Differentiation Of Leukemia Cells Research Articles

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.

Novel Role of Mitochondrial Folate Metabolism in Cell Fate Decisions and Leukaemogenesis

Folate-mediated one carbon (1C) metabolism sustains proliferation of rapidly dividing cells through its direct contribution to nucleotide biosynthesis. In brief, new 1C units are donated from the amino acid serine (the primary source of 1C units), while folate molecules function as 1C unit carriers. Accordingly, antifolates have been used in the treatment of several malignancies, particularly in the field of haemato-oncology. Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder that arises in a single haematopoietic stem cell with the introduction of the BCR::ABL1 oncoprotein. Although it is generally accepted that CML is driven by leukaemic stem cells (LSCs) that are insensitive to standard therapy due to their quiescent/slow-cycling status, the role of folate metabolism in LSCs remains undescribed. Transcriptomic analysis of patient-derived CML LSCs (CD34 +38 -) revealed a significant upregulation of folate metabolism genes, including the mitochondrial hydroxymethyltransferase (SHMT2; p≤0.05), when compared with normal counterparts. Furthermore, CML stem/progenitor (CD34 +) cells displayed a significant increase in the exchange rate of formate (folate intermediate necessary for nucleotide synthesis) compared to the secretome of normal CD34 + cells (p<0.05), suggesting an upregulation of 1C metabolism in therapy resistant CML cells. Following SHMT2 knockout (KO), we observed a strong antiproliferative effect and significantly impaired tumour formation in CML cell line xenograft models (p<0.01), verifying that mitochondrial 1C metabolism is not dispensable for CML cells. Metabolic characterisation using liquid chromatography-mass spectrometry revealed that both genetic and pharmacological inhibition (using the SHMT1/2 inhibitor, SHIN1) of 1C metabolism results in a significant decrease in de novo purine synthesis. Furthermore, we uncovered activation of AMPK signalling and suppression of mTORC1 activity following mitochondrial 1C metabolism inhibition. Notably, formate supplementation or reconstitution of purine levels was sufficient to reverse the effect on AMPK and mTORC1. Phenotypically, SHIN1 treatment induced the expression of erythropoiesis markers CD71 and Glycophorin A in CML cells, including patient derived CD34 + cells. AMPKα1/α2 KO revealed that the increased expression of these markers was independent of AMPK activity. Mechanistically, we discovered that reconstitution of purine levels (adenine) and mTORC1 activity prevented erythroid differentiation following SHMT1/2 inhibition, highlighting that depletion of 1C units drives differentiation of leukaemic cells through mTORC1-mediated purine sensing. Of clinical relevance, combination of SHIN1 with imatinib, a frontline treatment for CML patients, decreased the number of therapy-resistant CML LSCs in a patient-derived xenograft model (p<0.05). Lastly, stable isotope-assisted metabolomics using 13C 315N 1-serine revealed that imatinib reduced labelling into purine nucleotides, but it maintained labelling into glutathione (GSH), the most abundant antioxidant in the cell, and its oxidised derivate (GSSG). Through generation of glycine, folate metabolism directly contributes to the GSH pool. These data suggest that standard therapy does not affect the incorporation of serine into GSH, implying that the synergistic effect with folate metabolism inhibition might be related to impeded antioxidant defence. Overall, our findings highlight a novel role for mitochondrial folate metabolism in stem cell fate decisions and leukaemogenesis. This work is available as a preprint: https://www.biorxiv.org/content/10.1101/2022.12.21.521404v1

Pyrimidine Starvation Is a Targetable Cancer Vulnerability: Mechanisms of Nucleotide Homeostasis

