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Epigenetic programs are dysregulated in acute myeloid leukemia (AML) and help enforce an oncogenic state of differentiation arrest. To identify key epigenetic regulators of AML cell fate, we performed a differentiation-focused CRISPR screen in AML cells. This screen identified the histone acetyltransferase KAT6A as a novel regulator of myeloid differentiation that drives critical leukemogenic gene-expression programs. We show that KAT6A is the initiator of a newly described transcriptional control module in which KAT6A-catalyzed promoter H3K9ac is bound by the acetyl-lysine reader ENL, which in turn cooperates with a network of chromatin factors to induce transcriptional elongation. Inhibition of KAT6A has strong anti-AML phenotypes in vitro and in vivo, suggesting that KAT6A small-molecule inhibitors could be of high therapeutic interest for mono-therapy or combinatorial differentiation-based treatment of AML. Significance: AML is a poor-prognosis disease characterized by differentiation blockade. Through a cell-fate CRISPR screen, we identified KAT6A as a novel regulator of AML cell differentiation. Mechanistically, KAT6A cooperates with ENL in a “writer–reader” epigenetic transcriptional control module. These results uncover a new epigenetic dependency and therapeutic opportunity in AML. This article is highlighted in the In This Issue feature, p. 587

Supplementary Tables 1, 2, and 3

Fragility of regulatory T (Treg) cells manifested by the loss of neuropilin-1 (NRP1) and expression of IFNγ undermines the immune suppressive functions of Treg cells and contributes to the success of immune therapies against cancers. Intratumoral Treg cells somehow avoid fragility; however, the mechanisms by which Treg cells are protected from fragility in the tumor microenvironment are not well understood. Here, we demonstrate that the IFNAR1 chain of the type I IFN (IFN1) receptor was downregulated on intratumoral Treg cells. Downregulation of IFNAR1 mediated by p38α kinase protected Treg cells from fragility and maintained NRP1 levels, which were decreased in response to IFN1. Genetic or pharmacologic inactivation of p38α and stabilization of IFNAR1 in Treg cells induced fragility and inhibited their immune suppressive and protumorigenic activities. The inhibitor of sumoylation TAK981 (Subasumstat) upregulated IFNAR1, eliciting Treg fragility and inhibiting tumor growth in an IFNAR1-dependent manner. These findings describe a mechanism by which intratumoral Treg cells retain immunosuppressive activities and suggest therapeutic approaches for inducing Treg fragility and increasing the efficacy of immunotherapies.

Supplementary Figure from Protection of Regulatory T Cells from Fragility and Inactivation in the Tumor Microenvironment

Supplementary Data from Protection of Regulatory T Cells from Fragility and Inactivation in the Tumor Microenvironment

S1. Clinical and molecular profile of gliomas by ASNS status S2. CDKN2A and ASNS KM survival plots by IDH status S3. ASNS copy-number gain compared to ASNS mRNA & protein expression S4. IDH mutation inversely correlate to ASNS copy number gain S5. GSCs and ASNS expression levels Supp Table 1. Clinical & Molecular Profile for Parental GSC604 & GSC827 cell lines

Asparagine synthetase (ASNS) is a gene on the long arm of chromosome 7 that is copy-number amplified in the majority of glioblastomas. ASNS copy-number amplification is associated with a significantly decreased survival. Using patient-derived glioma stem cells (GSC), we showed that significant metabolic alterations occur in gliomas when perturbing the expression of ASNS, which is not merely restricted to amino acid homeostasis. ASNS-high GSCs maintained a slower basal metabolic profile yet readily shifted to a greatly increased capacity for glycolysis and oxidative phosphorylation when needed. This led ASNS-high cells to a greater ability to proliferate and spread into brain tissue. Finally, we demonstrate that these changes confer resistance to cellular stress, notably oxidative stress, through adaptive redox homeostasis that led to radiotherapy resistance. Furthermore, ASNS overexpression led to modifications of the one-carbon metabolism to promote a more antioxidant tumor environment revealing a metabolic vulnerability that may be therapeutically exploited.Implications:This study reveals a new role for ASNS in metabolic control and redox homeostasis in glioma stem cells and proposes a new treatment strategy that attempts to exploit one vulnerable metabolic node within the larger multilayered tumor network.

Colorectal cancer (CRC) is the 2nd-leading cause of cancer-related deaths worldwide and metastatic disease remains a major unmet clinical need. Nevertheless, the molecular underpinnings of metastatic spread and colonization of distant sites remain elusive. To systematically identify functional genes contributing to metastasis in a physiologically relevant model of CRC, we developed a novel platform enabling robust, pooled CRISPR/Cas9 screening in primary colonic organoids engineered with common CRC driver mutations in Apc, Kras, and Tp53. We performed parallel in vitro invasion and in vivo metastasis gain-of-function CRISPR/Cas9 knockout screens with high-content gRNA libraries to uncover novel suppressors of metastasis. We confirmed resulting hits as bona-fide metastatic suppressors using both pooled validation screens and single-gene approaches. Genes scoring in both in vitro and in vivo screens included previously identified regulators of migration and invasion, such as Ctnna1, and genes scoring only in the in vivo screen included novel negative regulators of metastasis such as Bcl2l13. These hits in the murine system were supported by human TCGA data, indicating that low expression of these genes is associated with worse 5-year survival in colon and rectal adenocarcinoma patients (COAD and READ). Utilizing transwell and live-imaging assays, we found that sgRNAs targeting Ctnna1 markedly enhanced invasion and migration of CRC organoids while sgRNAs targeting Bcl2l13 had no effect, an observation consistent with our in vitro screen. Therefore, while loss of Ctnna1 enhances metastasis by augmenting invasion and migration, loss of Bcl2l13 acts through alternative mechanisms. Single cell RNA sequencing of primary and metastatic lesions derived from Bcl2l13 knockout and control CRC tumors identified novel putative pathways promoting metastatic spread via migration-independent mechanisms, including upregulation of hypoxia signalling and recruitment of pro-tumorigenic macrophages. Thus, our CRISPR/Cas9 screening pipeline is a viable approach for the identification of novel regulators of CRC metastasis in vitro and in vivo, uncovering both motility and migration-dependent and independent paths to metastatic success. These findings will help untangle the complex web of molecular events underlying CRC metastasis and provide clinically-relevant insights into the process of human CRC metastasis. Citation Format: Zvi Cramer, Xin Wang, Nicolae Adrian Leu, Keara Monaghan, Joshua H. Rhoades, Yuhua Tian, Joshua Rico, Diego Mendez, Ricardo Petroni, Melissa S. Kim, Ning Li, Christopher J. Lengner, M. Andres Blanco. In vitro and in vivo CRISPR screens in colorectal cancer organoids identify suppressors of metastasis [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr PR004.