Abstract PR004: High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia


 Several leukemia-associated oncoproteins activate transcriptional circuits resembling a stem-like state in acute myeloid leukemia (AML). This activation of “stemness” genes is achieved by enlisting the activity of specialized components of the epigenetic machinery. We sought to comprehensively map epigenetic regulators critical for perpetuating these stemness networks in AML, as they may represent new therapeutic targets. To this end, we used a GFP-reporter knocked into the endogenous locus of the key leukemia oncogene and self-renewal-associated gene MEIS1 and conducted a pooled domain-focused CRISPR screen targeting >600 epigenetic modifiers. Through this screen, we identified and validated multiple members of eight distinct chromatin-modifying complexes that were required for sustaining MEIS1 expression in diverse AML subtypes. These included several novel MEIS1 regulators such as TAF6, LDB1, KAT2A, AFF2, JADE3, casein kinase 2 (CK2), ENY2 and SGF29, in addition to previously characterized regulators such as DOT1L, AF10, ENL and HBO1. A secondary pooled CRISPR screen, coupled with a single-cell transcriptome readout (CROP-seq) revealed that the deletion of several of these MEIS1 activators not only reversed MEIS1 activation but also reduced expression of stem-cell associated genes, including genes of the HOXA cluster, BMI1, SATB1 and Musashi 2, and concomitantly activated expression of differentiation-associated genes. Of particular interest to us was the TUDOR domain chromatin reader SGF29, a key component of the SAGA (Spt-Ada-Gcn5 acetyltransferase) complex. CRISPR-ko of SGF29 significantly reduced the proliferation of cells with distinct HOX-activating mutations including KMT2A rearranged and AF10-rearranged AML cells. ChIP-seq studies showed that SGF29 occupied the promoters and enhancers of key leukemia-oncogenes in a TUDOR-domain-dependent manner and SGF29 deletion selectively attenuated their transcription. Importantly, chromatin proteomics showed that SGF29 deletion led to the eviction of the SAGA complex subunit KAT2A from chromatin and into the cytoplasm. Further, SGF29 knockout impaired blast colony formation and induced differentiation in the KMT2A-MLLLT3, KMT2A-AF10, and CALM-AF10 mouse AML models but did not affect normal hematopoietic colony formation. Lastly, SGF29 deletion delayed disease latency in two distinct human AML cell line models as well as a patient-derived xenograft model in vivo and resulted in striking antiproliferative effects. Our study revealed several new attractive nodes for therapeutic targeting of leukemia stem cells in AML, including the chromatin reader SGF29, and provides a framework to identify vulnerabilities against recalcitrant oncogenic networks.
 Citation Format: Karina O. Barbosa Guerra, Anagha Deshpande, Ping Xiang, Anna Minkina, Fiorella Schischlik, Adam Brown, Neil A. Robertson, John Doench, Peter D. Adams, Keith Humphries, Eytan Ruppin, Jay Shendure, Prashant Mali, Aniruddha Deshpande. High-density CRISPR screens reveal mechanisms of chromatin regulation of stemness networks in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR004.