Proliferation Of Leukemia Cells Research Articles

Overexpression of PTPN21 promotes proliferation of EGF-stimulated acute lymphoblastic leukemia cells via the MAPK signaling pathways

ABSTRACT Objectives This study aims to investigate the role of excessive Protein Tyrosine Phosphatase Non-Receptor Type 21 (PTPN21) in the proliferation of Acute Lymphoblastic Leukemia (ALL) cells with EGF stimulation. Methods PTPN21 was overexpressed in ALL cell lines by lentiviral transfection. Apoptosis was assayed by Annexin V/7-AAD staining. The proliferation and cell cycle of EGF-treated ALL cells were assessed by MTT and Ki-67/7-AAD staining respectively. The phosphorylation of Src tyrosine kinase and mediators of distinct MAPK pathways were assessed by Western blot. Results Overexpression of PTPN21 had minimal effect on the apoptosis of ALL cells, but significantly promoted the proliferation and cell cycle progression of ALL cells stimulated with EGF. The activity of Src tyrosine kinase and the MAPK pathways was elevated. Inhibition of MAPK pathways by specific inhibitors mitigated this pro-proliferative effect of excessive PTPN21 on EGF-stimulated ALL cells. Conclusion PTPN21 may facilitate ALL progression by promoting cell proliferation via the Src/MAPK signaling pathways.

MDS and AML show elevated fractions of CD34-positive blast cell populations with a high anti-apoptotic versus proliferation ratio

This study investigates the intertwined processes of (anti-)apoptosis and cell proliferation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Utilizing antibodies to Bcl-2 and Ki-67, the CD34-positive blast cell compartments in bone marrow aspirates from 50 non-malignant cases, 25 MDS patients, and 25 AML patients were analyzed for their anti-apoptotic and proliferative cell fractions through ten-color flow cytometry. MDS patients exhibited a significantly increased anti-apoptotic (p=0.0014) and reduced proliferative cell fraction (p=0.0030) in their blast cell population as compared to non-malignant cases. AML patients showed an even more exacerbated trend than MDS patients. The resulting Bcl-2:Ki-67 cell fraction ratios in MDS and AML were significantly increased as compared to the non-malignant cases (p=0.0004 and p<0.0001, respectively). AML patients displayed, however, a high degree of variability in their anti-apoptotic and proliferation index, attributed to heterogeneity in maturation stage and severity of the disease at diagnosis. Using double-labeling for Bcl-2 and Ki-67 it could be shown that besides blast cells with a mutually exclusive Ki-67 and Bcl-2 expression, also blast cells concurrently exhibiting anti-apoptotic and proliferative marker expression were found.Integrating these two dynamic markers into MDS and AML diagnostic workups may enable informed conclusions about their biological behavior, facilitating individualized therapy decisions for patients.

Discovery of (−)-epigallocatechin gallate, a novel olfactory receptor 2AT4 agonist that regulates proliferation and apoptosis in leukemia cells

Olfactory receptors (ORs), the largest family of G protein-coupled receptors (GPCRs), are ectopically expressed in cancer cells and are involved in cellular physiological processes, but their function as anticancer targets is still potential. OR2AT4 is expressed in leukemia cells, influencing the proliferation and apoptosis, yet the limited number of known OR2AT4 agonists makes it challenging to fully generalize the receptor's function. In this study, we aimed to identify new ligands for OR2AT4 and to investigate their functions and mechanisms in K562 leukemia cells. After producing the recombinant OR2AT4 protein, immobilizing it on a surface plasmon resonance chip, and conducting screening to confirm binding activity using 258 chemicals, five novel OR2AT4 ligands were discovered. As a result of examining changes in intracellular calcium by five ligands in OR2AT4-expressing cells and K562 cells, (−)-epigallocatechin gallate (EGCG) was identified as an OR2AT4 agonist in both cells. EGCG reduced the viability of K562 cells and induced apoptosis in K562 cells. EGCG increased the expression of cleaved caspase 3/8 and had no effect on the expression of Bax and Bcl-2, indicating that it induced apoptosis through the extrinsic pathway. Additionally, the initiation of the extrinsic apoptosis pathway in EGCG-induced K562 cells was due to the activation of OR2AT4, using an OR2AT4 antagonist. This study highlights the potential of EGCG as an anti-cancer agent against leukemia and OR2AT4 as a target, making it a new anti-cancer drug.

