Differentiation In Acute Myeloid Leukemia Research Articles

Pyrimidine depletion enhances targeted and immune therapy combinations in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.

The CDK4/6 inhibitor Palbociclib synergizes with ATRA to induce differentiation in AML.

Differentiation therapy based on ATRA almost cured acute promyelocytic leukemia (APL). However, it is disappointing that ATRA is not effective against other acute myeloid leukemia (AML) subtypes. Developing new and effective anti-AML therapies that promote leukemia differentiation is necessary. The CDK4/6-cyclin D pathway is a key initiator of the G1/S phase transition, which determines cell fate. Herein, we investigated whether the CDK4/6 inhibitor palbociclib would synergize with ATRA to promote leukemia differentiation in vitro and in vivo. Our findings revealed that CDK4/6-cyclin D pathway genes were aberrantly expressed in AML, and we observed that palbociclib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes indicative of myeloid differentiation. The combination of palbociclib and ATRA attenuated AML cell expansion in vivo. These enhanced differentiation effects may be associated with the regulation of transcription factors, including RARα, E2F1, and STAT1. Overall, our findings demonstrate that CDK4/6 inhibition sensitizes AML cells to ATRA and could guide the development of novel therapeutic strategies for AML patients.

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.

Automated Machine Learning-Based Diagnosis and Molecular Characterization of Acute Leukemias using Flow Cytometry Data

Abstract Current flow cytometric analysis of blood and bone marrow samples for the diagnosis of acute leukemias relies heavily on manual intervention in both the processing and analysis steps, introducing significant subjectivity into the resulting diagnosis and increasing diagnostic turn-around time. Additionally, concurrent molecular characterization of these samples via cytogenetics and targeted sequencing panels can take multiple days, thereby delaying patient diagnosis and treatment. Attention-based multi-instance learning models are machine learning models that can make accurate predictions and generate interpretable insights regarding the classification of a sample from multiple events/cells; while these models have been developed for anatomic pathology applications, they have yet to be applied to flow cytometry data. By utilizing 1,820 flow cytometry samples from 2019-2022 at Brigham and Women’s Hospital, we developed attention-based multi-instance machine learning models for automated diagnosis of acute leukemia, including differentiation of acute myeloid leukemia (AML) from B-lymphoblastic leukemia/lymphoma (B-ALL). Additionally, using concurrent cytogenetic and targeted sequencing data from 674 acute leukemia samples, machine learning models for prediction of molecular aberrancies from flow cytometry data were developed. Machine learning models were created using the TabNet deep learning architecture and VIME self-supervised training algorithm, which are state-of-the-art approaches towards machine learning from tabular data including flow cytometry data. Attention-based multi-instance models demonstrated strong performance for the automated diagnosis of acute leukemia versus non-leukemia samples (AUROC 0.869), as well as the separation of AML from B-ALL samples (AUROC 0.971). These models also accurately predicted cytogenetic aberrancies among AML samples, including t(15;17);PML::RARA (AUROC 1.00), as well as mutations including NPM1 (AUROC 0.725). These models do not require any manual intervention including compensation or gating, and additionally provide quantitative scores for the relative importance of different flow cytometry events and markers for the diagnosis of a particular sample. These importance scores can be integrated into flow cytometry analysis software for visualization and interpretation by hematopathologists. In this study, we have demonstrated the capability of machine learning models to provide automated diagnoses of acute leukemia, as well as accurately predict cytogenetic and molecular aberrancies in blood and bone marrow samples using flow cytometry data. This automated workflow can significantly decrease diagnostic turn-around time and ultimately improve patient outcomes.

Dissecting Lineage and Cell State-Specific Epigenetic Evolution in Relapsed Acute Myeloid Leukemia

