FLT3-ITD Mutations Research Articles

Plm-102, a Next Generation FLT3 Inhibitor, Shows Potent Anti-Leukemic Activity on Resistance to Gilteritinib in FLT3 Mutated Acute Myeloid Leukemia

Introduction: Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 25 ~ 30% of all acute myeloid leukemia (AML) cases, with FLT3-ITD mutations having a poor prognosis. In 2022, the FDA granted approval to Gilteritinib, a 2nd generation FLT3 inhibitor, for the treatment of relapsed or refractory AML. Although Gilteritinib has demonstrated efficacy in R/R AML patients, its response duration is limited due to the development of secondary resistance. Herein, we report a next-generation drug candidate, PLM-102, which has shown promising pre-clinical results to overcome the current unmet needs of Gilteritinib resistance, including FLT3-ITD-TKD and Ras mutations in FLT3-mutated AML. Aims: The main objective of this study is to investigate the effect of PLM-102 in in-vitro and in-vivo acquired Gilteritinib-resistant models and to elucidate the underlying signaling pathways. Methods: To establish Gilteritinib-resistant cell lines, we exposed the AML cells to FLT3 ligand at 10 ng/ml and Gilteritinib at concentrations ranging from 10 nM to 100 nM for four months. Subsequently, for the following four months, we treated the cells with Gilteritinib alone at 100 nM, omitting FLT3 ligand from the treatment. Whole Exome Sequencing (WES) and mRNA sequencing were performed on primary FLT3 mutated AML cells and Gilteritinib resistance FLT3 mutated AML cells. Subsequently, cell viability assays were carried out on both parent FLT3-mutated AML cells and Gilteritinib-resistant FLT3-mutated AML cells. To assess the activities of PLM-102 at the on-target and off-target resistant mechanisms, immunoblot studies were conducted. An in vivo study was performed using a Gilteritinib-resistant MOLM-14 AML xenograft mouse model. Results: We conducted Whole Exome Sequencing on the Gilteritinib-resistant MOLM-13 and MOLM-14 cell lines. The analysis revealed the presence of KRAS G13D and FLT3-ITD-C828G mutations in the Gilteritinib-resistant MOLM-13 cell line, and NRAS G13D and FLT3-ITD-G697R mutations in the Gilteritinib-resistant MOLM-14 cell line. In the evaluation of the efficacies of Gilteritinib and PLM-102 in Gilteritinib-resistant MOLM-14 cell lines. The GI 50 values for Gilteritinib and PLM-102 were determined to be 427 nM and 6.17 nM, respectively. In the studies with a xenograft model with Gilteritinib-resistant MOLM-14 cells (20 mg/kg, p.o.), the Tumor Growth Inhibition (TGI, %) values were determined as 71.9% vs 17.1% for PLM-102 vs Gilteritinib, respectively. These results demonstrated the superior in-vivo efficacies of PLM-102 in Gilteritinib-resistant cells. mRNA sequencing was performed on both MOLM-14 cells and Gilteritinib-resistance MOLM-14 cells, resulting in an upregulation of the cytokine-mediated signaling pathway, including chemokines, in the Gilteritinib-resistant MOLM-14 cells. Additionally, we conducted mRNA sequencing on Gilteritinib-resistant MOLM-14 cells treated with and without PLM-102 at a concentration of 10 nM. Interestingly, in the group treated with PLM-102 at 10 nM, we observed a notable decrease in the cytokine-mediated signaling pathway, including chemokines. These findings provide valuable insights into the potential mechanism by which PLM-102 exerts its anti-leukemic effect in Gilteritinib-resistant cells by modulating the cytokine-mediated signaling pathway. In Gilteritinib-resistant MOLM-14 cells harboring the NRAS G13D mutation, we confirmed hyperactivation of the MEK-ERK signaling pathway. The significantly improved effects in the combination of PLM-102 and Trametinib, a MEK inhibitor, were observed through cell viability assays and a xenograft model, further supporting their potential as a synergistic therapeutic approach. Currently, the GLP toxicological studies of PLM-102 has successfully completed, and the Investigational New Drug (IND) for human clinical studies will be submitted near future. Conclusions: Our results have confirmed that RAS-MEK-ERK signaling pathway and cytokine signaling pathway were activated by Gilteritinib resistance. Notably, PLM-102 exhibits exceptional anti-leukemic activity in Gilteritinib-resistant cell lines through the unique mechanism of inhibiting the RAS-MEK-ERK-cytokine signaling pathway. These results highlight the potential of PLM-102 as a third-generation inhibitor with favorable efficacies in patients with resistance to current FLT3 inhibitors.

Validation of the Prognostic Risk Signatures for Older Patients with Acute Myeloid Leukemia Treated with Hypomethylating Agents and Venetoclax

Introduction: Hypomethylating agents (HMA) with Venetoclax (Ven) is the standard of care for patients with acute myeloid leukemia (AML) ≥ 75 yrs of age or not candidates to intensive treatment. Döhner, et al. (ASH 2022) demonstrated that the European LeukemiaNet (ELN) risk classifications do not accurately predict prognosis in HMA-Ven treated patients. A prognostic risk signature classification (PRSc) was proposed, stratifying patients by RAS, FLT3 and TP53 mutations alone. We report a validation of this PRSc in a large cohort of patients treated with HMA-Ven. Methods: This unicentric analysis included patients older than 60 years old with newly diagnosed treated with HMA-Ven in different treatment protocols. Responses were assessed with the ELN-2022 criteria. The PRSc allocated patients with [ K/N]- RAS or FLT3-ITD mutations in the intermediate benefit group, patients with TP53 in the lower benefit group, whereas patients with AML lacking these mutations were allocated in the higher benefit group. Cumulative incidence (CI) was calculated for pts achieving remission with death and relapse as competing events. Results: A total of 179 patients were included, diagnosed between 11/2014 and 12/2021. Median age was 74 yrs old (range, 61-89) and 58% were male. 31% of patients had diploid cytogenetics and 35% had complex karyotype. According to the ELN 2022 risk category, 30 (17%), 15 (8%) and 114 (64%) patients were allocated in the favorable, intermediate and adverse risk group, respectively. According to the PRSc, 75 (42%), 31 (17%) and 54 (30%) were allocated in the higher, intermediate and lower benefit group, respectively. In the PRSc intermediate benefit group, 21 patients had NRAS mutations, 9 patients had KRAS mutation and 7 patients had FLT3-ITD. Patients received treatment with decitabine 10 days with Ven (n=89, 50%), decitabine 5 days with Ven (n=69, 38%), oral decitabine 5 days with Ven (n=14, 8%) and Azacitidine 7 days with Ven (n=7, 4%). The overall response rate (ORR) was 70% (126/179), including 96 pts (54%) achieving complete remission (CR) rate and 30 (17%) having CR with incomplete hematologic recovery (CRi). The ORR for patients allocated in the ELN 2022 favorable, intermediate and adverse risk was 90% (27/30), 93% (14/15) and 63% (72/114). According to the PRSc, the ORR was 87% (65/75), 55% (17/31) and 59% (32/54) for patients of the higher, intermediate and lower benefit group, respectively. The median number of cycles to best response was 1 (1-8), and 22 patients (12%) underwent allogeneic stem cell transplantation. After a median follow-up of 35 months, the median overall survival (OS) was 11 months. According to the ELN 2022 classification, the mOS was 44, 13 and 8 months (p<0.001, C-index=0.6). When applying the PRSc, the median OS was 30, 12 and 5 months for higher, intermediate and lower benefit group (p<0.001, C-index=0.66). Median event-free survival (EFS) was 8 months, being 30, 8, and 5 months for ELN 2022 favorable, intermediate and adverse group, respectively (p<0.001, C-index=0.62). When applying the PRSc, the median EFS was 18, 8 and 4 months for higher, intermediate and lower benefit group, respectively (p<0.001, C-index=0.65). The 2-yr CI of relapse and death was 47% and 22%, respectively. By the ELN 2022 subgroups, the 2-yr CI of relapse was 29%, 43% and 55% for favorable, intermediate and adverse groups, respectively (p=0.1). The 2-yr CI of death was 12%, 21% and 25% for favorable, intermediate and adverse groups, respectively (p=0.4). By the PRSc, the 2-yr CI of relapse was 26%, 70% and 60% for higher, intermediate and lower benefit group, respectively (p=0.004). The 2-yr CI of death was 20%, 6% and 33% for higher, intermediate and lower benefit group, respectively (p=0.1). Conclusion: The PRSc classification can stratify 3 different groups of patients with distinct outcomes. In the present study the 3 categories defined by the PRSc have very similar survival than the median OS previously reported (27, 12 and 5 months for higher, intermediate and lower benefit group), with a better performance according to the C-index. Signaling mutations ( RAS or FLT3-ITD) as well as TP53 mutations represent a challenge with HMA-Ven, in which novel treatments or combinations are warranted to improve outcomes.

