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The role of mesenchymal stromal cells (MSC) in the development of acute myeloid leukemia

ABSTRACT: Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic stem cells with a poor prognosis. It has been reported in many tumors that mesenchymal stem/ stromal cells (MSCs) support the growth of the malignant cells. In this study we analyzed the interaction between MSCs and AML cells in patients and in murine models of human AML. We found that MSCs from AML patients or leukemic mice supported the growth of AML cells in vitro better than MSCs originating from healthy mice or control persons. Interestingly, MSCs from AML patients who achieved complete remission didn’t support the growth of leukemia cells in vitro anymore. Due to the difficulty to obtain sufficient human MSCs, we developed a novel method to immortalize human MSCs. Immortalized MSC cells derived from AML patients also supported the growth of human AML cells better than MSC cells derived from a healthy donor. Among others, AMSCs increased entering of leukemic cells into cell cycle and at the same time protected the leukemia cells against exogenous toxic events such as chemotherapy or irradiation. In vivo, we observed an increase in the number of MSCs in the bone marrow of leukemic mice compared to healthy mice. Microarray data of AMSCs show upregulation of epithelial mesenchymal trasition genes. The polarization of MSCs towards an AML-supporting state depends on upregulated expression of the transcription factor Growth factor independence 1 (Gfi1). Loss of Gfi1 abrogated the tumor-supporting state of AML-associated MSCs. In summary, we report that MSCs support the growth of AML cells in a Gfi1-dependent manner.

ORGANISM(S): Mus musculus

PROVIDER: GSE102928 | GEO | 2020/08/22

REPOSITORIES: GEO

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Project description:Transcriptional repressor Growth factor independence 1 (GFI1) is a key regulator of haematopoiesis. We previously established that the germline variant GFI1-36N promotes acute myeloid leukemia (AML) development, however the mechanism is not full elucidated. Here using multi-omics approach, we show GFI1-36N expression impedes DNA repair in leukemic cells. We demonstrate the presence of GFI1-36N is associated with increased frequency of chromosomal aberrations and mutational burden in murine and human AML cells. In particular, GFI1-36N modulates DNA repair pathways, O6-methylguanine-DNA-methyltransferase (MGMT) and homologous recombination repair (HR). Mechanistically, GFI1-36N exhibits impaired binding to Ndrg1 promoter element compared to GFI1-36S (wild type), causing decreased NDRG1 levels consequently leading to suppression of MGMT expression, imprinted at the transcriptome and proteome, thus leaving the AML cells vulnerable to DNA damaging agents. Targeting MGMT via temozolomide and HR via olaparib caused specifically extensive lethality in in vitro and ex vivo human and AML samples expressing GFI1-36N. Whereas the effects were insignificant on non-malignant GFI1-36S or GFI1-36N cells. Further, mice transplanted with GFI1-36N leukemic cells treated with combination of temozolomide and olaparib had a significantly longer AML-free survival than mice transplanted with GFI1-36S leukemic cells. In summary, we show that GFI1-36N disturbs DNA repair activity via NDRG1-MGMT axis and thus provides critical insights into novel therapeutic option for AML presented with GFI1-36N variant. Key Points Presence of GFI1-36N impedes Homologous DNA and MGMT DNA repair selectively in AML cells via the NDRG1-MGMT axis. Use of temozolomide and olaparib allows selectively targeting GFI1-36N leukemic cells. Introduction Gfi1 is a transcription factor which regulates the development of haematopoietic cells as well as neuronal and intestinal epithelial cells 1-5. We reported that a variant of GFI1, denominated GFI1-36N (characterized by an exchange of serine to asparagine at position 36), has a prevalence of 5-7% in a healthy control population but is found at an increased frequency of 10-15% among MDS and AML patients 6,7. The expression of germline variant GFI1-36N predisposes the carriers to develop de novo AML and MDS and correlates with a poor prognosis 6,7. Recently, we and other showed that malignant cells with GFI1-36N variant have increased H3K9-acetylation at target genes resulting in higher expression of genes required for cell survival and proliferation 8. GFI1 exerts its repressive role by recruiting histone-modifying enzymes (deacetylases HDAC1-3, demethylase LSD1, methyl transferase G9a) and regulates the accessibility of DNA to its target genes such as Hoxa9, Pbx1, Meis1, CSF1 and CSFR1 9-15. We also showed that GFI1 regulates apoptosis through its regulation of p53 in lymphoblastic leukemia 16 and we have demonstrated that GFI1 facilitates DNA repair 17. However, it is not known how these activities are affected in the GFI1-36N variant and whether the ability of GFI1 to regulate DNA repair pathways is maintained and how this might affect the development of myeloid malignancies. In this study, we leveraged multi-omics profiling to gain mechanistic insights into the molecular architecture that drives leukemia in the presence of GFI1-36N. We provide evidence that GFI1-36N interferes with DNA in leukemic myeloid cells, which leads to a higher frequency of genetic aberrations in MDS/AML cells. We also show that GFI1-36N myeloid leukemic cells are more sensitive to targeting MGMT and HR repair deficient cells, which opens a new selective therapeutic window to treat AML/MDS.

