SummaryAcute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify additional therapeutic targets in AML, we optimize a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. We identify 492 AML-specific cell-essential genes, including several established therapeutic targets such as DOT1L, BCL2, and MEN1, and many other genes including clinically actionable candidates. We validate selected genes using genetic and pharmacological inhibition, and chose KAT2A as a candidate for downstream study. KAT2A inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs of diverse genotypes while sparing normal hemopoietic stem-progenitor cells. Our results propose that KAT2A inhibition should be investigated as a therapeutic strategy in AML and provide a large number of genetic vulnerabilities of this leukemia that can be pursued in downstream studies.

The N6-methyladenosine (m 6 A) is an abundant internal RNA modification 1,2 catalysed predominantly by the METTL3-METTL14 methyltransferase complex 3,4 . The m 6 A writer METTL3 has been linked to the initiation and maintenance of acute myeloid leukaemia (AML), but its true therapeutic importance is still unknown [5][6][7] . Here we present the identification and characterisation of a highly potent and selective first-in-class catalytic inhibitor of METTL3 (STM2457) and its co-crystal structure bound to METTL3/METTL14. Treatment with (Extended Data Fig. 2f). These data demonstrate that STM2457 is a highly potent, specific and bioavailable inhibitor of METTL3, suitable for in vivo investigations. Cellular and molecular effects of STM2457To study the anti-leukaemic potential of STM2457 we examined the proliferation of a panel of human AML cell lines post-treatment and detected significant growth reduction in a concentration-dependent manner (Fig. 2a) while we found that STM2457 did not affect the colony-forming ability of normal human cord blood CD34 + cells (Extended Data Fig. 3a). We also observed no impact on the proliferation of MOLM-13 cells treated with the control small molecule STM2120, unlike our observations with STM2457 (Extended Data Fig. 3b). Additionally, treatment with STM2457 significantly reduced the clonogenic potential of primary murine AML cells (Fig. 2b and Extended Data Fig. 3c), while having no effect on normal haematopoietic stem and progenitor cells (HSPCs) (Fig. 2c). Pharmacological inhibition of METTL3 also caused significant myeloid differentiation 6,11 and cell cycle arrest in MOLM-13 and primary murine AML cells (Fig. 2d, e). In contrast, the same effects were not identified using the non-leukaemic haemopoietic cell line HPC7 (Fig. 3e and Extended Data Fig. 3d). Moreover, treatment with STM2457 induced apoptosis in human and mouse AML models but not in normal non-leukaemic haemopoietic cells (Fig. 2f and Extended Data Fig. 3e). To assess the impact of pharmacological inhibition of METTL3 on two known METTL3 biomarkers associated with AML, SP1 6,12 and BRD4 13,14 , we treated MOLM-13 cells with STM2457 and observed a dose-dependent reduction of SP1 and BRD4 protein levels (Fig. 2g). Notably, ectopic expression of SP1 significantly reduced the sensitivity of MOLM-13 cells to STM2457 (Extended Data Fig. 3f, g). These data establish that the catalytic function of METTL3 is important for leukaemia growth, in line with previous findings 6,7,15 . We next sought to investigate the molecular mechanism by which STM2457 affects AML. RNAseq analysis of MOLM-13 cells treated with STM2457 revealed 1,338 up-regulated and 489 down-regulated genes (Extended Data Fig. 4a and Supplementary Table 1). Gene ontology (GO) analysis of the differentially expressed genes showed enrichment in pathways related to myeloid differentiation, cell cycle and leukaemia progression (Extended Data Fig. 4b, c) in close agreement with our phenotypic observations (Supplementary Table 2). To examine the impact of the pharmac...

