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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.

Acute myeloid leukemia (AML) with nucleophosmin 1 (NPM1) mutations exhibits distinct biological and clinical features, accounting for approximately one-third of AML. Recently, the

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.

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.

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.

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.

Antifolate antimalarials, such as pyrimethamine, have experienced a dramatic reduction in therapeutic efficacy as resistance has evolved in multiple malaria species. We present evidence from one such species, Plasmodium vivax, which has experienced sustained selection for pyrimethamine resistance at the dihydrofolate reductase (DHFR) locus since the 1970s. Using a transgenic Saccharomyces cerevisiae model expressing the P. vivax DHFR enzyme, we assayed growth rate and resistance of all 16 combinations of four DHFR amino acid substitutions. These substitutions were selected based on their known association with drug resistance, both in natural isolates and in laboratory settings, in the related malaria species P. falciparum. We observed a strong correlation between the resistance phenotypes for these 16 P. vivax alleles and previously observed resistance data for P. falciparum, which was surprising since nucleotide diversity levels and common polymorphic variants of DHFR differ between the two species. Similar results were observed when we expressed the P. vivax alleles in a transgenic bacterial system. This suggests common constraints on enzyme evolution in the orthologous DHFR proteins. The interplay of negative trade-offs between the evolution of novel resistance and compromised endogenous function varies at different drug dosages, and so too do the major trajectories for DHFR evolution. In simulations, it is only at very high drug dosages that the most resistant quadruple mutant DHFR allele is favored by selection. This is in agreement with common polymorphic DHFR data in P. vivax, from which this quadruple mutant is missing. We propose that clinical dosages of pyrimethamine may have historically been too low to select for the most resistant allele, or that the fitness cost of the most resistant allele was untenable without a compensatory mutation elsewhere in the genome.