Glucose induced-AKT/mTOR activation accelerates glycolysis and promotes cell survival in acute myeloid leukemia.

Multiple studies have demonstrated that excessive glucose utilization is a common feature of cancer cells to support malignant phenotype. Acute myeloid leukemia (AML) is recognized as a heterogeneous disorder of hematopoietic stem cells characterized by altered glucose metabolism. However, the role of glucose metabolic dysfunction in AML development remains obscure. In this study, glucose and 2-Deoxy-D-glucose (2-DG) treatment were applied to analyze the relationship between glucose metabolism and cell survival. Cell Counting Kit-8 (CCK-8) and flow cytometry (FCM) assays were used to examine the cell viability and apoptosis rate. Glucose consumption and lactate production were measured to assess the glucose metabolism pathway. The results demonstrated that abnormally increased glucose effectively promoted proliferation of leukemic cells and inhibited cell apoptosis, while 2-DG ameliorated leukemic phenotypes. Importantly, glucose exposure induced active glycolysis by increasing glucose consumption and lactate production. Furthermore, the levels of key glycolysis-related genes glucose transporter 1 (GLUT1) and monocarboxylate transporter 1 (MCT1) were upregulated. Mechanistic investigations revealed that AKT/mTOR signaling pathway was activated in glucose condition. In conclusion, our findings indicate that glucose induced-AKT/mTOR activation plays a growth-promoting role in AML, highlighting that inhibition of glycolysis would be a vital adjuvant therapy strategy for AML.

Cytoplasmic Expression of TP53INP2 Modulated by Demethylase FTO and Mutant NPM1 Promotes Autophagy in Leukemia Cells.

Acute myeloid leukemia (AML) with a nucleophosmin 1 (NPM1) mutation is a unique subtype of adult leukemia. Recent studies show that NPM1-mutated AML has high autophagy activity. However, the mechanism for upholding the high autophagic level is still not fully elucidated. In this study, we first identified that tumor protein p53 inducible nuclear protein 2 (TP53INP2) was highly expressed and cytoplasmically localized in NPM1-mutated AML cells. Subsequent data showed that the expression of TP53INP2 was upregulated by fat mass and obesity-associated protein (FTO)-mediated m6A modification. Meanwhile, TP53INP2 was delocalized to the cytoplasm by interacting with NPM1 mutants. Functionally, cytoplasmic TP53INP2 enhanced autophagy activity by promoting the interaction of microtubule-associated protein 1 light chain 3 (LC3) - autophagy-related 7 (ATG7) and further facilitated the survival of leukemia cells. Taken together, our study indicates that TP53INP2 plays an oncogenic role in maintaining the high autophagy activity of NPM1-mutated AML and provides further insight into autophagy-targeted therapy of this leukemia subtype.

Targeted activation of GPER enhances the efficacy of venetoclax by boosting leukemic pyroptosis and CD8+ T cell immune function in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a rapidly progressing and often fatal hematopoietic malignancy. Venetoclax (VEN), a recent FDA-approved BCL-2 selective inhibitor, has high initial response rates in elderly AML patients, but the majority of patients eventually acquire resistance. Multiple studies have demonstrated that the female sex is associated with better outcomes in patients with AML, which are predominantly attributed to estrogen signaling. As a novel membrane estrogen receptor, G protein-coupled estrogen receptor (GPER)-mediated-rapid estrogen effects have attracted considerable attention. However, whether targeting GPER enhances the antileukemic activity of VEN is unknown. In this study, we first demonstrated that GPER expression was dramatically reduced in AML cells owing to promoter hypermethylation. Furthermore, pharmacological activation of GPER by G-1 combined with VEN resulted in synergistic antileukemic activity in vitro and in vivo. Mechanistically, G-1/VEN combination synergistically triggered concurrent mitochondria-related apoptosis and gasdermin E (GSDME)-dependent pyroptosis by activating p38-MAPK/myeloid cell leukemia 1 (MCL-1) axis. Importantly, leukemic pyroptosis heightened CD8+ T cell immune function by releasing interleukin (IL)-1ß/18 into the tumor microenvironment. Our study corroborates that GPER activation shows a synergistic antileukemic effect with VEN, making it a promising therapeutic regimen for AML.

