Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of SRSF2 mutant cells. RKI-1447 targeted SRSF2 mutated primary human samples in xenografts models. RKI-1447 induced mitotic catastrophe and induced major reorganization of the microtubule system and severe nuclear deformation. Transmission electron microscopy and 3D light microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation that are apparently driven by microtubule-rich cytoplasmic intrusions, which are exacerbated by RKI-1447. The severe nuclear deformation in RKI-1447-treated SRSF2 mutant cells prevents cells from completing mitosis. These findings shed new light on the interplay between microtubules and the nucleus and offers new ways for targeting pre-leukemic SRSF2 mutant cells.

Purpose of reviewBoth aging and reduced diversity at the hematopoietic stem cells (HSCs) level are ubiquitous. What remains unclear is why some individuals develop clonal hematopoiesis (CH), and why does CH due to specific mutations occur in specific individuals. Much like aging, reduced diversity of HSCs is a complex phenotype shaped by numerous factors (germline & environment). The purpose of the current review is to discuss the role of two other age-related ubiquitous processes that might contribute to the dynamics and characteristics of losing HSC diversity and the evolution of CH. These processes have not been reviewed in depth so far and include the accumulation of fatty bone marrow (FBM), and the decline in sex hormones.Recent findingsInterestingly, sex hormone decline can directly shape HSC function, but also reshape the delicate balance of BM supporting cells, with a shift towards FBM. FBM accumulation can shape the clonal expansion of preleukemic mutations, particularly DNMT3A mutations, through IL-6 mediation. DNMT3A mutations are one of the only preleukemic mutations which is more prevalent in women, and especially in women with early menopause, demonstrating an association between age-related hormone decline and CH evolution, the mechanisms of which are yet to be discovered.SummaryAging is a multifactorial phenotype and the same is true for the aging of the blood system. While many factors which can shape CH have been discussed, we shed more light on FBM and sex hormone decline.

To understand human longevity, inherent aging processes must be distinguished from known etiologies leading to age-related chronic diseases. Such deconvolution is difficult to achieve because it requires tracking patients throughout their entire lives. Here, we used machine learning to infer health trajectories over the entire adulthood age range using extrapolation from electronic medical records with partial longitudinal coverage. Using this approach, our model tracked the state of patients who were healthy and free from known chronic disease risk and distinguished individuals with higher or lower longevity potential using a multivariate score. We showed that the model and the markers it uses performed consistently on data from Israeli, British and US populations. For example, mildly low neutrophil counts and alkaline phosphatase levels serve as early indicators of healthy aging that are independent of risk for major chronic diseases. We characterize the heritability and genetic associations of our longevity score and demonstrate at least 1 year of extended lifespan for parents of high-scoring patients compared to matched controls. Longitudinal modeling of healthy individuals is thereby established as a tool for understanding healthy aging and longevity.

RNA splicing factors are recurrently mutated in clonal blood disorders, but the impact of dysregulated splicing in hematopoiesis remains unclear. To overcome technical limitations, we integrated genotyping of transcriptomes (GoT) with long-read single-cell transcriptomics and proteogenomics for single-cell profiling of transcriptomes, surface proteins, somatic mutations, and RNA splicing (GoT-Splice). We applied GoT-Splice to hematopoietic progenitors from myelodysplastic syndrome (MDS) patients with mutations in the core splicing factor SF3B1. SF3B1^mut cells were enriched in the megakaryocytic-erythroid lineage, with expansion of SF3B1^mut erythroid progenitor cells. We uncovered distinct cryptic 3′ splice site usage in different progenitor populations and stage-specific aberrant splicing during erythroid differentiation. Profiling SF3B1-mutated clonal hematopoiesis samples revealed that erythroid bias and cell-type-specific cryptic 3′ splice site usage in SF3B1^mut cells precede overt MDS. Collectively, GoT-Splice defines the cell-type-specific impact of somatic mutations on RNA splicing, from early clonal outgrowths to overt neoplasia, directly in human samples.

Donor clonal hematopoiesis may be transferred to the recipient through allogeneic hematopoietic stem cell transplantation (HSCT), but the potential for adverse long-term impact on transplant outcomes remains unknown. A total of 744 samples from 372 recipients who received HSCT and the corresponding donors were included. Bar-coded error-corrected sequencing using a modified molecular inversion probe capture protocol was performed, which targeted 33 genes covering mutations involved in clonal hematopoiesis with indeterminate potential (CHIP) and other acute myeloid leukemia-related mutations. A total of 30 mutations were detected from 25 donors (6.7%): the most frequently mutated gene was TET2 (n=7, 28%), followed by DNMT3A (n=4, 16%), SMC3 (n=3, 12%) and SF3B1 (n=3, 12%). With a median follow-up duration of 13 years among survivors, the presence of CHIP in the donor was not associated with recipient overall survival (P=0.969), relapse incidence (P=0.600) or non-relapse mortality (P=0.570). Donor CHIP did not impair neutrophil (P=0.460) or platelet (P=0.250) engraftment, the rates of acute (P=0.490), or chronic graft-versus-host disease (P=0.220). No significant difference was noted for secondary malignancy following HSCT between the two groups. The present study suggests that the presence of CHIP in allogeneic stem donors does not adversely affect transplant outcomes after HSCT. Accordingly, further study is warranted to reach a clearer conclusion on whether molecular profiling to determine the presence of CHIP mutations is necessary for the pretransplant evaluation of donors prior to stem cell donation.

