Acute Myeloid Leukemia Proliferation Research Articles

Dissection of the MEF2D-IRF8 transcriptional circuit dependency in acute myeloid leukemia

SummaryTranscriptional dysregulation is a prominent feature in leukemia. Here, we systematically surveyed transcription factor (TF) vulnerabilities in leukemia and uncovered TF clusters that exhibit context-specific vulnerabilities within and between different subtypes of leukemia. Among these TF clusters, we demonstrated that acute myeloid leukemia (AML) with high IRF8 expression was addicted to MEF2D. MEF2D and IRF8 form an autoregulatory loop via direct binding to mutual enhancer elements. One important function of this circuit in AML is to sustain PU.1/MEIS1 co-regulated transcriptional outputs via stabilizing PU.1’s chromatin occupancy. We illustrated that AML could acquire dependency on this circuit through various oncogenic mechanisms that results in the activation of their enhancers. In addition to forming a circuit, MEF2D and IRF8 can also separately regulate gene expression, and dual perturbation of these two TFs leads to a more robust inhibition of AML proliferation. Collectively, our results revealed a TF circuit essential for AML survival.

MiR-140 targets lncRNA FAM230B to suppress cell proliferation in acute myeloid leukemia running title: MiR-140 targets FAM230B in AML

ABSTRACT Background FAM230B serves as an oncogenic lncRNA in both gastric cancer and papillary thyroid cancer, while its role in acute myeloid leukemia (AML) is unclear. We predicted that FAM230B could be a target of miR-140, a well-characterized tumor suppressor, and analyzed their interaction in AML. Methods Differential expressions of FAM230B and miR-140 in bone marrow mononuclear cells (BMMNCs) were determined by RT-qPCR. Correlations were analyzed by Pearson’s correlation coefficient. Subcellular FAM230B location was determined using cellular fractionation assay. The direct interaction between FAM230B and miR-140 was confirmed by RNA pull-down assay. The roles of FAM230B and miR-140 in cell proliferation were explored using BrdU assay. Results High FAM230B expression level and low miR-140 expression level were observed in AML. FAM230B and miR-140 were inversely correlated and directly interacted with each other. FAM230B could be detected in both cytoplasm and nuclear samples. MiR-140 overexpression downregulated FAM230B expression and suppressed the enhancing effects of FAM230B overexpression on cell proliferation. Conclusion MiR-140 may target FAM230B to suppress cell proliferation in AML.

KMT2A Activates Akt/mTOR Signaling Pathway Through LncRNA-HOTAIR to Influence the Biological Behavior of AML Cells

To investigate the expression of lysine methyltransferase 2A (KMT2A) in acute myeloid leukemia (AML) cells and its molecular mechanism affecting the proliferation of AML cells. Co-immunoprecipitation assay was used to detect the binding of KMT2A to long non-coding RNA-HOX transcript antisense RNA (lncRNA-HOTAIR). AML cell proliferation was detected by 5-ethynyl-2'-deoxyuridine (EdU) assay. The PCR amplification signal of KMT2A group was significantly stronger than that of the negative control group and IgG group (P<0.01). Compared with the negative control group and KMT2A-OE + lncRNA-HOTAIR-KD group, the ratio of EdU+ cells in both KMT2A-OE group and lncRNA-HOTAIR-OE group significantly increased (P<0.01). Compared with negative control group, the ratio of EDU+ cells in KMT2A-KD group and lncRNA-HOTAIR-KD group significantly decreased (P<0.01), the expression levels of p-Akt and p-mTOR in both KMT2A-OE group and lncRNA-HOTAIR-OE group significantly increased (P<0.01). KMT2A can interact with lncRNA-HOTAIR to promote the activation of Akt/mTOR signaling pathway, thus promoting the proliferation of AML cells.

Republication: Targeting PI3KC2β Impairs Proliferation and Survival in Acute Leukemia, Brain Tumours and Neuroendocrine Tumours.