Exploiting leucine metabolism for therapeutic benefit in acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) presents significant challenges in treatment, especially for elderly patients. Conventional therapies have limitations, necessitating the exploration of novel strategies. Metabolic reprogramming in cancer cells offers promise in this regard, with growing evidence pointing to dysregulated leucine metabolism in AML. Leucine deprivation has shown potential in inhibiting leukemic cell proliferation, inducing cell cycle arrest, and enhancing differentiation. Combination therapies involving leucine deprivation and chemotherapy have exhibited synergistic effects. The precise molecular mechanisms of leucine deprivation's anti-leukemic effects, particularly its potential impact on the mTOR signaling pathway, require further elucidation. Targeting BCAAs and other amino acids, such as methionine and lysine, holds promise in disrupting leukemia cell proliferation and promoting apoptosis. These findings underscore the potential of exploiting leucine metabolism and amino acid pathways for therapeutic benefits in AML.

Discovery of a Novel Orally Bioavailable FLT3-PROTAC Degrader for Efficient Treatment of Acute Myeloid Leukemia and Overcoming Resistance of FLT3 Inhibitors.

Fms-like tyrosine receptor kinase 3 (FLT3) proteolysis-targeting chimeras (PROTACs) represent a promising approach to eliminate the resistance of FLT3 inhibitors. However, due to the poor druggability of PROTACs, the development of orally bioavailable FLT3-PROTACs faces great challenges. Herein, a novel orally bioavailable FLT3-ITD degrader A20 with excellent pharmacokinetic properties was discovered through reasonable design. A20 selectively inhibited the proliferation of FLT3-ITD mutant acute myeloid leukemia (AML) cells and potently induced FLT3-ITD degradation through the ubiquitin-proteasome system. Notably, oral administration of A20 resulted in complete tumor regression on subcutaneous AML xenograft models. Furthermore, on systemic AML xenograft models, A20 could completely eliminate the CD45+CD33+ human leukemic cells in murine and significantly prolonged the survival time of mice. Most importantly, A20 exerted significantly improved antiproliferative activity against drug-resistant AML cells compared to existing FLT3 inhibitors. These findings suggested that A20 could serve as a promising drug candidate for relapsed or refractory AML.

Bone Marrow Adipose Tissue.

Bone marrow (BM) acts as a dynamic organ within the bone cavity, responsible for hematopoiesis, skeletal remodeling, and immune system control. Bone marrow adipose tissue (BMAT) was long simply considered a filler of space, but now it is known that it instead constitutes an essential element of the BM microenvironment that participates in homeostasis, influences bone health and bone remodeling, alters hematopoietic stem cell functions, contributes to the commitment of mesenchymal stem cells, provides effects to immune homeostasis and defense against infections, and participates in energy metabolism and inflammation. BMAT has emerged as a significant contributor to the development and progression of various diseases, shedding light on its complex relationship with health. Notably, BMAT has been implicated in metabolic disorders, hematological malignancies, and skeletal conditions. BMAT has been shown to support the proliferation of tumor cells in acute myeloid leukemia and niche adipocytes have been found to protect cancer cells against chemotherapy, contributing to treatment resistance. Moreover, BMAT's impact on bone density and remodeling can lead to conditions like osteoporosis, where high levels of BMAT are inversely correlated with bone mineral density, increasing the risk of fractures. BMAT has also been associated with diabetes, obesity, and anorexia nervosa, with varying effects on individuals depending on their weight and health status. Understanding the interaction between adipocytes and different diseases may lead to new therapeutic strategies.

SUCLG1 restricts POLRMT succinylation to enhance mitochondrial biogenesis and leukemia progression.

Mitochondria are cellular powerhouses that generate energy through the electron transport chain (ETC). The mitochondrial genome (mtDNA) encodes essential ETC proteins in a compartmentalized manner, however, the mechanism underlying metabolic regulation of mtDNA function remains unknown. Here, we report that expression of tricarboxylic acid cycle enzyme succinate-CoA ligase SUCLG1 strongly correlates with ETC genes across various TCGA cancer transcriptomes. Mechanistically, SUCLG1 restricts succinyl-CoA levels to suppress the succinylation of mitochondrial RNA polymerase (POLRMT). Lysine 622 succinylation disrupts the interaction of POLRMT with mtDNA and mitochondrial transcription factors. SUCLG1-mediated POLRMT hyposuccinylation maintains mtDNA transcription, mitochondrial biogenesis, and leukemia cell proliferation. Specifically, leukemia-promoting FMS-like tyrosine kinase 3 (FLT3) mutations modulate nuclear transcription and upregulate SUCLG1 expression to reduce succinyl-CoA and POLRMT succinylation, resulting in enhanced mitobiogenesis. In line, genetic depletion of POLRMT or SUCLG1 significantly delays disease progression in mouse and humanized leukemia models. Importantly, succinyl-CoA level and POLRMT succinylation are downregulated in FLT3-mutated clinical leukemia samples, linking enhanced mitobiogenesis to cancer progression. Together, SUCLG1 connects succinyl-CoA with POLRMT succinylation to modulate mitochondrial function and cancer development.

