FLT3-ITD Acute Myeloid Leukemia Research Articles

Emerging DNA Methylome Targets in FLT3-ITD-Positive Acute Myeloid Leukemia: Combination Therapy with Clinically Approved FLT3 Inhibitors.

The internal tandem duplication (ITD) mutation of the FMS-like receptor tyrosine kinase 3 (FLT3-ITD) is the most common mutation observed in approximately 30% of acute myeloid leukemia (AML) patients. It represents poor prognosis due to continuous activation of downstream growth-promoting signaling pathways such as STAT5 and PI3K/AKT. Hence, FLT3 is considered an attractive druggable target; selective small FLT3 inhibitors (FLT3Is), such as midostaurin and quizartinib, have been clinically approved. However, patients possess generally poor remission rates and acquired resistance when FLT3I used alone. Various factors in patients could cause these adverse effects including altered epigenetic regulation, causing mainly abnormal gene expression patterns. Epigenetic modifications are required for hematopoietic stem cell (HSC) self-renewal and differentiation; however, critical driver mutations have been identified in genes controlling DNA methylation (such as DNMT3A, TET2, IDH1/2). These regulators cause leukemia pathogenesis and affect disease diagnosis and prognosis when they co-occur with FLT3-ITD mutation. Therefore, understanding the role of different epigenetic alterations in FLT3-ITD AML pathogenesis and how they modulate FLT3I's activity is important to rationalize combinational treatment approaches including FLT3Is and modulators of methylation regulators or pathways. Data from ongoing pre-clinical and clinical studies will further precisely define the potential use of epigenetic therapy together with FLT3Is especially after characterized patients' mutational status in terms of FLT3 and DNA methlome regulators.

Structure-Based Optimization of Pyrazinamide-Containing Macrocyclic Derivatives as Fms-like Tyrosine Kinase 3 (FLT3) Inhibitors to Overcome Clinical Mutations.

Secondary mutations in Fms-like tyrosine kinase 3-tyrosine kinase domain (FLT3-TKD) (e.g., D835Y and F691L) have become a major on-target resistance mechanism of FLT3 inhibitors, which present a significant clinical challenge. To date, no effective drugs have been approved to simultaneously overcome clinical resistance caused by these two mutants. Thus, a series of pyrazinamide macrocyclic compounds were first designed and evaluated to overcome the secondary mutations of FLT3. The representative 8v exhibited potent inhibitory activities against FLT3D835Y and FLT3D835Y/F691L with IC50 values of 1.5 and 9.7 nM, respectively. 8v also strongly suppressed the proliferation against Ba/F3 cells transfected with FLT3-ITD, FLT3-ITD-D835Y, FLT3-ITD-F691L, FLT3-ITD-D835Y-F691L, and MV4-11 acute myeloid leukemia (AML) cell lines with IC50 values of 12.2, 10.5, 24.6, 16.9, and 6.8 nM, respectively. Furthermore, 8v demonstrated ideal anticancer efficacy in a Ba/F3-FLT3-ITD-D835Y xenograft model. The results suggested that 8v can serve as a promising macrocycle-based FLT3 inhibitor for the treatment of AML.

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction.

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

Unveiling the signaling network of FLT3-ITD AML improves drug sensitivity prediction

Currently, the identification of patient-specific therapies in cancer is mainly informed by personalized genomic analysis. In the setting of acute myeloid leukemia (AML), patient-drug treatment matching fails in a subset of patients harboring atypical internal tandem duplications (ITDs) in the tyrosine kinase domain of the FLT3 gene. To address this unmet medical need, here we develop a systems-based strategy that integrates multiparametric analysis of crucial signaling pathways, and patient-specific genomic and transcriptomic data with a prior knowledge signaling network using a Boolean-based formalism. By this approach, we derive personalized predictive models describing the signaling landscape of AML FLT3-ITD positive cell lines and patients. These models enable us to derive mechanistic insight into drug resistance mechanisms and suggest novel opportunities for combinatorial treatments. Interestingly, our analysis reveals that the JNK kinase pathway plays a crucial role in the tyrosine kinase inhibitor response of FLT3-ITD cells through cell cycle regulation. Finally, our work shows that patient-specific logic models have the potential to inform precision medicine approaches.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

Abstract 1829: Brusatol overcomes tyrosine kinase inhibitors resistance in myeloid leukemia cells by inhibiting translation of fast turnover protein cyclin D1 and Mcl-1

Abstract Myeloid leukemia is characterized with fast proliferation and apoptosis evasion, which are controlled by mutant or activated tyrosine kinase signalings. Tyrosine kinase inhibitors (TKI) targeting BCR-ABL in chronic myeloid leukemia (CML) and FLT3-ITD in acute myeloid leukemia (AML) significantly improved the therapy, but not cure, due to the general resistance. Overactivated mTOR protein translation pathway is believed to be one of the mechanisms causing resistance and that targeting protein translation should be one effective approach to overcome resistance. Brusatol is a quassinoid extracted from the fruit of Brucea javanica and an inhibitor of protein translation. We tested its antiproliferation and apoptosis induction in a variety of AML and CML cell lines as well as in TKI resistant cell lines. Brusatol effectively inhibits the growth of seven leukemia cell lines with GI50 values less than 20 nM. Brusatol induces apoptosis in AML cell lines and induces G0/G1 phase arrest in CML cell lines. Brusatol decreases the levels of fast turnover protein cyclin D1 and Mcl-1. In FLT3-ITD inhibitor sorafenib resistant MOLM-13/Sor cell line brusatol exhibits similar growth inhibition and apoptosis induction with the parental MOLM-13 cells, associated with inhibition of Mcl-1 and FLT3-ITD proteins. In BCR-ABL inhibitor imatinib resistant K562/STI cell line brusatol is effective as in parental K562 cells, associated with inhibition of cyclin D1 and BCR-ABL proteins. Brusatol at 2 mg/kg effectively inhibit tumor growth of K562 xenografts without causing toxicity to normal organs. These data indicate that brusatol inhibits oncogenic protein translation and has potential to be used to treat TKI resistant leukemia. Citation Format: Huiming Hua, Ying Yang, Guangfuxin Lin, Jingyi Zhang, Samuel Waxman, Yongkui Jing. Brusatol overcomes tyrosine kinase inhibitors resistance in myeloid leukemia cells by inhibiting translation of fast turnover protein cyclin D1 and Mcl-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1829.