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease, though all AML exhibit a characteristic block in differentiation. Hence, much effort has been made to develop small molecules that promote leukemic cell differentiation in the treatment of AML. In 2016 we identified the enzyme dihydroorotate dehydrogenase (DHODH) as a therapeutic target in AML. DHODH is a mitochondrial enzyme that converts dihydroorotate to orotate in the 4th step of de novo pyrimidine synthesis. Without DHODH enzymatic activity, a cell is forced to rely on autophagy or extracellular salvage to maintain its intracellular pyrimidine pool. DHODH inhibitor therapy has shown broad preclinical efficacy across multiple cancer types including, glioma, neuroblastoma, breast, small cell lung and pancreatic cancer suggesting that malignant cells are specifically impaired in their ability to maintain nucleotide homeostasis during periods of limited availability. Despite these very encouraging pre-clinical results, the translation into human clinical trials has been met with limited anti-cancer activity and therapeutic success. This discordance between pre-clinical and clinical benefit likely speaks to our limited understanding of how normal and malignant cells respond to pyrimidine starvation. Methods: Using in vitro and in vivo assays to compare normal hematopoietic progenitor and leukemic cells we interrogated key nodes in pyrimidine synthesis to identify the mechanisms behind this selective, anti-leukemic activity in the context of nucleotide starvation: [1] Nucleotide measurement by HPLC, [2] Flux experiments by isotopic tracing, [3] Polysome profiling to compare the transcriptome and translatome, [4] Quantification of ribosome recycling by examining the lysosomes following ultracentrifugation and [5] Direct visualization of lysosomes using new techniques in cryogenic electron microscopy. Results: Treatment of leukemia cells with brequinar blocked de novo synthesis and led to the rapid depletion of intracellular pyrimidines. In comparison, normal hematopoietic CD34 positive progenitor cells had less depletion, suggesting a selective vulnerability of leukemic cells over normal cells. We used isotopically labeled glutamine (15N) and uridine (13C) to quantify the contribution of de novo synthesis and extracellular salvage under control and starvation conditions. The leukemia cell line THP1 showed a higher reliance on de novo synthesis than normal CD34 positive hematopoietic progenitors ( Figure 1A, left graphs). Furthermore, following DHODH inhibition, THP1 cells were less capable of extracellular salvage compared to normal cells ( Figure 1A, right graphs). Polysome profiling analysis in THP1 cells showed a dramatic decrease in translating ribosomes as well as dramatic decrease in ribosomal RNA. We identified a short-list of 22 genes whose translation is prioritized during nucleotide deprivation. Following DHODH inhibition and nucleotide starvation, cells upregulate their number of lysosomes, as quantified by flow cytometry. In addition, the contents of the lysosome include ribosomal proteins, whose abundance increased during nucleotide starvation, suggesting an ongoing recycling of ribosomes to meet the nucleotide demand of leukemic cells during starvation. Finally, for the first time we have been able to directly visualize (by cryogenic electron microscopy) ribosomes within lysosomes during periods of nucleotide starvation, a finding that was completely absent in leukemia cells cultured under normal conditions ( Figure 1B). Conclusion: We hypothesized that the transformation from normal to malignant cell is accompanied by changes in nucleotide metabolism that render the transformed cells more reliant on de novo synthesis, establishing a metabolic vulnerability (and inherent therapeutic window) in the use of DHODH inhibitors. Our work confirms that leukemic blasts differ in their dependence on de novo nucleotide synthesis and its ability to garner nucleotides via salvage or autophagy pathways. This balance appears to be disturbed in the setting of malignancy such that leukemic cells are empirically more sensitive to treatment targeting pyrimidine synthesis. Understanding the differences in how leukemic and normal cells handle their nucleotide pools at baseline, and under starvation conditions, will guide the clinical utility of this class of inhibitors.

Targeting EYA1 Activity in MLL- Rearranged Leukemia

The Mixed Lineage Leukemia gene (also known as MLL or KMT2A) is involved in chromosomal translocations in a subtype of acute myeloid (AML) and lymphoblastic leukemias (ALL). MLL translocations have been identified with more than 80 partner genes, and the most common AML translocation partner is MLLT3 (AF9). MLL-rearranged (MLL-r) leukemia is associated with a sudden onset, aggressive progression, and poor prognosis in comparison to non-MLL-r (MLL-nr) leukemias, and there is a need for more effective therapies. Previous work determined that AF9 directly interacts with multiple different regulatory proteins, including the BCL6 corepressor (BCOR). An AF9 point mutation (E531R) was identified that selectively disrupts BCOR binding to AF9 and MLL-AF9. The loss of BCOR binding to MLL-AF9 abrogated its leukemogenic ability in a mouse transplant model. RNA-seq analysis determined that of the MLL-AF9 direct target genes, EYA1 was the most down-regulated in the MLL-AF9 (E531R) point mutant-transformed cells. Addback of EYA1 into MLL-AF9 (E531R) cells partially rescued its leukemogenic ability, suggesting that EYA1 has an important role in MLL-AF9 leukemogenesis. EYA1 is a transcriptional coactivator and protein tyrosine phosphatase that is expressed during embryonic development and its re-expression has been implicated in several cancers, including MLL-r leukemia. The objective of this study is to investigate the in vitroand in vivo effects of EYA1 inhibition in MLL-AF9 transformed murine bone marrow stem/progenitor cells and in human MLL-rearranged (MLL-r) and non-rearranged (MLL-nr) leukemia cell lines. Cells were treated with small molecule inhibitors at varied concentrations and time points. Effects of inhibition were assessed with viability, cell morphology, cell cycle, surface and intracellular marker expression analyses, and in vivo transplant studies. EYA1 inhibition reduced cell viability by 50% - 90% in MLL-AF9 transformed murine bone marrow (mBM) cells and multiple human AML cell lines. EYA1 inhibition did not affect some human B-ALL cell lines, which also express undetectable levels of EYA1, indicating a correlation between the response to EYA1 inhibition and EYA1 expression. In MLL-r and MLL-nr cells, cell cycle analysis indicated a reduction in cells entering S-phase, stalling in G0/G1, with EYA1 inhibition as well as an induction in cellular senescence. Inhibiting EYA1 activity induced cell morphology changes toward a more differentiated state in MLL-AF9 mBM and several MLL-r and -nr cell lines. To confirm these qualitative results, cells were stained for myeloid differentiation markers, and cell surface expression of CD11b, Gr1, and CD14 increased in a dose- and time-dependent manner in all cell types, while HSC markers CD117 (c-KIT) and CD34 were reduced in MLL-AF9 mBM and human AML cells, respectively. In assessing the mechanism of EYA1 in MLL-r leukemia, MLL-AF9 cells treated with EYA1 inhibitors showed increased RNA pol II CTD Tyr1 phosphorylation levels (known EYA1 substrate) compared to vehicle-treated cells. Thus, unregulated EYA1 phosphatase activity targeting RNA Pol II CTD may contribute to leukemia development. Mice that received MLL-AF9 mBM cells treated with an EYA1 inhibitor had a significantly decreased rate of disease progression and increased survival compared to control. Bone marrow cells isolated from these mice showed increased expression of myeloid differentiation markers CD11b and Gr1 compared to vehicle-treated mice, confirming that EYA1 activity contributes significantly to leukemogenesis. The inhibition of EYA1 induces leukemia cell differentiation, cell cycle arrest and senescence, and cell death. Ongoing experiments include additional in vivo murine leukemia, further mechanistic analyses, and combinatorial experiments to evaluate the efficacy of a multi-target approach with EYA1 inhibition and other targeted-therapies. This work may provide a promising, novel therapeutic approach to MLL-r and MLL-nr leukemias that will help improve patient outcomes.