UBE2C promotes the proliferation of acute myeloid leukemia cells through PI3K/AKT activation

This study aims to investigate the role and mechanism of tubiquitin-conjugating enzyme E2 C (UBE2C) in acute myeloid leukemia (AML). Initially, UBE2C expression in leukemia was analyzed using the Cancer Genome Atlas database. Further, we silenced UBE2C expression using small-hairpin RNA (sh-RNA). UBE2C expression was detected via the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot analysis. Apoptotic events and reactive oxygen species (ROS) levels were detected by flow cytometry. A xenograft model of leukemia cells were established, and the protein levels of UBE2C, KI-67, and cleaved-caspase 3 were detected by immunohistochemistry. We reported an overexpression of UBE2C in leukemia patients and cell lines (HL60, THP-1, U937, and KG-1 cells). Moreover, a high expression level of UBE2C was correlated with a dismal prognosis in AML patients. UBE2C knockdown inhibited the viability and promoted apoptosis in AML cells by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Furthermore, UBE2C knockdown increased cellular Fe2+ and ROS levels, and enhanced erastin-induced ferroptosis in a proteasome-dependent manner. UBE2C knockdown also suppressed the tumor formation of AML cells in the mouse model. In summary, our findings suggest that UBE2C overexpression promotes the proliferation and inhibits ferroptosis in AML cells by activating the PI3K/AKT pathway.

Study of Patterns of Leukaemia in a Tertiary Care Hospital

Introduction: Leukaemia arises from abnormal proliferation of hematopoietic or lymphoid cells, categorized as acute or chronic and originating from lymphoid, myeloid, or mixed lineages. Clinical manifestations result from leukemic cell proliferation and tissue infiltration. Objectives: This study aimed to determine the frequency and clinicopathological profile of leukaemia cases. Material and Methods: A three-year cross-sectional observational study was carried out in the Department of Pathology, Indira Gandhi Medical College, Shimla. Diagnosis of Leukaemia was based on complete blood count, peripheral smear examination, bone marrow analysis and cytochemistry wherever required. Data comprising of patient details, clinical examination and haematological parameters were analysed. Results: A total of 163 leukaemia cases were identified, including 102 (63%) acute and 61(37%) cases of chronic leukaemia. Acute leukaemia consisted of 58 cases of Acute Myeloid Leukaemia (AML), 25 Acute Lymphoblastic Leukaemia (ALL), and 19 undifferentiated leukaemia (UL), while chronic cases comprised 47 Chronic Myeloid Leukaemia (CML) and 14 Chronic Lymphocytic Leukaemia (CLL). Most patients were in the age group of 31-60 years, with a male predominance (M:F 1.7:1). Conclusions: Early diagnosis and typing are vital for prompt leukaemia treatment. Despite advanced technologies, cytomorphological examination with cytochemistry remains crucial, especially in resource-limited settings like India. Keywords: Leukaemia, acute, chronic, cytomorphology, observational study

Linc01588 deletion inhibits the malignant biological characteristics of hydroquinone-induced leukemic cells via miR-9-5p/SIRT1

