Aberrant activation of TCL1A promotes stem cell expansion in clonal haematopoiesis.

Mutations in a diverse set of driver genes increase the fitness of haematopoietic stem cells (HSCs), leading to clonal haematopoiesis1. These lesions are precursors for blood cancers2-6, but the basis of their fitness advantage remains largely unknown, partly owing to a paucity of large cohorts in which the clonal expansion rate has been assessed by longitudinal sampling. Here, to circumvent this limitation, we developed a method to infer the expansion rate from data from a single time point. We applied this method to 5,071 people with clonal haematopoiesis. A genome-wide association study revealed that a common inherited polymorphism in the TCL1A promoter was associated with a slower expansion rate in clonal haematopoiesis overall, but the effect varied by driver gene. Those carrying this protective allele exhibited markedly reduced growth rates or prevalence of clones with driver mutations in TET2, ASXL1, SF3B1 and SRSF2, but this effect was not seen in clones with driver mutations in DNMT3A. TCL1A was not expressed in normal or DNMT3A-mutated HSCs, but the introduction of mutations in TET2 or ASXL1 led to the expression of TCL1A protein and the expansion of HSCs in vitro. The protective allele restricted TCL1A expression and expansion of mutant HSCs, as did experimental knockdown of TCL1A expression. Forced expression of TCL1A promoted the expansion of human HSCs in vitro and mouse HSCs in vivo. Our results indicate that the fitness advantage of several commonly mutated driver genes in clonal haematopoiesis may be mediated by TCL1A activation.

Transition to a mesenchymal state in neuroblastoma confers resistance to anti-GD2 antibody via reduced expression of ST8SIA1.

Immunotherapy with anti-GD2 antibodies has advanced the treatment of children with high-risk neuroblastoma, but nearly half of patients relapse, and little is known about mechanisms of resistance to anti-GD2 therapy. Here, we show that reduced GD2 expression was significantly correlated with the mesenchymal cell state in neuroblastoma and that a forced adrenergic-to-mesenchymal transition (AMT) conferred downregulation of GD2 and resistance to anti-GD2 antibody. Mechanistically, low-GD2-expressing cell lines demonstrated significantly reduced expression of the ganglioside synthesis enzyme ST8SIA1 (GD3 synthase), resulting in a bottlenecking of GD2 synthesis. Pharmacologic inhibition of EZH2 resulted in epigenetic rewiring of mesenchymal neuroblastoma cells and re-expression of ST8SIA1, restoring surface expression of GD2 and sensitivity to anti-GD2 antibody. These data identify developmental lineage as a key determinant of sensitivity to anti-GD2 based immunotherapies and credential EZH2 inhibitors for clinical testing in combination with anti-GD2 antibody to enhance outcomes for children with neuroblastoma.

Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence.

T cell exhaustion limits antitumor immunity, but the molecular determinants of this process remain poorly understood. Using a chronic stimulation assay, we performed genome-wide CRISPR-Cas9 screens to systematically discover regulators of T cell exhaustion, which identified an enrichment of epigenetic factors. In vivo CRISPR screens in murine and human tumor models demonstrated that perturbation of the INO80 and BAF chromatin remodeling complexes improved T cell persistence in tumors. In vivo Perturb-seq revealed distinct transcriptional roles of each complex and that depletion of canonical BAF complex members, including Arid1a, resulted in the maintenance of an effector program and downregulation of exhaustion-related genes in tumor-infiltrating T cells. Finally, Arid1a depletion limited the acquisition of exhaustion-associated chromatin accessibility and led to improved antitumor immunity. In summary, we provide an atlas of the genetic regulators of T cell exhaustion and demonstrate that modulation of epigenetic state can improve T cell responses in cancer immunotherapy.

Differential usage of transcriptional repressor Zeb2 enhancers distinguishes adult and embryonic hematopoiesis.

The transcriptional repressor ZEB2 regulates development of many cell fates among somatic, neural, and hematopoietic lineages, but the basis for its requirement in these diverse lineages is unclear. Here, we identified a 400-basepair (bp) region located 165 kilobases (kb) upstream of the Zeb2 transcriptional start site (TSS) that binds the E proteins at several E-box motifs and was active in hematopoietic lineages. Germline deletion of this 400-bp region (Zeb2Δ-165mice) specifically prevented Zeb2 expression in hematopoietic stem cell (HSC)-derived lineages. Zeb2Δ-165 mice lacked development of plasmacytoid dendritic cells (pDCs), monocytes, and B cells. All macrophages in Zeb2Δ-165 mice were exclusively of embryonic origin. Using single-cell chromatin profiling, we identified a second Zeb2 enhancer located at +164-kb that was selectively active in embryonically derived lineages, but not HSC-derived ones. Thus, Zeb2 expression in adult, but not embryonic, hematopoiesis is selectively controlled by the -165-kb Zeb2 enhancer.

National Hospital Ambulatory Medical Care Survey: 2000 outpatient department summary.

OBJECTIVES: This report describes ambulatory care visits to hospital outpatient departments (OPDs) in the United States. Statistics are presented on selected hospital, clinic, patient, and visit characteristics. Highlights of trends in OPD utilization from 1997 through 2000 are also presented. METHODS: The data presented in this report were collected from the 2000 National Hospital Ambulatory Medical Care Survey (NHAMCS). NHAMCS is part of the ambulatory care component of the National Health Care Survey that measures health care utilization across various types of providers. NHAMCS is a national probability sample survey of visits to emergency and outpatient departments of non-Federal, short-stay, and general hospitals in the United States. Sample data are weighted to produce annual national estimates. Trends are based on NHAMCS data from 1997 through 2000. RESULTS: During 2000, an estimated 83.3 million visits were made to hospital OPDs in the United States, about 30.4 visits per 100 persons. Females had higher OPD visit rates than males (35.3 versus 25.2 visits per 100 persons). The OPD utilization rate for black persons was higher than for white persons (48.3 versus 28.0 visits per 100 persons). Of all visits made to hospital OPDs in 2000, private insurance (38.5 percent), Medicaid (22.1 percent), and Medicare (16.9 percent) were listed as the leading primary expected source of payment. Approximately 21 percent of OPD visits reported that patients belonged to an HMO. There were an estimated 9.5 million injury-related OPD visits in 2000. Since 1997, the percent of OPD visits that were for injuries increased by 24% (from 9.2 percent to 1.4 percent). Most of these visits were for unintentional injuries (57.6 percent), including those caused by falls (12.9 percent). Medications were prescribed at 64.0 percent of visits. On average, 1.6 medications were ordered at each OPD visit. In 2000, patients saw one or more physicians (i.e., staff physician, resident/intern, or other physician) at approximately 79 percent of visits. Most patients were given an appointment to return to the clinic (57.2 percent).