Introduction: The prognosis for relapsed Acute Myeloid Leukemia (AML) following initial treatment and remission remains poor, largely due to a limited number of therapies with proven clinical efficacy. Epigenetic states and evolution are known to contribute to the initiation of AML, however, the role of epigenetic evolution in relapse remains understudied, and the effects of lineage-specific epigenetic changes at relapse are unknown. We hypothesized that epigenetic modifications confined to specific lineages or differentiation states may contribute to disease resistance and relapse. To test this, we generated single cell ATACseq data from four AML patients at initial diagnosis and at relapse post-induction chemotherapy. We utilized mapping of AML scATACseq to scATAC-derived healthy hematopoietic chromatin states, lineage trajectory analysis, and gene and transcription factor accessibility analysis to identify epigenetic changes occurring at discrete points in different AML differentiation trajectories. Methods: Malignant AML cells were purified from paired samples collected from four patients at diagnosis and relapse via FACS and were sequenced using the 10x chromium scATAC platform. ArchR, Monocle3, and custom R scripts were used for all analyses. AML cell scATAC data was mapped to a healthy hematopoietic scATAC reference using Latent Semantic Indexing (LSI) and projection onto a single cell ATAC-seq manifold derived from healthy hematopoietic cell types. Closest healthy cell types were identified for each AML cell, differentiation trajectories were generated using monocole3, and gene and transcription factor accessibility was quantified across the different AML differentiation states and lineages. Results: Our analysis showed varying patterns of lineage bias across AMLs. Some samples exhibited changes in differentiation state at relapse, becoming either less differentiated or less myeloid-like. Analysis of differences in TF activity between diagnosis and relapse within myeloid and lymphoid trajectories revealed lineage-specific and differentiation state-specific changes in TF activity. In patient SU360, overall differentiation states did not change, and AML cells at both diagnosis and relapse were primarily myeloid-like with a smaller lymphoid-like population. In this sample, GATA family TFs, which were active in progenitor-like cells at diagnosis, became significantly more active in terminally differentiated AML cells at relapse. No specific chromatin changes were observed in lymphoid-like AML cells at relapse. In patient SU484, where diagnosis cells were myeloid-differentiated and relapse cells were more progenitor-like, GATA and RUNX family TF activity, which was restricted to progenitor cells at diagnosis, showed broad activation in all cells at relapse, aligning with the overall de-differentiation observed in the AML. In SU142, a case with no significant change in overall chromatin or differentiation state at relapse, multiple SP family transcription factors showed significant activation specifically in progenitor-like AML cells at relapse. Conclusion: Our study of four distinct AMLs suggests the existence of lineage-specific epigenetic evolution in some relapsed AMLs. We found that epigenetic activation of specific transcription factors, such as GATA and RUNX, was restricted to certain steps of myeloid differentiation, suggesting that epigenetic changes contributing to resistance and relapse are relevant in specific AML lineages and differentiation states. Notably, we found that even in AMLs where overall epigenetic state remains stable, there is activation of specific TFs (e.g. SP1) in progenitor cells at relapse, consistent with literature demonstrating SP family TF relevance in Leukemic Stem Cell (LSC) drug resistance. These observations suggest that epigenetic modifications in less-differentiated AML cells may reflect changes occurring in LSCs, potentially contributing to resistance and relapse. Our study is limited due to a small sample size that precludes broad generalizations, but our findings provide a foundation for further characterization of AML relapse epigenetics. Ultimately, these findings suggest the existence of specific epigenetic changes at certain points in the AML differentiation hierarchy that contribute to relapse, warranting further exploration of cell state-specific epigenetic evolution.

Single-Cell RNA-Seq Reveals Intermediate Cell States and Identifies Features Defining Cellular Heterogeneity in Inv(16) Acute Myeloid Leukemia (AML)

AML is an aggressive hematological malignancy with heterogeneous genetic abnormalities. One recurrent chromosomal translocation common in AML is chromosome 16 inversion, inv(16)(p13.1q22), which results in the leukemogenic fusion gene CBFB- MYH11. Using a conditional Cbfb-MYH11 (CM) knock-in mouse model to mimic the somatic expression of the CM fusion gene, we demonstrated that CM expression leads to impaired hematopoietic differentiation as well as accumulation of phenotypic hematopoietic stem cells (HSC) and pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitors, which are predisposed to leukemic transformation. Flow cytometry-based phenotypic analysis revealed that the transformed leukemic blasts are predominantly characterized as pre-granulocyte-macrophage progenitor (pre-GM) (Lin −cKit +Sca1 −CD16/32 -/loCD150 −CD105 −). However, cell type characterization via conventional surface markers is limited by known cell markers and is incapable of resolving pathologic heterogeneities. We previously reported that modeling AML in a continuum of differentiation states using single-cell RNA-seq (scRNA-seq) data predicts intermediate states of differentiation in AML. Herein, we utilized scRNA-seq to characterize CM leukemic blasts, and identified transcriptomic alternations and features that defined the leukemic cell states and heterogeneities. CM expression was induced by poly (I:C) and mice (n=3) were monitored for leukemia progression. At the leukemic end-stage (detection of >50% of circulating cKit + cells), we collected unfractionated and ckit + sorted peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells. Similarly poly(I:C)More than 105,000 cells harvested from the WT and CM leukemic mice were analyzed by scRNA-seq. We applied uniform manifold approximation and projection (UMAP) for dimensionality reduction and performed cell type annotation by SingleR. First, we mapped mature hematopoietic cell types in the WT PB and hematopoietic stem/progenitor cell populations (HSPC) in the WT BM, and in contrast, leukemic PB and BM were highly similar and showed distinct cell clusters compared to WT. To characterize HSPC subpopulations, we focused on cKit+ sorted WT and leukemic BM cells, visualized them via UMAP, and performed clustering and cell type annotation. The leukemic cKit+ cells were mostly identified as megakaryocytic-erythroid progenitor (MEP)-like HSC, revealing a discrepancy between flow cytometry-based phenotypic characterization and transcriptome-based cell type assignment. These leukemic MEP-like HSCs showed up-regulation of erythroid differentiation genes, including Hba-a1, Hba-a2, Hbb-bs, Hbb-bt and Sox6, together with decreased expression of granulocytic markers such as Elane, Prtn3, S100a8, S100a9 and Mpo. Comparison of leukemic MEP-like HSCs vs WT MEP clusters via gene set enrichment analysis (GSEA) with a threshold of nominal p-value ≤ 0.05 and FDR ≤ 0.05 in 50 hallmark gene sets (MSigDB) indicated dysregulated pathways, including up-regulation of “TNFα signaling pathway via NFκB”, “inflammatory response”, and “UV responsive downregulated genes”; and down-regulation of “MYC target V1”, “MYC target V2”, “E2F targets”, “G2M checkpoint”, and “oxidative phosphorylation”. Similarly, GSEA in Gene Ontology (GO) showed downregulation of pathways relevant to mitochondrial protein translation and ribosomal biogenesis/assembly in leukemic MEP-like HSCs, compared to WT MEP. In addition, cKit+ BM cells can be separated into 31 clusters (C0-C30), of which 9 leukemic clusters were identified. Through gene expression-based cell cycle scoring, single sample gene set enrichment (ssGSEA), and marker identification, these 9 leukemic clusters can be defined by several features, including high heme-metabolism (C0, C1, and C2), high proliferation (C1, C8), S phase (C4, C10), low oxidative phosphorylation (C2, C5, C17), high Cd9 (C10), high Egfl7 (C7, C8). Altogether, these data reveal intermediate differentiation cell states in inv(16) leukemic cells which are highly heterogeneous populations with distinct cell cycle stages, metabolic activities, and marker expression.

