Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurement allowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

Evaluating the role of Day 14 bone marrow biopsy and European LeukemiaNet risk classification in predicting overall and relapse-free survival in acute myeloid leukemia.

Multivariable analysis for overall survival and relapse-free survival demonstrating lack of significant for D14 BM status.

JAK inhibitors in the treatment of myelofibrosis.

Myelofibrosis (MF) is a myeloproliferative neoplasm driven by constitutive activation of the JAK/STAT pathway, resulting in clonal hematopoiesis, fibrotic replacement of the bone marrow, extramedullary hematopoiesis, splenomegaly, and debilitating constitutional symptoms. The advent of JAK inhibitors has changed the landscape of treatment options for patients with MF, providing relatively tolerable drug options that control symptoms, reduce splenomegaly, and improve quality of life, but often at the expense of worsening cytopenias. JAK inhibitors do not appear to halt the progression of disease or prevent leukemic transformation, and their effect on survival is debated. Here, we review both the US Food and Drug Administration-approved JAK inhibitors and those in late-phase clinical trials, with a focus on clinical activity and unique adverse effects. We also provide a schema for choosing among these options for patients with MF.

Alternatives to intensive treatment in patients with AML.

A significant proportion of patients with acute myeloid leukemia (AML) are unable to tolerate standard induction chemotherapy regimens. This is particularly true for patients who are of advanced age, have a poor performance status, and/or have significant medical comorbidities. Recent advances in understanding the genetic and molecular properties of AML have led to a spate of new treatment options for patients considered ineligible for standard chemotherapy. Here, we discuss these new treatment options, provide an overview of the completed and ongoing trials of the new agents, and highlight promising future directions in the treatment of AML in patients ineligible for intensive induction chemotherapy.

A Novel t(1;9)(p36;p24.1) JAK2 Translocation and Review of the Literature.

The JAK2V617F point mutation has been implicated in the pathogenesis of the vast majority of myeloproliferative neoplasms (MPNs), but translocations involving JAK2 have increasingly been identified in patients with JAK2V617F-negativeMPNs. Here, we present a case of a patient diagnosed with JAK2V617F-negativepolycythemia vera (PV) that transformed to the MPN-blast phase. Cytogenetic and FISH analysis revealed a novel translocation of t(1;9)(p36;p24.1), causing a PEX14-JAK2 gene fusion product. The t(1;9)(p36;p24.1) represents a new addition to the list of known translocations involving JAK2that have been identified in hematologic malignancies. Although the prognostic and treatment implications of JAK2 translocations in MPNs have not been elucidated, positive outcomes have been described in case reports describing the use of JAK inhibitors in these patients. Further research into the role of JAK2 translocations in the pathogenesis and outcomes of hematologic malignancies is warranted.

Management of advanced phase myeloproliferative neoplasms.

The BCR-ABL1-negative myeloproliferative neoplasms (MPNs), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, can evolve into a form of secondary acute myeloid leukemia termed MPN in blast phase (MPN-BP). MPN in accelerated phase (MPN-AP), which is defined by 10% to 19% myeloid blasts in the peripheral blood or bone marrow, is a precursor to MPN-BP. Alternative definitions of MPN-AP exist based on studies identifying clinical variables that portend a poor prognosis and high risk for progression to MPN-BP. Allogeneic hematopoietic stem cell transplant remains the only curative therapeutic option; however, advanced age and high comorbidity index preclude the majority of patients from receiving this treatment modality. This article reviews management considerations for the advanced-phase MPNs (MPN-AP and MPN-BP), with a special focus on MPN-AP, and highlights novel experimental therapies.

Distinguishing autoimmune myelofibrosis from primary myelofibrosis.

Bone marrow fibrosis (BMF) is a histologic finding in a wide range of diseases, including malignancies, endocrine disorders, autoimmune diseases, and infections. Autoimmune myelofibrosis (AIMF) is an uncommon etiology of BMF; it can be secondary to a defined autoimmune disease, or it can be primary in the absence of a clinically diagnosed autoimmune disease but the presence of serologic evidence of autoantibodies. Distinguishing between primary myelofibrosis (PMF) and non-neoplastic AIMF is of the utmost importance because the prognosis and therapeutic options are different. This distinction, however, can be complicated by overlapping findings in the 2 disease entities. Here, using the case of a patient with BMF in the setting of idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia, we present a systematic approach to distinguishing between PMF and AIMF.

Role of the hypothalamus in mediating protective effects of dietary restriction during aging.

Dietary restriction (DR) can extend lifespan and reduce disease burden across a wide range of animals and yeast but the mechanisms mediating these remarkably protective effects remain to be elucidated despite extensive efforts. Although it has generally been assumed that protective effects of DR are cell-autonomous, there is considerable evidence that many whole-body responses to nutritional state, including DR, are regulated by nutrient-sensing neurons. In this review, we explore the hypothesis that nutrient sensing neurons in the ventromedial hypothalamus hierarchically regulate the protective responses of dietary restriction. We describe multiple peripheral responses that are hierarchically regulated by the hypothalamus and we present evidence for non-cell autonomous signaling of dietary restriction gathered from a diverse range of models including invertebrates, mammalian cell culture, and rodent studies.

