Contribution of mutant HSC clones to immature and mature cells in MDS and CMML, and variations with AZA therapy.

Myelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of individual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.

Cooperation between KDM6B overexpression and TET2 deficiency in the pathogenesis of chronic myelomonocytic leukemia.

Loss-of-function TET2 mutations are recurrent somatic lesions in chronic myelomonocytic leukemia (CMML). KDM6B encodes a histone demethylase involved in innate immune regulation that is overexpressed in CMML. We conducted genomic and transcriptomic analyses in treatment naïve CMML patients and observed that the patients carrying both TET2 mutations and KDM6B overexpression constituted 18% of the cohort and 42% of patients with TET2 mutations. We therefore hypothesized that KDM6B overexpression cooperated with TET2 deficiency in CMML pathogenesis. We developed a double-lesion mouse model with both aberrations, and discovered that the mice exhibited a more prominent CMML-like phenotype than mice with either Tet2 deficiency or KDM6B overexpression alone. The phenotype includes monocytosis, anemia, splenomegaly, and increased frequencies and repopulating activity of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). Significant transcriptional alterations were identified in double-lesion mice, which were associated with activation of proinflammatory signals and repression of signals maintaining genome stability. Finally, KDM6B inhibitor reduced BM repopulating activity of double-lesion mice and tumor burden in mice transplanted with BM-HSPCs from CMML patients with TET2 mutations. These data indicate that TET2 deficiency and KDM6B overexpression cooperate in CMML pathogenesis of and that KDM6B could serve as a potential therapeutic target in this disease.

Role of the bone marrow immune microenvironment in chronic myelomonocytic leukemia pathogenesis: novel mechanisms and insights into clonal propagation.

Recent studies in chronic myelomonocytic leukemia (CMML) involving clonal dendritic cell (DC) aggregates and association with systemic immune dysregulation have highlighted novel and potentially targetable pathways of disease progression. CMML DC aggregates are populated by heterogeneous cell types such as CD123+ plasmacytoid dendritic cells (pDCs), CD11c + myeloid-derived DCs (mDCs), myeloid-derived suppressor cells (MDSCs), monocytes, and associate with an immune checkpoint called indoleamine 2,3-dioxygenase (IDO). Systemically, these IDO + DC aggregates are associated with immune tolerance marked by regulatory T cell expansion, likely mediated by aberrant DC-T cell interactions occurring within the bone marrow (BM) microenvironment. Somatic mutational events in CMML such as ASXL1 and NRAS mutations cooperate to induce T cell exhaustion and contribute toward disease progression to acute myeloid leukemia (AML). In this review, we explore the role of aging-induced alterations in the BM immune microenvironment, aberrant innate immune and proinflammatory signaling, and the adaptive immune system in CMML.

RAS mutations drive proliferative chronic myelomonocytic leukemia via a KMT2A-PLK1 axis.

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

Cut-like homeobox 1 (CUX1) tumor suppressor gene haploinsufficiency induces apoptosis evasion to sustain myeloid leukemia.

While oncogenes promote tumorigenesis, they also induce deleterious cellular stresses, such as apoptosis, that cancer cells must combat by coopting adaptive responses. Whether tumor suppressor gene haploinsufficiency leads to such phenomena and their mechanistic basis is unclear. Here, we demonstrate that elevated levels of the anti-apoptotic factor, CASP8 and FADD-like apoptosis regulator (CFLAR), promotes apoptosis evasion in acute myeloid leukemia (AML) cells haploinsufficient for the cut-like homeobox 1 (CUX1) transcription factor, whose loss is associated with dismal clinical prognosis. Genome-wide CRISPR/Cas9 screening identifies CFLAR as a selective, acquired vulnerability in CUX1-deficient AML, which can be mimicked therapeutically using inhibitor of apoptosis (IAP) antagonists in murine and human AML cells. Mechanistically, CUX1 deficiency directly alleviates CUX1 repression of the CFLAR promoter to drive CFLAR expression and leukemia survival. These data establish how haploinsufficiency of a tumor suppressor is sufficient to induce advantageous anti-apoptosis cell survival pathways and concurrently nominate CFLAR as potential therapeutic target in these poor-prognosis leukemias.

