Highlights d Integrated analysis provides insight into the molecular classification in NKTCL d EBV lytic genes play an important role on NKTCL pathogenesis d Genomic alteration-based molecular subtypes associate with clinical outcomes d MYC, histone acetylation, and PD-L1/2 are potential therapeutic targets of NKTCL

T-cell acute lymphoblastic leukemia (T-ALL) is a clonal malignancy of immature T cells. Recently, the next-generation sequencing approach has allowed systematic identification of molecular features in pediatric T-ALL. Here, by performing RNA-sequencing and other genomewide analysis, we investigated the genomic landscape in 61 adult and 69 pediatric T-ALL cases. Thirty-six distinct gene fusion transcripts were identified, with SET-NUP214 being highly related to adult cases. Among 18 previously unknown fusions, ZBTB16-ABL1, TRA-SALL2, and involvement of NKX2-1 were recurrent events. ZBTB16-ABL1 functioned as a leukemogenic driver and responded to the effect of tyrosine kinase inhibitors. Among 48 genes with mutation rates >3%, 6 were newly found in T-ALL. An aberrantly overexpressed short mRNA transcript of the SLC17A9 gene was revealed in most cases with overexpressed TAL1, which predicted a poor prognosis in the adult group. Up-regulation of HOXA, MEF2C, and LYL1 was often present in adult cases, while TAL1 overexpression was detected mainly in the pediatric group. Although most gene fusions were mutually exclusive, they coexisted with gene mutations. These genetic abnormalities were correlated with deregulated gene expression markers in three subgroups. This study may further enrich the current knowledge of T-ALL molecular pathogenesis.

ObjectiveInnate immunity plays important roles in pancreatic ductal adenocarcinoma (PDAC), as non-T-cell-enriched tumour. Neutrophils are major players in innate immune system. Here, we aimed to explore the heterogeneity and pro-tumour mechanisms of neutrophils in PDAC.DesignWe analysed single-cell transcriptomes of peripheral blood polymorphonuclear leucocytes (PMNs) and tumour-infiltrating immune cells from five patients with PDAC, and performed immunofluorescence/immunohistochemistry staining, multi-omics analysis and in vitro experiments to validate the discoveries of bioinformatics analysis.ResultsExploration of the heterogeneity of tumour-associated neutrophils (TANs) revealed a terminally differentiated pro-tumour subpopulation (TAN-1) associated with poor prognosis, an inflammatory subpopulation (TAN-2), a population of transitional stage that have just migrated to tumour microenvironment (TAN-3) and a subpopulation preferentially expressing interferon-stimulated genes (TAN-4). Glycolysis signature was upregulated along neutrophil transition trajectory, and TAN-1 was featured with hyperactivated glycolytic activity. The glycolytic switch of TANs was validated by integrative multi-omics approach of transcriptomics, proteomics and metabolomics analysis. Activation of glycolytic activity by LDHA overexpression induced immunosuppression and pro-tumour functions in neutrophil-like differentiated HL-60 (dHL-60) cells. Mechanistic studies revealed BHLHE40, downstream to hypoxia and endoplasmic reticulum stress, was a key regulator in polarisation of neutrophils towards TAN-1 phenotype, and direct transcriptional regulation of BHLHE40 on TAN-1 marker genes was demonstrated by chromatin immunoprecipitation assay. Pro-tumour and immunosuppression functions were observed in dHL-60 cells overexpressing BHLHE40. Importantly, immunohistochemistry analysis of PDAC tissues revealed the unfavourable prognostic value of BHLHE40+ neutrophils.ConclusionThe dynamic properties of TANs revealed by this study will be helpful in advancing PDAC therapy targeting innate immunity.

T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive leukemia that is primarily caused by aberrant activation of the NOTCH1 signaling pathway. Recent studies have revealed that posttranslational modifications, such as ubiquitination, regulate NOTCH1 stability, activity, and localization. However, the specific deubiquitinase that affects NOTCH1 protein stability remains unestablished. Here, we report that ubiquitin-specific protease 7 (USP7) can stabilize NOTCH1. USP7 deubiquitinated NOTCH1 in vivo and in vitro, whereas knockdown of USP7 increased the ubiquitination of NOTCH1. USP7 interacted with NOTCH1 protein in T-ALL cells, and the MATH and UBL domains of USP7 were responsible for this interaction. Depletion of USP7 significantly suppressed the proliferation of T-ALL cells in vitro and in vivo, accompanied by downregulation of the NOTCH1 protein level. Similarly, pharmacologic inhibition of USP7 led to apoptosis of T-ALL cells. More importantly, we found that USP7 was significantly upregulated in human T-ALL cell lines and patient samples, and a USP7 inhibitor exhibited cell cytotoxicity toward primary T-ALL cells, indicating the clinical relevance of these findings. Overall, our results demonstrate that USP7 is a novel deubiquitinase that stabilizes NOTCH1. Therefore, USP7 may be a promising therapeutic target in the currently incurable T-ALL.

