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.

Keywords: Cell biology; Hematology; Hematopoietic stem cells; Leukemias; Mouse models; Oncology.

Figure 1. The hematopoietic stem cell MYB enhancer (H-Me) is a top-ranked essential regulatory element in T-ALL cells that appears relatively quiescent in the developmental stages from which T-ALL initiates.

(A) Schematic of the CRISPRi screen to identify essential ETS1-dependent enhancers in the THP-6 T-ALL cell line. (B) CRISPRi essentiality screen results. MAGeCK analysis provided in Supplemental Table 2. (C) ATAC-seq profiles of the murine H-Me in LT-HSCs through T cell development (Immgen). (D) ATAC-seq profiles of the human H-Me in sorted thymocyte subsets (GSE151075) (105). D1, donor 1; HSC, CD34⁺ cord blood; ETP/DN2, CD34⁺CD4^–CD1^–; DN3/DN4, CD34⁺CD4^–CD1⁺; ISP, CD28⁺CD4⁺CD3^–CD8^–; DP, CD4⁺CD8⁺; SP, single positive. (E) H3K27ac ChIP-seq profiles of normal thymocyte subsets at the H-Me and the N-Me in 50 kb windows (St. Jude Cloud, https://viz.stjude.cloud/mullighan-lab/collection/the-genomic-basis-of-childhood-t-lineageacute-lymphoblastic-leukemia~29). (F) Histone chromatin profiles of DP cells of 4 donors (D1–D4; BLUEPRINT project) comparing a silenced region with the MYB-AHI1 region.

Figure 2. The H-Me normally restricts HSC numbers and maintains ETPs under steady-state conditions.

(A) Schematic of the floxed H-Me allele (fl). SA, rabbit β-globin splice acceptor sequence (103). (B) RT-qPCR of Myb and Ahi1 in sorted LT-HSCs (CD150⁺CD48^– LSK cells) from Mx1Cre H-Me^Δ/Δ (Δ/Δ) and littermate control Mx1Cre (+/+) mice. (C–G) Representative BM Lineage^– flow cytometry plots (C) and absolute numbers of LT-HSCs (D), LSK cells (E), MPP/ST-HSCs (CD150⁺CD48^– LSKs) (F), and LMPPs (Lineage^–Sca-1⁺Kit^hiFlt3^hi) (G). (H–M) Representative thymus Lineage^– flow cytometry plots (H) and absolute numbers of ETP (Lineage^–CD44⁺CD25^–c-Kit^hi) (I) and DN2a (Lineage^–CD44⁺CD25⁺cKit^hi) (J), DN2b (Lineage^–CD44⁺CD25⁺c-Kit^lo) (K), DN3 (Lineage^–CD44^–CD25⁺) (L), and DN4 (Lineage^–CD44^–CD25^–) (M) cells. (N) Myb RT-qPCR in sorted DN cells from Il7rCre H-Me^Δ/Δ (Δ/Δ) and littermate control Il7rCre (+/+) mice. Unpaired 2-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. The H-Me is important for long-term HSC self-renewal under stress conditions.

(A) Schematic of serial competitive BMT experiment. (B–I) Peripheral blood analysis of indicated subsets tracking percent donor-derived (CD45.2⁺) cells after the first BMT (B–E) and the second BMT (F–I). (J–L) Representative Lineage^– flow cytometry plots (J) and percent donor-derived (CD45.2⁺) analysis of LT-HSCs (K) and MPP/ST-HSCs (L) at 16 weeks after the second BMT. Unpaired 2-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 4. The H-Me limits myeloid expansion and HSC loss with aging.

(A–J) WBC counts (A and F), neutrophil counts (B and G), monocyte counts (C and H), hemoglobin concentrations (D and I), and platelet counts (E and J) comparing the indicated mice at 8 (A–E) and 12 (F–J) months of age. (K–O) Representative flow cytometric plots (K) and absolute numbers of LT-HSCs (L), MPP/ST-HSCs (M), hematopoietic stem and progenitor cells (LSK) N), and total BM cells (O) in 1-year old mice as defined for Figure 2. (P–V) Representative flow cytometric plots (P) and absolute numbers of ETPs (Q) and DN2a (R), DN2b (S), DN3 (T), DN4 (U), and total thymocyte cells (V) as defined for Figure 1. Unpaired 2-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. The H-Me is an essential regulatory element for T-ALL leukemogenesis in multiple genetically engineered mouse models.