Leukemia caused by environmental chemical pollutants has attracted great attention, the malignant leukemic transformation model of TK6 cells induced by hydroquinone (HQ) has been previously found in our team. However, the type of leukemia corresponding to this malignant transformed cell line model needs further study and interpretation. Furthermore, the molecular mechanism of malignant proliferation of leukemic cells induced by HQ remains unclear. This study is the first to reveal the expression of aberrant genes in leukemic cells of HQ-induced malignant transformation, which may correspond to chronic lymphocytic leukemia (CLL). The expression of Linc01588, a long non-coding RNA (lncRNA), was significantly up-regulated in CLL patients and leukemic cell line model which previously described. After gain-of-function assays and loss-of-function assays, feeble cell viability, severe apoptotic phenotype and the increased secretion of TNF-α were easily observed in malignant leukemic TK6 cells with Linc01588 deletion after HQ intervention. The tumors derived from malignant TK6 cells with Linc01588 deletion inoculated subcutaneously in nude mice were smaller than controls. In CLL and its cell line model, the expression of Linc01588 and miR-9–5p, miR-9–5p and SIRT1 were negative correlation respectively in CLL and cell line model, while the expression of Linc01588 and SIRT1 were positive correlation. The dual-luciferase reporter assay showed that Linc01588 & miR-9–5p, miR-9–5p & SIRT1 could bind directly, respectively. Furthermore, knockdown of miR-9–5p successfully rescued the severe apoptotic phenotype and the increased secretion of TNF-α caused by the Linc01588 deletion, the deletion of Linc01588 in human CLL cell line MEC-2 could also inhibit malignant biological characteristics, and the phenotype caused by the deletion of Linc01588 could also be rescued after overexpression of SIRT1. Moreover, the regulation of SIRT1 expression in HQ19 cells by Linc01588 and miR-9–5 P may be related to the Akt/NF-κB pathway. In brief, Linc01588 deletion inhibits the malignant biological characteristics of HQ-induced leukemic cells via miR-9–5p/SIRT1, and it is a novel and hopeful clue for the clinical targeted therapy of CLL.

Effects of BMAL2 on Aerobic Glycolysis and Cell Proliferation in Acute Myeloid Leukemia Cells

To explore the expression of basic helix-loop-helix ARNT like 2 (BMAL2) in acute myeloid leukemia (AML) patients and its correlation with prognosis, and analyze its effects on the aerobic glycolysis and proliferation of AML cells. The expressions of BMAL2 in bone marrow mononuclear cells (BMMCs) of AML patients and normal control group were detected by RT-qPCR. The correlation of BMAL2 expression with prognosis of AML patients was analyzed using public database of National Center for Biotechnology Information (NCBI). The interfering in BMAL2 expression of HL-60 and Kasumi-1 cells was performed using lentiviral vector-mediated shRNA. Cell glucose metabolism and proliferation were detected by using glucose uptake experiment, lactate content test, CCK-8 assay and cell colony formation test. The expression level of BMAL2 mRNA in BMMCs of AML patients was significantly higher than normal control group (P < 0.01). The overall survival time of AML patients with high expression of BMAL2 was significantly shorter than those with low expression of BMAL2 (P < 0.05). Knockdown of BMAL2 significantly reduced glucose uptake and lactate production in AML cell line HL-60 and Kasumi-1 cells. The results of RT-PCR and Western blot showed that BMAL2 promoted aerobic glycolysis by enhancing the expression of HIF1A in AML cells, thereby promoting cell proliferation. BMAL2 is highly expressed in AML patients, and promotes aerobic glycolysis by enhancing the expression of HIF1A, thereby promoting cell proliferation.

Elesclomol-Cu Induces Cuproptosis in Human Acute Myeloid Leukemia Cells

To investigate the effects of elesclomol-Cu (ES-Cu) on the proliferation and cuproptosis of human acute myeloid leukemia (AML) cells. The effects of ES-Cu on the proliferation of AML cells and the AML cells pre-treated with ammonium tetrathiomolybdate (TTM) were examined by CCK-8 assay. The Calcein/PI kit was used to detected the changes in activity and cytotoxicity of AML cells induced by ES-Cu. Flow cytometry and Cytation3 fully automated cell imaging multifunctional detection system were used to analyze DCFH-DA fluorescence intensity, so as to determine the level of reactive oxygen species (ROS). The GSH and GSSG detection kits were used to measure the intracellular GSH content. Western blot was used to detected the expression of cuproptosis-related proteins ATP7B, FDX1, DLAT and DPYD. ES-Cu inhibited the proliferation of Kasumi-1 and HL-60 cells in a concentration-dependent manner (r Kasumi-1=-0.99， r HL-60=-0.98). As the concentration of ES-Cu increased, the level of intracellular ROS also increased (P <0.01-0.001). TTM could significantly reverse the inhibitory effect of ES-Cu on cell proliferation and its promoting effect on ROS. With the increase of ES-Cu concentration, the content of GSH was decreased (r =-0.98), and Western blot showed that the protein expressions of ATP7B, FDX1, DLAT and DPYD were significantly reduced (P <0.05). ES-Cu can induce cuproptosis in AML cells, which provides a new idea for the treatment of AML.