t(8;21)(q22;q22) acute myelogenous leukemia (AML) is morphologically characterized by a continuum of heterogeneous leukemia cells from myeloblasts to differentiated myeloid elements. Thus, t(8;21) AML is an excellent model for studying heterogeneous cell populations and cellular evolution during disease progression. Using integrative analyses of immunophenotype, RNA-sequencing (RNA-seq), and single-cell RNA-sequencing (scRNA-seq), we identified three distinct intrapatient leukemic cell populations that were arrested at different stages of myeloid differentiation: CD34+CD117dim blasts, CD34+CD117bri blasts, and abnormal myeloid cells with partial maturation (AM). CD117 is also known as c-KIT protein. CD34+CD117dim cells were blocked in the G0/G1 phase at disease onset, presenting with the regular morphology of myeloblasts showing features of granulocyte-monocyte progenitors (GMP), and were drug-resistant to chemotherapy. Genes associated with cell migration and adhesion (LGALS1, EMP3, and ANXA2) were highly expressed in the CD34+CD117dim population. CD34+CD117bri blasts were blocked a bit later than the CD34+CD117dim population in the hematopoietic differentiation stage and displayed high proliferation ability. AM cells, which bear abnormal myelocyte morphology, especially overexpressed granule genes AZU1, ELANE, and PRTN3 and were sensitive to chemotherapy. scRNA-seq at different time points identified CD34+CD117dim blasts as an important leukemic cluster that expanded at postrelapse refractory stage after several cycles of chemotherapy. Patients with t(8;21) AML with a higher proportion of CD34+CD117dim cells had significantly worse clinical outcomes than those with a lower CD34+CD117dim proportion. Univariate and multivariate analyses identified CD34+CD117dim proportion as an independent factor for poor disease outcome. Our study provides evidence for the multidimensional heterogeneity of t(8;21)AML and may offer new tools for future disease stratification.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by genetic alterations with high heterogeneity. Precise subtypes with distinct genomic and/or gene expression patterns have been recently revealed using high-throughput sequencing technology. Most of these profiles are associated with recurrent non-overlapping rearrangements or hotspot point mutations that are analogous to the established subtypes, such as DUX4 rearrangements, MEF2D rearrangements, ZNF384/ZNF362 rearrangements, NUTM1 rearrangements, BCL2/MYC and/or BCL6 rearrangements, ETV6-RUNX1-like gene expression, PAX5alt (diverse PAX5 alterations, including rearrangements, intragenic amplifications, or mutations), and hotspot mutations PAX5 (p.Pro80Arg) with biallelic PAX5 alterations, IKZF1 (p.Asn159Tyr), and ZEB2 (p.His1038Arg). These molecular subtypes could be classified by gene expression patterns with RNA-seq technology. Refined molecular classification greatly improved the treatment strategy. Multiagent therapy regimens, including target inhibitors (e.g., imatinib), immunomodulators, monoclonal antibodies, and chimeric antigen receptor T-cell (CAR-T) therapy, are transforming the clinical practice from chemotherapy drugs to personalized medicine in the field of risk-directed disease management. We provide an update on our knowledge of emerging molecular subtypes and therapeutic targets in BCP-ALL.

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Glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) plays a critical role in cancer metabolism by coordinating glycolysis and biosynthesis. A well-validated PGAM1 inhibitor, however, has not been reported for treating pancreatic ductal adenocarcinoma (PDAC), which is one of the deadliest malignancies worldwide. By uncovering the elevated PGAM1 expressions were statistically related to worse prognosis of PDAC in a cohort of 50 patients, we developed a series of allosteric PGAM1 inhibitors by structure-guided optimization. The compound KH3 significantly suppressed proliferation of various PDAC cells by down-regulating the levels of glycolysis and mitochondrial respiration in correlation with PGAM1 expression. Similar to PGAM1 depletion, KH3 dramatically hampered the canonic pathways highly involved in cancer metabolism and development. Additionally, we observed the shared expression profiles of several signature pathways at 12 h after treatment in multiple PDAC primary cells of which the matched patient-derived xenograft (PDX) models responded similarly to KH3 in the 2 wk treatment. The better responses to KH3 in PDXs were associated with higher expression of PGAM1 and longer/stronger suppressions of cancer metabolic pathways. Taken together, our findings demonstrate a strategy of targeting cancer metabolism by PGAM1 inhibition in PDAC. Also, this work provided “proof of concept” for the potential application of metabolic treatment in clinical practice.