Eight human catalytic phosphoinositide 3-kinase (PI3K) isoforms exist which are subdivided into three classes. While class I isoforms have been well-studied in cancer, little is known about the functions of class II PI3Ks. The expression pattern and functions of the class II PI3KC2β isoform were investigated in a panel of tumour samples and cell lines. Overexpression of PI3KC2β was found in subsets of tumours and cell lines from acute myeloid leukemia (AML), glioblastoma multiforme (GBM), medulloblastoma (MB), neuroblastoma (NB), and small cell lung cancer (SCLC). Specific pharmacological inhibitors of PI3KC2β or RNA interference impaired proliferation of a panel of human cancer cell lines and primary cultures. Inhibition of PI3KC2β also induced apoptosis and sensitised the cancer cells to chemotherapeutic agents. Together, these data show that PI3KC2β contributes to proliferation and survival in AML, brain tumours and neuroendocrine tumours, and may represent a novel target in these malignancies.

Reductive TCA cycle catalyzed by wild-type IDH2 promotes acute myeloid leukemia and is a metabolic vulnerability for potential targeted therapy

BackgroundIsocitrate dehydrogenase-2 (IDH2) is a mitochondrial enzyme that catalyzes the metabolic conversion between isocitrate and alpha-ketoglutarate (α-KG) in the TCA cycle. IDH2 mutation is an oncogenic event in acute myeloid leukemia (AML) due to the generation of 2-hydroxyglutarate. However, the role of wild-type IDH2 in AML remains unknown, despite patients with it suffer worse clinical outcome than those harboring mutant type.MethodsIDH2 expression in AML cell lines and patient samples was evaluated by RT-qPCR, western blotting and database analyses. The role of wild-type IDH2 in AML cell survival and proliferation was tested using genetic knockdown and pharmacological inhibition in AML cells and animal models. LC–MS, GC–MS, isotope metabolic tracing, and molecular analyses were performed to reveal the underlying mechanisms.ResultsWe found that wild-type IDH2 was overexpressed in AML and played a major role in promoting leukemia cell survival and proliferation in vitro and in vivo. Metabolomic analyses revealed an active IDH2-mediated reductive TCA cycle that promoted the conversion of α-KG to isocitrate/citrate to facilitate glutamine utilization for lipid synthesis in AML cells. Suppression of wild-type IDH2 by shRNA resulted in elevated α-KG and decreased isocitrate/citrate, leading to reduced lipid synthesis, a significant decrease in c-Myc downregulated by α-KG, and an inhibition of AML viability and proliferation. Importantly, pharmacological inhibition of IDH2 showed significant therapeutic effect in mice inoculated with AML cells with wt-IDH2 and induced a downregulation of C-MYC in vivo.ConclusionsWt-IDH2 is an essential molecule for AML cell survival and proliferation by promoting conversion of α-KG to isocitrate for lipid synthesis and by upregulating c-Myc expression and could be a potential therapeutic target in AML.

H3K9 dimethylation safeguards cancer cells against activation of the interferon pathway.

Activation of interferon genes constitutes an important anticancer pathway able to restrict proliferation of cancer cells. Here, we demonstrate that the H3K9me3 histone methyltransferase (HMT) suppressor of variegation 3–9 homolog 1 (SUV39H1) is required for the proliferation of acute myeloid leukemia (AML) and find that its loss leads to activation of the interferon pathway. Mechanistically, we show that this occurs via destabilization of a complex composed of SUV39H1 and the two H3K9me2 HMTs, G9A and GLP. Indeed, loss of H3K9me2 correlated with the activation of key interferon pathway genes, and interference with the activities of G9A/GLP largely phenocopied loss of SUV39H1. Last, we demonstrate that inhibition of G9A/GLP synergized with DNA demethylating agents and that SUV39H1 constitutes a potential biomarker for the response to hypomethylation treatment. Collectively, we uncovered a clinically relevant role for H3K9me2 in safeguarding cancer cells against activation of the interferon pathway.