Outcomes and prognosis of haploidentical haematopoietic stem cell transplantation in children with FLT3-ITD mutated acute myeloid leukaemia.

The presence of internal tandem duplication mutations in the FMS-like tyrosine kinase 3 receptor (FLT3-ITD) is a poor prognostic predictor in paediatric patients with acute myeloid leukaemia (AML). We evaluated the treatment outcomes and prognostic factors of 45 paediatric patients with FLT3-ITD AML who achieved complete remission before haploidentical haematopoietic stem cell transplantation (haplo-HSCT) at our institution from 2012 to 2021. Among the 45 patients, the overall survival (OS), event‑free survival (EFS), and cumulative incidence of relapse (CIR) rates were 74.9% ± 6.6%, 64.1% ± 7.2%, and 31.4% ± 7.1%, respectively, with 48.8 months of median follow-up. Univariate and multivariate analyses associated positive minimal residual disease (MRD) at pre-HSCT and non-remission (NR) after introduce 1 with inferior long-term survival. The 100-day cumulative incidence of grade II-IV acute graft-versus-host disease (aGVHD) was 35.6% ± 5.2%, and that of grade III-IV aGVHD was 15.6% ± 3.0% The overall 4-year cumulative incidence of chronic graft-versus-host disease after transplantation was 35.7% ± 9.8%, respectively. In conclusion, haplo-HSCT may be a feasible strategy for paediatric patients with FLT3-ITD AML, and pre-HSCT MRD status and NR after introduce 1 significantly affected the outcomes.

Red blood cell membrane-coated FLT3 inhibitor nanoparticles to enhance FLT3-ITD acute myeloid leukemia treatment

Red blood cell membrane-coated FLT3 inhibitor nanoparticles to enhance FLT3-ITD acute myeloid leukemia treatment

FLT3 tyrosine kinase inhibition modulates PRC2 and promotes differentiation in acute myeloid leukemia.

Internal tandem duplication mutations in fms-like tyrosine kinase 3 (FLT3-ITD) are recurrent in acute myeloid leukemia (AML) and increase the risk of relapse. Clinical responses to FLT3 inhibitors (FLT3i) include myeloid differentiation of the FLT3-ITD clone in nearly half of patients through an unknown mechanism. We identified enhancer of zeste homolog 2 (EZH2), a component of polycomb repressive complex 2 (PRC2), as a mediator of this effect using a proteomic-based screen. FLT3i downregulated EZH2 protein expression and PRC2 activity on H3K27me3. FLT3-ITD and loss-of-function mutations in EZH2 are mutually exclusive in human AML. We demonstrated that FLT3i increase myeloid maturation with reduced stem/progenitor cell populations in murine Flt3-ITD AML. Combining EZH1/2 inhibitors with FLT3i increased terminal maturation of leukemic cells and reduced leukemic burden. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the clinical observations. These results demonstrate that in addition to its known cell survival and proliferation signaling, FLT3-ITD has a second, previously undefined function to maintain a myeloid stem/progenitor cell state through modulation of PRC2 activity. Our findings support exploring EZH1/2 inhibitors as therapy for FLT3-ITD AML.

Metabolomic profile of acute myeloid leukaemia parallels of prognosis and response to therapy

The heterogeneity of acute myeloid leukemia (AML), a complex hematological malignancy, is caused by mutations in myeloid cells affecting their differentiation and proliferation. Thus, various cytogenetic alterations in AML cells may be characterized by a unique metabolome and require different treatment approaches. In this study, we performed untargeted metabolomics to assess metabolomics differences between AML patients and healthy controls, AML patients with different treatment outcomes, AML patients in different risk groups based on the 2017 European LeukemiaNet (ELN) recommendations for the diagnosis and management of AML, AML patients with and without FLT3-ITD mutation, and a comparison between patients with FLT3-ITD, CBF-AML (Core binding factor acute myelogenous leukemia), and MLL AML (mixed-lineage leukemia gene) in comparison to control subjects. Analyses were performed in serum samples using liquid chromatography coupled with mass spectrometry (LC–MS). The obtained metabolomics profiles exhibited many alterations in glycerophospholipid and sphingolipid metabolism and allowed us to propose biomarkers based on each of the above assessments as an aid for diagnosis and eventual classification, allowing physicians to choose the best-suited treatment approach. These results highlight the application of LC–MS-based metabolomics of serum samples as an aid in diagnostics and a potential minimally invasive prognostic tool for identifying various cytogenetic and treatment outcomes of AML.