Extended exposure to low doses of azacitidine induces differentiation of leukemic stem cells through activation of myeloperoxidase.

Oral azacitidine (oral-Aza) treatment results in longer median overall survival (OS) (24.7 vs. 14.8 months in placebo) in patients with acute myeloid leukemia (AML) in remission after intensive chemotherapy. The dosing schedule of oral-Aza (14 days/28-day cycle) allows for low exposure of Aza for an extended duration thereby facilitating a sustained therapeutic effect. However, the underlying mechanisms supporting the clinical impact of oral-Aza in maintenance therapy remain to be fully understood. In this preclinical work, we explore the mechanistic basis of oral-Aza/extended exposure to Aza through in vitro and in vivo modeling. In cell lines, extended exposure to Aza results in sustained DNMT1 loss, leading to durable hypomethylation, and gene expression changes. In mouse models, extended exposure to Aza, preferentially targets immature leukemic cells. In leukemic stem cell (LSC) models, the extended dose of Aza induces differentiation and depletes CD34+CD38- LSC. Mechanistically, LSC differentiation is driven in part by increased myeloperoxidase (MPO) expression. Inhibition of MPO activity either by using an MPO-specific inhibitor or blocking oxidative stress, a known mechanism of MPO, partly reverses the differentiation of LSC. Overall, our preclinical work reveals novel mechanistic insights into oral-Aza and its ability to target LSC.

Decoding the Epigenetic Drivers of Menin-MLL Inhibitor Resistance in KMT2A-Rearranged Acute Myeloid Leukemia

Menin inhibitors that disrupt Menin-MLL interaction are candidate therapeutic agents for several acute myeloid leukemia (AML) subtypes such as KMT2A rearrangement and NPM1 mutation. Several Menin inhibitors are currently being assessed in clinical trials and have demonstrated promising initial results. However, the development of both innate and acquired resistance to Menin inhibitors could limit their therapeutic potential. While studies have found that around 40% of these resistance cases can be traced back to mutations in the Menin gene, the Menin gene remains unaltered in other majorities, suggesting the presence of other mechanisms driving resistance. Here, we developed a unique chromatin-focused CRISPR knockout library, named Advanced Catalogue of Epigenetic Regulators (ACER). The ACER library contains approximately 800 traditional and newly-predicted epigenetic regulators using epigenetic network analyses that integrated publicly available gene codependency (DepMap) and protein-protein interaction data (BioPlex) (Figure A). To understand the chromatin mechanisms responsible for Menin-MLL inhibition resistance, we employed the ACER library for CRISPR screen in the context of Menin inhibition. Notably, our screen identified non-canonical polycomb repressive complex PRC1.1 (PCGF1, BCOR, and RYBP) and PRC2.2 (EZH2, SUZ12, and EED) among the top hits that conferred resistance to MLL-Menin inhibition upon gene depletion (Figure B). Consistent with our screen results, the depletion of PRC1.1 components led to a markedly increase in IC50 values when treated with the Menin inhibitor VTP50469 in both human and murine KMT2A-rearranged cell models. While treatment with Menin inhibitors typically induces cellular differentiation in leukemia cells, depletion of PRC1.1 genes resulted in an almost complete blockage in this differentiation process. Menin inhibitors evict Menin from chromatin and repress the expression levels of Menin-MLL target genes like MEIS1, PBX3, and MEF2C. Strikingly, our ChIP-seq and RNA-seq data indicatethat the loss of PRC1.1 components doesn't impact Menin's displacement from chromatin or the repression of KMT2A target genes by the Menin inhibitor. This is consistent with recent data from phase I clinical trial of revumenib (SNDX-5613) and preclinical patient derived xenograft models, wherein the Menin inhibitor persistently inhibitedkey KMT2A targets, even in resistant samples. These findings highlightthe possibility that resistance to Menin inhibition arising from mechanisms independent of canonical KMT2A target genes. Importantly, our unbiased transcriptomic analysis revealed that MYC signatures as the most affected pathway in PRC1.1-depleted cells following Menin inhibition. Furthermore, we show that both the transcriptionally active Menin-MLL and the repressive PRC1.1 complexes coexist at the MYC promoter, and the interplay between these two complexes determines MYC transcription output in AML. Moreover, enforced expression of MYC leads to resistance against Menin inhibition, while genetic and pharmacologic targeting MYC in PRC1.1 deficient cells rescued the resistance to Menin inhibition. Finally, in our RNA-seq datasets, we observed a diminished monocyte differentiation signature after PRC1.1 depletion. Considering AML cells with monocytic signatures, typically seen in KMT2A-rearranged AML, often exhibit resistance to BCL-2 inhibitor venetoclax, we hypothesized that AML cells lacking PRC1.1 could be more susceptible to BCL2 inhibition. This was confirmed in our ACER library screen with venetoclax treatment, where the PRC1.1 components PCGF1 and BCOR stood out as top epigenetic factors rendering drug sensitivity. Our subsequent studies using murine AML models and human primary samples further validated that PRC1.1-deficient AML cells are markedly sensitive to venetoclax. Furthermore, we show that venetoclax can be employed to overcome the resistance to Menin-MLL inhibition. In summary, our study identifiesthe non-canonical PRC1.1 as a key epigenetic driver of Menin-MLL resistance through a KMT2A target gene-independent mechanism which involves aberrant activation of MYC. We have further provided evidence that AML cells with loss of PRC1.1 are hypersensitive to BCL-2 inhibitor Venetoclax, opening a new therapeutic avenue for tackling Menin-resistant AMLs driven by polycomb inactivation.

