Central Nervous System Involvement in a Xenograft Model of Pre-B Acute Lymphoblastic Leukaemia Is Associated with Dysfunctional CXCR4 Expression and Upregulation of the Neurotropic Chemokine Receptors CCR6 and CX3CR1,
Authors: Halsey Christina , Williams Mark TS , Yousafzai Yasar , Rehe Klaus , Heidenreich Olaf , Vormoor Josef , Gibson Brenda , Graham Gerard 
Published: October 2019

Abstract
Abstract 3449
Despite huge advances in the treatment of paediatric acute lymphoblastic leukaemia (ALL) challenges remain. Disease in the central nervous system (CNS) continues to pose difficulties in diagnosis, prevention and treatment. Understanding the biological mechanisms of leukaemic cell entry into the CNS should allow better detection and monitoring of leukemia and may identify novel therapeutic targets for resistant and relapsed disease. We hypothesize that leukaemic cell dissemination to the CNS is associated with the abnormal expression of molecules governing physiological leukocyte trafficking i.e. chemokine receptors, selectins and integrins. To address this hypothesis we have developed a xenograft model of CNS disease using IV tail vein injection of human leukaemic cell lines and primary cells into immunodeficient (NOD/SCID/IL2R gamma null) mice.
Pre-B leukaemic cell lines were seen to differ in their capacity to home to the CNS. Using Taqman low density array plates quantitative expression of a panel of chemokine receptors, selectins and integrins was compared between these cell lines. Rapid onset of CNS disease was associated with significantly higher expression of P-selectin glycoprotein ligand 1 and the chemokine receptor CCR6 both known to be essential for blood:CSF barrier transit of leukocytes (Kivisakk et al PNAS 2003, 100, 8389–8394, Reboldi et al Nat Immunology 2009, 10, 514–523). Other genes upregulated in CNS homing cells included (1) the integrins alphaM beta2 and beta 7 (2) ICAM-1 and −3 and (3) the chemokine receptors CCR1, CCR7 and CXCR3.
Interestingly the chemokine receptor CXCR4 showed down-regulation when measured by qPCR and flow cytometry in CNS homing cells, with levels of receptor expression inversely proportional to the rapidity of onset of CNS disease. Furthermore in vitro studies showed that the migratory response of CXCR4 to its ligand CXCL12 was blunted or absent in cell lines which produced a more rapid onset of CNS disease. Two of the cell lines were Philadelphia positive, raising the possibility that p190 bcr-abl could be interfering with CXCR4 signalling or receptor levels as previously demonstrated for the p210 bcr-abl fusion protein (Geay et al Cancer Res 2005, 65, 2676–2683). Although non-migratory cells had higher levels of bcr-abl expression than migratory cells the blunted responses could not be reversed by treatment with the bcr-abl inhibitor imatinib. CXCR4 mutations were excluded by direct sequencing. Since functional CXCR4-CXCL12 interactions are known to be important for retention of cells in the bone marrow microenvironment (Ma et al, Immunity 1999, 10, 463–71), disruption of this interaction may be a necessary pre-requisite for cell migration to other sites.
To examine potential micro-environmental influences on gene expression patterns in vivo, cells were retrieved from the CNS, bone marrow, liver, spleen and kidneys of engrafted mice using anti-human CD19 magnetic bead sorting and species specific primers for qPCR. Cells derived from the CNS had higher levels of CCR6 and the neurochemokine CX3CR1 (fractalkine receptor) compared to the original cell line in vitro and cells retrieved from other sites. Increased CCR6 may represent sub-clonal selection of CCR6 high expressors during transit across the BCSFB. Fractalkine and its receptor are highly expressed in the central nervous system and are important for maintenance of microglial-neuronal communication with fractalkine activating pro-survival signaling pathways in cells bearing its receptor (Meucci et al PNAS 2000, 97, 8075–8080). This provides a possible mechanism by which fractalkine expression would provide a competitive advantage to cells and allow survival of pre-B cells in this normally hostile microenvironment.
In conclusion, we present a xenograft model of CNS leukemia and its utilization to identify increased CCR6 and P-selectin glycoprotein ligand 1 expression and reduced CXCR4 expression (and/or function) as candidate mechanisms by which leukaemic pre-B cells cross the blood:CSF barrier. In addition we propose that the Fractalkine receptor CX3CR1 may act as a potential pro-survival mechanism for pre-B cells residing in the CNS. As well as shedding light on the biology of CNS disease in pre-B cell ALL these molecules may be valid novel therapeutic targets for resistant or relapsed CNS disease.

Disclosures:
No relevant conflicts of interest to declare.