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TNFα-induced alterations in stromal progenitors in CML BM result in enhanced LSC growth via CXCL1-CXCR2 signaling

ABSTRACT: Leukemia stem cells (LSC) are not eradicated in chronic myeloid leukemia (CML) patients responding to tyrosine kinase inhibitor treatment. Bone marrow (BM) mesenchymal niches play an essential role in hematopoietic stem cell (HSC) maintenance. Here, we examined leukemia-induced alterations in mesenchymal progenitor populations using a murine CML model. 6C3+ stroma-forming progenitors were increased in CML BM, and demonstrated enhanced LSC but reduced HSC support compared to their normal counterparts. CML 6C3 cells showed enhanced TNFa-related gene signatures, and upregulated CXCL1 expression. TNFα signaling contributed to expansion and increased CXCL1 expression by 6C3 cells in CML BM. The CXCL1 receptor CXCR2 was upregulated in in LSC, and CXCR2 inhibition significantly reduced LSC growth both in vitro and in vivo. In conclusion, TNFα-induced alterations in stromal progenitors in CML BM result in enhanced CML LSC growth via CXCL1-CXCR2 signaling. Targeting this axis represents a novel therapeutic strategy to inhibit BM niche-protected LSC.

ORGANISM(S): Mus musculus

PROVIDER: GSE126547 | GEO | 2021/06/16

REPOSITORIES: GEO

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Project description:Tyrosine kinase inhibitors (TKI) are highly effective in treatment of chronic myeloid leukemia (CML) but do not eliminate leukemia stem cells (LSC), which remain a potential source of relapse. TKI treatment effectively inhibits BCR-ABL kinase activity in CML LSC, suggesting that additional kinase-independent mechanisms contribute to LSC preservation. We investigated whether signals from the bone marrow (BM) microenvironment protect CML LSC from TKI treatment. Coculture with human BM mesenchymal stromal cells (MSC) significantly inhibited apoptosis and preserved CML stem/progenitor cells following TKI exposure, maintaining colony forming ability and engraftment potential in immunodeficient mice. We found that the N-Cadherin receptor plays an important role in MSC-mediated protection of CML progenitors from TKI. N-Cadherin-mediated adhesion to MSC was associated with increased cytoplasmic N-Cadherin-M-NM-2-catenin complex formation, as well as enhanced M-NM-2-catenin nuclear translocation and transcriptional activity. Increased exogenous Wnt-mediated M-NM-2-catenin signaling played an important role in MSC-mediated protection of CML progenitors from TKI treatment. Our results reveal a close interplay between N-Cadherin and the Wnt-M-NM-2-catenin pathway in protecting CML LSC during TKI treatment. Importantly, these results reveal novel mechanisms of resistance of CML LSC to TKI treatment, and suggest new targets for treatment designed to eradicate residual LSC in CML patients. RNA was obtained from CML CD34+ cells treated with or without IM (5M-NM-<M) and MSC for 96 hours, amplified, labeled and hybridized to GeneChip 1.0 arrays (Affymetrix, Santa Clara, CA). Microarray data analysis was performed using R (version 2.9) with genomic analysis packages from Bioconductor (version 2.4). The 33297 probes represented on the microarray were filtered by cross-sample mean, and for standard deviation of greater than the 25% quantile, yielding 18624 probes representing 12553 genes. Linear regression was used to model the gene expression with the consideration of a 2x2 factorial design and matched samples. Differentially expressed genes were identified by calculating empirical Bayes moderated t-statistic, and p-values were adjusted by FDR using the M-bM-^@M-^\LIMMAM-bM-^@M-^] package. Gene Set Enrichment Analysis (GSEA) was performed using GSEA software version 2.04 to detect enrichment of predetermined gene sets using t-scores from all genes for 1263 gene sets in the C2 (curated gene sets) category from the Molecular Signature Database (MsigDB).

Project description: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.

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TNFα-induced alterations in stromal progenitors in CML BM result in enhanced LSC growth via CXCL1-CXCR2 signaling

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