FLT3 Testing and Clonal Evolution in Patients with Acute Myeloid Leukemia: A Retrospective Cohort Study in Taiwan

INTRODUCTION: Mutation of the FMS-like tyrosine kinase 3 ( FLT3) gene is one of the most common genetic alterations in acute myeloid leukemia (AML), while FLT3 internal tandem duplication (ITD) is a predictor of poor disease prognosis in patients. Timing of FLT3 mutation testing and the pattern of FLT3 clonal evolution are critical issues for ensuring that patients with FLT3 mutation-positive AML receive targeted therapy promptly, but data from real-world clinical practice are limited. AIM: This retrospective cohort study aimed to assess the turnaround time of FLT3 testing in two patient cohorts with non-M3 primary AML: newly diagnosed (ND) patients and those with relapsed or refractory disease (R/R). A further aim in the R/R cohort was to assess the pattern of FLT3 clonal evolution. METHODS: Eligible adult patients had evidence of new diagnosis of AML or R/R AML between 1 January 2009 and 31 December 2019 and had undergone at least one FLT3 test at National Taiwan University Hospital. Patients were followed up until either the end of the data period, loss to follow up (no records for 6 months), or death. We analyzed the time from the date of ordering FLT3 test to receipt of results (both cohorts) and the acquisition (negative at diagnosis, positive at R/R), loss (positive at diagnosis, negative at R/R), or maintenance (no change in status) of FLT3 mutation at R/R compared with status at diagnosis. We also explored the association between acquisition/loss/maintenance of FLT3 mutation at R/R and overall survival (OS) in the R/R cohort. RESULTS: From a total AML dataset of 1,789 patients, 659 and 223 were included in the ND and R/R cohorts, respectively ( Table 1). Demographics and clinical characteristics of the ND and R/R cohorts were similar. The most common type of AML was recurrent genetic abnormalities in both ND and R/R cohorts (50.7% and 67.7%, respectively); 45.8% and 48.0% of patients, respectively, had a normal karyotype; and NPM1 (21.1% and 16.6%, respectively), and IDH1/ IDH2 (18.5% and 15.2%, respectively) were the most common types of co-mutations detected. There was no difference in the turnaround time of FLT3 testing between the ND and R/R cohorts ( Table 1). Since 2020, FLT3 testing protocols have been further optimized to meet a 7-day turnaround time; additionally, almost all chemotherapy-fit patients with FLT3 mutation receive midostaurin in combination with 3+7 chemotherapy after reimbursement of midostaurin was implemented in Taiwan in February 2020. Of 164 patients with FLT3 tests at both diagnosis and R/R, most (75.6%, n=124) were FLT3 mutation-negative at diagnosis ( Table 1). At R/R, 27/124 (21.8%) acquired an FLT3 mutation and 12/40 (30.0%) patients lost their FLT3 mutation ( Table 1). Risk of mortality was increased in patients who acquired an FLT3 mutation (HR [95% CI] 1.59 [0.97, 2.62]), maintained their FLT3 mutation (1.52 [0.58, 2.56]), or lost their FLT3 mutation (1.22 [0.58, 2.56]), compared with those who were FLT3-mutation negative at both diagnosis and R/R ( Figure 1). CONCLUSIONS: This study provided information on the real-world landscape of FLT3 testing among patients with AML in Taiwan. The turnaround time of FLT3 testing in the current study was longer than the time reported in clinical trials, likely owing to different procedures in sampling arrangement and report delivery between real world and clinical trial settings. The distribution of FLT3 mutation and pattern of clonal evolution were largely consistent with previously published clinical studies. The higher prevalence of FLT3 mutation in the R/R cohort versus the ND cohort is in accordance with previous research showing that FLT3-ITD mutation is predictive of relapse. Patients with FLT3 mutation at R/R had a shorter OS, which indicates the importance of FLT3 testing at R/R diagnosis to optimize outcomes through targeted therapy.

Differences in Molecular Alterations and Clinical Outcomes By Ethnicity for Patients with Acute Myeloid Leukemia Treated with Hypomethylating Agents and Venetoclax

Introduction: Clinical outcomes for non-Hispanic Black and Hispanic patients with acute myeloid leukemia (AML) have been reported to be worse compared with non-Hispanic white patients despite increasing therapy options. Socioeconomic factors play a large role in causing these disparities. However, whether specific molecular alterations are associated with a particular ethnic group is understudied in adult populations. Prior studies either involve the pediatric population or focus on a few selected ethnic groups. Our primary objective is to determine whether particular molecular alterations associated with specific ethnic groups are associated with higher mortality. Methodology: Retrospective data was collected for 142 patients with AML diagnosed from July 2018 to December 2022 and treated in the Northwell Health system with a hypomethylating agent (HMA) and venetoclax. Demographic information was manually obtained from the electronic medical record (EMR) and ethnicity was categorized as non-Hispanic white (NHW), non-Hispanic black (NHB), Hispanic, Asian, or other/multiracial. Overall survival (OS) was calculated as the time difference between date of definitive diagnosis prior to starting venetoclax to death or date of last contact. Molecular alterations were detected by next-generation sequencing (NGS) testing. We looked at the presence of TP53, TET2, RUNX1, NPM1, FLT3-ITD, IDH1, IDH2, and several other mutations/deletions. Kaplan-Meier analysis with log-rank (Mantel-Cox) testing was used to compare overall survival between ethnic groups. Results: Of 142 patients, 87 (61%) were NHW, 23 (16%) were NHB, 7 (5%) were Asian, 10 (7%) were Hispanic, and 15 (11%) were other/multiracial. Median age was 77 years. The most common molecular alterations were TP53 mutations/deletions (25%) for the NHW group, TET2 (27%) for the NHB group, NPM1 without FLT3-ITD (43%) for the Asian group, ASXL1 and RUNX1 (both 40%) for the Hispanic group, and RUNX1 (40%) in the other/multiracial group. The NHB group had a similar incidence of TP53 mutations/deletions (21%) as the NHW group did. Median number of cycles of HMA and venetoclax tolerated was 2 for NHW, 3 for NHB, 5 for Asians, 3 for Hispanics, and 4 for other/multiracial. Kaplan-Meier analysis comparing all groups simultaneously did not show a significant difference in OS, X 2 (4, N = 142) = 5.37, p = 0.25. Individual comparisons between the NHB (X 2 (1, N = 110) = 0.61, p = .43), Hispanic (X 2 (1, N = 97) = 0.27, p = .61), and other/multiracial (X 2 (1, N = 102) = 0.90, p = .34) groups with the NHW group did not show any significant difference either. Asians had better OS when compared individually with the NHW group (X 2 (1, N = 94) = 4.29, p = .038). However, no significant difference was observed when OS for Asians was compared with OS for any other group. Conclusions: In this cohort of patients, the only significant OS benefit was in favor of Asians compared with NHW group. Based on molecular alterations, this may reflect the increased incidence of NPM1 mutations without FLT3-ITD mutations and decreased incidence of TP53 mutations in Asian patients. Additionally, adverse molecular alterations (TP53, RUNX1) were increased in the non-Asian groups. Asians were able to tolerate more cycles of HMA and venetoclax than any other group. Though prior studies suggested that Asians have molecular alterations with favorable risk, the sample size in this study for the Asian group (N = 7) was too small to make any definitive conclusions. Future studies should build on these findings.

Activation of GCN2 By HC-7366 Results in Significant Anti-Tumor Efficacy As Monotherapy and Overcomes Resistance Mechanisms When Combined with Venetoclax in AML

Venetoclax (anti-BCL2) is approved for elderly patients with acute myeloid leukemia (AML) in combination with hypomethylating agents such as azacitidine or decitabine. However, several resistance mechanisms arise, impeding responses in patients. These resistance mechanisms include TP53 or FLT3-ITD mutations, suppression of pro-apoptotic proteins NOXA or PUMA, increases in oxidative phosphorylation and energy metabolism, or elevated expression of S100A8/A9. Recent reports have highlighted that hyperactivation of the integrated stress response (ISR) pathway overcomes venetoclax resistance through NOXA activation and glycolytic activity inhibition. However, these studies used drugs that indirectly activate the ISR with other off-target effects. General Control Nonderepressible 2 (GCN2) is an ISR kinase that senses and responds to nutrient stress conditions, and activation of GCN2 results in antitumor activity. We are developing HC-7366, a first-in-class, first-in-human direct GCN2 activator, and are currently evaluating this agent in a phase 1 clinical trial in solid tumors (NCT05121948). Here we present a series of studies characterizing the antitumor effects of HC-7366 in AML, and how it impacts venetoclax resistance. AML patients with TP53 mutations are known to have lower response rates to venetoclax.The in vivo efficacy of HC-7366 in both CDX and PDX models was independent of TP53 mutation, resulting in 100% complete response and 100% tumor growth inhibition in TP53-mutated MOLM-16 and KG-1 tumor models, respectively. Analysis of tumors from treated mice by IHC demonstrated activation of the ISR as evidenced by increased expression of the ATF4 targets ASNS and PSAT1. A genome-wide CRISPR screen in KG-1 cells revealed that GCN2 sgRNAs were significantly enriched in HC-7366-treated cells, highlighting the importance of GCN2 activation in mediating the mechanism of action of HC-7366. Interestingly, BCL2 sgRNAs were significantly depleted in HC-7366 treated cells, suggesting a synthetic lethal relationship with BCL2 inhibitors, such as venetoclax, when combined with HC-7366. Indeed, in the MV4-11 FLT3-ITD mutant model (a differentiated subtype of AML that shows limited response to venetoclax), the combination of HC-7366 and venetoclax produced substantial benefit resulting in 26% tumor regression and enhanced activation of the ISR pathway. HC-7366 treatment also increased mRNA and protein levels of NOXA and PUMA while reducing mitochondrial respiration and glycolysis in a GCN2-dependent manner, leading to a low energetic state. Consistent with its impact on cellular energetics, global metabolomic analyses of AML xenograft tumors showed that HC-7366 significantly altered metabolites associated with glycolysis and the TCA cycle. Additionally, HC-7366 reduced protein levels of S100A8/A9, also known to correlate with venetoclax resistance. Our in vitro and in vivo results demonstrate that HC-7366 is a potent GCN2 activator with strong antitumor activity in AML as a single agent and in combination with venetoclax. The potential of HC-7366 to counteract multiple known resistance mechanisms to venetoclax could provide a viable treatment option for patients with relapsed/refractory AML when used in combination with venetoclax.