Interleukin-4 gene intron 3 VNTR polymorphism in adult acute myeloid leukemia

BackgroundThe proliferation of acute myeloid leukemia (AML) blast into the bone marrow microenvironment is controlled by cytokines. Interleukin-4 (IL-4) has recently been discovered to suppress the development and persistence of AML cells selectively. Intron three of the Interleukin-4 (IL-4) gene contains a 70-bp minisatellite region polymorphism that may influence gene transcriptional activity and subsequently affect the production level of IL4. We investigated the IL-4 gene intron three variable number tandem repeat (VNTR) polymorphism as a molecular marker in AML associated with clinical and laboratory variables and a prognostic factor for therapeutic response and disease outcome.ResultsIL-4 gene intron three minisatellite regions polymorphism was assessed in 60 adult AML patients and 60 healthy controls, comparable concerning age and gender, using polymerase chain reaction. Three study marker genotypes were detected in AML patients; P1/P1 (3%), P1/P2 (40%), and P2/P2 (56.7%). The frequency of P2 alleles was significantly more in AML patients than in healthy controls (76.7% versus 25%; P < 0.001). Compared to the heterozygous group and P1/P1 carriers, AML patients with the homozygous P2/P2 genotype had a higher total leucocytic count and increased blast percentages in bone marrow or peripheral blood, besides a lower platelet count. P2P2 genotype was also significantly associated with poor therapeutic response, higher susceptibility to disease recurrence and shorter overall survival and disease-free survival.ConclusionThe IL-4 intron 3 VNTR polymorphism could be included in the molecular risk stratification of AML to predict poor disease. This information can be utilized in incorporating biological therapy into the present therapeutic protocols to enhance chemotherapy regimens’ current low response rates.

The role and mechanism of lncRNA C9ORF139 targeting miR-24-3P/TAOK1 in regulating the proliferation of acute myeloid leukemia cells

Objective: To investigate the role and mechanism of long non-coding RNA (lncRNA) C9ORF139 targeting micro RNA(miR)-24-3P/TAOK1 in regulating the proliferation of acute myeloid leukemia (AML) cells. Methods: AML cells HL-60 and THP-1 were purchased from the Chinese Academy of Sciences and divided into 4 groups:group A was negative control group (siNC group), group B was interference C9ORF139 group (siC9ORF139 group), group C was siC9ORF139+miR-24-3p inhibitor group, and group D was miR-24-3P+TAOK1 overexpression group (oe-TAOK1 group). Real-time fluorescence quantitative reverse transcription PCR was used to detect the expression levels of AML cell lines of HL-60 and THP-1 in four groups. Cell Counting Kit-8 assay was performed to measure cell proliferation. Flow cytometry was applied to analyze cell apoptosis. Transwell test was applied to detect cell migration and invasion ability. Western blot was used to detect p-serine/threonine kinase (p-raf) and p-mitogen activation proteinkinase (p-MEK), p-extracellular regulatory protein kinase (p-ERK) expression. The luciferase reporter gene plasmid was constructed to verify the binding ability of C9ORF139,miR-24-3P and TAOK1.Nude mice were inoculated with subcutaneous tumor cells of HL-60 (group A) and HL-60 (group B). Results: After the C9ORF139 gene was knocked down and cultured for 120 h, The cell proliferation ability (0.62±0.02, 0.82±0.02), migration ability (0.22±0.03, 0.05±0.01), invasion ability (0.20±0.02, 0.13±0.03) of group B were all lower than that of group A (1.30±0.02, 1.83±0.07; 0.99±0.02, 0.99±0.02; 1.00±0.01, 1.00±0.01) (all P<0.05). When co-transfected with miR-24-3 inhibitor, cell proliferation ability, migration ability and invasion ability were all higher in group B (all P<0.05). When co-transfected with miR-24-3P and oe-TAOK1 plasmid, cell proliferation ability, migration ability and invasion ability were all higher than group B (all P<0.05).When the C9ORF139 gene in the cells was knocked down, the apoptosis level of group B (28.56±8.07, 17.74±1.91) were higher than those of group A (0.31±0.27, 2.49±0.33)(all P<0.05); when co-transfected with miR-24-3P inhibitor, the apoptosis level (2.34±0.09, 3.06±0.06) were lower than those in group B (all P<0.05); when co-transfected with miR-24-3P and oe-TAOK1 in the plasmid group, the apoptosis level (2.16±1.29, 4.80±0.37) were also lower than those of group B (all P<0.05). In HL-60 and THP-1 cells, when C9ORF139 was not mutated, the luciferase activity of miR-24-3P group was lower than that of the miR-NC group (P<0.05). When the binding site with miR-24-3p in C9ORF139 sequence was mutated, the luciferase activity in miR-24-3p group was equivalent to that in miR-NC group (P>0.05).When TAOK1 was not mutated; the luciferase activity of miR-24-3P group was lower than that of group A (P<0.05). When the binding site with miR-24-3p in TAOK1 sequence was mutated, the luciferase activity in miR-24-3p group was equivalent to that in miR-NC group (P>0.05).When the C9ORF139 gene in HL-60 cells was knocked down and cultured for 72 h, the phosphorylation expression levels of Raf, MEK and ERK molecules in group B were significantly lower than those in group A (all P<0.05). By day 14, the tumor volume in the group A was greater than the tumor cell volume in the group B [(284.49±57.61) vs (125.70±18.64) mm3, P=0.017]. The tumor weight of HL-60 in group A was heavier than that of group B [(847.80±159.36) vs (408.40±113.16) mg, P=0.001]. Conclusions: LncRNA C9ORF139 regulates TAOK1 by sponging miR-24-3P to promote the proliferation, invasion and migration of acute myeloid leukemiacell.In vivo experiments have confirmed that the expression of C9ORF139 can promote the growth of subcutaneous tumors in AML nude mice.

