Acute Myeloid Leukemia Proliferation Research Articles

B7-H3 in acute myeloid leukemia: From prognostic biomarker to immunotherapeutic target.

B7-H3 (CD276), an immune checkpoint protein of the B7 family, exhibits significant upregulation in solid tumors and hematologic malignancies, exerting a crucial role in their pathophysiology. The distinct differential expression of B7-H3 between tumors and normal tissues and its multifaceted involvement in tumor pathogenesis position it as a promising therapeutic target for tumors. In the context of acute myeloid leukemia (AML), B7-H3 is prominently overexpressed and closely associated with unfavorable prognoses, yet it has remained understudied. Despite various ongoing clinical trials demonstrating the potential efficacy of immunotherapies targeting B7-H3, the precise underlying mechanisms responsible for B7-H3-mediated proliferation and immune evasion in AML remain enigmatic. In view of this, we comprehensively outline the current research progress concerning B7-H3 in AML, encompassing in-depth discussions on its structural attributes, receptor interactions, expression profiles, and biological significance in normal tissues and AML. Moreover, we delve into the protumor effects of B7-H3 in AML, examine the intricate mechanisms that underlie its function, and discuss the emerging application of B7-H3-targeted therapy in AML treatment. By juxtaposing B7-H3 with other molecules within the B7 family, this review emphasizes the distinctive advantages of B7-H3, not only as a valuable prognostic biomarker but also as a highly promising immunotherapeutic target in AML.

AC024896.1/miR-363-3p Axis Regulates the Malignant Progression of Acute Myeloid Leukemia by Cuproptosis-Related Gene MYO1B.

Acute myeloid leukemia (AML) is a hematological malignancy with poor patient prognosis. Cuprotosis is a newly discovered cell death that regulates the proliferation and progression of tumor cells. Long non-coding RNAs (lncRNAs) are key molecules and potential biomarkers for the diagnosis and treatment of various diseases. However, the effect of cuprotosis-associated lncRNAs on AML remains unclear. The aim of this study was to investigate the relationship between the expression of cuprotosis-related gene and the prognosis of AML. Consensus cluster analysis was performed on AML patients according to the cuprotosis-related gene expression matrix, and survival analysis and differential gene analysis were performed. Then lncRNA and miRNA related to AML tumor progression were screened according to univariate COX regression analysis. After that, Kaplan-Meier analysis, correlation analysis, and AUC curve were used to determine the ceRNA network that might regulate AML. The regulatory relationship of ceRNA was verified in AML cell lines by RT-qPCR and Western blotting. The AC024896.1/miR-363-3p axis drives MYO1B to promote the malignant progression of AML. First, a change in the expression of AC024896.1 and miR-363-3p can affect the proliferation of AML by regulating MYO1B. Mechanistically, AC024896.1 regulates the expression of MYO1B as the ceRNA of miR-363-3p. Moreover, the regulation of AC024896.1 in the malignant progression of AML depends partly on miR-363-3p. In summary, our study reveals AC024896.1/miR-363-3p/MYO1B Axis in AML, which can be regarded as a new potential target for the diagnosis and treatment of AML.

Super enhancer related gene ANP32B promotes the proliferation of acute myeloid leukemia by enhancing MYC through histone acetylation

BackgroundAcute myeloid leukemia (AML) is a malignancy of the hematopoietic system, and childhood AML accounts for about 20% of pediatric leukemia. ANP32B, an important nuclear protein associated with proliferation, has been found to regulate hematopoiesis and CML leukemogenesis by inhibiting p53 activity. However, recent study suggests that ANP32B exerts a suppressive effect on B-cell acute lymphoblastic leukemia (ALL) in mice by activating PU.1. Nevertheless, the precise underlying mechanism of ANP32B in AML remains elusive.ResultsSuper enhancer related gene ANP32B was significantly upregulated in AML patients. The expression of ANP32B exhibited a negative correlation with overall survival. Knocking down ANP32B suppressed the proliferation of AML cell lines MV4-11 and Kasumi-1, along with downregulation of C-MYC expression. Additionally, it led to a significant decrease in H3K27ac levels in AML cell lines. In vivo experiments further demonstrated that ANP32B knockdown effectively inhibited tumor growth.ConclusionsANP32B plays a significant role in promoting tumor proliferation in AML. The downregulation of ANP32B induces cell cycle arrest and promotes apoptosis in AML cell lines. Mechanistic analysis suggests that ANP32B may epigenetically regulate the expression of MYC through histone H3K27 acetylation. ANP32B could serve as a prognostic biomarker and potential therapeutic target for AML patients.

Enhanced Expression of Glycolytic Enzymes and Succinate Dehydrogenase Complex Flavoprotein Subunit A by Mesothelin Promotes Glycolysis and Mitochondrial Respiration in Myeloblasts of Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive malignancy characterized by rapid growth and uncontrolled proliferation of undifferentiated myeloid cells. Metabolic reprogramming is commonly observed in the bone marrow of AML patients, as leukemia cells require increased ATP supply to support disease progression. In this study, we examined the potential role of mesothelin as a metabolic modulator in myeloid cells in AML. Mesothelin is a well-known marker of solid tumors that promotes cancer cell proliferation and survival. We initially analyzed alterations in mesothelin expression in the myeloblast subpopulations, defined as SSC-Alow/CD45dim, obtained from the bone marrow of AML patients using flow cytometry. Our results showed overexpression of mesothelin in 34.8% of AML patients. Subsequently, metabolic changes in leukemia cells were evaluated by comparing the oxygen consumption rates (OCR) of bone marrow samples derived from adult AML patients. Notably, a higher OCR was observed in the mesothelin-positive compared to the mesothelin-low and non-expressing groups. Treatment with recombinant human mesothelin protein enhanced OCR and increased the mRNA expression of glycolytic enzymes and mitochondrial complex II in KG1α AML cells. Notably, siRNA targeting mesothelin in KG1α cells led to the reduction of glycolysis-related gene expression but had no effect on the mitochondrial complex gene. The collective results demonstrate that mesothelin induces metabolic changes in leukemia cells, facilitating the acquisition of a rapid supply of ATP for proliferation in AML. Therefore, the targeting of mesothelin presents a potentially promising approach to mitigating the progression of AML through the inhibition of glycolysis and mitochondrial respiration in myeloid cells.

