Proteomic and phosphoproteomic landscapes of acute myeloid leukemia.

We have developed a deep-scale proteome and phosphoproteome database from 44 representative acute myeloid leukemia (AML) patients from the LAML TCGA dataset and 6 healthy bone marrow-derived controls. After confirming data quality, we orthogonally validated several previously undescribed features of AML revealed by the proteomic data. We identified examples of posttranscriptionally regulated proteins both globally (ie, in all AML samples) and also in patients with recurrent AML driver mutations. For example, samples with IDH1/2 mutations displayed elevated levels of the 2-oxoglutarate-dependent histone demethylases KDM4A/B/C, despite no changes in messenger RNA levels for these genes; we confirmed this finding in vitro. In samples with NPMc mutations, we identified several nuclear importins with posttranscriptionally increased protein abundance and showed that they interact with NPMc but not wild-type NPM1. We identified 2 cell surface proteins (CD180 and MRC1/CD206) expressed on AML blasts of many patients (but not healthy CD34+ stem/progenitor cells) that could represent novel targets for immunologic therapies and confirmed these targets via flow cytometry. Finally, we detected nearly 30 000 phosphosites in these samples; globally, AML samples were associated with the abnormal phosphorylation of specific residues in PTPN11, STAT3, AKT1, and PRKCD. FLT3-TKD samples were associated with increased phosphorylation of activating tyrosines on the cytoplasmic Src-family tyrosine kinases FGR and HCK and related signaling proteins. PML-RARA-initiated AML samples displayed a unique phosphorylation signature, and TP53-mutant samples showed abundant phosphorylation of serine-183 on TP53 itself. This publicly available database will serve as a foundation for further investigations of protein dysregulation in AML pathogenesis.

Enhanced Efficacy and Increased Long-Term Toxicity of CNS-Directed, AAV-Based Combination Therapy for Krabbe Disease.

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a demyelinating disease caused by the deficiency of the lysosomal enzyme galactosylceramidase (GALC) and the progressive accumulation of the toxic metabolite psychosine. We showed previously that central nervous system (CNS)-directed, adeno-associated virus (AAV)2/5-mediated gene therapy synergized with bone marrow transplantation and substrate reduction therapy (SRT) to greatly increase therapeutic efficacy in the murine model of Krabbe disease (Twitcher). However, motor deficits remained largely refractory to treatment. In the current study, we replaced AAV2/5 with an AAV2/9 vector. This single change significantly improved several endpoints primarily associated with motor function. However, nearly all (14/16) of the combination-treated Twitcher mice and all (19/19) of the combination-treated wild-type mice developed hepatocellular carcinoma (HCC). 10 out of 10 tumors analyzed had AAV integrations within the Rian locus. Several animals had additional integrations within or near genes that regulate cell growth or death, are known or potential tumor suppressors, or are associated with poor prognosis in human HCC. Finally, the substrate reduction drug L-cycloserine significantly decreased the level of the pro-apoptotic ceramide 18:0. These data demonstrate the value of AAV-based combination therapy for Krabbe disease. However, they also suggest that other therapies or co-morbidities must be taken into account before AAV-mediated gene therapy is considered for human therapeutic trials.

Single-Cell Transcriptomics of a Human Kidney Allograft Biopsy Specimen Defines a Diverse Inflammatory Response.

Background Single-cell genomics techniques are revolutionizing our ability to characterize complex tissues. By contrast, the techniques used to analyze renal biopsy specimens have changed little over several decades. We tested the hypothesis that single-cell RNA-sequencing can comprehensively describe cell types and states in a human kidney biopsy specimen.Methods We generated 8746 single-cell transcriptomes from a healthy adult kidney and a single kidney transplant biopsy core by single-cell RNA-sequencing. Unsupervised clustering analysis of the biopsy specimen was performed to identify 16 distinct cell types, including all of the major immune cell types and most native kidney cell types, in this biopsy specimen, for which the histologic read was mixed rejection.Results Monocytes formed two subclusters representing a nonclassical CD16+ group and a classic CD16- group expressing dendritic cell maturation markers. The presence of both monocyte cell subtypes was validated by staining of independent transplant biopsy specimens. Comparison of healthy kidney epithelial transcriptomes with biopsy specimen counterparts identified novel segment-specific proinflammatory responses in rejection. Endothelial cells formed three distinct subclusters: resting cells and two activated endothelial cell groups. One activated endothelial cell group expressed Fc receptor pathway activation and Ig internalization genes, consistent with the pathologic diagnosis of antibody-mediated rejection. We mapped previously defined genes that associate with rejection outcomes to single cell types and generated a searchable online gene expression database.Conclusions We present the first step toward incorporation of single-cell transcriptomics into kidney biopsy specimen interpretation, describe a heterogeneous immune response in mixed rejection, and provide a searchable resource for the scientific community.

Genomic landscape of TP53 -mutated myeloid malignancies.