Post-Hoc Analysis of Measurable Residual Disease from BMT-CTN 1506/Morpho: FLT3-ITD Variant Allele Frequency and Survival Are Highly Correlated

Background: BMT CTN 1506/MORPHO was a randomized study of the FLT3 inhibitor gilteritinib versus placebo as post-transplant (HCT) maintenance therapy for patients (pts) with FLT3-ITD acute myeloid leukemia (AML). Patients with FLT3-ITD AML in first remission underwent HCT and then were randomized, in double-blind fashion, to either gilteritinib or placebo for 24 months. The primary endpoint was relapse-free survival (RFS), and a pre-specified secondary endpoint was the effect of measurable residual disease (MRD) on survival as detected by a highly sensitive assay for FLT3-ITD mutations. In the primary analysis, while RFS was higher for pts randomized to gilteritinib, the difference did not reach the pre-determined threshold for significance (HR: 0.679; 95% CI: 0.459, 1.005; 2-sided p-value: 0.0518). However, in the secondary analysis, the 50.6% of pts who had MRD detectable pre- or post-HCT had significantly higher RFS with gilteritinib (HR of relapse or death=0.515, 95% CI: 0.316, 0.838, p = 0.0065). In this post-hoc analysis, we examined 1) the impact on RFS of different levels of FLT3-ITD variant allele frequency (VAF) pre-randomization (immediately before or after HCT but prior to randomization to gilteritinib or placebo); 2) the impact of the presence of multiple mutations detected as MRD pre-HCT; and 3) the eradication of FLT3-ITD clones detected post-HCT during follow up on gilteritinib or placebo. Methods: First-pull marrow aspirates were collected from pts at two time points pre-randomization (pre-HCT and between 30-90 days post-HCT), as well as at 3, 6, 12, 18, and 24 months post-randomization. For MRD detection (performed at Invivoscribe; San Diego, CA), 700 ng input DNA was amplified by polymerase chain-reaction (PCR) using 25 cycles and primers flanking exons 14 and 15 of FLT3, followed by next-generation sequencing (NGS) analysis of the amplicons. Variant allele frequency (VAF) was calculated as FLT3 mutant reads/total FLT3 reads. For pts with multiple FLT3-ITD mutations, the VAF used in analysis was the sum of the VAFs for each FLT3-ITD variant. The lower limit of blank (LOB) of the assay is estimated to be 1 x 10 -6 VAF. For terminology, VAF &amp;gt; 1 x 10 -6 and &amp;lt; 1 x 10 -5 is referred to as MRD6, VAF &amp;gt; 1 x 10 -5 and &amp;lt; 1 x 10 -4 is MRD5, VAF &amp;gt; 1 x 10 -4 or greater is MRD4, and MRD0 equals no detectable MRD. Results: MRD was evaluated in 350/356 (98.3%) pts pre-HCT and 347/356 (97.5%) pts post-HCT. MRD was detected in 46% of pts pre-HCT and 19.9% prior to randomization, including 4.5% who did not have detectable MRD pre-HCT. The variant allele frequency (VAF) of detectable MRD ranged from a low of 1.09 x 10 -6 to a high of 3.0 x 10 -1. More than a single mutation was detected in 51 pts pre-HCT. Most of these 51 pts had 2 clones, but the total ranged from 2-9. The presence of more than one mutation detected as MRD immediately pre-HCT was associated with worse RFS compared with all other pts (MRD-positive with only one clone and MRD-negative), irrespective of treatment arm (Figure 1A; HR=2.428, 95% CI: 1.555, 3.792, P &amp;lt; 0.001). There was no significant difference in age, mutation length, VAF, or karyotype between pts with more than one mutation and those with single mutations. However, if the VAFs of pts with multiple FLT3-ITD mutations were summed and treated as a single value, then the median VAF for pts with multiple mutations was higher than for those with single mutations (2.14 x 10 -4 vs. 3.12 x 10 -5). The VAF (including the summed VAFs of pts with multiple mutations) at multiple levels correlated very closely with RFS after HCT. To illustrate this quantitative effect of peri-HCT FLT3-ITD VAF on RFS, Kaplan-Meier analysis of pts on the placebo arm grouped by MRD level is displayed in Figure 1B. In the 71 pts with MRD detectable post-HCT, the MRD was eradicated in 69% of pts on gilteritinib versus 44% of pts on placebo. Conclusions Pts with multiple FLT3-ITD mutations have a worse prognosis, which may simply be a reflection of increased disease burden (VAF). The quantity of FLT3-ITD MRD appears to highly correlate with outcome. Gilteritinib appears to augment the effect of HCT, as evidenced by increased eradication of MRD post-HCT. These data illustrate the potential utility of the PCR-NGS FLT3-ITD MRD assay in the management of pts with FLT3-ITD AML.

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.

FLT3-ITD Induces Immune Escape in AML Via up-Regulating CD47 Expression and Decreased Phagocytic Ability of Macrophages