Inhibitory Effect of Metformin and Arsenic Trioxide on KG1a Cell Proliferation

To investigate the effect of metformin and arsenic trioxide on KG1a cells proliferation of acute myeloid leukemia and its possible mechanism. CCK-8 method was used to detect the killing effect of metformin, arsenic trioxide and combined application on KG1a cells. Annexin V-FITC/PI Dual Stain Flow Cytometry was used to detect the effect of combined application on apoptosis of KG1a cells. Western blot was used to detect the expression of intracellular apoptosis-,autophagy-related protein. Metformin and arsenic trioxide alone or in combination could inhibit the proliferation of KG1a cells and induce apoptosis of KG1a cells, and the proliferation inhibition rate and apoptosis rate in the combined drug group were higher than those in the drug group alone(P <0.05). The combination of drugs induced upregulation of Caspase 8 protein and P62 protein expression and was higher than that in the drug group alone(P <0.05). Metformin can synergize with arsenic trioxide to kill KG1a cells, and its mechanism of action may be related to inducing apoptosis and enhancing autophagy.

Histone lysine demethylase KDM5B facilitates proliferation and suppresses apoptosis in human acute myeloid leukemia cells through the miR-140-3p/BCL2 axis.

The histone lysine demethylase KDM5B is frequently up-regulated in various human cancer cells. However, its expression and functional role in human acute myeloid leukemia (AML) cells remain unclear. Here, we found that the expression level of KDM5B is high in primary human AML cells. We have demonstrated that knocking down KDM5B leads to apoptosis and impairs proliferation in primary human AML and some human AML cell lines. We further identified miR-140-3p as a downstream target gene of KDM5B. KDM5B expression was inversely correlated with the miR-140-3p level in primary human AML cells. Molecular studies showed that silencing KDM5B enhanced H3K4 trimethylation (H3K4me3) at the promoter of miR-140-3p, leading to high expression of miR-140-3p, which in turn inhibited B-cell CLL/lymphoma 2 (BCL2) expression. Finally, we demonstrate that the defective proliferation induced by KDM5B knockdown (KD) can be rescued with the miR-140-3p inhibitor or enhanced by combining KDM5B KD with a BCL2 inhibitor. Altogether, our data support the conclusion that KDM5B promotes tumorigenesis in human AML cells through the miR-140-3p/BCL2 axis. Targeting the KDM5B/miR-140-3p/BCL2 pathway may hold therapeutic promise for treating human AML.

Discovery of Novel Phenoxyaryl Pyridones as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with High Selectivity for the Second Bromodomain (BD2) to Potentially Treat Acute Myeloid Leukemia.

Bromodomain-selective BET inhibition has emerged as a promising strategy to improve the safety profiles of pan-BET inhibitors. Herein, we report the discovery of potent phenoxyaryl pyridones as highly BD2-selective BET inhibitors. Compound 23 (IC50 = 2.9 nM) exhibited a comparable BRD4 BD2 inhibitory activity relative to 10 (IC50 = 1.0 nM) and remarkably improved selectivity over BRD4 BD1 (23: 2583-fold; 10: 344-fold). This lead compound significantly inhibited the proliferation of acute myeloid leukemia (AML) cell lines through induction of G0/G1 arrest and apoptosis in vitro. Excellent in vivo antitumor efficacy with 23 was achieved in an MV;411 mouse xenograft model. Pleasingly, compound 23 (hERG IC50 > 30 μM) mitigated the inhibition of the human ether-à-go-go-related gene (hERG) ion channel compared with 10 (hERG IC50 = 2.8 μM). This work provides a promising BD2-selective lead for the development of more effective and safe BET inhibitors as anticancer agents.

Discovery of a PROTAC degrader for METTL3-METTL14 complex

N6-methyladenosine (m6A) methylation is the most abundant type of RNA modification that is mainly catalyzed by the METTL3-METTL14 methyltransferase complex. This complex has been linked to multiple cancers and is considered a promising therapeutic target for acute myeloid leukemia (AML). However, only a few METTL3 inhibitors targeting the catalytic activity were developed recently. Here, we present the discovery of WD6305 as the potent and selective proteolysis-targeting chimera (PROTAC) degrader of METTL3-METTL14 complex. WD6305 suppresses m6A modification and the proliferation of AML cells, and promotes apoptosis much more effectively than its parent inhibitor. WD6305 also affects a variety of signaling pathways related to the development and proliferation of AML. Collectively, our study reveals PROTAC degradation of METTL3-METTL14 complex as a potential anti-leukemic strategy and provides desirable chemical tool for further understanding METTL3-METTL14 protein functions.

LILRB3 Modulates Acute Myeloid Leukemia Progression and Acts as an Effective Target for CAR T-cell Therapy

Abstract Identifying novel cell surface receptors that regulate leukemia cell differentiation and can be targeted to inhibit cellular proliferation is crucial to improve current treatment modalities in acute myeloid leukemia (AML), especially for relapsed or chemotherapy-refractory leukemia. Leukocyte immunoglobulin-like receptor type B (LILRB) is an immunomodulatory receptor originally found to be expressed in myeloid cells. In this study, we found that LILRB receptors can be induced under inflammatory stimuli and chemotherapy treatment conditions. Blockade of LILRB3 inhibited leukemia cell proliferation and leukemia progression. In addition, treatment with LILRB3 blocking antibodies upregulated myeloid lineage differentiation transcription factors, including PU.1, C/EBP family, and IRF, whereas phosphorylation of proliferation regulators, for example, AKT, cyclin D1, and retinoblastoma protein, was decreased. Conversely, transcriptomic analysis showed LILRB3 activation by agonist antibodies may enhance leukemia survival through upregulation of cholesterol metabolism, which has been shown to promote leukemia cell survival. Moreover, LILRB3-targeted CAR T cells exhibited potent antitumor effects both in vitro and in vivo. Taken together, our results suggest that LILRB3 is a potentially potent target for multiple treatment modalities in AML. Significance: LILRB3 regulates differentiation and proliferation in acute myeloid leukemia and can be targeted with monoclonal antibodies and CAR T cells to suppress leukemia growth.

