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. 2017 Mar 2;20(3):329-344.e7.
doi: 10.1016/j.stem.2016.11.018. Epub 2017 Jan 12.

Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease

Mark P Chao  1 , Andrew J Gentles  2 , Susmita Chatterjee  3 , Feng Lan  3 , Andreas Reinisch  3 , M Ryan Corces  3 , Seethu Xavy  3 , Jinfeng Shen  3 , Daniel Haag  3 , Soham Chanda  3 , Rahul Sinha  3 , Rachel M Morganti  3 , Toshinobu Nishimura  3 , Mohamed Ameen  3 , Haodi Wu  3 , Marius Wernig  3 , Joseph C Wu  4 , Ravindra Majeti  5
Affiliations

Human AML-iPSCs Reacquire Leukemic Properties after Differentiation and Model Clonal Variation of Disease

Mark P Chao et al. Cell Stem Cell. .

Abstract

Understanding the relative contributions of genetic and epigenetic abnormalities to acute myeloid leukemia (AML) should assist integrated design of targeted therapies. In this study, we generated induced pluripotent stem cells (iPSCs) from AML patient samples harboring MLL rearrangements and found that they retained leukemic mutations but reset leukemic DNA methylation/gene expression patterns. AML-iPSCs lacked leukemic potential, but when differentiated into hematopoietic cells, they reacquired the ability to give rise to leukemia in vivo and reestablished leukemic DNA methylation/gene expression patterns, including an aberrant MLL signature. Epigenetic reprogramming was therefore not sufficient to eliminate leukemic behavior. This approach also allowed us to study the properties of distinct AML subclones, including differential drug susceptibilities of KRAS mutant and wild-type cells, and predict relapse based on increased cytarabine resistance of a KRAS wild-type subclone. Overall, our findings illustrate the value of AML-iPSCs for investigating the mechanistic basis and clonal properties of human AML.

Keywords: MLL; acute myeloid leukemia; epigenetics; induced pluripotent stem cells; reprogramming.