The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and confer a poor prognosis. FLT3 inhibitors such as Quizartinib could prolong survival. However, very few relapsed patients are cured. Little attention has been paid to the characteristics of the immune microenvironment in FLT3-ITD AML. Within the bone marrow, Flt3/ITD animals demonstrated significantly increased dendritic cells and T cells levels compared to Flt3/WT mice, which suggested that FLT3-ITD can modulate the immune microenvironment in AML. Here, we found that FLT3-ITD cell lines (MV-4-11 and MOLM-13) induced less LDH release compared to FLT3-WT cell lines (HEL and K562) ( Figure a), indicating less macrophage killing. CD47 gene is an integrin related protein, which acts as an “anti phagocytosis” signal, and it interacts with signal-regulatory protein alpha (SIRPalpha) on macrophages, so that tumor cells expressing CD47 can escape being destroyed by macrophages. Further Flow Cytometry results showed that the CD47 Mean fluorescent intensity (MFI) of the FLT3-ITD group was significantly higher than those of the control group ( Figure b). Therefore, we can ask a hypothesis that FLT3-ITD in tumor cells may leads to decreased phagocytic capacity of macrophages and immune tolerance. To test our hypothesis, the FLT3 wildtype (FLT3-OE, FLT3 over expression) or ITD mutant (FLT3-ITD) stable overexpression HEL/K562 cells were constructed by means of lentiviral transfections. In the FLT3-ITD group, the MFI levels of CD47 was higher than FLT3-OE group and FLT3 NC (FLT3 Normal control) group ( Figure c and d). Green-fluorescent (CFSE-stained) Hel and K562 were then added to cultures of macrophages induced by Peripheral Blood Mononuclear Cell stained with the red dye PKH26 (ratio tumor cell:macrophages, 5:1). Flow Cytometry results showed FLT3-ITD decreased the Phagocytic Capacity of Macrophages ( Figure e and f). The TRANSFAC database was used to predict the transcription factor binding sites of the CD47 promoter. We found that HOXB5 may regulate the expression of CD47 at the transcriptional level. Compared with the FLT3-NC and FLT3-OE group, the relative expression level of the CD47 gene in the FLT3-ITD group was significantly upregulated ( Figure g, p &amp;lt; 0.05). To ascertain the mechanism that HOXB5 regulated CD47, we analysed the location CD47 gene on the chromosome, as well as the sequence of promoter and first non-encoding exon of HOXB5 (−2000 ~ 100 bp) by Jaspar online tool (http://jaspar.genereg.net/). Mechanistically, Dual luciferase reporter gene assay and ChIP experiments suggest HOXB5 directly activate CD47( Figure h and i). Compared to the monotherapy group, Combination therapy with CD47 inhibitor and Quizartinib (AC220) significantly promoted the phagocytic ability of M1-like macrophages against MOLM13 cells ( Figure j and k). The mouse model of AML was constructed by injection of 5x106 FLT3-ITD cells via the tail vein. Mouse models further demonstrate that combination therapy with CD47 inhibitor and Quizartinib significantly reduces the tumor burden and improves survival rate. We first revealed that FLT3-ITD can up-regulates CD47 to promote immune escape of macrophages. These evidences indicates that combination therapy with CD47 inhibitor and FLT3-ITD inhibitor can be a promising therapeutic method for impressive treatment of FLT3-ITD AML patients.

Integrated Methylation and Transcriptional Landscape and Evolution of Pediatric AML

Acute myeloid leukemia (AML) is an aggressive hematopoietic malignancy associated with poor outcomes despite intensive therapies. The genomic landscape of pediatric AML has been well characterized and has led to translational benefits, such as improved relapse predication and effective targeted therapies for a subset of individuals (e.g., FLT3-ITD AML). Despite these advances, the majority of patients continue to receive therapeutic interventions that have not changed in three decades. This underscores the need to advance our understanding of AML biology beyond genomic alterations (e.g., somatic mutations). Here, we present the largest (N = 858 patients) integrative analysis of pediatric AML paired transcriptomes and methylomes to date, performed at diagnosis for patients enrolled on Children's Oncology Group clinical trials AAML0531 (NCT00372593) or AAML1031 (NCT01371981). We also analyzed N = 400 and 253 corresponding transcriptomes and methylomes at relapse, respectively. Additionally, we performed comprehensive mutation and fusion calling from NGS data and integrated clinical covariates and outcomes. This robust dataset allowed us to uncover how pediatric AML epigenetics are perturbed from normal hematopoiesis, characterize the evolution of transcriptional and methylation changes from leukemia diagnosis to relapse, and identify more accurate relapse prediction models for pediatric AML. We initially performed unbiased hierarchical clustering on CpG sites that are differentially methylated between AML and normal bone marrow to identify distinct AML-restricted methylation (ARM) signatures. This analysis demonstrated that that these ARM clusters were enriched in specific cytomolecular subtypes of pediatric AML. Specifically, we identified methylation signatures that recapitulated their adult counterparts (e.g., IDH1/2, DNMT3A, WT1) and pediatric specific methylation signatures that clustered with RUNX1::RUNX1T1, CBFB::MYH11, KMT2A-rearrangement, NUP98::NSD1, NUP98::KDM5A, and CEBPA mutant AML. Next, we investigated the transcriptional and methylation signatures for the relapsed cases and compared them to diagnosis. We demonstrated that relapsed transcriptional and methylation signatures clustered within their cytomolecular subtype counterparts at diagnosis (i.e., AMLs at relapse more closely resemble AMLs at diagnosis with the same underlying gene fusion than other relapsed AMLs) ( Fig. 1A). However, analyzing our data in the context of these cytomolecular subtypes, we found that leukemias uniformly acquired a more stem-like state at relapse. We focused our biomarker discovery efforts on KMT2A-rearranged AMLs since (as a cohort) these patients have an intermediate risk of relapse and existing molecular biomarkers (e.g., KMT2A fusion partner and measurable residual disease detection) result in only modest improvements in relapse prediction. First, we found that CpG islands associated with CD34 and HOXA gene expression were frequently (but variably) hypermethylated, which was also associated with downregulation of CD34 and HOXA gene expression and favorable survival outcomes. Next, we performed non-negative matrix factorization on diagnosis and relapsed KMT2A-rearranged AMLs and identified a novel gene set associated with relapse and poor outcomes. These three distinct methylation-transcriptional findings (i.e., CD34, HOXA, and relapsed signatures) were subsequently transformed into a novel methylation-transcription biomarker score (MTBS) using machine learning algorithms (i.e., LASSO Cox regression). We used KMT2A-rearranged AML patients from AAML1031 as our training cohort (N = 129) to generate a MTBS that was able to distinguish KMT2A-rearranged cases with disparate outcomes. This algorithm was validated in an independent cohort from AAML0531 (N = 28) ( Fig. 1B). Additional methylation-transcriptional biomarker discovery efforts for pediatric AML are underway. Since pediatric AML is associated with a low mutational burden, understanding leukemia-specific transcriptional and epigenetic alterations is of utmost importance. Our study demonstrates the translational promise offered by deep functional genomic characterization of pediatric AML and we believe that further interrogation may provide additional novel insights into leukemia initiation, relapse prediction, and novel treatment approaches.