Both fatty bone marrow (FBM) and somatic mutations in hematopoietic stem cells (HSCs), also termed clonal hematopoiesis (CH) accumulate with human aging. However it remains unclear whether FBM can modify the evolution of CH. To address this question, we herein present the interaction between CH and FBM in two preclinical male mouse models: after sub-lethal irradiation or after castration. An adipogenesis inhibitor (PPARγ inhibitor) is used in both models as a control. A significant increase in self-renewal can be detected in both human and rodent DNMT3A^Mut-HSCs when exposed to FBM. DNMT3A^Mut-HSCs derived from older mice interacting with FBM have even higher self-renewal in comparison to DNMT3A^Mut-HSCs derived from younger mice. Single cell RNA-sequencing on rodent HSCs after exposing them to FBM reveal a 6-10 fold increase in DNMT3A^Mut-HSCs and an activated inflammatory signaling. Cytokine analysis of BM fluid and BM derived adipocytes grown in vitro demonstrates an increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduce the selective advantage of DNMT3A^Mut-HSCs exposed to FBM. Overall, paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 pathway.

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly, a misfolded P53 was described in AML blasts that do not harbor mutations in TP53, emphasizing the dynamic equilibrium between wild-type (WT) and “pseudo-mutant” conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation, in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3A^R882H-mutated HSPC, suggesting that while a pre-leukemic mutation can predispose for P53 misfolding, additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53, specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention.

AML cells are arranged in a hierarchy with stem/progenitor cells giving rise to more differentiated bulk cells. Despite the importance of stem/progenitors in the pathogenesis of AML, the determinants of the AML stem/progenitor state are not fully understood. Through a comparison of genes that are significant for growth and viability of AML cells by way of a CRISPR screen, with genes that are differentially expressed in leukemia stem cells (LSC), we identified importin 11 (IPO11) as a novel target in AML. Importin 11 (IPO11) is a member of the importin β family of proteins that mediate transport of proteins across the nuclear membrane. In AML, knockdown of IPO11 decreased growth, reduced engraftment potential of LSC, and induced differentiation. Mechanistically, we identified the transcription factors BZW1 and BZW2 as novel cargo of IPO11. We further show that BZW1/2 mediate a transcriptional signature that promotes stemness and survival of LSC. Thus, we demonstrate for the first time how specific cytoplasmic-nuclear regulation supports stem-like transcriptional signature in relapsed AML.

Motivation: Single-molecule molecular inversion probes (smMIPs) provide an exceptionally cost-effective and modular approach for routine or large-cohort next-generation sequencing. However, processing the derived raw data to generate highly accurate variants calls remains challenging. Results: We introduce SmMIP-tools, a comprehensive computational method that promotes the detection of single nucleotide variants and short insertions and deletions from smMIP-based sequencing. Our approach delivered near-perfect performance when benchmarked against a set of known mutations in controlled experiments involving DNA dilutions and outperformed other commonly used computational methods for mutation detection. Comparison against clinically approved diagnostic testing of leukaemia patients demonstrated the ability to detect both previously reported variants and a set of pathogenic mutations that did not pass detection by clinical testing. Collectively, our results indicate that increased performance can be achieved when tailoring data processing and analysis to its related technology. The feasibility of using our method in research and clinical settings to benefit from low-cost smMIP technology is demonstrated.

Background: Clonal hematopoiesis of indeterminate potential (CHIP) is a novel cardiovascular disease (CVD) risk factor in individuals without acute myeloid leukemia (AML). Objectives: The aim of this study was to examine the association between mutations associated with CHIP (CHIP-related mutations) identified in patients at AML diagnosis and the risk for cardiovascular events (CVEs). Methods: This was a retrospective cohort study of 623 patients with AML treated between 2015 and 2018 who underwent DNA analysis. Cause-specific hazard regression models were used to study the associations between pathogenic mutations in common CHIP-related genes (DNMT3A, TET2, ASXL1, JAK2, TP53, SRSF2, and SF3B1) and the rate of CVEs (heart failure hospitalization, acute coronary syndrome, coronary artery revascularization, ischemic stroke, venous thromboembolism, and CVD death) and between CVE development and all-cause mortality. Results: Patients were 64.6 ± 15.3 years of age, 265 (42.5%) were women, and 63% had at least 1 CHIP-related mutation. Those with CHIP-related mutations were older (69.2 ± 12.3 vs 56.6 ± 16.6 years; P

Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals (n = 4417), with a panel of 616 MIPs, at high depth in duplicates. To improve the previous state-of-the-art statistical model for low variant allele frequency, we selected 4635 potentially positive variants and validated them using amplicon sequencing. Using machine learning prediction tools, we significantly improved precision of 10–56.25% (P 0.005. We further developed biochemically modified MIP protocol and improved its turn-around-time to ∼4 h. Our new biochemistry significantly improved uniformity, GC-Rich regions coverage, and enabled 95% on target reads in a large MIP panel of 8349 genomic targets. Overall, we demonstrate an enhancement of the MIP targeted sequencing approach in both detection of low frequency variants and in other key parameters, paving its way to become an ultrafast cost-effective research and clinical diagnostic tool.

