Erythroleukemia Cells Research Articles

SLC25A38 is required for mitochondrial pyridoxal 5’-phosphate (PLP) accumulation

Many essential proteins require pyridoxal 5’-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5’-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5’-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5’-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5’-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5’-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5’-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia.

Nootkatone Derivative Nootkatone-(E)-2-iodobenzoyl hydrazone Promotes Megakaryocytic Differentiation in Erythroleukemia by Targeting JAK2 and Enhancing JAK2/STAT3 and PKCδ/MAPK Crosstalk.

Erythroleukemia, a complex myeloproliferative disorder presenting as acute or chronic, is characterized by aberrant proliferation and differentiation of erythroid cells. Although nootkatone, a sesquiterpene derived from grapefruit peel and Alaska yellow cedar, has shown anticancer activity predominantly in solid tumors, its effects in erythroleukemia remain unexplored. This study aimed to investigate the impact of nootkatone and its derivatives on erythroleukemia. Our results demonstrate that the nootkatone derivative nootkatone-(E)-2-iodobenzoyl hydrazone (N2) significantly inhibited erythroleukemia cell proliferation in a concentration- and time-dependent manner. More importantly, N2 induced megakaryocytic differentiation, as evidenced by significant morphological changes, and upregulation of megakaryocytic markers CD41 and CD61. In vivo, N2 treatment led to a marked increase in platelet counts and megakaryocytic cell counts. Mechanistically, N2 activated a crosstalk between the JAK2/STAT3 and PKCδ/MAPK signaling pathways, enhancing transcriptional regulation of key factors like GATA1 and FOS. Network pharmacology and experimental validation confirmed that N2 targeted JAK2, and knockdown of JAK2 abolished N2-induced megakaryocytic differentiation, underscoring JAK2's critical role in erythroleukemia differentiation. In conclusion, N2 shows great promise as a differentiation therapy for erythroleukemia, offering a novel approach by targeting JAK2-mediated signaling pathways to induce megakaryocytic differentiation.

P2 Receptor Antagonists Rescue Defective Heme Content in an In Vitro SLC25A38-Associated Congenital Sideroblastic Anemia Cell Model.

Mutations in the SLC25A38 gene are responsible for the second most common form of congenital sideroblastic anemia (CSA), a severe condition for which no effective treatment exists. We developed and characterized a K562 erythroleukemia cell line with markedly reduced expression of the SLC25A38 protein (A38-low cells). This model successfully recapitulated the main features of CSA, including reduced heme content and mitochondrial respiration, increase in mitochondrial iron, ROS levels and sensitivity to oxidative stress. Notably, our study uncovered a new role for extracellular pyridoxal 5'-phosphate (PLP) and other P2 receptor antagonists in rescuing the altered parameters of A38-low cells (for example, the heme content of the A38-low cells was increased from about 50% to about 80% by the P2 receptor antagonists treatment compared with the value of the controls). These findings suggest that targeting P2 receptors could represent a promising therapeutic approach for SLC25A38-associated CSA.

Rocaglamide reprograms glucose metabolism in erythroleukemic cells via c-MYC transcriptional regulation of TXNIP and HK2

