Leukemia stem cells in T-ALL require active Hif1α and Wnt signaling

Abstract

The Wnt signaling pathway has been shown to play important roles in normal hematopoietic stem cell biology and in the development of both acute and chronic myelogenous leukemia. Its role in maintaining established leukemia stem cells, which are more directly relevant to patients with disease, however, is less clear. To address what role Wnt signaling may play in T-cell acute lymphoblastic leukemia (T-ALL), we used a stably integrated fluorescent Wnt reporter construct to interrogate endogenous Wnt signaling activity in vivo. In this study, we report that active Wnt signaling is restricted to minor subpopulations within bulk tumors, that these Wnt-active subsets are highly enriched for leukemia-initiating cells (LICs), and that genetic inactivation of β-catenin severely reduces LIC frequency. We show further that β-catenin transcription is upregulated by hypoxia through hypoxia-inducible factor 1α (Hif1α) stabilization, and that deletion of Hif1α also severely reduces LIC frequency. Of note, the deletion of β-catenin or Hif1α did not impair the growth or viability of bulk tumor cells, suggesting that elements of the Wnt and Hif pathways specifically support leukemia stem cells. We also confirm the relevance of these findings to human disease using cell lines and patient-derived xenografts, suggesting that targeting these pathways could benefit patients with T-ALL.

Figure 1

LICs are enriched within a small tumor subpopulation with active Wnt signaling. (A) Schematic diagram of the integrated fluorescent Wnt reporters, 7TGP and 7TGC, used to mark leukemia cells with active Wnt signaling. The reporter element is composed of 7 Tcf/Lef-binding sites upstream of a minimal promoter and GFP, followed by a separate SV40-puromycin (7TGP) or SV40-Cherry (7TGC) selection marker. The construct backbone is a self-inactivating lentiviral vector. (B) Schematic diagram of experimental approach. Primary mouse NOTCH1-ΔE leukemias were explanted and transduced with 7TGP or 7TGC lentivirus. 7TGP-transduced cells were selected with 1 μg/ml puromycin on MS5-DL1 puro feeder cells for 5 days beginning on day 2 posttransduction. 7TGC-transduced cells were FACS-sorted on day 2 posttransduction. Transduced cells were then transplanted into recipient mice, all of which subsequently developed leukemia. These secondary 7TGP/7TGC-transduced leukemias were then analyzed by flow cytometry for GFP expression. GFP⁺ and GFP⁻ subsets were then FACS sorted (also Cherry⁺ in 7TGC experiments) and transplanted at limiting dilution into tertiary recipients. (C) Summary of GFP⁺ cell abundance in Wnt reporter-transduced leukemias after passage in vivo. Freshly explanted BM from clinically morbid recipients of 7TGP or 7TGC-transduced primary leukemias was analyzed by flow cytometry for GFP expression within gated NGFR⁺ (7TGP) or NGFR⁺ Cherry⁺ (7TGC) leukemia cell populations (labeled “2° leukemia” in [B]). Results depicted are compiled from 10 independent primary leukemias (26 recipients in total; 11 × 7TGP and 15 × 7TGC). Each data point represents an individual mouse. Mean ± standard deviation (SD) values are indicated by horizontal lines. (D) Survival of recipient mice after transplantation with FACS-sorted Wnt active (GFP⁺) or Wnt inactive (GFP⁻) subsets from 7TGP and 7TGC-transduced secondary leukemias. Each of 4 recipient animals (n = 4) was injected with each of the cell doses as indicated in parentheses. Two of 3 transplant experiments using independent primary leukemias are depicted. ****P < .0001 (log-rank test). LTR, long terminal repeat; sinLTR, self-inactivating LTR.

Figure 2

Inhibition of Wnt signaling eliminates LICs. (A) Survival of recipient mice after transplantation with leukemias transduced by dnTCF lentivirus to block β-catenin signaling. Primary mouse NOTCH1-ΔE leukemias were explanted and transduced with dnTCF/GFP or empty virus control. Two days later, GFP⁺ leukemia cells were FACS-sorted and injected into each of 4 recipient animals (n = 4) at a dose of 1 × 10⁵ cells per mouse. Results depicted are compiled from 2 separate transplant experiments using independent primary leukemias (#V10-1 and #V12-1). **P < .01 (log-rank test). (B) Proliferation of mouse leukemias following transduction with dnTCF lentivirus vs nontransduced cells present in the same culture as measured by 5-bromo-2′-deoxyuridine incorporation. G1/G0, S, G2/M, and subG1 populations are gated as shown. Results depicted are representative of 2 independent leukemias analyzed (#V10-4 and #V12-1;3). (C) CFC assay for clonogenic activity. Primary mouse leukemias were transduced with dnTCF or empty control lentiviruses, FACS-sorted for the linked GFP marker, and plated in methylcellulose-containing media. *P < .05; **P < .01; ***P < .001 (Student t test). (D) Survival of recipient mice after transplantation with leukemias deleted of β-catenin. Primary mouse NOTCH1-ΔE leukemias on a Ctnnb1^loxP/loxP background were explanted, transduced with CreERT2/GFP lentivirus, and then treated with 4-OHT or vehicle (mock) control in vitro for 2 days. GFP⁺ leukemia cells were then FACS-sorted and injected into each of 4 recipient mice (n = 4) at each of the cell doses indicated in parentheses. Two separate experiments are depicted using independent primary leukemias. ****P < .0001 (log-rank test). (E-F) Cell growth/viability assays. Primary mouse NOTCH1-ΔE leukemias generated on a Ctnnb1^loxP/loxP background were transduced with CreERT2/GFP lentivirus, and then treated with 4-OHT or vehicle (mock) in vitro. In (E), relative cell growth was assessed by tracking the % GFP⁺ cells over time in culture by flow cytometry. A decreasing GFP⁺ fraction indicates transduced (GFP⁺) cells are growth disadvantaged compared with nontransduced (GFP⁻) cells in the same culture. In (F), absolute viable cell numbers were tracked over a 3-day culture period by flow cytometry with admixed polystyrene counting beads. Mean ± SD values are plotted.