The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway. Importantly, we provide evidence that these recurrent deletions originate in pre-leukemic stem cells. While DNA polymerase theta (POLQ) is considered a key component in MMEJ repair, we provide evidence that pre-leukemic MMEJ (preL-MMEJ) deletions can be generated in POLQ knockout cells. In contrast, aphidicolin (an inhibitor of replicative polymerases and replication) treatment resulted in a significant reduction in preL-MMEJ. Altogether, our data indicate an association between POLQ independent MMEJ and clonal hematopoiesis and elucidate mutational mechanisms involved in the very first steps of leukemia evolution.

Background. Coronavirus disease 2019 (COVID-19) and dengue fever are difficult to distinguish given shared clinical and laboratory features. Failing to consider COVID-19 due to false-positive dengue serology can have serious implications. We aimed to assess this possible cross-reactivity. Methods. We analyzed clinical data and serum samples from 55 individuals with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. To assess dengue serology status, we used dengue-specific antibodies by means of lateral-flow rapid test, as well as enzyme-linked immunosorbent assay (ELISA). Additionally, we tested SARS-CoV-2 serology status in patients with dengue and performed in-silico protein structural analysis to identify epitope similarities. Results. Using the dengue lateral-flow rapid test we detected 12 positive cases out of the 55 (21.8%) COVID-19 patients versus zero positive cases in a control group of 70 healthy individuals (P = 2.5E−5). This includes 9 cases of positive immunoglobulin M (IgM), 2 cases of positive immunoglobulin G (IgG), and 1 case of positive IgM as well as IgG antibodies. ELISA testing for dengue was positive in 2 additional subjects using envelope protein directed antibodies. Out of 95 samples obtained from patients diagnosed with dengue before September 2019, SARS-CoV-2 serology targeting the S protein was positive/ equivocal in 21 (22%) (16 IgA, 5 IgG) versus 4 positives/equivocal in 102 controls (4%) (P = 1.6E−4). Subsequent in-silico analysis revealed possible similarities between SARS-CoV-2 epitopes in the HR2 domain of the spike protein and the dengue envelope protein. Conclusions. Our findings support possible cross-reactivity between dengue virus and SARS-CoV-2, which can lead to false-positive dengue serology among COVID-19 patients and vice versa. This can have serious consequences for both patient care and public health.

Standardized lab tests are central for patient evaluation, differential diagnosis and treatment. Interpretation of these data is nevertheless lacking quantitative and personalized metrics. Here we report on the modeling of 2.1 billion lab measurements of 92 different lab tests from 2.8 million adults over a span of 18 years. Following unsupervised filtering of 131 chronic conditions and 5,223 drug–test pairs we performed a virtual survey of lab tests distributions in healthy individuals. Age and sex alone explain less than 10% of the within-normal test variance in 89 out of 92 tests. Personalized models based on patients’ history explain 60% of the variance for 17 tests and over 36% for half of the tests. This allows for systematic stratification of the risk for future abnormal test levels and subsequent emerging disease. Multivariate modeling of within-normal lab tests can be readily implemented as a basis for quantitative patient evaluation.

Age-related clonal hematopoiesis (ARCH) is characterized by age-associated accumulation of somatic mutations in hematopoietic stem cells (HSCs) or their pluripotent descendants. HSCs harboring driver mutations will be positively selected and cells carrying these mutations will rise in frequency. While ARCH is a known risk factor for blood malignancies, such as Acute Myeloid Leukemia (AML), why some people who harbor ARCH driver mutations do not progress to AML remains unclear. Here, we model the interaction of positive and negative selection in deeply sequenced blood samples from individuals who subsequently progressed to AML, compared to healthy controls, using deep learning and population genetics. Our modeling allows us to discriminate amongst evolutionary classes with high accuracy and captures signatures of purifying selection in most individuals. Purifying selection, acting on benign or mildly damaging passenger mutations, appears to play a critical role in preventing disease-predisposing clones from rising to dominance and is associated with longer disease-free survival. Through exploring a range of evolutionary models, we show how different classes of selection shape clonal dynamics and health outcomes thus enabling us to better identify individuals at a high risk of malignancy.

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In acute myeloid leukemia (AML), molecular heterogeneity across patients constitutes a major challenge for prognosis and therapy. AML with NPM1 mutation is a distinct genetic entity in the revised World Health Organization classification. However, differing patterns of co-mutation and response to therapy within this group necessitate further stratification. Here we report two distinct subtypes within NPM1 mutated AML patients, which we label as primitive and committed based on the respective presence or absence of a stem cell signature. Using gene expression (RNA-seq), epigenomic (ATAC-seq) and immunophenotyping (CyToF) analysis, we associate each subtype with specific molecular characteristics, disease differentiation state and patient survival. Using ex vivo drug sensitivity profiling, we show a differential drug response of the subtypes to specific kinase inhibitors, irrespective of the FLT3-ITD status. Differential drug responses of the primitive and committed subtype are validated in an independent AML cohort. Our results highlight heterogeneity among NPM1 mutated AML patient samples based on stemness and suggest that the addition of kinase inhibitors to the treatment of cases with the primitive signature, lacking FLT3-ITD, could have therapeutic benefit.