RUNX1::RUNX1T1 Impairs the Differentiation Potential of Primary AML Cells

Acute myeloid leukemias (AMLs) are often driven by genomic translocations, of which t(8;21) is the most frequent. The resulting gene fusion RUNX1::RUNX1T has been shown in cell line and transgenic expression models to impede cell differentiation and promote leukemic self-renewal. However, it has remained unclear if and how RUNX1::RUNX1T1 influences leukemic propagation in patient-derived AML cells. To address the role of RUNX1::RUNX1T1 in the differentiation status and heterogeneity of patient-derived leukemic blasts, we developed an siRNA lipid nanoparticle (LNP) delivery system for the fusion transcript knockdown (Issa et al, Leukemia 2023). For efficient delivery to primary cells, the LNPs were functionalized with a Leu-Asp-Val (LDV) tripeptide binding the Very Late antigen-4 receptor on leukocytes. Primary AML cells were cultured and expanded for several weeks using a serum-free co-culture system with human bone marrow-derived mesenchymal stromal cells (MSCs) (Swart et al, Pharmaceutics 2023). Treatment of t(8;21) patient-derived xenograft (PDX) and primary bone marrow aspirates depleted RUNX1::RUNX1T1 transcript and protein, as determined by RT-qPCR and western blotting. We unraveled the transcriptional repercussions of RUNX1::RUNX1T1 knockdown using bulk RNAseq and ATACseq in a PDX and identified 1068 up- and 1521 downregulated genes upon RUNX1::RUNX1T1 knockdown, as compared to mismatched siRNA-treated cells (padj < 0.001, log2FC > 2). Gene set enrichment analysis revealed loss of self-renewal potential and enhanced myeloid differentiation. Similarly, ATACseq revealed an increased GMP/monocyte-like chromatin accessibility and a decreased HCS/MPP-like one upon RUNX1::RUNX1T1 knockdown (Figure 1). Surprisingly and in contrast to previous cell line results, RUNX1::RUNX1T1 depletion correlated with increased cell cycle and DNA repair signatures. These combined data demonstrate that RUNX1::RUNX1T1 perturbance promotes differentiation of leukemic stem cells towards granulocytic and monocytic lineages. Furthermore, they suggest that the initial stages of differentiation are linked to increased proliferation in line with the increased proliferation capacity of GMPs compared to more immature progenitor states. To investigate the impact of RUNX1::RUNX1T1 on leukemic blast populations at greater granularity, we knocked RUNX1::RUNX1T1 down in 2 primary bone marrow aspirates and 1 PDX and performed single-cell (sc) RNAseq. Notably, principal component analysis revealed that loss of RUNX1::RUNX1T1 shifted all leukemic populations, while hardly affecting normal hematopoietic cells or MSCs (Figure 2). In all samples, pseudotime analysis showed a more differentiated state of cells upon RUNX1::RUNX1T1 depletion. For cell type annotation, we projected our datasets onto a reference normal bone marrow dataset (van Galen et al, Cell 2019). This projection confirmed differentiation toward granulocyte-monocyte progenitors (GMPs), granulocytes and monocytes. Strikingly, the confidence of cell type prediction according to the reference normal bone marrow was significantly and substantially higher upon RUNX1::RUNX1T1 depletion than in cells with unimpaired fusion transcript level, suggesting restoration of normal myeloid differentiation. In addition to that, primary RUNX1::RUNX1T1 AML samples also contained a new, distinct cell subpopulation that emerged upon RUNX1::RUNX1T1 depletion. That subpopulation could not be annotated as resembling any normal bone marrow cell type. However, it expressed eosinophil marker genes such as RNASE3 and EPX. We, therefore, labeled this cell subpopulation as aberrant eosinophilic. This reveals RUNX1::RUNX1T1's role in disrupting differentiation towards multiple lineages. Our work illustrates a strong dependency of the differentiation potential of t(8;21) AML cells on their driver gene RUNX1::RUNX1T1 in an ex vivo setting with PDX or primary cells. We demonstrate that RUNX1::RUNX1T1 hijacks more than one differentiation trajectory of the AML blasts. Our findings also highlight siRNA-loaded LNPs as an innovative therapeutic approach for fusion gene-driven AML by directly targeting the fusion gene as the leukemic driver.