Update Results of a Phase II Trial of Venetoclax in Combination with Azacitidine and Chidamide in Relapsed/Refractory Acute Myeloid Leukemia

Background: Therapies for relapsed or refractory (R/R) AML remain limited, with low response rates and short duration of response, especially in patients ineligible for intensive cytotoxic chemotherapy. Combinations of venetoclax (VEN), chidamide (CHI) plus azacytidine (AZA) have shown to be synergistic with low intensity and intensive chemotherapy in preclinical data and in relapsed or refractory (R/R) setting (Phase II, 2022 ASH). Here we present updated results of this Phase II trial. Methods: Eligible patients were >18 yrs old with R/R AML, defined as lack of response rate after at least one cycle of induction or relapsed disease with any prior number of treatments. Patients received AZA 100mg on D1-7, VEN 200mg on D1-14, and CHI 30mg biweekly in 28-day cycles. The primary objective was evaluation of CR/CRi. Secondary objectives included event-free survival (EFS), overall survival (OS) and adverse events (AEs). Clinical responses were determined using the 2017 ELN criteria. Results: Between January 2022 and April 2023, 53 patients with R/R AML were enrolled in the study. The median age was 51 years (range 18-73). This regimen was salvage 1 in 79% (42) of patients, salvage 2 in 11% (6) and salvage 3 in 9% (5). 17%, 36%, and 47% of patients were classified as favorable, intermediate, and adverse ELN risk, respectively ( Table 1). 11% of patients had TP53 mutation, 22% had DNMT3A mutation, 21% had FLT3-ITD mutation, 9% had ASXL1 mutation, 19% had NPM1 mutation, 9% had IDH1 mutation, 11% had IDH2 mutation ( Fig. A). Among the 53 patients, the ORR of these patients was 68% with CRc rate of 53% after one cycle, and 81% with CRc rate of 72% after two cycles ( Fig. B). By ELN risk, ORR and CRc rates were 70% and 50% for favorable risk, 40% and 40% for intermediate risk, and 43% and 30% for adverse risk. Pretreatment IDH1 and IDH2 mutations were associated with higher CRc rates (75% and 50%, respectively). The CRc rate in patients with TP53 mutations was 67%, with response noted among patients with concurrent IDH1/ IDH2 mutations. With a median follow-up of 6.7 months, the median EFS and OS were 6 (95%CI 3.3-8.6) months and not reached (NR), respectively. Among patients with CRc, the 6-month EFS and OS were 64% and 88%, respectively, among those receiving this regimen as first salvage had encouraging outcomes (median EFS, NR) ( Fig. C-D). Of the 8 patients that proceeding with allogeneic hematopoietic stem cell transplant (allo-HSCT), the median EFS and OS were not reached after transplant ( Fig. E-F) No non-hematologic AEs of grade ≥3 were observed. The most common grade 1-2 non-hematologic AEs included nausea (28%), fatigue (28%), hypokalemia (17%), hypoproteinemia (17%), elevated transaminase levels (33%) and hyperbilirubinemia (11%). The occurrence rates of grade 3/4 hematologic adverse events (AEs) were as follows: white blood cell decrease (87%), median time to white blood cell recovery 23 (range 4-29) days; neutropenia (96%), neutropenia with fever (17%), median time to neutrophil recovery 25 (range 10-29) days; platelet decrease (68%), median time to platelet recovery 28.5 (range 7-44) days; anemia (64%), median time to hemoglobin recovery 29 (range 13-56) days. 30-day and 60-day mortality were 0% and 5.7%, respectively. 3 deaths occurred within 60 days in patients with persistent leukemia. Conclusions: Thecombination of VEN and CHI plus AZA (VCA) demonstrated manageable safety and encouraging efficacy in patients with R/R AML. IDH1/ IDH2 mutations were associated with the sensitivity of VCA, even in some patients with concurrent TP53 mutations.

A Phase I/II Trial of the FLT3 Kinase Inhibitor XY0206 in Patients with Relapsed/Refractory Acute Myeloid Leukemia

Abstract Background: The FMS-like tyrosine kinase 3 (FLT3) mutations occur in approximately 30% of acute myeloid leukemia (AML) patients, which are associated with worse survival and higher relapse risk. XY0206 is a novel oral FLT3 inhibitor with preclinical activity against FLT3 internal tandem duplication mutations (FLT3-ITD). The Phase Ⅰ/Ⅱ clinical trail with XY0206 (NCT04471064) is currently underway to explore the initial efficacy, safety and pharmacokinetic (PK) of XY0206 in patients with relapsed/refractory AML (R/R AML). Methods: This open-label, multicenter, dose-escalation and dose-expansion Phase Ⅰ/Ⅱ trail enrolled subjects from August 2020 to September 2022 who were aged ≥18 years with R/R AML. Six dose-escalation or dose-expansion cohorts (12.5, 25, 37.5, 50, 62.5 mg per day, or 25 mg twice a day) were set up to explore the safety/tolerability and antileukemic activity of XY0206, and to determine the maximal tolerated dose (MTD) of XY0206. Safety and tolerability were assessed by monitoring dose-limiting toxicities (DLT), treament-emergent adverse events (TEAE) and adverse drug reaction (ADR). Cohort expansion was based on safety and antileukemic activity. Results: As of December 14, 2022, a total of 61 subjects were enrolled (37 with FLT3 mutation-positive). Demographics and baseline characteristics of patients are presented in Table 1. MTD of XY0206 was established as 50 mg when two of the three patients enrolled in the 62.5 mg dose-escalation cohort could not tolerate continuous medication. The 37.5 mg, 50 mg and 25 mg BID dose cohorts were further expanded. Antileukemic activity was assessed in the Full Analysis Set (FAS, n=61), with overall response rate (ORR) was 34.4% (n=21/61), composite complete remission (CRc) was 32.8% (n=20/61), complete remission (CR) was 6.6% (n=4/61) and CR with partial hematologic recovery (CRh) was 9.8% ( n=6/61 ). Among the patients with FLT3 mutation-positive (FLT3 mut+, n= 37), ORR was 48.6%(n=18/37), CRc was 45.9% ( n=17/37), CR was 2.7% (n=1/37) and CRh was 16.2% (n=6/37). Among the patients with FLT3-ITD mutation (n=30), ORR was 56.7% (n=17/30), CRc was 53.3% (n=16/30), CR was 3.3% (n=1/30) and CRh was 20%( n=6/30). The 37.5 mg dose cohort as the target dose was further expanded to include FLT3 mut+ patients only. CRc in FLT3 mut+ patients ( n= 19 ) and FLT3-ITD mutation patients (n=16) was 57.9% (n=11/19) and 68.8% (n=11/16 ), CR was 5.3% (n=1/19) and 6.3% (n=1/16), CRh was 26.3% (n=5/19 ) and 31.3% (n=5/16), respectively. In terms of safety, the overall incidence and severity of TEAE/ADR in 61 subjects did not correlate with dose significantly. The most common grade 3 and above adverse events (≥ 50% ) were leukopenia (65.6% ), thromobocytopenia (63.9%), neutropenia (62.3%), animia (52.5%) and hypokalemia (50.8%). The fatal adverse event was disease progression, which was mostly related to AML. Conclusion: XY0206 is a novel FLT3 inhibitor that showed a good safety profile, a potent anti-leukemic activity and improved efficacy in patients with FLT3 mut+ R/R AML.

NK-2 Homeobox Transcription Factor NKX2-3 Confers Differentiation Block in NPM1-Mutated in Acute Myeloid Leukaemia