Figure 3

Hypoxia potentiates Wnt signaling and supports clonogenic activity. (A) Flow cytometric analysis of pimonidazole staining, a marker of hypoxia. Mice were transplanted with 7TGP-transduced leukemia cells and, after developing clinically morbid disease, were injected by tail vein with pimonidazole (60 mg/kg). One hour later, mice were euthanized and BM cells harvested, fixed, and stained with anti-pimonidazole antibody. Gated NGFR⁺ leukemia cells are shown. The marrow contained >90% tumor cells (of which 40% to 70% were GFP⁺). Results depicted are representative of 2 different leukemic animals. (B) Immunohistochemical staining for GFP in BM sections of mice following transplantation with 7TGP-transduced leukemia cells. Mice were clinically morbid with disease at the time they were euthanized and contained >90% tumor cells (of which 40% to 70% were GFP⁺) within the marrow. Red arrowheads indicate individual GFP⁺ cells. Images depicted are representative of 2 different leukemic animals. Scale bar = 60 μm. (C-D) Analysis of β-catenin protein expression level in NOTCH1-ΔE mouse leukemia cells cultured in vitro under reduced oxygen conditions. Cells were cultured in complete growth media under 20% or 5% oxygen for 3 days in plastic tissue culture dishes, then harvested for analysis by western blot (C) and flow cytometry (D). The numbers below the β-catenin blot panel in (C) indicate relative band intensities after normalization to the β-actin loading control. Gated leukemia cells are depicted in (D). Results from at least 2 independent leukemias are shown. (E-F) CFC assay for in vitro clonogenic activity. Primary leukemia cells were plated in methylcellulose-containing media supplemented or not with Wnt3a ligand or Dickkopf-related protein 1 and cultured in either 20% or 5% oxygen. Each colored data point depicted represents an individual culture dish. Mean ± SD values are indicated by horizontal lines. Results in (F) are representative of 2 different leukemias. *P < .05; **P < .01; ****P < .0001 (Student t test in [E], 2-way analysis of variance [ANOVA] with Bonferroni multiple comparisons test in [F]). ns, not significant.

Figure 4

Hif1α contributes to β-catenin transcription directly. (A) Flow cytometric analysis of β-catenin protein levels. 7TGC-transduced mouse NOTCH1-ΔE leukemias were transduced with Hif1αTM (normoxia-stable, active Hif1α P402A/P577A/N813A triple mutant) or empty virus control, and cultured in vitro for 2 days on MS5 stromal feeders. Leukemia cells carrying the Hif1αTM or empty lentivirus were FACS-sorted by the linked hCD8 marker, then stained for intracellular β-catenin protein and analyzed by flow cytometry. Results depicted are representative of 3 different leukemias. (B) Flow cytometric analysis for Wnt reporter GFP expression. 7TGC-transduced leukemia cells were transduced with Hif1αTM/hCD8 or empty lentivirus and cultured in vitro for 2 days on MS5 stromal feeders. Cells were then harvested and assayed by flow cytometry. Gated Cherry⁺ hCD8⁺ leukemia cells are shown. Results depicted are representative of 3 different leukemias. (C) Real-time quantitative reverse transcription PCR analysis of β-catenin mRNA levels. The 144CLP mouse T-ALL cell line and 2 independent mouse leukemias were transduced with Hif1αTM or empty virus control, FACS-sorted for the linked GFP marker 2 days later, and then lysed for RNA. Mean ± SD values of triplicate assays are plotted. *P < .05; ***P < .001 (Student t test). (D) Map of the mouse Ctnnb1 locus with putative Hif1α binding sites indicated with asterisks. Primer pairs used for quantitative local ChIP assays are indicated by arrowheads with associated map coordinates. (E) Alignment of putative Hif1α binding sites within the mouse Ctnnb1 locus. Six regions containing Hif1α consensus binding sites are shown with the core RCGTGC motif indicated by underlined text in large font. Map coordinates corresponding to the positions assayed by local ChIP-PCR in (F) are indicated by underlined numbers in large font. Locus coordinates are from the GRCm38/mm10 genome assembly. (F) ChIP for Hif1α occupancy over the mouse Ctnnb1 locus. The 144CLP cell line was transduced with Myc epitope-tagged Hif1αTM or empty virus control, and FACS-sorted for the linked GFP marker 3 days later. Sheared chromatin was immunoprecipitated with anti-Myc tag (TAG) or IgG control antibody. Quantitative PCR was performed using primer pairs flanking putative Hif1α binding sites as indicated in (C). Data were normalized to their respective input DNA controls. Mean ± SD values of triplicate assays are plotted. **P < .01; ****P < .0001 (Student t test). FSC, forward light scatter.