Tuspetinib Myeloid Kinase Inhibitor Safety and Efficacy As Monotherapy and Combined with Venetoclax in Phase 1/2 Trial of Patients with Relapsed or Refractory (R/R) Acute Myeloid Leukemia (AML)

INTRODUCTION: Tuspetinib is a potent once daily oral myeloid kinase inhibitor of SYK, FLT3, JAK1/2, RSK1/2, mutant KIT, and TAK1-TAB1 kinases that mediate dysregulated cellular proliferation in AML. In an orthotopic mouse model of FLT3-mutant AML, tuspetinib exhibited greater antitumor activity than gilteritinib, entospletinib, venetoclax (VEN), and azacitidine (AZA) and combined favorably with VEN and AZA individually. Tuspetinib is being evaluated clinically as monotherapy (TUS) and in combination (VEN/TUS) in a global Phase 1/2 trial of patients with R/R AML. AIMS: Study objectives are to assess the safety, tolerability, and pharmacokinetics (PK) of TUS and VEN/TUS, to support selection of a recommended phase 2 dose (RP2D). METHODS: Dose escalation (Part A) and exploration (Part B) of single agent TUS have completed dosing of patients treated at 20 mg to 200 mg once daily. In dose expansion (Part C), 80 mg of TUS is administered continuously as once daily oral monotherapy in 28-day cycles, or in combination with daily VEN with a C1D15 marrow assessment and planned 28-day cycles. Treatment emergent adverse events (TEAE) and clinical responses are evaluated using CTCAE and Revised IWG criteria, respectively. RESULTS: As of May 30, 2023, 100 patients received TUS in Parts A/B (20mg to 200mg; n=77) and in Part C (n=23: TUS 120 mg (n=14) and TUS 80 mg + VEN 400 mg dose equivalent (n=9)). Patients are predominantly Asian (46%) and White (41%) with a median age of 63 (range 18 to 84); 60% FLT3 wild-type (FLT3-WT), 35% FLT3-mutated (FLT3m) of which 18 (51%) received prior FLT3 inhibitors, and 5% FLT3 undetermined. Patients received a mean of 2.6 prior lines of therapy (range 1-6) (Table 1). Sixty percent of patients have received prior venetoclax. In Parts A/B, clinical responses with TUS monotherapy (composite complete remission [CRc; including CR, CRh, CRp and CRi]; see Figure 1 for definitions), were observed at 40 mg (15.4%), 80 mg (29.4%), 120 mg (7.1%), and 160 mg (16.7%) in both FLT3-WT (n=33, CRc:15.2%,) and FLT3-mutated (n=23, CRc:21.7%) patients. Overall response rate (ORR, including CRc and PR) was 14.3% in prior VEN treated patients (n=28), 23.1% in FLT3-mutated patients with previous FLT3i treatment (n=13), and 66.7% in NPM1 and FLT3-mutated patients (n=6). Notably, responses were also seen in patients with TP53 (n=5; CRc:40%) and RAS (n=9; CRc:22.2%) mutations frequently associated with resistance to VEN and standard therapies. TUS was well tolerated, with no dose limiting toxicity (DLT) observed at doses ranging from 20 mg to 160 mg. One patient experienced a DLT (Grade 3 muscular weakness) at 200 mg which demonstrated partial improvement at the time of treatment discontinuation (9 days after AE onset). Related TEAEs of Grade ≥3 were low (10.4%) with only neutrophil count decreased and muscular weakness (n=2 each; 2.6%) reported in more than 1 patient. No treatment-related non-hematologic SAEs, deaths, or discontinuations were observed. PK exposure was linear (20 mg-160 mg). The RP2D of 80 mg was selected for TUS monotherapy. In Part C, all 9 patients receiving VEN/TUS have tolerated treatment to date and remain on study. Eight (89%) patients had prior VEN treatment. Preliminary responses have been observed at the first post-dose bone marrow assessment (C1D15), including patients with prior VEN treatment. No treatment-related SAEs or TEAEs of Grade ≥3 have been reported. Updated enrollment, safety, PK, and response data will be presented at the meeting along with findings on the association between somatic mutations and TUS sensitivity as well as results for the TUS food-effect absorption study. CONCLUSIONS: Tuspetinib was well-tolerated and delivered single agent clinical responses across four dose levels among diverse AML genotypes; with a RP2D of 80 mg chosen for future single agent studies. Although early, the VEN/TUS combination has been well tolerated with preliminary objective responses noted.

Addiction to Small Gtpase ARF6-Mediated Sphingolipid Homeostasis By AML but Not the Host