Dysregulation of HOX homeobox family transcription factor (TF) gene expression is important in the pathogenesis of AML in particular in NPM1-mutated and MLL rearranged molecular subtypes. However, little is known about the role of NK2-homeobox (NKX2) family TF genes. NKX2 genes have critical roles in normal development of the central nervous system, thyroid, lung and pancreas. We observed high expression of NKX2-3 in patient AML samples and hypothesised the transcription factor might have a functional role in the disease. By qPCR, we found that NKX2-3 is highly expressed in normal HSCs but is rapidly down regulated as cells differentiate. In primary AML samples, it is highly expressed in 21-37% cases, including within the immunophenotypic LSC compartment, in particular in cases with NPM1 and/or FLT3-ITD mutations. Forced expression of NKX2-3 in normal murine KIT + bone marrow (BM) HSPCs transiently enhanced their clonogenic activity and impaired differentiation: NKX2-3-expressing HSPCs expressed higher levels of E2F or MYC target genes, and leukemia stem cell (LSC) maintenance genes. Congenic transplant experiments revealed that forced expression of NKX2-3 in normal BM HSPCs was sufficient to enhance multilineage engraftment across four months of follow up without inducing leukemia. In keeping with this, transplantation of Nkx2-3 -/- knockout BM cells led to significantly inferior engraftment compared with BM cells from litter mate control mice. Thus, while increased expression of Nkx2-3 enhances engraftment of normal stem cells in transplantation assays, loss of Nkx2-3 impairs it. Analysis of NKX2-3 high primary human AML cases demonstrated that HOXA9 was the most highly upregulated transcription factor gene compared with NKX2-3 low cases. We expressed both factors singly and together in murine KIT+ BM HSPCs: Hoxa9/NKX2-3 cells exhibited significantly higher clonogenic activity and reduced morphologic and immunophenotypic differentiation compared with Hoxa9/MTV cells. Congenic BM transplant experiments demonstrated that Hoxa9/NKX2-3 recipients developed leukemias significantly earlier than Hoxa9/MTV recipients (median 82 vs 155 days). BM morphology and immunophenotyping confirmed that Hoxa9/NKX2-3AMLs exhibited a greater degree of differentiation block than Hoxa9/MTV AMLs. Leukemia cells from Hoxa9/NKX2-3 recipients coordinately expressed higher levels of E2F or MYC target genes, and LSC maintenance genes. NKX2-3 KD to ~30% of control values reduced proliferation and induced morphologic and immunophenotypic differentiation in primary patient NPM1-mutated AML cells. NKX2-3 KD led to down regulation of E2F and MYC target genes and normal human CD34 + stem cell genes; and up regulation of myeloid differentiation genes. Using a CRISPR ribonucleofection approach to relocate NPM1c to the nucleus we confirmed that NKX2-3 expression in NPM1-mutant primary AML cells is sustained by mutant NPM1c. ChIPseq in NPM1-mutated primary patient and KO52 AML cells identified NKX2-3 binding peaks. As expected, DNA sequences under peak apices were strongly enriched for NKX2-3 binding motifs; there was also significant enrichment for RUNX and ETS family motifs, but not HOX/MEIS/PBX motifs. To identify genes potentially regulated by NKX2-3, genomic coordinates of strong binding peaks were mapped to the single nearest gene. Remarkably, among genes up regulated following NKX2-3 knockdown in primary human AML cells there was a highly significant enrichment of genes with putative regulatory elements bound by NKX2-3. Likewise, among genes down regulated in NKX2-3-expressing KIT+ murine HSPCs, there was a similar significant enrichment of genes with putative regulatory elements bound by NKX2-3. These data suggest that NKX2-3 functions as a transcription repressor at bound regulatory elements in primary AML cells. In summary, NKX2-3 is highly expressed in NPM1 & FLT3 mutated molecular subtypes of AML and serves to enhance self-renewal and the level of differentiation block through binding to and repressing regulatory elements of myeloid differentiation genes.

Intensive Induction Chemotherapy (IC) Vs Hypomethylating Agents + Venetoclax (HMA/VEN) in IDH1- or IDH2-Mutant Newly Diagnosed Acute Myeloid Leukemia (AML) - a Multicenter Cohort Study

Introduction: Intensive induction chemotherapy (IC) remains the standard of care for younger and fit patients (pts) with acute myeloid leukemia (AML). Post-hoc analyses from clinical trials have associated IDH1 and IDH2 mutations with high rates of durable responses to venetoclax (VEN)-based therapies. However, these trials were conducted in pts ≥75 years of age or unfit for IC. As there are currently no trials comparing hypomethylating agents (HMA)+VEN and IC directly in IDH-mutant AML, we conducted a large multicenter retrospective cohort study of pts with newly diagnosed AML with IDH1 and IDH2 mutations to assess the impact of clinical and molecular characteristics on response and survival to IC versus HMA+VEN. Methods: We included pts with newly diagnosed IDH1- and IDH2-mutant AML who were treated at 4 large academic centers with IC (conventional 7+3 or liposomal cytarabine/daunorubicin) or HMA/VEN. Detection of IDH1 and IDH2 mutations was performed by next-generation sequencing per local standards at the participating sites. Responses were recorded according to European LeukemiaNet 2017 criteria. Composite complete response (cCR) was defined as complete response (CR) + CRi (CR with incomplete count recovery). Overall survival (OS) was compared between groups by log-rank test. Cox regression for OS was fitted to evaluate the effect of treatment, allogeneic stem cell transplantation (allo-SCT) as a time-varying covariate and additional covariates. All variables with p<0.1 in the univariate model were included in the multivariable stepwise backward selection model. Results: We reviewed 1132 pts with AML and included 98 and 131 pts with IDH1/2-mutant AML, respectively (9 pts had IDH1 and IDH2 co-mutations). As pts with IDH1-and IDH2-mutant AML had similar baseline demographic and disease characteristics, we combined both groups for further analyses. Of the 220 ptswith IDH1/2-mutant AML, 150 and 70 received IC and HMA+VEN, respectively ( Table 1). Twenty pts in the IC group received concurrent treatment with the IDH1 inhibitor ivosidenib. Compared to pts treated with IC, pts who received HMA+VEN were older (median age 75 years with HMA+Ven [Range: 60 - 88 years] vs 63 years [R: 24 -76] with IC; p<0.001) and had higher rates of molecular defined secondary AML (57% vs 41%; p<0.001), CEBPA mutations (10.0% vs 2.7%; p=0.040), and TP53 mutations (13% vs 2.7%; p=0.005) Despite these differences in baseline characteristics, rates of cCR were similar for IC and HMA+VEN-treated pts (68% vs 67%; p=0.90) with higher rates of CR seen with IC (64% vs 46%; p=0.010). The rate of allo-SCT was higher in pts treated with IC than with HMA+VEN (63% vs 23%; p<0.001). The median OS for the entire cohort was 32 months (95% CI: 24 months [mo] - not reached [NR]) with a significant difference in OS by treatment type favoring IC over HMA+VEN (52 mo [95% CI: 30 mo - NR] vs 14 mo [95% CI: 11 - 39 mo]; p=0.002). However, in pts > 60 years (n=168 pts), median OS was 21 mo (95% CI: 16 - 32 mo) and not statistically significant ( p=0.11) between IC (27 mo [95% CI: 20-57 mo]) and HMA+VEN (15 mo [95% CI: 11 mo - NR]). Comparable OS was seen among pts undergoing allo-SCT (n=111 pts; 24-mo OS 75% [95% CI: 66 - 84%] with IC vs 24-mo OS 79% [95% CI: 61 - 100% with HMA+VEN, p=0.71), as well as when censored at allo-SCT (IC 12 mo [95% CI: 7.7 - 25 mo]; HMA+VEN 12 mo [95% CI: 8.2 - 18 mo], p=0.58) Although treatment with HMA+VEN vs IC was found to be associated with worse OS in univariable analysis ( Table 2) it did not retain its prognostic value in the multivariable analysis (HR: 1.28; 95% CI: 0.74 - 2.19; p=0.37). Conversely, prior HMA, monosomal karyotype, JAK2 and FLT3-ITD mutations were associated with worse survival suggesting that these factors are more relevant for OS than the type of treatment. Conclusion: In pts with newly diagnosed IDH1/2-mutant AML, cCR rates were similar for pts treated with IC and HMA+VEN. While there was a difference in OS favoring treatment with IC over HMA+VEN in the unselected patient cohort, this difference was no longer present in subgroup analyses based on age >60 years and in pts who received an allo-SCT. Furthermore, treatment type did not impact OS in multivariable regression models suggesting that differences in OS between treatment groups are more likely to be explained by differences in patient and disease characteristics rather than treatment type. Due to the limitations of retrospective, real-world studies, a randomized clinical trial is needed.

Comprehensive Molecular Stratification of Patients with AML Treated with CPX-351

Background: Liposomal cytarabine-daunorubicin (CPX-351) is approved for treatment of acute myeloid leukemia (AML) with myelodysplasia (MDS)-related changes and therapy-related AML. CPX-351 induction has been observed to result in longer periods of bone marrow suppression than with 7+3 therapy. Thus, careful consideration is warranted when selecting patients for whom CPX-351 would provide an optimal risk-to-benefit ratio. It is unknown whether any specific genetic mutations impact outcomes from CPX-351 therapy. Methods: Forty-one subsequent patients with AML at VCU Massey Comprehensive Cancer Center from 2017 to 2022 were treated with CPX-351 (liposomal cytarabine-daunorubicin). Genetic mutation profiles were obtained from our institutional multi-gene panel next generation sequencing assay. Multivariate analysis was performed using Cox proportional hazards regression. Overall survival (OS) was analyzed via the Kaplan-Meier method. Composite complete response (CCR) was defined as CR + CR with incomplete hematologic recovery + CR with partial hematologic recovery. Results: The median age of patients treated with CPX-351 was 65.2 years (range, 20.1-78.5). Median ECOG performance status was 1 (range, 0-2) and Charlson Comorbidity Index was 4 (range,2-14). The majority (65.9%) of patients had adverse risk per ELN 2022 criteria, mostly with MDS-related and complex cytogenetics (17.1% favorable risk; 14.6% intermediate risk). Multivariate analysis identified mutated PTPN11 ( PTPN11mut)and IDH2 ( IDH2mut) as independent risk factors for decreased OS in patients treated with CPX-351. The hazard ratio (HR) for death of PTPN11 mut was 2438 (p = 0.017). The HR for IDH2mut was 124.2 (p = 0.022). Mutated TP53 was also a risk factor for reduced OS, with HR 3.21, but this was not statistically significant. Mutated SRSF2 ( SRSF2mut) was an independent prognostic factor for improved OS with CPX-351, with a HR of 0.0019 (p = 0.017). ASXL1, TET2, IDH1 and FLT3-ITD mutations were associated with improved OS, but these results did not reach statistical significance. For patients with SRSF2mut treated with CPX-351, median OS was 20.5 months, compared to 8.9 months for those without SRSF2mut (HR 0.49 [95% CI 0.28-0.88], p=0.037). The CCR rate was not significantly different between patients with and without SRSF2mut (p=0.66), and measurable residual disease (MRD) status could not be assessed due to unavailable historical data. Patients with PTPN11mut had a median OS of 11.2 months, whereas those without PTPN11mut had a median OS of 21.6 months, although this comparison was not statistically significant (HR 1.51 [95% CI 0.70-3.26], p=0.207). CCR rates were not significantly different between patients with and without PTPN11 mut. The median OS for patients with IDH2mut was 16.4 months, compared to 21.6 months for patients without IDH2mut but this difference was not statistically significant. Conclusions: Among patients with AML treated with CPX-351, PTPN11 and IDH2 mutations were independently associated with inferior overall survival compared to those without these mutations. The PTPN11 gene is involved in RAS pathway regulation and has also been associated with shorter survival in AML in other studies. Here, mutated SRSF2 was associated with improved survival after CPX-351 induction. SRSF2 is an RNA splicing factor gene that is frequently found in AML with antecedent MDS but has previously been associated with adverse outcomes after conventional intensive induction chemotherapy. Interestingly, CCR and MRD negativity rates did not differ for patients with these mutations compared to those without at our institution despite the noted changes in survival. These findings remain speculative and warrant further investigation with a greater number of patients in the era of routine MRD assessment. Moreover, controlling for confounders (such as death due to toxicity rather than disease or relapse), the impact of HCT, and frailty were not accounted for in this retrospective analysis. Future work includes identifying the potential impact of cooperating mutations.