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Figures

Figure 1
Figure 1. Human AML Cells Can Be Reprogrammed into iPSCs while Retaining the Original Patient Mutations and Cytogenetic Abnormalities
(A) SU042 AML patient cells were transduced with SKOM factors via Sendai virus. Resulting clones were assessed by light microscopy (clone SU042-2 is shown). Scale bar, 200 μm. (B and C) Candidate iPSC clone SU042-2 was analyzed for pluripotency markers by flow cytometry (B) and immunofluorescence (C). Scale bar, 500 μm. (D) SU042-2 cells were transplanted subcutaneously into NSG mice and resulting tumors were assessed for teratoma formation by H&E staining. Scale bar, 50 μm. (E) AML patient cells and iPSC clones were assessed for MLL rearrangement by FISH using break apart probes of the 5′ and 3′ ends of MLL (arrows). (F) The variant allele frequencies of leukemia mutations in AML patient cells and AML-iPSC clones were determined by DNA sequencing. Note that clone SU042-3 (arrow) harbored all leukemic mutations except KRAS-G13D. (G) Karyotypic analysis of AML-iPSCs was performed, and arrows denote t(10;11) for SU042 and t(9;11) for SU223.
Figure 2
Figure 2. Hematopoietic Cells Differentiated from AML-iPSCs Demonstrate an Aberrant Myeloid-Restricted Phenotype and Serially Replate In Vitro
(A) Morphologic changes by light microscopy are shown for AML-iPSCs differentiated into hematopoietic cells. Scale bar, 100 μm. (B and C) On day 12, cells were harvested and analyzed by flow cytometry for the presence of CD34+CD43+CD45+ hematopoietic cells as shown for SU042-2 (B) and summarized across multiple iPSC clones (C). (D) Pluripotency marker expression was determined by flow cytometry on day 0 and 12 of differentiation. (E and F) Differentiated CD43+CD45+ cells derived from AML-iPSCs were assessed for hematopoietic colony formation in methylcellulose. Representative colony morphology is shown (E) with results quantified (F). Scale bar, 100 μm. (G) Serial replating of cells from (F) is shown. BFU-E, burst-forming unit erythroid; CFU-E, colony-forming unit-erythroid; G, granulocyte; GM, granulocyte, monocyte/macrophage; GEMM, granulocyte, erythroid, monocyte/macrophage, megakaryocyte; M, macrophage. Error bars represent SD.
Figure 3
Figure 3. Hematopoietic Cells Differentiated from AML-iPSCs Give Rise to an Aggressive Myeloid Leukemia In Vivo
(A) Differentiated CD43+CD45+ cells derived from AML-iPSCs were transplanted intravenously (IV) into sublethally irradiated NSG mice or directly into humanized ossicle-bearing NSG mice with human leukemic engraftment in the bone marrow shown at 8 weeks. May-Grunwald-Giemsa staining of CD45+CD33+ cells from engrafted mouse bone marrow is demonstrated (right). Scale bar, 10 μm. (B) Engraftment of CD43+CD45+ cells derived from the indicated AML-iPSCs is shown. (C) H&E staining (left) and human CD45 immunohistochemistry (right) of engrafted bone marrow demonstrated leukemic infiltrates. Scale bar, 50 μm. (D and E) Gross representative images of spleens from engrafted and non-engrafted mice are shown (D), with quantification of spleen weights (E). (F and G) Cells isolated from the bone marrow of engrafted mice were analyzed for leukemia-associated mutations by DNA sequencing (F) and FISH for MLL rearrangement (G). (H) Human CD45+CD33+ cells isolated from the bone marrow of engrafted mice were transplanted into secondary NSG recipients. Bone marrow evaluation for leukemia is shown at 8 weeks. (I) Overall survival of secondary transplants is indicated with Kaplan-Meier analysis (n ≥ 5 per group).
Figure 4
Figure 4. Reprogramming AML Blasts into iPSCs Resets AML-Associated DNA Methylation Changes that Are Reacquired upon Hematopoietic Differentiation
(A) Methylation arrays (Infinium 450K BeadChip) were conducted on undifferentiated AML-iPSCs (undiff AML iPSC), CD43+CD45+ hematopoietic cells differentiated from AML-iPSCs and engrafted into NSG mice (engrafted diff AML iPSC), CD33+ primary AML cells (primary AML), primary AML cells engrafted into NSG mice (engrafted primary AML), undifferentiated healthy control ESC (undiff control ESC), undifferentiated healthy control iPSC (undiff control iPSC), and germline control undifferentiated iPSCs derived from AML patient T cells (undiff T cell iPSC). The 1,000 most variable CpG sites across all samples are displayed in heatmap form with unsupervised hierarchical clustering. Beta value signifies degree of methylation (red, hypomethylated; white, hypermethylated). (B) Multi-dimensional scaling analysis (MDS) plot of methylation data demonstrates clustering according to the two most impactful coordinates. Patient-specific SNPs were filtered from this analysis. (C) Pairwise comparisons of methylation values show individual CpGs and highlight significant DMRs. The density of blue regions is proportional to the density of individual CpG sites. Red dots represent the average β-value of CpGs within statistically significant (p < 0.01) DMRs between the sample types being compared, as identified by Bumphunter analysis. (D) The degree of methylation (beta value) at CpG sites overlapping selected pluripotent (left) and hematopoietic (right) genes is shown comparing iPSC and AML samples. Symbols represent individual CpG loci positions; lines are smoothed Loess fits across loci for the sample types indicated by color. (E) The 1,000 most variably expressed genes determined by RNA-seq were used for unsupervised clustering and are displayed in heatmap form. TPM, transcripts per million. (F) Multi-dimensional scaling analysis plot of gene expression data demonstrates clustering according to the two most impactful coordinates (pluripotent versus non pluripotent axis and samples according to patient axis).
Figure 5
Figure 5. Differentiated Hematopoietic Cells from AML iPSCs Display Activation of an Aberrant MLL Gene Signature
(A) Methylation arrays were conducted on hematopoietic cells differentiated from healthy control iPSC/ESCs and compared against hematopoietic cells differentiated from AML-iPSCs. The 1,000 most variable CpG sites across all samples are displayed in heatmap form with unsupervised hierarchical clustering. (B) The 1,000 most variably expressed genes determined by RNA-seq were used for unsupervised clustering and are displayed in heatmap form. Expression levels were adjusted so that the mean across normal-derived samples was zero for each gene, to facilitate comparison to AML-derived samples. (C) Gene set enrichment analyses were conducted for gene sets that were hypomethylated in the indicated pairwise comparison according to q values. (D) CpG sites overlapping MLL fusion signature genes from Ross et al. (2004) were used for unsupervised clustering of hematopoietic cells differentiated from control and AML-iPSCs. (E) Mean methylation values are presented for CpG sites within target genes known to be aberrantly activated by MLL fusions in leukemia for differentiated hematopoietic cells from normal iPSC/ESCs and AML-iPSCs. P values are shown for each indicated gene comparing the two populations by t test.
Figure 6
Figure 6. AML-iPSCs Enable Mutation-Specific Targeting of AML Blasts and Leukemic Subclones
(A) Differentiated CD43+CD45+ cells derived from AML-iPSCs were plated in methylcellulose in the presence of 10 μM EPZ-5676 or DMSO control and analyzed on day 14. (B and C) Undifferentiated AML-iPSCs from SU042-4 (B) and SU223-A7 (C) were cultured in iPSC medium in the presence of increasing concentrations of EPZ-5676 or DMSO and assessed for proliferation by trypan blue staining exclusion. Differentiated CD43+CD45+ cells derived from the same AML-iPSC clone were plated in methylcellulose with similar concentrations of EPZ-5676 or DMSO. (D and E) Hematopoietic colonies derived from AML-iPSCs were incubated in myeloid expansion media with or without cytokine (GM-CSF) for KRAS wild-type (D) and KRAS mutant clones (E). Total cell number was determined over 11 days of culture by trypan blue exclusion. (F) CD43+CD45+ cells differentiated from AML-iPSCs from KRAS mutant and wild-type clones were transplanted intravenously into NSG mice and assessed for leukemic engraftment 8–12 weeks later.
Figure 7
Figure 7. AML iPSCs Model Clonal Evolution and Relapse in an AML Patient
(A) Variant allele frequencies of leukemic mutations were determined for primary SU042 patient blasts at diagnosis (SU042, black arrow), SU042-3 iPSC clone lacking the KRAS-G13D mutation from diagnosis (blue arrow), and primary SU042 patient blasts at relapse (SU042R, red arrow). (B and C) CD43+CD45+ cells derived from AML-iPSCs were plated in methylcellulose in the presence of 0.1 μM of cytarabine, and colony formation (B) and IC50 values (C) were determined. (D) KRAS G13D and wild-type MLL-rearranged AML patient samples were treated in liquid culture with cytarabine or DMSO control. (E) Schematic is shown of the clonal evolution of AML patient SU042 across disease course. New mutations present at relapse are shown in red. Student’s t test was conducted for (B) and (D). Error bars represent SD. Two-way ANOVA analyses were conducted for (C). *p < 0.05 (G–I) CD43+CD45+ cells differentiated from KRAS mutant or wild-type AML-iPSCs were plated in methylcellulose in the presence of 10 μM EPZ-5676, PD98059 (a MEK inhibitor). DMSO control and colony formation (G and H) and IC50 values (I) were determined. (J and K) CD43+CD45+ cells differentiated from AML-iPSCs were plated in methylcellulose in the presence of 0.1 μM of the MEK inhibitor trametinib, and colony formation (J) and IC50 values (K) were determined. (L) KRAS G13D and wild-type MLL-rearranged AML patient samples were treated in liquid culture with PD98059 or DMSO control. Student’s t test was conducted for (A), (H), (J), and (L). Error bars represent SD. Two-way ANOVA analyses were conducted for (B)–(E), (G), (I), and (K). NS, non-significant. *p < 0.05, **p < 0.01, ***p < 0.005.
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References