The Combination of Lanraplenib, a Selective SYK Inhibitor, and Gilteritinib, a FLT3 Inhibitor, Targets Aberrant Proliferation and Differentiation Blockade in Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is an aggressive disease characterized by uncontrolled clonal proliferation of poorly differentiated myeloid progenitor cells in the bone marrow and blood. Deregulated spleen tyrosine kinase (SYK) impairs myeloid differentiation and has been implicated in the pathogenesis of AML driven by FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutation (FLT3-ITD). While SYK is rarely mutated, it is highly activated in FLT3-ITD-mutated AML and directly phosphorylates FLT3-ITD, resulting in aberrant expression of multiple oncogenic pathways. Lanraplenib (LANRA) is a selective, next-generation, SYK inhibitor that has shown favorable PK, PD, and safety in healthy volunteers and patients with autoimmune diseases. LANRA is currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). By jointly inhibiting the leukemogenic network created by SYK and mutant FLT3, LANRA and gilteritinib potentially provide a new treatment regimen in a patient population with few treatment options. Method: AML cell viability in response to LANRA, gilteritinib or their combination was assessed by CellTiter Glo (CTG) at 5 days. Apoptosis was evaluated by measuring Annexin V and propidium iodide staining by flow cytometry. Myeloid differentiation was assessed by flow cytometry using CD11b and CD14 cell surface markers and/or immunohistochemistry. In addition, RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. In vivo studies were conducted using the MV4;11 cell line-derived xenograft model. LANRA was administered twice daily at a dose of 75 mg/kg via subcutaneous injections, either alone or in combination with gilteritinib at a dose of 2 mg/kg dosed once daily by oral gavage, for 28 days. Results: Treatment of FLT3-ITD mutated AML cells with LANRA results in dose- and time-dependent increase in myeloid differentiation and reductions in leukemic cell viability. RNA-seq analysis of MV4;11 cells revealed that LANRA abrogates multiple downstream FLT3-ITD leukemogenic signaling pathways including JAK/STAT3/5, MAPK, mTOR and OXPHOS. Combined pharmacologic inhibition of SYK with LANRA and FLT3-ITD with gilteritinib results in robust antileukemic effects in preclinical models of FLT3 ITD-driven AML. Additionally, the combination of LANRA and gilteritinib demonstrated increased differentiation and apoptosis induction in vitro. LANRA in combination with gilteritinib showed significant tumor growth inhibition compared to either single agent in a subcutaneous FLT3-ITD AML cell line-derived xenograft mouse model. In line with these observations, a retrospective analysis of bone marrow-engrafted AML cells from a disseminated FLT3-ITD AML patient-derived xenograft mouse model showed evidence of increased myeloid differentiation and inhibition of cell proliferation. Conclusion: In preclinical models, LANRA demonstrates compelling anti-leukemic activity in combination with gilteritinib. By jointly targeting hyperproliferation and differentiation blockade, LANRA and gilteritinib may provide a new therapeutic approach that can be exploited in AML. Additionally, LANRA disrupts downstream signaling of key pathways often associated with resistance to gilteritinib, providing additional mechanistic support for continued testing of LANRA in combination with gilteritinib in FLT3-ITD-mutated AML patients. These studies helped motivate the ongoing clinical combination with gilteritinib in patients with (R/R) FLT3-mutated AML (NCT05028751).

NUP98-Rearranged Acute Myeloid Leukemia Stem Cells Superimpose Type I Interferon Signaling with Canonical Self-Renewal Programs

The clinical outcomes of pediatric acute myeloid leukemia (AML) have shown improvement with combination therapies; however, relapse remains a significant concern due to the resistance of leukemia cells to conventional treatments. In some cases, epigenetic heterogeneity within leukemias can contribute to therapy resistance. For example, leukemia stem cells (LSCs) have been shown maintain AML, both before and after therapy. This study aims to identify LSCs within a mouse model of high-risk, NUP98-rearranged pediatric AML, and to identify mechanisms that sustain this population. To identify and isolate a LSC subpopulation, we characterized murine NUP98-HOXD13/Flt3-ITD-driven AML in mice by performing cellular indexing of transcriptomes and epitopes (CITE-seq) analysis at two stages: pre-AML and fully transformed AML. Uniform Manifold Approximation and Projection (UMAP) revealed a highly reproducible pattern of heterogeneity within each AML specimen. Each AML had a small percentage of cells that resembled normal and pre-leukemic multipotent progenitors (MPPs), as well as additional populations with B-cell or myeloid gene expression. Pseudotime analysis indicated a trajectory from MPPs to mature cell-like lineages, implicating the MPP-like population as a potential LSC. This population expressed type I interferon (IFN-1) target genes more highly than non-LSC AML cells and pre-AML cells. This finding is significant because we previously identified IFN-1 as an effector of Flt3-ITD/NUP98-HOXD13 synergy during AML initiation, and in human NUP98-rearranged AML (PMID: 36395068). Differential gene expression analysis also identified several established self-renewal genes, including Hlf, Myct1, Mpl, Slc7a8, and Gata2, as highly expressed in putative LSCs relative to non-LSCs (figure B). This suggests that AML LSCs superimpose inflammatory signaling programs, particularly IFN-1, and core stem cell self-renewal programs to maintain a malignant state. The data raise the question of how IFN-1 shapes LSC identity. We next sought to isolate and validate the putative LSC population. CITE-seq feature barcode data indicated high expression of CD117 and CD48, medium expression of B220, and low expression of Gr1 and CD11B in putative LSCs. We isolated LSCs based on these markers (Gr1-CD11b-B220 MidCD117 highCD48+). Several non-LSC populations were isolated for comparison (e.g., Gr1+CD11B+ and Gr1-CD11B-B220 highCD117 Mid populations. Cytospin observation revealed no morphological differences among the three populations, but cell culture assays showed that only the Gr1-CD11b-B220 MidCD117 highCD48+ cells exhibited proliferative capacity after multiple passages. Limit dilution transplant assays showed that the Gr1-CD11b-B220 MidCD117 highCD48+ population was greatly enriched for LSC function relative to whole bone marrow (figure A). Altogether, we have shown that we can prospectively isolate a LSC population from NUP98-HOXD13/Flt3-ITD AML. The LSC population is highly reproducible across specimens and it demonstrates high degrees of IFN-1 activity. Further work will establish the role of IFN-1/self-renewal gene interactions in LSC maintenance.