Therapeutic targeting in pediatric acute myeloid leukemia with aberrant HOX/MEIS1 expression

Despite advances in the clinical management of childhood acute myeloid leukemia (AML) during the last decades, outcome remains fatal in approximately one third of patients. Primary chemoresistance, relapse and acute and long-term toxicities to conventional myelosuppressive therapies still constitute significant challenges and emphasize the unmet need for effective targeted therapies. Years of scientific efforts have translated into extensive insights on the heterogeneous spectrum of genetics and oncogenic signaling pathways of AML and identified a subset of patients characterized by upregulation of HOXA and HOXB homeobox genes and myeloid ecotropic virus insertion site 1 (MEIS1). Aberrant HOXA/MEIS1 expression is associated with genotypes such as rearrangements in Histone-lysine N-methyltransferase 2A (KMT2A-r), nucleoporin 98 (NUP98-r) and mutated nucleophosmin (NPM1c) that are found in approximately one third of children with AML. AML with upregulated HOXA/MEIS1 shares a number of molecular vulnerabilities amenable to recently developed molecules targeting the assembly of protein complexes or transcriptional regulators. The interaction between the nuclear scaffold protein menin and KMT2A has gained particular interest and constitutes a molecular dependency for maintenance of the HOXA/MEIS1 transcription program. Menin inhibitors disrupt the menin-KMT2A complex in preclinical models of KMT2A-r, NUP98-r and NPM1c acute leukemias and its occupancy at target genes leading to leukemic cell differentiation and apoptosis. Early-phase clinical trials are either ongoing or in development and preliminary data suggests tolerable toxicities and encouraging efficacy of menin inhibitors in adults with relapsed or refractory KMT2A-r and NPM1c AML. The Pediatric Acute Leukemia/European Pediatric Acute Leukemia (PedAL/EUPAL) project is focused to advance and coordinate informative clinical trials with new agents and constitute an ideal framework for testing of menin inhibitors in pediatric study populations. Menin inhibitors in combination with standard chemotherapy or other targeting agents may enhance anti-leukemic effects and constitute rational treatment strategies for select genotypes of childhood AML, and provide enhanced safety to avoid differentiation syndrome. In this review, we discuss the pathophysiological mechanisms in KMT2A-r, NUP98-r and NPM1c AML, emerging molecules targeting the HOXA/MEIS1 transcription program with menin inhibitors as the most prominent examples and future therapeutic implications of these agents in childhood AML.

Hematopoietic stem cells suppress proliferation and enhance differentiation of leukemia cells through regulating apoptotic and inflammatory genes.

Stem cells are known to play an important role in tumor treatment and many of them have shown tumor-suppressing ability in different cancers; however, whether hematopoietic stem cells (HSCs) have growth-inhibiting effects on leukemia cells has not been fully evaluated. Herein, we aimed to demonstrate the growth-restraining function of HSCs in acute leukemia treatment. Cell fusion experiment was conducted by PEG-1500. The viability, proliferation, apoptosis and differentiation of leukemia cells were evaluated by cell counting, CCK-8 and flow cytometry analysis. The morphological changes were imaged using a fluorescence microscope. The expression of genes was detected by quantitative reverse transcription PCR (qRT-PCR). We observed that HSCs and their lytic extracts had the capability to suppress leukemia cells proliferation, promote apoptosis and especially induce acute myelogenous leukemia (AML) cells differentiation, which might have an effect on differentiation therapy to leukemia especially AML treatment. The expression levels of Bcl-2, Survivin decreased and Bax increased following HSCs extracts treatment. Furthermore, the expression of inflammatory cytokines also changed in AML cells which might have to do with the mechanism of HSCs/extracts suppressing effect. HSCs and their extracts can suppress the proliferation of leukemia cells and enhance the differentiation of AML cells and using the extracts of HSCs might be a probable therapeutic option for acute leukemia.

Targeting cis-regulatory elements of FOXO family is a novel therapeutic strategy for induction of leukemia cell differentiation

Differentiation therapy has been proposed as a promising therapeutic strategy for acute myeloid leukemia (AML); thus, the development of more versatile methodologies that are applicable to a wide range of AML subtypes is desired. Although the FOXOs transcription factor represents a promising drug target for differentiation therapy, the efficacy of FOXO inhibitors is limited in vivo. Here, we show that pharmacological inhibition of a common cis-regulatory element of forkhead box O (FOXO) family members successfully induced cell differentiation in various AML cell lines. Through gene expression profiling and differentiation marker-based CRISPR/Cas9 screening, we identified TRIB1, a complement of the COP1 ubiquitin ligase complex, as a functional FOXO downstream gene maintaining an undifferentiated status. TRIB1 is direct target of FOXO3 and the FOXO-binding cis-regulatory element in the TRIB1 promoter, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), played a critical role in differentiation blockade. Thus, we designed a DNA-binding pharmacological inhibitor of the FOXO-FRE-T interface using pyrrole-imidazole polyamides (PIPs) that specifically bind to FRE-T (FRE-PIPs). The FRE-PIPs conjugated to chlorambucil (FRE-chb) inhibited transcription of TRIB1, causing differentiation in various AML cell lines. FRE-chb suppressed the formation of colonies derived from AML cell lines but not from normal counterparts. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. In conclusion, targeting cis-regulatory elements of the FOXO family is a promising therapeutic strategy that induces AML cell differentiation.

