Ablation of Wnt signaling in bone marrow stromal cells overcomes microenvironment-mediated drug resistance in acute myeloid leukemia.

The survival of leukemic cells is significantly influenced by the bone marrow microenvironment, where stromal cells play a crucial role. While there has been substantial progress in understanding the mechanisms and pathways involved in this crosstalk, limited data exist regarding the impact of leukemic cells on bone marrow stromal cells and their potential role in drug resistance. In this study, we identify that leukemic cells prime bone marrow stromal cells towards osteoblast lineage and promote drug resistance. This biased differentiation of stroma is accompanied by dysregulation of the canonical Wnt signaling pathway. Inhibition of Wnt signaling in stroma reversed the drug resistance in leukemic cells, which was further validated in leukemic mice models. This study evaluates the critical role of leukemic cells in establishing a drug-resistant niche by influencing the bone marrow stromal cells. Additionally, it highlights the potential of targeting Wnt signaling in the stroma by repurposing an anthelmintic drug to overcome the microenvironment-mediated drug resistance.

RIO-kinase 2 is essential for hematopoiesis.

Regulation of protein synthesis is a key factor in hematopoietic stem cell maintenance and differentiation. Rio-kinase 2 (RIOK2) is a ribosome biogenesis factor that has recently been described an important regulator of human blood cell development. Additionally, we have previously identified RIOK2 as a regulator of protein synthesis and a potential target for the treatment of acute myeloid leukemia (AML). However, its functional relevance in several organ systems, including normal hematopoiesis, is not well understood. Here, we investigate the consequences of RIOK2 loss on normal hematopoiesis using two different conditional knockout mouse models. Using competitive and non-competitive bone marrow transplantations, we demonstrate that RIOK2 is essential for the differentiation of hematopoietic stem and progenitor cells (HSPCs) as well as for the maintenance of fully differentiated blood cells in vivo as well as in vitro. Loss of RIOK2 leads to rapid death in full-body knockout mice as well as mice with RIOK2 loss specific to the hematopoietic system. Taken together, our results indicate that regulation of protein synthesis and ribosome biogenesis by RIOK2 is essential for the function of the hematopoietic system.

SMS121, a new inhibitor of CD36, impairs fatty acid uptake and viability of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common among children. AML is characterized by aberrant proliferation of myeloid blasts in the bone marrow and impaired normal hematopoiesis. Despite the introduction of new drugs and allogeneic bone marrow transplantation, patients have poor overall survival rate with relapse as the major challenge, driving the demand for new therapeutic strategies. AML patients with high expression of the very long/long chain fatty acid transporter CD36 have poorer survival and very long chain fatty acid metabolism is critical for AML cell survival. Here we show that fatty acids are transferred from human primary adipocytes to AML cells upon co-culturing. A drug-like small molecule (SMS121) was identified by receptor-based virtual screening and experimentally demonstrated to target the lipid uptake protein CD36. SMS121 reduced the uptake of fatty acid into AML cells that could be reversed by addition of free fatty acids and caused decreased cell viability. The data presented here serves as a framework for the development of CD36 inhibitors to be used as future therapeutics against AML.

Vitamin D Mitigates Hepatic Fat Accumulation and Inflammation and Increases SIRT1/AMPK Expression in AML-12 Hepatocytes.

Emerging evidence has demonstrated a strong correlation between vitamin D status and fatty liver disease. Aberrant hepatic fat infiltration contributes to oxidant overproduction, promoting metabolic dysfunction, and inflammatory responses. Vitamin D supplementation might be a good strategy for reducing hepatic lipid accumulation and inflammation in non-alcoholic fatty liver disease and its associated diseases. This study aimed to investigate the role of the most biologically active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), in hepatic fat accumulation and inflammation in palmitic acid (PA)-treated AML-12 hepatocytes. The results indicated that treatment with 1,25(OH)2D significantly decreased triglyceride contents, lipid peroxidation, and cellular damage. In addition, mRNA levels of apoptosis-associated speck-like CARD-domain protein (ASC), thioredoxin-interacting protein (TXNIP), NOD-like receptor family pyrin domain-containing 3 (NLRP3), and interleukin-1ß (IL-1ß) involved in the NLRP3 inflammasome accompanied by caspase-1 activity and IL-1ß expression were significantly suppressed by 1,25(OH)2D in PA-treated hepatocytes. Moreover, upon PA exposure, 1,25(OH)2D-incubated AML-12 hepatocytes showed higher sirtulin 1 (SIRT1) expression and adenosine monophosphate-activated protein kinase (AMPK) phosphorylation. A SIRT1 inhibitor alleviated the beneficial effects of 1,25(OH)2D on PA-induced hepatic fat deposition, IL-1ß expression, and caspase-1 activity. These results suggest that the favorable effects of 1,25(OH)2D on hepatic fat accumulation and inflammation may be, at least in part, associated with the SIRT1.

