PML::RARA and GATA2 proteins interact via DNA templates to induce aberrant self-renewal in mouse and human hematopoietic cells.

The underlying mechanism(s) by which the PML::RARA fusion protein initiates acute promyelocytic leukemia is not yet clear. We defined the genomic binding sites of PML::RARA in primary mouse and human hematopoietic progenitor cells with V5-tagged PML::RARA, using anti-V5-PML::RARA chromatin immunoprecipitation sequencing and CUT&RUN approaches. Most genomic PML::RARA binding sites were found in regions that were already chromatin-accessible (defined by ATAC-seq) in unmanipulated, wild-type promyelocytes, suggesting that these regions are "open" prior to PML::RARA expression. We found that GATA binding motifs, and the direct binding of the chromatin "pioneering factor" GATA2, were significantly enriched near PML::RARA binding sites. Proximity labeling studies revealed that PML::RARA interacts with ~250 proteins in primary mouse hematopoietic cells; GATA2 and 33 others require PML::RARA binding to DNA for the interaction to occur, suggesting that binding to their cognate DNA target motifs may stabilize their interactions. In the absence of PML::RARA, Gata2 overexpression induces many of the same epigenetic and transcriptional changes as PML::RARA. These findings suggested that PML::RARA may indirectly initiate its transcriptional program by activating Gata2 expression: Indeed, we demonstrated that inactivation of Gata2 prior to PML::RARA expression prevented its ability to induce self-renewal. These data suggested that GATA2 binding creates accessible chromatin regions enriched for both GATA and Retinoic Acid Receptor Element motifs, where GATA2 and PML::RARA can potentially bind and interact with each other. In turn, PML::RARA binding to DNA promotes a feed-forward transcriptional program by positively regulating Gata2 expression. Gata2 may therefore be required for PML::RARA to establish its transcriptional program.

Rapid and accurate remethylation of DNA in Dnmt3a-deficient hematopoietic cells with restoration of DNMT3A activity.

Here, we characterize the DNA methylation phenotypes of bone marrow cells from mice with hematopoietic deficiency of Dnmt3a or Dnmt3b (or both enzymes) or expressing the dominant-negative Dnmt3aR878H mutation [R882H in humans; the most common DNMT3A mutation found in acute myeloid leukemia (AML)]. Using these cells as substrates, we defined DNA remethylation after overexpressing wild-type (WT) DNMT3A1, DNMT3B1, DNMT3B3 (an inactive splice isoform of DNMT3B), or DNMT3L (a catalytically inactive "chaperone" for DNMT3A and DNMT3B in early embryogenesis). Overexpression of DNMT3A for 2 weeks reverses the hypomethylation phenotype of Dnmt3a-deficient cells or cells expressing the R878H mutation. Overexpression of DNMT3L (which is minimally expressed in AML cells) also corrects the hypomethylation phenotype of Dnmt3aR878H/+ marrow, probably by augmenting the activity of WT DNMT3A encoded by the residual WT allele. DNMT3L reactivation may represent a previously unidentified approach for restoring DNMT3A activity in hematopoietic cells with reduced DNMT3A function.

Stabilizing the Proteomes of Acute Myeloid Leukemia Cells: Implications for Cancer Proteomics.