Epigenetic targeting to enhance acute myeloid leukemia-directed immunotherapy.

AML is a malignant disease of hematopoietic progenitor cells with unsatisfactory treatment outcome, especially in patients that are ineligible for intensive chemotherapy. Immunotherapy, comprising checkpoint inhibition, T-cell engaging antibody constructs, and cellular therapies, has dramatically improved the outcome of patients with solid tumors and lymphatic neoplasms. In AML, these approaches have been far less successful. Discussed reasons are the relatively low mutational burden of AML blasts and the difficulty in defining AML-specific antigens not expressed on hematopoietic progenitor cells. On the other hand, epigenetic dysregulation is an essential driver of leukemogenesis, and non-selective hypomethylating agents (HMAs) are the current backbone of non-intensive treatment. The first clinical trials that evaluated whether HMAs may improve immune checkpoint inhibitors' efficacy showed modest efficacy except for the anti-CD47 antibody that was substantially more efficient against AML when combined with azacitidine. Combining bispecific antibodies or cellular treatments with HMAs is subject to ongoing clinical investigation, and efficacy data are awaited shortly. More selective second-generation inhibitors targeting specific chromatin regulators have demonstrated promising preclinical activity against AML and are currently evaluated in clinical trials. These drugs that commonly cause leukemia cell differentiation potentially sensitize AML to immune-based treatments by co-regulating immune checkpoints, providing a pro-inflammatory environment, and inducing (neo)-antigen expression. Combining selective targeted epigenetic drugs with (cellular) immunotherapy is, therefore, a promising approach to avoid unintended effects and augment efficacy. Future studies will provide detailed information on how these compounds influence specific immune functions that may enable translation into clinical assessment.

Antileukemic potential of Nile blue-mediated photodynamic therapy on HL60 human myeloid leukemia cells.

Photodynamic therapy (PDT) has received great attention over the past decade in the treatment of diseases such as leukemia which is a cancer of the blood and bone marrow cells that causes a significant number of deaths worldwide. In this study, it was aimed to investigate the effects of Nile blue-mediated PDT (NB-mediated PDT) on HL60 cells. The effect of NB-mediated PDT on cell proliferation was evaluated with cell volume analysis using flow cytometry at 24 h. Cell apoptosis, ROS production, mitochondrial membrane potential, and cell cycle analysis were evaluated using annexin V-FITC, H2DCFDA, JC-1, and PI staining, respectively, by flow cytometry and fluorescence microscopy. The morphological and ultrastructural analyses were examined by Giemsa staining and SEM. CD11b staining is used to determine the differentiation of leukemia cells. NB-mediated PDT induced an apoptotic response at 12.5 μM in HL60 cells. When Giemsa staining and SEM images were evaluated, apoptotic bodies, holes, and occasional folds were detected on the surfaces of cells in the NB-mediated PDT group. The NB-mediated PDT had no effect on the differentiation of leukemia cells, but this therapy affects the growth of HL60 cells in vitro, which may provide a new idea for removing leukemic cells from bone marrow intended for autologous transplant.

Elucidating the cell metabolic heterogeneity during hematopoietic lineage differentiation based on Met-Flow.

Cell metabolism is critically involved in the differentiation of the hematopoietic lineage and, therefore, has attracted the attention of researchers, however, in-depth studies on cellular metabolic activity of hematopoietic cells (HCs) require attention. This investigation compared the metabolic activity of HCs at critical lineage differentiation stages, including hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and differentiated blood cells, via multiple methods and basic reference values. Primary metabolic processes of HCs, including anabolism, catabolism, phosphate, and glucose metabolism, were analyzed, and their maps were drawn. The data revealed that GLUT1 expression in HSCs was substantially higher than in all progenitor cells and mature myeloid blood cells, indicating their strong glucose uptake capacity. In myeloid differentiation, the ACAC expression of HPC2 was markedly higher than in neutrophils and monocytes. The ACAC, ASS1, ATP5A, and PRDX2 of HPC2 expression in lymphoid differentiation was substantially greater than in B and Natural-killer cells. CLP, CMP, GMP, MEP, and HPC1 inherit increased glucose uptake stem cell properties. In lymphocyte subsets, the expression of ACAC, ASS1, ATP5A, CPT1A, and PRDX2 in CD4+ T subgroups (naive and memory CD4+ T and nTreg) were elevated than in B subgroups (pro-, pre-, immature and mature Bs) and CD8+ T subgroups. Furthermore, leukemia stem cells (LSCs) had increased levels of ACAC, CPT1A, G6PD, IDH2, and PRDX2 than leukemia cells, indicating a stronger metabolic capacity of LSCs than differentiated leukemia cells.

