Structural variation cooperates with permissive chromatin to control enhancer hijacking-mediated oncogenic transcription.

Structural variants (SVs) involving enhancer hijacking can rewire chromatin topologies to cause oncogene activation in human cancers, including hematologic malignancies; however, because of the lack of tools to assess their effects on gene regulation and chromatin organization, the molecular determinants for the functional output of enhancer hijacking remain poorly understood. Here, we developed a multimodal approach to integrate genome sequencing, chromosome conformation, chromatin state, and transcriptomic alteration for quantitative analysis of transcriptional effects and structural reorganization imposed by SVs in leukemic genomes. We identified known and new pathogenic SVs, including recurrent t(5;14) translocations that cause the hijacking of BCL11B enhancers for the allele-specific activation of TLX3 in a subtype of pediatric leukemia. Epigenetic perturbation of SV-hijacked BCL11B enhancers impairs TLX3 transcription, which are required for the growth of t(5;14) leukemia cells. By CRISPR engineering of patient-derived t(5;14) in isogenic leukemia cells, we uncovered a new mechanism whereby the transcriptional output of SV-induced BCL11B enhancer hijacking is dependent on the loss of DNA hypermethylation at the TLX3 promoter. Our results highlight the importance of the cooperation between genetic alteration and permissive chromatin as a critical determinant of SV-mediated oncogene activation, with implications for understanding aberrant gene transcription after epigenetic therapies in patients with leukemia. Hence, leveraging the interdependency of genetic alteration on chromatin variation may provide new opportunities to reprogram gene regulation as targeted interventions in human disease.

Disabling Uncompetitive Inhibition of Oncogenic IDH Mutations Drives Acquired Resistance.

Mutations in IDH genes occur frequently in acute myeloid leukemia (AML) and other human cancers to generate the oncometabolite R-2HG. Allosteric inhibition of mutant IDH suppresses R-2HG production in a subset of patients with AML; however, acquired resistance emerges as a new challenge, and the underlying mechanisms remain incompletely understood. Here we establish isogenic leukemia cells containing common IDH oncogenic mutations by CRISPR base editing. By mutational scanning of IDH single amino acid variants in base-edited cells, we describe a repertoire of IDH second-site mutations responsible for therapy resistance through disabling uncompetitive enzyme inhibition. Recurrent mutations at NADPH binding sites within IDH heterodimers act in cis or trans to prevent the formation of stable enzyme-inhibitor complexes, restore R-2HG production in the presence of inhibitors, and drive therapy resistance in IDH-mutant AML cells and patients. We therefore uncover a new class of pathogenic mutations and mechanisms for acquired resistance to targeted cancer therapies. SIGNIFICANCE: Comprehensive scanning of IDH single amino acid variants in base-edited leukemia cells uncovers recurrent mutations conferring resistance to IDH inhibition through disabling NADPH-dependent uncompetitive inhibition. Together with targeted sequencing, structural, and functional studies, we identify a new class of pathogenic mutations and mechanisms for acquired resistance to IDH-targeting cancer therapies. This article is highlighted in the In This Issue feature, p. 1.

Metabolon formation regulates branched-chain amino acid oxidation and homeostasis.

The branched-chain aminotransferase isozymes BCAT1 and BCAT2, segregated into distinct subcellular compartments and tissues, initiate the catabolism of branched-chain amino acids (BCAAs). However, whether and how BCAT isozymes cooperate with downstream enzymes to control BCAA homeostasis in an intact organism remains largely unknown. Here, we analyse system-wide metabolomic changes in BCAT1- and BCAT2-deficient mouse models. Loss of BCAT2 but not BCAT1 leads to accumulation of BCAAs and branched-chain α-keto acids (BCKAs), causing morbidity and mortality that can be ameliorated by dietary BCAA restriction. Through proximity labelling, isotope tracing and enzymatic assays, we provide evidence for the formation of a mitochondrial BCAA metabolon involving BCAT2 and branched-chain α-keto acid dehydrogenase. Disabling the metabolon contributes to BCAT2 deficiency-induced phenotypes, which can be reversed by BCAT1-mediated BCKA reamination. These findings establish a role for metabolon formation in BCAA metabolism in vivo and suggest a new strategy to modulate this pathway in diseases involving dysfunctional BCAA metabolism.

Convergence of oncogenic cooperation at single-cell and single-gene levels drives leukemic transformation.

Cancers develop from the accumulation of somatic mutations, yet it remains unclear how oncogenic lesions cooperate to drive cancer progression. Using a mouse model harboring NRasG12D and EZH2 mutations that recapitulates leukemic progression, we employ single-cell transcriptomic profiling to map cellular composition and gene expression alterations in healthy or diseased bone marrows during leukemogenesis. At cellular level, NRasG12D induces myeloid lineage-biased differentiation and EZH2-deficiency impairs myeloid cell maturation, whereas they cooperate to promote myeloid neoplasms with dysregulated transcriptional programs. At gene level, NRasG12D and EZH2-deficiency independently and synergistically deregulate gene expression. We integrate results from histopathology, leukemia repopulation, and leukemia-initiating cell assays to validate transcriptome-based cellular profiles. We use this resource to relate developmental hierarchies to leukemia phenotypes, evaluate oncogenic cooperation at single-cell and single-gene levels, and identify GEM as a regulator of leukemia-initiating cells. Our studies establish an integrative approach to deconvolute cancer evolution at single-cell resolution in vivo.

Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing.

Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe CRISPR/dCas9-based enhancer-targeting epigenetic editing systems, enCRISPRa and enCRISPRi, for efficient analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the improved systems display more robust perturbations of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Single or multi-loci perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoiesis. Hence, enhancer-targeting CRISPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.

The mechanical and pharmacological regulation of glioblastoma cell migration in 3D matrices.

The invasion of glioblastoma is a complex process based on the interactions of tumor cells and the extracellular matrix. Tumors that are engineered using biomaterials are more physiologically relevant than a two-dimensional (2D) cell culture system. Matrix metalloproteinases and the plasminogen activator generated by tumor cells regulate a tumor's invasive behavior. In this study, microtumors were fabricated by encapsulating U87 glioma cells in Type I collagen and then glioma cell migration in the collagen hydrogels was investigated. Crosslinking of collagen with 8S-StarPEG increased the hydrogel viscosity and reduced the tumor cell migration speed in the hydrogels. The higher migration speed corresponded to the increased gene expression of MMP-2, MMP-9, urokinase plasminogen activator (uPA), and tissue plasminogen activator (tPA) in glioma cells grown in non-crosslinked collagen hydrogels. Inhibitors of these molecules hindered U87 and A172 cell migration in collagen hydrogels. Aprotinin and tranexamic acid did not inhibit U87 and A172 migration on the culture dish. This study demonstrated the differential effect of pharmacologic molecules on tumor cell motility in either a 2D or three-dimensional culture environment.