Discovery of Selective and Potent ATR Degrader for Exploration its Kinase-Independent Functions in Acute Myeloid Leukemia Cells.

ATR has emerged as a promising target for anti-cancer drug development. Several potent ATR inhibitors are currently undergoing various stages of clinical trials, but none have yet received FDA approval due to unclear regulatory mechanisms. In this study, we discovered a potent and selective ATR degrader. Its kinase-independent regulatory functions in acute myeloid leukemia (AML) cells were elucidated using this proteolysis-targeting chimera (PROTAC) molecule as a probe. The ATR degrader, 8 i, exhibited significantly different cellular phenotypes compared to the ATR kinase inhibitor 1. Mechanistic studies revealed that ATR deletion led to breakdown in the nuclear envelope, causing genome instability and extensive DNA damage. This would increase the expression of p53 and triggered immediately p53-mediated apoptosis signaling pathway, which was earlier and more effective than ATR kinase inhibition. Based on these findings, the in vivo anti-proliferative effects of ATR degrader 8 i were assessed using xenograft models. The degrader significantly inhibited the growth of AML cells in vivo, unlike the ATR inhibitor. These results suggest that the marked anti-AML activity is regulated by the kinase-independent functions of the ATR protein. Consequently, developing potent and selective ATR degraders could be a promising strategy for treating AML.

Differential Gene Regulatory Network Analysis between Azacitidine-Sensitive and -Resistant Cell Lines.

Azacitidine, a DNA methylation inhibitor, is employed for the treatment of acute myeloid leukemia (AML). However, drug resistance remains a major challenge for effective azacitidine chemotherapy, though several studies have attempted to uncover the mechanisms of azacitidine resistance. With the aim to identify the mechanisms underlying acquired azacitidine resistance in cancer cell lines, we developed a computational strategy that can identify differentially regulated gene networks between drug-sensitive and -resistant cell lines by extending the existing method, differentially coexpressed gene sets (DiffCoEx). The technique specifically focuses on cell line-specific gene network analysis. We applied our method to gene networks specific to azacitidine sensitivity and identified differentially regulated gene networks between azacitidine-sensitive and -resistant cell lines. The molecular interplay between the metallothionein gene family, C19orf33, ELF3, GRB7, IL18, NRN1, and RBM47 were identified as differentially regulated gene network in drug resistant cell lines. The biological mechanisms associated with azacitidine and AML for the markers in the identified networks were verified through the literature. Our results suggest that controlling the identified genes (e.g., the metallothionein gene family) and "cellular response"-related pathways ("cellular response to zinc ion", "cellular response to copper ion", and "cellular response to cadmium ion", where the enriched functional-related genes are MT2A, MT1F, MT1G, and MT1E) may provide crucial clues to address azacitidine resistance in patients with AML. We expect that our strategy will be a useful tool to uncover patient-specific molecular interplay that provides crucial clues for precision medicine in not only gastric cancer but also complex diseases.

Ankrd26 is a retinoic acid-responsive plasma membrane-binding and -shaping protein critical for proper cell differentiation.

