PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells

Abstract

Myelodysplastic syndrome (MDS) is a clonal malignancy arising in hematopoietic stem cells (HSCs). The mechanisms of MDS initiation in HSCs are still poorly understood. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway is frequently activated in acute myeloid leukemia, but in MDS, PI3K/AKT is often down-regulated. To determine whether PI3K down-regulation can perturb HSC function, we generated a triple knockout (TKO) mouse model with Pik3ca, Pik3cb, and Pik3cd deletion in hematopoietic cells. Unexpectedly, PI3K deficiency caused cytopenias, decreased survival, and multilineage dysplasia with chromosomal abnormalities, consistent with MDS initiation. TKO HSCs exhibit impaired autophagy, and pharmacologic autophagy induction improved HSC differentiation. Using intracellular LC3 and P62 flow cytometry and transmission electron microscopy, we also observed abnormal autophagic degradation in patient MDS HSCs. Therefore, we have uncovered an important protective role for PI3K in maintaining autophagic flux in HSCs to preserve the balance between self-renewal and differentiation and to prevent MDS initiation.

Fig. 1.. Class IA PI3K deletion leads to cell-autonomous pancytopenia and abnormal self-renewal.

(A) Analysis of PTEN, INPP4B, and SHIP1 expression in the MDS gene set GSE 19429 classified by French-American-British (FAB) subtype versus control healthy CD34⁺ cells. RARS, refractory anemia with ringed sideroblasts. (B) Relative frequencies of BM colonies plated in methylcellulose supplemented with myeloid growth factors (N_WT = 4, N_δKO = 4, and N_TKO = 4). BFU-E, burst-forming units-erythroid; GM, granulocyte-macrophage. (C) Quantitative analysis of WT and TKO BM serial replating assay round 1 through round 6 (R1 to R6) in methylcellulose (N_WT = 12 and N_TKO = 10). (D) Experimental design of noncompetitive BM transplantation (BMT; created with Biorender.com). (E) Longitudinal analysis of blood counts from WT, δ KO, and TKO BM recipients (N_WT = 7, N_δKO = 9, and N_TKO = 9). WBC, white blood cells. (F) Representative flow cytometry plots gated on the CD45.2⁺ lineage-low population from WT, δ KO, and TKO BM at 8 weeks after polyI-polyC (PIPC) from noncompetitive BM transplant mice. (G and H) Absolute numbers of donor-derived CD45.2⁺ cells per femur of (G) LT-HSCs, ST-HSCs, MPPs (MPP2 and MPP3), (H) Flk2⁺ LSK, myeloid progenitors (MPROG), and common lymphoid progenitors (CLP). Representative graphs from each experiment are shown. Each experiment was performed at least three times. WT;Mx1-Cre (WT) and p110δ KO (δ KO) mice were used as controls for TKO;Mx1-Cre mice (TKO). Immunophenotypic populations were defined as follows: LT-HSCs, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁻CD150⁺; ST-HSCs, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁻CD150⁻; MPP2, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁺CD150⁺; MPP3, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁺CD150⁻; MPROG, Lin⁻cKit⁺Sca1⁻; LSK, Lin⁻cKit⁺Sca1⁺; and CLP, Lin⁻cKit^midSca1^midFlk2⁺IL7R⁺. Significance was determined using t test (B and C) or one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test (E, G, and H). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. ns, not significant.

Fig. 2.. Class IA PI3K deletion leads to a competitive disadvantage in multilineage differentiation.

(A) Experimental design of competitive BMT (created with Biorender.com). (B) Longitudinal analysis of total donor chimerism and myeloid and lymphoid donor chimerism in the PB (N_WT = 7, N_δKO = 9, and N_TKO = 9). (C) Absolute numbers of donor-derived CD45.2⁺ cells per femur at 16 weeks after PIPC of LT-HSCs, ST-HSCs, multipotent progenitors (MPP2 and MPP3), and MPROG. Representative graphs from one experiment are shown. The experiment was performed three times. WT;Mx1-Cre (WT) and p110δ KO (δKO) mice were used as controls for TKO;Mx1-Cre mice (TKO). Immunophenotypic populations were defined as follows: LT-HSCs, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁻CD150⁺; ST-HSCs, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁻CD150⁻; MPP2, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁺CD150⁺; MPP3, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁺CD150⁻; and MPROG, Lin⁻cKit⁺Sca1⁻. Significance was determined using one-way ANOVA with Tukey’s multiple comparisons test *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Fig. 3.. Class IA PI3K deletion in the BM leads to decreased survival, myelodysplasia, and gene expression changes associated with MDS.

(A) Kaplan-Meier survival curve of noncompetitively transplanted animals (N_WT = 25, N_δKO = 14, and N_TKO = 27). Significance was determined using the log-rank (Mantel-Cox) test. (B to G) Photomicrographs of Wright-Giemsa–stained BM cytospins from a WT primary transplant recipient (control) and TKO primary transplant recipients at 8 weeks after transplantation (arrows point to dysplastic cells). (H) Experimental design for noncompetitive BMT and RNA sequencing sample preparation (created with Biorender.com). (I) GSEA of the TKO versus WT HSC signature with LT-HSC and progenitor signatures. NES, normalized enrichment score. (J) GSEA comparison between the TKO versus δKO HSC gene set and patient MDS versus healthy CD34⁺ cell signatures [GSE 19429; Pellagatti et al. (13)].

Fig. 4.. Serial transplantation of TKO BM cells promotes cytogenetic changes and progression to AML.