Background Cancer cells' unique metabolic demand has emerged as a promising therapeutic target in Acute Myeloid Leukemia (AML). The vesicular trafficking and signaling gatekeeper, ADP-Ribosylation Factor 6 (ARF6), belongs to the RAS superfamily of small GTPases and has been shown to regulate the homeostasis of lipids such as PIP2, phosphatidic acids, and cholesterol. Here we describe AML cells' unusual addiction to ARF6-mediated sphingolipid homeostasis and its clinical significance. Experiments and Results ARF6 knockdown (KD) or CRISPR-mediated ARF6 knockout (KO) inhibited the proliferation of AML cell lines, THP-1 and MV4-11, as well as the colony formation of primary AML CD34 + cells. Surprisingly, Tamoxifen-induced whole body KO of Arf6 in adult Arf6 f/f;Rosa-CreERT 2 mice led to healthy and grossly normal mice with a normal lifespan. Adult mice lacking ARF6 show no abnormality in complete blood count, urine test, or histology. Hence, ARF6 is genetically indispensable for AML, but not for the host. To confirm AML's addition to ARF6 in vivo, we introduced Arf6 KO in an MLL-AF9-induced AML model in C57BL6/J mice. We are currently following up with the disease progression between Arf6-KO AML group and control AML group, and expect to obtain results before the 2023 ASH meeting. After discovering that ARF6 is genetically necessary for AML but not needed for the host, we analyzed its clinical significance. Since ARF6 mutations are not detected in AML samples, we focused on analyzing its expression. With univariate analysis, we show that high ARF6 expression (top 50%) is significantly associated with worse overall survival (OS) and worse disease-free survival (DFS) of AML patients in the TCGA-NEJM-2013 (TCGA) cohort (N=200) and the Münchner Leukämielabor (MLL) cohort (N=579). In multi-variate analysis, high ARF6 expression is significantly associated with worse OS in the BeatAML cohort (N=562) and shows a trend towards worse OS and DFS in the TCGA cohort. Following a hypothesis-driven approach, we investigated the role of ARF6 in regulating sphingolipid homeostasis by HPLC-MS lipidomic analyses of AML cells (THP-1) with or without ARF6 depletion. Lipidomic profiles revealed that ARF6 KD led to an increase of the overall ceramide/sphingomyelin ratio, suggesting a defect in sphingolipid homeostasis. We then analyzed the expression of sphingolipid metabolic enzymes and observed that ARF6 KD downregulated sphingomyelin synthase 1 and 2 (SGMS1 and SGMS2) expression, hindering ceramide-to-sphingomyelin conversion. Supporting this, we found a positive linear correlation between ARF6 and SGMS1/2 expression among the patients in TCGA cohort, confirming their correlation. High expression of the ARF6 effectors SGMS1/2 is also associated with worse OS and DFS in the TCGA cohort and the MLL cohort in univariate analysis, resembling that of ARF6. Downstream of ceramide accumulation, ARF6 KD inhibited the pro-survival AKT pathway, confirming the pro-apoptotic roles of ceramide in cancers. The anti-proliferative effect of ARF6 depletion can be rescued by sphingomyelinase inhibitor GW4869 (blocking ceramide production) or by expressing a constitutively active form of AKT (AKT1 E17K). To pharmacologically target ARF6, we developed two generations of ARF6 small molecular inhibitor pro-drugs, named NAV-2729 and A6-4471. NAV-2729 and A6-4471 showed high specificity to ARF6 without inhibiting other small GTPases, with in vitro IC 50 in the lower μM range. Both NAV-2729 and A6-4471 inhibited AML cell line proliferation and reduced primary AML cell colony formation in lower μM range. Cord blood normal CD34 + cells are not sensitive to ARF6 inhibitors, but non-specific toxicity was observed at higher concentrations. When tested in vivo with THP-1 xenografts in NSG mice, both inhibitors reduced circulating AML cells without obvious toxicity to the host. Conclusion ARF6 is necessary for AML proliferation and its higher expression is associated with worse patient survival. However, ARF6 is dispensable for normal host physiology. Mechanistically, ARF6 maintains critical ceramide/sphingomyelin balance in AML by regulating SGMS1/2 expression. Pharmacologically inhibiting ARF6 suppressed AML proliferation in vitro and in vivo in a genotype-agnostic fashion. Our study implicates ARF6 as a potentially actionable and safe target in AML.

Self-Supporting Human Acute Myeloid Leukemia (AML) Bone Marrow Organoids Established from Patient-Derived Xenografts Mimic the Human AML Microenvironment, with Distinct Cytokine Kinetics Correlating with Dynamic Cell Proliferation and Metabolic Shifts

Hallmarks of acute myeloid leukemia (AML) include (1) formation of a self-supporting and self-regulating tumor microenvironment (TME) in the bone marrow (BM) rendering pathological niches that orchestrate retention, proliferation and survival of AML blasts, and (2) a dynamic metabolism that promotes therapy resistance and disease progression. Efforts to understand AML biology and enhance current therapies are hampered by the paucity of state-of-the-art human models that can recreate the three-dimensional (3D) BM TME and recapitulate the heterogeneity, complexity, dynamics, and metabolic shifts observed in human disease. Previously, we described the development of the first long-term self-propagating serum- and cytokine-free human BM organoid generated from an AML patient-derived xenograft (PDX) that captured metabolic shifts during AML progression. Here, we characterize the dynamic changes observed in the self-constituted TME with respect to AML proliferation, metabolism, and upon reconstitution of secondary organoids. BM was collected from NSG mice engrafted with human primary AML cells (PDX-AML model) or naïve control mice and seeded into polyurethane scaffolds in serum- and cytokine-free medium at 4e6 cells per scaffold, optimized previously. Scaffolds were recharged with autologous PDX-AML BM on day (D) 13 and cultured until D70. Organoids were assessed for (1) inter- and intra-scaffold AML kinetics, (2) the TME, (3) metabolic composition and (4) propagation into secondary organoids. AML cell kinetics from organoid culture supernatants reflected those of scaffold-extracted populations (n=3). CD33 +CD44 + AML cells expanded and/or were maintained from 7% (D0) to &amp;gt;10% (D70), being &amp;gt;15% at most culture timepoints. Confocal microscopy revealed reconstitution of typical AML-TME niche interactions within organoids, including expression of fibronectin, VCAM-1 and N-Cadherin, and formation of proliferative niches composed of AML cells with heterogeneous Osteopontin- and Osterix-expressing support cells. Human AML-supportive cytokines were consistently observed in organoid supernatants (N=3), with SDF-1, IL-1α, IL-8, GM-CSF, FGF-2 and Osteopontin present throughout the culture; some cytokines had biphasic kinetics with factors critical for TME formation present early (e.g. Osteopontin, GM-CSF), and those required for AML proliferation dominant later (e.g. FLT3L, IL-8), coinciding also with metabolic shifts during culture. Despite the absence of IL-6, single molecule RNA fluorescence in situ hybridization confirmed IL-6 expression by niche-resident AML blasts suggesting autocrine signaling networks beyond those identified in supernatants. Cells from D70 organoids were able to re-establish secondary organoids (n=2) which also exhibited long-term (D70) AML maintenance (&amp;gt;11% blasts), and regenerated the AML TME. Previously, we identified 3 culture stages defined by metabolic shifts: 1) pre/early recharge (D4-D19), (2) intermediate (D25-D40) and, (3) late (from D55) where organoids exhibited highest proliferation. To further characterize these shifts, intracellular metabolites were extracted from organoids (n=5) at D10, D25, D40, D55 and D70, analyzed with gas-chromatography mass spectrometry (GC-MS) and evaluated through unsupervised hierarchical clustering and multivariate analysis. Significance Analysis of Microarrays (SAM) analysis demonstrated dysregulation of alanine, serine, isoleucine, glycolate, hexanoic acid and glycolysis when comparing stage 1 with stage 2, and in isoleucine, leucine, serine, succinate and tryptophan metabolism when comparing stage 2 with stage 3. When proliferative and non-proliferative timepoints were compared, amino acid and glycolysis-driven metabolic switches were identified, typical of AML metabolism. We have characterized a long-term 3D human AML BM biomimicry in serum- and cytokine-free conditions with a self-organizing TME that recapitulates many features of AML BM, including robust human AML cell maintenance within niches, cytokine and metabolic kinetics to support different phases of progression, and the ability to propagate into secondary organoids. This dynamic, physiologically-relevant ex vivo platform can enable studies in human AML pathogenesis/progression and is poised to test novel therapeutics directed towards AML cells, the TME and metabolic targets.