Morphogens are important triggers for differentiation processes. Yet, downstream effectors that organize cell shape changes in response to morphogenic cues, such as retinoic acid, largely remain elusive. Additionally, derailed plasma membrane-derived signaling often is associated with cancer. We identify Ankrd26 as a critical player in cellular differentiation and as plasma membrane-localized protein able to self-associate and form clusters at the plasma membrane in response to retinoic acid. We show that Ankrd26 uses an N-terminal amphipathic structure for membrane binding and bending. Importantly, in an acute myeloid leukemia-associated Ankrd26 mutant, this critical structure was absent, and Ankrd26's membrane association and shaping abilities were impaired. In line with this, the mutation rendered Ankrd26 inactive in both gain-of-function and loss-of-function/rescue studies addressing retinoic acid/brain-derived neurotrophic factor (BDNF)-induced neuroblastoma differentiation. Our results highlight the importance and molecular details of Ankrd26-mediated organizational platforms for cellular differentiation at the plasma membrane and how impairment of these platforms leads to cancer-associated pathomechanisms involving these Ankrd26 properties.

A novel HDAC6 inhibitor attenuate APAP-induced liver injury by regulating MDH1-mediated oxidative stress.

Glutathione (GSH) depletion, mitochondrial damage, and oxidative stress have been implicated in the pathogenesis of acetaminophen (APAP) hepatotoxicity. Here, we demonstrated that the expression of histone deacetylase 6 (HDAC6) is highly elevated, whereas malate dehydrogenase 1 (MDH1) is downregulated in liver tissues and AML-12 cells induced by APAP. The therapeutic benefits of LT-630, a novel HDAC6 inhibitor on APAP-induced liver injury, were also substantiated. On this basis, we demonstrated that LT-630 improved the protein expression and acetylation level of MDH1. Furthermore, after overexpression of MDH1, an upregulated NADPH/NADP+ ratio and GSH level and decreased cell apoptosis were observed in APAP-stimulated AML-12 cells. Importantly, MDH1 siRNA clearly reversed the protection of LT-630 on APAP-stimulated AML-12 cells. In conclusion, LT-630 could ameliorate liver injury by modulating MDH1-mediated oxidative stress induced by APAP.

C/EBPα-p30 confers AML cell susceptibility to the terminal unfolded protein response and resistance to Venetoclax by activating DDIT3 transcription.

BACKGROUND: Acute myeloid leukemia (AML) with biallelic (CEBPAbi) as well as single mutations located in the bZIP region is associated with a favorable prognosis, but the underlying mechanisms are still unclear. Here, we propose that two isoforms of C/EBPα regulate DNA damage-inducible transcript 3 (DDIT3) transcription in AML cells corporately, leading to altered susceptibility to endoplasmic reticulum (ER) stress and related drugs. METHODS: Human AML cell lines and murine myeloid precursor cell line 32Dcl3 cells were infected with recombinant lentiviruses to knock down CEBPA expression or over-express the two isoforms of C/EBPα. Quantitative real-time PCR and western immunoblotting were employed to determine gene expression levels. Cell apoptosis rates were assessed by flow cytometry. CFU assays were utilized to evaluate the differentiation potential of 32Dcl3 cells. Luciferase reporter analysis, ChIP-seq and ChIP-qPCR were used to validate the transcriptional regulatory ability and affinity of each C/EBPα isoform to specific sites at DDIT3 promoter. Finally, an AML xenograft model was generated to evaluate the in vivo therapeutic effect of agents. RESULTS: We found a negative correlation between CEBPA expression and DDIT3 levels in AML cells. After knockdown of CEBPA, DDIT3 expression was upregulated, resulting in increased apoptotic rate of AML cells induced by ER stress. Cebpa knockdown in mouse 32Dcl3 cells also led to impaired cell viability due to upregulation of Ddit3, thereby preventing leukemogenesis since their differentiation was blocked. Then we discovered that the two isoforms of C/EBPα regulate DDIT3 transcription in the opposite way. C/EBPα-p30 upregulated DDIT3 transcription when C/EBPα-p42 downregulated it instead. Both isoforms directly bound to the promoter region of DDIT3. However, C/EBPα-p30 has a unique binding site with stronger affinity than C/EBPα-p42. These findings indicated that balance of two isoforms of C/EBPα maintains protein homeostasis and surveil leukemia, and at least partially explained why AML cells with disrupted C/EBPα-p42 and/or overexpressed C/EBPα-p30 exhibit better response to chemotherapy stress. Additionally, we found that a low C/EBPα p42/p30 ratio induces resistance in AML cells to the BCL2 inhibitor venetoclax since BCL2 is a major target of DDIT3. This resistance can be overcome by combining ER stress inducers, such as tunicamycin and sorafenib in vitro and in vivo. CONCLUSION: Our results indicate that AML patients with a low C/EBPα p42/p30 ratio (e.g., CEBPAbi) may not benefit from monotherapy with BCL2 inhibitors. However, this issue can be resolved by combining ER stress inducers.