Ethnopharmacological relevanceThe theory of traditional Chinese medicine (TCM) views leukemia as an imbalance between cell growth and death mainly caused by blood stasis. Medicinal plants Aglaia Lour. (family Meliaceae) are traditionally used as folk medicine in China. It possesses the effects of removing blood stasis and swelling for treatment of cancer. Rocaglamide (RocA) is the main active phytochemical component of the genus Aglaia Lour. Possessing highly anti-leukemia properties. However, the molecular mechanisms by which RocA exerts its anti-growth effect on erythroleukemia cells are largely unknown. Aim of the studyThis study aimed to explore the underlying mechanism and glucose metabolism regulation effects of RocA responsible for its anti-erythroleukemia activity. Materials and methodsHuman erythroleukemic cells were tested for glucose metabolism and treated with glucose deprivation and RocA. MTT assay, cell cycle and apoptosis were used to elucidate growth inhibition. Glucose uptake, glucose consumption and lactate production were evaluated for identification of glucose metabolism. Luciferase assay and ChIP were used to examine the transcriptional activity of c-MYC on the conserved E-boxes binding of the TXNIP (thioredoxin-interacting protein) and HK2 (hexokinase 2) genes. siRNA, shRNA and exogenous transfection were employed to elucidate the effects of TXNIP and HK2 on glucose metabolism. ResultsWe find that glucose deprivation results in growth inhibition, cell cycle arrest and extensive apoptosis in erythroleukemic cells accompanied by downregulation of c-MYC and HK2, responsible for glucose metabolism. The similar results emerged in RocA treated erythroleukemic cells in presence of glucose. RocA is shown to decrease glucose uptake, glucose consumption and lactate production. Mechanistically, RocA dramatically increases TXNIP expression through interference with c-MYC binding to the promoter of the TXNIP gene. RocA also represses c-MYC transcriptional recognition of conserved E-boxes in the HK2 first intron, resulting in HK2 loss. These results implicate c-MYC as an important regulator of TXNIP and HK2 after RocA treatment. TXNIP overexpression or knockdown of HK2 suppresses the proliferation of erythroleukemic cells. Ectopic TXNIP expression restricts glucose uptake and HK2 suppression decreases glucose utilization. Further, our data suggests that loss of HK2 weakens the RocA-driven inhibition effects. We propose repression of c-MYC or the binding by RocA upregulates TXNIP and downregulates HK2, possibly contributes to growth inhibition in human erythroleukemic cells. ConclusionsThis study uncovers molecular mechanism of RocA against leukemic cells proliferation, linking the anti-erythroleukemia properties of RocA to glucose metabolism.

Engineered bacterial lipoate protein ligase A (lplA) restores lipoylation in cell models of lipoylation deficiency

Protein lipoylation, a vital lysine post-translational modification, plays a crucial role in the function of key mitochondrial tricarboxylic acid cycle enzymatic complexes. In eukaryotes, lipoyl post-translational modification synthesis occurs exclusively through de novo pathways, relying on lipoyl synthesis/transfer enzymes, dependent upon mitochondrial fatty acid and Fe-S cluster biosynthesis. Dysregulation in any of these pathways leads to diminished cellular lipoylation. Efficient restoration of lipoylation in lipoylation deficiency cell states using either chemical or genetic approaches has been challenging because of pathway complexity and multiple upstream regulators. To address this challenge, we explored the possibility that a bacterial lipoate protein ligase A (lplA) enzyme, which can salvage free lipoic acid bypassing the dependency on de novo synthesis, could be engineered to be functional in human cells. Overexpression of the engineered lplA in lipoylation null cells restored lipoylation levels, cellular respiration, and growth in low glucose conditions. Engineered lplA restored lipoylation in all tested lipoylation null cell models, mimicking defects in mitochondrial fatty acid synthesis (MECR KO), Fe-S cluster biosynthesis (BOLA3 KO), and specific lipoylation-regulating enzymes (FDX1 [ferredoxin 1], LIAS [lipoyl synthase], and LIPT1 [lipoyl (octanoyl) transferase 1] KOs). Furthermore, we describe a patient with a homozygous c.212C>T variant LIPT1 with a previously uncharacterized syndromic congenital sideroblastic anemia. K562 erythroleukemia cells engineered to harbor this missense LIPT1 allele recapitulate the lipoylation-deficient phenotype and exhibit impaired proliferation in low glucose that is completely restored by engineered lplA. This synthetic approach offers a potential therapeutic strategy for treating lipoylation disorders.