TP53 -mutated myeloid malignancies are most frequently associated with complex cytogenetics. The presence of complex and extensive structural variants complicates detailed genomic analysis by conventional clinical techniques. We performed whole genome sequencing of 42 AML/MDS cases with paired normal tissue to characterize the genomic landscape of TP53 -mutated myeloid malignancies. The vast majority of cases had multi-hit involvement at the TP53 genetic locus (94%), as well as aneuploidy and chromothripsis. Chromosomal patterns of aneuploidy differed significantly from TP53 -mutated cancers arising in other tissues. Recurrent structural variants affected regions that include ETV6 on chr12p, RUNX1 on chr21, and NF1 on chr17q. Most notably for ETV6 , transcript expression was low in cases of TP53 -mutated myeloid malignancies both with and without structural rearrangements involving chromosome 12p. Telomeric content is increased in TP53 -mutated AML/MDS compared other AML subtypes, and telomeric content was detected adjacent to interstitial regions of chromosomes. The genomic landscape of TP53 -mutated myeloid malignancies reveals recurrent structural variants affecting key hematopoietic transcription factors and telomeric repeats that are generally not detected by panel sequencing or conventional cytogenetic analyses. Key Points: WGS comprehensively determines TP53 mutation status, resulting in the reclassification of 12% of cases from mono-allelic to multi-hit Chromothripsis is more frequent than previously appreciated, with a preference for specific chromosomes ETV6 is deleted in 45% of cases, with evidence for epigenetic suppression in non-deleted cases NF1 is mutated in 48% of cases, with multi-hit mutations in 17% of these cases TP53 -mutated AML/MDS is associated with altered telomere content compared with other AMLs.

Genomic landscape of TP53-mutated myeloid malignancies.

TP53-mutated myeloid malignancies are associated with complex cytogenetics and extensive structural variants, which complicates detailed genomic analysis by conventional clinical techniques. We performed whole-genome sequencing (WGS) of 42 acute myeloid leukemia (AML)/myelodysplastic syndromes (MDS) cases with paired normal tissue to better characterize the genomic landscape of TP53-mutated AML/MDS. WGS accurately determines TP53 allele status, a key prognostic factor, resulting in the reclassification of 12% of cases from monoallelic to multihit. Although aneuploidy and chromothripsis are shared with most TP53-mutated cancers, the specific chromosome abnormalities are distinct to each cancer type, suggesting a dependence on the tissue of origin. ETV6 expression is reduced in nearly all cases of TP53-mutated AML/MDS, either through gene deletion or presumed epigenetic silencing. Within the AML cohort, mutations of NF1 are highly enriched, with deletions of 1 copy of NF1 present in 45% of cases and biallelic mutations in 17%. Telomere content is increased in TP53-mutated AMLs compared with other AML subtypes, and abnormal telomeric sequences were detected in the interstitial regions of chromosomes. These data highlight the unique features of TP53-mutated myeloid malignancies, including the high frequency of chromothripsis and structural variation, the frequent involvement of unique genes (including NF1 and ETV6) as cooperating events, and evidence for altered telomere maintenance.

Genetic and Transcriptional Contributions to Relapse in Normal Karyotype Acute Myeloid Leukemia.

To better understand clonal and transcriptional adaptations after relapse in patients with acute myeloid leukemia (AML), we collected presentation and relapse samples from six normal karyotype AML cases. We performed enhanced whole-genome sequencing to characterize clonal evolution, and deep-coverage single-cell RNA sequencing on the same samples, which yielded 142,642 high-quality cells for analysis. Identifying expressed mutations in individual cells enabled us to discriminate between normal and AML cells, to identify coordinated changes in the genome and transcriptome, and to identify subclone-specific cell states. We quantified the coevolution of genetic and transcriptional heterogeneity during AML progression, and found that transcriptional changes were significantly correlated with genetic changes. However, transcriptional adaptation sometimes occurred independently, suggesting that clonal evolution does not represent all relevant biological changes. In three cases, we identified cells at diagnosis that likely seeded the relapse. Finally, these data revealed a conserved relapse-enriched leukemic cell state bearing markers of stemness, quiescence, and adhesion. SIGNIFICANCE: These data enabled us to identify a relapse-enriched leukemic cell state with distinct transcriptional properties. Detailed case-by-case analyses elucidated the complex ways in which the AML genome, transcriptome, and immune microenvironment interact to evade chemotherapy. These analyses provide a blueprint for evaluating these factors in larger cohorts.This article is highlighted in the In This Issue feature, p. 1.

Functional and epigenetic phenotypes of humans and mice with DNMT3A Overgrowth Syndrome.

Germline pathogenic variants in DNMT3A were recently described in patients with overgrowth, obesity, behavioral, and learning difficulties (DNMT3A Overgrowth Syndrome/DOS). Somatic mutations in the DNMT3A gene are also the most common cause of clonal hematopoiesis, and can initiate acute myeloid leukemia (AML). Using whole genome bisulfite sequencing, we studied DNA methylation in peripheral blood cells of 11 DOS patients and found a focal, canonical hypomethylation phenotype, which is most severe with the dominant negative DNMT3AR882H mutation. A germline mouse model expressing the homologous Dnmt3aR878H mutation phenocopies most aspects of the human DOS syndrome, including the methylation phenotype and an increased incidence of spontaneous hematopoietic malignancies, suggesting that all aspects of this syndrome are caused by this mutation.