Biology and Therapeutic Properties of Mesenchymal Stem Cells in Leukemia.

This comprehensive review delves into the multifaceted roles of mesenchymal stem cells (MSCs) in leukemia, focusing on their interactions within the bone marrow microenvironment and their impact on leukemia pathogenesis, progression, and treatment resistance. MSCs, characterized by their ability to differentiate into various cell types and modulate the immune system, are integral to the BM niche, influencing hematopoietic stem cell maintenance and functionality. This review extensively explores the intricate relationship between MSCs and leukemic cells in acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia. This review also addresses the potential clinical applications of MSCs in leukemia treatment. MSCs' role in hematopoietic stem cell transplantation, their antitumor effects, and strategies to disrupt chemo-resistance are discussed. Despite their therapeutic potential, the dual nature of MSCs in promoting and inhibiting tumor growth poses significant challenges. Further research is needed to understand MSCs' biological mechanisms in hematologic malignancies and develop targeted therapeutic strategies. This in-depth exploration of MSCs in leukemia provides crucial insights for advancing treatment modalities and improving patient outcomes in hematologic malignancies.

Development of Immune Cell Therapy Using T Cells Generated from Pluripotent Stem Cells.

In the field of cancer immunotherapy, the effectiveness of a method in which patient-derived T cells are genetically modified ex vivo and administered to patients has been demonstrated. However, problems remain with this method, such as (1) time-consuming, (2) costly, and (3) difficult to guarantee the quality. To overcome these barriers, strategies to regenerate T cells using iPSC technology are being pursued by several groups in the last decade. The authors have been developing a method by which specific TCR genes are introduced into iPSCs and T cells are generated from those iPSCs (TCR-iPSC method). At present, our group is preparing this approach for clinical trial, where iPSCs provided from the iPSC project are transduced with WT1 antigen-specific TCR that had been already clinically tested, and killer T cells are generated from such TCR-iPSCs, to be administered to acute myeloid leukemia patients. While the adoptive T cell therapies have been mainly directed to be used in cancer immunotherapy, it is possible to apply these approaches to viral infections. Strategies by other groups to regenerate various types of T cells from iPSCs will also be introduced.

Prognostic implications of cGAS and STING gene expression in acute myeloid leukemia.

Acute myeloid leukemia (AML) is one of the most threatening hematological malignances. cGAS-STING pathway plays an important role in tumor immunity and development. However, the prognostic role of cGAS-STING pathway in AML remains unknown. Firstly, The expression of cGAS and STING was analyzed by bioinformatics analysis. Subsequently, Bone marrow samples were collected from 120 AML patients and 15 healthy individuals in an independent cohort. The cGAS and STING expression was significantly elevated in AML patients compared with healthy controls. Patients with high cGAS and STING expression had a higher NRAS/KRAS mutation rate and lower complete remission (CR) rate. High cGAS and STING expression was significantly associated with lower overall survival (OS) and disease-free survival (DFS). Our findings revealed that the expression levels of cGAS and STING in AML are elevated. High expression of cGAS and STING correlated with worse OS and DFS and may be a useful biomarker for inferior prognosis in AML patients.

miR-26b-5p Affects the Progression of Acute Myeloid Leukemia by Regulating the USP48-Mediated Wnt/ß-Catenin Pathway.