JK-1, a useful erythroleukemic cell line model to study a controlled erythroid differentiation from progenitors to terminal erythropoiesis

New hematopoietic cell models have recently emerged through immortalization of CD34 cells to study and understand various molecular mechanisms of erythropoiesis. Here, we characterize the JK-1 CML-derived cell line, previously shown to spontaneously differentiate without cytokines. Using an epigenetic differentiation inhibitor that keeps JK-1 in an early differentiation phase, we characterized 2 progenitor stages: BFU-E JK-1 and CFU-E JK-1 with CD34+/CD36− and CD34−/CD36 + phenotypes respectively. Then, using the PFI-1 inducer known to synchronously control the terminal differentiation of JK-1 cells, 5 precursor stages were obtained (ProE, Baso1-2, PolyC, OrthoC) and characterized via cell morphology, CD49a and Band3 markers. Enlarged phenotyping was carried out for the earlier phase, and expression kinetics of membrane proteins such as RhAG, RhD/CE, CD47, DARC and CD44 were performed on each stage of the terminal phase. Furthermore, since JK-1 offers the unique property of covering a broad spectrum of differentiation stages, we explored deeper the GATA2/GATA1 and the non-erythroid/erythroid spectrin ‘switching’. The possibility of obtaining large quantities of JK-1 cells at each stage of differentiation, as shown in this study, as well as the potential to genetically modify these cells, via CrisprCas9, makes their use of considerable interest for studying pathologies occurring during erythropoiesis.

Hydroxyurea reduces the levels of the fetal globin gene repressors ZBTB7A/LRF and BCL11A in erythroid cells in vitro

Abstract Objectives Hydroxyurea (HU) is the most widely used therapy for adults and children with sickle cell disease (SCD). It is believed to act largely by inducing the transcription of fetal γ-globin genes to generate fetal hemoglobin (HbF), which inhibits the pathological polymerization of sickle hemoglobin (HbS). The mechanisms by which hydroxyurea elevates HbF are unclear. We explored the hypothesis that hydroxyurea induces HbF expression by inhibiting the expression of 2 γ-globin gene repressors, BCL11A and ZBTB7A (also known as LRF), which normally bind the γ-globin gene promoters to inhibit their expression after birth. Methods We treated immortalized murine erythroleukemia cells and normal human donor CD34+ hematopoietic stem and progenitor cell-derived erythroblasts with hydroxyurea and measured the effects on globin, BCL11A and ZBTB7A protein and mRNA expression. Results Treating murine erythroleukemia cells or human CD34+ hematopoietic stem and progenitor cell-derived erythroblasts with hydroxyurea reduced the protein levels of BCL11A and ZBTB7A compared to the vehicle-treated control. BCL11A mRNA levels were reduced in both cell types upon hydroxyurea treatment. However, ZBTB7A mRNA levels were only reduced in human CD34+ hematopoietic stem and progenitor cell-derived erythroblasts. Conclusions Hydroxyurea can act in erythroid cells to reduce the levels and activity of two direct fetal γ-globin transcriptional repressors with accompanying de-repression of the γ-globin genes and induction of HbF, which may explain the mechanism of action leading to amelioration of symptoms in SCD patients treated with this drug.

Growth hormone is involved in GATA1 gene expression via STAT5B in human erythroleukemia and monocytic cell lines

GATAs are a family of transcription factors consisting of six members. Particularly, GATA1 and GATA2 have been reported to promote the development of erythrocytes, megakaryocytes, eosinophils, and mast cells. However, little information is available on the extracellular ligands that promote GATA1 expression. We evaluated whether growth hormone (GH) is an extracellular stimulator that participates in the signal transduction of GATAs, focusing on GATA1 expression in hematopoietic cell lineages. We used a reporter assay, RT-PCR, real-time quantitative PCR, and western blotting to evaluate GH-induced expression of GATA1 and GATA2 in the human erythroleukemic cell line K562 and the non-erythroid cell line U937. GATA1 expression in these hematopoietic cell lines increased at the transcriptional and protein levels in the presence of GH, and was inhibited by a STAT5 specific inhibitor. Cells transfected with activated STAT5B showed increased expression of GATA1. We identified functional STAT5B consensus sequences as binding site-158 bp from the transcription starting site in the GATA1 promoter region. These results suggest that GH directly induces GATA1 expression via GHR/JAK/STAT5 and is related to hematopoietic cell proliferation.

