Abstract
Infant acute myeloid leukaemia (AML) is an aggressive form of paediatric leukaemia diagnosed in children under two years of age. Infants with AML display distinctive clinical features, such as extramedullary infiltration, and experience high rates of relapse. Over 50% of infant AML cases are associated with rearrangements affecting the KMT2A (lysine methyltransferase 2A) gene, with the gene coding for the MLLT3super elongation complex subunit the most frequent fusion partner.
Bone marrow (BM) is a highly complex tissue containing many stromal and endothelial cell types which tightly regulate normal haematopoiesis, through secretion of soluble factors and cytokines to and from haematopoietic cells, as well as via direct cell-cell contact. It is becoming increasingly well recognised that these bidirectional cellular communication pathways are subverted in adult AML to support leukemic survival at the dispense of normal haematopoiesis. Given the prenatal origins of infant AML, it has been difficult to investigate these microenvironmental interactions due to the lack of faithful in vivo models which can recapitulate its prenatal origins.
We employed the previously described doxycycline-inducible KMT2A-MLLT3mouse model (Stavropolou et al., 2016) and induced KMT2A-MLLT3expression from embryonic day (E)12.5 onwards. KMT2A-MLLT3-expressing progeny succumbed to a lethal myelo-monocytic AML with leptomeningeal infiltration – a clinical feature enriched in the infant AML patient cohort (Blais et al., 2019).
Single-cell RNA-sequencing (scRNA-Seq) was performed via the 10x Genomics platform on BM from control and sick leukemic mice to characterise the cellular microenvironment and signalling networks. Multiple microenvironmental populations were captured and transcriptomically characterised including mesenchymal stromal cells, osteolineage stromal cells, pericytes, and both arterial- and venous-like endothelial cells. AML development was characterised by a generalised loss of stromal sub-populations and the emergence of a fibroblast-like population with enrichment of biological processes involved in niche remodelling. Secretory stromal populations which produce haematopoietic niche support factors were particularly severely depleted, suggestive that leukaemic stroma in infants is less supportive to haematopoietic stem and progenitor cells. Alterations in expression of genes coding for extracellular matrix (ECM) proteins by stromal cells were identified with increased secretion of collagens, in keeping with leukaemic niche remodelling.
Furthermore, RNAMagnet (Baccin et al., 2020) inferred that KMT2A-MLLT3blasts closely associated with LepR+ mesenchymal stromal cells and venous/sinusoidal endothelium suggesting preferential localisation to the perivascular niche.
Cell-to-cell communication (CCC) inference (Jin et al., 2021; Browaeys et al., 2020) was utilised to dissect bidirectional communication between KMT2A-MLLT3blasts and stromal populations and highlighted multiple signalling pathways, including various collagen encoding genes and Cd44, Cxcl12-Cxcr4 and inflammatory pathways. Transforming growth factor β1 (TGF-β1) was noted to be one of the most active ligands between blasts and stromal populations, and is known to be a key mediator in the development of an immunosuppressive tumour microenvironment.
CCC inference between KMT2A-MLLT3blasts and T-lymphocytes further corroborated this with high probability receptor-ligand (R-L) interactions including thrombospondin1 (Thsb1)-Cd47, galectin-9 (Lgals9)-Cd45 and selectin P ligand-L-selectin (SelpIg-Sell) signalling, all of which have been implicated in inducing T cell dysfunction, and supports the development of an immunosuppressive niche, facilitating leukaemic survival.
A major challenge when treating infant patients with leukaemia are their unique vulnerabilities to therapy-related toxicities from chemotherapy and haematopoietic stem cell transplantation. Immunotherapeutic agents, such as bispecific antibodies, have therefore emerged as attractive therapeutic options for these patients. Our data suggest that these T-cell mediated immunotherapies may have limited efficacy as single agents in KMT2A-MLLT3driven infant AML, and novel agents which can block these immunosuppressive signalling pathways in the leukaemic BM will be crucial for enhancing the effects of future immunotherapies in this challenging disease.
