 underexpression was significantly correlated with bone marrow blasts, and WBC counts. Furthermore, decreased expression of SOX6 was more common in core binding factor AML (CBF-AML), rarely found in complex karyotype AML (CK-AML), and correlated with FLT3 mutations. By survival analyses, low-expression of SOX6 was associated with shorter overall survival (OS) and event-free survival (EFS) among cytogenetic normal AML (CN-AML) patients. Moreover, both univariate and multivariate analyses showed that low SOX6 expression was an independent unfavorable prognostic biomarker for CN-AML. Our findings indicated that SOX6 underexpression, as a frequent event in AML, was associated with genetic abnormalities and prognosis in AML. SOX6 might be a valuable biomarker for risk stratification, predictingprognosisand relapse of AML.

Clinical and molecular profiling of AML patients with chromosome 7 or 7q deletions in the context of TP53 alterations and venetoclax treatment

Deletions in chromosome 7 (del(7)) or its long arm (del(7q)) constitute the most common adverse cytogenetic events in acute myeloid leukemia (AML). We retrospectively analyzed 243 treatment-naive patients with AML and del(7) (168/243; 69%) or del(7q) (75/243; 31%) who did not receive any myeloid-directed therapy prior to AML diagnosis. This is the largest comprehensive clinical and molecular analysis of AML patients with del(7) and del(7q). Our results show that relapse-free survival was significantly longer for AML patients with del(7q) compared to del(7), but the overall survival and remission duration were similar. TP53 mutations and del5/5q were the most frequent co-occurring mutations and cytogenetic abnormalities, and conferred worse outcomes in del(7) and del(7q) patients. Venetoclax-based treatments were associated with worse outcomes in TP53 mutated AML patients with del(7) or del(7q), as well as del(7) with TP53 wildtype status, requiring further investigation.

Abstract 5788: Genomic and transcriptomic profiles of DNA damage response genes in acute myeloid leukemia

Abstract The DNA damage response (DDR) pathway is frequently deregulated in cancer and it represent an attractive therapeutic opportunity. In acute myeloid leukemia (AML), different mechanisms of DDR deregulation have been identified, but a systematic investigation on DDR alterations is missing. To understand how the DDR pathways contribute to leukemogenesis, we studied the gene expression and mutational profiles of 274 DDR genes by analysing 539 AML cases profiled by whole genome (WGS) and RNA sequencing. WGS data were used to identify mutations in genes of the DDR and in a panel of genes known to be mutated in AML (n=73). Transcriptomic data were analysed through unsupervised clustering, differential expression and enrichment analysis. We detected 150 single nucleotide variants (SNVs) in 130 patients (24%, average 0.3 SNVs/case). Genes mutated in more than 1% of cases were ATM, BLM, BRCA2, POLG and POLQ. The most frequently altered pathway was the homologous recombination/Fanconi Anemia (HR) pathway (29%), followed by the genes that coordinates the DDR pathway (20%). We detected a trend toward mutual exclusivity between mutations in TP53 and mutations in genes of HR pathway or the genes that coordinates the DDR pathway (adj-p &amp;lt;0.02). To further investigate the interplay between TP53 mutations and the HR pathway, we analysed the expression profiles of HR genes in 539 patients. We identified two groups of patients having higher (HR-high) or lower (HR-low) expression levels of HR genes. A panel of 5 genes was able to discriminate patients between the two groups (BRCA1, RAD54B, RMI2, UBE2T and XRCC2; AUC=0.9). Enrichment analysis on differentially expressed genes and gene set enrichment analysis showed that the cell cycle pathway, together with the G2/M transition/mitotic phase, E2F targets and the fatty acid metabolism pathways were upregulated in HR-high patients, while the pRB, EZH2, RPS14 and HOXA9 pathways were downregulated. Moreover, we observed that AML expressing CBFB-MYH1, RUNX1-RUNXT1 or carrying RAD21 mutations had higher chances to express lower levels of HR genes (HR-low), while patients with STAG2, SRSF2, U2AF1, FLT3-ITD alterations had higher chances of having higher expression of HR genes (p&amp;lt;0.05). NPM1-mutated cases without FLT3-ITD clustered within the HR-low profile (adj-p&amp;lt;0.05), while TP53 mutated cases tended to cluster in the HR-high group, although statistical significance was not reached. In conclusion, our data showed the presence of alterations in the DDR pathway that might be the reflection of driver events in AML. Functional studies will elucidate the functional impact of these alterations. The results suggested the presence of a therapeutic window that might be exploited with DDR inhibitors in molecularly-defined subgroups of patients. Supported by the Torsten Haferlach-Leukämiediagnostik-Stiftung and AIRC IG 2019 (project 23810). Citation Format: Antonella Padella, Stephan Hutter, Wencke Walter, Constance Baer, Irene Azzali, Andrea Ghelli Luserna Di Rorà, Martina Ghetti, Lorenzo Ledda, Matteo Paganelli, Claudia Haferlach, Wolfgang Kern, Giorgia Simonetti, Giovanni Martinelli, Torsten Haferlach. Genomic and transcriptomic profiles of DNA damage response genes in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5788.

Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

BackgroundIsocitrate dehydrogenase-2 (IDH2) is a mitochondrial enzyme that catalyzes the metabolic conversion between isocitrate and alpha-ketoglutarate (α-KG) in the TCA cycle. IDH2 mutation is an oncogenic event in acute myeloid leukemia (AML) due to the generation of 2-hydroxyglutarate. However, the role of wild-type IDH2 in AML remains unknown, despite patients with it suffer worse clinical outcome than those harboring mutant type.MethodsIDH2 expression in AML cell lines and patient samples was evaluated by RT-qPCR, western blotting and database analyses. The role of wild-type IDH2 in AML cell survival and proliferation was tested using genetic knockdown and pharmacological inhibition in AML cells and animal models. LC–MS, GC–MS, isotope metabolic tracing, and molecular analyses were performed to reveal the underlying mechanisms.ResultsWe found that wild-type IDH2 was overexpressed in AML and played a major role in promoting leukemia cell survival and proliferation in vitro and in vivo. Metabolomic analyses revealed an active IDH2-mediated reductive TCA cycle that promoted the conversion of α-KG to isocitrate/citrate to facilitate glutamine utilization for lipid synthesis in AML cells. Suppression of wild-type IDH2 by shRNA resulted in elevated α-KG and decreased isocitrate/citrate, leading to reduced lipid synthesis, a significant decrease in c-Myc downregulated by α-KG, and an inhibition of AML viability and proliferation. Importantly, pharmacological inhibition of IDH2 showed significant therapeutic effect in mice inoculated with AML cells with wt-IDH2 and induced a downregulation of C-MYC in vivo.ConclusionsWt-IDH2 is an essential molecule for AML cell survival and proliferation by promoting conversion of α-KG to isocitrate for lipid synthesis and by upregulating c-Myc expression and could be a potential therapeutic target in AML.

ERG amplification is a secondary recurrent driver event in myeloid malignancy with complex karyotype and TP53 mutations.

ERG is a transcription factor encoded on chromosome 21q22.2 with important roles in hematopoiesis and oncogenesis of prostate cancer. ERG amplification has been identified as one of the most common recurrent events in acute myeloid leukemia with complex karyotype (AML-CK). In this study, we uncover three different modes of ERG amplification in AML-CK. Importantly, we present evidence to show that ERG amplification is distinct from intrachromosomal amplification of chromosome 21 (iAMP21), a hallmark segmental amplification frequently encompassing RUNX1 and ERG in a subset of high-risk B-lymphoblastic leukemia. We also characterize the association with TP53 aberrations and other chromosomal aberrations, including chromothripsis. Lastly, we show that ERG amplification can initially emerge as subclonal events in low-grade myeloid neoplasms. These findings demonstrate that ERG amplification is a recurrent secondary driver event in AML and raise the tantalizing possibility of ERG as a therapeutic target.

