Inherited cancer predisposing mutations in patients with therapy-related myeloid neoplasms.

Some patients with therapy-related myeloid neoplasms (t-MN) may have unsuspected inherited cancer predisposition syndrome (CPS). We propose a set of clinical criteria to identify t-MN patients with high risk of CPS (HR-CPS). Among 225 t-MN patients with an antecedent non-myeloid malignancy, our clinical criteria identified 52 (23%) HR-CPS patients. Germline whole-exome sequencing identified pathogenic or likely pathogenic variants in 10 of 27 HR-CPS patients compared to 0 of 9 low-risk CPS patients (37% vs. 0%, p = 0.04). These simple clinical criteria identify t-MN patients most likely to benefit from genetic testing for inherited CPS.

EGR1 Haploinsufficiency Confers a Fitness Advantage to Hematopoietic Stem Cells Following Chemotherapy.

Therapy-related myeloid neoplasms (t-MNs) share many clinical and molecular characteristics with AML de novo in the elderly. One common factor is that they arise in the setting of chronic inflammation, likely because of advanced age or chemotherapy-induced senescence. Here, we examined the effect of haploinsufficient loss of the del(5q) tumor suppressor gene, EGR1, commonly deleted in high-risk MNs. In mice, under the exogenous stress of either serial transplant or successive doses of the alkylating agent N-ethyl-nitrosourea (ENU), Egr1-haploinsufficient hematopoietic stem cells (HSCs) exhibit a clonal advantage. Complete loss of EGR1 function is incompatible with transformation; mutations of EGR1 are rare and are not observed in the remaining allele in del(5q) patients, and complete knockout of Egr1 in mice leads to HSC exhaustion. Using chromatin immunoprecipitation sequencing (ChIP-seq), we identified EGR1 binding sites in human CD34+ cord blood-derived stem and progenitor cells (HSPCs) and found that EGR1 binds genes critical for stem cell differentiation, inflammatory signaling, and the DNA damage response. Notably, in the chromosome 5 sequences frequently deleted in patients, there is a significant enrichment of innate and inflammatory genes, which may confer a fitness advantage in an inflammatory environment. Short hairpin RNA (shRNA)-mediated silencing of EGR1 biases HSPCs toward a self-renewal transcriptional signature. In the absence of EGR1, HSPCs are characterized by upregulated MYC-driven proliferative signals, downregulated CDKN1A (p21), disrupted DNA damage response, and downregulated inflammation-adaptations anticipated to confer a relative fitness advantage for stem cells especially in an environment of chronic inflammation.

International Consensus Classification of Myeloid Neoplasms and Acute Leukemias: integrating morphologic, clinical, and genomic data.

The classification of myeloid neoplasms and acute leukemias was last updated in 2016 within a collaboration between the World Health Organization (WHO), the Society for Hematopathology, and the European Association for Haematopathology. This collaboration was primarily based on input from a clinical advisory committees (CACs) composed of pathologists, hematologists, oncologists, geneticists, and bioinformaticians from around the world. The recent advances in our understanding of the biology of hematologic malignancies, the experience with the use of the 2016 WHO classification in clinical practice, and the results of clinical trials have indicated the need for further revising and updating the classification. As a continuation of this CAC-based process, the authors, a group with expertise in the clinical, pathologic, and genetic aspects of these disorders, developed the International Consensus Classification (ICC) of myeloid neoplasms and acute leukemias. Using a multiparameter approach, the main objective of the consensus process was the definition of real disease entities, including the introduction of new entities and refined criteria for existing diagnostic categories, based on accumulated data. The ICC is aimed at facilitating diagnosis and prognostication of these neoplasms, improving treatment of affected patients, and allowing the design of innovative clinical trials.

Oncogenic and Tumor Suppressor Functions for Lymphoid Enhancer Factor 1 in E2a-/- T Acute Lymphoblastic Leukemia.

T lymphocyte acute lymphoblastic leukemia (T-ALL) is a heterogeneous disease affecting T cells at multiple stages of their development and is characterized by frequent genomic alterations. The transcription factor LEF1 is inactivated through mutation in a subset of T-ALL cases but elevated LEF1 expression and activating mutations have also been identified in this disease. Here we show, in a murine model of T-ALL arising due to E2a inactivation, that the developmental timing of Lef1 mutation impacts its ability to function as a cooperative tumor suppressor or oncogene. T cell transformation in the presence of LEF1 allows leukemic cells to become addicted to its presence. In contrast, deletion prior to transformation both accelerates leukemogenesis and results in leukemic cells with altered expression of genes controlling receptor-signaling pathways. Our data demonstrate that the developmental timing of Lef1 mutations impact its apparent oncogenic or tumor suppressive characteristics and demonstrate the utility of mouse models for understanding the cooperation and consequence of mutational order in leukemogenesis.