Circulating plasma exosomal long non-coding RNAs LINC00265, LINC00467, UCA1, and SNHG1 as biomarkers for diagnosis and treatment monitoring of acute myeloid leukemia.

Exosomal long non-coding RNAs (lncRNAs) have emerged as a cell-free biomarker for clinical evaluation of cancers. However, the potential clinical applications of exosomal lncRNAs in acute myeloid leukemia (AML) remain unclear. Herein, we attempted to identify plasma exosomal lncRNAs as prospective biomarkers for AML. In this study, plasma exosomes were first successfully extracted from AML patients and healthy donors (HD). Subsequently, the downregulated plasma exosomal lncRNAs (LINC00265, LINC00467, and UCA1) and the upregulated plasma exosomal lncRNA (SNHG1) were identified in AML patients (n=65) compared to HD (n=20). Notably, individual exosomal LINC00265, LINC00467, UCA1, or SNHG1 had a capability for discriminating AML patients from HD, and their combination displayed better efficiency. Furthermore, exosomal LINC00265 and LINC00467 were increased expressed in patients achieving complete remission after chemotherapy. Importantly, there was upregulation of exosomal LINC00265 and downregulation of exosomal SNHG1 upon allogeneic hematopoietic stem cell transplantation. Additionally, these lncRNAs were high stability in plasma exosomes. Exosomal LINC00265, LINC00467, UCA1, and SNHG1 may act as promising cell-free biomarkers for AML diagnosis and treatment monitoring and provide a new frontier of liquid biopsy for this type of cancer.

Mutant NPM1-Regulated FTO-Mediated m6A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis.

Acute myeloid leukemia (AML) with nucleophosmin 1 (NPM1) mutations exhibits distinct biological and clinical features, accounting for approximately one-third of AML. Recently, the N 6-methyladenosine (m6A) RNA modification has emerged as a new epigenetic modification to contribute to tumorigenesis and development. However, there is limited knowledge on the role of m6A modifications in NPM1-mutated AML. In this study, the decreased m6A level was first detected and high expression of fat mass and obesity-associated protein (FTO) was responsible for the m6A suppression in NPM1-mutated AML. FTO upregulation was partially induced by NPM1 mutation type A (NPM1-mA) through impeding the proteasome pathway. Importantly, FTO promoted leukemic cell survival by facilitating cell cycle and inhibiting cell apoptosis. Mechanistic investigations demonstrated that FTO depended on its m6A RNA demethylase activity to activate PDGFRB/ERK signaling axis. Our findings indicate that FTO-mediated m6A demethylation plays an oncogenic role in NPM1-mutated AML and provide a new layer of epigenetic insight for future treatments of this distinctly leukemic entity.

Tumour-derived small extracellular vesicles suppress CD8+ T cell immune function by inhibiting SLC6A8-mediated creatine import in NPM1-mutated acute myeloid leukaemia.

Acute myeloid leukaemia (AML) carrying nucleophosmin (NPM1) mutations has been defined as a distinct entity of acute leukaemia. Despite remarkable improvements in diagnosis and treatment, the long-term outcomes for this entity remain unsatisfactory. Emerging evidence suggests that leukaemia, similar to other malignant diseases, employs various mechanisms to evade killing by immune cells. However, the mechanism of immune escape in NPM1-mutated AML remains unknown. In this study, both serum and leukemic cells from patients with NPM1-mutated AML impaired the immune function of CD8+ T cells in a co-culture system. Mechanistically, leukemic cells secreted miR-19a-3p into the tumour microenvironment (TME) via small extracellular vesicles (sEVs), which was controlled by the NPM1-mutated protein/CCCTC-binding factor (CTCF)/poly (A)-binding protein cytoplasmic 1 (PABPC1) signalling axis. sEV-related miR-19a-3p was internalized by CD8+ T cells and directly repressed the expression of solute-carrier family 6 member 8 (SLC6A8; a creatine-specific transporter) to inhibit creatine import. Decreased creatine levels can reduce ATP production and impair CD8+ T cell immune function, leading to immune escape by leukemic cells. In summary, leukemic cell-derived sEV-related miR-19a-3p confers immunosuppression to CD8+ T cells by targeting SLC6A8-mediated creatine import, indicating that sEV-related miR-19a-3p might be a promising therapeutic target for NPM1-mutated AML.

[miR-148b-3p inhibits the proliferation and autophagy of acute myeloid leukemia cells by targeting ATG14].