Chip-AML22 Master Protocol: An Open-Label Clinical Trial in Newly Diagnosed Pediatric De Novo Acute Myeloid Leukemia (AML) Patients Including a Linked Phase II Trial with Quizartinib in FLT3-ITD/ NPM1wt Patients - a Study By the NOPHO-DB-SHIP Consortium

Background and Significance Pediatric AML is a heterogeneous disease with current rates for event-free survival (EFS) of 50-60% and for overall survival (OS) of 70-80%, for newly diagnosed patients. In patients with refractory and relapsed disease, and in subsets as defined by AML biology and treatment response, prognosis is dismal. To further improve EFS, the NOPHO-DB-SHIP consortium has initiated trial CHIP-AML22. Building on the successes achieved by the recent trial from this consortium (NOPHO-DBH AML-2012, preliminary data as per June 2023, n=878: 5-years pEFS 63%, 5-years pOS 78%), CHIP-AML22 will incorporate innovative elements, mainly the FLT3-inhibitor quizartinib (Vanflyta®) that was recently approved by the FDA and PMDA in combination with chemotherapy for the treatment of newly diagnosed adult AML patients with FLT3-ITD mutations, and gemtuzumab ozogamicin (GO, Mylotarg ®) that improved outcome in adult and pediatric AML when added to chemotherapy. CHIP-AML22 will maintain the approach of treatment-response driven risk-adapted treatment, intensified induction treatment for patients with ≥5% AML cells in the bone marrow (BM) after course 1, and consolidation with allogeneic stem cell transplantation (allo-SCT) for high-risk patients after the first consolidation course HAM. Study Design and Methods This clinical trial will enroll newly diagnosed de novo patients aged ≥1 day to ≤18 years to 2 randomized studies in the Master protocol, to the linked quizartinib trial, or the standard arm of the Master (Figure 1). Innovations include: (1) a phase II, single arm, open-label study on the safety, efficacy, pharmaco-kinetics and -dynamics of quizartinib in combination with chemotherapy, and as single-agent after allo-SCT, in FLT3-ITD/ NPM1wt AML; All patients will receive quizartinib monotherapy for 6 months post-SCT, except those that were negative for measurable residual disease determined with multiparameter flow cytometry (MFC-MRD) in BM1 and remained negative (study CHIP-AML22/Quizartinib); (2) a phase III, open-label randomization with gemtuzumab ozogamicin (GO; Mylotarg®) added to induction therapy MEC for pediatric CD33-positive newly diagnosed AML patients aimed at improved outcome (study Ri), (3) a phase III, open-label randomization with three (standard of care) versus two (investigational) courses of consolidation chemotherapy for standard-risk patients as non-inferiority trial (study Rc), and (4) refining risk-group adapted treatment, aimed at improved outcome. Novel high-risk categories are patients with RAM-phenotype and/or CBFA2T3::GLIS2, and patients with KMT2A-rearranged AML (excluding KMT2A::MLLT3) with ≥0.1% AML cells after course 1 in BM1. Patients with ≥15% AML cells on day 22 BM after course 1, or ≥0.1-5% AML cells by MFC-MRD after course 2 in BM2 (end-of-induction), or those with FLT3-ITD/NPM1wt remain to be high-risk, similar to AML-2012. Further, dexrazoxane is recommended to all patients before receiving daunorubicin or mitoxantrone, aiming to prevent late-onset cardiotoxicity. The quizartinib trial includes a safety run-in with a modified rolling-6 design and two dose levels with 6 evaluable patients per dose level. In study Ri, patients assigned to the experimental GO-arm will receive GO twice during induction with MEC at a dose of 3 mg/m 2 (max 5 mg) with an interim-analysis for superiority of any of these two arms. The investigational arm of the Rc randomization will omit HA 3E. Both the quizartinib trial and each randomization will also be compared to historical controls from the AML-2012 trial. CHIP-AML22 expects to enroll 130-140 patients per year and it will take 7-8 years to reach the total number of 905 patients needed to answer all study questions. Patients will be followed-up for at least 5 years from diagnosis and thus, the total study duration is expected to be 12-13 years. The Master protocol (EU CT: 2023-504999-25) and linked quizartinib trial (EU CT: 2023-505000-27-00) were approved by the Dutch Ethical Review Authority and as of July 2023, enrollment in the Master protocol including study Rc has started in The Netherlands. Study Ri and the linked quizartinib trial are expected to open in Q4 2023. At least 15 other countries with nearly 60 sites will participate in CHIP-AML22: Belgium, Denmark, Estonia, Finland, Hong Kong, Iceland, Israel, Latvia, Lithuania, Norway, Portugal, Spain, Sweden, Switzerland and Uruguay.

The Impact of DNMT3A Mutation on Survival of AML Patients Receiving Allogeneic Hematopoietic Cell Transplantation in First Remission Depends on the Karyotype and Co-Occurring Mutations: On Behalf of the EBMT Acute Leukemia Working Party

DNMT3A gene mutations are more frequent in cytogenetically normal acute myeloid leukemia (AML) and frequently associated with NPM1 mutation and internal tandem duplication (ITD) of the FLT3 gene. However, DNMT3A mutation is not included as a distinct prognostic group in the recent European Leukemia Net (ELN) 2022 genetic risk classification of AML. In the context of allogeneic hematopoietic cell transplantation (allo-HCT), the prognostic value of DNMT3A is still not well studied. We evaluated, through the European Society for Blood and Marrow Transplantation (EBMT) registry, the impact of DNMT3A mutation, according to the karyotype, and NPM1 and FLT3-ITD mutation status, on post-transplant outcomes in AML patients receiving allo-HCT in first complete remission (CR1). We identified a total of 1374 adult AML patients (51% female, median age 57 years; range 18-78 years) with ELN-2022 intermediate- or poor-risk cytogenetics and available DNMT3A, FLT3ITD and NPM1 mutation status, who underwent their first allo-HCT in CR1 between 2013 and 2021. Of these, 631 (46%) patients had DNMT3A mutation. Patients with mutant DNMT3A were more likely to be female (57% versus 46%, p<0.001), to have normal cytogenetics (75% versus 54%, p<0.001), FLT3-ITD co-mutation (50% vs 23%, p<0.001), NPM1 co-mutation (60% vs 21%, p<0.001), and to receive a myeloablative conditioning and in vivo T cell depletion. After a median follow-up of 2.3 years, the 2-year leukemia free survival (LFS) and overall survival (OS) were 62% and 69%, respectively. The subsequent analysis was split according to baseline karyotype (normal or abnormal). Among 870 patients with normal cytogenetics, 472 (54%) patients had DNMT3A mutation. The 2-year relapse incidence (RI), LFS and OS were 24%, 63% and 70% respectively, in patients with DNMT3A mutation compared to 18%, 69% and 74% in patients with wild type DNMT3A. Among these patients with normal karyotype, multivariable analyses (MVA) were done within the 4 groups of FLT3-ITD and NPM1 mutations. Within the group of 324 patients with both FLT3-ITD and NPM1 positives, 244 were triple-positive (75%), LFS and OS were not significantly different with and without DNMT3A mutation (2y LFS: 62% vs 63%, HR: 0.98, p=0.95; 2y OS: 70% vs 73%, HR: 1.17, p=0.58). Conversely, among 146 patients with NPM1 mutation without FLT3-ITD, LFS was significantly lower for 91 patients with DNMT3A mutation (2y LFS: 68% vs 87%, HR: 2.70, p=0.02) but OS was not significantly different (2y OS: 79% vs 87%, HR: 2, p=0.13). On the other hand, among 318 with wild type NPM1 and without FLT3-ITD, LFS and OS were not significantly different for 108 patients with DNMT3A mutation compared to 210 patients with wild type DNMT3A (2y LFS: 62% vs 67%, HR: 1, p=0.98; 2y OS: 65% vs 71%, HR: 1.02, p=0.94). Conversely, among 82 patients with FLT3-ITD and wild type NPM1, 2-year LFS and OS were 53% and 58% respectively, for 29 patients with DNMT3A mutation compared to 71% and 76% respectively, for 53 patients with wild type DNMT3A. The number of patients didn't allow to perform MVA in this group. Finally, among 504 patients with abnormal cytogenetics, 159 (31%) patients had DNMT3A mutation. On MVA, DNMT3A mutation did not significantly affect LFS or OS. Poor-risk cytogenetics was associated with worse LFS, OS and increased RI. Older age negatively affected LFS and OS. In conclusion, in AML patients allografted in CR1, the impact of DNMT3A mutation depends on the karyotype and co-occurring mutations. Our data suggest that DNMT3A mutation negatively affects post-transplant survival selectively in patients with normal karyotype and either NPM1 mutation without FLT3-ITD, or patients with normal karyotype, FLT3-ITD and wild type NPM1. DNMT3A mutation shows no impact on post-transplant outcomes in other settings.