Improvement in Survival of Patients with FLT3 Mutated Acute Myeloid Leukemia: Results from a Retrospective Canadian Cohort

Introduction: FLT3 internal tandem duplication (ITD) mutations have been historically associated with inferior outcomes in patients with acute myeloid leukemia (AML) and an intermediate risk status as per the European LeukemiaNet (ELN) 2022 guidelines. In 2017, the US FDA granted approval to midostaurin for frontline treatment and a year later, gilteritinib for use in relapsed/refractory (R/R) AML. These agents began to be widely used in our provincial leukemia program in early 2018 as per the label indications. In this study, we compared clinical outcomes for patients with FLT3-ITD mutated AML before and after this change in British Columbia. Methods: We identified patients in our program database diagnosed with FLT3-ITD mutated AML from January 1, 2010 to December 31, 2021. Only patients receiving intensive chemotherapy with cytarabine and anthracycline were included. Patients were divided into two eras: 2010-2017 (era 1) and 2018-2021 (era 2). The two eras were compared in terms of patient characteristics, genetics, rate of complete remission (CR) and CR with incomplete count recovery (CRi), receipt of allogeneic stem cell transplant (alloSCT) and the overall and relapse free survival (OS and RFS). OS was calculated from diagnosis to death from any cause and was not censored at the time of alloSCT. RFS was calculated from CR/CRi to relapse. Patients without an event during the study period were censored at the time of last follow-up. Categorical variables were compared using Chi-square and Fisher exact tests and continuous variable were compared using paired T tests. Kaplan-Meier survival method and log rank test were used to estimate and compare survival. Uni- and multivariate analyses were performed in a Cox proportional regression model and alloSCT was considered as a time dependent covariate. Results: 169 patients were included (era 1=101, era 2=68). The median duration of follow up of the entire cohort was 17 months (range 0.5-148.6 months) and follow-up of surviving patients (n=70) was longer in era 1 vs. era 2 (median 88 months vs. 32 months, p&amp;lt;0.001). The median age of the entire cohort was 60 years (range 26-79 years) and it was comparable in both eras. Most patients had normal cytogenetics (era 1: 76%; era 2: 69%). NPM1 was mutated in 69/101 (68%) and 38/68 (56%) patients in eras 1 and 2 respectively. Due to lack of genetic data for era 1, we did not risk stratify based on the recent ELN classification. The rate of CR/CRi was 58/101 (57%) vs. 40/68 (59%) after induction chemotherapy and 78/101 (77%) vs. 58/67 (87%) (p=0.09) at any time in eras 1 and 2 respectively. AlloSCT was performed in 58% and 72% of patients in the two cohorts. Midostaurin was used in 82% and gilteritinib was used in 26% of era 2 patients in the second era. OS was significantly longer in era 2 (median OS 73 months vs. 11 months, HR (death) 6.2, p=0.012) (Figure). The 2-year OS rate was higher in era 2 (57%) than in era 1 (39%). In patients achieving CR/CRi, median RFS was longer in era 2 (51 vs. 16 months, p=0.43), although this was not statistically significant. In univariate Cox regression analysis the following factors were associated with improved OS: use of midostaurin (HR 0.487, 95% CI 0.304-0.779, p=0.003), diagnosis in era 2 (HR 0.583, 95% CI 0.38-0.895, p=0.014) and receipt of alloSCT (HR 0.346,) 95% CI 0.219-0.545, p&amp;lt;0.001). Other parameters including age, sex, NPM1 status and cytogenetics were not significant. In multivariate analysis including era, midostaurin and alloSCT, only the receipt of alloSCT was associated with significantly improved OS (HR 0.362, 95% CI 0.229 - 0.572, p&amp;lt;0.001) whereas use of midostaurin (HR=0.465, CI 0.207-1.042, p=0.063) and era (HR 1.12, 95% CI 0.538-2.34, p=0.76) were not significant. Conclusions: Our results show thatOS for patients with FLT3-ITD AML has improved in patients diagnosed in a more recent treatment era. Patients diagnosed after 2018 were more frequently treated with AlloSCT and FLT3-inhibitors, which may have resulted in improvements in OS. Our findings support the assignment of FLT3-ITD AML to theintermediate risk category as per the current ELN guidelines. A limitation of this study is the differences in follow-up time between eras, which may influence the event rate between the groups.