Figure 5

Deletion of Hif1α eradicates LICs. (A) Survival of recipient mice after transplantation with leukemias deleted of Hif1α. Two independent mouse NOTCH1-ΔE leukemias on Hif1α^loxP/loxP background were explanted, transduced with CreERT2/GFP only or CreERT2/GFP and Hif1αTM/Cherry viruses, and then treated with 4-OHT or vehicle (mock) control in vitro. GFP⁺ or GFP⁺ Cherry⁺ leukemia cells, respectively, were then FACS-sorted and injected into each of 4 recipient animals (n = 4) at a dose of 1 × 10⁵ cells per mouse. ***P < .001 (log-rank test). (B-C) Growth/survival of Hif1α^Δ/Δ leukemia cells cultured in vitro. Primary mouse Hif1α^loxP/loxP leukemias were explanted, transduced with CreERT2/GFP virus, and then treated with 4-OHT or vehicle (mock) in vitro. In (B), relative cell growth was assessed by tracking the % GFP⁺ cells over time, in culture by flow cytometry. A decreasing GFP⁺ fraction indicates transduced (GFP⁺) cells are growth disadvantaged compared with nontransduced (GFP⁻) cells in the same culture. In (C), transduced (GFP⁺) cells were FACS-sorted 2 days after initiation of 4-OHT treatment, then overall culture viability was assessed by flow cytometry for propidium iodide (PI) exclusion.

Figure 6

Effects of WNT inhibition on human T-ALL. (A) Survival of recipient NSG mice after transplantation with xenograft-expanded primary human T-ALL leukemias transduced by lentivirus encoding dnTCF or empty vector control. Leukemia cells were transduced with virus, cultured on MS5-DL1 feeders for 3 days, FACS-sorted for the viral GFP marker, and then injected into each of 4 recipient animals (n = 4) at a dose of 1 × 10⁴ cells per mouse. Results are depicted for 2 different patient leukemias (M71 and F1313) in each experiment. *P < .05; **P < .01 (log-rank test). (B-D) Cell growth as measured by resazurin reduction. Human T-ALL cell lines (HPBALL and RPMI 8402) or PDX tumor cells (M71) were treated in vitro with XAV-939 and/or the γ-secretase inhibitor (GSI) compound E at the doses indicated for 3 to 5 days prior to assay. For the PDX culture, results were normalized against MS5-DL1 feeder-only wells. Mean ± SD values of triplicate assays are plotted. **P < .01; ****P < .0001 (one-way ANOVA). In (D), GSI accounted for 51% of the variation (P < .0001), XAV for 39% of the variation (P < .0001), and their interaction for 3% of the variation (P = .0125) by 2-way ANOVA.

Figure 7

Effects of HIF inhibition on patient-derived T-ALL xenografts. (A) Survival of recipient NSG mice after transplantation with xenograft-expanded primary human T-ALL leukemias transduced by lentivirus encoding shRNAs against HIF1α or scrambled shRNA control. Leukemia cells were transduced with virus, cultured on MS5-DL1 feeders for 3 days, FACS-sorted for the viral GFP marker, and then injected into each of 4 recipient animals (n = 4) at a dose of 1 × 10⁴ cells per mouse. Results are depicted for 2 different patient leukemias (M71 and F1313) in each experiment. **P < .01 (log-rank test). (B-C) Cell growth/viability assays. Xenograft-expanded primary human T-ALL cells were transduced with 2 different lentiviral shRNAs against HIF1α or scrambled shRNA control. In (B), transduced (GFP⁺) cells were FACS-sorted 2 days after transduction and cultured in vitro on MS5-DL1 feeders. Cell viability was tracked daily by flow cytometry for PI dye exclusion. In (C), cells were cultured in vitro on MS5-DL1 feeders following viral transduction. Absolute numbers of viable GFP⁺ cells from the unsorted culture were tracked daily by flow cytometry for PI dye exclusion with admixed polystyrene counting beads. Mean ± SD values are plotted. ns, not significant.