Fig 2: Spi1 expression is maintained by β-catenin-SPI1-HAVCR2 regulatory circuit.(A) q-PCR analysis of SPI1 and SPI1-regulated HAVCR2 and c-MYC expressions after the overexpression of active β-catenin in the Jurkat T-ALL cell line (red bars). The data are normalized to that of empty plasmid controls (blue bars); (B–E) Upper panels: quantitative intracellular FACS analyses of P-GSK-3β, non-phospho-β-catenin, P-p65 and SPI1 levels in the HAVCR2high, HAVCR2low and blast subgroups; lower panels: representative intracellular FACS analysis of P-GSK-3β, non-phospho-β-catenin, P-p65 and SPI1 levels in the HAVCR2high, HAVCR2low and blast subgroups. Gray line, isotype control;(F) FACS analysis shows cells in the HAVCR2high subgroup at the ETP/DN1 stage, which are absent in WT and dKO mice; (G) Representative FACS plots show the number of cells in the HAVCR2high subgroup in the different drug treatment groups. The data in A, B, C, D and E are the means ± S.Ds of 3 independent tests; *p≤0.05; **p≤0.01; ***p≤0.001.

Fig 3: SPI1 is the master regulator of LSC signature genes and controls HAVCR2 and c-MYC expression.(A) Nearly 70% of the genes highly expressed in the HAVCR2high subgroup—the LSC signature genes—are potential SPI1 target genes (purple);(B) Pseudotemporal ordering of single cells based on Spi1 or Myc expression; (C) FACS analysis shows the correlation of HAVCR2 cell surface expression and intracellular SPI1 and c-MYC levels in the LSC-enriched (Lin-CD3+KITmid; red) and blast (Lin-CD3+KIT-; blue) subpopulations. Gray, isotype control; (D–E) ChIP-qPCR analysis identifies SPI1 binding regions in the HAVCR2 promoter (left) and Tcra/d enhancer A(EA) region (right), using Blast-SPI1 cells; (F) ChIP analysis identifies a SPI1 binding site in the endogenous Myc enhancer;(G) q-PCR shows the fold changes in Havcr2, Itgax, Lmo2 and Myc expression between Blast-SPI1 cells (red) and Blast-EGFP cells (blue); (H) Western blotting shows the SPI1, HAVCR2 and c-Myc protein levels in WT thymus, Blast-EGFP and Blast-SPI1 cells. The fold changes relative to expression in the WT thymus are shown above each lane; (I) q-PCR analysis shows the fold changes in HAVCR2, ITGAX, LMO2 and MYC expression in control shRNA (blue) and shSPI1 knockdown human T-ALL KE-37 cells (red); (D–I) All experiments were performed at least three independent times, and the data in D, E, F, G, and I are the means ± S.Ds; *p≤0.05; **p≤0.01; ***p≤0.001.

Fig 4: Gal-9 is essential for the Pten null T-ALL LSC formation and leukemia development.(A). Deletion of the HAVCR2 ligand Gal-9 delays or prevents the Pten null T-ALL development in vivo. (B). HAVCR2high LSCs at ETP stage in the Pten null T-ALL are eliminated by SPI1 or Gal-9 deletion in vivo.

Fig 5: ETP and non-ETP T-ALL have different HAVCR2 and SPI1 expression levels and leukemia reconstitution abilities.(A). The HAVCR2 and SPI1 expression levels are significant higher in ETP T-ALL samples as compared to non-ETP T-ALL samples. ***, p<0.001. (B). Bone marrow cells (1X106) from human T-ALL patients were i.v. injected into the NSG mice. Leukemia reconstitution was determined when >25% of the cells in the peripheral blood were human CD45+;CD7+ leukemic cells.