mTOR complex 1 plays critical roles in hematopoiesis and Pten-loss-evoked leukemogenesis

Abstract

The mechanistic target of rapamycin (mTOR) pathway serves as a key sensor of cellular-energetic state and functions to maintain tissue homeostasis. Hyperactivation of the mTOR pathway impairs hematopoietic stem cell (HSC) function and is associated with leukemogenesis. However, the roles of the unique mTOR complexes (mTORCs) in hematopoiesis and leukemogenesis have not been adequately elucidated. We deleted the mTORC1 component, regulatory-associated protein of mTOR (Raptor), in mouse HSCs and its loss causes a nonlethal phenotype characterized by pancytopenia, splenomegaly, and the accumulation of monocytoid cells. Furthermore, Raptor is required for HSC regeneration, and plays largely nonredundant roles with rapamycin-insensitive companion of mTOR (Rictor) in these processes. Ablation of Raptor also significantly extends survival of mice in models of leukemogenesis evoked by Pten deficiency. These data delineate critical roles for mTORC1 in hematopoietic function and leukemogenesis and inform clinical strategies based on chronic mTORC1 inhibition.

Figure 1. Deletion of Raptor Ablates mTORC1 Activity and Leads to HSPC Pertubations

(A) Representative flow-cytometry plots of sorted Lin⁻ PI⁻ cells post fixation and permeabilization from pIpC-treated Raptor^Fl/Fl (+/+) and Raptor ^Fl/Fl , Mx-Cre (−/−) mice (Lin⁻ PI⁻ Sca-1⁺c-Kit⁺ [LSK], Lin⁻ PI⁻ Sca-1⁻c-Kit⁺[LK], Lin⁻ PI⁻ Sca-1⁻c-Kit⁺CD34⁺ [GMP/CMP], Lin⁻ PI⁻ Sca-1⁻c-Kit⁺CD34⁻ [MEP], and Lin⁻ PI⁻ Sca-1⁻c-Kit[L⁻ S⁻ K⁻]). (B) Flow cytometry was performed on sorted Lin⁻PI⁻ cells from pIpC-treated Raptor^Fl/Fl (+/+) and Raptor ^Fl/Fl, Mx-Cre (−/−) mice that were stimulated ex vivo with 20 ng/mL of Scf for the indicated time points in minutes (mins), and then processed for flow cytometry. Representative histograms are shown (% maximal value on the y-axis) and for quantification see Figure S1E (*p<0.05 relative to unstimulated +/+ controls). (C) Quantification of CD48/CD150/CD34/Flt3/LSK subsets from BM and spleen (D) of Raptor^Fl/Fl (+/+) and Raptor ^Fl/Fl, Mx-Cre (−/−) mice 5–7 months post pIpc. (n=5 +/+, n=6 −/−). For gating see Figure S1N. (E) Quantification of immunophenotypic HPC subsets from mice described in (C), from BM (left panel) and spleen (right panel). Immunophenotypes are defined as in Pronk et al., 2007. (F) CFC formation from pIpC-treated Raptor^Fl/Fl (+/+) and Raptor ^Fl/Fl, Mx-Cre (−/−) mouse BM and spleen cells. (n=3–4). (G) Assessment of EdU incorporation in cells from pIpC-treated Raptor^Fl/Fl (+/+) and Raptor ^Fl/Fl , Mx-Cre (−/−) mice. (n=3). (H) Ho 33342/Ki-67 staining from the indicated FACS-sorted LSK populations or whole BM (WBM) and pIpC-treated genotypes. G₀ (2N DNA-Ki67^Lo), G₁ (2N DNA-Ki67^Hi), and SG₂M (>2N DNA-Ki67^Hi) (n=3). (I) Ho 33342/Pyronin Y in the indicated surface-stained populations of the indicated genotypes (as in [B]) by flow cytometry). Ho^Lo = 2N DNA. (n=8–9) (J) Heat-map of metabolite measurements from pIpC-treated Raptor^Fl/Fl (WT [1–3]) and Raptor ^Fl/Fl, Mx-Cre (KO [4–6]) LSK cells. (p<0.057, citrate p=0.077) (A–J) Data are expressed as mean or mean ± SEM (*p<0.05, n.s. p>0.05).