Myeloid ecotropic viral integration site-1 inhibition promotes apoptosis, suppresses proliferation of acute myeloid leukemia cells, accentuates the effects of anticancer drugs

ABSTRACT To investigate the effects of myeloid ecotropic viral integration site-1 (MEIS1) on the proliferation and apoptosis of acute myeloid leukemia (AML) cells and the anticancer effects of the drug, we screened Kasumi-6, KG-1, and Kasumi-1 cells using quantitative reverse transcription polymerase chain reaction. Kasumi-6 and Kasumi-1 cells were subjected to human antigen R (HuR)-mediated interference (IV). Hexokinase 2 (HK2) expression and phosphorylation of protein kinase B (p-AKT) and mammalian target of rapamycin (p-mTOR) were observed with Western blotting. Cell proliferation was assessed using Cell Counting Kit-8, apoptosis was examined using Hoechst 33,258 staining, and glucose uptake was detected with a colorimetric biochemical assay kit. We found that, among the three cell lines tested, MEIS1 expression was highest in Kasumi-1 cells, which were therefore selected for subsequent experiments. Kasumi-1 cells receiving IV showed significantly decreased proliferation (p < 0.05) and increased apoptosis compared to the control group. Compared with the controls, IV significantly increased the expression of HK2, p-AKT, p-mTOR, multidrug resistance-associated protein 1 and P-glycoprotein (P < 0.05), but decreased glucose uptake. Treatment with adriamycin, daunorubicin and imatinib resulted in a progressive increase in inhibition of cell proliferation, with the IV group showing the highest inhibition rate among the three groups (P < 0.05). Thus, inhibition of MEIS1 activity promoted apoptosis, inhibited the proliferation of Kasumi-1 and Kasumi-6 cells, and increaseed the anticancer effect of the drugs, suggesting that inhibition of MEIS1 may be a potential strategy for the treatment of AML.

MiR-140 Targets lncRNA DNAJC3-AS1 to Suppress Cell Proliferation in Acute Myeloid Leukemia.