Autophagy Induction Constitutes a Targetable Vulnerability in FLT3-Mutant Acute Myeloid Leukemia Cells Treated with FLT3-Inhibitors

Recently, we and others identified autophagy induction as a protective mechanism in FLT3-ITD mutant acute myeloid leukemia (AML) cells upon treatment with FLT3-inhibitors (FLT3i), constituting an increasing issue in clinical practice. Here, we investigated the molecular consequences of autophagy induction upon FLT3i treatment, exploring resistance mechanisms and interrogated whether autophagy inhibition can improve FLT3i antileukemic therapy. Drug response of primary FLT3-ITD AML samples ex vivo treated with FLT3i, quizartinib (AC220, n= 59) and midostaurin (PKC412, n=64), was associated with their transcriptional profile to unravel which molecular programs were enriched in poor responders. Both gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA) revealed enriched molecular signatures compatible with autophagy activation in poor responders to FLT3i, while sensitive samples enriched molecular signatures consonant with mitochondrial function. Enrichment scores for programs associated with “regulation of autophagy” and “autophagosome organization” were positively correlated to ex vivo response area under the curve values for AC220 and PKC412. To functionally address the molecular pathways associated with autophagy activation upon FLT3i treatment, MOLM-13 and MV4-11 AML cells ( FLT3-ITD) and primary AML samples, were treated with AC220 and PKC412 alone or in combination with the autophagy inhibitor chloroquine (CQ, 5-10μM). In FLT3-ITD AML models, treatment with FLT3i significantly induced the formation of acidic vesicular organelles, suggesting autophagy induction. We also observed a reduction of autophagy regulators in a dose- and time-dependent manner, evidenced by the conversion of LC3B-I to LC3B-II, with degradation of LC3B-II and p62. Furthermore, FLT3i treatment resulted in phosphorylation inhibition of AKT Ser473, mTOR Ser2448 and its downstream protein p70S6K Thr421/Ser424, and phosphorylation reduction of ULK1 Ser757. Combination of FLT3i with CQ (10 μM) significantly reduced cell viability and survival for both PKC412 and AC220 inhibitors. To rule out the off target effects associated with CQ treatment, we performed a knockdown (KD) on the key autophagy mediator ATG5 using shRNA-mediated inhibition in FLT3-ITD cells. In MOLM13 cells, FLT3i treatment in ATG5-KD cells significantly reduced the IC50 (control: 31nM vs. ATG5-KD: 17nM) and increased apoptosis induction (control: 41% vs. ATG5-KD: 58%). We validated these observations in ex vivo treated primary AML samples, where we observed that in FLT3-ITD but not in FLT3 wild-type samples, the combination of FLT3i (PKC412 and AC220) plus autophagy inhibitors (CQ) reduced cell proliferation and survival. In summary, we demonstrated that autophagy induction is a biological process intrinsically associated with drug resistance in FLT3-ITD mutant AMLs when treated with FLT3i. Hence, autophagy inhibition can be leveraged to increase the antileukemic effect of FLT3i and highlights the autophagy as a major acquired biological process associated with FLT3i therapy.

Establishing an AML Ex Vivo Platform to Generate Physiologically Relevant Models

Acute myelogenous leukemia (AML) comprises a highly polyclonal and heterogeneous disease containing leukemic stem cells, progenitors, and differentiated blasts. Despite advances in treatment, the long-term relapse-free survival for AML remains poor. Due to complex mutational profiles and highly divergent subtypes in AML, the use of physiologically relevant, patient-derived models is needed. Traditional cell line models of AML almost exclusively represent differentiated blasts, are homogenous in clonality, and often do not encompass major genetic subtypes. To address these gaps, we have established a culture system that supports the growth of higher-order AML samples ex vivo. By optimizing culture conditions, we successfully cultivated 70% (10/14) of AML disseminated PDX models ex vivo and demonstrated they continue to proliferate after long term cultures. We established a 16-color flow panel to characterize their cell maturity state and show they maintain cell surface heterogeneity. Importantly, these models have not ‘drifted’ transcriptomically after multiple passages. Our platform includes 2 NPM1c mutant and 3 IDH1/2 mutant ex vivo lines, in addition to other subtypes underrepresented in traditional culture methods. Furthermore, we treated AML PDXs in vivo with a clinically-relevant dose of venetoclax + azacytidine (Ven/AZA) for 2 cycles (14 days) and then evaluated post-VEN/AZA cells in our ex vivo assay. Growing these models ex vivo, we show using our flow panel that Ven/AZA treatment shifts the differentiation status from a stem-like state to a more mature-like cell state which is consistent with literature evidence of treatment resistance. Additionally, a second PDX model treated with three cycles of Ven/AZA in vivo and harvested in ex vivo culture conditions maintained both Ven and AZA resistance. Interestingly, compared with the vehicle treatment, Ven/AZA treatment caused resistance to other cell death agents including selective MCL1 and dual Bcl2/xL inhibitors. Lastly, we performed a 7-day cell compound panel screen using common standard of care agents for the treatment of AML. Many of the targeted agent activity correlated with expected genetic subtypes, such as FLT3 inhibitors in FLT3-ITD mutants and venetoclax in WT TP53 models. This established ex vivo platform provides physiologically relevant models to explore novel target biology, better understand compound mechanism of action (MoA), and generate drug resistant models through in vivo driven clinically relevant dose scheduling using standard of care agents.

Evaluation of Neddylation and Apoptosis-Related Gene Expression in Patients with Acute Myeloid Leukemia

Introduction Regulated cell death (RCD) is a process commonly dysregulated in patients (pts) with acute myeloid leukemia (AML), resulting in the survival of genetically unstable cells. Apoptosis, as the best-known form of RCD, interacts with neddylation, a process that contributes to tumorigenesis and apoptosis resistance. Aims This study was conducted to determine the expression of the selected genes involved in the neddylation pathway and to assess their clinical importance along with selected interacting proapoptotic genes. In addition, we correlated the expression levels of these genes with known prognostic factors in AML and determined their impact on the survival of AML pts. Methods The expression of selected proapoptotic genes BIK, BAX, BAK, BCL2L11, BBC3 , PMAIP1 CASP3, CASP7, and neddylation-related genes: CUL1, CUL2 , CUL4A, CUL5, CUL7, CUL9, NEDD8 was determined in duplicates in bone marrow samples of newly diagnosed AML pts. Real-Time PCR was carried out using the TaqMan chemistry and the QuantStudio7 thermal cycler (Applied Biosystems-Thermo Fisher Scientific). Gene expression was normalized using reference genes selected by the Normirazor tool. The normalization was performed by ∆Ct = Ct (reference) - Ct (mRNA of interest). This approach results in higher values for higher mRNA expression. Differential expression analysis was performed using a Welch t-test. Kaplan-Meier method with the log-rank test was used to assess overall survival (OS). Results In a prospective study, we included 40 newly diagnosed AML pts (mean age 62; range 26-87) with a median (m) follow-up of 6.7 months (95% CI: 4.5-9.1). High-risk AML was present in 52.5%, intermediate-risk in 32.5%, and low-risk in 15% of pts, according to European LeukemiaNet (ELN) 2022 risk stratification. NPM1 mutation was present in 23%, whereas FLT3 ITD or TKD mutation in 25% of pts. Intensive treatment was introduced in 43% of pts, while 25% were treated with azacitidine+venetoclax. Non-intensive treatment with azacitidine in monotherapy was administered to the remaining pts. BAK expression was detected in 70%, BIK in 98%, while expression of the other genes was found in all pts. In the high-risk group, we detected lower expression of both proapoptotic BAX (fold change - FC=0.7, p=0.01) and BIK gene (FC=0.4, p=0.01), as well as lower expression of CUL9 (FC=0.7, p=0.03). Accordingly, in pts with NPM1 mutation we revealed upregulation of BIK (FC=6.1, p=0.001), and CUL9 (FC=1.6, p=0.01). Whereas pts with FLT3 mutation had lower expression of proapoptotic BBC3 (FC=0.5, p=0.01), and neddylation-related CUL1 (FC=0.6, p=0.01). In cases of initial bone marrow blasts infiltration above median value (>49%) BCL2L11 CUL5 , NEDD8 and PMAIP1were downregulated ( p=0.001-0.02), while CUL7 ( p=0.00) and CASP3 ( p=0.04) were upregulated. Patients with initial white blood cell count >20 G/l had lower expression of BCL2L11 ( p=0.005), and CUL4A ( p=0.02), while higher expression of CASP7 ( p=0.04). The mOS in the study cohort was not reached. OS was longer in pts with higher (> -2.061, cutoff was determined with CutOff Finder) BAX expression (log-rank: p=0.03) [Figure 1]. The mOS was not reached in this group, whereas the mOS in pts with lower BAX expression was 1.3 months (95% CI: 0.6-1.3). In univariate Cox regression analysis, higher BAX expression (HR 0.31, 95%CI: 0.11-0.84, p=0.022), higher albumin level (HR 0.13, 95%CI: 0.04-0.41, p=0.0004), and intensive treatment (HR 0.18, 95%CI: 0.04-0.69, p=0.012) were factors influencing the outcome. In the multivariate model for OS, higher expression of BAX (HR 0.03, 95%CI: 0.003-0.33, p=0.004) retained its significant protective effect in the context of established prognostic factors [Table 1]. Conclusions To our knowledge, we were the first to evaluate such a complex spectrum of neddylation and apoptosis-related genes in AML. We revealed dysregulation of these genes in AML. Our data indicate that upregulation of BAX at the diagnosis is associated with longer OS. Moreover, we reported significantly lower BAX expression in ELN high-risk group. Thus, indicating BAX may be a potential prognostic factor in AML. Our preliminary results did not prove significant differences in the effect of neddylation gene expression levels on the prognosis of AML patients. As studies are emerging on the potential of neddylation-targeted therapies, we are conducting further research to verify the effect of neddylation on AML.