Fatty Acid Binding Protein FABP5 Inhibition Activates Retinoic Acid Signaling and Induces Differentiation in Acute Myeloid Leukemia

Introduction. Accumulation of undifferentiated immature blood cells is a hallmark of myeloid neoplasia (MN), and indicates decoupling of terminal-differentiation from proliferation. Therefore, reinstating to a programmed state of differentiation (lineage-maturation) is a treatment approach, exemplified by all-trans retinoic acid (ATRA) treatment of acute promyelocytic leukemias that contain t(15;17) (q24;q21). This translocation inactivates retinoic acid receptor alpha (RARA) due to its fusion with promyelocytic leukemia PML resulting in PML-RARA. This fusion gene responsible for cellular transformation confers sensitivity to treatment with ATRA. However, ATRA's therapeutic-success in APLs has not extended to other AML phenotypes. We and others have shown that RA is a ligand, not just for RARs, but also pro-survival peroxisome proliferator-activated receptor β/δ (PPARβ/δ), a member of the nuclear hormone receptor superfamily transcription factors that regulate several metabolic pathways. RA is transported by two carrier proteins, cellular retinoid-binding protein CRABP2, or fatty-acid-binding protein 5 (FABP5), depending on cellular context. These carriers preferentially deliver RA to RARs versus PPARβ/δ respectively. Somatic lesions such as loss of function TET2 or Tp53 mutation, NPM1 and ASXL1 mutations, are associated with upregulated FABP5 and downregulated CRABP2. In AML cells with these mutations, therefore, the high FABP5 can be expected to deliver RA to PPARβ/δ instead of RAR, to potentially contribute to decoupling of differentiation from proliferation. We therefore investigated this possibility and its treatment implications. Methods. Overexpression, gene knockdown/knockout (ko/kd) of key regulators of RAR and PPARβ/δ were generated in specific genotypes of AMLs. Small molecule inhibitors of FABP5 were used to further interrogate the pathways in a translationally relevant manner, both in vitro, in both malignant and normal cells, and in pre-clinical in vivo models. Results. Gene expression analysis of AMLs in the Beat AML and TCGA databases demonstrated upregulation of pro-survival PPARβ/δ target genes including FABP5 and downregulation of differentiation promoting RAR pathway genes including CRABP2in AMLs 1. In particular, FABP5 was highly expressed in AMLs containing TET2 NPM1, TP53 and ASXL1 mutations. AML cell lines OCIAML3 (NPM1 ins/DNMT3a R882c), OCIAML5 (ASXL1 Y591*/TET2 S825*), THP1 (TP53 del) and isogenic CRISPR engineered K562 (TET2 +/+/TET2 -/-) demonstrated 4-100 fold higher FABP5 expression compared to normal myeloid precursors. These models did not differentiate in response to pharmacologic concentrations of ATRA. To test if FABP5 is necessary and sufficient to maintain the undifferentiated state of these cells in the presence of ATRA, we genetically inactivated FABP5 either using RNAi (kd) or CRISPR (ko). FABP5 knockdown or knockout cells created sensitivity to ATRA, resulting in terminal-differentiation as observed by flow cytometry for terminal-differentiation markers ( See Figure). FABP5 was then inhibited using highly specific small molecule FABP5 inhibitors with a 56-fold higher affinity compared to ATRA 1: combination of iFABP5 and ATRA induced terminal-differentiation with significant upregulation of the monocyte lineage markers CD14 and granulocyte lineage marker CD11b measured by flow cytometry at 72h. Toinvestigatewhether knocking down FABP5 and ATRA treatment affects gene expression changes we performed transcriptomic profiling of OCIMAL3 Scr, shFABP5 cells treated with or without ATRA using whole exome RNA sequencing. Treatment of FABP5 kd OCIMAL3 cells with ATRA resulted in the upregulation of RAR pathways genes and downregulation of proliferation promoting genes. Conclusion. In sum, recoupling of lineage-maturation to proliferation can be achieved in non-APL AMLs that highly express the RA carrier FABP5, by inhibiting FABP5 using small molecules. This candidate treatment modality is distinct from cytotoxic chemotherapy, and importantly, can spare normal hematopoiesis.

