Gene expression profiling of CD34/CD38 sorted CML cells
ABSTRACT: BACKGROUND: BCR-ABL1+ chronic myeloid leukemia (CML) is characterized by abnormal production of leukemic stem (LSC) and progenitor cells and their spread from the bone marrow into the blood resulting in extramedullary myeloproliferation. So far, little is known about specific markers and functions of LSC in CML. METHODS: We examined the phenotype and function of CD34+/CD38─/Lin─ CML LSC by a multi-parameter screen approach employing antibody-phenotyping, mRNA expression profiling, and functional studies, including LSC repopulation experiments in irradiated NOD-SCID-IL-2Rgamma-/- (NSG) mice, followed by marker-validation using diverse control-cohorts and follow-up samples of CML patients treated with imatinib. RESULTS: Of all LSC markers examined, dipeptidylpeptidase IV (DPPIV=CD26) was identified as specific and functionally relevant surface marker-enzyme on CD34+/CD38─ CML LSC. CD26 was not detected on normal CD34+/CD38─ stem cells or LSC in other hematopoietic malignancies. The percentage of CD26+ CML LSC decreased to undetectable levels during successful treatment with imatinib in all patients (p<0.001). Whereas the sorted CD26─ stem cells obtained from CML patients engrafted irradiated NSG mice with multilineage BCR-ABL1-negative hematopoiesis, CD26+ LSC engrafted NSG mice with BCR-ABL1+ cells. Functionally, CD26 was identified as target-enzyme disrupting the SDF-1alpha-CXCR4-axis by cleaving SDF-1alpha a chemotaxin for CXCR4+ stem cells. Whereas CD26 was found to inhibit SDF-1alpha-induced migration, CD26-targeting gliptins reverted this effect and blocked the mobilization of CML LSC in a stroma co-culture assay. CONCLUSIONS: CD26 is a robust biomarker of LSC and a useful tool for their quantification and isolation in patients with BCR/ABL1+ CML. Moreover, CD26 expression may explain the extramedullary spread of LSC in CML. To define specific mRNA expression patterns and to identify specific LSC markers in CML LSC, gene array analyses were performed. RNA was isolated from sorted CD34+/CD45+/CD38─ CML LSC, CD34+/CD45+/CD38+ CML progenitor cells, CML MNC, sorted CD34+/CD38─ cord blood (CB) SC, CB-derived CD34+/CD38+ progenitor cells, and CB MNC. Total RNA was extracted from sorted cells using RNeasy Micro-Kit (Qiagen) and used (100 ng total RNA) for Gene Chip analyses. Preparation of terminal-labeled cRNA, hybridization to genome-wide human PrimeView GeneChips (Affymetrix, Santa Clara, CA, USA) and scanning of arrays were carried out according to the manufacturer's protocols (https://www.affymetrix.com). Robust Multichip Average (RMA) signal extraction and normalization were performed according to http://www.bioconductor.org/ as described.18 Differences in mRNA expression levels (from multiple paired samples) were calculated as mRNA ratio of i) CML LSC versus CB SC, ii) CML LSC versus CD34+/CD38+ CML progenitors, and normal cord blood SC versus cord blood progenitors. To calculate differential gene expression between individual sample groups where appropriate, we performed a statistical comparison using the LIMMA package as described previously. Briefly, LIMMA estimates the fold change between predefined sample groups by fitting a linear model and using an empirical Bayes method to moderate the standard errors of the estimated log-fold changes for each probe set.
Project description:In chronic myeloid leukemia (CML) neoplastic stem cells (NCS) represent a critical target of therapy. However, little is known about markers and targets expressed in CML NSC. We examined the phenotype and function of CD34+/CD38─/Lin─ CML LSC by a multi-parameter screen approach employing antibody-phenotyping, mRNA expression profiling, and functional studies, followed by marker-validation using diverse control-cohorts and follow-up samples of CML patients treated with imatinib. We here show that in contrast to normal stem cells, CD34+/CD38− CML NSC express IL2RA (CD25), and that STAT5 induces expression of CD25 in Lin−/Sca-1+/Kit+ NSC (LSK) in C57/Bl6 mice. Correspondingly, expression of CD25 decreased in the human BCR/ABL1+ stem cell line KU812 upon shRNA-induced STAT5-depletion. The BCR/ABL1-inhibitors nilotinib and ponatinib were also found to decrease STAT5 activity and CD25-expression in KU812 cells and primary CML NSC. A CD25-targeting shRNA augmented the proliferation of KU812 cells in vitro and in vivo in NOD/SCID-IL2R-/- mice. In consecutive experiments the PI3K/mTOR-blocker BEZ235 was found to promote STAT5- and CD25 expression and to produce synergistic anti-neoplastic effects with nilotinib and ponatinib in CML cells. Together, CD25 is a novel STAT5-dependent marker and target in CML NSC. Overall design: To define differences in mRNA expression patterns in KU812 cells transduced with a random control shRNA or a shRNA against CD25, gene array analyses were performed.
