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Genome editing technologies have transformed our ability to engineer desired genomic changes within living systems. However, detecting precise genomic modifications often requires sophisticated, expensive, and time-consuming experimental approaches. Here, we describe DTECT (Dinucleotide signaTurE CapTure), a rapid and versatile detection method that relies on the capture of targeted dinucleotide signatures resulting from the digestion of genomic DNA amplicons by the type IIS restriction enzyme AcuI. DTECT enables the accurate quantification of marker-free precision genome editing events introduced by CRISPR-dependent homology-directed repair, base editing, or prime editing in various biological systems, such as mammalian cell lines, organoids, and tissues. Furthermore, DTECT allows the identification of oncogenic mutations in cancer mouse models, patient-derived xenografts, and human cancer patient samples. The ease, speed, and cost efficiency by which DTECT identifies genomic signatures should facilitate the generation of marker-free cellular and animal models of human disease and expedite the detection of human pathogenic variants., Competing Interests: Declaration of Interests P.B. and A.C. have filed a provisional patent for the invention described in this paper. R.L.L. is on the supervisory board of QIAGEN and is a scientific advisor to Loxo, Imago, C4 Therapeutics, and Isoplexis, each of which includes an equity interest. R.L.L. receives research support from and has consulted for Celgene and Roche; he has received research support from Prelude Therapeutics, and he has consulted for Incyte, Novartis, Astellas, Morphosys, and Janssen. He also has received honoraria from Lilly and Amgen for invited lectures and from Gilead for grant reviews., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glucocorticoid resistance represents a major challenge in treating acute lymphoblastic leukemia. In this issue of Cancer Cell, Jing and colleagues show epigenetic deregulation of glucocorticoid-induced BIM activation in glucocorticoid-resistant leukemia cells, and restore glucocorticoid-receptor-induced BIM upregulation with DNA demethylating agents to effectively enhance glucocorticoid response., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML) led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Sequencing efforts led to the identification of somatic mutations that could affect the self-renewal and differentiation of cancer-initiating cells. One such recurrent mutation targets the binding pocket of the ubiquitin ligase Fbxw7. Missense FBXW7 mutations are prevalent in various tumors, including T cell acute lymphoblastic leukemia (T-ALL). To study the effects of such lesions, we generated animals carrying regulatable Fbxw7 mutant alleles. Here, we show that these mutations specifically bolster cancer-initiating cell activity in collaboration with Notch1 oncogenes but spare normal hematopoietic stem cell function. We were also able to show that FBXW7 mutations specifically affect the ubiquitylation and half-life of c-Myc protein, a key T-ALL oncogene. Using animals carrying c-Myc fusion alleles, we connected Fbxw7 function to c-Myc abundance and correlated c-Myc expression to leukemia-initiating activity. Finally, we demonstrated that small-molecule-mediated suppression of MYC activity leads to T-ALL remission, suggesting an effective therapeutic strategy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Here, we have investigated the role of the Notch pathway in the generation and maintenance of Kras(G12V)-driven non-small cell lung carcinomas (NSCLCs). We demonstrate by genetic means that γ-secretase and RBPJ are essential for the formation of NSCLCs. Of importance, pharmacologic treatment of mice carrying autochthonous NSCLCs with a γ-secretase inhibitor (GSI) blocks cancer growth. Treated carcinomas present reduced HES1 levels and reduced phosphorylated ERK without changes in phosphorylated MEK. Mechanistically, we show that HES1 directly binds to and represses the promoter of DUSP1, encoding a dual phosphatase that is active against phospho-ERK. Accordingly, GSI treatment upregulates DUSP1 and decreases phospho-ERK. These data provide proof of the in vivo therapeutic potential of GSIs in primary NSCLCs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Constitutive phosphoinositide 3-kinase (PI3K)/Akt activation is common in T cell acute lymphoblastic leukemia (T-ALL). Although four distinct class I PI3K isoforms (α, β, γ, δ) could participate in T-ALL pathogenesis, none has been implicated in this process. We report that in the absence of PTEN phosphatase tumor suppressor function, PI3Kγ or PI3Kδ alone can support leukemogenesis, whereas inactivation of both isoforms suppressed tumor formation. The reliance of PTEN null T-ALL on the combined activities of PI3Kγ/δ was further demonstrated by the ability of a dual inhibitor to reduce disease burden and prolong survival in mice as well as prevent proliferation and promote activation of proapoptotic pathways in human tumors. These results support combined inhibition of PI3Kγ/δ as therapy for T-ALL., (Copyright © 2012 Elsevier Inc. All rights reserved.)