Single cell transcriptomics of matched cerebrospinal fluid and bone marrow patient samples highlights the role for CXCR4 in acute lymphoblastic leukemia CNS disease

Abstract Acute lymphoblastic leukemia (ALL) disseminates to the central nervous system (CNS) with high prevalence, and a very high proportion of ALL relapses involve the CNS. While experimental studies have suggested a role for several proteins in CNS involvement and relapse of ALL, studies of leukemic cells in the cerebrospinal fluid (CSF) of patients are lacking. In addition, it is unknown whether these proteins are selectively upregulated in subsets of leukemic cells to facilitate CNS invasion. In this study, we have characterized the gene expression profile of leukemic cells on a single cell level in matched cerebrospinal fluid, bone marrow (BM), and peripheral blood (PB) samples from patients with ALL, to uncover potential mechanisms for CNS involvement and relapse. We specifically investigate 16 genes, coding for proteins shown in xenograft models to play a role for CNS invasion or persistence of leukemic cells in ALL. The study included 34 patients with ALL, represented by bone marrow samples from all patients (n=34), matched CSF samples from patients with CNS involvement at either diagnosis or relapse (n=7), and peripheral blood samples from patients with CNS involvement and leukemic cells present in blood (n=5). Single cell RNA sequencing was performed on single cell FACS sorted leukemic cells from fresh material. We investigated differentially regulated genes in leukemic cells of the CNS compared with those in BM/PB for each patient based on the Wilcoxon rank sum test (Seurat FindMarkers function). Next, we selected the top differentially expressed genes based on a combination of p-values and the number of patients where the gene was differentially regulated. Of the 16 genes known from literature to play a role in xenograft models, only CXCR4 was among the top 100 upregulated genes. We then investigated the expression of the 16 genes in CSF leukemic cells of patients with CNS relapse (n=4) compared to CSF leukemic cells from patients with CNS involvement at diagnosis (n=3), using DeSeq2 on pseudobulk data. This revealed that CXCR4 (log2FC 2,1, p=0.00096) and VEGFA (log2FC 3,8, p=0.0047) levels were higher in CSF leukemic cells from patients with CNS relapse than in those from patients analyzed at diagnosis, while ITGA6 (log2FC -2,1. P=0.017) was downregulated. Since CXCR4 was the only gene significantly upregulated in CSF cells compared with BM/PB, and also in CSF cells of patients with CNS relapse compared with patients without relapse at diagnosis, we then measured CSF protein levels of the CXCR4 ligand CXCL12 by ELISA in 36 patients with ALL at time of diagnosis or relapse. Of the included patients, 26 did not experience relapse within the follow-up period and 10 had a relapse involving the CNS, sampled either at time of diagnosis or relapse. We found higher levels of CXCL12 in the CSF of patients with CNS relapse compared with those with no relapse (Mann-Whitney U test, p=0.044). We also investigated the CXCR4 gene expression level in bone marrow leukemic cells of the 30 patients at diagnosis comparing patients with and without detectable leukemic cells in the CSF measured by flow cytometry and found no significant difference.In conclusion, our study identifies CXCR4 as the only gene upregulated in CSF leukemic cells among known candidates for ALL CNS disease. CXCR4 was further upregulated in CSF leukemic cells of patients with relapse, as was CSF levels of the CXCR4 ligand CXCL12. Thus, our study highlights for the first time through single cell transcriptomics of matched CSF and BM patient samples a potential role for the CXCR4-CXCL12 axis in ALL CNS disease and suggests that therapies inhibiting this axis should be investigated for prevention of CNS relapse in ALL.