Project description:Understanding leukemia heterogeneity is critical for the development of curative treatments as the failure to eliminate therapy-persistent leukemic stem cells (LSCs) may result in disease relapse. Here we have combined high-throughput immunophenotypic screens with large-scale single-cell gene expression analysis to define the heterogeneity within the LSC-population in chronic phase (CP) chronic myeloid leukemia (CML) patients at diagnosis and following conventional tyrosine kinase inhibitor (TKI) treatment. Our results reveal substantial heterogeneity within the putative LSC population in CP-CML and demonstrate differences in response to subsequent TKI-treatment between distinct subpopulations. Importantly, LSC subpopulations with myeloid and proliferative molecular signatures are proportionally reduced at a higher extent in response to TKI-therapy compared to subfractions displaying primitive and quiescent signatures. Additionally, cell surface expression of the CP-CML stem cell markers CD25, CD26 and IL1RAP is high on all subpopulation at diagnosis, but downregulated and unevenly distributed across subpopulations in response to TKI-treatment. The most TKI-insensitive cells of the LSC-compartment can be captured within the CD45RA- fraction and further defined as positive for CD26 in combination with an aberrant lack of cKIT expression. Thus, our results reveal the heterogeneity of the CML stem cell population and propose a Lin-CD34+CD38-/lowCD45RA-cKIT-CD26+ population to be targeted for improved therapy response. Overall design: qPCR gene expression profiling of the HSC compartment of CML patients at diagnosis (Dx) and after TKI treatment (TKI). In total 29 96-well plates with pre-amplified HSC single-cell cDNA was loaded on to a 96.96 Dynamic Array Chip for Gene Expression. Sample IDs; nBM, Dx and TKI represents the clinical phase of patients, with nBM as a healthy control, Dx as the cells taken at diagnosis and finally TKI as the cells taken after 1-3 moths of TKI-treatment.
Project description:Expression of P190 and P210 BCR/ABL1 in normal human CD34(+) cells induces similar gene expression profiles and results in a STAT5-dependent expansion of the erythroid lineage The P190 and P210 BCR/ABL1 fusion genes are mainly associated with different types of hematologic malignancies, but it is presently unclear whether they are functionally different following expression in primitive human hematopoietic cells. We investigated and systematically compared the effects of retroviral P190 BCR/ABL1 and P210 BCR/ABL1 expression on cell proliferation, differentiation, and global gene expression in human CD34(+) cells from cord blood. Expression of either P190 BCR/ABL1 or P210 BCR/ABL1 resulted in expansion of erythroid cells and stimulated erythropoietin-independent burst-forming unit-erythroid colony formation. By using a lentiviral anti-signal transducer and activator of transcription 5 (STAT5) short-hairpin RNA, we found that both P190 BCR/ABL1- and P210 BCR/ABL1-induced erythroid cell expansion were STAT5-dependent. Under in vitro conditions favoring B-cell differentiation, neither P190 nor P210 BCR/ABL1-expressing cells formed detectable levels of CD19-positive cells. Gene expression profiling revealed that P190 BCR/ABL1 and P210 BCR/ABL1 induced almost identical gene expression profiles, and we identified a common set of 222 differentially expressed genes. Our data suggest that the early cellular and transcriptional effects of P190 BCR/ABL1 and P210 BCR/ABL1 expression are very similar when they are expressed in the same human progenitor cell population, and that STAT5 is an important regulator of BCR/ABL1-induced erythroid cell expansion. Keywords: global gene expression profiling, BCR/ABL1, CD34+ cord blood cells, CML, Ph+ ALL Overall design: The transcriptional effects of retroviral expression of P190 and P210 BCR/ABL1 in human cord blood (CB) CD34+ cells were compared to the expression profiles of CD34+ control cells transduced with an empty vector. Total RNA was isolated from sorted GFP+ cells isolated at day 2, 3, and 4 posttransduction in two independent experiments. RNA extraction, labeling, hybridization, washing, scanning, and feature analysis were performed as described (Järås et al., 2009, Exp Hematol).