Previous work has shown that inhibition of abundant myeloid azurophil granule-associated serine proteases (ELANE [neutrophil elastase], PRTN3 [protease 3], and CTSG [Cathepsin G]) is required to stabilize some proteins in myeloid cells. We therefore hypothesized that effective inhibition of these proteases may be necessary for quantitative proteomic analysis of samples containing myeloid cells. To test this hypothesis, we thawed viably preserved acute myeloid leukemia cells from cryovials in the presence or the absence of diisopropyl fluorophosphate (DFP), a cell-permeable and irreversible serine protease inhibitor. Global proteomic analysis was performed, using label-free and isobaric peptide-labeling quantitation. The presence of DFP resulted in an increase of tryptic peptides (14-57%) and proteins (9-31%). In the absence of DFP, 11 to 31% of peptide intensity came from nontryptic peptides; 52 to 75% had cleavage specificity consistent with activities of ELANE-PRTN3. Treatment with DFP reduced the intensity of nontryptic peptides to 4-8% of the total. ELANE inhibition was 95%, based on diisopropyl phosphate modification of active site serine residue. Overall, the relative abundance of 20% of proteins was significantly altered by DFP treatment. These results suggest that active myeloid serine proteases, released during sample processing, can skew quantitative proteomic measurements. Finally, significant ELANE activity was also detected in Clinical Proteomics Tumor Analysis Consortium datasets of solid tumors (many of which have known myeloid infiltration). In the pancreatic cancer dataset, the median percentage of nontryptic intensity detected across patient samples was 34%, with many patient samples having more than half of their detected peptide intensity from nontryptic cleavage events consistent with ELANE-PRTN3 cleavage specificity. Our study suggests that in vitro cleavage of proteins by myeloid serine proteases may be relevant for proteomic studies of any tumor that contains infiltrating myeloid cells.

Proteogenomic analysis reveals cytoplasmic sequestration of RUNX1 by the acute myeloid leukemia-initiating CBFB::MYH11 oncofusion protein.

Several canonical translocations produce oncofusion genes that can initiate acute myeloid leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase (TurboID) in-frame with 3 favorable-risk AML oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11) and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID-interacting proteins were largely cytoplasmic, probably because of an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.

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.

Melanoma in a patient with DNMT3A overgrowth syndrome.

Alterations in epigenetic regulators are increasingly recognized as early events in tumorigenesis; thus, patients with acquired or inherited variants in epigenetic regulators may be at increased risk for developing multiple types of cancer. DNMT3A overgrowth syndrome (DOS), caused by germline pathogenic variants in the DNA methyltransferase gene DNMT3A, has been associated with a predisposition toward development of hematopoietic and neuronal malignancies. DNMT3A deficiency has been described to promote keratinocyte proliferation in mice. Although altered DNA methylation patterns are well-recognized in melanoma, the role of DNA methyltransferases in melanoma pathogenesis is not clear. We report the case of an adult DOS patient with a germline DNMT3A loss-of-function mutation, who developed an early-onset melanoma with regional lymph node metastatic disease. Exome sequencing of the primary tumor identified an additional acquired, missense DNMT3A mutation in the dominant tumor clone, suggesting that the loss of DNMT3A function was relevant for the development of this tumor.

Persistent Molecular Disease in Adult Patients With AML Evaluated With Whole-Exome and Targeted Error-Corrected DNA Sequencing.

PURPOSE: Persistent molecular disease (PMD) after induction chemotherapy predicts relapse in AML. In this study, we used whole-exome sequencing (WES) and targeted error-corrected sequencing to assess the frequency and mutational patterns of PMD in 30 patients with AML. MATERIALS AND METHODS: The study cohort included 30 patients with adult AML younger than 65 years who were uniformly treated with standard induction chemotherapy. Tumor/normal WES was performed for all patients at presentation. PMD analysis was evaluated in bone marrow samples obtained during clinicopathologic remission using repeat WES and analysis of patient-specific mutations and error-corrected sequencing of 40 recurrently mutated AML genes (MyeloSeq). RESULTS: WES for patient-specific mutations detected PMD in 63% of patients (19/30) using a minimum variant allele fraction (VAF) of 2.5%. In comparison, MyeloSeq identified persistent mutations above 0.1% VAF in 77% of patients (23/30). PMD was usually present at relatively high levels (>2.5% VAFs), such that WES and MyeloSeq agreed for 73% of patients despite differences in detection limits. Mutations in DNMT3A, ASXL1, and TET2 (ie, DTA mutations) were persistent in 16 of 17 patients, but WES also detected non-DTA mutations in 14 of these patients, which for some patients distinguished residual AML cells from clonal hematopoiesis. Surprisingly, MyeloSeq detected additional variants not identified at presentation in 73% of patients that were consistent with new clonal cell populations after chemotherapy. CONCLUSION: PMD and clonal hematopoiesis are both common in patients with AML in first remission. These findings demonstrate the importance of baseline testing for accurate interpretation of mutation-based tumor monitoring assays for patients with AML and highlight the need for clinical trials to determine whether these complex mutation patterns correlate with clinical outcomes in AML.