Haematopoietic stem and progenitor cells (HSPCs) are defined as unspecialized cells that give rise to more differentiated cells. In a similar way, leukaemic stem and progenitor cells (LSPCs) are defined as unspecialized leukaemic cells, which can give rise to more differentiated cells. Leukaemic cells carry leukaemic mutations/variants and have clear differentiation abnormalities. Pre‐leukaemic HSPCs (PreL‐HSPCs) carry pre‐leukaemic mutations/variants (pLMs) and are capable of producing mature functional cells, which will carry the same variants. Under the roof of LSPCs, one can find a broad range of cell types genetic and disease phenotypes. Present‐day knowledge suggests that this phenotypic heterogeneity is the result of interactions between the cell of origin, the genetic background and the microenvironment background. The combination of these attributes will define the LSPC phenotype, frequency, differentiation capacity and evolutionary trajectory. Importantly, as LSPCs are leukaemia‐initiating cells that sustain clinical remission and are the source of relapse, an improved understanding of LSPCs phenotype would offer better clinical opportunities for the treatment and hopefully prevention of human leukaemia. The current review will focus on LSPCs attributes in the context of human haematologic malignancies.

Novel targeted therapies demonstrate improved survival in specific subgroups (defined by genetic variants) of acute myeloid leukemia (AML) patients, validating the paradigm of molecularly targeted therapy. However, identifying correlations between AML molecular attributes and effective therapies is challenging. Recent advances in high-throughput in vitro drug sensitivity screening applied to primary AML blasts were used to uncover such correlations; however, these methods cannot predict the response of leukemic stem cells (LSCs). Our study aimed to predict in vitro response to targeted therapies, based on molecular markers, with subsequent validation in LSCs. We performed ex vivo sensitivity screening to 46 drugs on 29 primary AML samples at diagnosis or relapse. Using unsupervised hierarchical clustering analysis we identified group with sensitivity to several tyrosine kinase inhibitors (TKIs), including the multi-TKI, dasatinib, and searched for correlations between dasatinib response, exome sequencing and gene expression from our dataset and from the Beat AML dataset. Unsupervised hierarchical clustering analysis of gene expression resulted in clustering of dasatinib responders and non-responders. In vitro response to dasatinib could be predicted based on gene expression (AUC=0.78). Furthermore, mutations in FLT3/ITD and PTPN11 were enriched in the dasatinib sensitive samples as opposed to mutations in TP53 which were enriched in resistant samples. Based on these results, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. Our results demonstrate that in a subgroup of FLT3/ITD AML (4 out of 9) dasatinib significantly inhibits LSC engraftment. In summary we show that dasatinib has an anti-leukemic effect both on bulk blasts and, more importantly, LSCs from a subset of AML patients that can be identified based on mutational and expression profiles. Our data provide a rational basis for clinical trials of dasatinib in a molecularly selected subset of AML patients.

Acute myeloid leukemia (AML) is one of the extreme outcomes of age-related clonal hematopoiesis (ARCH)1. With aging, mutations accumulate in hematopoietic stem and progenitor cells (HSPCs)2,3. Based on the estimated number of HSPCs (~50,000) in the human body and the number of somatic mutations in adult single cells (~1000)4, it is predicted that every ~100 nucleotides, a somatic mutation will occur at a low variant allele frequency (VAF).

Sensitive mutation detection by next-generation sequencing is critical for early cancer detection, monitoring minimal/measurable residual disease (MRD), and guiding precision oncology. Nevertheless, because of artifacts introduced during library preparation and sequencing, the detection of low-frequency variants at high specificity is problematic. Here, we present Espresso, an error suppression method that considers local sequence features to accurately detect single-nucleotide variants (SNVs). Compared to other advanced error suppression techniques, Espresso consistently demonstrated lower numbers of false-positive mutation calls and greater sensitivity. We demonstrated Espresso’s superior performance in detecting MRD in the peripheral blood of patients with acute myeloid leukemia (AML) throughout their treatment course. Furthermore, we showed that accurate mutation calling in a small number of informative genomic loci might provide a cost-efficient strategy for pragmatic risk prediction of AML development in healthy individuals. More broadly, we aim for Espresso to aid with accurate mutation detection in many other research and clinical settings.

Majewski Osteodysplastic Primordial Dwarfism type II (MOPDII) is a form of dwarfism associated with severe microcephaly, characteristic skeletal findings, distinct dysmorphic features and increased risk for cerebral infarctions. The condition is caused by bi-allelic loss-of-function variants in the gene PCNT. Here we describe the identification of a novel founder pathogenic variant c.3465-1G > A observed in carriers from multiple Druze villages in Northern Israel. RNA studies show that the variant results in activation of a cryptic splice site causing a coding frameshift. The study was triggered by the diagnosis of a single child with MOPDII and emphasizes the advantages of applying next generation sequencing technologies in community genetics and the importance of establishing population-specific sequencing databases.