Objective To investigate the effect of miR-148b-3p on the proliferation and autophagy of acute myeloid leukemia (AML) cells and its molecular mechanism. Methods Based on GEO and TCGA databases, the expression of miR-148b-3p in AML cells and its association with clinical prognosis of patients were analyzed with the bioinformatics software. The expression of miR-148b-3p in AML cells was detected by real-time quantitative PCR. The miR-148b-3p mimic and the miR-148b-3p inhibitor were transiently transfected into AML cell lines THP-1 and NB4 by liposome-mediated transfection, respectively. The proliferation of leukemia cells was evaluated by CCK-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) labeling, and the protein levels of Bcl2, Bcl2-associated X protein (BAX), autophagy marker LC3, P62, and autophagy-related gene 14 (ATG14) were detected by Western blotting. The targeted binding of miR-148b-3p to ATG14 was measured by dual-luciferase reporter gene assay, and the effect of miR-148b-3p/ATG14 axis on the phenotype of AML cells was observed in the rescue experiments. Results A decreased expression of miR-148b-3p was found in leukemia blasts of AML patients, and the overall survival rate of AML patients with low expression of miR-148b-3p was significantly lower than that of the control group. Overexpression of miR-148b-3p inhibited THP-1 cells proliferation, promoted their apoptosis, downregulated the LC3II and ATG14 protein levels, and upregulated the P62 protein levels, while inhibiting the expression of miR-148b-3p in NB4 cells had the opposite effect. miR-148b-3p significantly reduced the luciferase activity of the wild-type ATG14 expression vector. The results of rescue experiments showed that overexpression of ATG14 reversed the inhibitory effect of miR-148b-3p upregulation on cell proliferation and autophagy, while inhibition of ATG14 expression weakened the promotive effect of miR-148b-3p downregulation on cell phenotype. Conclusion The miR-148b-3p inhibits the in vitro proliferation and autophagy of AML cells by targeting ATG14.

Mutant NPM1-regulated lncRNA HOTAIRM1 promotes leukemia cell autophagy and proliferation by targeting EGR1 and ULK3.

BACKGROUND: Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1), which displays a distinct long noncoding RNA (lncRNA) expression profile, has been defined as a unique subgroup in the new classification of myeloid neoplasms. However, the biological roles of key lncRNAs in the development of NPM1-mutated AML are currently unclear. Here, we aimed to investigate the functional and mechanistic roles of the lncRNA HOTAIRM1 in NPM1-mutated AML. METHODS: The expression of HOTAIRM1 was analyzed with a public database and further determined by qRT-PCR in NPM1-mutated AML samples and cell lines. The cause of upregulated HOTAIRM1 expression was investigated by luciferase reporter, chromatin immunoprecipitation and ubiquitination assays. The functional role of HOTAIRM1 in autophagy and proliferation was evaluated using western blot analysis, immunofluorescence staining, a Cell Counting Kit-8 (CCK-8) assay, a 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay, flow cytometric analyses and animal studies. The action mechanism of HOTAIRM1 was explored through RNA fluorescence in situ hybridization, RNA pulldown and RNA immunoprecipitation assays. RESULTS: HOTAIRM1 was highly expressed in NPM1-mutated AML. High HOTAIRM1 expression was induced in part by mutant NPM1 via KLF5-dependent transcriptional regulation. Importantly, HOTAIRM1 promoted autophagy and proliferation both in vitro and in vivo. Mechanistic investigations demonstrated that nuclear HOTAIRM1 promoted EGR1 degradation by serving as a scaffold to facilitate MDM2-EGR1 complex formation, while cytoplasmic HOTAIRM1 acted as a sponge for miR-152-3p to increase ULK3 expression. CONCLUSIONS: Taken together, our findings identify two oncogenic regulatory axes in NPM1-mutated AML centered on HOTAIRM1: one involving EGR1 and MDM2 in the nucleus and the other involving the miR-152-3p/ULK3 axis in the cytoplasm. Our study indicates that HOTAIRM1 may be a promising therapeutic target for this distinct leukemia subtype.

NPM1 mutant maintains ULK1 protein stability via TRAF6-dependent ubiquitination to promote autophagic cell survival in leukemia.