ObjectivesMiR-140 and DNAJC3-AS1 have been demonstrated to play critical roles in cancer biology, while their participation in acute myeloid leukemia (AML) is unclear. This study aimed to explore the role of miR-140 and DNAJC3-AS1 in AML.MethodsThe expression of DNAJC3-AS1 and miR-140 were detected by RT-qPCR. Then, the role of DNAJC3-AS1 and miR-140 in regulating each other was explored by overexpression assay. Next, the direct interaction between DNAJC3-AS1 and miR-140 was analyzed using an RNA pull-down assay. Next, the subcellular location of DNAJC3-AS1 was explored using cellular, subcellular fractionation assay. Finally, cell proliferation analysis was evaluated with BrdU assay.ResultsIncreased expression levels of DNAJC3-AS1 and decreased expression levels of miR-140 were observed in AML patients. DNAJC3-AS1 was detected in nuclear and cytoplasm samples and direct interaction between DNAJC3-AS1 and miR-140 was observed.DiscussionReduced expression levels of DNAJC3-AS1 were observed after overexpression of miR-140 in AML cells. DNAJC3-AS1 increased cell proliferation and inhibited the role of miR-140 in suppressing cell proliferation.ConclusionIn conclusion, miR-140 may target DNAJC3-AS1 to suppress cell proliferation in AML.

SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

SUMMARYAcute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we use domain-focused CRISPR screening and identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML.

The expression and clinical significance of CD59 and FLAER in Chinese adult AML patients.

BackgroundThe role of CD59 and fluorescently labeled aerolysin (FLAER) in acute myeloid leukemia (AML) remains unclear and requires further investigation. To explore the relationship between CD59, FLAER, and AML, we investigated CD59 and FLAER expression in AML and analyzed their relationship with clinical characteristics of AML patients.MethodsWe employed flow cytometry (FCM) to analyze CD59 and FLAER expression in 161 AML patients at Tianjin Medical University General Hospital and evaluated its association with sex, white blood cell (WBC) count, platelet (PLT) count, thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), D‐Dimer(D‐D), and lactate dehydrogenase (LDH), followed by analyzing its connection with disease progression and complete remission (CR).ResultsCD59 and FLAER deficiencies were identified in AML patients. Compared with CR group, non‐CR group patients revealed more CD59 and FLAER deficiency. Compared with non‐acute promyelocytic leukemia (M3) group, M3 group patients had more CD59 and FLAER deficiency. CD59− level in primordial cells of M3 patients was positively correlated with primordial cell ratio (r = 0.660, p = 0.003). Additionally, we discovered that the decline in CD59 and FLAER levels might be linked to higher D‐D and LDH in AML patients. The difference was statistically significant (p < 0.05).ConclusionsWe demonstrated that the decline in CD59 and FLAER levels was associated with leukemia cell proliferation and abnormal coagulation function in AML, suggesting that they could serve as a predictor of AML coagulation dysfunction, particularly in M3.

Metabolic Rewiring Is Essential for AML Cell Survival to Overcome Autophagy Inhibition by Loss of ATG3.

Simple SummaryThe importance of autophagy in leukemia progression and survival has been studied previously. However, little is known about the development of resistance mechanisms to autophagy inhibition in leukemia. Here, we present data on the mechanisms by which leukemia cells maintain their cell survival after inhibition of autophagy by the loss of ATG3. After the loss of ATG3, leukemia cells upregulated their energy metabolism by increasing glycolysis and mitochondrial metabolism, in particular oxidative phosphorylation, which resulted in higher ATP levels. Moreover, inhibition of mitochondrial function strongly impaired cell survival in ATG3 deficiency, thus demonstrating the importance of ATG3 in the regulation of metabolism and survival of leukemic cells. Therefore, our data provide a rationale for combining autophagy inhibitors with inhibitors targeting mitochondrial metabolism for the development of leukemia therapy to overcome the potential obstacle of emerging resistance to autophagy inhibition.Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.

Lin28A/CENPE Promoting the Proliferation and Chemoresistance of Acute Myeloid Leukemia.

The prognosis of chemoresistant acute myeloid leukemia (AML) is still poor, mainly owing to the sustained proliferation ability of leukemic cells, while the microtubules have a major role in sustaining the continuity of cell cycle. In the present study, we have identified CENPE, a microtubular kinesin-like motor protein that is highly expressed in the peripheral blood of patients with chemoresistant AML. In our in vitro studies, knockdown of CENPE expression resulted in the suppression of proliferation of myeloid leukemia cells and reversal of cytarabine (Ara-C) chemoresistance. Furthermore, Lin28A, one of the RNA-binding oncogene proteins that increase cell proliferation and invasion and contribute to unfavorable treatment responses in certain malignancies, was found to be remarkably correlated with CENPE expression in chemoresistance AML. Overexpression of LIN28A promoted the proliferation and Ara-C chemoresistance of leukemic cells. RIP assay, RNA pull-down, and dual luciferase reporter analyses indicated that LIN28A bound specifically to the promoter region GGAGA of CENPE. In addition, the impacts of LIN28A on cell growth, apoptosis, cell cycle progression, and Ara-C chemoresistance were reverted by the knockdown of CENPE. Hence, Lin28A/CENPE has enhanced the proliferation and chemoresistance of AML, and therefore, it could be a prospective candidate for AML treatment.