Retinoic Acid and Ascorbate Remodel the Epigenome of Leukemia Cells to Improve Therapeutic Efficacy By Enhancing TET Activity

Ten-eleven translocation (TET) enzymes are commonly mutated in leukemia, leading to aberrant DNA methylation and impaired hydroxymethylation. All-trans retinoic acid (ATRA) is a vitamin A derivative and cofactor of retinoic acid receptors (RARs) which, in combination with arsenic trioxide, can curatively treat acute promyelocytic leukemia. ATRA has also been shown to upregulate expression of Tet2 and Tet3 in murine embryonic stem cells, and retinol and ascorbic acid (vitamin C) have been shown to enhance pluripotent stem cell reprogramming through increasing hydroxymethylcytosine and promoting DNA demethylation. Our previous work has demonstrated ascorbate can slow disease progression in vivo and, combined with the PARP inhibitor Olaparib, promote cell cycle dysregulation and induce cellular differentiation in acute myeloid leukemia (AML) models both in vitro and in vivo. Here, we show ATRA can induce TET2 and/or TET3 expression in AML cells, and, combined with ascorbate, increase genome-wide oxidized methylcytosine in a TET-dependent manner. Combination treatment with ATRA and ascorbate also induces transcriptional reprogramming, leading to upregulation of DNA repair genes and pathways leading to increased expression of myeloid differentiation markers. Additionally, we observe chromatin remodeling associated with an enrichment for ETS motifs including genes and regions regulated by the transcription factor PU.1, an essential modulator of hematopoietic cell fate. We also show enrichment of retinoic acid receptor A (RARA) at the TET2 locus and at several RAR elements in response to ATRA in AML cells. Using very low dose ascorbate and ATRA, we show that the combination treatment can enhance the efficacy of Olaparib and Venetoclax to alter the cell cycle, promote differentiation, and induce apoptosis of AML cells. These data demonstrate the potential for ATRA and ascorbate to increase TET activity and drive myeloid differentiation and death of AML cells that can be exploited as an adjuvant therapy for the treatment leukemia.

Single-Cell Proteomic Analysis Reveals Menin Inhibition-Induced Proteomic Alterations in AML Patients Treated with Revumenib