Innovative Combinations, Cellular Therapies and Bispecific Antibodies for Chronic Lymphocytic Leukemia: A Narrative Review.

In the last few years, several agents targeting molecules that sustain the survival and the proliferation of chronic lymphocytic leukemia (CLL) cells have become clinically available. Most of these drugs target surface proteins, such as CD19 or CD20, via monoclonal or bispecific monoclonal antibodies (BsAbs), CAR T cells, intracellular proteins like BTK by using covalent or non-covalent inhibitors or BCL2 with first or second generation BH3-mimetics. Since the management of CLL is evolving quickly, in this review we highlighted the most important innovative treatments including novel double and triple combination therapies, CAR T cells and BsAbs for CLL. Recently, a large number of studies on novel combinations and newer strategic options for CLL therapy have been published or presented at international conferences, which were summarized and linked together. Although the management of treatment with a single continuous agent is easier, the emergence of protein mutations, long-term toxicities and costs are important concerns that favor the use of a fixed duration therapy. In the future, a measurable residual disease (MRD)-guided treatment cessation and MRD-based re-initiation of targeted therapy seems to be a more feasible approach, allowing identification of the patients who might benefit from continuous therapy or who might need a consolidation with BsAbs or CAR T cells to clear the neoplastic clone.

LSP1 promotes the progression of acute myelogenous leukemia by regulating KSR/ERK signaling pathway and cell migration

ABSTRACT We aimed to investigate the role and mechanism of LSP1 in the progression of acute myelogenous leukemia. In this study, we established shLSP1 cell line to analyze the function of LSP1 in AML. We observed high expression of LSP1 in AML patients, whereas it showed no expression in normal adults. Furthermore, we found that LSP1 expression was associated with disease prognosis. Our results indicate that LSP1 plays a crucial role in mediating proliferation and survival of leukemia cells through the KSR/ERK signaling pathway. Additionally, LSP1 promotes cell chemotaxis and homing by enhancing cell adhesion and migration. We also discovered that LSP1 confers chemotactic ability to leukemia cells in vivo. Finally, our study identified 12 genes related to LSP1 in AML, which indicated poor survival outcome in AML patients and were enriched in Ras and cell adhesion signaling pathways. Our results revealed that the overexpression of LSP1 is related to the activation of the KSR/ERK signaling pathway, as well as cell adhesion and migration in AML patients. Reducing LSP1 expression impair AML progression, suggesting that LSP1 may serve as a potential drug therapy target for more effective treatment of AML.

Nilotinib Exerts a Therapeutic Approach via JAK/STAT Pathway and Cytokine Network in Chronic Myeloid Leukemia Cells

Aim: Chronic myeloid leukemia (CML) displays a constitutive tyrosine kinase (TK) activity which in turn leads to the activation of various signaling pathways and the outcome of leukemic phenotype. Activated STAT5A and STAT5B from JAK/STAT pathway induce cell growth, proliferation, differentiation, and survival of leukemic cells which are promoted by a cytokine network. Since the second-generation tyrosine kinase inhibitor nilotinib has the advantage of inhibiting this oncogenic TK activity; we aimed to investigate the underlying mechanism of its therapeutic approach and how it induced apoptosis via analyzing the forthcoming molecular targets of the pathway. &#x0D; Methods: By Nilotinib treatments, cell viability and proliferation assays, apoptotic analysis, expressional regulations of STAT5A&amp;5B mRNA transcripts, protein expression levels, and also cytokines’ expressional assessments were determined in CML model K562 cells, in vitro.&#x0D; Results: Nilotinib treatment in a time and dose-dependent manner assessed a therapeutic approach by decreasing leukemic cell proliferation and survival; inducing leukemic cell apoptosis, down-regulating STAT5A&amp;5B mRNA, and protein expression levels, and regulating cytokine expressional network. &#x0D; Conclusion: Nilotinib-mediated therapeutics could be dependent on targeting JAK/STAT pathway members STAT5A and STAT5B, besides; regulating the cytokine network might be another underlying mechanism for sensitization and response of K562 cells to nilotinib in leukemia pathogenesis.

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.

Effect of Ferroptosis Inducers and Inhibitors on Cell Proliferation in Acute Leukemia.