Clonal hematopoiesis (CH) is a fundamental process in the aging of the blood system; however, it remains enigmatic why some individuals of the same age carry mutations, whereas others do not or carry different mutations with different variant allele frequencies (VAF). In this issue of Blood, Fabre et al(1) and Hansen et al(2) address the differential roles of nature (genotype/heritability) vs nurture (environment) in the evolution of age-related clonal hematopoiesis (ARCH).(3) In these studies, twin concordance analysis was used to determine the germline contribution to ARCH.(4) In both studies, no hereditary component of ARCH could be detected. Therefore, it is most likely that the variability of ARCH in the population can be explained by differential environmental exposures. Although such a conclusion is reasonable, a few specific aspects of the nature vs nurture dilemma in CH need to be considered.

CKI alpha wablation induces p53 activation, and CKI alpha degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKI alpha inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKI alpha-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven on-cogenes. We show that blocking CKI alpha together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2(-/-); Flt3(ITD) AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

The incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without any detectable early symptoms and patients usually present with the acute complications of bone marrow failure1. The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in preleukaemic haematopoietic stem and progenitor cells (HSPCs) that undergo clonal expansion2,3. However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH)4-8. Here we use deep sequencing to analyse genes that are recurrently mutated in AML to distinguish between individuals who have a high risk of developing AML and those with benign ARCH. We analysed peripheral blood cells from 95 individuals that were obtained on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls and had more mutations per sample, higher variant allele frequencies, indicating greater clonal expansion, and showed enrichment of mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AML cases and 262 controls. Because AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide proof-of-concept that it is possible to discriminate ARCH from pre-AML many years before malignant transformation. This could in future enable earlier detection and monitoring, and may help to inform intervention.

Age-related alterations in the human blood system occur in B cells, T cells, cells of the innate system, as well as hematopoietic stem and progenitor cells (HSPCs). Interestingly, age-related, reduced genetic diversity can be identified at the stem cell level and also independently in B cells and T cells. This reduced diversity is most probably related to somatic mutations or to changes in the microenvironmental niche. Either process can select for specific clones or cause repeated evolutionary bottlenecks. This review discusses the age-related clonal expansions in the human HSPC pool, which was termed in the past age-related clonal hematopoiesis (ARCH). ARCH is defined as the gradual, clonal expansion of HSPCs carrying specific, disruptive, and recurrent genetic variants, in individuals without clear diagnosis of hemato-logical malignancies. ARCH is associated not just with chronological aging but also with several other, age-related pathological conditions, including inflammation, vascular diseases, cancer mortality, and high risk for hematological malignancies. Although it remains unclear whether ARCH is a marker of aging or plays an active role in these various pathophysiologies, it is suggested here that treating or even preventing ARCH may prove to be beneficial for human health. This review also describes a decision tree for the diagnosis and follow-up for ARCH in a research setting.

In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.

Acute myeloid leukemia (AML) is characterized by accumulation of myeloid cells in the bone marrow because of impaired differentiation and proliferation, resulting in hematopoietic insufficiency. NPM1 is one of the most commonly mutated genes in AML, present in 20-30% of cases. Mutations in NPM1 represent a distinct entity in the World Health Organization (WHO) classification and commonly indicate a better risk prognosis. In this review, we discuss the many functions of NPM1, the consequence of mutations in NPM1 and possible mechanisms through which mutations lead to leukemogenesis. We also discuss clinical consequences of mutations, associated gene expression patterns and the role of NPM1 mutations in informing prognosis and therapeutic decisions and predicting relapse in AML.

The molecular determinants governing escape of Acute Myeloid Leukemia (AML) cells from DNA damaging therapy remain poorly defined and account for therapy failures. To isolate genes responsible for leukemia cells regeneration following multiple challenges with irradiation we performed a genome-wide shRNA screen. Some of the isolated hits are known players in the DNA damage response (e.g. p53, CHK2), whereas other, e.g. SMYD2 lysine methyltransferase (KMT), remains uncharacterized in the AML context. Here we report that SMYD2 knockdown confers relative resistance to human AML cells against multiple classes of DNA damaging agents. Induction of the transient quiescence state upon SMYD2 downregulation correlated with the resistance. We revealed that diminished SMYD2 expression resulted in the upregulation of the related methyltransferase SET7/9, suggesting compensatory relationships. Indeed, pharmacological targeting of SET7/9 with (R)-PFI2 inhibitor preferentially inhibited the growth of cells expressing low levels of SMYD2. Finally, decreased expression of SMYD2 in AML patients correlated with the reduced sensitivity to therapy and lower probability to achieve complete remission. We propose that the interplay between SMYD2 and SET7/9 levels shifts leukemia cells from growth to quiescence state that is associated with the higher resistance to DNA damaging agents and rationalize SET7/9 pharmacological targeting in AML.