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.

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.

Convergent Clonal Evolution of Signaling Gene Mutations Is a Hallmark of Myelodysplastic Syndrome Progression.

Progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML) is associated with the acquisition and expansion of subclones. Our understanding of subclone evolution during progression, including the frequency and preferred order of gene mutation acquisition, remains incomplete. Sequencing of 43 paired MDS and secondary AML samples identified at least one signaling gene mutation in 44% of MDS and 60% of secondary AML samples, often below the level of standard sequencing detection. In addition, 19% of MDS and 47% of secondary AML patients harbored more than one signaling gene mutation, almost always in separate, coexisting subclones. Signaling gene mutations demonstrated diverse patterns of clonal evolution during disease progression, including acquisition, expansion, persistence, and loss of mutations, with multiple patterns often coexisting in the same patient. Multivariate analysis revealed that MDS patients who had a signaling gene mutation had a higher risk of AML progression, potentially providing a biomarker for progression. SIGNIFICANCE: Subclone expansion is a hallmark of progression from MDS to secondary AML. Subclonal signaling gene mutations are common at MDS (often at low levels), show complex and convergent patterns of clonal evolution, and are associated with future progression to secondary AML. See related article by Guess et al., p. 316 (33). See related commentary by Romine and van Galen, p. 270. This article is highlighted in the In This Issue feature, p. 265.

IL-1ß expression in bone marrow dendritic cells is induced by TLR2 agonists and regulates HSC function.

Hematopoietic stem/progenitor cells (HSPCs) reside in localized microenvironments, or niches, in the bone marrow that provide key signals regulating their activity. A fundamental property of hematopoiesis is the ability to respond to environmental cues such as inflammation. How these cues are transmitted to HSPCs within hematopoietic niches is not well established. Here, we show that perivascular bone marrow dendritic cells (DCs) express a high basal level of Toll-like receptor-1 (TLR1) and TLR2. Systemic treatment with a TLR1/2 agonist induces HSPC expansion and mobilization. It also induces marked alterations in the bone marrow microenvironment, including a decrease in osteoblast activity and sinusoidal endothelial cell numbers. TLR1/2 agonist treatment of mice in which Myd88 is deleted specifically in DCs using Zbtb46-Cre show that the TLR1/2-induced expansion of multipotent HPSCs, but not HSPC mobilization or alterations in the bone marrow microenvironment, is dependent on TLR1/2 signaling in DCs. Interleukin-1ß (IL-1ß) is constitutively expressed in both murine and human DCs and is further induced after TLR1/2 stimulation. Systemic TLR1/2 agonist treatment of Il1r1-/- mice show that TLR1/2-induced HSPC expansion is dependent on IL-1ß signaling. Single-cell RNA-sequencing of low-risk myelodysplastic syndrome bone marrow revealed that IL1B and TLR1 expression is increased in DCs. Collectively, these data suggest a model in which TLR1/2 stimulation of DCs induces secretion of IL-1ß and other inflammatory cytokines into the perivascular niche, which in turn, regulates multipotent HSPCs. Increased DC TLR1/2 signaling may contribute to altered HSPC function in myelodysplastic syndrome by increasing local IL-1ß expression.

Recurrent transcriptional responses in AML and MDS patients treated with decitabine.