Ferroptosis refers to an iron-dependent mechanism of regulated cell death that is attributable to lipid peroxidation. Ferroptosis has been documented as a therapeutic target for various solid cancers; nonetheless, its implication in leukemia remains ambiguous. Therefore, this study aimed at investigating the impact of ferroptosis inducers and inhibitors on in vitro leukemia cell line proliferation. Six leukemia cell lines, including acute myeloid leukemia (AML)-derived MV4-11, THP-1, HL-60, and U-937, and T-lymphoblastic leukemia (T-ALL)-derived Jurkat and KOPT-K1 with activating NOTCH1 mutations, were assessed. Erastin, which interrupts cystine uptake and depletes intracellular glutathione, and RAS-selective lethal 3 (RSL3), which suppresses glutathione peroxidase 4 (GPX4), were employed as ferroptosis inducers. Lipid peroxidation-arresting ferrostatin-1 and deferoxamine were used as ferroptosis inhibitors. Cells were cultured with these compounds and cell proliferation was assessed using a colorimetric assay. Additionally, signaling protein expression was monitored using immunoblotting, and the outcome of GPX4 knockdown was evaluated. Ferroptosis inducers suppressed proliferation in all cell lines except THP-1 for Erastin and THP-1 and Jurkat for RSL3. Although the ferroptosis inhibitors did not affect cell proliferation, they rescued inducer-mediated growth suppression. Ferroptosis inducers impeded MYC and cyclin D3 expression in certain cell lines and NOTCH1 signaling in T-ALL cells. GPX4 knockdown and RSL3 treatment interrupted MYC and cyclin D3 expression, respectively, in four cell lines. Ferroptosis inducers may serve as potential candidates for novel molecular therapy against AML and T-ALL.

MiRNA-126 as a Biomarker for Cancer Stem Cells: Role in Chemotherapy Resistance in Iraqi Patients with Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is characterized as an aggressive blood cancer with rapid growth of immature leukemic cells. It appears that each subtype of AML displays a distinct miRNA profile. miRNAs play a role in regulating gene expression that is implicated in AML pathogenesis. Objective: This study was designed to assess the level of miRNA-126 gene expression in relation to chemotherapy resistance in various AML groups with the hope of developing a novel marker for targeted therapy and the early diagnosis and prognosis of cancer stem cells in AML patients. Methods: 120 AML cases were studied. Based on the chemotherapy stage, 40 patients were assigned to each group (newly diagnosed, under treatment, or relapsed). Baghdad Teaching Hospital, Iraq, provided the cases and samples from February 2022 to April 2023. This study also included 40 healthy controls. We used the qRT-PCR method to count the genes after setting them to the same level as a housekeeping gene (GAPDH). This method uses the ∆Ct-value and fold change (2-∆∆Ct). Results: In this study, there were significant elevated levels of miRNA-126 in AML patients compared to controls, with a higher fold change detected in the newly diagnosed group. Conclusions: The miRNA-126 upregulation is suggested to be linked to AML development and relapse, with a contribution to leukemic stem cell proliferation and treatment failure. We hypothesized that miR-126 could be an effective target for eradicating the LSC in AML.

Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6’s role in hematopoietic differentiation and leukemia

BackgroundLong non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources).MethodsINFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo.ResultsThe INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo.ConclusionsOur approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine.

PHF6 loss reduces leukemia stem cell activity in an acute myeloid leukemia mouse model

Acute myeloid leukemia (AML) is a malignant hematologic disease caused by gene mutations and genomic rearrangements in hematologic progenitors. The PHF6 (PHD finger protein 6) gene is highly conserved and located on the X chromosome in humans and mice. We found that PHF6 was highly expressed in AML cells with MLL rearrangement and was related to the shortened survival time of AML patients. In our study, we knocked out the Phf6 gene at different disease stages in the AML mice model. Moreover, we knocked down PHF6 by shRNA in two AML cell lines and examined the cell growth, apoptosis, and cell cycle. We found that PHF6 deletion significantly inhibited the proliferation of leukemic cells and prolonged the survival time of AML mice. Interestingly, the deletion of PHF6 at a later stage of the disease displayed a better anti-leukemia effect. The expressions of genes related to cell differentiation were increased, while genes that inhibit cell differentiation were decreased with PHF6 knockout. It is very important to analyze the maintenance role of PHF6 in AML, which is different from its tumor-suppressing function in T-cell acute lymphoblastic leukemia (T-ALL). Our study showed that inhibiting PHF6 expression may be a potential therapeutic strategy targeting AML patients.