The cell fate determinant Llgl1 influences HSC fitness and prognosis in AML

Abstract

A unique characteristic of hematopoietic stem cells (HSCs) is the ability to self-renew. Several genes and signaling pathways control the fine balance between self-renewal and differentiation in HSCs and potentially also in leukemia stem cells. Recently, studies have shed light on developmental molecules and evolutionarily conserved signals as regulators of stem cells in hematopoiesis and leukemia. In this study, we provide evidence that the cell fate determinant Llgl1 (lethal giant larvae homolog 1) plays an important role in regulation of HSCs. Loss of Llgl1 leads to an increase in HSC numbers that show increased repopulation capacity and competitive advantage after transplantation. This advantage increases upon serial transplantation or when stress is applied to HSCs. Llgl1(-/-) HSCs show increased cycling but neither exhaust nor induce leukemia in recipient mice. Llgl1 inactivation is associated with transcriptional repression of transcription factors such as KLF4 (Krüppel-like factor 4) and EGR1 (early-growth-response 1) that are known inhibitors of HSC self-renewal. Decreased Llgl1 expression in human acute myeloid leukemia (AML) cells is associated with inferior patient survival. Thus, inactivation of Llgl1 enhances HSC self-renewal and fitness and is associated with unfavorable outcome in human AML.

Figure 1.

Loss of Llgl1 increases the amount and competitive advantage of HSCs. (A) Peripheral blood counts after conditional deletion of Llgl1 during steady-state hematopoiesis. (B and C) Numbers of HSCs (CD34^lo, Flk2⁻ LSK and CD34^lo, Flk2⁻ CD48⁻ SLAM⁺ LSK) 16 wk after deletion. (D and G) Llgl1^−/− cells competed better against WT competitor cells (Llgl1^+/+ littermate controls) in peripheral blood (D)– and BM-HSC chimerism (G; two independent cohorts; n > 4 per cohort). (E, F, H, and I) Increase of competitive advantage over time throughout serial transplantations in total BM and HSCs of secondary (E and H) and tertiary (F and I) recipient mice (secondary and tertiary recipients received total unfractionated BM from primary recipient mice). (E and H) Horizontal lines represent the mean of each group. Error bars indicate the standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 2.

Enhanced HSC fitness after genetic inactivation of Llgl1 is independent of engraftment and detectable after ex vivo culture. (A) Conditional deletion of Llgl1 4 wk after transplantation of previously undeleted cells. Competitive advantage of Llgl1^−/− cells (compared with WT controls) in vivo in primary (left) and secondary (right) recipient mice (two independent cohorts, n > 3 per cohort) is shown. (B) Limiting dilution assay. Increased frequency of HSCs in Llgl1^−/− cohorts compared with WT controls 14 wk after transplantation is shown. (C) Homing of LSK cells in lethally irradiated recipient mice. (D and E) Repopulation capacity of Llgl1^−/− cells after ex vivo culture of HSCs for 5 d. (D) Peripheral blood chimerism at 4 wk. *, P < 0.05; **, P < 0.01. (E) BM and HSC chimerism 16 wk after transplantation (two independent cohorts; >7 mice/dilution). (F) Radioprotection of lethally irradiated recipient mice (survival after ex vivo culture of transplanted HSCs). Error bars indicate the standard deviation.

Figure 3.

Llgl1 protects quiescence of LT-HSCs. (A and B) HSC cell cycle in steady-state hematopoiesis after genetic inactivation of Llgl1. (B) Distribution of cell cycle phases in HSCs (34^lo LSK), MPPs, GMPs, or MEPs (2 cohorts, n = 6) after genetic inactivation of Llgl1. Error bars indicate the standard deviation. *, P < 0.05. (C) Short-term BrdU incorporation confirms stem cell cycling in vivo.

Figure 4.

Llgl1 acts upstream of transcriptional activators of self-renewal capacity, and its repression is associated with leukemia development and an adverse prognosis in CN-AML. (A) GEP on HSCs (Flk2⁻ LSK) after conditional deletion of Llgl1 in steady-state hematopoiesis (14-wk-old mice, 10 wk after pIpC administration). The top 25 up- or down-regulated genes (by fold change) are displayed. (B) Hierarchical clustering of 436 AML cases along with the Llgl1^−/− signature. hc groups, hierarchical cluster–defined groups. (C) Clustering based on the overlap for the top 200 genes reveals a significant grouping into karyotype-associated groups (P < 0.001). The Llgl1^−/− signature clusters with a CN-AML predominated subgroup. (D) The Llgl1^−/− signature–associated subgroup is associated with inferior outcome (P = 0.0089). (E) GEP during development of LMPP and GMP into their malignant counterparts reveals Llgl1 to be the top down-regulated cell fate determinant. Expression of Llgl1 is reduced in LMPP-AML compared with LMPP and in GMP-AML compared with GMP. (F) Primary AML samples show a high variability in Llgl1 expression. AML patient samples reveal a decreased expression in AML when compared with the MPD cases or normal granulocytes. Error bars indicate the standard deviation. (G) Low expression of Llgl1 as measured by Affymetrix gene arrays was associated with decreased survival in 83 karyotypic normal AML (CN-AML) patients below the age of 60 yr (AMLSG). (H) This finding could be confirmed in an independent CN-AML cohort (n = 80; OSHO study group) by qRT-PCR (all patient samples measured in triplicate).