Myeloid neoplasms in inflammatory bowel disease: A case series and review of the literature.

Patients with inflammatory bowel disease (IBD) are exposed to chronic systemic inflammation and are at risk for secondary malignancies. Here we review the literature on the risk of myeloid neoplasms (MN) in IBD and present the disease profiles of patients at a single institution with IBD who later developed MN, comparing them to those in the literature. No IBD characteristic was found to associate with MN disease severity, including the previously-identified association between MNs and thiopurine exposure. Of the somatic mutations identified in out cohort's MN, mutations in TET2 were most prevalent, followed by FLT3-ITD, BCR-ABL, and NPM1 mutations.

Discontinuation Syndrome With JAK2 Selective Agents: Case Presentation and Mechanistic Insights.

We report the first case of pacritinib-withdrawal syndrome with in vitro elucidation of underlying mechanisms.

Impact of gender representativeness in online symptom survey and clinical trial participation among patients with myeloproliferative neoplasms.

Patients with myeloproliferative neoplasms (MPNs) face chronic symptom burden. Online symptom assessment studies allow for recruitment of large numbers of motivated patients, but patient self-selection can lead to sampling bias. This study evaluated how gender representativeness in MPN symptom surveys and trials impacted symptom score mean estimates, using data from 4825 survey respondents and 291 trial participants with MPNs. The survey data showed that men participated at a rate roughly 50% less than what would be expected based on prevalence, and women reported higher scores than men on average for six of 10 symptoms. Together, this led to potential over estimation in six of 10 symptom score means (ranging from 5.8% to 15.3% overestimated). The trial data showed less gender-based sampling bias compared to the survey data. Studies utilizing online symptom surveys should implement study design features to recruit more men, assess for gender participation imbalances, and provide weighted estimates where appropriate.

The reparative immunologic consequences of stem cell transplantation as a cellular therapy for refractory Crohn's disease.

Background: Treatment strategies for Crohn's disease (CD) suppress diverse inflammatory pathways but many patients remain refractory to treatment. Autologous hematopoietic stem cell transplantation (SCT) has emerged as a therapy for medically refractory CD. SCT was developed to rescue cancer patients from myelosuppressive chemotherapy but its use for CD and other immune diseases necessitates reimagining SCT as a cellular therapy that restores appropriately responsive immune cell populations from hematopoietic progenitors in the stem cell autograft (i.e. immune "reset"). Here we present a paradigm to understand SCT as a cellular therapy for immune diseases and reveal how SCT re-establishes cellular immunity utilizing high-dimensional cellular phenotyping and functional studies of the stem cell grafts. Methods: Immunophenotyping using CyTOF, single cell RNA sequencing (scRNA-seq) and T cell receptor (TCR) sequencing was performed on peripheral blood and intestinal tissue samples from refractory CD patients who underwent SCT. The stem cell graft from these patients was analyzed using flow cytometry and functionally interrogated using a murine model for engraftment. Results: Our study revealed a remodeling of intestinal macrophages capable of supporting mucosal healing that was independently validated using multimodal studies of immune reconstitution events including CyTOF and scRNA-seq. Functional interrogation of hematopoietic stem cells (HSCs) using a xenograft model demonstrated that HSCs shape the timing of immune reconstitution, the selected reconstitution of specific cell lineages and potentially the clinical efficacy of SCT. Conclusions: These studies indicate that SCT serves as a myeloid-directed cellular therapy re-establishing homeostatic intestinal macrophages that support intestinal healing and suggest refractory CD evolves from impairment of restorative functions in myeloid cells. Furthermore, we report heterogeneity among HSCs from CD patients which may drive SCT outcomes and suggests an unrecognized impact of CD pathophysiology on HSC in the marrow niche.

Clonal hematopoiesis and inflammation: A review of mechanisms and clinical implications.

Clonal hematopoiesis (CH) is defined by the presence of somatic mutations in hematopoietic stem and progenitor cells (HSPC). CH is associated primarily with advancing age and confers an elevated risk of progression to overt hematologic malignancy and cardiovascular disease. Increasingly, CH is associated with a wide range of diseases driven by, and sequelae of, inflammation. Accordingly, there is great interest in better understanding the pathophysiologic and clinical relationship between CH, aging, and disease. Both observational and experimental findings support the concept that CH is a potential common denominator in the inflammatory outcomes of aging. However, there is also evidence that local and systemic inflammatory states promote the growth and select for CH clones. In this review, we aim to provide an up-to-date summary of the nature of the relationship between inflammation and CH, which is central to unlocking potential therapeutic opportunities to prevent progression to myeloid malignancy.

