Hypomethylating agents are used as frontline therapy for myelodysplastic neoplasms (MDS), but clinical response is unpredictable. To determine whether response was associated with in vivo dynamics of DNA hypomethylation, we conducted a phase 2 trial for MDS using both injection and oral azacitidine (AZA). We established that global DNA methylation levels in peripheral blood and bone marrow mononuclear cells were comparable in AZA responders and non-responders during their course of treatment. However, there were distinct baseline and early drug induced differences in CpG methylation in haematopoietic stem and progenitor cells (HSPCs) in responders compared to non-responders that overlapped with regulatory regions of genes associated with tissue patterning, cell migration and myeloid differentiation. Following six cycles of therapy when clinical response typically manifests, differential hypomethylation in responder HSPCs pointed to marrow adaptation as a driver of enhanced haematopoiesis. Taken together, CpG methylation differences in HSPCs may explain variable response to AZA. ### Competing Interest Statement F.V. is affiliated with OmniOmics.AI Pty Ltd. C.F. is an advisory board member at Amgen, AbbVie, Adaptive Biotech, BeiGene, Pfizer, Otsuka, and Jazz, a consultant at Novotech, and received speaker fees from Amgen, Pfizer, Servier, BMS, and Astella. D.H. has consultancy agreements with GlaxoSmithKline and Pharming Corp. M.H. is a consultant/advisory board member at Roche, Gilead, Otsuka, Janssen, Beigene, and Takeda. M.N.P. received research funding and/or provision of drug for clinical trials (to institution) from AstraZeneca, BRII Biosciences, Celgene/BMS, CSL Behring, Eli Lilly, Emergent Biosciences, Gilead Pharmaceuticals, GlaxoSmithKline, Grifols, Janssen/Johnson and Johnson, Takeda, ViiV Pharmaceuticals and has advisory roles with Celgene/BMS, Gilead Pharmaceuticals, and ViiV Pharmaceuticals. J.E.P. received research funding and/or provision of drug for clinical trials (to institution) from Celgene/BMS, Astex, Verastem Oncology and received honoraria from Abbvie as an advisory board member. The remaining authors declare no competing financial interests. ### Clinical Trial NCT03493646 ### Funding Statement The investigator initiated clinical trial was funded in part by Celgene/BMS (RG172029) with research support from the National Health and Medical Research Council (RG170246, RG211412), Anthony Rothe Memorial Trust (RG182042, RG202657, RG213236), Leukaemia Foundation (RG231257). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The trial protocol received ethical approval from the South Eastern Sydney Local Health District Human Research Ethics Committee, and participating sites received Institution approval to conduct the trial prior to commencing recruitment. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Determinants of clinical response to hypomethylating agents (HMAs) in MDS/CMML are unclear. To address this knowledge gap, we enrolled 40 patients to NCT03493646 where they received 6 cycles of injection AZA (vidaza) or oral AZA (CC486) delivered for 7 or 21 days respectively in a 28-day cycle to study the kinetics of drug incorporation and DNA hypomethylation during treatment, and noted the following(i) Average DAC incorporation in DNA was higher with vidaza than CC486, but global DNA hypomethylation was greater with CC486. Although DAC incorporation was higher in responders (R) than in non-responders (NR) with vidaza, global DNA demethylation did not differ between response groups with either drug.(ii) DAC incorporation after vidaza correlated with the fraction of HPCs progressing through S/G2/M pre-treatment, but there were no pre-treatment differences between R and NR in the fraction of HSCs and HPCs progressing through S/G2/M. However, vidaza induced HSCs and HPCs to exit quiescence in R but not NR.(iii) Baseline CpG methylation was higher in CD34+ bone marrow cells in NR vs. R, and CpG sites that were associated with regulation of pattern specification (HOX cluster), epithelial cell migration/EMT and mesenchymal stromal cell differentiation were differentially hypomethylated in R vs. NR. Following vidaza, early drug-induced hypomethylation was seen at CpG sites in CD34+ that regulated genes associated with myeloid cell differentiation. By contrast, late drug-induced CpG hypomethylation in R was at sites associated with leukocyte development in response to marrow environmental changes.(iv) Variant alleles persist in R and NR following vidaza or CC486, and there was no association between change in variant allele frequencies and the degree of DNA hypomethylation.Site-specific differences in CpG hypomethylation in HSPCs may explain clinical response to HMAs.

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 naive 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.

Supplementary Figure 3, related to main Figure 5.