(A) Kaplan-Meier survival curve of secondary BM transplant recipients. Significance was determined by the log-rank (Mantel-Cox) test (N_WT = 12 and N_TKO = 10). (B) Representative images of SKY chromosomal painting of donor-derived cKit⁺ WT or TKO cells from secondary transplant recipient mice. White boxes outline chromosomal abnormalities. N = 3 animals per genotype, with total cells analyzed per genotype: N_WT = 30 and N_TKO = 46. (C) Representative flow cytometry plots of BM and spleen (SP) and (D) photomicrographs of hematoxylin and eosin (H&E)–stained BM and spleen sections of a TKO secondary transplant recipient with MDS and extramedullary hematopoiesis. (E) Representative flow cytometry plots of BM and spleen (SP) and (F) photomicrographs of H&E-stained sections of the BM, spleen, and liver of a TKO secondary transplant recipient with AML.

Fig. 5.. Loss of class IA PI3K leads to decreased autophagy in HSCs.

(A) Representative LC3 flow cytometry analysis histograms and quantification of median fluorescent intensity (MFI) of LC3 in WT, δKO littermate control, and TKO HSCs and MPROG after serum and cytokine starvation (N_WT = 3, N_δKO = 5, and N_TKO = 3). (B) Quantification of P62 degradation in serum- and cytokine-starved WT and TKO HSCs with and without chloroquine (CQ) treatment, calculated as the ratio of P62 MFI treated with CQ/P62 MFI without CQ treatment (N_WT = 9 and N_TKO = 9). (C) Representative confocal images of sorted LT-HSCs stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), anti-LC3 antibody (green), and anti-LAMP1 antibody (red). Colocalization of LC3 with LAMP1 appears yellow (see arrows). (D) Quantification of LC3⁺ events per cell and (E) colocalization events of LC3 with LAMP1 assessed by Pearson’s correlation (N = 20 cells per genotype from three independent samples). (F) Representative EM images of autophagic vesicles in sorted WT and TKO HSCs (N = 20 cells per genotype). (G) Quantification of the average area of autophagic vesicles (N_WT = 117 and N_TKO = 141) in WT versus TKO HSCs and of the average ratio of autophagic vesicles to cytoplasm per cell in WT and TKO HSCs (N_WT = 23 and N_TKO = 25). (A to H) Immunophenotypic populations were defined as follows: HSCs, Lin⁻cKit⁺Sca1⁺Flk2⁻CD48⁻; LT-HSC, Lin⁻Sca1⁺cKit⁺Flk2⁻CD48⁻CD150⁺; MPROG, Lin⁻cKit⁺Sca1⁻; and LSK, Lin⁻cKit⁺Sca1⁺. Significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (A) or t test (B, D, and F) *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. Representative data from individual experiments are shown. Each experiment was performed at least three times.

Fig. 6.. Pharmacologic up-regulation of autophagy in TKO HSCs improves differentiation and suppresses pathologic self-renewal.

(A) Representative LC3II flow cytometry histograms and LC3II MFI quantification of WT and TKO HSCs on the right. After starvation, cells were either not treated (NT, PBS only) or treated with rapamycin (RAPA). (B) Quantification of GEMM colonies formed by WT and TKO BM cells in methylcellulose upon rapamycin (RAPA; 20 ng/ml) or metformin (MET; 50 mM) treatment (N = 4 per group). NT, no drug treatment (PBS only). (C) Experimental design of noncompetitive BMT with in vivo treatment with RAPA or MET (created with Biorender.com). At each time point, at least five animals per treatment group were analyzed. (D) Absolute numbers of donor-derived WT (N = 8) or TKO (N = 5 per group) LT-HSC and ST-HSC after 8 weeks of treatment. (E) Representative flow cytometry plots and (F) quantification of the number of donor-derived FLK2⁺ LSK cells in the BM after 8 weeks of in vivo treatment with rapamycin (RAPA; 15 mg/ml) or metformin (MET; 5 mg/ml). (A and D to F) Immunophenotypic populations were defined as follows: Flk2⁺LSK, Lin⁻Sca1⁺cKit⁺Flk2⁺; HSCs, Lin⁻Sca1⁺cKit⁺Flk2⁻CD48⁻; ST-HSCs, Lin⁻Sca1⁺cKit⁺Flk2⁻CD48⁻CD150⁻; and LT-HSCs, Lin⁻Sca1⁺cKit⁺Flk2⁻CD48⁻CD150⁺. Significance was determined using t test (D and F) or one-way ANOVA with Tukey’s multiple comparisons test (B). *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Fig. 7.. Human MDS stem cells have abnormal autophagic degradation.

(A) Representative patient sample flow cytometry plots gated on Lin⁻ normal BM (NBM) and MDS BM. (B and C) Representative flow cytometry histograms and quantification of the MFI of (B) LC3 and (C) P62 (N_NBM = 3 and N_MDS = 6) in patient samples gated on the CD45Ra⁻CD123⁻ HSC population. (A to C) The experiment was performed three times with a total of NBM equal to 9 samples and 11 different MDS BM samples. (D) Representative electron microscopy images of autophagic vesicles from sorted CD34⁺ cells of NBM and MDS patient samples. (E and F) Quantification of the (E) area of autophagic vesicles (N_NBM = 92 and N_MDS = 143) and (F) ratio of total area of autophagic vesicles to area of the cytoplasm per cell in NBM versus MDS CD34⁺ cells (N_NBM = 15 and N_MDS = 18). (D to F) Analyzed images are from three different BM controls and three different patients with MDS. Significance was determined using Welsh’s t test *P ≤ 0.05 and ***P ≤ 0.001. (G) Current model: Under low cytokine and serum conditions, a functional PI3K/AKT pathway maintains autophagic degradation and supports functional HSCs. Inactivation of PI3K compromises autophagic degradation, leading to decreased HSC differentiation and MDS initiation. However, pharmacologic autophagy induction can bypass compromised PI3K/AKT activity, improving autophagic degradation and restoring HSC differentiation (image created with Biorender.com).