CXCR4 Upregulation Is a Biomarker Of Sensitivity To Targeted Inhibition Of B-Cell Receptor Signaling In Diffuse Large B-Cell Lymphoma

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell tumors including diffuse large B-cell lymphoma (DLBCL). BCR signaling activates proximal pathway components such as the spleen tyrosine kinase (SYK) and downstream effectors including PI3K/AKT and NF-κB. In recent studies, we characterized distinct SYK/PI3K/AKT-dependent viability pathways in BCR DLBCL cell lines and primary tumors with high- or low-baseline NF-κB activity and defined additional SYK/PI3K/AKT-dependent processes essential for BCR DLBCLs with low- or high-baseline NF-κB (Chen et al Cancer Cell 2013; 23:826-838). For example, SYK/PI3K/AKT-dependent cholesterol biosynthesis was identified as a feed-forward mechanism of preserving the integrity of BCRs in lipid rafts in all BCR-dependent DLBCLs.

In the current study, we sought to identify biomarkers of intact BCR signaling in DLBCLs with low or high baseline NF-κB. We found highly significant transcriptional upregulation of CXCR4 in all BCR-dependent DLBCLs treated with a chemical SYK inhibitor (R406). These results were of particular interest because BCR signaling inhibits stromal cell-derived factor (SDF)-1α chemotaxis and promotes intermobilization of the SDF-1α receptor, CXCR4.

To expand on our findings, we analyzed CXCR4 transcript abundance in a large panel of cell lines including BCR-dependent/ low NF-κB (GC type) and BCR-dependent/ high NF-κB (ABC type) and BCR-independent/ OxPhos DLBCLs. CXCR4 transcript abundance was significantly and selectively upregulated in all BCR-dependent DLBCL lines assessed by qRT-PCR following chemical SYK inhibition. Consistent with the qRT-PCR results, SYK blockade also selectively upregulated cell surface CXCR4 expression in all BCR-dependent DLBCLs. Similar results were obtained following SYK knockdown with multiple independent shRNAs; CXCR4 transcript abundance was significantly and selectively upregulated in all BCR-dependent/ low NF-κB (GCB) DLBCLs and BCR-dependent/ high NF-κB (ABC) DLBCLs but was not altered in BCR-independent/ OxPhos tumors. Following SYK blockade, we also observed significant CXCR4 induction (by qRT-PCR) in multiple primary BCR-dependent DLBCLs (NF-κB low and NF-κB high) and unchanged CXCR4 levels in primary BCR-independent tumors. These data confirm that the observations regarding SYK blockade and CXCR4 induction in BCR-dependent DLBCL cell lines translated to primary tumors.

Our recent studies indicate that all BCR-dependent DLBCLs exhibit SYK/PI3K/AKT signaling and modulation of FOXO1 regardless of baseline NF-κB levels. Given the recent identification of CXCR4 as a FOXO1 target, we asked whether modulation of CXCR4 in DLBCLs was via a PI3K/AKT pathway. In BCR-dependent DLBCLs with low or high baseline NF-κB, chemical PI3K inhibition (LY294002) phenocopied chemical SYK blockade (R406) with highly significant transcriptional upregulation of CXCR4. Moreover, in BCR-dependent DLBCL cell lines, enforced expression of a constitutively active form of AKT (myrAKT) abrogated CXCR4 upregulation resulting from SYK blockade. These data indicate that in BCR-dependent DLBCLs, CXCR4 is modulated by a SYK/PI3K/AKT-dependent pathway.

To assess the functional significance of CXCR4 upregulation in BCR-dependent DLBCLs, we performed a transwell chemotaxis assay and evaluated migration toward a SDF-1α gradient. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased migration toward the CXCR4 ligand, SDF-1α, whereas the BCR-independent/ OxPhos DLBCLs were unaffected. The enhanced SDF-1α dependent migration was abrogated when the chemotaxis assay was performed in the presence of the specific CXCR4 inhibitor, AMD3100, confirming the specificity of the observed effect.

Therefore, in BCR-dependent DLBCLs with low or high baseline NF-kB, SYK/PI3K inhibition is associated with significant and functionally relevant upregulation of CXCR4. For these reasons, CXCR4 may serve as a robust biomarker to assess the integrity of the BCR pathway and evaluate the efficacy of BCR inhibition in DLBCL.