An MDM2 degrader for treatment of acute leukemias.

In acute myeloid leukemia (AML), p53 tumor suppressor activity can be reduced due to enhanced expression of MDM2 which promotes the degradation of p53. In TP53 wild-type malignancies, therapy with small molecule antagonists of MDM2 results in antileukemic activity. Current treatment strategies, however, have been limited by poor tolerability and incomplete clinical activity. We have developed a proteolysis-targeting chimera (PROTAC) MS3227 that targets MDM2 by recruiting the E3 ligase Von Hippel-Lindau, resulting in proteasome-dependent degradation of MDM2. In WT TP53 leukemia cell lines, MS3227 led to activation of p53 targets p21, PUMA, and MDM2 and resulted in cell-cycle arrest, apoptosis, and decreased viability. The catalytic PROTAC MS3227 led to more potent activation when compared to a stoichiometric inhibitor, in part by dampening the negative feedback mechanism in the p53 - MDM2 circuit. The effectiveness of MS3227 was also observed in primary patient specimens with selectivity towards leukemic blasts. The addition of MS3227 enhanced the activity of other anti-leukemic agents including azacytidine, cytarabine, and venetoclax. In particular, MS3227 treatment was shown to downregulate MCL-1, a known mediator of resistance to venetoclax. A PROTAC-based approach may provide a means of improving MDM2 inhibition to gain greater therapeutic potential in AML.

Patient-Specific Pharmacokinetics and Dasatinib Nephrotoxicity.

BACKGROUND: Dasatinib has been associated with nephrotoxicity. We sought to examine the incidence of proteinuria on dasatinib and determine potential risk factors that may increase dasatinib-associated glomerular injury. METHODS: We examined glomerular injury through urine albumin-creatinine ratio (UACR) in 82 patients with chronic myelogenous leukemia who were on tyrosine-kinase inhibitor therapy for at least 90 days. t tests were used to compare mean differences in UACR, while regression analysis was used to assess the effects of drug parameters on proteinuria development while on dasatinib. We assayed plasma dasatinib pharmacokinetics using tandem mass spectroscopy and further described a case study of a patient who experienced nephrotic-range proteinuria while on dasatinib. RESULTS: Participants treated with dasatinib ( n =32) had significantly higher UACR levels (median 28.0 mg/g; interquartile range, 11.5-119.5) than participants treated with other tyrosine-kinase inhibitors ( n =50; median 15.0 mg/g; interquartile range, 8.0-35.0; P < 0.001). In total, 10% of dasatinib users exhibited severely increased albuminuria (UACR >300 mg/g) versus zero in other tyrosine-kinase inhibitors. Average steady-state concentrations of dasatinib were positively correlated with UACR ( ρ =0.54, P = 0.03) and duration of treatment ( P = 0.003). There were no associations with elevated BP or other confounding factors. In the case study, kidney biopsy revealed global glomerular damage with diffuse foot process effacement that recovered on termination of dasatinib treatment. CONCLUSIONS: Exposure to dasatinib was associated with a significant chance of developing proteinuria compared with other similar tyrosine-kinase inhibitors. Dasatinib plasma concentration significantly correlated with higher risk of developing proteinuria while receiving dasatinib. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/CJASN/2023_09_08_CJN0000000000000219.mp3.

Dasatinib nephrotoxicity correlates with patient-specific pharmacokinetics.

Introduction: Dasatinib has been associated with nephrotoxicity. We sought to examine the incidence of proteinuria on dasatinib and determine potential risk factors that may increase dasatinib-associated glomerular injury. Methods: We examine glomerular injury via urine albumin-to-creatinine ratio (UACR) in 101 chronic myelogenous leukemia patients who were on tyrosine-kinase inhibitor (TKI) therapy for at least 90 days. We assay plasma dasatinib pharmacokinetics using tandem mass spectroscopy, and further describe a case study of a patient who experienced nephrotic-range proteinuria while on dasatinib. Results: Patients treated with dasatinib (n= 32) had significantly higher UACR levels (median 28.0 mg/g, IQR 11.5 - 119.5) than patients treated with other TKIs (n=50; median 15.0 mg/g, IQR 8.0 - 35.0; p < 0.001). In total, 10% of dasatinib users exhibited severely increased albuminuria (UACR > 300 mg/g) versus zero in other TKIs. Average steady state concentrations of dasatinib were positively correlated with UACR (ρ = 0.54, p = 0.03) as well as duration of treatment ( p =0.003). There were no associations with elevated blood pressure or other confounding factors. In the case study, kidney biopsy revealed global glomerular damage with diffuse foot process effacement that recovered upon termination of dasatinib treatment. Conclusions: Exposure to dasatinib is associated a significant chance of developing proteinuria compared to other similar TKIs. Dasatinib plasma concentration significantly correlates with increased risk of developing proteinuria while receiving dasatinib. Screening for renal dysfunction and proteinuria is strongly advised for all dasatinib patients.