LC3-Associated Phagocytosis in Bone Marrow Macrophages Suppresses AML Progression through Mitochondrial DAMP Induced Sting Activation

The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation and chemotherapy resistance. Following cancer cell death, a growing body of evidence suggests an important role for uncleared apoptotic debris in regulating the immunologic response to, and growth of, solid tumors. LC3-associated phagocytosis (LAP) maintains tissue homeostasis by regulating immune responses, such as tumor immunity. Here we investigate the role of LAP in macrophage within the BM microenvironment of AML.We find that depletion of BM macrophages via clodronate liposomes increased AML growth in-vivo. We show that LAP is an important pathway in BM macrophage to process dead and dying cells in the AML microenvironment. We used two syngeneic leukemia models (HOXA9/Meis1 and MN1) to investigate the role of LAP on AML proliferation. AML cells were injected into LAP deficient (Atg16L1 E230-) and wild-type (Atg16L1 E230+) mice. Targeted inhibition of LAP leads to accumulation of apoptotic cells (AC) and apoptotic bodies (AB) in the tumor microenvironment resulting in accelerated leukemia growth and decreased animal survival.Mechanistically, we show, via cytokine arrays and gene analysis, that the phagocytosis of AML derived AB via LAP in BM macrophage resulted in STING pathway activation in the phagocytic cells. Furthermore, through inhibition of STING using H-151 STING inhibitor, we show that STING activation in vivo supressed leukemia growth. STING activation can lead to a type I IFN response and to recruitment of cytotoxic T-cells. We saw no increase in CD8 + T-cell numbers or activation, however, via ex vivo analysis found that STING activation is required for phagocytic functions in macrophages.Next, we found that leukemic AB can induce a STING response in BM derived macrophages and that leukemic AB have increased mitochondria content that are processed by macrophages. Moreover, we identify that mitochondrial damage associated molecular patterns (DAMPs) from leukemic AB are processed by BM macrophages via LAP. Additionally, the depletion of mitochondrial DNA (mtDNA) in AML derived AB identified that the mtDNA from leukemic AB is responsible for the induction of STING signalling in BM macrophages.In summary, we report that LAP in BM macrophage of apoptotic debris in the AML microenvironment suppresses leukemic growth, through mechanisms stimulated by AML apoptotic bodies which contain mtDNA in the BM microenvironment. This process is mediated by the activation of the STING pathway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Proteasome Inhibition Attenuates Self-Renewal in Human Acute Myeloid Leukemia By Targeting NF-Kappa B in Leukemia Stem Cells

