Crispri-Based Screening of Clustered Regulatory Elements Reveals Novel Leukemia Dependencies

In the recent years, massively parallel sequencing approaches allowed the identification of hundreds of mutated genes in Leukemia. Although these data gave unprecedented amount of information about mechanisms of leukemia cell maintenance and/or progression, the functional characterization of genes that are key player in regulating cancer development remain laborious. Analysis at the single gene level often fails to identify gene or pathway collaborations leading to transformation. Studies aimed at depicting new oncogene cooperation would involve the generation of challenging mouse models or the deployment of tedious screening pipelines, which would be inadequate to depict new oncogene circuitry in cancer. Genome wide mapping of epigenetic modifications on histone tails or binding of factors such as MED1 and BRD4 allowed identification of clusters of regulatory elements, also termed as Super Enhancers. Functional annotation of these regions revealed their high relevance during normal hematopoiesis and Leukemogenesis. We hypothesized that these regulatory regions could regulate simultaneously expression of genes cooperating to promote Leukemia development.

We thus developed a novel genome-wide CRISPRi-based screening approach to directly target these regulatory regions. CRISPRi technology relies on the use of deactivated Cas9 that can't cut the DNA and that is fused to the repressive KRAB domain (dCas9-KRAB). Therefore, properly targeted dCas9-KRAB by single guide RNAs will recruit chromatin modifying factors and trigger generation of heterochromatin thus inhibiting enhancer function. We performed this screen using acute megakaryoblastic leukemia model driven by the CBFA2T3-GLIS2 fusion, the most frequent fusion oncogene in this disease that we recently identified as being associated with Super Enhancers (Thirant et al, Cancer Cell 2017). To inhibit Super Enhancer activity we integrated ChIP-seq data of H3K27ac and ATAC-seq data to define open chromatin regions located in Super Enhancers. We designed a library of 7995 single guide RNAs targeting 450 Super Enhancer regions found active in CBFA2T3-GLIS2 bearing cell line M07e and primary AMKL patient samples.

This screening methodology allowed us to nominate Super Enhancer regions, which are functionally linked to leukemia progression. In particular, we pinpointed a novel Super Enhancer region, induced by CBFA2T3-GLIS2 fusion, regulating the expression of both tyrosine kinases associated receptors KIT and PDGFRA. We were able to show that this Super Enhancer region is normally not active in normal megakaryocytic development and aberrantly induced by CBFA2T3-GLIS2 expression. RNA-sequencing experiments and 4C-seq experiments (chromatin conformation capture) showed that this Super Enhancer is directly regulating KIT and PDGFRA expression. Whereas single inhibition of these genes using shRNA or small molecule inhibitors affects modestly leukemic cell growth, concomitant inhibition of these two receptors synergizes to impair AMKL cell lines and primary patient cells growth and survival.

In vivo targeting of this Super Enhancer activity in patient-derived xenograft models using CRISPRi showed significant reduction of tumor burden and increased overall survival.

Our results demonstrate that genome-wide screening of regulatory DNA elements can identify co-regulated genes collaborating to promote leukemia progression and could open new avenues for the design of combination therapies.

Reference:

Thirant C, Ignacimouttou C, Lopez CK, Diop M, Le Mouël L, Thiollier C, Siret A, Dessen P, Aid Z, Rivière J, Rameau P, Lefebvre C, Khaled M, Leverger G, Ballerini P, Petit A, Raslova H, Carmichael CL, Kile BT, Soler E, Crispino JD, Wichmann C, Pflumio F, Schwaller J, Vainchenker W, Lobry C, Droin N, Bernard OA, Malinge S, Mercher T (2017). ETO2-GLIS2 Hijacks Transcriptional Complexes to Drive Cellular Identity and Self-Renewal in Pediatric Acute Megakaryoblastic Leukemia. Cancer Cell. 31(3):452-465.