(A) Schematic showing generation of Notch-induced T-ALL by transducing ΔE/Notch1 retrovirus (54, 55) into hematopoietic stem and progenitor cells from H-Me^–/– mice or littermate controls, followed by transplantation. (B–D) Representative flow cytometry plots (B), GFP⁺ blast counts at 4 weeks after transplant (C), and leukemia-free survival curves (D) for the experiment in A. (E) Schematic showing generation of Notch-induced T-ALL by transducing ΔE/Notch1 retrovirus into hematopoietic stem and progenitor cells from Mx1Cre H-Me^fl/fl mice and Mx1Cre littermate controls, followed by transplantation and injection of pI-pC to delete the H-Me. (F–H) Representative flow cytometry plots (F), GFP⁺ blast counts at 4 weeks after injection of pI-pC (G), and leukemia-free survival curves (H) for the experiment in E. (I and J) Lmo2-tg H-Me^–/– and littermate control H-Me^+/+ mice were observed for survival (I) and development of T-ALL, which was confirmed by flow cytometry of thymic mass (mice #33, #98, and #61) or spleen mass (mouse #772) (J). Unpaired 2-tailed t test and log-rank test. ****P < 0.0001.

Figure 6. The H-Me is coamplified with MYB in nearly all T-ALL patients with MYB amplifications.

(A) Amplified region in primary human T-ALL in the WGS dataset from AALL0434 (n = 1,309 patients) (40). Each row represents a unique patient (n = 115). Bar shows the MYB–to–H-Me region. (B and C) WGS tracks showing sequence reads in the MYB-AHI1 region including the H-Me in THP-6 cells (B) and other T-ALL cell lines used in this study (C).

Figure 7. The H-Me is important in diverse models of human T-ALL maintenance.

(A) ATAC-seq profiles (green) of a panel of T-ALL cell lines and primary samples across the MYB TAD. ETS1 and H3K27ac tracks in THP-6 cells are shown in blue (GSE138516). MYB enhancers labeled A and B were previously described (–64). ATAC-seq datasets from GSE129086, GSE110630, GSE263585, GSE263977, and GSE225559; TAD datasets from GSE134761. (B–G) THP-6 (B and E), Jurkat (C and F), and MOLT14 (D and G) cells were transduced with constitutive (THP-6) or Dox-inducible (Jurkat, MOLT14) dCas9-KRAB and sgRNAs against the H-Me or the MYB promoter and then tested for expression of MYB (B–D) and measured for cell growth (E–G). HBS1L and AHI1 are flanking genes of MYB. (H–J) CEM cells, related to THP-6, transduced with the indicated sgRNAs were injected into NSG mice and 2–5 days later treated with Dox in drinking water to activate dCas9-KRAB–GFP. Representative GFP/hCD45.2 flow cytometry plots of peripheral blood at 4 weeks after injection (H), GFP⁺/hCD45⁺ blast counts (I), and survival (log-rank test P values) (J) were measured. n = 9 (control); n = 10 (PE); n = 10 (H-Me). NT, nontargeting; PE, pan-essential gene RPL34. 1-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 8. ETS1 recruits cBAF through the ETS motif in the H-Me to activate MYB expression in THP-6/CEM cells.

(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-FKBP^F36V 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 (–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-FKBP^F36V–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 P_adj < 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-FKBP^F36V–knockin THP-6 cells treated with 500 nM dTAG to degrade ETS1. (M) ATAC-seq profiles of the MYB TAD in ETS1-FKBP^F36V–knockin THP-6 cells treated with dTAG to degrade ETS1. DeSeq2 analysis. (N) MYB RT-qPCR in ETS1-FKBP^F36V–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.