Volume 29, Issue 1, Pages 61-74 (January 2016) GSK3 Deficiencies in Hematopoietic Stem Cells Initiate Pre-neoplastic State that Is Predictive of Clinical Outcomes of Human Acute Leukemia  Borhane Guezguez, Mohammed Almakadi, Yannick D. Benoit, Zoya Shapovalova, Susann Rahmig, Aline Fiebig-Comyn, Fanny L. Casado, Borko Tanasijevic, Silvia Bresolin, Riccardo Masetti, Bradley W. Doble, Mickie Bhatia  Cancer Cell  Volume 29, Issue 1, Pages 61-74 (January 2016) DOI: 10.1016/j.ccell.2015.11.012 Copyright © 2016 Elsevier Inc. Terms and Conditions

Cancer Cell 2016 29, 61-74DOI: (10.1016/j.ccell.2015.11.012) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Combined Deletion of Gsk3α and β Alleles Leads to an AML-like Disease In Vivo| (A) Experimental design to generate conditional GSK-3α/β mutant mice (Gsk3α−/− β +/flx and Gsk3α−/− βflx/flx) and transplant strategy. (B) Kaplan-Meier survival curves of transplanted and tamoxifen-treated recipient mice with Gsk3αβ Lin−BM cells (n = 15 mice for each condition). Tam, tamoxifen. (C) BM and spleen cellularities of Gsk3αβ recipient mice (mean ± SD, n = 6 for each condition). (D) Leukocytes counts in peripheral blood (PB) and lymph nodes (LN) of Gsk3αβ recipient mice (mean ± SD, n = 6 for each condition). WBC, white blood cells. (E) Representative flow cytometry histograms denoting the composition of the myeloid cell compartment in Gsk3αβ BM recipient mice (n = 6 for each condition). (F) Wright-Giemsa-stained cytospins for BM samples and PB smears of indicated Gsk3αβ genotype recipient mice (n = 5 for each condition). (G and H) Myeloid lineage composition of (G) BM, spleen, (H) PB and lymph node (LN) of Gsk3αβ recipient mice (mean ± SD, n = 6 for each condition). (I) Effect of GSK-3α/β combined deletion on the frequencies of HSC (LSK) and GMP subsets (mean ± SD, n = 10 for each condition) and cell proliferation rate (mean ± SD, n = 5 for each condition). (J) Kaplan-Meier survival curves of secondary transplants of primary engrafting Gsk3αβ BM cells (n = 12 for each condition), ∗∗p < 0.01. See also Figure S1. Cancer Cell 2016 29, 61-74DOI: (10.1016/j.ccell.2015.11.012) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Gsk3β Deficiency Alone Leads to Myelodysplasia and Impaired Hematopoiesis (A) BM transplant strategy. (B) Kaplan-Meier survival curves of transplanted and tamoxifen-treated recipient mice with Gsk3β Lin−BM cells compared with the indicated control animals (n = 15 mice for each condition). Tam, tamoxifen. (C) BM cellularity of Gsk3β recipient mice (mean ± SD, n = 10 for each condition). (D) Wright-Giemsa-stained cytospins for BM samples of Gsk3β recipient mice with the presence of lobated or hypersegmented neutrophils (arrows) (n = 10 for each condition). (E) Representative flow cytometry histograms of Gsk3β recipient mice denoting mature blood cells and blasts frequencies (n = 10 for each condition). (F) Histological spleen sections delineating tissue structure (arrowheads) with high magnification of megakaryocytes and neutrophils (arrowheads) in PB of Gsk3β recipient mice (n = 11 for each condition). (G) BM serial transplantation strategy. (H) MDS disease occurrence in secondary recipients with different transplanted cell doses (n = 10 mice for each condition). (I) Kaplan-Meier survival curves of secondary transplants with primary engrafting Gsk3β cells (n = 10 for each condition). ∗∗p < 0.01. See also Figure S2. Cancer Cell 2016 29, 61-74DOI: (10.1016/j.ccell.2015.11.012) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Gsk3β-MDS BM Arises from HSCs and Displays MDS-IC Activity (A) Experimental design for competitive reconstitution assays using sorted HSCs (LSK) and progenitors (non-LSK) from both Gsk3β+/flx and Gsk3βflx/flx mice (n = 15 for each condition). (B) Kaplan-Meier survival curves of transplanted recipient mice (n = 15 for each condition). Tx, transplant. (C) BM and spleen cellularities of transplanted LSK mice (mean ± SD, n = 10 for each condition). (D) Wright-Giemsa-stained cytospins for BM samples with dysplastic neutrophils (arrowheads) and histological spleen sections with diffuse infiltration by myeloid and granulocytic