Menin inhibition is a promising therapeutic approach for acute myeloid leukemia (AML) with certain genetic alterations including KMT2A rearrangements or NPM1 mutations. The first-in-human Phase 1 clinical trial of revumenib (SNDX-5613) in patients with R/R AML showed promising results and induced complete remission in patients who refractory to multiple lines to therapy. These findings suggest that revumenib could provide a new therapeutic option for patients with AML with KMT2A and NPM1 genetic alterations. AML is characterized by the existence of distinct differentiation hierarchies, a phenomenon substantially contributing to disease heterogeneity. AML hierarchies exhibit distinct expressions of phenotypic profiles, apoptotic networks, signaling pathways, and metabolic profiles, leading to varying sensitivity towards targeted therapies, resulting in differential sensitivity to targeted therapies. Revumenib induces AML differentiation and thus shift the balance between AML hierarchies, resulting in drastic alterations in the overall AML proteomic profiles and enrichment of distinct leukemia phenotypes with differential sensitivity to targeted therapies. Detailed interrogation of revumenib-induced proteomic alterations could permit precise targeting of persistent cells and development of data-driven combinatorial approaches to achieve higher response rates. Moreover, the distinct combinations of genetic alterations can impact the efficacy of revumenib-induced differentiation and therapeutic outcomes. Thus, to better understand these complexities we performed multimodal single-cell (sc) analysis of AML landscape in R/R AML treated with revumenib (n=4) in Phase 1. We utilized two leukemia-focused 51-parameter CyTOF panels to analyze pre and post-treatment samples from R/R AML patients treated with revumenib. Sc proteogenomic profiling was performed using MissionBio platform using a panel covering 37 genes with recurrent AML-relevant mutations. UMAP and clustering analyses of CyTOF data identified AML blasts, unveiled quantitative changes in cell composition, and delineated proteomic shifts induced under revumenib therapy pressure. We observed enrichment of CD11b+/-CD68+, more differentiated monocytic cells situated at close proximity to AML blasts and depletion of immature AML blasts. Through unsupervised analysis, we discovered discrete AML differentiation hierarchies, delineated by a differentiation continuum that initiates with immature AML blasts (CD34+/-c-Kit+/-), progresses through intermediate AML blasts (CD68+/CD11b-/CD14-), and culminates with more differentiated monocytic cells (CD68++/CD11b+/CD14+/-). Notably, intermediate AML blasts and monocytic cells were enriched in post-treatment samples. Simultaneous ScDNA+protein analysis showed enrichment of differentiated AML blasts with monocytic/dendritic cell phenotype and harboring AML mutations. Differentiated AML blasts expressed higher levels of MCL1 and BCL-xL and upon differentiation we observed gradual downregulation of BCL2. Importantly, intermediate and more differentiated AML blasts under revumenib therapy expressed high levels of p53 protein, a feature that can partly attributed to NPM1 mutations. Sc proteomic analysis delineated proteomic shifts elicited by revumenib and identified potential therapeutic partners, by targeting MCL1, BCL-xL and MDM2, that could be used in combination with revumenib to improve therapeutic efficacy. We next performed differential expression analysis by comparing the proteomic profiles of pre- and post-therapy samples. We observed suppression of less differentiated AML phenotypic profiles (c-Kit, CD123, CD34) along with MEIS1 and PBX3. Importantly, we also observed significant alterations in immune landscape, suggesting that revumenib could rewire Myeloid-to-T and NK cell interaction/communication by inducing proteomic shifts and driving AML blast differentiation. Further analysis of an expanded cohort of R/R AML patients is currently underway. In conclusion, multimodal sc analysis revealed profound proteomic alterations induced by revumenib and identified potential therapeutic targets. These findings present new avenues for optimizing combinatorial treatment regimens, enhancing revumenib's efficacy, and illuminating survival mechanisms in persistent, differentiated AML blasts.

Epigenetic Silencing of CD9 Shapes a New Mechanism to Render Differentiation Block and Immune Evasion in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous hematologic malignancy characterized by clonal expansion of myeloid progenitor cells with impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain suboptimal. Emerging evidence suggest myeloblasts could evolve multiple machineries to evade immune patrol and hinder immunotherapies. Here, we present a new mechanism involving the tetraspanin protein CD9 in orchestrating differentiation arrest and immune escape in pediatric AML. We first examined CD9 expression and its prognostic impact in patient cohorts of childhood leukemia. The expression of CD9 on blasts of AML patients (12.2%, n=82) was significantly lower than those of ALL patients (90.4%, n=219, P<0.001) or stem cells from normal bone marrow donors (48.4%, n=22, P=0.014). Among AML cases, the blasts of 32 patients (39%) were CD9+. The expression pattern of CD9 was highly associated with cytogenetic/genetic anomalies and FAB subtypes, for instance enrichment of CD9 in M4, M5 and M7 AML. The 5-year relapse-free survival rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018), with its prognostic significance exhibited distinctively in M4/M5 AML. To elucidate the mechanism governing CD9 silencing, DNA and histone methylation status of CD9 were evaluated by bisulfite and ChIP sequencing for the classical histone marks H3K9/27me3, which indicated methylation may not contribute to CD9 repression. However, a marked decrease of H3K9/27Ac occupancy at the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P<0.05), and strongly correlated with CD9 repression ( r=0.585-0.719, P<0.01). Exposure of CD9- AML cell lines (n=8) or samples (n=9) to the histone deacetylase inhibitor panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P<0.05), restored activating histone acetylation marks (4.1-41.6-fold, P<0.05), and potently suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM). An epigenetic compound library screen confirmed histone hypoacetylation as the key mechanism driving CD9 silencing. Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation ( P<0.01) and colony formation ( P=0.002). NOD/SCID mice transplanted with CD9+ cells exhibited a drastically reduced leukemic load in hematopoietic organs by 70.7-91.8% ( P<0.05), a significantly prolonged survival duration ( P<0.001), and a marked regression of extramedullary myeloid sarcoma when compared with animals receiving CD9- cells. Global transcriptome profiling of pediatric AML (n=31) revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ samples. Concordantly, we observed a profound up-regulation of CD9 (9.4-51.1-fold, P<0.01) preceding the appearance of lineage markers (CD14, CD36, CD86, CD115, CD300e, iCD68 and iTNFα) in PMA-mediated monocyte/macrophage differentiation but not ATRA-mediated neutrophil differentiation of myeloblasts. Importantly, the gain of CD9 in THP-1 cells and primary myeloblasts (n=6, P<0.05) consistently augmented the expression of specific monocytic markers, corroborating its cell-intrinsic function in AML differentiation. Single-cell transcriptomic analyses of bone marrow cells from MV4-11-transplanted mice detected a significant enrichment of differentially regulated genes functioning in antigen processing and presentation. Confirmatory overexpression of CD9 promoted basal and IFNγ-induced MHC-I/II expression ( P<0.01) through the JAK2/STAT5 axis. Inter-patient comparisons (n=31) revealed a higher MHC-I expression in CD9+ AML ( P<0.001). Interestingly, CD9 physically bound to MHC-I/II and formed an immune complex at the cell membrane as revealed by IP-MS, co-IP and confocal microscopy. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13, P<0.05), concomitant with a robust bone marrow infiltration of cytotoxic T cells. Taken together, our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining monocytic differentiation and immune recognition in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy.