CDK9 phosphorylates RUNX1 to promote megakaryocytic fate in megakaryocytic-erythroid progenitors

AbstractThe specification of megakaryocytic (Mk) or erythroid (E) lineages from primary human megakaryocytic-erythroid progenitors (MEPs) is crucial for hematopoietic homeostasis, yet the underlying mechanisms regulating fate specification remain elusive. In this study, we identify RUNX1 as a key modulator of gene expression during MEP fate specification. Overexpression of RUNX1 in primary human MEPs promotes Mk specification, whereas pan-RUNX inhibition favors E specification. Although total RUNX1 levels do not differ between Mk progenitors (MkPs) and E progenitors (ErPs), there are higher levels of serine-phosphorylated RUNX1 in MkPs than ErPs, and mutant RUNX1 with phosphorylated-serine/threonine mimetic mutations (RUNX1-4D) significantly enhances the functional efficacy of RUNX1. To model the effects of RUNX1 variants, we use human erythroleukemia (HEL) cell lines expressing wild-type (WT), phosphomimetic (RUNX1-4D), and nonphosphorylatable (RUNX1-4A) mutants showing that the 3 forms of RUNX1 differentially regulate expression of 2625 genes. Both WT and RUNX1-4D variants increase expression in 40%, and decrease expression in another 40%, with lesser effects of RUNX1-4A. We find a significant overlap between the upregulated genes in WT and RUNX1-4D–expressing HEL cells and those upregulated in primary human MkPs vs MEPs. Although inhibition of known RUNX1 serine/threonine kinases does not affect phosphoserine RUNX1 levels in primary MEPs, specific inhibition of cyclin dependent kinase 9 (CDK9) in MEPs leads to both decreased RUNX1 phosphorylation and increased E commitment. Collectively, our findings show that serine/threonine phosphorylation of RUNX1 promotes Mk fate specification and introduce a novel kinase for RUNX1 linking the fundamental transcriptional machinery with activation of a cell type–specific transcription factor.

GPS2 promotes erythroid differentiation in K562 erythroleukemia cells primarily via NCOR1.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.

Development and Characterization of a Photocrosslinkable, Chitosan-Based, Nerve Growth Factor-Eluting Hydrogel for the Ocular Surface.

Recombinant human nerve growth factor (rhNGF; cenegermin-bkbj, OXERVATE) is the first and only U.S. Food and Drug Administration-approved treatment for moderate to severe neurotrophic keratopathy. The aim of this study was to determine the feasibility of incorporating a version of rhNGF in a mucoadhesive hydrogel capable of sustained drug release to the ocular surface. Hydrogels loaded with rhNGF were synthesized by conjugating chitosan with azidobenzoic acid (Az-Ch), adding rhNGF, and exposing the solution to ultraviolet (UV) radiation to induce photocrosslinking. Az-Ch hydrogels were evaluated for physical properties and rhNGF release profiles. Cytocompatbility of Az-Ch was assessed using immortalized human corneal limbal epithelial (HCLE) cells. TF1 erythroleukemic cell proliferation and HCLE cell proliferation and migration were used to assess the bioactivity of rhNGF released from Az-Ch hydrogels. Az-Ch formed hydrogels in <10 seconds of UV exposure and demonstrated high optical transparency (75-85 T%). Az-Ch hydrogels exhibited good cytocompatibility with no demonstratable effect on HCLE cell morphology or viability. rhNGF was released gradually over 24hours from Az-Ch hydrogels and retained its ability to induce TF1 cell proliferation. No significant difference was observed between rhNGF released from Az-Ch and freshly prepared rhNGF solutions on HCLE cell proliferation or percent wound closure after 12hours; however, both were significantly better than control (P < 0.01). rhNGF-loaded Az-Ch hydrogels exhibited favorable physical, optical, and drug-release properties, as well as retained drug bioactivity. This drug delivery system has the potential to be further developed for in vivo and translational clinical applications. Az-Ch hydrogels may be used to enhance rhNGF therapy in patients with NK.

