VEGFA- a New Therapeutic Target in CNS Leukemia

Abstract

Acute lymphoblastic leukemia (ALL) is the commonest cancer to affect children. Historically, central nervous system (CNS) involvement has been considered as a feature of high risk disease. Although this risk is mitigated by current intended CNS directed treatments based on risk stratification strategies, currently 10% or more of children will die from relapsing or unresponsive disease and the CNS is still a major site of relapse.CNS directed treatments bypass the blood brain barrier (BBB) but do not specifically target CNS leukemic cells. Consequently, although effective, they are associated both with serious short-term toxicities and debilitating long-term morbidities. Understanding the biology of those ALL cells which lodge in the CNS may lead to the development of drugs to specifically target them, and this could further improve outcomes but with fewer complications.We transplanted bone marrow (BM) derived leukemic cells from children with B-ALL into non-conditioned immune-deficient mice by tail-vein injection. We confirmed BM engraftment and evidence for CNS involvement was demonstrated by microscopy and flow cytometry analyses of the CSF, and was confirmed histo-pathologically in brain tissue. The pattern of leukemic infiltration of the CNS in xenograft recipients closely mimicked that seen post mortem in children with ALL. Furthermore, the MRI scans of xenograft mice showed a meningeal enhancement typical of the leukemic meningitis seen in patients with CNS involvement. Thus by using standard clinical techniques, we were able to demonstrate that the xenograft recipients of B-ALL cells faithfully recapitulates patterns of CNS disease that are seen in human.Interestingly, human leukemic cells isolated from either the CNS or BM of individual recipient mice demonstrated different kinetics of engraftment following serial passage into secondary and quaternary recipients. This functional observation implies cell intrinsic differences between leukemic cells lodged at the two sites.We next compared the gene expression profiles of leukemic cells isolated from the CNS and BM of affected children and xenograft recipients. Using gene set enrichment analysis, we demonstrate that leukemic cells that have infiltrated the CNS, in contrast to those in the BM, show features of adaptation to hypoxia including cell cycle quiescence and downregulation of oxidative phosphorylation. Consistent with these transcriptional data, we could demonstrated that fewer of the CNS-derived leukemic cells were in S/G2/M compared with BM-derived leukemic cells and that CNS-derived leukemic cells consumed less oxygen in in vitro assays.We found that CNS derived leukemic cells had a transcriptional signatures suggestive of physiological adaptation to hypoxic microenvironment and VEGFA is one of the most up-regulated genes in CNS-derived leukemic cells. We tested therapeutic potential of targeting VEGFA using bevacizumab, a VEGFA-neutralizing antibody. Bevacizumab or normal saline (NS) was injected intraperitoneally into the xenografted recipients of B-ALL cells. In the bevacizumab group, leukemic cell numbers in the CNS were significantly less at the end of treatment, and staining of brain sections confirmed reduced leukemic burden in CNS of bevacizumab-treated mice compared with control mice. This in vivo preclinical assay shows that VEGFA could be targeted to treat the patients with ALL who have CNS involvement.In conclusion, we have shown that B-ALL cells residing in the CNS have gene expression signatures and phenotypic features that are distinct from those in the BM, and that this can be effectively targeted in preclinical model of CNS leukemia. These observations may inform not only further mechanistic research, but also future clinical trials. DisclosuresOgawa:Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.