The histone H3 Lys27-specific demethylase UTX (or KDM6A) is targeted by loss-of-function mutations in multiple cancers. Here, we demonstrate that UTX suppresses myeloid leukemogenesis through noncatalytic functions, a property shared with its catalytically inactive Y-chromosome paralog, UTY (or KDM6C). In keeping with this, we demonstrate concomitant loss/mutation of KDM6A (UTX) and UTY in multiple human cancers. Mechanistically, global genomic profiling showed only minor changes in H3K27me3 but significant and bidirectional alterations in H3K27ac and chromatin accessibility; a predominant loss of H3K4me1 modifications; alterations in ETS and GATA-factor binding; and altered gene expression after Utx loss. By integrating proteomic and genomic analyses, we link these changes to UTX regulation of ATP-dependent chromatin remodeling, coordination of the COMPASS complex and enhanced pioneering activity of ETS factors during evolution to AML. Collectively, our findings identify a dual role for UTX in suppressing acute myeloid leukemia via repression of oncogenic ETS and upregulation of tumor-suppressive GATA programs.

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2nd and 3rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.

Epigenetic regulators, such as EZH2, are frequently mutated in cancer, and loss-of-function EZH2 mutations are common in myeloid malignancies. We have examined the importance of cellular context for Ezh2 loss during the evolution of acute myeloid leukemia (AML), where we observed stage-specific and diametrically opposite functions for Ezh2 at the early and late stages of disease. During disease maintenance, WT Ezh2 exerts an oncogenic function that may be therapeutically targeted. In contrast, Ezh2 acts as a tumor suppressor during AML induction. Transcriptional analysis explains this apparent paradox, demonstrating that loss of Ezh2 derepresses different expression programs during disease induction and maintenance. During disease induction, Ezh2 loss derepresses a subset of bivalent promoters that resolve toward gene activation, inducing a feto-oncogenic program that includes genes such as Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. Our data highlight the importance of cellular context and disease phase for the function of Ezh2 and its potential therapeutic implications.

We recently identified the splicing kinase gene SRPK1 as a genetic vulnerability of acute myeloid leukemia (AML). Here, we show that genetic or pharmacological inhibition of SRPK1 leads to cell cycle arrest, leukemic cell differentiation and prolonged survival of mice transplanted with MLL-rearranged AML. RNA-seq analysis demonstrates that SRPK1 inhibition leads to altered isoform levels of many genes including several with established roles in leukemogenesis such as MYB, BRD4 and MED24. We focus on BRD4 as its main isoforms have distinct molecular properties and find that SRPK1 inhibition produces a significant switch from the short to the long isoform at the mRNA and protein levels. This was associated with BRD4 eviction from genomic loci involved in leukemogenesis including BCL2 and MYC. We go on to show that this switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Our findings reveal that SRPK1 represents a plausible new therapeutic target against AML.

MAPK (mitogen-activated protein kinase) pathways are among the most frequently deregulated signalling events in cancer. Among the critical targets of MAPK activities are members of the AP-1 (activator protein 1) transcription factor, a dimeric complex consisting of Jun, Fos, Maf and ATF (activating transcription factor) family DNA-binding proteins. Depending on the cellular context, the composition of the dimeric complexes determines the regulation of growth, survival or apoptosis. JNK (c-Jun N-terminal kinase), p38 and a number of Jun and Fos family proteins have been analysed for their involvement in oncogenic transformation and tumour formation. These data are also emerging for the ATF components of the AP-1 factor. The aim of the present review is to provide an overview of the functions of two ATF family proteins, ATF2 and ATF7, in mammalian development and their potential functions in tumour formation.

JNK and p38 phosphorylate a diverse set of substrates and, consequently, can act in a context-dependent manner to either promote or inhibit tumor growth. Elucidating the functions of specific substrates of JNK and p38 is therefore critical for our understanding of these kinases in cancer. ATF2 is a phosphorylation-dependent transcription factor and substrate of both JNK and p38. Here, we show ATF2 suppresses tumor formation in an orthotopic model of liver cancer and cellular transformation in vitro. Furthermore, we find that suppression of tumorigenesis by JNK requires ATF2. We identify a transcriptional program activated by JNK via ATF2 and provide examples of JNK- and ATF2-dependent genes that block cellular transformation. Significantly, we also show that ATF2-dependent gene expression is frequently downregulated in human cancers, indicating that amelioration of JNK-ATF2-mediated suppression may be a common event during tumor development.