Pancreatic cancer, a highly aggressive tumour type with uniformly poor prognosis, exemplifies the classically held view of stepwise cancer development. The current model of tumorigenesis, based on analyses of precursor lesions, termed pancreatic intraepithelial neoplasm (PanINs) lesions, makes two predictions: first, that pancreatic cancer develops through a particular sequence of genetic alterations (KRAS, followed by CDKN2A, then TP53 and SMAD4); and second, that the evolutionary trajectory of pancreatic cancer progression is gradual because each alteration is acquired independently. A shortcoming of this model is that clonally expanded precursor lesions do not always belong to the tumour lineage, indicating that the evolutionary trajectory of the tumour lineage and precursor lesions can be divergent. This prevailing model of tumorigenesis has contributed to the clinical notion that pancreatic cancer evolves slowly and presents at a late stage. However, the propensity for this disease to rapidly metastasize and the inability to improve patient outcomes, despite efforts aimed at early detection, suggest that pancreatic cancer progression is not gradual. Here, using newly developed informatics tools, we tracked changes in DNA copy number and their associated rearrangements in tumour-enriched genomes and found that pancreatic cancer tumorigenesis is neither gradual nor follows the accepted mutation order. Two-thirds of tumours harbour complex rearrangement patterns associated with mitotic errors, consistent with punctuated equilibrium as the principal evolutionary trajectory. In a subset of cases, the consequence of such errors is the simultaneous, rather than sequential, knockout of canonical preneoplastic genetic drivers that are likely to set-off invasive cancer growth. These findings challenge the current progression model of pancreatic cancer and provide insights into the mutational processes that give rise to these aggressive tumours.

In this issue of Blood, Mossner et al took an important step in delineating the evolution of myelodysplastic syndrome (MDS). They were able to estimate mutation acquisition order and to demonstrate that, in the majority of MDS cases, earlier preexisting minor clones survived therapy.

Chronological human aging is associated with a number of changes in the hematopoietic system, occurring at many levels from stem to mature cells, and the marrow microenvironment as well. This review will focus mainly on the aging of hematopoietic stem and progenitor cells (HSPCs), and on the associated increases in the incidence of hematological malignancies. HSPCs manifest reduced function and acquire molecular changes with chronological aging. Furthermore, while for many years it has been known that the human hematopoietic system becomes increasingly clonal with chronological aging (clonal hematopoiesis), only in the last few years has it become clear that clonal hematopoiesis may result from the accumulation of preleukemic mutations in HSPCs. Such mutations confer a selective advantage that leads to clonal hematopoiesis, and that may occasionally result in the development of leukemia, and define the existence of both preleukemic stem cells, and of `preleukemia' as a clinical entity. While it is well appreciated that clonal hematopoiesis is very common in the elderly, several questions remain unanswered: why and how does clonal hematopoiesis develop? How is clonal hematopoiesis related to the age-related changes observed in the hematopoietic system? And why do only some individuals with clonal hematopoiesis develop leukemia?.

Dismal outcomes of acute myeloid leukemia (AML), especially in the elderly, are mainly associated with leukemia relapse and primary no response to initial therapy. This review will focus on AML relapse, and how a better understanding of the evolutionary stages that lead to relapse might help us improve disease outcome. The fact that the relapse rate for some AMLs is so high indicates that we do not truly understand the biology of relapse or possibly that we are not implementing our current understanding into, clinical practice. Therefore, this review will also aim to explore some of the current understanding of AML relapse biology in order to identify the gaps in our knowledge and translation. Accumulating evidence suggests that the root of relapse evolves even before the time of diagnosis, meaning that the complex clonal structure of AML is created before patients present to the clinic. Some of the clones that exist at diagnosis can survive chemotherapy and give rise to relapse. Accordingly, in order to better understand the mechanisms of relapse, we must consider both early and late steps in AML evolution.

Somatic/clonal evolution is the process of sequential acquisition of vertically transmittable genetic/epigenetic elements in multicellular organisms. Cancer is the result of somatic evolution. Understanding the processes that shape the evolution of individual tumors might help us to treat cancer more efficiently. The initiating genetic/epigenetic events occur in functional cells and provide the cell of origin a selective advantage under a changing environment. The initiating genetic events tend to be enriched in specific tissues (and are sometimes specific for those tissues), as different tissues undergo different changes in the environment that will activate selective forces on different cells of origin. For the initial clonal expansion to occur premalignant clones need to have a relative fitness advantage over their competitors. It is estimated that the premalignant phase can take several years. Once the premalignant clonal expansion is established, the premalignant cells will contribute to the changing environment and will start competing among themselves. In late stages of cancer evolution the environmental changes might be similar across different tissues, including a lack of physical space, a shortage of energy, and activation of the immune system, and more and more of the hallmarks of cancer will evolve. In this review we will explore the possible clinical relevance of the heterogeneity that evolves during this long somatic evolution. Above all, it should be stressed that the earlier the clonal expansion is recognized, the less diverse and less fit for survival the cells in the population are.