The molecular events responsible for decitabine responses in myelodysplastic syndrome and acute myeloid leukemia patients are poorly understood. Decitabine has a short serum half-life and limited stability in tissue culture. Therefore, theoretical pharmacologic differences may exist between patient molecular changes in vitro and the consequences of in vivo treatment. To systematically identify the global genomic and transcriptomic alterations induced by decitabine in vivo, we evaluated primary bone marrow samples that were collected during patient treatment and applied whole-genome bisulfite sequencing, RNA-sequencing, and single-cell RNA sequencing. Decitabine induced global, reversible hypomethylation after 10 days of therapy in all patients, which was associated with induction of interferon-induced pathways, the expression of endogenous retroviral elements, and inhibition of erythroid-related transcripts, recapitulating many effects seen previously in in vitro studies. However, at relapse after decitabine treatment, interferon-induced transcripts remained elevated relative to day 0, but erythroid-related transcripts now were more highly expressed than at day 0. Clinical responses were not correlated with epigenetic or transcriptional signatures, although sample size and interpatient variance restricted the statistical power required for capturing smaller effects. Collectively, these data define global hypomethylation by decitabine and find that erythroid-related pathways may be relevant because they are inhibited by therapy and reverse at relapse.

Physician-scientists in the United States at 2020: Trends and concerns.

Physician-scientists comprise a unique and valuable part of the biomedical workforce, but for decades there has been concern about the number of physicians actively engaged in research. Reports have outlined the challenges facing physician-scientists, and programs have been initiated to encourage and facilitate research careers for medically trained scientists. Many of these initiatives have demonstrated successful outcomes, but there has not been a recent summary of the impact of the past decade of effort. This report compiles available data from surveys of medical education and physician research participation to assess changes in the physician-scientist workforce from 2011-2020. Several trends are positive: rising enrollments in MD-PhD programs, greater levels of interest in research careers among matriculating medical students, more research experience during medical school and rising numbers of physicians in academic medicine, and an increase in first R01 grants to physician-scientists. However, there are now decreased levels of interest in research careers among graduating medical students, a steady decline in MDs applying for NIH loan repayment program support, an increased age at first R01 grant success for physicians, and fewer physicians reporting research as their primary work activity: all of these indicators create concern for the stability of the career path. Despite a recommendation by the Physician-Scientist Workforce in 2014 to create "real-time" reporting on NIH grants and grantees to help the public assess trends, this initiative has not been completed. Better information is still needed to fully understand the status of the physician-scientist workforce, and to assess efforts to stabilize this vulnerable career path.

Somatic Dnmt3a inactivation leads to slow, canonical DNA methylation loss in murine hematopoietic cells.

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) are the most common cause of clonal hematopoiesis and are among the most common initiating events of acute myeloid leukemia (AML). Studies in germline and somatic Dnmt3a knockout mice have identified focal, canonical hypomethylation phenotypes in hematopoietic cells; however, the kinetics of methylation loss following acquired DNMT3A inactivation in hematopoietic cells is essentially unknown. Therefore, we evaluated a somatic, inducible model of hematopoietic Dnmt3a loss, and show that inactivation of Dnmt3a in murine hematopoietic cells results in a relatively slow loss of methylation at canonical sites throughout the genome; in contrast, remethylation of Dnmt3a deficient genomes in hematopoietic cells occurs much more quickly. This data suggests that slow methylation loss may contribute, at least in part, to the long latent period that characterizes clonal expansion and leukemia development in individuals with acquired DNMT3A mutations in hematopoietic stem cells.

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.

Failure to Detect Mutations in U2AF1 due to Changes in the GRCh38 Reference Sequence.

The U2AF1 gene is a core part of mRNA splicing machinery and frequently contains somatic mutations that contribute to oncogenesis in myelodysplastic syndrome, acute myeloid leukemia, and other cancers. A change introduced in the GRCh38 version of the human reference build prevents detection of mutations in this gene, and others, by variant calling pipelines. This study describes the problem in detail and shows that a modified GRCh38 reference build with unchanged coordinates can be used to ameliorate the issue.