Syntheses, characterization of Imidazo[4,5-f][1,10]phenanthroline derivative and As (III) complex, In vitro evaluation, the determine of apoptosis mechanism; theoretical and quantum studies

In the present work, a novel ligand, (4-amino-3-methoxybenzaldehyde)-1H-imidazo[4,5-f][1,10] phenanthroline (AMIP) based on the formation of an imidazole ring and [As(AMIP)](Cl)]Cl2 was synthesized and characterized through Fourier-transform infrared (FT-IR) spectroscopy. In addition, the anticancer activity of [As(AMIP)](Cl)]Cl2 complex was tested against human acute promyelocytic leukemia cells (HL-60) and granulocytic differentiation of human promyelocytic leukemia cells (NB4). The MTT results showed IC50 values of 7.31 ± 15 and 8.4 ± 62 μM for As (III) complex against NB4 and HL-60, respectively. In a study on both cell lines, As (III) reduced gene expression Bcl-2 and Bcl-xl, increased Bax and Bad, and activated caspase-3, resulting in apoptosis. The dye Hoechst 33342 showed increased apoptosis induction in the NB4 cell line than in the HL-60 cell line. BrdU test exhibited an inhibitory effect on DNA synthesis. The protein levels of caspase-3 were enhanced in a dose-dependent manner in both HL-60 and NB-4 cell lines. Based on the Annexin V-FITC/PI assay, apoptosis was the primary mechanism of cell death. The real-time polymerase chain reaction (real-time PCR) results demonstrated that the expression level of Atg 12, Atg 7, Lc 3, and Beclin-1 genes, antiapoptotic genes including Bcl-2 and Bcl-xl, and apoptotic genes containing BAD and BAX was improved. Furthermore, the hemolysis assay showed the anti-hemolytic activity of the complex. Moreover, quantum chemical calculations were utilized to optimize all structures, and the output results were employed to study molecular docking of compounds with caspase-3 tethered to irreversible inhibitor (PDB ID: 1NMS) and DNA with sequence d(CCGTCGACGG) (PDB ID:423D). The results showed that As (III) complex had the lowest-energy conformation, suggesting the optimal interaction.

Role of miR‑let‑7c‑5p/c‑myc signaling axis in the committed differentiation of leukemic THP‑1 cells into monocytes/macrophages.

In a preliminary experiment, it was found that c-myc expression was decreased following the differentiation of THP-1 cells into monocytes/macrophages induced by phorbol 12-myristate 13 acetate (PMA) + lipopolysaccharide (LPS) + interferon (IFN)-γ. The expression of miR-let-7c-5p was then found to be elevated by cross-sectional analysis using TargetScan and PubMed and differential microarray analysis. The present study aimed to investigate the role of the miR-let-7c-5p/c-myc signaling axis in the committed differentiation of THP-1 leukemic cells into monocytes/macrophages induced by PMA + LPS + IFN-γ. Human THP-1 leukemic cells were induced to differentiate into monocytes/macrophages by PMA + LPS + IFN-γ. Following induction for 48 h, the growth density of the THP-1 cells was observed directly under an inverted microscope, cell proliferation was measured using Cell Counting Kit-8 assay and the cell cycle and the expression of differentiation-related antigens (CD11b and CD14) were measured using flow cytometry. The mRNA expression of miR-let-7c-5p and c-myc was detected using reverse transcription-quantitative PCR and the protein expression of c-myc was detected using western blot analysis. Dual luciferase reporter gene analysis was used to detect the targeted binding of miR-let-7c-5p on the 3'UTR of c-myc. The relative expression of miR-let-7c-5p and c-myc genes in THP-1 cells induced by PMA + LPS + IFN-γ was found to be up- and downregulated respectively, and expression of miR-let-7c-5p was negatively correlated with the expression of c-myc gene. Dual luciferase reporter gene assays confirmed that miR-let-7c-5p targeted the 3'UTR of c-myc and inhibited luciferase activity. Following transfection with miR-let-7c-5p mimics, the expression of c-myc was markedly downregulated and the proliferative ability of the THP-1 cells was decreased, while the expression rate of CD11b and CD14 was significantly increased. The rescue experiment revealed that the effects of miR-let-7c-5p mimics on the proliferation and differentiation of THP-1 cells were attenuated by transfection with c-myc overexpression vector. Together, the findings of the present study demonstrated that miR-let-7c-5p can target the 3'UTR region of c-myc and that the miR-let-7c-5p/c-myc signaling axis is one of the critical pathways involved in the directional differentiation of leukemic cells into monocytes/macrophages.

The Effects of Green Tea Catechins in Hematological Malignancies.

Green tea catechins are bioactive polyphenol compounds which have attracted significant attention for their diverse biological activities and potential health benefits. Notably, epigallocatechin-3-gallate (EGCG) has emerged as a potent apoptosis inducer through mechanisms involving caspase activation, modulation of Bcl-2 family proteins, disruption of survival signaling pathways and by regulating the redox balance, inducing oxidative stress. Furthermore, emerging evidence suggests that green tea catechins can modulate epigenetic alterations, including DNA methylation and histone modifications. In addition to their apoptotic actions, ROS signaling effects and reversal of epigenetic alterations, green tea catechins have shown promising results in promoting the differentiation of leukemia cells. This review highlights the comprehensive actions of green tea catechins and provides valuable insights from clinical trials investigating the therapeutic potential of green tea catechins in leukemia treatment. Understanding these multifaceted mechanisms and the outcomes of clinical trials may pave the way for the development of innovative strategies and the integration of green tea catechins into clinical practice for improving leukemia patient outcomes.