PURPOSE OF REVIEW: In the present review, we will define the preleukemic state. We aim at increasing awareness and research in the field of preleukemia that will nurture targeted therapy for the earlier steps of leukemia evolution. RECENT FINDINGS: Emerging evidence supports the role of hematopoietic stem/progenitor cells carrying recurrent leukemia-related mutations as the cell of origin of both myeloid and lymphoid malignancies. The preleukemic stem cells can maintain at least to some extent their functionality; however, they have increased fitness endowed by the preleukemic mutations that lead to clonal expansion. SUMMARY: The latent preleukemic period before overt leukemia presents can take years, and the majority of carriers will never develop leukemia in their lifetime. The preleukemic state is not rare, with greater than 1% of individuals having acquired one or more of the recognized preleukemic lesions. The high frequency of such abnormalities in the population may be the cost of growing old; however, another view could be that in order to survive to old age, the hematopoietic system must adapt to create robust hematopoietic stem/progenitor cells with an increased fitness and clonal expansion. Hence, leukemia does not necessarily start as a disease, but rather as a need, with the normally functioning preleukemic hematopoietic stem cells trying to maintain health for years but in time succumbing to their own acquired virtues.

FMS-like tyrosine kinase 3 receptor internal tandem duplication (FLT3-ITD) commonly occurs in acute myeloid leukemia and is considered rare in acute lymphocytic leukemia. Acute leukemia has poor prognosis, mainly due to relapse. Standard FLT3-ITD diagnostic techniques are based on genomic polymerase chain reaction and have recently incorporated GeneScan (Applied Biosystems, Foster City, CA) to identify variations of the FLT3 gene. As this is an average-based assay utilized in a heterogeneous leukemic cell population, we hypothesized that cells of acute leukemia, considered FLT3-ITD-negative by standard methods, could possess a fraction of FLT3-ITD-positive cells. The present study employed single cell mutation analysis to evaluate the FLT3-ITD status in newly diagnosed acute myeloid leukemia (n = 5) and acute lymphocytic leukemia (n = 3) patients. A total of 541 single leukemic cells and 36 mononuclear cells from healthy volunteers were analyzed. Seven patients, considered FLT3-ITD-negative according to bulk DNA analysis, appeared to possess a small fraction of FLT3-ITD-positive cells based on single cell analysis. Moreover, this approach revealed the heterogeneity of the tumor as evident by different FLT3-ITD mutations present in the same patient. The presence of a minor clone carrying FLT3-ITD in almost all patients tested provides evidence that this lesion is a common late event in leukemogenesis. Additionally, 3 relapsed patients demonstrated loss of heterozygosity of the normal allele, affecting 25 %-100% of the cells found to be FLT3-ITD-positive. Though further clinical testing is warranted, these findings may have implications on the prognostic significance of FLT3-ITD and the use of targeted therapy.

Organism cells proliferate and die to build, maintain, renew and repair it. The cellular history of an organism up to any point in time can be captured by a cell lineage tree in which vertices represent all organism cells, past and present, and directed edges represent progeny relations among them. The root represents the fertilized egg, and the leaves represent extant and dead cells. Somatic mutations accumulated during cell division endow each organism cell with a genomic signature that is unique with a very high probability. Distances between such genomic signatures can be used to reconstruct an organism's cell lineage tree. Cell populations possess unique features that are absent or rare in organism populations (e. g., the presence of stem cells and a small number of generations since the zygote) and do not undergo sexual reproduction, hence the reconstruction of cell lineage trees calls for careful examination and adaptation of the standard tools of population genetics. Our lab developed a method for reconstructing cell lineage trees by examining only mutations in highly variable microsatellite loci (MS, also called short tandem repeats, STR). In this study we use experimental data on somatic mutations in MS of individual cells in human and mice in order to validate and quantify the utility of known lineage tree reconstruction algorithms in this context. We employed extensive measurements of somatic mutations in individual cells which were isolated from healthy and diseased tissues of mice and humans. The validation was done by analyzing the ability to infer known and clear biological scenarios. In general, we found that if the biological scenario is simple, almost all algorithms tested can infer it. Another somewhat surprising conclusion is that the best algorithm among those tested is Neighbor Joining where the distance measure used is normalized absolute distance. We include our full dataset in Tables S1, S2, S3, S4, S5 to enable further analysis of this data by others.

Human cancers display substantial intratumoral genetic heterogeneity, which facilitates tumor survival under changing microenvironmental conditions. Tumor substructure and its effect on disease progression and relapse are incompletely understood. In the present study, a high-throughput method that uses neutral somatic mutations accumulated in individual cells to reconstruct cell lineage trees was applied to hundreds of cells of human acute leukemia harvested from multiple patients at diagnosis and at relapse. The reconstructed cell lineage trees of patients with acute myeloid leukemia showed that leukemia cells at relapse were shallow (divide rarely) compared with cells at diagnosis and were closely related to their stem cell subpopulation, implying that in these instances relapse might have originated from rarely dividing stem cells. In contrast, among patients with acute lymphoid leukemia, no differences in cell depth were observed between diagnosis and relapse. In one case of chronic myeloid leukemia, at blast crisis, most of the cells at relapse were mismatch-repair deficient. In almost all leukemia cases, > 1 lineage was observed at relapse, indicating that diverse mechanisms can promote relapse in the same patient. In conclusion, diverse relapse mechanisms can be observed by systematic reconstruction of cell lineage trees of patients with leukemia.