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.

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<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<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<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<0.0001; TNFα: 5.456% +/- 1.3 AML vs 1.307% +/- 1.3 WT, n=10, p<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<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.

Transcription Factor Redistributors Pharmacologically Actuate Non-Canonical Gene Networks to Drive AML Differentiation

Aberrant transcriptional networks are hallmarks of aging and cancer, yet our ability to target these aberrations is poor. PU.1 is one such transcription factor (TF) who's transcriptional networks are corrupted in disease, including in >50% of Acute Myeloid Leukemia (AML) cases. In this study we investigate an unappreciated pharmacological approach to target the aberrant PU.1 network, by employing novel small molecules which competitively inhibit PU.1:DNA interactions. We performed an extensive multiomics-driven, molecular characterization of AML cells following exposure to PU.1-DNA binding inhibitors (e.g. DB2115). Unexpectedly, we found that such compounds not only led to a reduction of many canonical PU.1 transcripts, but also (seemingly paradoxically) caused increases in alternative PU.1-driven transcripts. We discovered that this two-sided response was a result of robust cistromic repositioning of PU.1 chromatin binding rather than global inhibition. From kinetic CUT&Tag and ATAC sequencing studies, we identified that PU.1 redistribution involved rapid PU.1 loss (~1hr) followed by delayed PU.1 gain (~4-12hrs), causing subsequent opening of the chromatin, thus demonstrating endogenous PU.1's powerful pioneering ability. Through development of CLICK-on-CUT&Tag, we identified exclusive drug binding at displaced PU.1 binding sites, and identified unique, sequence-specific ETS motifs dictating PU.1 gain or loss. By employing these motifs in eGFP reporter assays and CRISPRd systems, we identified direct PU.1-mediated transcriptional control of known and novel functionally relevant genes, including MYC, POMP and STRAP. Finally, we establish that primary AML samples are sensitive to pharmacological PU.1 redistribution upon exposure to DB2115, and the rewiring of the PU.1 network in these cells drives differentiation down the myeloid lineage (Figure 1). Overall, we uncover a novel biological phenomenon we describe as: Pharmacological transcription factor (TF) redistribution. “TF Redistributors”, such as DB2115 and next generation derivatives, are an unprecedented investigative and pharmacological tool which allow the study of complex and fast transcription factor dynamics without disrupting the structure or levels of the TF itself. Furthermore, we discover that pharmacological binding-site restriction and consecutive repositioning of the PU.1 network, cascades into an extensive rewiring of transcriptional circuits which ultimately drives myeloid differentiation of AML cells, potentially providing a novel therapeutic intervention for PU.1-corrupted AML.

Bone marrow stromal cells reduce low-dose cytarabine-induced differentiation of acute myeloid leukemia.

Low-dose cytarabine (LDAC) is a standard therapy for elderly acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. While high doses of cytarabine induce cytotoxicity, the precise mechanism of action of LDAC in AML remains elusive. In vitro studies have demonstrated LDAC-induced differentiation; however, such differentiation is seldom observed in vivo. We hypothesize that this discrepancy may be attributed to the influence of bone marrow (BM) stromal cells on AML cells. Thus, this study aimed to investigate the impact of BM stromal cells on LDAC-induced differentiation of AML cell lines and primary samples. Our results demonstrate that the presence of MS-5 stromal cells prevented LDAC-induced cell cycle arrest, DNA damage signaling and differentiation of U937 and MOLM-13 cell lines. Although transcriptomic analysis revealed that the stroma reduces the expression of genes involved in cytokine signaling and oxidative stress, data obtained with pharmacological inhibitors and neutralizing antibodies did not support the role for CXCL12, TGF-β1 or reactive oxygen species. The presence of stromal cells reduces LDAC-induced differentiation in primary samples from AML-M4 and myelodysplastic syndrome/AML patients. In conclusion, our study demonstrates that BM stroma reduces differentiation of AML induced by LDAC. These findings provide insights into the limited occurrence of terminal differentiation observed in AML patients, and suggest a potential explanation for this observation.