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis.

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron-sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron-sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

PI3K/HSCB axis facilitates FOG1 nuclear translocation to promote erythropoiesis and megakaryopoiesis

Erythropoiesis and megakaryopoiesis are stringently regulated by signaling pathways. However, the precise molecular mechanisms through which signaling pathways regulate key transcription factors controlling erythropoiesis and megakaryopoiesis remain partially understood. Herein, we identified heat shock cognate B (HSCB), which is well known for its iron–sulfur cluster delivery function, as an indispensable protein for friend of GATA 1 (FOG1) nuclear translocation during erythropoiesis of K562 human erythroleukemia cells and cord-blood-derived human CD34+CD90+hematopoietic stem cells (HSCs), as well as during megakaryopoiesis of the CD34+CD90+HSCs. Mechanistically, HSCB could be phosphorylated by phosphoinositol-3-kinase (PI3K) to bind with and mediate the proteasomal degradation of transforming acidic coiled-coil containing protein 3 (TACC3), which otherwise detained FOG1 in the cytoplasm, thereby facilitating FOG1 nuclear translocation. Given that PI3K is activated during both erythropoiesis and megakaryopoiesis, and that FOG1 is a key transcription factor for these processes, our findings elucidate an important, previously unrecognized iron–sulfur cluster delivery independent function of HSCB in erythropoiesis and megakaryopoiesis.

A Carbon 21 Steroidal Glycoside with Pregnane Skeleton from Cynanchum atratum Bunge Promotes Megakaryocytic and Erythroid Differentiation in Erythroleukemia HEL Cells through Regulating Platelet-Derived Growth Factor Receptor Beta and JAK2/STAT3 Pathway.

Erythroleukemia is a rare form of acute myeloid leukemia (AML). Its molecular pathogenesis remains vague, and this disease has no specific therapeutic treatments. Previously, our group isolated a series of Carbon 21 (C-21) steroidal glycosides with pregnane skeleton from the root of Cynanchum atratum Bunge. Among them, we found that a compound, named BW18, can induce S-phase cell cycle arrest and apoptosis via the mitogen-activated protein kinase (MAPK) pathway in human chronic myeloid leukemia K562 cells. However, its anti-tumor activity against erythroleukemia remains largely unknown. In this study, we aimed to investigate the anti-erythroleukemia activity of BW18 and the underlying molecular mechanisms. Our results demonstrated that BW18 exhibited a good anti-erythroleukemia activity in the human erythroleukemia cell line HEL and an in vivo xenograft mouse model. In addition, BW18 induced cell cycle arrest at the G2/M phase and promoted megakaryocytic and erythroid differentiation in HEL cells. Furthermore, RNA sequencing (RNA-seq) and rescue assay demonstrated that overexpression of platelet-derived growth factor receptor beta (PDGFRB) reversed BW18-induced megakaryocytic differentiation in HEL cells, but not erythroid differentiation. In addition, the network pharmacology analysis, the molecular docking and cellular thermal shift assay (CETSA) revealed that BW18 could inactivate Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway, which might mediate BW18-induced erythroid differentiation. Taken together, our findings elucidated a novel role of PDGFRB in regulating erythroleukemia differentiation and highlighted BW18 as an attractive lead compound for erythroleukemia treatment.