Project description:ABL1 kinase inhibitors such as imatinib mesylate (IM) are effective in managing chronic myelogenous leukemia (CML) but incapable of eliminating leukemia stem cells (LSCs), suggesting that kinase−independent pathways support LSC survival. Given that the bone marrow hypoxic microenvironment supports hematopoietic stem cells, we investigated if hypoxia similarly contributes to LSC persistence. Importantly, we found that while BCR−ABL1 kinase remained effectively inhibited by IM under hypoxia, apoptosis became partially suppressed. Furthermore, hypoxia enhanced the clonogenicity of CML cells, as well as their efficiency in repopulating immunodeficient mice, both in the presence and absence of IM. HIF1−α, which is the master regulator of the hypoxia transcriptional response is expressed in the bone marrow specimens of CML individuals. In vitro, HIF1−α is stabilized during hypoxia and its expression and transcriptional activity can be partially attenuated by concurrent IM treatment. Expression analysis demonstrates at the whole transcriptome level that hypoxia and IM regulate distinct subsets of genes. Functionally, knockdown of HIF1−α abolished the enhanced clonogenicity during hypoxia. Taken together, our results suggest that in the hypoxic microenvironment, HIF1−α signaling supports LSC persistence independently of BCR−ABL1 kinase activity. Thus targeting HIF1−α and its pathway components may be therapeutically important for the complete eradication of LSCs. 24 samples consisting CD34+ bone marrow aspirates of 3 chronic phase patients that were subjected to 24h or 96h of DMSO/Normoxia (21% oxygen, 5% carbon dioxide) control, 2 µM Imatinib, hypoxia (0.5% oxygen, 5% carbon dioxide) or combined Imatinib/hypoxia treatments in triplicate cultures.
Project description:Chronic myeloid leukemia is a disease originated at the level of hematopoietic stem cell, characterized by the abnormal overproduction and accumulation, both in blood and bone marrow, of myeloid cells. Treatment options include tyrosine kinase inhibitors that inhibit BCR-ABL activity, however some patients develop resistance to these drugs and has been asociated to the stem cells We have performed a comparative analysis of the global gene expression profiles between CML and normal HSCs. Our goal was to identify key genes and pathways –preferentially, or solely, expressed by LSCs- that could be used as markers for the identification and selection of LSC LSC(CD34+ CD38- lineage-negative cells), and as targets for inhibiting the growth of such cells. We have also analyzed the population of progenitor cells (CD34+ CD38+ Lin- cells) since it has been clearly shown that these cells play an important role in the pathophysiology of CML Overall design: Stem and progenitor cells of chronic myeloid leukemia patients and normal bone marrow were isolated by FACS, stem cells were lysed after FACS and one fraction of progenitor cells were lysed and other fraction were cultured with or without imatinib.