Nucleophosmin (NPM1) mutations are the most frequent genetic alteration in acute myeloid leukemia (AML) and aberrant cytoplasm-dislocated NPM1 mutant is a distinct biological characterization of this disease. Our group previously reported that NPM1 mutant elevated autophagy activity and autophagy activation contributed to leukemic cell survival. However, the molecular mechanisms by which cytoplasmic NPM1 mutant involving in the autophagy pathway has not been fully elucidated. Here, we showed that Unc-51-like kinase 1 (ULK1) as a core autophagy protein was highly expressed in NPM1-mA positive OCI-AML3 cells and primary NPM1-mutated AML blasts. Meanwhile, we found that NPM1-mA could interact with ULK1 protein and positively regulated ULK1 protein levels. Mechanically, NPM1-mA promoted TRAF6-dependent K63 ubiquitination and further maintained ULK1 stability and kinase activity via miR-146a. In addition, ULK1 high expression-mediated autophagy activation and facilitated to leukemic cell proliferation. Finally, we demonstrated that restoring ULK1 expression, ULK1 inhibitor SBI-0206965 treatment and using shULK1 partially rescued the effect of NPM1-mA on autophagy and cell survival. In conclusion, our findings suggest that NPM1 mutant interacts with ULK1, and thus, maintains its protein stability, which is required for NPM1 mutant-mediated autophagic cell survival. These data extend our understanding of the functions of NPM1 mutant in the regulation of autophagy activation in NPM1-mutated AML.

Exploration of Serum Exosomal LncRNA TBILA and AGAP2-AS1 as Promising Biomarkers for Diagnosis of Non-Small Cell Lung Cancer.

Non-small cell lung cancer is the most common type of cancer with a poor prognosis, and development of an effective diagnostic method is urgently needed. Exosomal lncRNAs, a class of transcripts longer than 200 nucleotides packaged into exosomes, have been defined as an ideal diagnostic biomarker for cancer. However, little is known about the clinical utility of exosomal lncRNAs in NSCLC. Here, we aimed to identify exosomal lncRNAs as promising biomarkers for NSCLC diagnosis. First, serum exosomes from NSCLC patients were successfully isolated by a polymer precipitation kit and then identified by TEM, NTA and western blot analysis. A total of nine candidate lncRNAs were detected by qRT-PCR in a training set. The two exosomal lncRNA TBILA and AGAP2-AS1 were screened out for the higher levels in NSCLC patients than that of healthy controls in a validation set. And there was a significant positive correlation between these exosomal lncRNAs levels and tumor size, lymph node metastasis and TNM stage. Additionally, we validated that these exosomal lncRNAs were stable in serum. Next, we evaluated the diagnostic efficiency of exosomal lncRNAs in NSCLC patients by ROC curve analysis. The data showed that individual TBILA or AGAP2-AS1 exhibited better diagnostic efficiency in NSCLC patients with different tumor pathologic subtypes and early stage, whereas the combination of lncRNAs did not provide better results than individual lncRNAs. Notably, the combination of two exosomal lncRNAs and the serum tumor biomarker Cyfra21-1 widely used in clinical practices further improved the diagnostic accuracy for NSCLC patients. This study suggests that exosomal lncRNA TBILA and AGAP2-AS1 may be promising biomarkers for diagnosis of NSCLC.

Up-regulation of EMT-related gene VCAN by NPM1 mutant-driven TGF-ß/cPML signalling promotes leukemia cell invasion.

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) is acknowledged as a distinct leukemia entity in the 2016 updated World Health Organization (WHO) classification. NPM1-mutated AML patients are correlated with higher extramedullary involvement. Epithelial-mesenchymal transition (EMT) is one of the key steps which cause distant metastasis in tumor. However, whether EMT-related programs contribute to cell invasion in NPM1-mutated AML remains unclear. In this study, we identified the EMT-related gene versican (VCAN) in NPM1-mutated AML across three patient datasets. Further experiments validated the elevated VCAN expression in NPM1-mutated AML primary blasts and OCI-AML3 cells with NPM1 mutation. Mechanistic studies revealed that increased VCAN expression was at least partially regulated by NPM1 mutant via TGF-ß/cPML/Smad signalling. Functional evaluations showed that silencing VCAN by shRNA significantly suppressed cell migration and invasion capacity, whereas increased VCAN by overexpressing NPM1-mA enhanced migration and invasion ability of leukemia cells. Finally, we found that high expression of VCAN was associated with poor prognosis in AML patients. These findings provide insights into the involvement of EMT-related gene VCAN in the pathogenesis of NPM1-mutated leukemia, which suggests that VCAN is an attractive target for novel diagnostic and therapeutic strategies in NPM1-mutated AML.

