The hematopoietic stem cell MYB enhancer is essential for and recurrently amplified during T cell leukemogenesis
Carea Mullin, Karena Lin, Elizabeth Choe, Cher Sha, Zeel Shukla, Koral Campbell, Anna C. McCarter, Annie Wang, Jannaldo Nieves-Salva, Sarah Khan, Theresa M. Keeley, Shannon Liang, Qing Wang, Ashley F. Melnick, Pearl Evans, Alexander C. Monovich, Ashwin Iyer, Rohan Kodgule, Yamei Deng, Felipe da Veiga Leprevost, Kelly R. Barnett, Petri Pölönen, Rami Khoriaty, Daniel Savic, David T. Teachey, Charles G. Mullighan, Marcin Cieslik, Alexey I. Nesvizhskii, Linda C. Samuelson, Morgan Jones, Qing Li, Russell J.H. Ryan, Mark Y. Chiang
Carea Mullin, Karena Lin, Elizabeth Choe, Cher Sha, Zeel Shukla, Koral Campbell, Anna C. McCarter, Annie Wang, Jannaldo Nieves-Salva, Sarah Khan, Theresa M. Keeley, Shannon Liang, Qing Wang, Ashley F. Melnick, Pearl Evans, Alexander C. Monovich, Ashwin Iyer, Rohan Kodgule, Yamei Deng, Felipe da Veiga Leprevost, Kelly R. Barnett, Petri Pölönen, Rami Khoriaty, Daniel Savic, David T. Teachey, Charles G. Mullighan, Marcin Cieslik, Alexey I. Nesvizhskii, Linda C. Samuelson, Morgan Jones, Qing Li, Russell J.H. Ryan, Mark Y. Chiang
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Research Article Cell biology Hematology Oncology

The hematopoietic stem cell MYB enhancer is essential for and recurrently amplified during T cell leukemogenesis

Abstract

There is an urgent need to find targeted agents for T cell acute lymphoblastic leukemia (T-ALL). NOTCH1 is the most frequently mutated oncogene in T-ALL, but clinical trials showed that pan-Notch inhibitors caused dose-limiting toxicities. Thus, we shifted our focus to ETS1, which is one of the transcription factors that most frequently co-bind Notch-occupied regulatory elements in the T-ALL context. To identify the most essential enhancers, we performed a genome-wide CRISPRi screen of the strongest ETS1-dependent regulatory elements. The top-ranked element is located in an intron of AHI1 that interacts with the MYB promoter and is amplified with MYB in approximately 8.5% of patients with T-ALL. Using mouse models, we showed that this enhancer promoted self-renewal of hematopoietic stem cells and T cell leukemogenesis, maintained early T cell precursors, and restrained myeloid expansion with aging. We named this enhancer the hematopoietic stem cell MYB enhancer (H-Me). The H-Me showed limited activity and function in committed T cell progenitors but was accessed during leukemogenesis. In one T-ALL context, ETS1 bound the ETS motif in the H-Me to recruit cBAF to promote chromatin accessibility and activation. ETS1 or cBAF degraders impaired H-Me function. Thus, we identified a targetable stem cell element that was co-opted for T cell transformation.

Authors

Carea Mullin, Karena Lin, Elizabeth Choe, Cher Sha, Zeel Shukla, Koral Campbell, Anna C. McCarter, Annie Wang, Jannaldo Nieves-Salva, Sarah Khan, Theresa M. Keeley, Shannon Liang, Qing Wang, Ashley F. Melnick, Pearl Evans, Alexander C. Monovich, Ashwin Iyer, Rohan Kodgule, Yamei Deng, Felipe da Veiga Leprevost, Kelly R. Barnett, Petri Pölönen, Rami Khoriaty, Daniel Savic, David T. Teachey, Charles G. Mullighan, Marcin Cieslik, Alexey I. Nesvizhskii, Linda C. Samuelson, Morgan Jones, Qing Li, Russell J.H. Ryan, Mark Y. Chiang

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Figure 8

ETS1 recruits cBAF through the ETS motif in the H-Me to activate MYB expression in THP-6/CEM cells.

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ETS1 recruits cBAF through the ETS motif in the H-Me to activate MYB exp...
(A and B) Western blot of ETS1 and MYB proteins (A) and MYB RT-qPCR (B) showing the effect of 500 nM dTAG in degrading ETS1 in ETS1-FKBPF36V knockin THP-6 cells. (C and D) Representative Western blots (C) and quantitative ImageJ analyses (D) showing effect of the ETS motif mutation on ETS1 binding (p54 and p42 isoforms) in reverse ChIP in THP-6 cells. (E) Normalized abundance plot of transcriptional regulators that were pulled down by reverse ChIP and identified by mass spectrometry comparing WT and ETS motif–mutated H-Me; and analyzed with PD (Thermo Fisher Proteome Discoverer) and FragPipe (106–108). Full results are provided in Supplemental Table 3. (F–H) Sequences of homozygous partially mutated ETS sites in the 3 H-Me alleles (F), ETS1 qChIP at the H-Me (G); and MYB RT-qPCR (H) in a subclone of ETS1-FKBPF36V–knockin THP-6 cells after CRISPR/Cas9 editing and HDR. (I) Venn diagram showing intersection of the H-Me and ETS1 interactomes ranked by strength of interaction with Flag-ETS1. The H-Me interactome was supplemented with transcriptional regulators that met Padj < 0.1/ LFC (Log2 Fold Change). > 0 criteria by Proteome Discoverer. Full results are provided in Supplemental Tables 4 and 5. (J) Flag co-IP assay in vector-transduced (Ctrl) and Flag-ETS1–transduced CEM cells showing interactions with endogenous SMARCC1 and SMARCB1. (K) Reciprocal co-IP assay comparing IgG and anti-SMARCC1 pulldowns in CEM cells to detect interactions with endogenous ETS1. (L) ARID1A and PBRM1 qChIP using primers at the H-Me peak center or a negative control site 1.25 kb downstream in ETS1-FKBPF36V–knockin THP-6 cells treated with 500 nM dTAG to degrade ETS1. (M) ATAC-seq profiles of the MYB TAD in ETS1-FKBPF36V–knockin THP-6 cells treated with dTAG to degrade ETS1. DeSeq2 analysis. (N) MYB RT-qPCR in ETS1-FKBPF36V–knockin THP-6 cells with mutated ETS-binding sites in the H-Me (F) treated with DMSO versus AU-15330. (O) Western blot for the indicated proteins in DMSO-treated or AU-15330–treated T-ALL cells. 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.