Acute myeloid leukemia (AML) is a highly heterogeneous disease. Exploring the pathogenesis of AML is still an important topic in the treatment of AML. The expression levels of miR-26b-5p and USP48 were measured by qRT-PCR. The expression levels of related proteins were detected by Western blot. Cell proliferation and apoptosis were detected by CCK-8 and flow cytometry, respectively. Coimmunoprecipitation was used to examine the interaction between USP48 and Wnt5a. Bioinformatics analysis showed that high levels of miR-26b-5p and low levels of USP48 were associated with poor prognosis in AML. miR-26b-5p can negatively regulate the expression of USP48. Downregulation of miR-26b-5p inhibited EMT, cell viability and proliferation of AML cells and accelerated apoptosis. Furthermore, the influence of miR-26b-5p inhibition and USP48 knockdown on AML progression could be reversed by a Wnt/ß-catenin signaling pathway inhibitor. This study revealed that miR-26b-5p regulates AML progression, possibly by targeting the USP48-mediated Wnt/ß-catenin molecular axis to affect AML cell biological behavior.

Lactate acid promotes PD-1+ Tregs accumulation in the bone marrow with high tumor burden of Acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) displayed poor response to programmed death-1 (PD-1) blockade therapy. Regulatory T cells (Tregs) was one of major immunosuppressive components in Tumor microenvironment and plays a vital role in the resistance of immunotherapy. Coinhibitory receptors regulate function of regulatory Tregs and are associated with resistance of PD-1 blockade. However, the coinhibitory receptors expression and differentiated status of Tregs in AML patients remain to be unclear. METHODS: Phenotypic determination of Tregs and CD8+ T cells in bone marrow of healthy donors and AML patients was performed by flow cytometry. Coculture experiments of AML and Tregs in vitro were performed and the concentrations of lactate acid (LA) in the supernatant were examined by ELISA. RESULTS: More Tregs differentiated into effector subsets in AML patients. However, PD-1 and T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) expression on Tregs were comparable in healthy donors and AML patients. Further analysis showed that PD-1+ and PD-1+TIGIT+Tregs are more abundant in the bone marrow of patients with higher leukemic load. Moreover, PD-1+ Tregs accumulation was associated with higher level of senescent CD4+ T cells and increased frequencies of exhausted CD4+ as well as CD8+ T cells. Notably, neither Tregs nor their effector subsets were decreased among patients in complete remission. PD-1 expression was significantly downregulated in Tregs after achieving complete remission. Mechanistically, both AML cell line (KG-1α) and primary AML blasts produced high concentration of LA. Blockade of LA by lactate transporter inhibitor abrogated the upregulation of PD-1 by AML cells. CONCLUSION: PD-1+ Tregs accumulation in bone marrow in higher leukemic burden setting was linked to lactate acid secreted by AML blasts and decreased after disease remission. Our findings provided a novel insight into Tregs in AML and possible mechanism for resistance of PD-1 blockade in AML.

The effect of STAT1, miR-99b, and MAP2K1 in alcoholic liver disease (ALD) mouse model and hepatocyte.

