Intratumoral heterogeneity arises through the evolution of genetically diverse subclones during tumor progression. However, it remains unknown whether cells within single genetic clones are ...functionally equivalent. By combining DNA copy number alteration (CNA) profiling, sequencing, and lentiviral lineage tracking, we followed the repopulation dynamics of 150 single lentivirus-marked lineages from 10 human colorectal cancers through serial xenograft passages in mice. CNA and mutational analysis distinguished individual clones and showed that clones remained stable upon serial transplantation. Despite this stability, the proliferation, persistence, and chemotherapy tolerance of lentivirally marked lineages were variable within each clone. Chemotherapy promoted the dominance of previously minor or dormant lineages. Thus, apart from genetic diversity, tumor cells display inherent functional variability in tumor propagation potential, which contributes to both cancer growth and therapy tolerance.
Tumor recurrence following treatment remains a major clinical challenge. Evidence from xenograft models and human trials indicates selective enrichment of cancer-initiating cells (CICs) in tumors ...that survive therapy. Together with recent reports showing that CIC gene signatures influence patient survival, these studies predict that targeting self-renewal, the key 'stemness' property unique to CICs, may represent a new paradigm in cancer therapy. Here we demonstrate that tumor formation and, more specifically, human colorectal CIC function are dependent on the canonical self-renewal regulator BMI-1. Downregulation of BMI-1 inhibits the ability of colorectal CICs to self-renew, resulting in the abrogation of their tumorigenic potential. Treatment of primary colorectal cancer xenografts with a small-molecule BMI-1 inhibitor resulted in colorectal CIC loss with long-term and irreversible impairment of tumor growth. Targeting the BMI-1-related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer.
Genetic and functional studies underscore the central role of JAK/STAT signaling in myeloproliferative neoplasms (MPNs). However, the mechanisms that mediate transformation in MPNs are not fully ...delineated, and clinically utilized JAK inhibitors have limited ability to reduce disease burden or reverse myelofibrosis. Here we show that MPN progenitor cells are characterized by marked alterations in gene regulation through differential enhancer utilization, and identify nuclear factor κB (NF-κB) signaling as a key pathway activated in malignant and non-malignant cells in MPN. Inhibition of BET bromodomain proteins attenuated NF-κB signaling and reduced cytokine production in vivo. Most importantly, combined JAK/BET inhibition resulted in a marked reduction in the serum levels of inflammatory cytokines, reduced disease burden, and reversed bone marrow fibrosis in vivo.
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•Inflammation is associated with changes in the chromatin landscape of MPN cells•NF-κB is constitutively active in MPNs and drives MPN-associated inflammation•JQ1 shows potent anti-fibrotic efficacy in MPN mouse models•BET inhibition cooperates with JAK inhibition to ameliorate MPN-driven inflammation
Kleppe et al. show that aberrant JAK2 signaling in myeloproliferative neoplasms (MPN) leads to chromatin changes that promote NF-κB signaling. BET inhibitors reduce NF-κB-induced inflammation and bone marrow fibrosis in MPN models, and combination treatment with BET and JAK inhibitors shows improved efficacy.
4-α-Rhamnosyloxy benzyl glucosinolate (glucomoringin) was isolated and identified as the main glucosinolate in 10 out of 13 screened Noccaea caerulescens populations. This is the first time that ...glucomoringin was identified in a member of the Brassicaceae. Display omitted
•4-α-Rhamnosyloxy benzyl glucosinolate (glucomoringin) was identified in Noccaea caerulescens.•This is the first report of glucomoringin in this species or the Brassicaceae.•In 10 out of 13 N. caerulescens populations it was the main glucosinolate.•The isothiocyanate is highly biologically active, this may confer benefits to plants.
Glucosinolates are secondary plant compounds typically found in members of the Brassicaceae and a few other plant families. Usually each plant species contains a specific subset of the ∼130 different glucosinolates identified to date. However, intraspecific variation in glucosinolate profiles is commonly found. Sinalbin (4-hydroxybenzyl glucosinolate) so far has been identified as the main glucosinolate of the heavy metal accumulating plant species Noccaea caerulescens (Brassicaceae). However, a screening of 13 N. caerulescens populations revealed that in 10 populations a structurally related glucosinolate was found as the major component. Based on nuclear magnetic resonance (NMR) and mass spectrometry analyses of the intact glucosinolate as well as of the products formed after enzymatic conversion by sulfatase or myrosinase, this compound was identified as 4-α-rhamnosyloxy benzyl glucosinolate (glucomoringin). So far, glucomoringin had only been reported as the main glucosinolate of Moringa spp. (Moringaceae) which are tropical tree species. There was no apparent relation between the level of soil pollution at the location of origin, and the presence of glucomoringin. The isothiocyanate that is formed after conversion of glucomoringin is a potent antimicrobial and antitumor agent. It has yet to be established whether glucomoringin or its breakdown product have an added benefit to the plant in its natural habitat.