Supplementary Methods - revised, plus Supplementary Tables 1-6

Supplementary Figure 1, related to main Figure 1

Supplementary Figure 2, related to main Figure 3

Introduction: Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSC) and characterized by the accumulation of blasts in the bone marrow and accompanying cytopenias. Hypomethylating agents (HMA) such as azacytidine (AZA) can modify the clinical course of MDS and CMML (Platzbecker et al Blood 2019). Although not curative, they are used as first-line therapies for high-risk patients who are ineligible or unable to access an allogeneic bone marrow transplant. Clinical improvement in response to HMAs is not accompanied by clearance or major shifts in mutant clones in the bone marrow (Unnikrishnan et al Cell Reports 2017, Merlevede et al Nature Communications 2016). However, it is unclear whether in patients with established MDS; (a) hematopoietic stem and progenitor cells with multiple mutations progress through to mature cells with comparable robustness to their counterparts with fewer or no mutations, or (b) the improvements in peripheral blood counts following HMA therapy is driven by residual wild-type hematopoietic stem and progenitor cells or by clones with particular combinations of mutations. Methods: We index sorted hematopoietic stem (HSC, MPP) and progenitor cells (CMP, MEP and GMP) and mature blood cells (neutrophils (Neut), monocytes (Mono), and naïve B-cells (nBC): short lived cell types representative of current stem/progenitor output) from 3 MDS/CMML patients (1 treatment naïve, 2 treated >10 years with AZA) and performed targeted amplicon sequencing on thousands of single cells (n = 4248). In a second cohort (9 MDS patients; 6 responders, 3 non-responders) we sorted Mono, natural killer (NK) cells, and CD33+ progenitors and measured variant allele fraction (VAF) before and after 6 cycles of AZA. Results: Single cell data showed the proportion of residual wild-type HSCs, and their contribution to mature myeloid cells, was minor (0 - 5.3% of HSCs, 0.3 - 4.5% of mature myeloid cells). Driver mutations were proportionately represented across multiple hematopoietic cell types, and even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature myeloid and, in some cases, lymphoid cell types in vivo, irrespective of AZA treatment (Fig A). In a second cohort composed of paired pre- and post-AZA samples, clonal composition differed slightly between cell types; e.g. NK cells often harbored lower VAFs compared to monocytes and progenitors. We also observed treatment-associated reduction in a minority of mutated clones in this cohort. However, in all patients, clonal composition was remarkably similar before and after treatment, with highly mutated progenitors making a significant contribution to mature cells, even in patients showing reduced blast plus improved blood counts (Fig B). Conclusions: Highly mutated immature cells contribute significantly to mature blood production in MDS and CMML, before and after AZA treatment. Our data highlight the key role of AZA therapy to promote output from mutant progenitors rather than simply eradicating them. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ABSTRACTProgressively acquired somatic mutations in hematopoietic stem cells are central to pathogenesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). They can lead to proliferative advantages, impaired differentiation and progressive cytopenias. MDS or CMML patients with high-risk disease are treated with hypomethylating agents including 5-azacytidine (AZA). Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. To address this, we characterised the somatic mutations of individual stem, progenitor (common myeloid progenitor, granulocyte monocyte progenitor, megakaryocyte erythroid progenitor), and matched circulating (monocyte, neutrophil, naïve B cell) haematopoietic cells in treatment naïve and AZA-treated MDS and CMML via high-throughput single cell genotyping. The mutational burden was similar across multiple hematopoietic cell types, and even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature myeloid and, in some cases, lymphoid cell types in vivo. Our data show that even highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.Key points*Highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.*The mutational burden in matched bone marrow and peripheral blood cells in MDS and CMML was similar throughout myelopoiesis.

Abstract Purpose: RNA splicing is a fundamental biological process that generates protein diversity from a finite set of genes. Recurrent somatic mutations of splicing factor genes are common in some hematologic cancers but are relatively uncommon in acute myeloid leukemia (AML, < 20% of patients). We examined whether RNA splicing differences exist in AML, even in the absence of splicing factor mutations. Experimental Design: We developed a bioinformatics pipeline to study alternative RNA splicing in RNA-sequencing data from large cohorts of patients with AML. Results: We have identified recurrent differential alternative splicing between patients with poor and good prognosis. These splicing events occurred even in patients without any discernible splicing factor mutations. Alternative splicing recurrently occurred in genes with specific molecular functions, primarily related to protein translation. Developing tools to predict the functional impact of alternative splicing on the translated protein, we discovered that approximately 45% of the splicing events directly affected highly conserved protein domains. Several splicing factors were themselves misspliced and the splicing of their target transcripts were altered. Studying differential gene expression in the same patients, we identified that alternative splicing of protein translation genes in ELNAdv patients resulted in the induction of an integrated stress response and upregulation of inflammation-related genes. Finally, using machine learning techniques, we identified a splicing signature of four genes which refine the accuracy of existing risk prognosis schemes and validated it in a completely independent cohort. Conclusions: Our discoveries therefore identify aberrant alternative splicing as a molecular feature of adverse AML with clinical relevance. See related commentary by Bowman, p. 3503