Project description:BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized treatment of Philadelphia chromosome-positive (Ph+) hematologic neoplasms. Nevertheless, acquired TKI resistance remains a major problem in chronic myeloid leukemia (CML), and TKIs are less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). GAB2, a scaffolding adaptor that binds and activates SHP2, is essential for leukemogenesis by BCR-ABL1, and a GAB2 mutant lacking SHP2 binding cannot mediate leukemogenesis. Using a genetic loss-of-function approach and bone marrow transplantation (BMT) models for CML and BCR-ABL1+ B-ALL, we show that SHP2 is required for BCR-ABL1-evoked myeloid and lymphoid neoplasia. Ptpn11 deletion impairs initiation and maintenance of CML-like myeloproliferative neoplasm, and compromises induction of BCR-ABL1+ B-ALL. SHP2, and specifically, its SH2 domains, PTP activity and C-terminal tyrosines, is essential for BCR-ABL1+, but not WT, pre-B cell proliferation. The MEK/ERK pathway is regulated by SHP2 in WT and BCR-ABL1+ pre-B cells, but is only required for the proliferation of BCR-ABL1+ cells. SHP2 is required for SRC family kinase (SFK) activation only in BCR-ABL1+ pre-B cells. RNAseq reveals distinct SHP2-dependent transcriptional programs in BCR-ABL1+ and WT pre-B cells. Our results suggest that SHP2, via SFKs and ERK, represses MXD3/4 to facilitate a MYC-dependent proliferation program in BCR-ABL1-transformed pre-B cells. Overall design: RNA-Seq expression profiling of 16 mouse pre-B cell samples: 4 BCR-ABL/SHP2+/+ 4 BCR-ABL/SHP2-/+ 4 BCR-ABL/SHP2+/- 4 BCR-ABL/SHP2-/-
Project description:Cord blood (CB) samples from normal donors were obtained with informed consent. Fresh CB samples were processed within 18-34h after collection. Mononuclear cells were isolated and CD34+ fraction was separated. CB CD34+ enriched fraction was lineage depleted by staining with purified anti-human CD2, CD3, CD4, CD7, CD8a, CD11b, CD14, CD19, CD20, CD56, CD235a followed by Qdot 605 conjugated goat F(ab')2 anti-mouse IgG (H+L). Cells were also stained with anti-human CD38-FITC, CD45RA-PE or -BV650, CD123-PE Cy7, CD90-biotin, CD34- PerCP and CD10-APC. Finally, cells were incubated with streptavidin-conjugated APC-eF780 and Hoechst 33258 (Invitrogen, final concentration: 1 g/ml). Populations were defined, as follows: HSC - Lin-CD34+CD38-CD90+CD45RA-CD10-, MPP - Lin-CD34+CD38-CD90-CD45RA-CD10-, LMPP - Lin-CD34+CD38-CD90-/loCD45RA+CD10-, MLP - Lin-CD34+CD38-CD90-/loCD45RA+CD10+, GMP - Lin-CD34+CD38+CD123+CD45RA+CD10-, CMP - Lin-CD34+CD38+CD123+CD45RA-CD10-, MEP - Lin-CD34+CD38+CD123-CD45RA-CD10-.
Project description:Single-cell whole transcriptome analysis of chronic myeloid leukemia stem cells, defined as Lin-CD34+CD38-. Overall design: We developed a method that combines high-sensitivity mutation detection with whole-transcriptome analysis of the same single cell. We applied this technique to analyze more than 2,000 single cells from patients with chronic myeloid leukemia (CML) throughout the disease course, revealing heterogeneity of CML stem cells, including the identification of a subgroup of CML stem cells with a distinct molecular signature that selectively persisted during prolonged therapy. We performed differential gene expression and the gene set enrichment analyses of BCR-ABL+ vs BCR-ABL- single cells from either patients or normal donors, analyzed at different stages of the disease.
Project description:Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GAB2 as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transduced Gab2-/- bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors PI3K or SHP2. GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph+ hematologic neoplasms. RNA-Seq expression profiling of 6 mouse bone marrow samples: 3 GAB2 WT (+/+) and 3 GAB2 NULL (-/-)
Project description:Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. Leukemia associated antigens (LAA) might be suitable structures to be attacked by immunotherapeutic agents. We performed primary AML sample enrichment and microarray studies to define LAA expression levels in AML. We compared the LAA expression in the enriched CD34+CD38- AML fraction to that of enriched HSC of healthy donors and AML bulk cells (CD34+CD38+, CD34-CD38+ and CD34-CD38). Furthermore, we investigated the expression patterns of co-stimulatory molecules in LSC, bulk AML cells and enriched HSC, Conclusion: We demonstrated the differential expression of several LAA in LSC, and their suitability as target structures. We enriched primary AML samples using CD34 and CD38 as markers to compare LAA expression levels of LSC, HSC and AML bulk. LAA expression profiles comparing LSC, HSC and leukemic bulk AML citation: Leukemic progenitor cells are susceptible to targeting by stimulated cytotoxic T cells against immunogenic leukemia-associated antigens Vanessa Schneider, Lu Zhang, Markus Rojewski, Natalie Fekete, Hubert Schrezenmeier, Alexander Erle, Lars Bullinger, Susanne Hofmann, Marlies Götz, Konstanze Döhner, Susann Ihme, Hartmut Döhner, Christian Buske, Michaela Feuring-Buske, Jochen Greiner