Adipogeneis in the Bone Marrow Microenvironment Confers a Proliferative Advantage in Residual Leukaemic Cell Populations in Acute Myeloid Leukaemia after Allogeneic Stem Cell Transplantation
Authors: May Felicity , Alvares Caroline , Ottmann Oliver G. , Zabkiewicz Joanna 
Published: October 2019

Abstract
Up to 40% of poor risk Acute Myeloid Leukaemia (AML) patients receiving an allogeneic bone marrow transplant (BMT) from a donor will suffer disease recurrence/relapse within a year, significantly shortening their overall survival. Drug development to date has focused on clearing the bulk of leukaemic cells and how to target residual 'Leukaemic stem cells,' which may give rise to relapse, however little work has been undertaken to discover the mechanisms by which the bone marrow niche may be unable to support normal haematopoietic stem cell growth and this may have a significant impact on the re-emergence of the patient's leukaemia. With the rise in the use of reduced intensity conditioning (RIC) regimes prior to transplant, the role of the patient microenvironment into which donor HSCs are engrafted warrants closer investigation.
Stromal layers (AML-MSC) were derived from a cohort of 45 serial diagnostic, early (1-3 month), mid (6-9 month) and late (12 month+) post-transplant AML patients. Uniformity of stromal immunophenotyping markers (CD29, CD73, CD90, CD105, CD166, CD44, CD146) was present from passage 2 with concomitant loss of CD45 and other myeloid lineage markers (CD14, CD13, CD33, CD34). Diagnostic AML-MSCs showed a significant increase in stromal multipotency (CFU-F colony assays and CD146 expression, p<0.001) compared to both NBM and post-transplant MSCs. In comparison, early post-transplant colony formation was significantly reduced compared to other timepoints.
AML-MSC supportive capacity for matched allogeneic donor (normal HSC) or autologous (malignant) blast populations was investigated using 14 day co-culture assays. Flow cytometric analysis of suspension and adherent co-culture fractions revealed AML blast cell numbers were consistently higher than comparative donor cell cultures at all post-transplant stages (p<0.001), suggesting patient AML-MSCs retain a preference for supporting their own blasts throughout different treatment stages. This observation was also confirmed in long-term LTC-IC assays in the stroma-adherent cobblestone fraction. Throughout serial samples, supportive capacity increased in later transplant AML-MSCs samples, confirming previous multipotency observations that stromal fitness to support stem cell growth may be compromised early post-transplant.
Cytokine induced differentiation of AML-MSCs to osteoblast and adipocyte lineages resulted in a significant enhancement of adhesion and supportive capacity in the adipocyte lineage for AML blasts at all timepoints (p<0.002), which was absent in donor cell cultures. Osteoblast cultures gave results similar to that of the undifferentiated stromal layers for both AML and donor blast support. Parallel experiments using donor NBM-MSC layers revealed reduced support for AML blasts compared to that of matched AML-MSC stroma, however the same predisposition for adipogenic support of AML blasts was also observed.
Luminex secretome profiling of serial AML-MSC supernatants using a panel of 105 pro-survival, inflammatory, migratory and immunomodulation cytokines revealed significant alterations in a number of targets associated with the diagnostic and post-transplant setting. Of the 80/105 targets detectable (>100pg/ml), high expression levels were seen in adhesion and ECM remodelling targets highlighting the ability of stromal secretory molecules to modify the surrounding environment. Hierarchical clustering analysis revealed post-transplant AML profiles correlated more closely with that of NBM-MSC at later time points, however significant increases in inflammatory/metabolic stress signalling molecules including IGFBP-3, Protein S, DKK1, VEGF, MMP1,MMP3, TPO (p,0.001), which have all been implicated HSC quiescence were associated with diagnostic and early post-transplant secretory profiles.
In conclusion we identified several changes in the molecular and functional behaviour of the patient bone marrow microenvironment that may promote pro-leukaemic cell survival in post-transplant AML. Alterations in stromal fitness, adipogenic preference and metabolic stress signalling may contribute to a failing ability to support normal donor blood cells. Further exploration of therapies that target these alterations and prime the niche in favour of donor cells may provide a critical window for early clinical intervention and improved patient outcome.

Disclosures
Ottmann: Novartis: Consultancy; Celgene: Consultancy, Research Funding; Pfizer: Consultancy; Amgen: Consultancy; Takeda: Consultancy; Fusion Pharma: Consultancy, Research Funding; Incyte: Consultancy, Research Funding.