Abstract 1461: ETV6 deficiency and microsatellite enhancers drive transcriptional dysregulation in B-lymphoblastic leukemia
Authors: Goldman Joshua W. , Kodgule Rohan , Monovich Alexander C. , Saari Travis , Hall Cody N. , Rajesh Niharika , Gupta Juhi , Aguilar Athalee , Brown Noah , Chiang Mark Y. , Cieslik Marcin P. , Ryan Russell J. 
Published: June 2022

Abstract
Subtypes of B-lymphoblastic leukemia (B-ALL) are defined by unique genetic aberrations and gene expression programs. ETV6-RUNX1+ (E-R+) B-ALL, a common pediatric subtype, shares a gene activation signature with “ETV6-RUNX1-like” B-ALL that lacks the E-R fusion gene. Both E-R+ and E-R-like B-ALL show frequent inactivating mutations or deletions of ETV6, which encodes an ETS family transcriptional repressor (core binding motif GGAA/T). Signature genes overexpressed in E-R+ B-ALL, such as EPOR and PIK3C3, were shown to sustain the malignant phenotype.
We performed H3K27ac ChIP-Seq and ATAC-Seq on 26 B-cell cancer cell lines to identify subtype-specific active enhancer-like elements. Unbiased K-means clustering identified distal elements that were selectively acetylated in four E-R+ B-ALL cell lines (all with inactivation of the second ETV6 allele) and in a fifth B-ALL cell line with biallelic ETV6 deletion. Motif analysis of this cluster showed strong enrichment for tandem repeats of the sequence “GGAA” (p&lt;10-100). Repeats with ≥6 tandem copies of “GGAA” were selectively acetylated and accessible in H3K27ac ChIP-Seq and ATAC-Seq datasets from primary patient samples of E-R+ B-ALL, but not from other genetic subtypes that lack ETV6 aberrations.
Doxycycline-inducible expression of ETV6 in ETV6-deficient, E-R+ B-ALL cells led to significant growth inhibition. Restored ETV6 bound extensively to GGAA repeats (80% of ETV6 binding sites), repressed repeat enhancer acetylation, and repressed expression of adjacent E-R+ signature genes (n=40). Repression of a published E-R+ signature gene set was significant by GSEA analysis (p&lt;0.001). Direct CRISPR-interference repression of 6 GGAA repeat enhancers led to significant downregulation of their associated E-R+ signature genes, including EPOR and PIK3C3.
GGAA repeat enhancers are a known driver of Ewing sarcoma, where they are activated by oncogenic fusions of the ETS activators FLI1 or ERG. Active GGAA repeat enhancers and repeat-regulated genes in E-R+ B-ALL show only partial overlap with repeat-regulated genes in Ewings, possibly due to tissue-specific factors. Wild-type ERG and FLI1 are highly expressed in B-ALL. We found that knockdown of ERG, but not FLI1, led to significant downregulation of repeat enhancer-regulated E-R+ signature genes in B-ALL. ChIP-Seq showed that the ERG binds nearly all active GGAA repeat enhancers in three ETV6-deficient B-ALL cell lines, but not in ETV6-intact B-ALL.
We identify GGAA repeat enhancers, sustained by ETV6 deficiency and strong ERG expression, as a unifying mechanism for a subtype-specific oncogenic gene expression program in B-ALL. Our findings may have crucial implications for E-R+ B-ALL modeling, as most GGAA repeats are not conserved between mice and humans. Mechanisms that activate leukemia-specific repeat enhancers may represent appealing targets for future therapeutic development.
Citation Format: Joshua W. Goldman, Rohan Kodgule, Alexander C. Monovich, Travis Saari, Cody N. Hall, Niharika Rajesh, Juhi Gupta, Athalee Aguilar, Noah Brown, Mark Y. Chiang, Marcin P. Cieslik, Russell J. Ryan. ETV6 deficiency and microsatellite enhancers drive transcriptional dysregulation in B-lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1461.