In acute myeloid leukemia (AML), relapse following standard chemotherapy is common, leading to 2-year survival rates of less than 30%. Relapse is caused by leukemia stem cells (LSCs), a rare population of mostly quiescent cells that are chemo-refractory and can recapitulate the disease. Our overall goal is to define mechanisms of self-renewal that could be targeted to cure AML and prevent relapse. Functionally, self-renewal is defined by the ability to propagate leukemia in vivo.We use transcriptional and protein profiling to define the functional states of LSCs. We previously demonstrated that NRAS G12V facilitates self-renewal in a mouse model of AML (Mll-AF9/NRAS G12V). Using single-cell RNA sequencing and in vivo leukemia assays, we showed that the stem cell compartment in this model has two distinct subpopulations which differ in their self-renewal and proliferative abilities. The subset of LSCs marked by CD36 LowCD69 High (CD69 High) expression can self-renew and are poorly proliferative. The CD36 HighCD69 Low (CD36 High) subset is unable to self-renew and is highly proliferative. These data demonstrated that self-renewal and rapid proliferation are mutually exclusive functions among LSCs. We demonstrated that the gene expression profiles associated with these functionally distinct LSC subsets are also similarly differentially expressed in primary human LSCs, at the single-cell level (Sachs Cancer Research 2020). Given the functional differences between the CD36 LowCD69 High and CD36 HighCD69 Low subsets in murine AML, we tested whether CD69 and CD36 likewise discriminate self-renewal and proliferation in human AML. We sorted primary human AML samples according to CD69 and CD36 expression and found that Lin -CD69 High cells formed more colonies than Lin -CD36 High cells in every sample tested (n=6). Notably, in three of the six samples, the Lin -CD36 High population was unable to form any colonies (Panel A). These data suggest that CD69 may identify a self-renewing subset of human AML as it does in murine AML. Next, we asked whether Lin -CD69 High and Lin -CD36 High subsets harbor unique signaling protein activation profiles that could potentially be targeted therapeutically. We used CyTOF (mass cytometry) to compare the levels of 12 intracellular signaling proteins between these subpopulations in 14 human AML samples with intermediate and poor risk genetics. CyTOF quantitatively measures a panel of proteins at the single-cell level and allows us to profile activated signaling pathways within well-defined immunophenotypic subpopulations. We found upregulation of both total and phosphorylated (t- and p-) NF-kappa B, pERK, p4EBP1, pMAPKAPKII, and its upstream activator, pP38 in the Lin -CD69 High subset relative to the Lin -CD36 High. Notably, CD69 is a NF-kappa B target gene. Indeed, our single-cell transcriptional data showed that NF-kappa B target genes were upregulated in the self-renewing, CD69 High subset of murine and human LSCs (Sachs Cancer Research 2020). NF-kappa B is a well-known mediator of AML-self-renewal. Next, we asked whether targeting NF-kappa B with proteasome inhibitors might reduce self-renewal capacity in human AML. We plated six primary human samples in colony-forming assays with pan-proteasome inhibitor, carfilzomib, immunoproteasome inhibitor, PR957, or vehicle and found that both inhibitors reduced colony formation in every sample tested. We harvested colonies from each of the inhibitor and vehicle groups and plated them in secondary colony-forming assays. Colonies harvested from vehicle-treated samples formed colonies but those harvested from either carfilzomib or PR957-treated samples were unable to form any secondary colonies (Panel B). Using CyTOF, we validated that both inhibitors reduce NF-kappa B levels in these samples.These data demonstrate that CD69 marks a self-renewing subset of human AML, as it does in our murine AML model, and suggest that the transcriptional mechanisms of self-renewal that we defined in our murine model at the single-cell level may be shared with human AML. Additionally, these data demonstrate enhanced NF-kappa B levels in this self-renewing subset and that targeting the NF-kappa B pathway with proteasome inhibitors attenuates self-renewal in primary human AML samples (Panel C). These findings suggest that proteasome inhibition may be an effective approach to treating and preventing relapse in AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity

BackgroundDisease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML.MethodsLeukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors.ResultsMolecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression.ConclusionsWe found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.

MiRNA-142-3P and FUS can be Sponged by Long Noncoding RNA DUBR to Promote Cell Proliferation in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) represents a frequently occurring adulthood acute leukemia (AL). Great progresses have been achieved in the treatment of AML, but its pathogenic mechanism remains unclear. This study reported the biological functions of lncRNA DUBR in AML pathogenic mechanism. As a result, lncRNA DUBR showed high expression level within AML, resulting in poor prognosis, especially in M4 AML. In vitro studies elucidated that knockdown of DUBR with small interfering RNA (siRNA) resulted in the suppression of survival and colony formation ability, as well as induction of apoptosis, in AML cells. RNA pull-down assay and computational revealed that DUBR could sponge with miRNA-142-3P and interact with FUS protein. MiRNA-142-3P have a negative correlation with DUBR and overexpression of miRNA-142-3P inhibited cell growth in AML. Meanwhile, DUBR promoted the expression of FUS protein, targeting inhibition of FUS significantly promoted cell apoptosis in AML cell lines. In conclusion, these results revealed new mechanism of lncRNA DUBR in AML malignant behavior, and suggested that the manipulation of DUBR expression could serve as a potential strategy in AML therapy.