    1. Afonja O, Smith JE, Jr, Cheng DM, Goldenberg AS, Amorosi E, Shimamoto T, Nakamura S, Ohyashiki K, Ohyashiki J, Toyama K, Takeshita K. MEIS1 and HOXA7 genes in human acute myeloid leukemia. Leuk Res. 2000;24:849–855. - PubMed
    1. Alharbi RA, Pettengell R, Pandha HS, Morgan R. The role of HOX genes in normal hematopoiesis and acute leukemia. Leukemia. 2013;27:1000–1008. - PubMed
    1. Alizadeh AA, Aranda V, Bardelli A, Blanpain C, Bock C, Borowski C, Caldas C, Califano A, Doherty M, Elsner M, et al. Toward understanding and exploiting tumor heterogeneity. Nat Med. 2015;21:846–853. - PMC - PubMed
    1. Amabile G, Di Ruscio A, Müller F, Welner RS, Yang H, Ebralidze AK, Zhang H, Levantini E, Qi L, Martinelli G, et al. Dissecting the role of aberrant DNA methylation in human leukaemia. Nat Commun. 2015;6:7091. - PMC - PubMed
    1. Argiropoulos B, Humphries RK. Hox genes in hematopoiesis and leukemogenesis. Oncogene. 2007;26:6766–6776. - PubMed

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