Type 3 Innate Lymphoid Cells Promote Acute Myeloid Leukemia Immune Evasion and Expansion

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy originated in the bone marrow with poor overall survival rates of less than 30%. The immune system has been shown to play an important role in detecting and eliminating leukemic cells. Recent studies have described a population of immune cells termed Innate lymphoid cells (ILCs). ILCs are known to regulate both innate and adaptive immunity in an antigen-independent manner and are believed to be among the first immune cells to interact with malignantly transformed cells. Natural killer (NK) cells are a subset of ILCs and are known to inhibit AML progression, however the impact of other ILCs is not yet fully understood. Type 3 ILCs (ILC3s) have key roles in maintaining homeostasis in mucosal immunity through their ability to secrete cytokines including IL-17, IL-22, TNFα and GM-CSF (pro-myeloid). An expansion of ILC3s has recently been observed in solid tumors, however the overall functional impact of ILC3s in the setting of AML remains poorly understood and warrants further investigation. We first utilized an MLL-partial tandem duplication and FLT3-internal tandem duplication Flt3-ITD double knock-in mutation murine model of AML that recapitulates two clinically relevant mutations in human AML. Using this AML model, we discovered an expansion of ILC3s in the bone marrow of leukemic mice compared to wild-type control mice (2.364% +/- 0.40% AML vs 0.045% +/- 0.40% WT, n=5, p&amp;lt;0.0001). We also observed an increase in ILC3s in the bone marrow of untreated AML patients relative to age matched donor controls (7.013% +/- 2.6% vs. 2.328% +/- 2.6%). Our published data have previously shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. AHR is an important transcription factor in ILC3 development. Therefore, we hypothesized that the ILC3 increase was due at least in part to AHR-mediated increased ILC3 differentiation. To address this hypothesis, we isolated the ILC precursor (ILCP) from normal human donors and co-cultured with AML cells. By regulating AHR activity in this system, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (28.02% +/- 3.89% without AML, 60% +/- 5.5% with AML and 27% +/- 5% AML+AHR inhibitor, n=15, p&amp;lt;0.001). Next, we sought to determine the direct impact of ILC3s on AML proliferation and differentiation that could contribute to AML relapse. We utilized a colony forming assay (CFU) assay in the presence or absence of ILC3s. Bone marrow from primary untreated AML patients was cultured alone or with ILC3s (Lin-CD94-NKp44+). AML samples co-cultured with ILC3s had significantly increased colony formation relative to AML alone (69.4 CFU AML+ILC3 vs 42.7 CFU AML alone, n=8, p&amp;lt;0.05). Post-culture, we observed significant increases in the transcript of GM-CSF and TNFα in ILC3s co-cultured with AML marrow as compared to ILC3s alone. Furthermore, in our immunocompetent murine AML model, we observed significantly higher production of GM-CSF and TNFα by ILC3s in the bone marrow of leukemic mice relative to nonleukemic controls (GM-CSF: 11.08% +/- 1.6 AML vs 1.128% +/- 1.6 WT, n=10, p&amp;lt;0.0001; TNFα: 5.456% +/- 1.3 AML vs 1.307% +/- 1.3 WT, n=10, p&amp;lt;0.05), suggesting a potential mechanism of ILC3-mediated increased colony formation. Lastly, to determine if ILC3 expansion impacted NK cell immune surveillance, we co-cultured normal donor human ILC3s with AML cells, separated by transwell, for 48 hours before subjecting the AML cells to an NK cell cytotoxicity assay. We observed significantly reduced NK cell killing of AML cells when co-cultured with ILC3s compared to controls, suggesting ILC3s protect AML cells from NK cell-mediated killing (24.3% killing +/- 3.81% AML alone vs 14.57% +/- 1.35% AML with ILC3, n=18, p&amp;lt;0.01). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion to both support AML proliferation and protect AML blasts from immune surveillance. Future studies will focus in exploring how to target these interactions to design effective therapeutic approaches and improve patient clinical outcomes.

