MLL-AF9- and HOXA9-mediated acute myeloid leukemia stem cell self-renewal requires JMJD1C

Abstract

Self-renewal is a hallmark of both hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs); therefore, the identification of mechanisms that are required for LSC, but not HSC, function could provide therapeutic opportunities that are more effective and less toxic than current treatments. Here, we employed an in vivo shRNA screen and identified jumonji domain-containing protein JMJD1C as an important driver of MLL-AF9 leukemia. Using a conditional mouse model, we showed that loss of JMJD1C substantially decreased LSC frequency and caused differentiation of MLL-AF9- and homeobox A9-driven (HOXA9-driven) leukemias. We determined that JMJD1C directly interacts with HOXA9 and modulates a HOXA9-controlled gene-expression program. In contrast, loss of JMJD1C led to only minor defects in blood homeostasis and modest effects on HSC self-renewal. Together, these data establish JMJD1C as an important mediator of MLL-AF9- and HOXA9-driven LSC function that is largely dispensable for HSC function.

Figure 1. In vivo shRNA screening of MLL-AF9 targets identifies JMJD1C as essential for MLL-AF9 leukemia.

(A) Schematics of in vivo shRNA screen. (B) Bar graph of log₂ fold change in the BM of all hairpins used in the screen. (C) Bar graph of log₂ fold change of hairpins against JMJD1C, showing positive (HOXA9, MEIS1, CTNNB1, MLL-AF9 [GFP]) and negative (LacZ, luciferase, and Rfp) hairpins in the screen. (D) Left panel: heat map of microarray gene-expression data (14) on MLL-AF9 target genes (28). Right panel: Jmjd1c expression data derived from microarray data (14). Data are represented as mean ± SEM in D. See also Supplemental Figure 1 and Supplemental Tables 1 and 2.

Figure 2. Loss of JMJD1C decreases LSC frequency in established MLL-AF9 leukemia.

(A) Schematics of conditional knockout allele of Jmjd1c. (B) Colony counts of serial replating of primary MLL-AF9 leukemia after transduction of CRE or MIT control viruses in methylcellulose. Duplicate samples of 2 independent Jmjd1c^f/f and 1 WT leukemia are shown. (C) Morphologic changes (left, colony in methylcellulose; right, Wright-Giemsa stain) in established MLL-AF9 leukemia 7 days after transduction with CRE. Scale bars: 100 μm (left panels). Original magnification, ×400 (right panels). (D and E) Flow cytometry analysis of apoptosis (D) and cell cycle by BrdU and sytox blue (E) in MLL-AF9 leukemia 6 days after transduction with CRE. Results from 3 independent leukemias for D and E. (F) Genotyping result of PB day 14 after pIpC and BM at the time of sacrifice. (G) Survival curve of secondary recipient mice that received Mx1-Cre (n = 7), Jmjd1c^f/f(n = 6), or Jmjd1c^f/f Mx1-Cre (n = 7) MLL-AF9 leukemia after pIpC administration. Data are represented as mean ± SEM in B, D, and E. *P < 0.05, Student’s t test. See also Supplemental Figure 2.

Figure 3. JMJD1C interacts with HOXA9 and modulates a HOXA9-controlled gene-expression program.

(A) Heat map of differentially expressed genes in MLL-AF9 leukemia cells 6 days after loss of JMJD1C. (B) GSEA analysis result showing enrichment of HOXA9 repressed genes (37) and HOXA9 bound and repressed genes (44). NES, normalized enrichment score. (C) Co-IP of HA-HOXA9 and KDM3A family members in 293T cells transfected with indicated plasmids. (D) GST-binding assay using bacteria expressed and purified GST-HOXA9 and baculovirus system expressed and purified flag-JMJD1C. See also Supplemental Figure 3 and Supplemental Table 4.

Figure 4. Loss of JMJD1C impairs leukemic transformation by HOXA9/MEIS1.