Fetal hemoglobin (HbF, α2γ2) induction is a well-validated strategy for sickle cell disease (SCD) treatment. Using a small-molecule screen, we found that UNC0638, a selective inhibitor of EHMT1 and ...EHMT2 histone methyltransferases, induces γ-globin expression. EHMT1/2 catalyze mono- and dimethylation of lysine 9 on histone 3 (H3K9), raising the possibility that H3K9Me2, a repressive chromatin mark, plays a role in silencing γ-globin expression. In primary human adult erythroid cells, UNC0638 and EHMT1 or EHMT2 short hairpin RNA–mediated knockdown significantly increased γ-globin expression, HbF synthesis, and the percentage of cells expressing HbF. At effective concentrations, UNC0638 did not alter cell morphology, proliferation, or erythroid differentiation of primary human CD34+ hematopoietic stem and progenitor cells in culture ex vivo. In murine erythroleukemia cells, UNC0638 and Ehmt2 CRISPR/Cas9-mediated knockout both led to a marked increase in expression of embryonic β-globin genes Hbb-εy and Hbb-βh1. In primary human adult erythroblasts, chromatin immunoprecipitation followed by sequencing analysis revealed that UNC0638 treatment leads to genome-wide depletion in H3K9Me2 and a concomitant increase in the activating mark H3K9Ac, which was especially pronounced at the γ-globin gene region. In RNA-sequencing analysis of erythroblasts, γ-globin genes were among the most significantly upregulated genes by UNC0638. Further increase in γ-globin expression in primary human adult erythroid cells was achieved by combining EHMT1/2 inhibition with the histone deacetylase inhibitor entinostat or hypomethylating agent decitabine. Our data provide genetic and pharmacologic evidence that EHMT1 and EHMT2 are epigenetic regulators involved in γ-globin repression and represent a novel therapeutic target for SCD.
•EHMT1/2 inhibition increases human γ-globin and HbF expression, as well as mouse embryonic β-globin gene expression.•EHMT1/2 inhibition decreases H3K9Me2 and increases H3K9Ac at the γ-globin gene locus in adult human erythroid cells.
Single-cell analysis methods are valuable tools; however, current approaches do not easily enable live cell retrieval. That is a particular issue when further study of cells that were eliminated ...during experimentation could provide critical information. We report a clonal molecular barcoding method, called SunCatcher, that enables longitudinal tracking and live cell functional analysis. From complex cell populations, we generate single cell-derived clonal populations, infect each with a unique molecular barcode, and retain stocks of individual barcoded clones (BCs). We develop quantitative PCR-based and next-generation sequencing methods that we employ to identify and quantify BCs in vitro and in vivo. We apply SunCatcher to various breast cancer cell lines and combine respective BCs to create versions of the original cell lines. While the heterogeneous BC pools reproduce their original parental cell line proliferation and tumor progression rates, individual BCs are phenotypically and functionally diverse. Early spontaneous metastases can also be identified and quantified. SunCatcher thus provides a rapid and sensitive approach for studying live single-cell clones and clonal evolution, and performing functional analyses.
Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit ...complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs. Top pairs that demonstrate either compensatory non-lethal interactions or synergistic lethality enrich for paralogs and targets that occupy the same protein complex. The screen highlights protein complex dependencies not apparent in single KO screens, for example MCM histone exchange, the nucleosome remodeling and deacetylase (NuRD) complex, and HBO1 (KAT7) complex. We explore two approaches to NuRD complex inactivation. Paralog and non-paralog combinations of the KAT7 complex emerge as synergistic lethal and specifically nominate the ING5 PHD domain as a potential therapeutic target when paired with other KAT7 complex member losses. These findings highlight the power of combinatorial screening to provide mechanistic insight and identify therapeutic targets within redundant networks.
In this issue of
, Li and colleagues provide a blueprint for the identification and functional validation of cancer-associated mutations in noncoding regions of the genome. Integration of ...whole-genome sequencing and high-throughput epigenome editing screens is starting to reveal the extent to which noncoding genetic lesions contribute to cancer.
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