Critical role of MXRA7 in differentiation blockade in human acute promyelocytic leukemia cells

The biology of the matrix remodeling associated 7 (MXRA7) gene had been ill-defined. Inspired by public datasets and domestic findings concerning MXRA7, we hypothesized that MXRA7 might be involved in the pathogenesis of leukemia. This study aimed to investigate the role and effect of MXRA7 expression in acute promyelocytic leukemia (APL) cells differentiation and behavior. Bioinformatics of public datasets revealed MXRA7 mRNA was expressed highly in acute myeloid leukemia (AML), especially in APL. High expression of MXRA7 was associated with poor overall survival of AML patients. We confirmed MXRA7 expression was upregulated in APL patients and cell line. Knockdown or overexpression of MXRA7 did not affect the proliferation of NB4 cells directly. Knockdown of MXRA7 in NB4 cells promoted drug induced cell apoptosis, while overexpression of MXRA7 had no obvious influence on the drug induced cell apoptosis. Lowering MXRA7 protein levels in NB4 cells promoted ATRA-induced cell differentiation possibly through decreasing PML-RARα level and increasing PML and RARα levels. Correspondingly, overexpression of MXRA7 showed the consistent results. We also demonstrated that MXRA7 altered the expression of genes involved in leukemic cell differentiation and growth. Knockdown of MXRA7 upregulated the expression levels of C/EBPB, C/EBPD and UBE2L6, and downregulated the expression levels of KDM5A, CCND2 and SPARC. Moreover, knockdown of MXRA7 inhibited the malignancy of NB4 cells in NOD-SCID mice model. In conclusion, this study demonstrated that MXRA7 influences the pathogenesis of APL via regulation of the cell differentiation. The novel findings about the role of MXRA7 in leukemia not only shed lights on the biology of this gene, but also proposed this gene as a new target for APL treatment.

NLRP3 participates in the differentiation and apoptosis of PMA‑treated leukemia cells.

Acute myeloid leukemia (AML) is a clonal malignant proliferative disease. In recent years, with the use of all‑trans retinoic acid to induce cancer cell differentiation in acute promyelocytic leukemia, and its advantages of high efficacy and low toxic side effects, tumor differentiation therapy has become a research hotspot; however, the mechanisms underlying its role remain to be fully established. Nod‑like receptor family pyridine domain containing 3 (NLRP3) is the most extensively studied and well‑characterized inflammasome, is involved in a variety of inflammation‑related diseases, including cancer, and is a very attractive potential target for the study of novel therapeutic agents. Activation of the NLRP3 inflammasome is a double‑edged sword in tumor therapy, with evidence of protective anti‑tumor and pro‑tumor effects in different types of cancer. Whether the NLRP3 inflammasome promotes disease progression or exerts a protective anti‑tumor effect in hematological malignancies remains contested. In the present study, the protective anti‑tumor effects of NLRP3 on leukemia cells during their differentiation and maturation were investigated. It was found that the upregulation of NLRP3 expression induced using Phorbol 12‑Myristate 13‑Acetate played a role in promoting the differentiation and maturation of leukemia cells into monocytes/macrophages, and it was directly involved in the apoptosis of leukemia cells and the differentiation and maturation of CD11b+ cells. These results provide novel theoretical evidence for exploring the mechanism of differentiation therapy in leukemia and improves our understanding of the role of NLRP3 in hematologic tumors.

The Role of Leukemia Inhibitory Factor in Counteracting the Immunopathology of Acute and Chronic Lung Inflammatory Diseases

Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is highly expressed throughout the body in multiple tissues and cell types. LIF is primarily known to induce the differentiation of myeloid leukemia cells, but recent studies show that LIF has many other functions, including playing multiple roles in cancer and normal physiology. LIF expression is linked to cellular proliferation, metastasis, inflammation, and chemoresistance. LIF expression and secretion are triggered by many means and its downstream signaling can vary based on tissue types. Recent publications suggest that LIF may play a role in pulmonary diseases and its regulation is altered through external factors, such as cigarette smoke, inflammation stimuli, or infections. This review outlines the current knowledge of the function of LIF protein, mediators of LIF expression, receptors it interacts with, downstream LIF signaling, and possible pulmonary outcomes mediated by LIF.

Discovery of new tetrahydroisoquinolines as potent and selective LSD1 inhibitors for the treatment of MLL-rearranged leukemia

Histone lysine-specific demethylase 1 (LSD1) is a promising target for cancer therapy. Here, we performed the design, synthesis, and extensive structure-activity relationship (SAR) studies based on our previously discovered natural LSD1 inhibitor, higenamine. We found that the tetracyclic tetrahydroisoquinoline FY-21 is a potent and selective inhibitor of LSD1 (IC50 = 340 nM). FY-21 inhibited leukemia cell proliferation and colony formation and increased the level of p53 expression. Meanwhile, FY-21 reduced the mRNA levels of the transcription factors HOXA9 and MEIS1. Furthermore, FY-21 significantly induced leukemia cell differentiation. In vivo studies showed that FY-21 prolonged the survival rate of leukemia mice. Collectively, FY-21 is a potent, selective LSD1 inhibitor and can serve as a lead compound for the development of novel and highly effective LSD1 inhibitors for AML treatment.