Metformin-Induced Ferroptosis Is a Therapeutic Vulnerability in IDH2-Mutant AML Linked to Metabolic Rewiring Towards Fatty Acid Oxidation

Metabolic rewiring is an essential feature of leukemic cells to sustain tumorigenesis and is largely influenced by mutational status. We previously demonstrated this phenomenon in FLT3-ITD acute myeloid leukemia (AML) upon combined inhibition of complex II activity and lactate import (Erdem et al., 2022). Mutations in isocitrate dehydrogenase ( IDH1/2 mut) occur in about 20% of AML cases, and despite the emergence of targeted therapies, patients often relapse. The molecular and epigenetic outcomes of 2-R-hydroxyglutarate accumulation and competitive inhibition of histone demethylases have been extensively studied in IDH1/2 mut AMLs, but the metabolic consequences are still largely unexplored. Here, we applied multi-omics studies on cell line models and primary AML samples combined with functional studies to unravel the metabolic rewiring of IDH1/2 mut AMLs. Transcriptional characterization of isogenic TF1 wt and IDH2 R140Q cell lines (10978 genes) revealed increased expression of CD36, a major fatty acid (FA) transporter, and gene set enrichment analysis (GSEA) associated IDH2 R140Q cells with terms related to FA processes and activity of mitochondrial complex I. Quantitative metabolome analysis of TF1 wt and IDH2 R140Q (180 metabolites) revealed lower levels of acylcarnitines of variable carbon lengths, suggesting increased FA oxidation, and upregulation of glycerophospholipid and glycerolipid metabolism in IDH2 R140Q cells. Next, we validated our findings by performing a metabolomic profiling (172 metabolites) on primary AML samples (n=26, including 4 IDH1 mut and 2 IDH2 mut), for which label-free quantitative proteome data (11272 proteins) was also generated. Proteome analysis confirmed increased CD36 expression and enrichment for FA and mitochondrial processes in IDH1/2 mut patients. In line, metabolome data also revealed increased oxidation of branched-chain FAs in this sample group. Altogether, these findings suggested a profound disturbance in lipid metabolism and supported the notion that IDH1/2 mutant cells rely on mitochondrial respiration, whereby FAs seem to be the preferred carbon source. Next, we performed an in vitro drug screen targeting the main metabolic pathways, which revealed increased sensitivity of IDH2 R140Q cells to the FDA-approved complex I inhibitor metformin. Extracellular flux analysis indicated no significant difference in the oxygen consumption rate (OCR) between TF1 wt and IDH2 R140Q upon metformin treatment, while the viability of mutant cells was significantly diminished. Contrarily, the basal extracellular acidification rate (ECAR) was increased in TF1 wt cells upon complex I inhibition, suggesting that these cells rewire their metabolism towards glycolysis more efficiently than mutant cells. Furthermore, we knocked down CD36 expression to investigate whether disrupting lipid metabolism in IDH2 R140Q cells would constitute a metabolic vulnerability. Notably, CD36 knockdown resulted in increased resistance to metformin in IDH2 R140Q cells. These data suggested that enhanced lipid uptake mediated by CD36 in IDH2 R140Q cells not only boosts OCR but may also disrupt lipid homeostasis, causing cells to become more susceptible to lipid peroxidation. Since the role of metformin-induced ferroptosis in AML is still unclear, we performed an RNA-seq analysis on TF1 wt and IDH2 R140Q cells treated with metformin (5 mM) for 72 hours (10326 genes). Single-sample GSEA associated the transcriptome of metformin-treated cells with ferroptosis signatures in both cell lines but to a significantly higher extent in IDH2 R140Q. We functionally validated this finding by using the BODIPY C11 probe, a lipid peroxidation sensor. After 24h of metformin treatment, both TF1 wt and IDH2 R140Q displayed increased lipid ROS. Lipid peroxidation levels were further enhanced by combining metformin with palmitate, a saturated FA, supporting the notion that elevated lipid availability and uptake may increase cell death via ferroptosis. Altogether, we identified a new metabolic vulnerability in IDH1/2 mut AMLs associated with a profound disturbance in lipid metabolism. Moreover, we show that treatment with metformin not only inhibits complex I but also induces cell death via ferroptosis in IDH2 mut cells providing an alternative treatment option in combination with available IDH2 mut targeted therapies for this subgroup of patients.

PHI-101 As a Potent Next-Generation FLT3 Inhibitor, Overcome Resistances in Previously Treated Patients with FLT3-ITD or TKD Acute Myeloid Leukemia: Results of a Phase Ia/Ib Clinical Trial

Background: The FLT3 receptor is mutated in about 30% of acute myeloid leukemia (AML) patients (pts) and has proven to be an attractive therapeutic target. The most prevalent alteration is internal tandem duplication (ITD) of the juxtamembrane domain, and several FLT3 inhibitors (e.g. gilteritinib, quizartinib, midostaurin) succeddfully target this mutation. However, patients frequently relapse due to resistance to these FLT3 inhibitors after initial clinical responses, often caused by the secondary mutations observed in the kinase domain (TKD) region. PHI-101, an orally bioavailable novel small molecule, is a next-generation FLT3 inhibitor that shows a potent anti-leukemic activity in primary AML samples harboring several FLT3-resistance mutations in preclinical studies. Global Phase I clinical trials with PHI-101 (NCT04842370) are currently underway in refractory or relapsed (R/R) AML pts. Study Design and Methods: The open-label, multicenter AML clinical trial consists of phase Ia, dose-escalation, and phase Ib, dose-expansion, utilizing oral PHI-101 tablets as a single-agent with R/R AML pts. If dose-escalation concludes with no dose-limiting toxicities (DLT), dose-expansion proceeds upon recommendation from the Safety Monitoring Committee (SMC). Response assessments with PHI-101 were reported according to 2017 ELN guidelines. The variant allelic frequency of mutations of FLT3 and other AML-related mutations was analyzed using NGS with bone marrow aspirates collected from pts before and after PHI-101 treatment. Blood samples were collected to evaluate pharmacokinetics (PK) and the plasma inhibitory assay (PIA) using FLT3-mutant cell lines. Together, these assessments were considered to determine the recommended dose for expansion (RDE) in phase Ib trials. Results: Phase Ia clinical trials of PHI-101 was designed to evaluate for safety and tolerability at five dose levels ranging from 40 mg to 200 mg. A total of 13 R/R AML pts were enrolled in the phase Ia and no DLTs were reported from daily doses of PHI-101 during the 28-day cycle. The target RDE at 160 mg was determined based on several considerations, including the PIA test achieving &amp;gt;85% inhibition of phosphor-FLT3, all available adverse events, PK data, and recommendation by the SMC. As of May 2023, with 6 pts enrolled in phase Ib, a total of 19 pts (5 FLT3-wild type (26.3%) and 14 FLT3 mutated (73.7%)), have been treated with PHI-101. Thirteen pts (68.4%) had more than 3 prior anti-leukemic treatments. Twelve of 13 pts (92%) assessed for the leukemic blast evaluation achieved clinical responses-4 pts with composite complete remissions [CRc, including complete remission with incomplete hematological recovery (CRi) and morphological leukemia free state (MLFS)] within 1 cycle of PHI-101 treatment. Ten pts were R/R following previous treatment with other FLT3 inhibitors (gilteritinib, quizartinib, midostaurin, or HM43239). 88 % of these pts achieved clinical responses including 3 CRc and 1 partial response (PR). Notably, all 5 R/R pts after gilteritinib treatment had clinical responses that included 2 with CRc. In addition, a patient with both FLT3-ITD and TKD (D835E and N676K) mutations who was resistant to 3 different FLT3 inhibitor treatments achieved PR and continued PHI-101 treatment for 6 cycles. Among pts who received 160 mg of PHI-101, 4 pts (57.1%) responded: 2 (28.6%) achieved CRi, 1(14.3%) achieved MLFS, and 1 (14.3%) achieved PR. Conclusion: In dose-escalating phase Ia clinical trials, PHI-101 was well tolerated at all dose levels with no DLTs. Phase Ib dose-expansion trials with 160 mg daily dosing are currently ongoing. PHI-101 has delivered CRcs at 120 mg and 160 mg. In this first-in-human, phase Ia/Ib trial of PHI-101, 92% of FLT3-wild or -mutated AML pts showed clinical responses even after having previously been treated with otherFLT3 inhibitors. More notably, 100% of pts who had relapsed after prior gilteritinib treatment showed clinical responses. The data from the clinical study of PHI-101to date demonstrates significant efficacy, particularly in R/R FLT3 mutant AML pts.