Stem cell dynamics in vivo are often being studied by lineage tracing methods. Our laboratory has previously developed a retrospective method for reconstructing cell lineage trees from somatic mutations accumulated in microsatellites. This method was applied here to explore different aspects of stem cell dynamics in the mouse colon without the use of stem cell markers. We first demonstrated the reliability of our method for the study of stem cells by confirming previously established facts, and then we addressed open questions. Our findings confirmed that colon crypts are monoclonal and that, throughout adulthood, the process of monoclonal conversion plays a major role in the maintenance of crypts. The absence of immortal strand mechanism in crypts stem cells was validated by the age-dependent accumulation of microsatellite mutations. In addition, we confirmed the positive correlation between physical and lineage proximity of crypts, by showing that the colon is separated into small domains that share a common ancestor. We gained new data demonstrating that colon epithelium is clustered separately from hematopoietic and other cell types, indicating that the colon is constituted of few progenitors and ruling out significant renewal of colonic epithelium from hematopoietic cells during adulthood. Overall, our study demonstrates the reliability of cell lineage reconstruction for the study of stem cell dynamics, and it further addresses open questions in colon stem cells. In addition, this method can be applied to study stem cell dynamics in other systems.

Background: Cellular senescence plays important roles in the aging process of complex organisms, in tumor suppression and in response to stress. Several markers can be used to identify senescent cells, of which the most widely used is the senescence-associated β-galactosidase (SABG) activity. The main advantage of SABG activity over other markers is the simplicity of the detection assay and the capacity to identify in situ a senescent cell in a heterogeneous cell population. Several approaches have been introduced to render the SABG assay quantitative. However none of these approaches to date has proven particularly amenable to quantitative analysis of SABG activity in situ. Furthermore the role of cellular senescence (CS) in vivo remains unclear mainly due to the ambiguity of current cellular markers in identifying CS of individual cells in tissues.Results: In the current study we applied a digital image analysis technique to the staining generated using the original SABG assay, and demonstrate that this analysis is highly reproducible and sensitive to subtle differences in staining intensities resulting from diverse cellular senescence pathways in culture. We have further validated our method on mouse kidney samples with and without diabetes mellitus, and show that a more accurate quantitative SABG activity with a wider range of values can be achieved at a pH lower than that used in the conventional SABG assay.Conclusions: We conclude that quantitative in situ SABG assay, is feasible and reproducible and that the pH at which the reaction is performed should be tailored and chosen, depending on the research question and experimental system of interest.

Many cross-sectional studies have tried to assess the in vivo effect of oxidative stress on organismal aging in general and on telomere length dynamics specifically. Here we followed telomere length dynamics over a 12-month interval, in divers exposed to intense hyperbaric oxygen in comparison with an age-matched control group. Both groups were exposed to extreme physical activity, as well. Among the divers following the oxidative stress, significant telomere elongation was observed in granulocytes and naïve T cells, but not in memory T cells and B cells. Telomere length in granulocytes was mildly elongated in the control group as well, a finding that may relate to the extreme physical activity to which they were exposed. While telomere elongation in naïve T cells may be attributed to telomerase activation, we suggest that in granulocytes the elongation results from undifferentiated hematopoietic cells carrying longer telomeres that repopulate the peripheral hematopoietic compartment. This event might be accompanied by enhanced cell division within the repopulating pool. Since the aging of mammalian tissues can be attributed in part to the reduction in the replicative potential of self renewing cells, enhanced cell turnover under conditions of hyperbaric oxidative stress might be directly relevant to tissue and organismal aging.

Telomeres are DNA repeats protecting chromosomal ends which shorten with each cell division, eventually leading to cessation of cell growth. We present a population mixture model that predicts an exponential decrease in telomere length with time. We analytically solve the dynamics of the telomere length distribution. The model provides an excellent fit to available telomere data and accounts for the previously unexplained observation of telomere elongation following stress and bone marrow transplantation, thereby providing insight into the nature of the telomere clock.

Background: Phylogenetic mitochondrial DNA haplogroups are highly partitioned across global geographic regions. A unique exception is the X haplogroup, which has a widespread global distribution without major regions of distinct localization. Principal Findings: We have examined mitochondrial DNA sequence variation together with Y-chromosome-based haplogroup structure among the Druze, a religious minority with a unique socio-demographic history residing in the Near East. We observed a striking overall pattern of heterogeneous parental origins, consistent with Druze oral tradition, together with both a high frequency and a high diversity of the mitochondrial DNA (mtDNA) X haplogroup within a confined regional subpopulation. Furthermore demographic modeling indicated low migration rates with nearby populations. Conclusions: These findings were enabled through the use of a paternal kindred based sampling approach, and suggest that the Galilee Druze represent a population isolate, and that the combination of a high frequency and diversity of the mtDNA X haplogroup signifies a phylogenetic refugium, providing a sample snapshot of the genetic landscape of the Near East prior to the modern age.