Review of Sacred Herbal Medicine Aegle marmelos: A Potent Metastasis Inhibitor

Abstract: Aegle marmelos (A. marmelos) appears to be a significantly used ayurvedic medicine. This is a brilliantly composed nutritious fruit with carbohydrates, proteins, vitamins like riboflavin, thiamine, niacin, fatty acids, and minerals. Scientific studies have proved that A. marmelos has phytochemicals: carotenoids, phenolic, alkaloids, pectins, tannins, coumarins, flavonoids and terpenoids. Recent research on the effects of A. marmelos proved its anticancer, antimicrobial, cardioprotective, antidiabetic, and hepatoprotective activities. Extracts of various parts of plants such as leaves, bark, stems, fruits, and pulp subjected to preclinical studies conducted on cell lines showed apoptosis induction, inhibition of cell cycle and inhibition of proliferation of cells. Anti-cancer activity of A. marmelos was studied on selected cell lines of lung cancer, breast cancer, colon cancer and leukaemia. A. marmelos reported significant inhibitory effect on cell proliferation in cell line studies on A549, Ehrlich Ascites Carcinoma, MNU, DMBA, MCF-7, k562 cells, t-lymphoid Jurkat cells, b lymphoid Raji cells, erythroleukemic Hel cells, melanoma Colo 38 cells which supports the anticancer potential of it. Imperatorin has shown an antiproliferative effect on several cancer cell lines. The hydroethanolic extract of A. marmelos leaves showed a decrease in cell viability on A549 Human lung cancer cell line which works by fold change over control of the β-Catenin-m RNA and Wnt-mRNA expression of A549 and tumour growth suppression in DMBA induced carcinogenesis in rats. In human promyelocytic leukaemia, HL-60 cells, imperatorin from A. marmelos has caused cytochrome c-dependent apoptosis. Drugs interfere with the proliferative and survival signals that suppress the growth of K562 cells by blocking BCR-ABL1. Proper awareness, development of formulation and consumption of it make this drug scale up commercially.

Abstract 5630: Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML

Abstract Acute Myeloid Leukemia (AML) represents a diverse category of blood cancers marked by the clonal proliferation of atypical myeloid precursor cells within the bone marrow. Lysine Specific Demethylase 1 (LSD1), an epigenetic regulator, exhibits elevated expression in AML. Consequently, inhibiting LSD1 has emerged as a promising therapeutic approach for addressing blood and bone marrow cancers. While initial emphasis centered on LSD1's enzymatic inhibition, compelling evidence highlights its pivotal role as a scaffolding protein, orchestrating transcriptional and chromatin remodeling complexes that control cellular differentiation and proliferation. However, our understanding of the precise mechanisms governing these complexes remains limited, necessitating further investigation. The K562 erythroleukemia cell line has served as a prominent model for investigating the molecular factors influencing cell fate determination. In response to specific cues, these cells exhibit a bi-potential capacity, mirroring the behavior of normal hematopoietic megakaryocyte/erythrocyte progenitors (MEP). Notably, when LSD1 is either depleted or inhibited, the erythroid differentiation of K562 cells is hindered, even when exposed to various pro-differentiating triggers, including the BCR-ABL inhibitor Imatinib. In order to gain deeper insights and identify potential new protein partners involved in the K562 differentiation process that interact with LSD1, we employed the erythroleukemic K562 cell model. Using unbiased, proteome-wide proximity labeling we discovered that LSD1 serves as a scaffold for recruitment of the ChAHP complex, comprised of ADNP, CBX3, and CHD4. The ChAHP complex serves fundamental roles in chromatin remodeling, RNA splicing, and transcriptional control. We confirmed these proximity relationships by immunoblotting biotinylated proteins generated in situ by the promiscuous biotin ligase, (BirA*) when anchored to LSD1. We show that depleting ADNP induces spontaneous erythroid differentiation in K562 erythroleukemia cells, which is augmented by BCR-ABL inhibitor Imatinib treatment. ADNP-deficient K562 cells exhibited accelerated erythroid differentiation, as evidenced by a four-fold increase in the surface expression of erythroid markers (CD71, CD235a) and a significant upregulation of beta-globin gene cluster markers. These findings collectively suggest an antagonistic functional relationship between ADNP and LSD1, potentially counterbalancing LSD1's role in lineage allocation. Furthermore, we demonstrate that the loss of the key ChAHP component, ADNP, substantially hampers proliferation and increases apoptosis in K562 cells. These results underscore the potential of ADNP/ChAHP as an appealing target to modulate the choice between erythroid and megakaryocytic fates and offer promise as a target for pro-differentiation therapies in AML Citation Format: Mehraju Din Lone, Mohd Sayeed, Alaa Habieb, Praveen Kumar, Michael E. Engel. Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5630.