Low doses of IFN-γ maintain self-renewal of leukemia stem cells in acute myeloid leukemia.

Conventional therapies for acute myeloid leukemia (AML) often fail to eliminate the disease-initiating leukemia stem cell (LSC) population, leading to disease relapse. Interferon-γ (IFN-γ) is a known inflammatory cytokine that promotes antitumor responses. Here, we found that low serum IFN-γ levels correlated with a higher percentage of LSCs and greater relapse incidence in AML patients. Furthermore, IFNGR1 was overexpressed in relapsed patients with AML and associated with a poor prognosis. We showed that high doses (5-10 μg/day) of IFN-γ exerted an anti-AML effect, while low doses (0.01-0.05 μg/day) of IFN-γ accelerated AML development and supported LSC self-renewal in patient-derived AML-LSCs and in an LSC-enriched MLL-AF9-driven mouse model. Importantly, targeting the IFN-γ receptor IFNGR1 by using lentiviral shRNAs or neutralizing antibodies induced AML differentiation and delayed leukemogenesis in vitro and in mice. Overall, we uncovered essential roles for IFN-γ and IFNGR1 in AML stemness and showed that targeting IFNGR1 is a strategy to decrease stemness and increase differentiation in relapsed AML patients.

The transcription factor HIF2α partakes in the differentiation block of acute myeloid leukemia.

One of the defining features of acute myeloid leukemia (AML) is an arrest of myeloid differentiation whose molecular determinants are still poorly defined. Pharmacological removal of the differentiation block contributes to the cure of acute promyelocytic leukemia (APL) in the absence of cytotoxic chemotherapy, but this approach has not yet been translated to non-APL AMLs. Here, by investigating the function of hypoxia-inducible transcription factors HIF1α and HIF2α, we found that both genes exert oncogenic functions in AML and that HIF2α is a novel regulator of the AML differentiation block. Mechanistically, we found that HIF2α promotes the expression of transcriptional repressors that have been implicated in suppressing AML myeloid differentiation programs. Importantly, we positioned HIF2α under direct transcriptional control by the prodifferentiation agent all-trans retinoic acid (ATRA) and demonstrated that HIF2α blockade cooperates with ATRA to trigger AML cell differentiation. In conclusion, we propose that HIF2α inhibition may open new therapeutic avenues for AML treatment by licensing blasts maturation and leukemia debulking.

Clonally resolved single-cell multi-omics identifies routes of cellular differentiation in acute myeloid leukemia

Inter-patient variability and the similarity of healthy and leukemic stem cells (LSCs) have impeded the characterization of LSCs in acute myeloid leukemia (AML) and their differentiation landscape. Here, we introduce CloneTracer, a novel method that adds clonal resolution to single-cell RNA-seq datasets. Applied to samples from 19 AML patients, CloneTracer revealed routes of leukemic differentiation. Although residual healthy and preleukemic cells dominated the dormant stem cell compartment, active LSCs resembled their healthy counterpart and retained erythroid capacity. By contrast, downstream myeloid progenitors constituted a highly aberrant, disease-defining compartment: their gene expression and differentiation state affected both the chemotherapy response and leukemia's ability to differentiate into transcriptomically normal monocytes. Finally, we demonstrated the potential of CloneTracer to identify surface markers misregulated specifically in leukemic cells. Taken together, CloneTracer reveals a differentiation landscape that mimics its healthy counterpart and may determine biology and therapy response in AML.

TREM2 acts as a receptor for IL-34 to suppress acute myeloid leukemia in mice

AbstractThe bone marrow microenvironment supports leukocyte mobilization and differentiation and controls the development of leukemias, including acute myeloid leukemia (AML). Here, we found that the development of AML xenotransplants was suppressed in mice with osteoclasts tuberous sclerosis 1 (Tsc1) deletion. Tsc1-deficient osteoclasts released a high level of interleukin-34 (IL-34), which efficiently induced AML cell differentiation and prevented AML progression in various preclinical models. Conversely, AML development was accelerated in mice deficient in IL-34. Interestingly, IL-34 inhibited AML independent of its known receptors but bound directly to triggering receptor expressed on myeloid cells 2 (TREM2), a key hub of immune signals. TREM2-deficient AML cells and normal myeloid cells were resistant to IL-34 treatment. Mechanistically, IL-34–TREM2 binding rapidly phosphorylated Ras protein activator like 3 and inactivated extracellular signal-regulated protein kinase 1/2 signaling to prevent AML cell proliferation and stimulate differentiation. Furthermore, TREM2 was downregulated in patients with AML and associated with a poor prognosis. This study identified TREM2 as a novel receptor for IL-34, indicating a promising strategy for overcoming AML differentiation blockade in patients with AML.