Lymphatic vessels collect interstitial fluid that has extravasated from blood vessels and return it to the circulatory system. Another important function of the lymphatic network is to facilitate immune cell migration and antigen transport from the periphery to draining lymph nodes. This migration plays a crucial role in immune surveillance, initiation of immune responses and tolerance. Here we discuss the significance and mechanisms of lymphatic migration of innate and adaptive immune cells in homeostasis, inflammation and cancer.

Immune therapy is rapidly gaining prominence in the clinic as a major weapon against cancer. Whereas much attention has been focused on the infiltration of tumors by immune cells, the subsequent fate of these infiltrates remains largely unexplored. We therefore established a photoconversion-based model that allowed us to label tumor-infiltrating immune cells and follow their migration. Using this system, we identified a population of tumor-experienced cells that emigrate from primary tumors to draining lymph nodes via afferent lymphatic vessels. Although the majority of tumor-infiltrating cells were myeloid, T cells made up the largest population of tumor-egressing leukocytes. Strikingly, the subset composition of tumor-egressing T cells was greatly skewed compared with those that had infiltrated the tumor and those resident in the draining lymph node. Some T-cell subsets such as CD8(+) T cells emigrated more readily; others including CD4(-)CD8(-)T cells were preferentially retained, suggesting that specific mechanisms guide immune cell egress from tumors. Furthermore, tumor-egressing T cells were more activated and displayed enhanced effector function in comparison with their lymph node counterparts. Finally, we demonstrated that tumor-infiltrating T cells migrate to distant secondary tumors and draining lymph nodes, highlighting a mechanism whereby tumor-experienced effector T cells may mediate antitumor immunity at metastatic sites. Thus, our results provide insights into migration and function of tumor-infiltrating immune cells and the role of these cells in tumor immunity outside of primary tumor deposits.

Secondary lymphoid organs provide a specialized microenvironment tailored to foster communication between cells of the innate and adaptive immune systems. These interactions allow immune cells to coordinate multilayered defense against pathogens. Until recently dendritic cells and macrophages were thought to comprise the main innate immune cell subsets responsible for delivering signals that drive the adaptive immune response, while the function of neutrophils was largely confined to the innate immune system. However, the discovery of neutrophils in lymph nodes has raised the question of whether neutrophils might play a more extensive role not only in innate immunity per se, but also in coordinating the interactions between innate and adaptive immune responses. In this review we discuss the mechanisms and consequences of neutrophil recruitment to lymph nodes and how this recruitment influences subsequent immune responses both in situ and at distant sites.

Recognition of peptidoglycan is integral to detection of gram-positive bacterial pathogens. In a recent issue of Cell, Wolf et al. (2016) report that detection of the N-acetylglucosamine component of peptidoglycan by the glycolytic enzyme hexokinase activates the NLRP3 inflammasome, revealing an intriguing interplay between pathogen detection and metabolism.

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.

Neutrophil recruitment to the site of injury is an essential first step of an anti-bacterial response. However, little is known about the basis for and relevance of neutrophil migration from inflamed tissue into lymphoid organs. We established a photoconversion-based system to monitor the fate of neutrophils recruited to inflamed skin. While neutrophils are efficiently recruited to sites of both microbial and sterile lesions, subsequent re-localization to draining lymph nodes happens only when bacteria are present in the primary lesion. Skin egress of neutrophils occurs via lymphatic vessels and is dependent on CD11b and CXCR4 but not CCR7. Neutrophils are the predominant immune cell to migrate from inflamed skin into lymph nodes where they augment lymphocyte proliferation. Furthermore, inhibition of neutrophil migration from skin reduces T-cell proliferation in draining lymph nodes. Thus neutrophils mediate rapid cellular communication between the initial injury site and secondary lymphoid organs and modulate immune responsiveness.