Project description:Heterogeneity and variable survival outcomes of Acute Myeloid Leukemia (AML), suggest that yet undiscovered genes and pathways contribute to AML. Mesenchymal stem cells (MSC), an important component of bone marrow/ stromal microenvironment has been shown to contribute to development and progression of various cancers. Current study was aimed at characterizing MSC from AML bone marrow (BM) and evaluate their therapeutic potential in controlling survival of AML leukemic blasts. MSCs were isolated and cultured from BM of high-risk AML patients. MSC were also obtained from BM of bi-lineage leukemia (ETP-ALL) patients as control MSC from precursor stage of leukemia. MSC derived from uninvolved BM (UnBM) of lymphoma patients were used as normal MSC control. Leukemic blasts were derived from BM of AML and ETP-ALL patients. Patient derived AML-MSC exhibited characteristic profile of MSC Type-II CD90+CD45lo expressing high percent of TLR3 than TLR4 receptors, indicating pro-tumorigenic nature. Gene expression profiles of freshly derived leukemia blasts, AML cell line and AML MSC exhibited deregulated and overactivated Aurora Kinase pathway and inflammasome innate immune pathway. These two pathways are known to be upregulated in many solid and hematological cancers. Further we observed few tumor suppressor genes were downregulated in these groups. In vitro, AML MSC supported survival of leukemic blasts and increased chemoresistance to standard anticancer drugs. Hence AML MSC and ETP-ALL MSC were treated with immunomodulatory drug cocktail (IMiD) containing TLR3 antibody, TLR4 ligand and CXCR4 antagonist peptide (Gift from Kyoto University, Japan), designated as MSC-IMiD. It was observed that MSC-IMiD reduced chemoresistance of leukemic blasts to certain extent in vitro. Further we evaluated if leukemia BM derived MSC, MSC culture supernatant or MSC-IMiD can provide therapeutic benefit to control growth of AML xenograft in mouse model. Human gene expression profile was mapped in human-mouse xenograft made as subcutaneous tumor in immunodeficient NOD-SCID mice model from AML cell line KG1. Comparison was done of all treated groups with tumor bearing control gene expression data. It was interesting to note that MSC and MSC-IMiD could reduce tumor growth in 50% of mice tested. Mice treated with MSC culture supernatant exhibited reduction in tumor growth at par with that seen in Adriamycin treated positive control group. Deregulation of Aurora kinase pathway, inflammasome pathway genes and tumor suppressor genes was found to be reversed in residual tumor tissue of mice treated with MSC and MSC culture supernatant. This gene expression profiling study has helped understanding pathways involved in chemoresistance and immune suppression observed in AML. Moreover, this study has provided leads that MSC or its conditioned media can be explored further to control abnormal myelopoiesis in AML and develop targeted therapy. Funding source: Grant ID: 53/13/2013/CMB/BMS. Grantee: Jyoti Kode Grant title: Understanding the cross-talk between mesenchymal stromal cells and leukemic stem cells in Acute Myeloid Leukemia: Implications in disease biology and therapy. Name of the funding source: Indian Council of Medical Research, Government of India, India.

Project description:Acute myeloid leukemia (AML) is a malignancy of transformed hematopoietic progenitors, which is curable in only 25-50% of patients thus the risk of relapse remains the major challenge. Considerable efforts in improving outcome through the understanding of AML biology elucidate that AML is not autonomous but rather supported by a complex multicellular-bone marrow microenvironment for leukemogenesis, leukemia expansion, and chemoresistance. Within the microenvironment, bone marrow mesenchymal stroma cells (BMSC) safeguard leukemia allowing rapid growth and enable resistance to therapy. Herein, we modeled leukemia-stroma interactions to unravel the reciprocal signaling processes regulating leukemic cell growth and survival. First, we conducted a proteomic analysis of primary AML samples (n=13) cocultured with non-malignant stroma cells by utilizing liquid chromatography mass spectrometry (LC-MS/MS) to understand how the stroma contributes to AML cellular signaling. In the analysis, 84 proteins were significantly affected including upregulation of rate-limiting metabolic enzymatic proteins NAMPT, FASN, and HK1 in AML-stroma cocultures relative to AML monoculture. Through an epigenetic drug screen, we modeled stroma mediated protection of leukemia and uncovered prominent drug resistance by histone deacytlase inhibitor (HDACi) treatment in cocultured leukemic cell lines and cocultured primary AML. We performed a quantitative phosphoproteomics analysis of AML-stroma interactions of leukemic KG1a cells in monoculture and in coculture with stroma (Hs5) treated with HDACi. We uncovered phosphorylation networks of stroma mediated protection enriched in pathways involved in AMP-activated signaling, glucose catabolic processes and EGF/EGFR signaling with the most potent changes in hyper-phosphorylation of acetyl-coenzyme A synthetase (ACSS2, S30) and of hypo-phosphorylation acetyl-coenzyme A carboxylase alpha, (ACACA, S80) both of which are involved in acetyl-CoA (acetyl-CoA) utilization for fatty acid synthesis pathway. Validating these findings, we found that ACSS2 substrate, acetate, contributed to leukemic proliferative growth and ACSS2 loss impacted leukemia metabolic fitness. In addition, we uncovered a novel subgroup of AML patients based on high expression of ACSS1 or ACSS2, which significantly correlated with inferior outcome. The differentially upregulated genes of this ACSS1/2-high subgroup revealed a metabolic gene signature related to mitochondrial processes suggesting the metabolic state of AML may aid in predicting outcome. Our findings elucidate phosphoproteome network of AML-stroma interactions whereby overriding stroma mediated protection, we conclude, requires intervening the metabolic support by ACSS2 in AML.