cells (n = 10 for each condition). (E and F) Blood cell counts and myeloid lineage composition of transplanted non-LSK mice mean ± SD, n = 4 for each condition). (G and H) Competitive reconstitution capacity (G) and cell proliferation rate (H) of both Gsk3β+/flx and Gsk3βflx/flx HSCs (n = 5 for each condition). (I) Representative flow cytometry histograms of diseased BM from Gsk3βflx/flx mice at different time points post-transplantation (n = 5 for each condition). (J and K) Kaplan-Meier survival curves of secondary transplants and regenerative capacity of primary engrafting Gsk3β HSCs (mean ± SD, n = 8 for each condition). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figure S3 and Table S1. Cancer Cell 2016 29, 61-74DOI: (10.1016/j.ccell.2015.11.012) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 HSCs Gsk3β-Null and α/β-Null Acquires Dysregulated Stem Cell and Myeloid Differentiation Gene Programs Driven by Progressive Activation of the Wnt/Akt/mTOR Signaling Axis (A) Principle component analysis (PCA) on all entities from global gene expression profiling of LSK HSCs from Gsk3α+/+ β +/flx (WT), Gsk3α−/− β +/+ (α KO), Gsk3α+/+ β flx/flx (β KO) and Gsk3α−/− β flx/flx (β + α DKO). (B) Gene set enrichment analysis (GSEA) showing the highest ranked gene signatures differentially expressed between Gsk3 genotypes. (C and D) GSEA plots (C) and quantitative PCR validation of Wnt signaling activation (D) in both Gsk3β KO and β + α DKO LSK cells. (E and F) Measurement of activity (E) and nuclear stabilization (F) of β-catenin levels in both Gsk3β LSK cells (all data are means ± SD, n = 5 for each condition). (G and H) GSEA plots (G) and qPCR validation of mTOR signaling activation (H) in both Gsk3β KO and β + α DKO LSK (mean ± SD, n = 5 for each condition). (I and J) Effect of mTOR complex inhibition (I) and PI3K/AKT/mTOR pathway inhibition (J) at different levels on total CFU-C derived from each Gsk3 genotype (mean ± SD, n = 5 for each condition). (K) GSEA plots of oncogenic APC/MYC and STAT5 gene targets in both Gsk3β KO and β + α DKO LSK cells. (L) GSEA plots of the PI3K/AKT pathway are also found in both Gsk3β KO and Gsk3β + α DKO LSK cells. (M) Representative western blots and frequency of phosphorylated AKT levels (active form) in β + α DKO LSK cells (mean ± SEM, n = 3). (N) Hierarchical cluster analysis of both glycolysis and oxidative phosphorylation gene signatures across all Gsk3 genotypes. (O–Q) Measurement of metabolic activity (O) and differential responses to mitochondrial stress of Gsk3β KO and β + α DKO cells (P and Q) with respect to WT cells (mean ± SD, n = 5). ∗p < 0.05 and ∗∗p < 0.01. See also Figure S4. Cancer Cell 2016 29, 61-74DOI: (10.1016/j.ccell.2015.11.012) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 Gsk3β-Null BM Expresses a Unique Gene Expression Profile and Predicts Pediatric MDS Disease Outcome (A) Hierarchical cluster analysis of global gene profile between mouse Gsk3β deleted BM cells and human MDS samples (MDS-LR: n = 60, MDS-HR: n = 44, 5q-: n = 11), CMML (n = 20) and AML (n = 30). (B) Gsk3β-deletion gene signature (63 genes, triangles) is determined between GSK-3 WT and BKO (p ≤ 0.05, fold change ≥5). (C) Hierarchical cluster analysis of human myeloid neoplasm subtypes using Gsk3β gene signature. (D) Hierarchical cluster analysis of both adult (normal: n = 6, stable-MDS: n = 23 and transformed-MDS: n = 12) and (E) pediatric myelodysplasia (RAEB: n = 8, RAEB-t: n = 8 and RC: n = 16) using either Gsk3β-deletion differential transcriptome (p ≤ 0.05, fold change ≥1.5) or 63-gene signature. (F–H) Assessment of disease risk status and overall disease progression to AML in a cohort of pediatric MDS samples using different criteria. The statistical significance was assessed by log rank Mantel-Cox test. See also Figure S5 and Tables S2, S3, S4, S5, and S6. Cancer Cell 2016 29, 61-74DOI: (10.1016/j.ccell.2015.11.012) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Model of MDS Onset and AML Progression by Allelic Dosage of Gsk3α/β. Cancer Cell 2016 29, 61-74DOI: (10.1016/j.ccell.2015.11.012) Copyright © 2016 Elsevier Inc. Terms and Conditions