UBE2N Regulates Oncoprotein Networks in Myeloid Malignancies

Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) originate from clonal mutant hematopoietic stem and progenitor cells, referred to as leukemic stem and progenitor cells (LSPCs). Ubiquitination is an essential process for various cellular maintenance functions, including protein degradation and scaffolding, which is often perturbed in a variety of cancers. Ubiquitination of protein substrates involves three related enzymes: the E1 activating enzyme, the E2 conjugating enzyme, and the E3 ligase. In humans, there are up to 40 E2 conjugating enzymes, among which Ubiquitin conjugating enzyme E2 N (UBE2N) stands out as one of the most crucial and overexpressed E2 enzymes in AML cells. Our recent study reported that UBE2N is a druggable target in MDS and AML (Barreyro et al., Science Translational Medicine, 2022), and we found that its catalytic active site cysteine 87 is required for various AML mouse models, including MLL-AF9, FLT3-ITD, AML1-ETO9a, and MN1 (unpublished). These observations indicate that the catalytic function of UBE2N is critical for AML cells but dispensable for normal hematopoiesis. To examine the active state of UBE2N, we investigated the “charged” version of UBE2N, which contains a ubiquitin bound to cysteine 87, in normal hematopoietic cells and in a AML cell line panels. Our findings confirmed that the activated form of UBE2N is highly expressed in human AML cells compared to normal hematopoietic cells. UBE2N exclusively synthesizes Lysine-63 (K63)-linked polyubiquitin chains on target proteins in conjunction with selected E3 ligases, leading to the stabilization and/or activation of protein substrates. To identify the relevant targets of UBE2N in AML, we conducted a ubiquitin-enrichment followed by a proteomic analysis in isogenic UBE2N-deficient AML cells compared to WT AML cells. We identified ~300 proteins that are ubiquitinated by UBE2N. Importantly, UBE2N regulates a network of leukemia-associated oncoproteins, including SYNCRIP, HUWE1, NPM1, TYK2, IKBKG, VAV1, BAX, IGF2BP2, IRF4, IRAK4, RBMX, STAT3, and BTK. UBE2N can interact with various E3 ligases in a cell- and context-dependent manner. To gain insight into the major E3 ligase that cooperates with activated UBE2N in AML, we performed a proximity-based labeling assay followed by mass spectrometry. This interactome screen identified Tripartite motif containing 21 (TRIM21) as the top E3 ligase that partners with UBE2N in AML cells. TRIM21 is overexpressed in AML patients compared to healthy controls. Moreover, deletion of TRIM21 in AML cells impaired cell viability and proliferation in vitro and prolonged survival in a xenograft model in vivo, which phenocopies the loss of UBE2N. Importantly, restoration of TRIM21 rescued the functional defect of UBE2N-deficient AML cells, indicating that the UBE2N-TRIM21 axis is operational in AML. To prioritize the UBE2N-dependent oncoprotein network, we focused on UBE2N-TRIM21 regulated signaling in AML. For this, a gene expression analysis was performed in UBE2N-deficient AML cells that were restored with TRIM21. The top UBE2N-TRIM21 oncoprotein network that emerged was related to interferon-STAT signaling. Additionally, both UBE2N and TRIM21 were found to regulate STAT3 ubiquitination. Deletion or inhibition of UBE2N catalytic activity significantly reduced total and activated STAT3 in AML cells. Finally, the expression of constitutively active STAT3 partially restored the functional defect of AML cells treated with a UBE2N inhibitor, indicating that STAT3 function, a known driver of LSPCs, is regulated via the UBE2N-TRIM21 axis in AML. In summary, our data reveal that UBE2N regulates oncoprotein networks in AML and that inhibiting UBE2N catalytic function, such as with small molecule inhibitors, effectively suppresses LSPCs.