Genetic and Functional Characterization of NUP98-Rearranged Leukemia

NUP98 rearrangements ( NUP98r) are recurrent alterations in pediatric acute myeloid leukemia (AML) and are associated with dismal outcomes (PMID:21813447). These structural variantsresult in fusion oncoproteins comprised of the N-terminus of NUP98 and the C-terminus of fusion partners. Among more than 30 fusion partners discovered so far, the associations between NUP98::NSD1 with myeloblastic AML and NUP98::KDM5A with acute megakaryocytic/erythroid leukemia (AMKL/AEL) have been appreciated, which often harbor distinct cooperating mutations (e.g., NUP98:: NSD1 with FLT3-ITD and WT1 mutations). In contrast, other fusions, such as NUP98:: RAP1GDS1 or NUP98:: HOXA9 can be found also in myelodysplastic syndrome (MDS) or T-cell acute lymphoblastic leukemia (T-ALL) (PMID:32766874). However, how these fusions and cooperating mutations contribute to the specific phenotypes of NUP98r leukemias is poorly understood. To investigate the genetic background of NUP98r leukemia, we collected RNA-seq, WGS, and WES data for 91 pediatric cases from St. Jude (n=71),AEIOP (n=3), and the TARGET study (PMID:29227476, n=17; Panel.A), which included AML (n=82), MDS (n=4), and T-ALL (n=5). NUP98::NSD1 (n=47, 51.6%) and NUP98::KDM5A (n=26, 28.6%) were the most common fusions, in line with previous observations. NUP98::RAP1GDS1 (n=6, 6.6%), an understudied NUP98 fusion, was the third most common fusion in this study. We also confirmed frequent mutations of WT1 and FLT3-ITD in NUP98::NSD1 AML and entire or partial chromosome 13 loss involving RB1 locus in NUP98::KDM5A AMKL, while we observed frequent mutations in RAS or epigenetics-related genes, CCND2/3, and RUNX1 preferentially in cases without alterations of WT1, FLT3 or RB1 loss. NUP98:: RAP1GDS1 and NOTCH1 mutations were strongly enriched in T-ALL. These findings suggest that fusions and cooperating alterations both contribute to the disease phenotypes. To study how NUP98 fusion oncoproteins (FOs) drive leukemogenesis and specific phenotypes, we applied Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to determine NUP98 FO binding for a primary sample (myeloblastic with NUP98::NSD1), CHRF-288-11 cell line (megakaryocytic with NUP98::KDM5A), and HA-tagged CD34 NUP98::KDM5A model (Panel.B-top). CUT&RUN using antibodies against the N-terminus NUP98 and histone modifications (H3K4me3, H3K27ac) revealed that these models shared NUP98 FO binding sites at the HOXA/B and RUNX1 loci, together with active histone marks. In contrast, binding patterns at differentiation-related gene loci were heterogenous. NUP98::NSD1 uniquely binds to CEBPA, which encodes a master regulator of myeloid differentiation, contrary to NUP98::KDM5A binding to GFI1B encoding regulator of erythromegakaryocytic differentiation . These data indicate that NUP98 FOs directly and uniquely regulate differentiation-related genes. We also performed CUT&RUN in AML cells with RUNX1::RUNX1T1 or UBTF tandem duplications, in which the N-terminus NUP98 antibody did not show any detectable peaks, confirming the specificity of the N-terminus NUP98 signals in NUP98r AML models. Experiments using a HA antibody in HA-tagged NUP98::KDM5A CD34 models confirmed that fusion oncoproteins are binding to these target regions. However, the C-terminus NUP98 antibody also detected similar signals as the N-NUP98 antibody, suggesting that wild-type NUP98 is also recruited to NUP98 FO target genes, possibly by a heteromer formation with NUP98 FOs and other NPC components. To understand the possible association between somatic mutations and the genomic binding of NUP98 FOs, we next investigated chromatin immunoprecipitation sequencing (ChIP-seq) data from the ENCODE study (PMID:22955991) for RB1, WT1, and STAT5 (downstream FLT3-ITD). Binding peaks of these proteins partly coincided with NUP98 peaks on representative target genes (Panel.B-bottom), suggesting that frequent mutations in NUP98r leukemia converge on the genomic interaction of NUP98 FOs. In conclusion, genomic characterization of NUP98r leukemia showed the association among fusion partners, cooperating mutations, and disease phenotypes. Epigenetic profiling revealed that NUP98 FOs directly regulate target genes in a fusion and differentiation-specific manner and provide a basis for further study to investigate how NUP98 FOs and cooperating partners drive leukemogenesis and disease phenotypes.

Optimizing FLT3 Inhibitor Use in Adult Acute Myeloid Leukemia with FLT3 Mutations Using Proteomics

CONCLUSION FLT3 mutations are the most frequent genetic alteration in adult Acute Myeloid Leukemia (AML). The use of FLT3 inhibitors (FLT3I) has improved outcome among FLT3 mutant patients, but not all benefit. We developed a proteomic-based approach to stratify patients according to their response to FLT3I therapy. The expression of 429 proteins (339 total, 90 post-translational modified) was measured by Reverse Phase Protein Arrays (RPPA) in fresh samples from 805 newly diagnosed AML patients, with levels normalized to normal CD34+ cells. Of 137 with FLT3 ITD or D835 mutations, 54 who were treated with FLT3I (Sorafenib=22, Quizartinib=11, Midostaurine=6, Gilteritinib=4 and Crenolanib=1) combined with other agents (Hypomethylating agents (HMA)=23, HMA+Venetoclax (VTX)=5, Idarrubicin+Cytarabine (AraC)+Purine Analogs (IA+Pur)=19, IA+Pur+VTX=2, AraC=3, Fludarabine+AraC (FA)=1, none=1) were selected for analyses. Proteins prognostic for Overall Survival (OS) and Complete Remission Duration (CRD) were identified using pairwise LogRank tests (p < 0.05) adjusted for False Discovery Rate, followed by unbiased hierarchical clustering. Continuous and categorial variables were compared with Wilcoxon, Kruskal-Wallis or Fisher's Exact test with simulated p-values (10000 replicates). Cox proportional hazards models (CoxPH) were developed for Uni-(UV) and Multi-variate (MV) analysis. Deep neural network analysis using random forest models identified proteins most predictive of cluster assignment. 28 proteins were individually prognostic for both OS and CRD and divided patients into 3 clusters (Fig. 1: C1=red, C2=blue, C3=green). The distribution of nearly all other clinical and molecular features did not differ between clusters, including: age, gender, race, cytogenetic risk and AML group. Co-occurrence with other mutations was not biased, except for FLT3+TET2 and FLT3+TET2+NPM1 mutants, more frequent in C1. OS was significantly longer (Fig. 2), in C1 (Median: C1>84mo, C2=14.8mo, C3=11.4mo; p<0.001) as was CRD (Median: C1>84mo, C2=8.6mo, C3=5.7mo; p<0.05). A similar OS pattern was observed for mutation combos, including with NPM1 or DNMT3a but not if IDH, CEBPa, TET2, or ASXL1 mutation were present. It was also seen when patients were divided by gender, age groups (except >70yo), primary, but not secondary AML, diploid karyotype and intermediary cytogenetic risk, and with Sorafenib or IA+PR therapies. In the UV CoxPH model for OS, C1, C2 and C3 were predictive (HR=1, 4.1, 7.5; p=ref., 0.037, 0.009), along with age, secondary AML, complex karyotype, Trisomy 6 and IDH1 mutation. In the MV model for OS, only C1, C2 and C3 (HR=1, 3.3, 4.8; p=ref., 0.011, <0.001), and age were predictive. In the CRD UV model, C1 and C3 (HR=1, 3.3, 4.8; p=ref., 0.072, 0.022), age, secondary AML, and mutations in ASLX1, PTPN11, RAS, TP53 and WT1 were predictive. Only C1 and C3 were predictive in the MV model for CRD (HR=1, 2.9, 6.3; p=ref., 0.16, 0.04). The predictive proteins, are biased towards specific cellular processes, mainly: proliferation and metabolism (RHEB, GSKA_B, SDHA, AKT1, AKT2 and TYRO3), cell cycle and DNA damage response (CDKN2A, DDB1, SIRT6, SLFN11 and WEE1), cellular adhesion and cytoskeleton organization (MYH9.pS1943, PAK4 and PXN), and apoptosis (AIFM1, BBC3 and BCL2L11). Key differences between clusters included: higher FOS in C1, and higher CDKN2A, MYH9.pS1943, PXN, PAK4, SDHA, BBC3 in both C2 and C3, with higher levels of these in C3. Since PAK4, PXN and MYH9 are connected to cellular motility/migration and inhibitors for those proteins are available, there is a therapeutic opportunity to overcome FLT3I resistance in C2/C3 patients. Moreover, we identified a set of 6 proteins (FOS, AKT1, PXN, SMARCA2, CDKN2A and DPF2) that predicted cluster assignment with 97.5% accuracy, making rapid patient stratification possible in the clinical setting. Protein profiling identified cohorts of FLT3 mutant patients that achieved the highest benefit from FLT3I addition, regardless of all other clinical or molecular characteristics. We identified targetable proteins that are elevated in adverse signatures, and hypothesized that these may be targets for modulation to improve FLT3I response. Moreover, a small set of 6 proteins accurately classifies patients and could be used clinically to triage patients for treatment recommendation.

