Key Points
An increase in the classical monocyte subset to >94% of total monocytes discriminates CMML from other monocytoses with high specificity. This characteristic increase in classical monocytes disappears in CMML patients who respond to hypomethylating agents.
The cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect.
These authors contributed equally to this work.Keywords: autophagy, CMML, CSF1, differentiation, primary monocyte, PRKAA1/AMPKa1, P2RY6Abbreviations: ACTB actin, b; CAMKK2, calcium/calmodulin-dependent protein kinase kinase 2, b; CASP8, caspase 8; apoptosisrelated cysteine peptidase; CFLAR CASP8 and FADD-like apoptosis regulator; CMML chronic myelomonocytic leukemia; CSF1 colony stimulating factor 1 (macrophage); CSF1R colony stimulating factor 1 receptor; DEFA1 defensin a 1; DEFA3 defensin a 3 neutrophil-specific; DRS; dorsomorphin; EMR1 EGF-like module-containing mucin-like hormone receptor-like 1; FADD Fas (TNFRSF6)-associated via death domain; ITGAM integrin a M; MAP1LC3B/LC3B microtubule-associated protein 1 light chain 3 b; P2RY6 pyrimidinergic receptor P2Y; G-protein coupled 6; PLCB3 phospholipase C; b 3 (phosphatidylinositol-specific); PLC phospholipase; PLCG2 phospholipase C gamma 2 (phosphatidylinositol-specific); PRKAA protein kinase AMP-activated; PRKAA1 protein kinase AMP-activated a 1 catalytic subunit; PRKAA2 protein kinase AMP-activated a 2 catalytic subunit; PRKAG1 protein kinase AMP-activated gamma 1 noncatalytic subunit; RIPK1 receptor (TNFRSF)-interacting serine-threonine kinase 1; STK11 serine/ threonine kinase 11; TFRC transferrin receptor; UDP uridine diphosphate; ULK1 unc-51 like autophagy activating kinase 1; WT wild-type.Autophagy is induced during differentiation of human monocytes into macrophages that is mediated by CSF1/CSF-1/M-CSF (colony stimulating factor 1 [macrophage]). However, little is known about the molecular mechanisms that link CSF1 receptor engagement to the induction of autophagy. Here we show that the CAMKK2-PRKAA1-ULK1 pathway is required for CSF1-induced autophagy and human monocyte differentiation. We reveal that this pathway links P2RY6 to the induction of autophagy, and we decipher the signaling network that links the CSF1 receptor to P2RY6-mediated autophagy and monocyte differentiation. In addition, we show that the physiological P2RY6 ligand UDP and the specific P2RY6 agonist MRS2693 can restore normal monocyte differentiation through reinduction of autophagy in primary myeloid cells from some but not all chronic myelomonocytic leukemia (CMML) patients. Collectively, our findings highlight an essential role for PRKAA1-mediated autophagy during differentiation of human monocytes and pave the way for future therapeutic interventions for CMML.
Peripheral blood monocytes include three subsets defined by CD14 and CD16 surface markers. An increase in the CD14++CD16− classical monocyte fraction ≥ 94% of the total monocytes was proposed to rapidly and efficiently distinguish chronic myelomonocytic leukemia from reactive monocytosis. The robustness of this assay required a multicenter validation. The flow cytometry assay designed to quantify peripheral blood monocyte subsets was implemented by multiple diagnosis laboratories in France. A nationwide survey was performed to evaluate its performance. All the 48 French laboratories answered the questionnaire, revealing that 63% use this assay routinely. Central blind reanalysis of 329 cytometry files collected from five laboratories demonstrated an excellent correlation in classical monocyte fraction measurement (r = 0.93; p < 0.0001). The cutoff value of 94% classical monocytes being the critical readout for diagnosis, we then compared 115 patients with classical monocytes ≥ 94% and 214 patients with a fraction < 94% between initial analysis and reanalysis. An agreement was obtained in 311 files. Finally, an overt diagnosis, available for 86 files, confirmed a good sensitivity (93.6%) and specificity (89.7%). This survey demonstrates the robustness of the flow assay with limited variability of classical monocyte percentage between centers, validates the 94% cutoff value, and confirms its sensitivity and specificity.
Non-classical monocyte subsets may derive from classical monocyte differentiation and the proportion of each subset is tightly controlled. Deregulation of this repartition is observed in diverse human diseases, including chronic myelomonocytic leukemia (CMML) in which non-classical monocyte numbers are significantly decreased relative to healthy controls. Here, we identify a down-regulation of hsa-miR-150 through methylation of a lineage-specific promoter in CMML monocytes. Mir150 knock-out mice demonstrate a cell-autonomous defect in non-classical monocytes. Our pulldown experiments point to Ten-Eleven-Translocation-3 (TET3) mRNA as a hsa-miR-150 target in classical human monocytes. We show that Tet3 knockout mice generate an increased number of non-classical monocytes. Our results identify the miR-150/TET3 axis as being involved in the generation of non-classical monocytes.
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