Alcoholic liver disease (ALD) serves as the leading cause of chronic liver diseases-related morbidity and mortality, which threatens the life of millions of patients in the world. However, the molecular mechanisms underlying ALD progression remain unclear. Here, we applied microarray analysis and experimental approaches to identify miRNAs and related regulatory signaling that associated with ALD. Microarray analysis identified that the expression of miR-99b was elevated in the ALD mouse model. The AML-12 cells were treated with EtOH and the expression of miR-99b was enhanced in the cells. The expression of miR-99b was positively correlated with ALT levels in the ALD mice. The microarray analysis identified the abnormally expressed mRNAs in ALD mice and the overlap analysis was performed with based on the differently expressed mRNAs and the transcriptional factors of miR-99b, in which STAT1 was identified. The elevated expression of STAT1 was validated in ALD mice. Meanwhile, the treatment of EtOH induced the expression of STAT1 in the AML-12 cells. The expression of STAT1 was positively correlated with ALT levels in the ALD mice. The positive correlation of STAT1 and miR-99b expression was identified in bioinformatics analysis and ALD mice. The expression of miR-99b and pri-miR-99b was promoted by the overexpression of STAT1 in AML-12 cells. ChIP analysis confirmed the enrichment of STAT1 on miR-99b promoter in AML-12 cells. Next, we found that the expression of mitogen-activated protein kinase kinase 1 (MAP2K1) was negatively associated with miR-99b. The expression of MAP2K1 was downregulated in ALD mice. Consistently, the expression of MAP2K1 was reduced by the treatment of EtOH in AML-12 cells. The expression of MAP2K1 was negative correlated with ALT levels in the ALD mice. We identified the binding site of MAP2K1 and miR-99b. Meanwhile, the treatment of miR-99b mimic repressed the luciferase activity of MAP2K1 in AML-12 cells. The expression of MAP2K1 was suppressed by miR-99b in the cells. We observed that the expression of MAP2K1 was inhibited by the overexpression of STAT1 in AML-12 cells. Meanwhile, the apoptosis of AML-12 cells was induced by the treatment of EtOH, while miR-99b mimic promoted but the overexpression of MAP2K1 attenuated the effect of EtOH in the cells. In conclusion, we identified the correlation and effect of STAT1, miR-99b, and MAP2K1 in ALD mouse model and hepatocyte. STAT1, miR-99b, and MAP2K1 may serve as potential therapeutic target of ALD.

Impact of measurable residual disease in combination with CD19 on postremission therapy choices for adult t(8;21) acute myeloid leukemia in first complete remission.

BACKGROUND: The post-remission therapy (PRT) choices for adult t(8;21) acute myeloid leukemia (AML) in first complete remission (CR1) need to be further explored. AIMS: We aimed to investigate the impact of measurable residual disease (MRD) combined with CD19 on PRT choices for adult t(8;21) AML in CR1. METHODS: A total of 150 t(8;21) AML patients were enrolled, including 67 underwent chemotherapy (CMT) and 83 allogeneic hematopoietic stem cell transplantation (allo-SCT) as PRT in CR1. Subgroup analyses were performed according to MRD level after three cycles of chemotherapy combined with CD19 expression. RESULTS: Multivariate analysis indicated MRDhigh after three courses of treatment (HR, 0.14 [95% CI, 0.03-0.66]; p = 0.013) and CD19 negativity (HR, 0.14 [95% CI, 0.02-0.96]; p = 0.045) were risk factors for relapse, while allo-SCT was protective factor for relapse (HR, 0.34 [95% CI, 0.15-0.75]; p = 0.008). Grouped by MRD after three courses of chemotherapy, allo-SCT had lower CIR (p < 0.001) and better OS (p = 0.003) than CMT for MRDhigh patients, CMT showed a higher CIR (35.99% vs. 15.34%, p = 0.100) but comparable OS (p = 0.588) than allo-SCT for MRDlow patients. Grouped by CD19 expression, allo-SCT demonstrated lower CIR (p < 0.001) and better OS (p = 0.002) than CMT for CD19- patients. CMT had a higher CIR (41.37% vs. 10.48%, p = 0.007) but comparable OS (p = 0.147) than allo-SCT for CD19+ patients. Grouped by MRD combined with CD19, MRDhigh /CD19+ subsets were identified out of CD19+ patients benefiting from allo-SCT with lower CIR (p = 0.002) and superior OS (p = 0.020) than CMT. CMT preserved comparable CIR (p = 0.939) and OS (p = 0.658) with allo-SCT for MRDlow /CD19+ patients. MRDlow /CD19- subsets were also identified from MRDlow patients requiring allo-SCT with lower CIR (p < 0.001) and superior OS (p = 0.008) than CMT. Allo-SCT maintained lower CIR (p < 0.001) and superior OS (p = 0.008) than CMT for MRDhigh /CD19- patients. CONCLUSIONS: MRD combined with CD19 might optimize PRT choices for adult t(8;21) AML patients in CR1.

Landscape of FLT3 Variations Associated with Structural and Functional Impact on Acute Myeloid Leukemia: A Computational Study.