Cytokine-like protein 1-induced survival of monocytes suggests a combined strategy targeting MCL1 and MAPK in CMML.

Mouse models of chronic myeloid malignancies suggest that targeting mature cells of the malignant clone disrupts feedback loops that promote disease expansion. Here, we show that in chronic myelomonocytic leukemia (CMML), monocytes that accumulate in the peripheral blood show a decreased propensity to die by apoptosis. BH3 profiling demonstrates their addiction to myeloid cell leukemia-1 (MCL1), which can be targeted with the small molecule inhibitor S63845. RNA sequencing and DNA methylation pattern analysis both point to the implication of the mitogen-activated protein kinase (MAPK) pathway in the resistance of CMML monocytes to death and reveal an autocrine pathway in which the secreted cytokine-like protein 1 (CYTL1) promotes extracellular signal-regulated kinase (ERK) activation through C-C chemokine receptor type 2 (CCR2). Combined MAPK and MCL1 inhibition restores apoptosis of monocytes from patients with CMML and reduces the expansion of patient-derived xenografts in mice. These results show that the combined inhibition of MCL1 and MAPK is a promising approach to slow down CMML progression by inducing leukemic monocyte apoptosis.

Genomic Landscape and Risk Stratification in Chronic Myelomonocytic Leukemia.

PURPOSE: The advent of next-generation sequencing has allowed for the annotation of a vast array of recurrent somatic mutations across human malignancies, ushering in a new era of precision oncology. Chronic myelomonocytic leukemia is recognized as a myelodysplastic/myeloproliferative neoplasm and displays heterogenous clinical and genetic features. Herein, we review what is currently understood regarding the genomic landscape of this disease and discuss how somatic mutations have impacted current risk stratification methods. RECENT FINDINGS: Genomic studies in chronic myelomonocytic leukemia have identified a characteristic spectrum of cytogenetic and molecular abnormalities. Chromosomal abnormalities are detected in ~30% of patients and somatic gene mutations in up to 90% of patients, most commonly in TET2, SRSF2, and ASXL1. While cytogenetic abnormalities have long been known to impact the prognosis of myeloid neoplasms, recent studies have identified that somatic mutations impact prognosis independent of cytogenetic and clinical variables. This is best exemplified by mutations in ASXL1, which have been uniformly associated with inferior survival. These findings have led to the development of three molecularly inspired prognostic models, in an attempt to more accurately prognosticate in the disease. Our understanding of the genomic landscape of chronic myelomonocytic leukemia continues to evolve, with somatic mutations demonstrating an expanding role in diagnosis, risk stratification, and therapeutic decision-making. Given these findings, molecular profiling by next-generation sequencing should be considered standard of care in all patients.

Asxl1 C-terminal mutation perturbs neutrophil differentiation in zebrafish.

ASXL1 is one of the most frequently mutated genes in malignant myeloid diseases. In patients with myeloid malignancies, ASXL1 mutations are usually heterozygous frameshift or nonsense mutations leading to C-terminal truncation. Current disease models have predominantly total loss of ASXL1 or overexpressed C-terminal truncations. These models cannot fully recapitulate leukemogenesis and disease progression. We generated an endogenous C-terminal-truncated Asxl1 mutant in zebrafish that mimics human myeloid malignancies. At the embryonic stage, neutrophil differentiation was explicitly blocked. At 6 months, mutants initially exhibited a myelodysplastic syndrome-like phenotype with neutrophilic dysplasia. At 1 year, about 13% of mutants further acquired the phenotype of monocytosis, which mimics chronic myelomonocytic leukemia, or increased progenitors, which mimics acute myeloid leukemia. These features are comparable to myeloid malignancy progression in humans. Furthermore, transcriptome analysis, inhibitor treatment, and rescue assays indicated that asxl1-induced neutrophilic dysplasia was associated with reduced expression of bmi1a, a subunit of polycomb repressive complex 1 and a reported myeloid leukemia-associated gene. Our model demonstrated that neutrophilic dysplasia caused by asxl1 mutation is a foundation for the progression of myeloid malignancies, and illustrated a possible effect of the Asxl1-Bmi1a axis on regulating neutrophil development.