Genome-wide association study identifies susceptibility loci for acute myeloid leukemia.

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we perform a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identify a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10-8; KMT5B). We also identify a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N = 1287) at 6p21.32 (rs3916765; P = 1.51 × 10-10; HLA). Our results inform on AML etiology and identify putative functional genes operating in histone methylation (KMT5B) and immune function (HLA).

Therapy-related myeloid neoplasms in 109 patients after radiation monotherapy.

Therapy-related myeloid neoplasms (t-MNs) are a late complication of cytotoxic therapy and are defined as a distinct entity by the World Health Organization. Although the link between chemotherapy exposure and risk of subsequent t-MN is well described, the association between radiation monotherapy (RT) and t-MN risk is less definitive. We analyzed 109 consecutive patients who developed t-MNs after RT and describe latencies, cytogenetic profile, mutation analyses, and clinical outcomes. The most common cytogenetic abnormality was a clonal abnormality in chromosome 5 and/or 7, which was present in 45% of patients. The median latency from RT to t-MN diagnosis was 6.5 years, with the shortest latency in patients with balanced translocations. One-year overall survival (OS) was 52% and 5-year OS was 22% for the entire cohort. Patients with chromosome 5 and/or 7 abnormalities experienced worse 1-year OS (37%) and 5-year OS (2%) compared with other cytogenetic groups (P < .0001). Sixteen patients underwent net-generation sequencing; ASXL1 and TET2 were the most commonly mutated genes (n = 4). In addition, 17 patients underwent germline variant testing and 3 carried pathogenic or likely pathogenic germline variants. In conclusion, patients with t-MN after RT monotherapy have increased frequencies of chromosome 5 and/or 7 abnormalities, which are associated with poor OS. In addition, pathogenic germline variants may be common in patients with t-MN after RT monotherapy.

Cytotoxic Therapy-Induced Effects on Both Hematopoietic and Marrow Stromal Cells Promotes Therapy-Related Myeloid Neoplasms.

Therapy-related myeloid neoplasms (t-MNs) following treatment with alkylating agents are characterized by a del(5q), complex karyotypes, alterations of TP53, and a dismal prognosis. To decipher the molecular pathway(s) leading to the pathogenesis of del(5q) t-MN and the effect(s) of cytotoxic therapy on the marrow microenvironment, we developed a mouse model with loss of two key del(5q) genes, EGR1 and APC, in hematopoietic cells. We used the well-characterized drug, N-ethyl-N-nitrosurea (ENU) to demonstrate that alkylating agent exposure of stromal cells in the microenvironment increases the incidence of myeloid disease. In addition, loss of Trp53 with Egr1 and Apc was required to drive the development of a transplantable leukemia, and accompanied by the acquisition of somatic mutations in DNA damage response genes. ENU treatment of mesenchymal stromal cells induced cellular senescence, and led to the acquisition of a senescence-associated secretory phenotype, which may be a critical microenvironmental alteration in the pathogenesis of myeloid neoplasms.

A phase 1 study of azacitidine with high-dose cytarabine and mitoxantrone in high-risk acute myeloid leukemia.

In this phase 1 study, azacitidine (AZA) was given before high-dose cytarabine (HiDAC) and mitoxantrone (mito) based on the hypothesis that epigenetic priming with a hypomethylating agent before cytotoxic chemotherapy would improve response rates in patients with high-risk acute myeloid leukemia (AML), including relapsed/refractory disease. The primary objective was to establish the recommended phase 2 dose of AZA given before standard HiDAC/mito. In a dose escalation scheme, 46 patients (median age, 66 years) received AZA at 37.5, 50, or 75 mg/m2 subcutaneously or IV once daily on days 1 to 5 followed by HiDAC (3000 mg/m2) and mitoxantrone (30 mg/m2) once each on days 6 and 10 (the HiDAC/mito dose was reduced 33% in elderly subjects). Two dose-limiting toxicities occurred (both in the same patient): acute liver failure and kidney injury at the 50 mg/m2 dose. The 30-day induction death rate was 2.2% (1 of 46). The overall response rate, including complete remission and complete remission with incomplete count recovery, was 61% (28 of 46). Previously untreated patients aged ≥60 years with therapy-related AML and de novo AML were more likely to respond than untreated patients with AML progressing from an antecedent hematologic disorder (myelodysplastic syndrome and chronic myelomonocytic leukemia). Patients with favorable European Leukemia Network risk (P = .008), NPM1 mutations (P = .007), or IDH2 mutations (P = .03) were more likely to respond, and those with TP53 mutations (P = .03) were less likely to respond. The recommended phase 2 dose of AZA is 75 mg/m2 per day on days 1 to 5 followed by HiDAC (3000 mg/m2) and mitoxantrone (30 mg/m2) once each on days 6 and 10. This trial was registered at www.clinicaltrials.gov as #NCT01839240.