3077 – FUNCTIONAL AND MOLECULAR IDENTIFICATION OF A FETAL-ASSOCIATED TUMOR SUPPRESSOR AXIS: ABROGATION OF MLL-ENL DRIVEN ACUTE MYELOID LEUKEMIA

MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r have been suggested to be sufficient to drive transformation. However, despite the prenatal origin of MLL-r, the incidence of congenital leukemia is very low with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific RNA binding protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-express MLL-ENL and LIN28B, we investigated the impact of LIN28B on AML. We show that LIN28B impedes the progression of MLL-r AML through a compromised leukemia initiating cell activity and a suppression of MYB signaling. Mechanistically, we found that LIN28B directly bound to MYBBP1A mRNA, resulting in elevated protein levels of this co-repressor of the transcription factor MYB. Overexpression of MYBBP1A in MLL-ENL driven leukemogenesis largely phenocopied the tumor suppressor effects of LIN28B. Thereby, our work proposes that the developmentally-restricted expression of LIN28B during fetal development provides a layer of protection against MYB-dependent AML. MLL-rearrangements (MLL-r) are recurrent genetic events in acute myeloid leukemia (AML) and frequently associate with poor prognosis. In infants, MLL-r have been suggested to be sufficient to drive transformation. However, despite the prenatal origin of MLL-r, the incidence of congenital leukemia is very low with transformation usually occurring postnatally. The influence of prenatal signals on leukemogenesis, such as those mediated by the fetal-specific RNA binding protein LIN28B, remains controversial. Here, using a dual-transgenic mouse model that co-express MLL-ENL and LIN28B, we investigated the impact of LIN28B on AML. We show that LIN28B impedes the progression of MLL-r AML through a compromised leukemia initiating cell activity and a suppression of MYB signaling. Mechanistically, we found that LIN28B directly bound to MYBBP1A mRNA, resulting in elevated protein levels of this co-repressor of the transcription factor MYB. Overexpression of MYBBP1A in MLL-ENL driven leukemogenesis largely phenocopied the tumor suppressor effects of LIN28B. Thereby, our work proposes that the developmentally-restricted expression of LIN28B during fetal development provides a layer of protection against MYB-dependent AML.

Microsatellite Instability Assessment by Immunohistochemistry in Acute Myeloid Leukemia: A Reappraisal and Review of the Literature

Microsatellite instability (MSI) is caused by defects in DNA mismatch repair (MMR) components. Inactivation of any MMR gene(s), including hMLH1, hMSH2, hMSH6, and hPMS2, can result in MSI. Immunohistochemistry (IHC) is a sensitive and specific screening tool for MSI that can detect loss of expression of one or more MMR components. Of the four MMR markers, hMLH1 and hMSH2 are considered most informative of MSI status. There has been renewed interest in MSI status in view of its favorable association with response to immune checkpoint inhibitors in some cancers. MMR expression patterns in acute myeloid leukemia (AML) have not been evaluated systematically. We used clinically-validated IHC assays to assess the expression of hMLH1, hMSH2, hMSH6, and/or hPMS2 in formalin-fixed paraffin-embedded tissue sections of bone marrow core biopsies from patients diagnosed with AML. Mutation profiling was performed using next-generation sequencing to assess for mutations in MMR genes. The study group included 236 patients with AML, including a cohort treated on a clinical trial of azacitidine and nivolumab (NCT02397720). In addition, hMSH6, and/or hPMS2 expression was assessed in 99 AML patients with diploid karyotype. All patients, except two, had retained expression of all MMR markers assessed: One patient from the azacytidine+nivolumab group had zonal patchy loss of staining of hMLH1 and, to a lesser extent, a similar staining pattern of hMSH2; and one patient from the AML with diploid karyotype group had loss of hMSH2 but retained expression of hMLH1, hMSH6 and hPMS2. In addition, a retrospective analysis on a separate cohort of 139 patients with primary AML, on which next generation sequencing profiling was performed, identified 14 cases with alterations in MMR genes. MMR loss is a rare event in AML, thus does not appear to underlie response patterns to anti-PD1 therapy.