Synthetic Lethal Interactions with IRAK4 Inhibition in Myeloid Malignancies

Overexpression of immune-related genes is widely reported in acute myeloid leukemia (AML). Research from our lab and others has demonstrated that AML leukemic stem and progenitor cells (LSPC) rely on innate immune signaling through IRAK4 (reviewed in Bennett et al., Blood 2023). Moreover, AML LSPCs express a hypermorphic isoform of IRAK4 (IRAK4-L), due to U2AF1- and SF3B1-dependent mis-splicing. Building on this preclinical evidence, several IRAK4 inhibitors are now undergoing clinical trials in AML patients. In Phase-1 findings with a selective IRAK4 inhibitor (CA-4948; Curis Therapeutics), it was found that MDS and AML patients with splicing factor mutations responded best to monotherapy IRAK4 inhibition, although the overall response rate with monotherapy was modest. These findings suggest that IRAK4 is a relevant target in myeloid malignancies, but the identification of synthetic lethal dependencies may be crucial in revealing effective combination therapies. In this study, we sought to identify synthetic lethal interactions upon IRAK4 inhibition in AML. For this, we first generated isogenic AML cells that are proficient (WT THP1) or deficient for IRAK4 (IRAK4 KO THP1) using site-directed mutagenesis with CRISPR-Cas9. Deletion of IRAK4 resulted in a modest (~50%) reduction in leukemic colony formation and negligible differences in growth potential. We used the isogenic WT and IRAK4 KO AML cells to screen for synthetic lethal interactions by performing a 2400+ drug in vitro screen. Hits were prioritized for potency in IRAK4 KO cells vs WT AML cells, degree of fitness, and overall effect. Among these hits, the Cereblon E3 ligase modulator (CELMoD) CC-885 emerged as the top target. Independent validation confirmed that CC-885 significantly decreased proliferation and viability of isogenic IRAK4 KO MDS (MDSL) and AML (THP1) cells by approximately 3-4-fold compared to WT MDS or AML cells. Colony formation in methylcellulose after treatment with CC-885 was also significantly reduced in IRAK4 KO compared to WT MDS and AML cells. Based on Caspase-3 activation and AnnexinV staining, CC-885 induced significantly more apoptosis of IRAK4 KO cells as compared to WT AML cells. We extended our synthetic lethal analysis to a clinical-stage IRAK4 inhibitor (CA-4948). MDS and AML cells were treated with CA-4948 concurrently with CC-885 or pretreated for 7 days prior to treatment with CC-885. Unexpectedly, we found that only prior exposure of the AML cells to the IRAK4 inhibitors resulted in sensitivity to CC-885. These findings indicate that IRAK4 inhibition reprograms AML cells so that they acquire a synthetic lethal interaction with CC-885. CC-885 is known to target multiple substrates for degradation by the ubiquitin proteasome, including IKZF1, IKZF3, CK1a, and GSPT1. We compared the effect of CC-885 to CC-90009, a CELMoD with GSPT1 specific activity in the isogenic IRAK4 KO and WT AML cell lines. CC-90009 did not result in complete cell death up to concentrations of 10mM in both WT and IRAK4 KO, suggesting that the selective sensitivity of IRAK4 KO AML cells to CC-885 is not due to inhibition of GSPT1. In support of these interpretations, IRAK4 deficient AML cells exhibit increased protein expression of IKZF2 and IKZF3, but not GSPT1 or CK1a. Given the unique responses of IRAK4 KO cells to CELMoDs, we posited that IRAK4 inhibition alters the neosubstrate profile of CELMoDs in AML cells. Total proteome characterization of CC-885 treated WT and IRAK4 KO THP1 cells by mass spectrometry identified a significant decrease in 90 protein substrates in IRAK4 KO cells treated with CC-885 as compared to WT cells treated with CC-885. These findings suggest that IRAK4 inhibition alters the pool of neosubstrates in AML cells for certain CELMoDs. Overall, our study demonstrates that IRAK4 is a therapeutic target in AML, but that combination therapies, such as with certain CELMoDs, will be necessary to achieve better clinical responses.

Wilms' Tumor 1 Functions As a Tumor Suppressor to Suppress FLT3-STAT Signaling and Epigenetic Remodeling in Acute Myeloid Leukemia (CALGB 8461, 9665 and 20202; Alliance)

The Wilms' Tumor 1 ( WT1) gene is a transcription factor that is recurrently mutated or commonly overexpressed in several cancer types. In acute myeloid leukemia (AML), frequent overexpression of WT1 and poor patient outcomes associated with WT1 mutations highlight its importance in the disease; however, there are no tailored treatments for these patients. Furthermore, WT1's fundamental role as either an oncogene or tumor-suppressor remains unresolved. Here we examine the roles of both wild-type and mutant WT1 in AML through epigenetic and mechanistic studies. Using our findings we propose a personalized treatment strategy. WT1 mutations are enriched in the NPM1 mut subset of AML; thus we first focused our study on a cohort of 581 patients with de novo AML and NPM1 mutations enrolled on Alliance for Clinical Trials in Oncology studies. Transcriptomic analyses revealed that WT1 mut patientsphenocopied a distinct, aberrant gene expression signature associated with FLT3 internal tandem duplications (ITD), a mutation known to activate STAT signaling and associated with poor outcome in AML. We observed that WT1 expression levels were remarkably elevated in FLT3-ITD patients and were driven by STAT5A binding to the WT1 promoter. STAT5A binding and subsequent WT1 upregulation were blocked by small molecule FLT3 inhibitors. These findings linking FLT3 activity with WT1 expression raise two possibilities: either WT1 is an oncogene cooperating with the FLT3-STAT pathway, or WT1 naturally functions to suppress FLT3 signaling in a negative feedback loop subverted by WT1 mutations. As WT1 interacts with TET2 to facilitate epigenetic remodeling and DNA binding, we performed genome-wide DNA methylation analysis on WT1 mut AML patients and observed selective hypermethylation of WT1 binding motifs consistent with loss of function. We confirmed that WT1 mutations in AML commonly cause truncation of the C-terminal DNA binding domain in our patient cohort. Using co-immunoprecipitation, ChIP-sequencing and luciferase reporter assays, we found that mutant WT1 functions as a dominant-negative, inhibiting WT1 derived from the wild-type allele. Investigation of WT1 target genes by intersecting transcriptomic and DNA methylation profiles identified hypermethylation and downregulation of miR-193a, leading to a significant downregulation of miR-193a-3p in WT1 mut AML patients. Overexpression of wild-type, but not mutated, WT1 rescued miR-193a expression in AML cell lines. Enforced expression of miR-193a significantly delayed AML onset in vivo. In addition, overexpression of miR-193a in multiple human AML cell lines and primary patient samples impaired AML cell growth and colony-forming capacity while promoting monocytic differentiation, underscoring its role as potent tumor suppressor. Therapeutic modulation of miRNA levels in cancer patients has been limited by inefficient delivery and tissue enrichment. To overcome this, we tested a novel lipid-nanoparticle (LNP) formulation of miR-193a-3p (INT-1B3), currently being investigated in a phase I clinical trial (NCT04675996). Biweekly i.v. treatments of INT-1B3 in the immunocompetent Hoxa9/Meis1 (H9M)-transduced model system prevented AML formation, highlighting the potent anti-leukemic activity of this miRNA based therapeutic. Overexpression of miR-193a-loaded LNPs downregulated FLT3 expression and suppressed STAT signaling in primary AML samples. Finally, treatment of primary AML cells with FLT3 inhibitors revealed enhanced sensitivity of WT1 mut cells in the absence of FLT3-ITD, highlighting the role of wild-type FLT3 in WT1 mut cells. In summary, we uncovered a critical negative-feedback loop maintained by WT1 to suppress FLT3 activity. Loss-of-function WT1 mutations subvert the tumor suppressor function of WT1 via failure to maintain miR-193a expression, leading to increased FLT3 expression and STAT5 signaling, subsequently impairing differentiation, increasing proliferation and disease aggressiveness in AML (Figure 1). Our findings advocate for use of FLT3 inhibition and miR-193a supplementation for treatment of WT1 mut patients, a subgroup with poor outcomes and no targeted treatment options.