Abstract 3628: Combined mek and jak inhibition in vitro is an effective strategy in overcoming jak inhibitor resistance in primary myelofibrosis (PMF)

Abstract Introduction Primary myelofibrosis (PMF) is a myeloproliferative neoplasm arising from various mutations in myeloid-committed hematopoietic progenitor cells. Treatment of PMF is challenging as Jak inhibitor resistance is common, making the expansion of molecular characterization and novel therapeutic target identification in PMF essential. The main goals of this study are to determine both the presence of Ras activity in PMF and the impact on Ras/MAP Kinase pathway targeting in the disease. Methods Integrated genomic-transcriptomic sequencing analyses were performed on peripheral blood mononuclear cells (PBMCs) purified from 27 patients with PMF at a single institution; protein was then obtained from whole cell lysates. Ras activity was then assessed by applying Ras binding domain (RBD) to sample in an ELISA and was compared between patients with PMF, essential thrombocythemia (ET), polycythemia vera (PV) and healthy donors. Statistical analyses were conducted using two-way unpaired t-tests. The next set of in vitro studies aimed to evaluate the effects of MEK inhibition in PMF. Cell viability studies were first performed on human erythroleukemia (HEL) cells to identify IC50 values. Colony forming assays in methylcellulose-enriched media formulated for the growth of CD34+ cells were then performed in 4 conditions: Ruxolitinib and Cobimetinib monotherapy, combined therapies and untreated sample. Results As previously reported in our initial study, Ras activity in 21 patients with MF was 1.74 fold higher than healthy donors (13 volunteers, p=0.0304) and 1.64 fold higher than in PV/ET (12 patients, p=0.0462). We also reported previously on Ras activity from differential enrichment of myeloid and lymphoid cells from PBMC samples, where a key observation was that myeloid contribution to Ras activity in PMF patients with RAS/MAP Kinase pathway mutations was 3.8 times higher in Ras/MAP Kinase WT patients (p=0.0012), potentially suggesting that RAS pathway mutations have a predominance in myeloid cells. In HEL cell viability assays, cobimetinib and ruxolitinib, when utilized in tandem, resulted in more cell growth inhibition than either agent individually. This same finding was recapitulated in colony forming assays in 4 different patients with varying levels of sensitivity to Jak inhibitors, in all of whom ruxolitinib and cobimetinib lead to a greater reduction in colony formation than ruxolitinib monotherapy. Discussion Data from colony forming assays of bone marrow from patients with PMF identifies at least an additive effect of combined Jak and Ras/MAP Kinase (specifically Mek) inhibition on CD34+ colony formation. The specific observation that Ras pathway inhibition may be utilized as a means of overcoming Jak inhibitor resistance in PMF is a novel finding in this field and may be a valuable avenue for future early-phase clinical trials. Citation Format: Samuel Benjamin Reynolds, Sho Matono, Malathi Kandarpa, Kristen Pettit, Mark Ribick, Moshe Talpaz, Qing Li. Combined mek and jak inhibition in vitro is an effective strategy in overcoming jak inhibitor resistance in primary myelofibrosis (PMF) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3628.

FLI1 induces erythroleukemia through opposing effects on UBASH3A and UBASH3B expression