Distinct associations of NEDD4L expression with genetic abnormalities and prognosis in acute myeloid leukemia

BackgroundThere is mounting evidence that demonstrated the association of aberrant NEDD4L expression with diverse human cancers. However, the expression pattern and clinical implication of NEDD4L in acute myeloid leukemia (AML) remains poorly defined.MethodsWe systemically determined NEDD4L expression with its clinical significance in AML by both public data and our research cohort. Moreover, biological functions of NEDD4L in leukemogenesis were further tested by in vitro experiments.ResultsBy the public data, we identified that low NEDD4L expression was correlated with AML among diverse human cancers. Expression of NEDD4L was remarkably decreased in AML compared with controls, and was confirmed by our research cohort. Clinically, low expression of NEDD4L was correlated with greatly lower age, higher white blood cells, and higher bone marrow/peripheral blood blasts. Moreover, NEDD4L underexpression was positively correlated with normal karyotype, FLT3 and NPM1 mutations, but negatively associated with complex karyotype and TP53 mutations. Importantly, the association between NEDD4L expression and survival was also discovered in cytogenetically normal AML patients. Finally, a number of 1024 RNAs and 91 microRNAs were identified to be linked to NEDD4L expression in AML. Among the negatively correlated microRNAs, miR-10a was also discovered as a microRNA that may directly target NEDD4L. Further functional studies revealed that NEDD4L exhibited anti-proliferative and pro-apoptotic effects in leukemic cell line K562.ConclusionsOur findings indicated that NEDD4L underexpression, as a frequent event in AML, was associated with genetic abnormalities and prognosis in AML. Moreover, NEDD4L expression may be involved in leukemogenesis with potential therapeutic target value.

Clinical implication and prognostic significance of FLT3-ITD and ASXL1 mutations in Egyptian AML patients: A single-center study.

Both Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) and Additional Sex Comb-like 1 (ASXL1) mutations are frequent and early genetic alteration events in acute myeloid leukemia (AML) patients. These genetic alterations may be associated with an unfavorable prognosis. Up to our knowledge, this is the first study performed to evaluate the clinical implication and prognostic significance of FLT3-ITD and ASXL1 mutations and their coexistence on the outcome of Egyptian AML patients. Our study included 83 patients with AML who were subjected to immunophenotyping and detection of FLT3-ITD and ASXL1 gene mutation by polymerase chain reaction (PCR) and real-time PCR, respectively. FLT3-ITD and ASXL1 mutations were detected in 20.5% and 18.1% of AML patients respectively. Seven patients (8.4%) had co-expression of both genes' mutations. FLT3-ITD mutation was significantly higher in younger age, higher WBCs count and poor cytogenetic risk patients (P= 0.01, < 0.001 and 0.008 respectively). ASXL1 mutation was significantly higher in intermediate cytogenetic risk patients (P= 0.2). The mean period of survival and relapse-free survival (RFS) were significantly reduced in FLT3-ITD and ASXL1 mutations compared with their non-mutant types (P= 0.01 and 0.03 respectively). Both mutations were independent risk factors for overall survival (OS) and (RFS) in univariate and multivariate analysis in AML patients. FLT3-ITD and ASXL1 gene mutations or their coexistence can predict a poor prognosis in AML patients.

Iadademstat in Combination with Azacitidine Generates Robust and Long Lasting Responses in AML Patients (ALICE Trial)

Iadademstat in Combination with Azacitidine Generates Robust and Long Lasting Responses in AML Patients (ALICE Trial)

Identification of survival-related alternative splicing signatures in acute myeloid leukemia.

Aberrant RNA alternative splicing (AS) variants play critical roles in tumorigenesis and prognosis in human cancers. Here, we conducted a comprehensive profiling of aberrant AS events in acute myeloid leukemia (AML). RNA AS profile, including seven AS types, and the percent spliced in (PSI) value for each patient were generated by SpliceSeq using RNA-seq data from TCGA. Univariate followed by multivariate Cox regression analysis were used to identify survival-related AS events and develop the AS signatures. A nomogram was developed, and its predictive efficacy was assessed. About 27,892 AS events and 3,178 events were associated with overall survival (OS) after strict filtering. Parent genes of survival-associated AS events were mainly enriched in leukemia-associated processes including chromatin modification, autophagy, and T-cell receptor signaling pathway. The 10 AS signature based on seven types of AS events showed better efficacy in predicting OS of patients than those built on a single AS event type. The area under curve (AUC) value of the 10 AS signature for 3-year OS was 0.91. Gene set enrichment analysis (GSEA) confirmed that these survival-related AS events contribute to AML progression. Moreover, the nomogram showed good predictive performance for patient's prognosis. Finally, the correlation network of AS variants with splicing factor genes found potential important regulatory genes in AML. The present study presented a systematic analysis of survival-related AS events and developed AS signatures for predicting the patient’s survival. Further studies are needed to validate the signatures in independent AML cohorts and might provide a promising perspective for developing therapeutic targets.