EP102: Pharmacological Inhibition of METTL3 Elicits Tumor Growth Inhibition In Vivo and Demonstrates Synergy with Venetoclax in Various AML Models

METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of acute myeloid leukemia (AML) 1. EPICS has discovered and optimized a small molecule inhibitor of METTL3 (“M3i”). M3i was shown to inhibit the enzymatic activity of METTL3 (IC 50 = 1.8 nM, SPA assay). The anti-proliferative effects of M3i was demonstrated in various AML cell lines (Kasumi, IC 50 = 63 nM; MV-411, IC 50 = 506 nM; KG1a, IC 50 = 99 nM). Biomarkers were evaluated following M3i treatment of Kasumi and MV-411 cells with consistent results demonstrating depletion of oncoproteins relevant to apoptotic (BCL-2, MCL1) and proliferative (MYC, BRD4, SP1) pathways. In addition, combined treatment with M3i and venetoclax showed clear synergistic effects 2 to inhibit proliferation on both cell lines consistent with the depletion of BCL-2 and MCL1, known targets for venetoclax activity and resistance mechanisms. M3i has been optimized for oral dosing with oral exposure confirmed in mouse, rat and minipig (oral bioavailability &amp;gt;80%, rat; &amp;gt;30%, minipig). In vivo efficacy was evaluated in a disseminated xenograft model using established systemic MV-411-Luc-mCh-Puro 3 in female NSG mice where M3i (10, 30 mg/kg, oral, QDx31 days) demonstrated dose-dependent tumor growth inhibition (p&amp;lt;0.01, relative to vehicle control) of cancer progression as measured by in-life imaging and confirmed by flow cytometry demonstrating significant, dose-dependent pharmacological knockdown of hCD45+ cells in bone marrow, blood and spleen (p&amp;lt;0.01, in all cases). These results confirm that the pharmacological inhibition of METTL3 arrests proliferation of AML and further provide a mechanistic basis for the synergistic activity of M3i in combination with venetoclax as is a viable strategy for the treatment of disease. 1Yankova E et al. (2021), Nature 593:597-601; 2Ianevski A et al. (2022), Nucl Acids Res 50:W739-W743; 3Trachet E (2016), LabCorp Drug Development website: model spotlight MV (4;11)

Differentiation of Acute Myeloid Leukemia Cells upon Pharmacological Inhibition of LSD1 Requires Its N-Terminal Intrinsically Disordered Region

Lysine-specific histone demethylase 1 (LSD1/KDM1A) governs hematopoiesis by regulating hematopoietic stem cell renewal and proper differentiation. Additionally, LSD1 is required for the maintenance of acute myeloid leukemia (AML) cells and has emerged as a key therapeutic target. Prior work from our group and others have revealed that the LSD1 demethylase activity is not required for AML proliferation. Instead, the interaction between LSD1 and GFI1/GFI1B is necessary to sustain AML cell survival. Thus, beyond its canonical demethylase activity, LSD1 has nonenzymatic, transcription factor (TF)-specific scaffolding functions critical for its function. Most LSD1 inhibitors function by blocking protein-protein interactions between LSD1 and TFs, even though they were originally designed as enzyme inhibitors. While studies indicate that LSD1 has important scaffolding functions, how LSD1 can coordinate with TFs to control gene expression programs is not well understood. Here, we used CRISPR-suppressor scanning to identify regions of LSD1 outside the active site that when mutated can lead to resistance to the covalent LSD1 inhibitor, GSK-LSD1. The understudied N-terminal intrinsically disordered region (IDR) of LSD1 was among the most enriched regions. To explore this further, we generated clonal AML cell lines harboring in-frame deletions within the IDR. These IDR-mutant cell lines were drug resistant and failed to differentiate upon GSK-LSD1 treatment. Moreover, knockdown of endogenous LSD1 followed by overexpression of LSD1 IDR-mutants rescued growth in the presence of GSK-LSD1, confirming that a fully functional IDR is necessary for differentiation. We performed LSD1 co-immunoprecipitation followed by mass spectrometry in wild-type and IDR-mutant cells to measure changes in LSD1 interactors upon GSK-LSD1 treatment. GSK-LSD1 treatment disrupted the LSD1-GFI1B interaction in both wild-type and mutant cells at comparable levels. Since GSK-LSD1 still induces dissociation of the mutant complex but has minimal effect on proliferation, our results suggest that the IDR-mutant cells no longer require GFI1B. In support, knockout of GFI1B inhibited growth of wild-type but not IDR-mutant cells. Strikingly, GSK-LSD1 treatment promoted LSD1's interaction with several master regulator TFs in hematopoiesis, including C/EBPα, PU.1, and RUNX1. Notably, C/EBPα was the most up-regulated interactor in both cell lines, with modest enrichment in IDR-mutant over wild-type. Using live-cell imaging assays, we found that deletions within the LSD1 IDR enhance association with these TFs. These results suggest that GSK-LSD1 reprograms LSD1-TF interactions, disrupting LSD1-GFI1B to induce LSD1-C/EBPα association, and that the IDR mutations alters this LSD1 redistribution to other TF partners. LSD1 is known to silence gene enhancers during differentiation in a process termed “enhancer decommissioning”. The mechanisms by which LSD1 occupies and controls enhancers remain elusive. To test if the LSD1 IDR regulates enhancer elements, we profiled LSD1 and H3K27ac using ChIP-seq in wild-type and IDR-mutant cells upon GSK-LSD1 treatment. Upon GSK-LSD1 treatment, disruption of LSD1-GFI1B binding was accompanied by increased LSD1 levels at other TF and H3K27ac sites, supporting the notion that loss of LSD1-GFI1B interaction induces LSD1 redistribution to other TF partner sites. A concomitant increase in H3K27ac levels was observed across LSD1 sites co-bound by C/EBPα, PU.1, or RUNX1 in wild-type cells. By contrast, in IDR-mutant cells, increases in H3K27ac levels were blocked at these sites upon GSK-LSD1 treatment, suggesting that full enhancer activation is impaired by mutations in the IDR, potentially due to altered association with master regulator TFs. This points to a function of LSD1 IDR in controlling enhancer activation for differentiation. Collectively, our study reveals a role for IDR in regulating LSD1-TF interactions, controlling enhancer activation that is necessary for AML cell differentiation. These data provide deep functional and mechanistic insights into the role of LSD1 IDR and offer important considerations for pharmacological targeting of protein-protein interactions.