Acute myeloid leukemia (AML) is hallmarked by the clonal proliferation of myeloid blasts. Mutations that result in the constitutive activation of the fms-like tyrosine kinase 3 (FLT3) gene, coding for a class III receptor tyrosine kinase, are significantly associated with this heterogeneous hematologic malignancy. The fms-related tyrosine kinase 3 ligand binds to the extracellular domain of the FLT3 receptor, inducing homodimer formation in the plasma membrane, leading to autophosphorylation and activation of apoptosis, proliferation, and differentiation of hematopoietic cells in bone marrow. In the present study, we evaluated the association of FLT3 as a significant biomarker for AML and tried to comprehend the effects of specific variations on the FLT3 protein's structure and function. We also examined the effects of I836 variants on binding affinity to sorafenib using molecular docking. We integrated multiple bioinformatics tools, databases, and resources such as OncoDB, UniProt, COSMIC, UALCAN, PyMOL, ProSA, Missense3D, InterProScan, SIFT, PolyPhen, and PredictSNP to annotate the structural, functional, and phenotypic impact of the known variations associated with FLT3. Twenty-nine FLT3 variants were analyzed using in silico approaches such as DynaMut, CUPSAT, AutoDock, and Discovery Studio for their impact on protein stability, flexibility, function, and binding affinity. The OncoDB and UALCAN portals confirmed the association of FLT3 gene expression and its mutational status with AML. A computational structural analysis of the deleterious variants of FLT3 revealed I863F mutants as destabilizers of the protein structure, possibly leading to functional changes. Many single-nucleotide variations in FLT3 have an impact on its structure and function. Thus, the annotation of FLT3 SNVs and the prediction of their deleterious pathogenic impact will facilitate an insight into the tumorigenesis process and guide experimental studies and clinical implications.

Synthesis, kinase inhibition and anti-leukemic activities of diversely substituted indolopyrazolocarbazoles.

Pyrazole analogues of the staurosporine aglycone K252c, in which the lactam ring was replaced by a pyrazole moiety, were synthesized. In this series, one or the other nitrogen atoms of the indolocarbazole scaffold was substituted by aminoalkyl chains, aiming at improving protein kinase inhibition as well as cellular potency toward acute myeloid leukemia (AML) cell lines. Compound 19a, substituted at the N12-position by a 3-(methylamino)propyl group, showed high cellular activity in the low micromolar range toward three AML cell lines (MOLM-13, OCI-AML3 and MV4-11) with selectivity over non-cancerous cells (NRK, H9c2). 19a is also a highly potent inhibitor of the three Pim kinase isoforms, Pim-3 being the most inhibited with an IC50 value in the nanomolar range. A selectivity screening toward a panel of 50 protein kinases showed that 19a also potently inhibited PRK2 and to a lower extent AMPK, MARK3, GSK3ß and JAK3. Our results enhance the understanding of the structural characteristics of indolopyrazolocarbazoles essential for potent protein kinase inhibition with therapeutic potential against AML.

Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth.

Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity.

SHP-1 inhibition targets leukaemia stem cells to restore immunosurveillance and enhance chemosensitivity by metabolic reprogramming.

Leukaemia stem cells (LSCs) in acute myeloid leukaemia present a considerable treatment challenge due to their resistance to chemotherapy and immunosurveillance. The connection between these properties in LSCs remains poorly understood. Here we demonstrate that inhibition of tyrosine phosphatase SHP-1 in LSCs increases their glycolysis and oxidative phosphorylation, enhancing their sensitivity to chemotherapy and vulnerability to immunosurveillance. Mechanistically, SHP-1 inhibition leads to the upregulation of phosphofructokinase platelet (PFKP) through the AKT-ß-catenin pathway. The increase in PFKP elevates energy metabolic activities and, as a consequence, enhances the sensitivity of LSCs to chemotherapeutic agents. Moreover, the upregulation of PFKP promotes MYC degradation and, consequently, reduces the immune evasion abilities of LSCs. Overall, our study demonstrates that targeting SHP-1 disrupts the metabolic balance in LSCs, thereby increasing their vulnerability to chemotherapy and immunosurveillance. This approach offers a promising strategy to overcome LSC resistance in acute myeloid leukaemia.