B-cell acute lymphoblastic leukemia (B-ALL) occurs most commonly in children, while chronic myeloid leukemia (CML) is more frequent in adults. The myeloid bias of hematopoiesis in elderly individuals has been considered causative, but the age of the bone marrow (BM) microenvironment (BMM) may be contributory. Using various murine models of B-ALL in young versus old mice, we recapitulated B-ALL preponderance in children versus adults. We showed differential effects of young versus old BM macrophages on B-ALL cell function. Molecular profiling using RNA- and ATAC-seq revealed pronounced differences in young versus old BMM-derived macrophages and enrichment for gene sets associated with inflammation. In concordance with the role of C-X-C motif chemokine (CXCL) 13 for disease-associated B cell chemoattraction, we found CXCL13 to be highly expressed in young macrophages on a translational compared to a transcriptional level. Inhibition of CXCL13 in BM macrophages impaired leukemia cell migration and decreased the proliferation of cocultured B-ALL cells, while recombinant CXCL13 increased pAKT and B-ALL cell expansion. Pretreatment of B-ALL-initiating cells with CXCL13 accelerated B-ALL progression. Deficiency of Cxcr5, the receptor for CXCL13, on B-ALL-initiating cells prolonged murine survival, while high expression of CXCR5 in pediatric B-ALL may predict central nervous system relapse. CXCL13 staining was increased in bone sections from pediatric compared to adult B-ALL patients. Taken together, our study shows that the age of the BMM and, in particular, BM macrophages influence the leukemia phenotype. The CXCR5-CXCL13-axis may act as prognostic marker and an attractive novel target for the treatment of B-ALL.
While B-cell acute lymphoblastic leukaemia (B-ALL) can be described as the leukaemia of childhood, chronic myeloid leukaemia (CML) mostly develops in elderly individuals. Understanding and utilising mechanisms involved in the development and persistence of these leukaemias as possible targets for treatment strategies has received particular interest. Processes that happen in the vicinity of the cancerous cells themselves could influence cancer growth and behaviour and hence can serve as novel targets, leading to the development of two-pronged therapies that act both on leukaemic cells directly as well as their niche. The niche in the case of leukaemia is the bone marrow microenvironment (BMM) where these cells are not only generated but also instructed and protected. As the BMM is situated inside bones that undergo drastic changes and growth processes during the ageing process, the BMM itself is also being altered throughout life. These alterations and the very process of expansion itself may therefore also provide distinct regulatory influences on the cells (healthy or malignant) that are generated inside this niche, leading to the question: Does the age of the bone marrow microenvironment differentially influence the development of (“childhood”) B-ALL versus (“adult”) CML by the release of cytokines? In previous studies by the host-laboratory the age distribution of B-ALL versus CML in a murine transduction/ transplantation model could be recapitulated; young mice which received the same number of leukaemia-initiating cells as their old counterparts died significantly earlier of B-ALL while showing a significantly delayed clinical course, when they were suffering from CML. The tumour load and other leukaemia-associated parameters also showed a clear disposition towards preferential induction of CML in elderly and B-ALL in younger mice. In this project we could support the hypothesis that the age of the BMM differentially influences the proliferation of leukaemic cells and thereby the development and persistence of different types of leukaemias by utilising different in vitro culture experiments. Specifically, we could show that young (compared to old) bone marrow 11 stroma cells (BMSC) support the growth of (BCR-ABL1+) B-ALL cells both in a direct, cell on cell co-culture setting, as well as in young BMSC-derived conditioned medium. This supports the hypothesis that varying factors are differentially released from a young versus an old BMM and influence the growth of the leukaemia cells. The opposite might be true for CML cells (BCR-ABL1+ 32D cells); BMSC obtained from old animals showed a tendency to support their growth more profoundly than cells acquired from young animals. Possible proteins responsible for the distinct regulation of myeloid versus lymphatic leukaemic cells by young versus old BMM have also been studied. We investigated C-X-C motif chemokine 13 (CXCL13) and growth differentiation factor 11 (GDF11) in their effect on leukaemia cells, as both proteins having previously been described to have tumour-modelling properties and age-dependent levels (see below). We identified an increased secretion of CXCL13, a B-cell chemotactic factor, into conditioned medium from young versus old BMSC. In accordance with this we found migration of B-ALL cells towards BMSC from young compared to old mice to be improved, while adhesion of both B-ALL and CML cells to young versus old BMSC did not show any differences. By blocking CXCL13 the proliferation-supporting effect of young BMSC on B-ALL cells could be diminished. Similar effects could be demonstrated by blocking GDF11. In the case of CML cells we could observe the opposite effect; blocking CXCL13 and GDF11 increased their proliferation in a co-culture with BMSC. This supported our hypothesis that both cytokines differentially regulate B-ALL and CML behaviour. After the completion of this thesis, another member of the host-laboratory convincingly demonstrated the role of BMM age in the regulation of B-ALL via CXCL13 signalling (see discussion).
