S6K1 regulates hematopoietic stem cell self-renewal and leukemia maintenance

Abstract

Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) functions and promotes leukemogenesis. mTORC1 and mTORC2 differentially control normal and leukemic stem cell functions. mTORC1 regulates p70 ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding (eIF4E-binding) protein 1 (4E-BP1), and mTORC2 modulates AKT activation. Given the extensive crosstalk that occurs between mTORC1 and mTORC2 signaling pathways, we assessed the role of the mTORC1 substrate S6K1 in the regulation of both normal HSC functions and in leukemogenesis driven by the mixed lineage leukemia (MLL) fusion oncogene MLL-AF9. We demonstrated that S6K1 deficiency impairs self-renewal of murine HSCs by reducing p21 expression. Loss of S6K1 also improved survival in mice transplanted with MLL-AF9-positive leukemic stem cells by modulating AKT and 4E-BP1 phosphorylation. Taken together, these results suggest that S6K1 acts through multiple targets of the mTOR pathway to promote self-renewal and leukemia progression. Given the recent interest in S6K1 as a potential therapeutic target in cancer, our results further support targeting this molecule as a potential strategy for treatment of myeloid malignancies.

Figure 1. S6K1 regulates HSC/Ps frequencies and absolute numbers in the BM.

(A) Relative expression level of S6k1 in purified BM-derived hematopoietic subsets as assessed by quantitative real-time reverse-transcription PCR (qRT-PCR). Data are from a representative experiment performed twice independently in replicates of 4; mean ± SD. *P < 0.001; **P < 0.0001. (B) Total BM MNCs derived from WT and S6k1^–/– mice; mean ± SEM; n = 4–5/group. *P < 0.002, t test. Data representative of 3 independent experiments. (C) Frequency and absolute number of LT-HSCs in the BM of WT and S6k1^–/– mice. n = 4–5/group; mean ± SEM. *P < 0.02, t test. (D) Frequency and absolute number of hematopoietic progenitor subpopulations in bone marrow of WT and S6k1^–/– mice; n = 4–5/group; mean ± SEM. *P < 0.02; **P < 0.005, t test. (E) Frequency of Ki-67–negative LT-HSCs in the BM of WT and S6k1^–/– mice; mean ± SEM. *P < 0.01, t test. n = 4–5 mice/group. (F) Expression levels of genes regulating quiescence in LT-HSCs of WT and S6k1^–/– mice; mean ± SEM. *P < 0.005. Data are representative of 2 independent experiments performed in replicates of 3.

Figure 2. S6K1 regulates quiescence of HSCs following regeneration from myeloablative stress.

(A) BM LSK frequency in WT and S6k1^–/– mice following a single dose of 5-FU; mean ± SEM. *P < 0.03, t test. n = 8–18/group. (B) Quantitative representation of BrdU⁺ LSK cells in the BM of WT and S6k1^–/– mice on the ninth day after 5-FU treatment; mean ± SEM. *P < 0.05, t test. n = 3/group. Data are representative of 2 independent experiments. (C) Expression levels of Ccng1 in BM LSK cells of WT and S6k1^–/– mice on ninth day after 5-FU treatment. Data are from a representative experiment performed twice independently. Experiments performed in quadruplicates; mean ± SD. *P < 0.01. (D) Kaplan-Meier survival curve of WT and S6k1^–/– mice treated with weekly doses of 5-FU. *P < 0.001. n = 11–15/group. (E) Kaplan-Meier survival curve of WT mice transplanted with WT and S6k1^–/– HSC/Ps and treated with weekly doses of 5-FU. *P < 0.007. n = 10/group. (F) WT and S6k1^–/– mice were treated with a single dose of 5-FU. After 6 days, MNCs from treated mice were transplanted into lethally irradiated recipients at a dilution of 1:8. Quantitative representation of donor-derived cells in PB of recipients is shown; mean ± SEM. n = 3/group. *P < 0.05, 1-way ANOVA.

Figure 3. S6K1 is a positive regulator of HSC self-renewal and prolongs the survival of leukemic mice.

(A) Quantitative analysis of donor-derived chimerism in PB of secondary recipients of WT and S6k1^–/– LT-HSCs; mean ± SEM. *P < 0.001, 1-way ANOVA. n = 9–10/group. (B) Quantitative analysis of donor-derived chimerism in PB of tertiary transplant recipients of WT and S6k1^–/– LT-HSCs; mean ± SEM. *P < 0.001, 1-way ANOVA. n = 7–10/group. (C) Expression of Cdkn1a in CD45.2⁺ LSK cells isolated from WT and S6k1^–/– secondary recipients depicted in part A; mean ± SD. *P < 0.001. Experiment performed in triplicate. (D) Kaplan-Meier survival curves of secondary recipients of MLL-AF9 fusion gene transduced with WT or S6k1^–/– cells derived from primary recipients. *P < 0.001 (n = 17/group). (E) Quantitative representation of Ki-67–negative GFP⁺ Gr1^–Mac1^– Sca1^–c-Kit⁺ cells in BM and spleen of secondary recipients (from part D) of WT or S6k1^–/– AML cells expressing MLL-AF9; mean ± SEM. *P < 0.01, t test. n = 5 mice/group. Data representative of 2 independent experiments. (F) Phosphorylation level of AKT and 4E-BP1 in WT and S6k1^–/– HSC/Ps expressing MLL-AF9; n = 2. (G) Phosphorylation level of mTOR and 4E-BP1 in MA9-3 cells following treatment with PF-4708671; n = 3. (H) Kaplan-Meier survival curves of NOD/SCID mice transplanted with MA9-3 cells treated with either vehicle or PF-4708671. *P < 0.05. n = 7/group.