FAT1 inhibits AML autophagy and proliferation via downregulating ATG4B expression

BackgroundEmerging studies have shown that FAT atypical cadherin 1 (FAT1) and autophagy separately inhibits and promotes acute myeloid leukemia (AML) proliferation. However, it is unknown whether FAT1 were associated with autophagy in regulating AML proliferation. MethodsAML cell lines, 6-week-old male nude mice and AML patient samples were used in this study. qPCR/Western blot and cell viability/3H-TdR incorporation assays were separately used to detect mRNA/protein levels and cell activity/proliferation. Luciferase reporter assay was used to examine gene promoter activity. Co-IP analysis was used to detect the binding of proteins. ResultsIn this study, we for the first time demonstrated that FAT1 inhibited AML proliferation by decreasing AML autophagy level. Moreover, FAT1 weakened AML autophagy level via decreasing autophagy related 4B (ATG4B) expression. Mechanistically, we found that FAT1 reduced the phosphorylated and intranuclear SMAD family member 2/3 (smad2/3) protein levels, thus decreasing the activity of ATG4B gene promoter. Furthermore, we found that FAT1 competitively bound to TGF-βR II which decreased the binding of TGF-βR II to TGF-βR I and the subsequent phosphorylation of TGF-βR I, thus reducing the phosphorylation and intranuclear smad2/3. The experiments in nude mice showed that knockdown of FAT1 promoted AML autophagy and proliferation in vivo. ConclusionsCollectively, these results revealed that FAT1 downregulates ATG4B expression via inhibiting TGFβ-smad2/3 signaling activity, thus decreasing the autophagy level and proliferation activity of AML cells. General significanceOur study suggested that the “FAT1-TGFβ-smad2/3-ATG4B-autophagy” pathway may be a novel target for developing new targeted drugs to AML treatment.

Tumor suppressor miR-520a inhibits cell growth by negatively regulating PI3K/AKT signaling pathway in acute myeloid leukemia.

Short regulatory RNAs, called microRNAs (miRNAs), have been found to possess regulatory functions in cancer and, as such, have recently been evaluated for their therapeutic role against various human malignancies. The present work aimed to investigate whether miR-520a can play a therapeutic role in the treatment of human acute myeloid leukemia. Human myeloid leukemia cell lines (Kasumi-1, Kasumi-3, Kasumi-6, BDCM, and K562) and a normal myeloid cell line (NCI-H5N6) were used for the study. Cell lines were subjected to real-time quantitative polymerase chain reaction (RT-qPCR), evaluation of cell viability and proliferation by MTT assay and colony formation assays. Dual acridine orange (AO)/ethidium bromide (EB) staining was applied for transfected K562 cells with miR-negative control (NC) or miR-520a mimics, and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) dual staining and flow cytometry were performed to analyze cancer cell apoptosis followed by western blot. Cancerous cell lines exhibited lower gene expression of miR-520a, and its overexpression significantly reduced (p < 0.05) the proliferation and viability of cancer cells. Cancer cells demonstrated the induction of Bax/Bcl-2-mediated apoptosis following miR-520a overexpression. The miR-520a was shown to target the PI3K/AKT signaling pathway in human acute myeloid leukemia to exercise its regulatory role in cancer. The study showed that miR-520a actively regulated cell proliferation in acute myeloid leukemia and illustrated the mechanism by which it exerts its regulatory role, emphasizing the possibility of targeting miR-520a as an efficient therapeutic strategy against human acute myeloid leukemia.

Genetic landscape of acute myeloid leukemias with leukocytosis

Background. Tumor cell proliferation in acute myeloid leukemia (AML) may manifest with high leukocyte counts. In our work, we evaluate the association of high leukocyte counts with individual mutations, as well as their total contribution to the development of leukocytosis in AML. The results obtained should improve our understanding of pathogenic mechanisms leading to the leukocytosis in AML.Aim. To study the genetic landscape of AML with leukocytosis.Materials and methods. The laboratory data of 214 AML patients admitted to the National Medical Research Center for Hematology (Moscow) from 2010 to 2022 were retrospectively examined. Real-time PCR, capillary electrophoresis and NGS (next generation sequencing) methods were used to detect mutations of FLT3, NPM1, CEBPA, IDH1/2, DNMT3A, TET2 genes, and CBFB::MYH11, RUNX1::RUNX1T1 chimeric gene transcripts.Results. Mutations of the FLT3 gene (odds ratio 5.45; p &lt; 0.0001), inv(16)/CBFB::MYH11 (odds ratio 10.03; p = 0.0009) are most associated with leukocyte counts higher than 30 × 109 / L in the debut of AML. Translocation t(8;21)/RUNX1::RUNX1T1 and adverse cytogenetic aberrations, such as -5/del(5q); -7 / del(7q); -17 / abn(17p), complex and monosomic karyotype were significantly associated with leukocyte counts lower than 30 × 109 / L at the time of disease manifestation (p &lt; 0.0001). In the group of patients with intermediate cytogenetic risk bearing only IDH1/2, DNMT3A, and TET2 gene mutations, leukocyte counts at AML debut were significantly lower, whereas the most pronounced leukocytosis was observed in patients with a combination of driver mutations with IDH1/2, DNMT3A, and TET2 gene mutations or FLT3, NPM1, and CEBPA gene mutations.Conclusion. In addition to the individual effect of certain genetic lesions and cytogenetic aberrations on the proliferative potential of tumor cells, there is a total contribution of various types of genetic events to the development of leukocytosis in AML. High leukocyte counts at the time of AML manifestation in patients with intermediate cytogenetic risk can serve as an indirect marker of the presence of a large number of genetic aberrations with a combination of IDH1/2, DNMT3A, and TET2 gene mutations or FLT3, NPM1, and CEBPA gene mutations.