Type I interferon upregulation and deregulation of genes involved in monopoiesis in chronic myelomonocytic leukemia.

Chronic myelomonocytic leukemia (CMML) is characterized by myelomonocytic bias and monocytic proliferation. Whether cell-intrinsic innate immune or inflammatory upregulation mediate disease pathogenesis and phenotype or whether the degree of aberrant monocytic differentiation influences outcomes remains unclear. We compared the transcriptomic features of bone marrow CD34+ cells from 19 patients with CMML and compared to healthy individuals. A total of 1495 genes had significantly differential expression in CMML (q<0.05, fold change>2), including 1271 genes that were significantly upregulated and 224 that were significantly downregulated in CMML. Top upregulated genes were associated with interferon (IFN) alpha and beta signaling, chemokine receptors, IFN gamma, G protein-coupled receptor ligand signaling, and genes involved in immunomodulatory interactions between lymphoid and non-lymphoid cells. Additionally, 6 gene sets were differentially upregulated and 139 were significantly downregulated in patients with myeloproliferative compared to myelodysplastic CMML. A total of 23 genes involved in regulation of monopoiesis were upregulated in CMML compared to healthy controls. We developed a prediction model using Cox regression including 3 of these genes, which differentiated patients into two prognostic subsets with distinct survival outcomes. This data warrants further evaluation of the roles and therapeutic potential of type I IFN signaling and monopoiesis in CMML.

Mast cell differentiation of leukemic blasts in diverse myeloid neoplasms: A potential pre-myelomastocytic leukemia condition.

INTRODUCTION: Myeloid neoplasm with blasts showing mast cell (MC)-differentiation and MC-component less than 10% of all nucleated cells but not fulfilling the criteria for systemic mastocytosis with associated hematological neoplasm (SM-AHN) or myelomastocytic leukemia (MML) has not been described in the literature. Herein, we report a study of diverse myeloid malignancies with blasts showing MC-differentiation but not meeting the criteria for SM-AHN or MML. We also evaluated the utility of flow-cytometric immunophenotyping (FCI) in the characterization of immature-MCs (iMCs). METHODS: We identified nine patients of myeloid neoplasms and studied their morphological, FCI, immunohistochemistry, cytogenetic and molecular characteristics. We also compared the immunophenotypic features of MCs from patient samples with control samples. RESULTS: The study included patients with newly-diagnosed acute myeloid leukemia (n = 4), chronic myelomonocytic leukemia (n = 1), and chronic myeloid leukemia on follow-up (n = 4) showing MC differentiation in leukemic-blasts. These patients had mildly increased MCs (range, 0.5%-3%) in bone-marrow morphology, including immature-forms and did not meet the criteria for either SM-AHN or MML. On FCI, iMCs were positive for bright-CD117, heterogeneous-CD34, dim-to-negative-HLADR, and moderate-CD203c expression. Expression-levels of CD123 and CD38 were higher (p < 0.001) but CD33 and CD45 were lower in iMCs compared to mature-MC from control samples (p = 0.019 and p = 0.0037). CONCLUSION: We reported a rare finding of MC differentiation of leukemic blasts in diverse myeloid neoplasms and proposed it as a potential pre-myelomastocytic leukemia condition. We described the distinct immunophenotypic signature of immature-MCs using commonly used markers and highlighted the utility of FCI for the diagnosis of this entity.

Oligo-monocytic CMML and other pre-CMML states: Clinical impact, prognostication and management.