Figure 2. Raptor Is Required for HSC Regeneration in a Cell-Autonomous Manner

(A) 8 × 10⁵ whole BM cells from pIpC-treated Raptor^Fl/Fl (+/+) or Raptor ^Fl/Fl, Mx-Cre (−/−) mice (both CD45.2⁺) were transplanted into lethally-irradiated CD45.1 mice and survival was monitored. Numbers of recipient mice are indicated (left panel). (p value was derived by Log-rank Test). BM from surviving mice was analyzed for CD45.2 cell contribution to the CD34⁻Flt3⁻LSK pool 20 weeks post pIpC (n=3) (right panel). (B) BM cells from Raptor^Fl/Fl (+/+) and Raptor ^Fl/Fl, Mx-Cre (−/−) mice, 6 weeks post pIpC treatment (both CD45.2+), were mixed at the indicated ratios with CD45.1⁺ BM cells and transplanted into lethally-irradiated CD45.1 recipients. Percentage of CD45.2⁺ cells in PB is shown over time (n=5) (left panel). 22 weeks post transplantation the percentage of CD45.2⁺ CD34LSK cells in BM was assessed (n=5) (right panel). (C) FACS-sorted DiD-labeled LSK-CD48⁻CD150⁺ from pIpC-treated Raptor^Fl/Fl(+/+) and Raptor ^Fl/Fl, Mx-Cre (−/−) mice were transplanted into lethally-irradiated Col2.3GFP mice and ~24 hrs later live imaging was performed to assess relative distance of HSPC to osteoblasts. Shown are results from 2 independent experiments performed with 5,000–10,000 HSPC. (D) 200 LT-HSC or ST-HSC from pIpC-treated Raptor^Fl/Fl (+/+) and Raptor ^Fl/Fl, Mx-Cre (−/−) mice were used to initiate cultures in serum-free conditions that promote HSC expansion ex vivo. 7–9 days post culture the number of cells was enumerated. Data are from 3 independent experiments performed in duplicate. (E) 8 × 10⁵ BM cells from non pIpC-treated Raptor^Fl/Fl or Raptor ^Fl/Fl, Mx-Cre mice were transplanted into lethally-irradiated CD45.1 recipients. 5 weeks post transplantation mice were injected with 3 doses of pIpC over 5 days. PB chimerism was assessed by measuring the contribution of CD45.2⁺ cells over the indicated time (n=9–10 recipients). (F) BM from mice from (E) was analyzed for CD45.2 chimerism in CD34⁻Flt3-LSK fractions 25 weeks post pIpC. Raptor ^Fl/Fl, Mx-Cre recipients were separated into two CD45.2⁺ groups, one with low CD45.2 chimerism that retained deletion (Group1) and ones with high CD45.2 chimerism that escaped deletion (Group 2). (n=4–5). (See Figure S2C). (G) 2.5 × 10⁵ BM cells from uninduced Raptor ^Fl/Fl, Mx-Cre were mixed with 2.5 × 10⁵ CD45.1 cells and transplanted into lethally-irradiated CD45.1 recipients. 5.7 weeks post transplantation ½ of the mice were injected with pIpC as in (D) (+pIpC). Contribution of CD45.2 cells to PB was assessed over the indicated time points. (n=6–8) (left panel). Recipient BM was analyzed for CD45.2 cell contribution to the CD34⁻Flt3-LSK pool 21 weeks post pIpC (n=6–8) (right panel). (H) Kaplan-Meier (KM) survival curve from pIpC-treated Raptor^Fl/Fl (+/+), n=10, and Raptor ^Fl/Fl, Mx-Cre (−/−), n=4, mice that received sub-lethal irradiation. The one surviving −/− mouse had escaped deletion (data not shown) (left panel). (p value was derived by Log-rank Test). pIpC-treated Raptor^Fl/Fl (+/+) and Raptor ^Fl/Fl, Mx-Cre (−/−) mice received sub-lethal irradiation and shown is the frequency of BM CD150⁺-LSK cells 10–12 days post irradiation (IR). (n=4). (A–H) Data are expressed as mean or mean ± SEM (*p<0.05).