Dhx15 regulates zebrafish definitive hematopoiesis through the unfolded protein response pathway

Gene alterations are recognized as important events in acute myeloid leukemia (AML) progression. Studies on hematopoiesis of altered genes contribute to a better understanding on their roles in AML progression. Our previous work reported a DEAH box helicase 15 (DHX15) R222G mutation in AML patients, and we showed DHX15 overexpression is associated with poor prognosis in AML patients. In this work, we further study the role of dhx15 in zebrafish developmental hematopoiesis by generating dhx15−/− zebrafish using transcription activator‐like effector nuclease technology. Whole‐mount in situ hybridization (WISH) analysis showed hematopoietic stem/progenitor cells were dramatically perturbed when dhx15 was deleted. Immunofluorescence staining indicated inhibited hematopoietic stem/progenitor cell (HSPC) proliferation instead of accelerated apoptosis were detected in dhx15−/− zebrafish. Furthermore, our data showed that HSPC defect is mediated through the unfolded protein response (UPR) pathway. DHX15 R222G mutation, a recurrent mutation identified in AML patients, displayed a compromised function in restoring HSPC failure in dhx15−/−; Tg (hsp: DHX15 R222G) zebrafish. Collectively, this work revealed a vital role of dhx15 in the maintenance of definitive hematopoiesis in zebrafish through the unfolded protein respone pathway. The study of DHX15 and DHX15 R222G mutation could hold clinical significance for evaluating prognosis of AML patients with aberrant DHX15 expression.

Incidence of Bleeding Events in Acute Myeloid Leukemia (AML) Patients Receiving Anticoagulation Therapy for Treatment of Deep Vein Thrombosis (DVT) or Pulmonary Embolism (PE) Who Are Also Undergoing Induction or Re-Induction Therapy