Chronic myelomonocytic leukemia (CMML) is defined by myelodysplasia, pathologic accumulation of monocytes and a substantial risk to transform to secondary acute myeloid leukemia (sAML). In recent years, minimal diagnostic criteria for classical CMML and CMML-variants were proposed. Moreover, potential pre-stages of CMML and interface conditions have been postulated. Oligomonocytic CMML is a condition where the absolute peripheral blood monocyte count does not reach a diagnostic level but all other criteria for CMML are fulfilled. Among potential pre-stages of CMML, clonal and non-clonal conditions have been described, including idiopathic monocytosis (IMUS) and clonal monocytosis of unknown significance (CMUS). Patients with myelodysplastic syndromes (MDS), clonal cytopenia of unknown significance (CCUS), clonal hematopoiesis of indeterminate potential (CHIP) and idiopathic cytopenia of undetermined significance (ICUS) may also progress to CMML. The current article provides an overview of pre-CMML conditions and oligomonocytic CMML, with special reference to diagnostic criteria, differential diagnoses, clinical outcomes and management.

Concordance among hematopathologists in classifying blasts plus promonocytes: A bone marrow pathology group study.

Enumeration of blasts and promonocytes is essential for World Health Organization (WHO) classification of myelomonocytic neoplasms. The accuracy of distinguishing blasts, promonocytes and monocytes, including normal vs abnormal monocytes, remains controversial. The objective of this analysis is to assess concordances between experienced hematopathologists in classifying cells as blasts, promonocytes, and monocytes according to WHO criteria. Each of 11 hematopathologists assessed glass slides from 20 patients [12 with chronic myelomonocytic leukemia (CMML) and 8 with acute myeloid leukemia (AML)] including blood and BM aspirate smears, and limited nonspecific esterase (NSE) stains. All cases were blindly reviewed. Fleiss' extension of Cohen's kappa for multiple raters was used on these variables, separately for peripheral blood (PB) and bone marrow (BM). Spearman's rank correlation was used to assess correlations between each pair of hematopathologists for each measurement. For the classification based on the sum of blasts and promonocytes in the BM, Fleiss' kappa was estimated as 0.744. For PB, categorizing patients according to the sum of blasts and promonocytes, Fleiss' kappa was estimated as 0.949. Distinction of abnormal monocytes from normal monocytes in PB did not achieve a good concordance and showed strong evidence of differences between hematopathologists (P < .0001). The hematopathologists achieved a good concordance rate of 74% in CMML vs AML classification and a high k rate, confirming that criteria for defining the blasts equivalents (blasts plus promonocytes) could be applied consistently. Identification of monocyte subtypes (abnormal vs normal) was not concordant. Our results support the practice of combining blasts/promonocytes into a single category.

Correlation of RAS-Pathway Mutations and Spontaneous Myeloid Colony Growth with Progression and Transformation in Chronic Myelomonocytic Leukemia-A Retrospective Analysis in 337 Patients.

Although the RAS-pathway has been implicated as an important driver in the pathogenesis of chronic myelomonocytic leukemia (CMML) a comprehensive study including molecular and functional analyses in patients with progression and transformation has not been performed. A close correlation between RASopathy gene mutations and spontaneous in vitro myeloid colony (CFU-GM) growth in CMML has been described. Molecular and/or functional analyses were performed in three cohorts of 337 CMML patients: in patients without (A, n = 236) and with (B, n = 61) progression/transformation during follow-up, and in patients already transformed at the time of sampling (C, n = 40 + 26 who were before in B). The frequencies of RAS-pathway mutations (variant allele frequency ≥ 20%) in cohorts A, B, and C were 30%, 47%, and 71% (p < 0.0001), and of high colony growth (≥20/105 peripheral blood mononuclear cells) 31%, 44%, and 80% (p < 0.0001), respectively. Increases in allele burden of RAS-pathway mutations and in numbers of spontaneously formed CFU-GM before and after transformation could be shown in individual patients. Finally, the presence of mutations in RASopathy genes as well as the presence of high colony growth prior to transformation was significantly associated with an increased risk of acute myeloid leukemia (AML) development. Together, RAS-pathway mutations in CMML correlate with an augmented autonomous expansion of neoplastic precursor cells and indicate an increased risk of AML development which may be relevant for targeted treatment strategies.