BackgroundFLI1 is an oncogenic transcription factor that promotes diverse malignancies through mechanisms that are not fully understood. Herein, FLI1 is shown to regulate the expression of Ubiquitin Associated and SH3 Domain Containing A/B (UBASH3A/B) genes. UBASH3B and UBASH3A are found to act as an oncogene and tumor suppressor, respectively, and their combined effect determines erythroleukemia progression downstream of FLI1.MethodsPromoter analysis combined with luciferase assays and chromatin immunoprecipitation (ChIP) analysis were applied on the UBASH3A/B promoters. RNAseq analysis combined with bioinformatic was used to determine the effect of knocking-down UBASH3A and UBASH3B in leukemic cells. Downstream targets of UBASH3A/B were inhibited in leukemic cells either via lentivirus-shRNAs or small molecule inhibitors. Western blotting and RT-qPCR were used to determine transcription levels, MTT assays to assess proliferation rate, and flow cytometry to examine apoptotic index.ResultsKnockdown of FLI1 in erythroleukemic cells identified the UBASH3A/B genes as potential downstream targets. Herein, we show that FLI1 directly binds to the UBASH3B promoter, leading to its activation and leukemic cell proliferation. In contrast, FLI1 indirectly inhibits UBASH3A transcription via GATA2, thereby antagonizing leukemic growth. These results suggest oncogenic and tumor suppressor roles for UBASH3B and UBASH3A in erythroleukemia, respectively. Mechanistically, we show that UBASH3B indirectly inhibits AP1 (FOS and JUN) expression, and that its loss leads to inhibition of apoptosis and acceleration of proliferation. UBASH3B also positively regulates the SYK gene expression and its inhibition suppresses leukemia progression. High expression of UBASH3B in diverse tumors was associated with worse prognosis. In contrast, UBASH3A knockdown in erythroleukemic cells increased proliferation; and this was associated with a dramatic induction of the HSP70 gene, HSPA1B. Accordingly, knockdown of HSPA1B in erythroleukemia cells significantly accelerated leukemic cell proliferation. Accordingly, overexpression of UBASH3A in different cancers was predominantly associated with good prognosis. These results suggest for the first time that UBASH3A plays a tumor suppressor role in part through activation of HSPA1B.ConclusionsFLI1 promotes erythroleukemia progression in part by modulating expression of the oncogenic UBASH3B and tumor suppressor UBASH3A.

Dachshund Homolog 1: Unveiling Its Potential Role in Megakaryopoiesis and Bacillus anthracis Lethal Toxin-Induced Thrombocytopenia.

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34-megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.

Altered platelet-megakaryocyte endocytosis and trafficking of albumin and fibrinogen in RUNX1 haplodeficiency

AbstractPlatelet α-granules have numerous proteins, some synthesized by megakaryocytes (MK) and others not synthesized but incorporated by endocytosis, an incompletely understood process in platelets/MK. Germ line RUNX1 haplodeficiency, referred to as familial platelet defect with predisposition to myeloid malignancies (FPDMMs), is associated with thrombocytopenia, platelet dysfunction, and granule deficiencies. In previous studies, we found that platelet albumin, fibrinogen, and immunoglobulin G (IgG) were decreased in a patient with FPDMM. We now show that platelet endocytosis of fluorescent-labeled albumin, fibrinogen, and IgG is decreased in the patient and his daughter with FPDMM. In megakaryocytic human erythroleukemia (HEL) cells, small interfering RNA RUNX1 knockdown (KD) increased uptake of these proteins over 24 hours compared with control cells, with increases in caveolin-1 and flotillin-1 (2 independent regulators of clathrin-independent endocytosis), LAMP2 (a lysosomal marker), RAB11 (a marker of recycling endosomes), and IFITM3. Caveolin-1 downregulation in RUNX1-deficient HEL cells abrogated the increased uptake of albumin, but not fibrinogen. Albumin, but not fibrinogen, partially colocalized with caveolin-1. RUNX1 KD resulted in increased colocalization of albumin with flotillin and fibrinogen with RAB11, suggesting altered trafficking of both proteins. The increased uptake of albumin and fibrinogen, as well as levels of caveolin-1, flotillin-1, LAMP2, and IFITM3, were recapitulated by short hairpin RNA RUNX1 KD in CD34+-derived MK. To our knowledge, these studies provide first evidence that platelet endocytosis of albumin and fibrinogen is impaired in some patients with RUNX1-haplodeficiency and suggest that megakaryocytes have enhanced endocytosis with defective trafficking, leading to loss of these proteins by distinct mechanisms. This study provides new insights into mechanisms governing endocytosis and α-granule deficiencies in RUNX1-haplodeficiency.