B-cell acute lymphoblastic leukaemia (B-ALL) occurs most commonly in children, while chronic myeloid leukaemia (CML) is more frequent in adults. So far, the myeloid bias of haematopoiesis in elderly people has been considered the main reason for these differences in leukaemia phenotype in different age groups. However, differential contribution of a young versus an old bone marrow (BM) microenvironment (BMM) to leukaemia development may have been underrecognized given the fact that the BMM undergoes constant remodelling during a lifetime. Using a murine transduction/transplantation model of BCR-ABL1+ B-ALL and CML, we recapitulated the human phenotype, whereby young (3-4 weeks old) recipient mice, which received the same number and type of leukaemia-initiating cells as their old (>30 weeks old) counterparts, died significantly earlier of B-ALL, while showing prolonged survival in the CML model. Engraftment of CML-initiating cells in young bone marrow (BM) was decreased and myeloid progenitors in young mice were reduced in the CML model compared to old mice. In contrast, homing of B-ALL-initiating cells to a young BMM was increased, and by in vivo 2-photon microscopy we observed that B-ALL-initiating cells homed to locations closer to bone and vessels and migrated faster in young compared to old mice. In in vitro coculture assays we showed that BCR-ABL1+ B-ALL cells proliferate, migrate and adhere significantly more in the presence of young BM macrophages or in conditioned medium from these macrophages, while CML cells showed stronger adhesion and proliferation in the presence of old BM macrophages. Old macrophages showed hallmarks of ageing such as an increase of reactive oxygen species, increased DNA damage, proliferative defects and mitochondrial alterations compared to young macrophages. In addition, genome-wide profiling of chromatin accessibility using ATAC-seq revealed strong differences between young and old bone marrow-derived macrophages and an enrichment of inflammatory response gene sets, which includes IL-6/Jak/Stat3 signalling, as well as the C-X-C motif chemokine (CXCL) 13. Cxcl13 is a chemoattractant for B cells and we showed its expression to be upregulated in young versus old BM macrophages and to have higher levels in a young versus an old BMM. Inhibition or knockdown of Cxcl13 in BMM-derived macrophages led to a decrease in proliferation of cocultured B-ALL cells and impaired migration of leukaemia cells towards young BMM-derived macrophages. Consistently, deficiency of Cxcr5, the receptor for Cxcl13, on B-ALL-initiating cells prolonged murine survival in our B-ALL model. In support of our murine data, decreased CXCR5 expression was associated with improved outcome in human hyperdiploid B-ALL and revealed a trend towards improved outcome in BCR-ABL1+ B-ALL and other subtypes. Taken together, our study shows that the age of the BMM and, in particular, BM macrophages influence the leukemia phenotype. The CXCL13-CXCR5 axis may act as prognostic marker or an attractive target for the treatment of B-ALL. Disclosures Kumar: Merck: Research Funding; European Patent No. 16187926.7: Other: USE OF FIBRONECTIN OR ILK INHIBITORS FOR USE IN THE TREATMENT OF LEUKEMIA. Stock:Agios: Membership on an entity's Board of Directors or advisory committees; UpToDate: Honoraria; Kite, a Gilead Company: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Daiichi: Membership on an entity's Board of Directors or advisory committees; Research to Practice: Honoraria. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding. Lipka:InfectoPharm GmbH: Employment. Krause:Merck KGaA: Research Funding; Patent: Patents & Royalties: European Patents No. 16187926.7-1401, EP18184430.9-.
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