Sex-associated differences in frequencies and prognostic impact of recurrent genetic alterations in adult acute myeloid leukemia (Alliance, AMLCG)

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients’ sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3-ITD, DNMT3A, NPM1 and WT1 mutations and less often complex karyotype, ASXL1, SRSF2, U2AF1, RUNX1, or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients’ sex in analyses of AML biology and prognostication.

Ubtf tandem Duplications Define a Novel Subtype of Acute Myeloid Leukemia Associated with Younger Age, WT1 Mutations and HOXA9 Marked Overexpression

Background Upstream Binding Transcription Factor ( UBTF) gene plays an important role in ribosomal RNA transcription. UBTF tandem duplications (TD) have been recently described as a recurrent genetic lesion in acute myeloid leukemia (AML). UBTF-TDs appear to be mutually exclusive with other class-defining lesions in AML and display a unique comutational signature. UBTF-mutated AML affects predominantly younger patients and is associated with trisomy 8, internal tandem duplication of FLT3 ( FLT3-ITD), mutations in WT1 and poor prognosis. Moreover, HOX and MEIS genes seem to be overexpressed in AML with UBTF-TD similarly to NPM1-mutated ( NPM1m) or KMT2A-rearragend ( KMT2Ar) AML, suggesting potential sensitivity to menin inhibitors. In this study we sought to characterize UBTF-TDs in our AML cohort. Methods We studied 456 adult patients (median age 54 years; range 18-70) diagnosed with de novo AML and included in AML-12 protocol from CETLAM group. All patients from the 2012-2017 period (n=403) were consecutively studied and we also selected 53 cases of WT1-mutated AML from the 2017-2020 period, given the strong association with UBTF-TD. Screening for UBTF-TD was performed on bone marrow (BM) or peripheral blood (PB) genomic DNA samples. Exon 13 of UBTF gene was amplified and analysed by Polymerase chain reaction (PCR) and subsequent fragment length analysis by capillary electrophoresis. Mutated samples were confirmed through Sanger sequencing. Further, RNA from 9 UBTF-TD cases was available and HOXA9 and MEIS1 gene expression analysis was performed by Real-Time Polymerase Chain Reaction (RT-qPCR). Clinical and biological data were obtained from CETLAM registry. Results UBTF-TD was found in 13 patients from our cohort whose characteristics are detailed in Table 1. UBTF-TDs range from 45 base pairs (bp) to 365 bp, being the 48 bp the most recurring size. As described, although small UBTF-TDs are in frame insertions, the largest ones are not multiple of 3. Notably, most UBTF-TDs presented small insertions and deletions among the duplication. Presence of UBTF-TD was associated with younger age (36 vs 55 years; p&amp;lt;0.001) and BM dysplastic changes (80% of U BTFMUT). UBTF-TD was particularly frequent (8.9% of all cases) in patients below 40 years, compared to other lesions as CEBPA mutations (1.1%). Eleven (85%) U BTFMUT patients presented intermediate cytogenetics, being trisomy 8 (5), normal karyotype (3) and 9q deletion (2) the most recurrent findings. Two cases of adverse karyotype with MECOM-rearrangements were found. As previously described, there was a strong association between UBTF-TD and of FLT3-ITD (38.5%). Other recurrent genetic lesions in AML like NPM1 and CEBPA mutations or core-binding factor (CBF) rearrangements were exclusive of UBTF wild type patients. Further, we found that UBTFMUT leukemias showed marked overexpression of HOXA9 compared with other AML subtypes (22.1-fold vs 5.0-fold; p&amp;lt;0.001; Fig. 1) and similar to NPM1m AML (22.3-fold). On the other hand, MEIS expression did not differ significantly among groups. Conclusions In our cohort of adult AML patients, UBTF-TDs are found in 1.8% of cases and are especially frequent in younger individuals in absence of other class-defining genetic lesions. The presence of UBTF-TD is strongly associated with cytomorphological dysplasia and co-occurrence of FLT3-ITD and WT1 mutations, while NPM1 and CEBPA mutations and CBF rearrangements appear to be mutually exclusive. HOXA9 upregulation could be a leukemogenic event in UBTF-TD AML, as described in NPM1-m and KMT2A-r AML, suggesting potential utility of menin inhibitors in this subset of patients. Screening for UBTF-TD is going to be incorporated in our molecular diagnostic panel for new AML cases.

Impact of Outpatient Administration of Cytarabine Consolidation Chemotherapy on Hospital Length of Stay in Adults with Acute Myeloid Leukemia

Background: Administration of 3-4 cycles of high/intermediate dose cytarabine (H/IDAC) reduces relapse of acute myeloid leukemia (AML) and bridges patients (pts) to allograft. There is no accepted standard model of safely delivering H/IDAC in these pts. To reduce reliance on inpatient (inpt) chemotherapy administration and safely avoid or forestall hospitalization during cycles, we developed an outpatient (outpt) protocol to deliver H/IDAC. Pts were assigned outpt or inpt treatment at their hematologist's discretion. We aimed to evaluate the benefits and safety of our model in this retrospective observational study. Methods: Pts diagnosed with AML and treated with intensive induction chemotherapy between 2015 and 2020 were identified; included pts received ≥1 consolidation cycle of 1-3g/m² cytarabine over 3 hours BID, dose adjusted for age and renal function, on days 1, 3 &amp; 5 of a planned 28-day cycle. Outpts received H/IDAC via computerized ambulatory delivery device (CADD) pump and central venous access device (CVAD), attended days 1-6, then 3 times a week for blood product support until count recovery; elective readmission was not mandated. Pts were educated to present to hospital if unwell. Inpts were admitted for H/IDAC but could be discharged prior to count recovery. Antimicrobial prophylaxis consisted of antifungal agents and valaciclovir. The primary endpoint was mean duration of hospital admission (LOS) per consolidation cycle. As pts may have been treated on an inpt and/or outpt basis, generalized estimating equations were used to compare LOS clustered by pt. Secondary outcomes included death during H/IDAC; intensive care unit (ICU) admission; time to admission; blood product use; cycle length; bacteremia; time to count recovery; relapse free survival (RFS) and overall survival (OS). Results: Of 109 pts identified, 62 pts were included and 47 pts excluded (not AML=3; induction death=7; refractory leukemia=14; alternative=13; no consolidation=10). Median age was 55.2 years (range 18.2-70.3); 31 (50%) males; ELN 2010 fav=23 (37.1%), Int-2/3 = 30 (48.4%), Adv=8 (12.9%); denovo AML in 60 (96.7%); and therapy related in 2 (3.2%). FLT3-ITD, NPM1 and bi-allelic CEBPA mutations detected in 11 (17.7%), 27 (43.5%) and 2 (3.2%) pts, respectively. Fifty-nine pts (95.1%) received 7+3 idarubicin induction and 7 (11.3%) received midostaurin. A total of 131 cycles of H/IDAC were administered; 81 (61.8%) were commenced as outpt and 50 (38.2%) as inpt. Twenty-seven (43.6%) pts commenced all cycles as an outpt. Those commencing consolidation as an outpt spent, on average, 10.3 fewer days in hospital per cycle. The mean (SD) LOS for outpt and inpt was 11 (8.3) versus 21.3 (8) days (p&amp;lt;0.001), respectively. For each consolidation cycle, outpts spent less time in hospital (mean, [SD]): cycle 1, 12.6 (10.3) vs 20.7 (8.0) days (RR 1.6, p=0.007); cycle 2, 11.6 (7.2) vs 21.4 (8.3) days (RR 1.8, p=0.001); cycle 3, 8.4 (5.8) vs 24.6 (7.4) days (RR 2.9, p&amp;lt;0.001). There were no deaths among inpts and one death in outpts (septic shock). ICU admission occurred in 8/81 (9.9%) cycles in outpts vs 1/50 cycles (2%) in inpts (p=0.12). Mean ICU LOS for outpts vs inpts in cycle 1 was 17.6 days vs 1.6; cycle 2, 5.1 vs 0 days; and cycle 3, 4.2 vs 0 days. Bacteremia was detected in 41 cycles (31.3%), with 38 isolates (gram pos=17, gram neg=21) in 31 outpt cycles (38.2%) and 15 isolates (gram pos=6, gram neg=9) in 10 inpt cycles (20%). Median time to admission in outpts was 14 days (IQR, 3-21); 15 (18.5%) cycles did not result in pt admission. There were no differences in timing of admissions by cycle number nor time-point to prompt elective admission (Fig.1). There was no difference in packed cell or platelet transfusion requirement between inpts and outpts. Time to blood count recovery was similar. No improvement was seen in cycle length. The proportion undergoing allogeneic transplant in first remission was 37.1%, and at any time, 59.7%. Over a median follow up of 3.3 years, median RFS was 2 years and OS, not reached. Conclusion: In selected pts, outpt based AML H/IDAC consolidation therapy significantly reduced hospital length of stay compared with inpts. There was no impact on cycle length. Bacteremia and ICU admission rates were higher in outpts, and strategies such as remote monitoring or pre-emptive hospitalization could be considered. Our findings suggest outpt consolidation should be considered in a supported clinical setting.