The Harmful Consequences of Increased Fitness in Hematopoietic Stem Cells.

Clonal hematopoiesis of indeterminate potential (CHIP) describes clonal selection of a hematopoietic stem cell with a somatic mutation that confers increased fitness, influenced by a selective environment such as aging, inflammation, or therapeutic exposure. In this issue of Cell Stem Cell, Hsu et al. (2018) explore the role of cytotoxic therapy in disease-relevant CHIP.

Differing clinical features between Japanese and Caucasian patients with myelodysplastic syndromes: Analysis from the International Working Group for Prognosis of MDS.

Clinical features of myelodysplastic syndromes (MDS) could be influenced by many factors, such as disease intrinsic factors (e.g., morphologic, cytogenetic, molecular), extrinsic factors (e.g, management, environment), and ethnicity. Several previous studies have suggested such differences between Asian and European/USA countries. In this study, to elucidate potential differences in primary untreated MDS between Japanese (JPN) and Caucasians (CAUC), we analyzed the data from a large international database collected by the International Working Group for Prognosis of MDS (300 and 5838 patients, respectively). JPN MDS were significantly younger with more severe cytopenias, and cytogenetic differences: less del(5q) and more +1/+1q, -1/del(1p), der(1;7), -9/del(9q), del(16q), and del(20q). Although differences in time to acute myeloid leukemia transformation did not occur, a significantly better survival in JPN was demonstrated, even after the adjustment for age and FAB subtypes, especially in lower, but not in higher prognostic risk categories. Certain clinical factors (cytopenias, blast percentage, cytogenetic risk) had different impact on survival and time to transformation to leukemia between the two groups. Although possible confounding events (e.g., environment, diet, and access to care) could not be excluded, our results indicated the existence of clinically relevant ethnic differences regarding survival in MDS between JPN and CAUC patients. The good performance of the IPSS-R in both CAUC and JP patients underlines that its common risk model is adequate for CAUC and JP.

Clonal architecture in patients with myelodysplastic syndromes and double or minor complex abnormalities: Detailed analysis of clonal composition, involved abnormalities, and prognostic significance.

The study analyzes the clonal architecture and the abnormalities involved in a series of 191 patients with myelodysplastic syndromes (MDS) and 2-3 clonal abnormalities. All patients were extracted from an international database. The patients were classified into six clonal subtypes (2A-3C) based on the number of abnormalities and the presentation of unrelated clones (UC) and/or a clonal evolution. UC were detected in 23/191 patients (12%). The composition of UC showed great variability. The only recurrent combination of abnormalities was del(5q) and + 8 in 8 of 23 patients (35%). In patients with clonal evolution, the clone size of the primary and secondary clone varied: Patients with -7 and + 8 in the primary clone showed a larger primary and a smaller secondary clone (-7: median 74% vs 10%; +8 73% vs 18%) while patients with del(5q) in the primary clone showed a smaller primary and a larger secondary clone (33% vs 61%). Univariate and multivariate analyses showed no significant differences regarding overall or AML-free survival between the clonal subtypes. Only the subtype 3C (3 abnormalities and clonal evolution) was an independent risk factor for developing AML (Hazard Ratio 5.5 as compared to subtype 2A, P < .05). Finally, our study confirms that the number of abnormalities clearly defines a significant risk factor for overall- as well as AML-free survival. Importantly, in patients with more than one clone, the calculation of the number of abnormalities in the entire sample instead of the number of abnormalities per clone allows a higher prognostic accuracy.

Regulated Capture of Vκ Gene Topologically Associating Domains by Transcription Factories.