Abstract 1594: SMARCA2 (BRM) degraders promotes differentiation and inhibit proliferation in AML models

Abstract Dysregulated cellular differentiation is a major pathological feature of myeloid malignancies such as acute myeloid leukemia (AML). Targeting cellular differentiation programs has emerged as a novel therapeutic approach to treat patients with AML. Advantages of such differentiation therapy may include fewer systemic side-effects as well as opportunities to target leukemic stem cells (LSCs) and a broader range of clonal populations, likely resulting in lower frequencies of resistance and relapse in AML patients. The success of ATRA and decitabine in subsets of AML patients has proven that inducing differentiation can play a critical role in long-term durable responses. More agents targeting epigenetic regulators have been increasingly studied as differentiation inducers, including LSD1, DNMT1, Menin, and BET inhibitors. Recently, targeting SWI/SNF chromatin remodeling complexes has also been shown to regulate key leukemic gene expression signatures and induce AML differentiation. Small molecule inhibitors as well as gene knockdown for ATP-dependent SWI/SNF subunits SMARCA2 (BRM) and SMARCA4 (BRG1) are associated with re-direction of oncogenic transcriptional regulation to drive cellular differentiation and apoptosis in AML models. We have previously described the activity of highly potent, bispecific SMARCA2 degraders that efficiently promote SMARCA2 protein degradation in preclinical models. In the present study, we investigated the effects of our SMARCA2 selective degraders in AML models. Treatment with SMARCA2 degraders significantly inhibits AML cell line proliferation in vitro with IC50 ranging from 10 to 50 nM. In the SMARCA2 degrader treated cells, expression of PU.1 (SPI1), a key transcription factor in myeloid leukemias, was downregulated. In an in vivo OCI-AML3 xenograft model, treatment with a SMARCA2 selective degrader showed moderate tumor growth inhibition accompanied by robust increases in monocytic maturation markers CD11b and CD14. Further analyses of SMARCA2 degrader effects on global transcriptome and AML immunophenotypes as well as combination effects with other therapies are currently in progress. These findings highlight the potential of SMARCA2 degraders to target AML differentiation blocks and to improve the effectiveness of other therapeutic agents such as decitabine and venetoclax in AML patients. Citation Format: Anjana Agarwal, Olusola Peace Osinubi, Komali Vykuntam, Norman Fultang, Neha Bhagwat, Diane Heiser, Kris Vaddi, Koichi Ito, Peggy Scherle. SMARCA2 (BRM) degraders promotes differentiation and inhibit proliferation in AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1594.

LSD1 inhibition modulates transcription factor networks in myeloid malignancies.

Acute Myeloid Leukemia (AML) is a type of cancer of the blood system that is characterized by an accumulation of immature hematopoietic cells in the bone marrow and blood. Its pathogenesis is characterized by an increase in self-renewal and block in differentiation in hematopoietic stem and progenitor cells. Underlying its pathogenesis is the acquisition of mutations in these cells. As there are many different mutations found in AML that can occur in different combinations the disease is very heterogeneous. There has been some progress in the treatment of AML through the introduction of targeted therapies and a broader application of the stem cell transplantation in its treatment. However, many mutations found in AML are still lacking defined interventions. These are in particular mutations and dysregulation in important myeloid transcription factors and epigenetic regulators that also play a crucial role in normal hematopoietic differentiation. While a direct targeting of the partial loss-of-function or change in function observed in these factors is very difficult to imagine, recent data suggests that the inhibition of LSD1, an important epigenetic regulator, can modulate interactions in the network of myeloid transcription factors and restore differentiation in AML. Interestingly, the impact of LSD1 inhibition in this regard is quite different between normal and malignant hematopoiesis. The effect of LSD1 inhibition involves transcription factors that directly interact with LSD1 such as GFI1 and GFI1B, but also transcription factors that bind to enhancers that are modulated by LSD1 such as PU.1 and C/EBPα as well as transcription factors that are regulated downstream of LSD1 such as IRF8. In this review, we are summarizing the current literature on the impact of LSD1 modulation in normal and malignant hematopoietic cells and the current knowledge how the involved transcription factor networks are altered. We are also exploring how these modulation of transcription factors play into the rational selection of combination partners with LSD1 inhibitors, which is an intense area of clinical investigation.