(A) Colony counts of HOXA9/MEIS1 transformed LSKs from Jmjd1c^f/f BM after transduction of CRE or MIT control viruses in methylcellulose. (B) Colony counts of AML-ETO9a transformed LIN^– Jmjd1c^f/f BM cells after transduction of CRE or MIT control viruses in methylcellulose. Results from 2 to 3 independent experiments for A and B. (C) Morphologic changes (left, colony in methylcellulose; right, Wright-Giemsa stain) in HOXA9/MEIS1 preleukemia cells 10 days after transduction with CRE. Scale bars: 100 μm (left panels). Original magnification, ×400 (right panels). (D–F) Flow cytometry analysis of c-Kit expression (D), cell-cycle analysis by BrdU and sytox blue (E), apoptosis analysis (F) in HOXA9/MEIS1 leukemic cells day 5 after transduction with CRE. Results from 3 independent leukemias. (G) Survival curves of secondary recipient mice that received 100,000; 20,000 or 5,000 double-sorted GFP⁺Tomato⁺ HOXA9/MEIS1 leukemia cells 2 days after transduction with CRE or MIT control viruses (n = 5 per group). Right panel: genotyping result of BM at the time of sacrifice. Data are represented as mean ± SEM in A, B, and D–F. *P < 0.05; **P < 0.01 Student’s t test.

Figure 5. Loss of JMJD1C affected a HOXA9-controlled gene-expression program in HOXA9/MEIS1 leukemia.

(A) Heat map showing differentially expressed genes in HOXA9/MEIS1 leukemia cells 6 days after deleting Jmjd1c. (B) GSEA analysis result showing enrichment of HOXA9 repressed genes (37) and HOXA9 bound and repressed genes (44). See also Supplemental Table 5. (C) Venn diagram of overlapping genes that are upregulated upon loss of JMJD1C in MLLL-AF9 and HOXA9/MEIS1 leukemia cells. P value calculated by exact hypergeometric probability.

Figure 6. Effect of loss of JMJD1C in steady-state hematopoiesis.

(A) Genotyping result of BM, SPL, and LSK (Lin^–Sca-1⁺ c-Kit⁺) cells from Jmjd1c^f/f Vav1-Cre mice. (B) PB counts and (C) BM (from tibias, femurs, and pelvic bones) and SPL cellularity of 6- to 8-week-old Vav1-Cre and Jmjd1c^f/f Vav1-Cre mice. (D and E) HSPC (D) and mature cell (E) numbers in 8-week-old Vav1-Cre and Jmjd1c^f/f Vav1-Cre mice (n = 3–4 per genotype ). Data are represented as mean ± SEM in B–E. *P < 0.05, Student’s t test. See also Supplemental Figure 4.

Figure 7. Effect of loss of JMJD1C on regenerative function of HSC.

(A–F) Flow cytometry analysis shows results of chimerism of recipient mice from serial transplantation experiments. (A–C) One million BM cells from Vav1-Cre or Jmjd1c^f/f Vav1-Cre mice were mixed 1:1 with CD45.1 BM and transplanted into lethally irradiated CD45.1 recipients. (n = 10). (D–F) Two million BM cells from primary competitive transplantation were serially transplanted into secondary CD45.1 recipients (n = 5). Chimerism in PB (A) and (D); BM and SPL (B) and (E); stem and progenitor populations in BM (C) and (F). (G) Heat map of differentially expressed genes in Vav1-Cre and Jmjd1c^f/f Vav1-Cre LSK cells. (H) GSEA analysis result showing enrichment of E2F1, Myc, and cyclin D1 signature. (I) Percentage of cells in S/G₂/M phase of cell cycle determined by Sytox blue staining (n = 3 per genotype). (J) Time course of wbc counts after 5-Fu administrations (indicated by arrows, n = 5 per genotype). (K) Percentage of apoptotic cells determined by annexin V staining (n = 3 per genotype). (L) HSPC numbers 2 weeks after 5-Fu treatment (n = 3–4 per genotype). Data are represented as mean ± SEM in A–F and I–L. *P < 0.05. See also Supplemental Figures 5 and 6 and Supplemental Table 6.