Expression of vast repertoires of antigen receptors by lymphocytes, with each cell expressing a single receptor, requires stochastic activation of individual variable (V) genes for transcription and recombination. How this occurs remains unknown. Using single-cell RNA sequencing (scRNA-seq) and allelic variation, we show that individual pre-B cells monoallelically transcribe divergent arrays of Vκ genes, thereby opening stochastic repertoires for subsequent Vκ-Jκ recombination. Transcription occurs upon translocation of Vκ genes to RNA polymerase II arrayed on the nuclear matrix in transcription factories. Transcription is anchored by CTCF-bound sites or E2A-loaded Vκ promotors and continues over large genomic distances delimited only by topological associating domains (TADs). Prior to their monoallelic activation, Vκ loci are transcriptionally repressed by cyclin D3, which prevents capture of Vκ gene containing TADs by transcription factories. Cyclin D3 also represses protocadherin, olfactory, and other monoallelically expressed genes, suggesting a widely deployed mechanism for coupling monoallelic gene activation with cell cycle exit.

Author Correction: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia.

In the originally published version of this Article, the GAPDH loading control blot in Fig. 1a was inadvertently replaced with a duplicate of the DNMT2 blot in the same panel during assembly of the figure. This has now been corrected in both the PDF and HTML versions of the Article.

Publisher Correction: miR-196b directly targets both HOXA9/MEIS1 oncogenes and FAS tumour suppressor in MLL-rearranged leukaemia.

This corrects the article DOI: 10.1038/ncomms1681.

RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia.

The roles of RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) in lineage-associated chromatin organization and drug response/resistance are unclear. Here we demonstrate that the RCMTs, namely NSUN3 and DNMT2, directly bind hnRNPK, a conserved RNA-binding protein. hnRNPK interacts with the lineage-determining transcription factors (TFs), GATA1 and SPI1/PU.1, and with CDK9/P-TEFb to recruit RNA-polymerase-II at nascent RNA, leading to formation of 5-Azacitidine (5-AZA)-sensitive chromatin structure. In contrast, NSUN1 binds BRD4 and RNA-polymerase-II to form an active chromatin structure that is insensitive to 5-AZA, but hypersensitive to the BRD4 inhibitor JQ1 and to the downregulation of NSUN1 by siRNAs. Both 5-AZA-resistant leukaemia cell lines and clinically 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukaemia specimens have a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin. This study reveals novel RNA:m5C/RCMT-mediated chromatin structures that modulate 5-AZA response/resistance in leukaemia cells, and hence provides a new insight into treatment of leukaemia.

Therapy-related myeloid neoplasms: when genetics and environment collide.

Therapy-related myeloid neoplasms (t-MN) arise as a late effect of chemotherapy and/or radiation administered for a primary condition, typically a malignant disease, solid organ transplant or autoimmune disease. Survival is measured in months, not years, making t-MN one of the most aggressive and lethal cancers. In this Review, we discuss recent developments that reframe our understanding of the genetic and environmental aetiology of t-MN. Emerging data are illuminating who is at highest risk of developing t-MN, why t-MN are chemoresistant and how we may use this information to treat and ultimately prevent this lethal disease.

Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apcdel/+ MDS mouse model.

There is accumulating evidence that functional alteration(s) of the bone marrow (BM) microenvironment contribute to the development of some myeloid disorders, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In addition to a cell-intrinsic role of WNT activation in leukemia stem cells, WNT activation in the BM niche is also thought to contribute to the pathogenesis of MDS and AML. We previously showed that the Apc-haploinsufficient mice (Apcdel/+ ) model MDS induced by an aberrant BM microenvironment. We sought to determine whether Apc, a multifunctional protein and key negative regulator of the canonical ß-catenin (Ctnnb1)/WNT-signaling pathway, mediates this disease through modulating WNT signaling, and whether inhibition of WNT signaling prevents the development of MDS in Apcdel/+ mice. Here, we demonstrate that loss of 1 copy of Ctnnb1 is sufficient to prevent the development of MDS in Apcdel/+ mice and that altered canonical WNT signaling in the microenvironment is responsible for the disease. Furthermore, the US Food and Drug Administration (FDA)-approved drug pyrvinium delays and/or inhibits disease in Apcdel/+ mice, even when it is administered after the presentation of anemia. Other groups have observed increased nuclear CTNNB1 in stromal cells from a high frequency of MDS/AML patients, a finding that together with our results highlights a potential new strategy for treating some myeloid disorders.