Background/Rationale:Venous thromboembolic events (VTE) remain a prevalent complication in cancer patients. Despite this increased risk, the decision on whether or not to treat and/or use prophylactic therapy remains highly debatable among clinicians. Induction and re-induction therapy, in combination with the pathophysiology of the disease can cause profound thrombocytopenia. Additionally, leukemic cells can cause an inappropriate release of procoagulants directly into the bloodstream, initiating the clotting cascade and causing simultaneous clot formation and hemorrhage leading to disseminated intravascular coagulation (DIC). Another major factor to consider in this patient population is central venous access. Although the National Comprehensive Cancer Network (NCCN) guidelines recommend ≥3 months of therapeutic anticoagulation for catheter-associated VTEs, clinicians use anticoagulation cautiously in the AML population due to severe thrombocytopenia and the increased risk of bleeding. Data that can be used to optimize treatment strategies for catheter-associated VTE in patients with acute leukemia are needed as the risk versus benefit for anticoagulation is currently unclear. A recent article by Rajasekhar and colleagues investigated this risk and noted that among patients with severe thrombocytopenia (platelets <50,000/μL) who received therapeutic anticoagulation, 27% experienced a significant bleeding event compared to only 2% of patients who did not receive therapeutic anticoagulation. In order to establish the bleeding complications associated with patients receiving induction or re-induction therapy for AML, we conducted a retrospective chart review evaluating the incidence of bleeding in patients receiving therapeutic anticoagulation for treatment of VTE.Objective:The primary outcome of this study is incidence of bleeding events in AML patients receiving anticoagulation therapy for treatment of DVT or PE who are also undergoing induction or re-induction therapy. Secondary outcomes include incidence of chemical and non-chemical DVT prophylaxis, as well as the difference in incidence of bleeding events among agents.Methods:This is a retrospective chart review evaluating the incidence of bleeding in AML patients receiving anticoagulation for treatment of VTE during induction or re-induction therapy at WFBH from October 2012 to October 2016. AML patients who were >18 years of age who had undergone induction or re-induction chemotherapy and developed a VTE were included in the study. Clinical diagnosis of VTE was confirmed by objective imaging procedures. The following induction or re-induction regimens were excluded: azacitidine or decitabine. Descriptive statistics were used for data analysis.Results: Out of 523 patients screened, 24 met eligibility criteria for a total VTE incidence of 4.6%. The age range for the patients included was 22 to 77, with 14 patients (58.3%) being female and 10 patients (41.7%) being male. Of the patients who developed a VTE, 18 (75%) received non-chemical DVT prophylaxis. Twenty (83.3%) of the 24 VTEs were catheter-associated DVTs. Twenty-two of 24 patients received therapeutic anticoagulation with either heparin (n=21) or enoxaparin (n=1). The 2 patients who did not receive anticoagulation did not have a bleeding event. Patients who developed a bleed were more likely to have supratherapeutic PTTs (defined as PTTs ≥ 90 seconds); however, supratherapeutic levels were isolated and not associated with the time of the bleed. Of the patients who received therapeutic anticoagulation, 8 (36.4%) had a major bleed and 9 (40.9%) had a minor bleed, for an overall bleeding rate of 77.3%. There were no deaths as a result of bleeding events. Of the 20 patients with catheter-associated VTEs, 7 (35%) had a major bleed; 4 of these patients required anticoagulation reversal with protamine.Conclusions:AML patients who received induction or re-induction therapy had a low incidence of VTE overall. However, when patients received anticoagulation for VTE treatment there was a high incidence of bleeding. The results found in this retrospective chart review showed that for patients with catheter-associated VTEs, the risk of bleeding outweighed the risk of VTE. We recommend a risk-adapted strategy where patients do not receive therapeutic anticoagulation until platelet recovery. DisclosuresPowell:Rafael Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ellis:Alexion: Speakers Bureau. Pardee:Rafael Pharmaceuticals: Employment, Research Funding.

Adaptive immunity-related gene expression profile is correlated with clinical phenotype in patients with acute myeloid leukemia.

BackgroundAcute myeloid leukemia (AML) is a common and lethal hematopoietic malignancy that is highly dependent on the immune microenvironment. However, light has yet to be shed on the landscape of adaptive immunity-related genes. This work aimed to uncover the novel molecular events in AML and potential therapeutic strategies for AML treatment.MethodsFor the current research, the transcriptional information of 732 genes that participate in adaptive immunity was collected from 173 patients with AML, and the patients were grouped into different cohorts based on the different expression patterns. The correlations between gene expression and clinical characteristics, including prognosis, were studied.ResultsAccording to the notably different expressions of adaptive immunity-related genes, the 173 patients were divided into 2 clusters and 3 subclusters. No significant differences in overall survival (OS) or progression-free survival (PFS) were detected between the clusters or subclusters. There were obvious discrepancies found in age, peripheral blood (PB) blast percentage, and French-American-British (FAB) classification between each cluster or subcluster. The patients in cluster 1 were older and more of them had M5 type; the patients in cluster 2 were younger and more of them had M2 type. Further, 81 genes were significantly correlated with age and 101 genes were significantly correlated with PB blast percentage. Comparison of the prognosis between each FAB type revealed that patients with M3 type displayed the most favorable OS and PFS. Among the differentially expressed genes (DEGs), CLEC2B expression was much lower in M2 patients than in patients with other types (P<0.001), and its high expression indicated a worse outcome (12.4 vs. 46.5 months of OS).ConclusionsThis study has uncovered the expression profile of adaptive immunity-related genes in AML. The different gene expression patterns are not associated with survival, but are significantly correlated the FAB types. CLEC2B expression is low in patients with M2 type and is negatively correlated with prognosis, thus revealing a potential therapeutic target for AML.