LukS-PV Induces Apoptosis via the SET8-H4K20me1-PIK3CB Axis in Human Acute Myeloid Leukemia Cells.

Evidence suggests that histone modification disorders are involved in leukemia pathogenesis. We previously reported that LukS-PV, a component of Panton–Valentine leukocidin (PVL), has antileukemia activities that can induce differentiation, increase apoptosis, and inhibit proliferation of acute myeloid leukemia (AML) cells. Furthermore, LukS-PV inhibited hepatoma progression by regulating histone deacetylation, speculating that LukS-PV may exert antileukemia activity by targeting histone modification regulators. In this study, the results showed that LukS-PV induced apoptosis by downregulating the methyltransferase SET8 and its target histone H4 monomethylated at Lys 20 (H4K20me1). Furthermore, chromatin immunoprecipitation sequencing and polymerase chain reaction identified the kinase PIK3CB as a downstream target gene for apoptosis mediated by SET8/H4K20me1. Finally, our results indicated that LukS-PV induced apoptosis via the PIK3CB-AKT-FOXO1 signaling pathway by targeting SET8. This study indicates that SET8 downregulation is one of the mechanisms by which LukS-PV induces apoptosis in AML cells, suggesting that SET8 may be a potential therapeutic target for AML. Furthermore, LukS-PV may be a drug candidate for the treatment of AML that targets epigenetic modifications.

Downregulation of GNA15 Inhibits Cell Proliferation via P38 MAPK Pathway and Correlates with Prognosis of Adult Acute Myeloid Leukemia With Normal Karyotype.

BackgroundThe prognosis of acute myeloid leukemia (AML) with a normal karyotype is highly heterogonous, and the current risk stratification is still insufficient to differentiate patients from high-risk to standard-risk. Changes in some genetic profiles may contribute to the poor prognosis of AML. Although the prognostic value of G protein subunit alpha 15 (GNA15) in AML has been reported based on the GEO (Gene Expression Omnibus) database, the prognostic significance of GNA15 has not been verified in clinical samples. The biological functions of GNA15 in AML development remain open to investigation. This study explored the clinical significance, biological effects and molecular mechanism of GNA15 in AML.MethodsReverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression level of GNA15 in blasts of bone marrow specimens from 154 newly diagnosed adult AML patients and 26 healthy volunteers. AML cell lines, Kasumi-1 and SKNO-1, were used for lentiviral transfection. Cell Counting Kit-8 (CCK8) and colony formation assays were used to determine cell proliferation. Cell cycle and apoptosis were analyzed by flow cytometry. The relevant signaling pathways were evaluated by Western blot. The Log-Rank test and Kaplan-Meier were used to evaluate survival rate, and the Cox regression model was used to analyze multivariate analysis. Xenograft tumor mouse model was used for in vivo experiments.ResultsThe expression of GNA15 in adult AML was significantly higher than that in healthy individuals. Subjects with high GNA15 expression showed lower overall survival and relapse-free survival in adult AML with normal karyotype. High GNA15 expression was independently correlated with a worse prognosis in multivariate analysis. Knockdown of GNA15 inhibited cell proliferation and cell cycle progression, and induced cell apoptosis in AML cells. GNA15-knockdown induced down-regulation of p-P38 MAPK and its downstream p-MAPKAPK2 and p-CREB. Rescue assays confirmed that P38 MAPK signaling pathway was involved in the inhibition of proliferation mediated by GNA15 knockdown.ConclusionsIn summary, GNA15 was highly expressed in adult AML, and high GNA15 expression was independently correlated with a worse prognosis in adult AML with normal karyotype. Knockdown of GNA15 inhibited the proliferation of AML regulated by the P38 MAPK signaling pathway. Therefore, GNA15 may serve as a potential prognostic marker and a therapeutic target for AML in the future.