Figure 3. Rictor is Required for mTORC2 Activity in HSPC, but Not LT-Hematopoietic Regeneration

(A) Flow cytometry was performed on sorted LIN⁻PI⁻ cells from pIpC-treated Rictor^Fl/Fl (+/+) and Rictor ^Fl/Fl, Mx-Cre (−/−) mice that were treated with Scf for the indicated time (mins). Shown are data from fixed/permeablized LSK-gated events and fold change in MFI for the indicated p-protein is shown relative to untreated +/+ cells. (n=3–4 mice). (B) Frequency of BM LSK subsets from Rictor^Fl/Fl (+/+) and Rictor ^Fl/Fl, Mx-Cre (−/−) mice 4 months post pIpC treatment. (n=4–5). (C) 5 × 10⁵ whole BM cells from pIpC-treated Rictor^Fl/Fl (+/+) and Rictor ^Fl/Fl, Mx-Cre (−/−) mice, (both CD45.2+), were mixed with the same amount of CD45.1⁺ BM cells and transplanted into lethally-irradiated CD45.1 recipients. The percentage of CD45.2⁺ cells in PB is shown over the indicated time (n=6–7 recipients). Panels on the right show the % contribution of CD45.2⁺ cells to the indicated BM populations 30 weeks post transplantation. Data are from 1 of 2 experiments. (D) The levels of indicated p-proteins were assessed as in (A) in Scf-treated LSK-gated cells from pIpC-treated mice of the indicated genotypes. (n=3–4 mice per genotype) (E) BM cell number (n=2–9), spleen mass (F) (n=5–9) and percentage of Mac-1 ⁺Gr-1^Mid/Lo splenic cells (F right panel) (n=3–5) are shown from mice of the indicated genotype 1–3 months post pIpC treatment. (A–F) Data are expressed as mean ± SEM (*p<0.05, n.s. p>0.05).

Figure 4. Raptor is Required for Efficient Leukemogenesis Evoked by Pten Loss

(A) KM curve from Pten^Fl/Fl, MxCre mice of the indicated Raptor genotypes that were treated with pIpC. Number of mice is indicated (*p<0.05 Log-rank Test). (B) Western blots were performed with BM and spleen extracts from mice of the indicated genotypes ~6 months post pIpC using the indicated antibodies. Shown are samples from 2 individual pIpC-treated Raptor^Fl/Fl, Pten^Fl/Fl, MxCre mice. (C) The frequency of BM and Splenic (D) LSK-CD48⁻CD150⁺ cells was assessed from mice of the indicated genotypes 2.5–5 weeks post pIpC treatment (n=6–10). (E) 1 × 10⁶ BM cells from non pIpC-treated mice of the indicated genotypes (CD45.2) were mixed with 5 × 10⁵ CD45.1 cells and transplanted into lethally-irradiated CD45.1 mice. 5.5 weeks post transplant mice received 3 injections of pIpC over 6 days. Survival (left) and chimerism (right) were then assessed (n=4–6, *p<0.05 Log-rank Test). (F) Chimerism was assessed from mice described in (E). (n=4) (G) Assessment of p-protein levels in LSK-gated events from Scf-treated LIN-PI-cells of the indicated gentoypes (n=2–3). (H) BM cells from mice of the indicated genotypes (all from Pten^Fl/Fl, MxCre backgrounds) were transplanted and treated as in (D). Number of transplant recipients is indicated. (A–H). Data are expressed as mean or mean ± SEM. (*p<0.05, n.s. p>0.05).