Glycolytic Enzyme PKM2 Mediates Autophagic Activation to Promote Cell Survival in NPM1-Mutated Leukemia.

Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been defined as a distinct leukemia entity in the 2016 updated WHO classification of myeloid neoplasm. Our previous report showed that autophagic activity was elevated in NPM1-mutated AML, but the underlying molecular mechanisms remain elusive. Mount of study provides evidence that glycometabolic enzymes are implicated in the autophagic process. Pyruvate kinase isoenzyme M2 (PKM2), a key glycolytic enzyme, has been recently reported as a tumor supporter in leukemia. However, little is known about the roles of PKM2 in autophagic activity in NPM1-mutated AML. In this study, PKM2 highly expressed in NPM1-mutated AML, and partially, high levels of PKM2 were upregulated by PTBP1. Further experiments demonstrated that PKM2 mediated autophagic activation and increased the phosphorylation of key autophagy protein Beclin-1. Importantly, functional experiments demonstrated that PKM2 contributed to cell survival via autophagic activation. Ultimately, high PKM2 expression was associated with short overall and event-free survival time in NPM1-mutated AML patients. Our findings indicate for the first time that glycolytic enzyme PKM2 mediates autophagic activation and further contributes to cell survival in NPM1-mutated AML, suggesting that PKM2 may serve as a promising target for treatment of NPM1-mutated AML.

INPP4B promotes cell survival via SGK3 activation in NPM1-mutated leukemia.

BACKGROUND: Acute myeloid leukemia (AML) with mutated nucleophosmin (NPM1) has been recognized as a distinct leukemia entity in the 2016 World Health Organization (WHO) classification. The genetic events underlying oncogenesis in NPM1-mutated AML that is characterized by a normal karyotype remain unclear. Inositol polyphosphate 4-phosphatase type II (INPP4B), a new factor in the phosphoinositide-3 kinase (PI3K) pathway-associated cancers, has been recently found a clinically relevant role in AML. However, little is known about the specific mechanistic function of INPP4B in NPM1-mutated AML. METHODS: The INPP4B expression levels in NPM1-mutated AML primary blasts and AML OCI-AML3 cell lines were determined by qRT-PCR and western blotting. The effect of INPP4B knockdown on OCI-AML3 leukemia cell proliferation was evaluated, using the Cell Counting Kit-8 and colony formation assay. After INPP4B overexpression or knockdown, the activation of serum and glucocorticoid-regulated kinase 3 (SGK3) and AKT was assessed. The effects of PI3K signaling pathway inhibitors on the levels of p-SGK3 in OCI-AML3 cells were tested. The mass of PI (3,4) P2 and PI (3) P was analyzed by ELISA upon INPP4B overexpression. Knockdown of SGK3 by RNA interference and a rescue assay were performed to confirm the critical role of SGK3 in INPP4B-mediated cell survival. In addition, the molecular mechanism underlying INPP4B expression in NPM1-mutated leukemia cells was explored. Finally, Kaplan-Meier survival analysis was conducted on the NPM1-mutated AML cohort stratified into quartiles for INPP4B expression in The Cancer Genome Atlas (TCGA) dataset. RESULTS: High expression of INPP4B was observed in NPM1-mutated AML. Knockdown of INPP4B repressed cell proliferation in OCI-AML3 cells, whereas recovered INPP4B rescued this inhibitory effect in vitro. Mechanically, INPP4B enhanced phosphorylated SGK3 (p-SGK3) status, but did not affect AKT activation. SGK3 was required for INPP4B-induced cell proliferation in OCI-AML3 cells. High levels of INPP4B were at least partially caused by the NPM1 mutant via ERK/Ets-1 signaling. Finally, high expression of INPP4B showed a trend towards lower overall survival and event-free survival in NPM1-mutated AML patients. CONCLUSIONS: Our results indicate that INPP4B promotes leukemia cell survival via SGK3 activation, and INPP4B might be a potential target in the treatment of NPM1-mutated AML.