Page 203 - Haematologica May 2020

BCR/PI3K blockade upregulates CXCR4 in DLBCL
Results
SYK inhibition selectively induces CXCR4 expression in BCR-dependent DLBCL cell lines and primary tumors
To identify potential compensatory signaling pathways in DLBCL treated with chemical BCR inhibitors, we reviewed the transcriptional profiles of five BCR-depen- dent DLBCL cell lines treated with the chemical SYK inhibitor, R406, or vehicle (DMSO).3 Differential analysis of treated versus untreated samples revealed that CXCR4 transcripts were significantly upregulated in all five BCR-
AB
dependent DLBCL cell lines (DHL4, DHL6, LY7, HBL1, U- 2932) following 6-24 h of R406 treatment (P-value=0.00052 at 24 h; Online Supplementary Figure S1).
To expand on these findings, we treated an extended panel of BCR-dependent and BCR-independent DLBCL cell lines with R406 (or vehicle) and evaluated CXCR4 transcript abundance by qRT-PCR. The extended DLBCL cell line panel included five BCR-dependent DLBCL cell lines (DHL4, DHL6, LY7 [low NF-κB, GCB]; and HBL1 and U-2932 [high NF-κB, ABC])3 and an additional ABC DLBCL cell line, TMD8, that is sIgM+, BCR-dependent,
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Figure 1. CXCR4 is upregulated in BCR-dependent DLBCL cell lines following SYK inhibition. (A) CXCR4 transcript abundance in diffuse large B-cell lymphoma (DLBCL) cell lines treated with 1 mM R406 (upper panel) or 2 μM GS-9973 (lower panel) for 24 hours (h) was determined by quantitative RT-PCR (qRT-PCR) relative to PPIA. The P-values for vehicle versus R406 treated or GS-9973 treated were determined with a one-sided Welch t-test. ***P<0.0001; *P<0.01. Error bars repre- sent the SD of three independent assays in a representative experiment. (B) CXCR4 transcript abundance in SYK-depleted DLBCL cell lines (72 h following completion of puromycin selection) was determined by qRT-PCR relative to PPIA. NC (negative control) shRNA. The P-values for NC versus shSYK constructs were determined with a one-sided Welch t-test. ***P<0.0001; **P<0.001; *P<0.01; #P<0.05. Error bars represent the SD of three independent assays in a representative experi- ment. (C) Cell surface expression of CXCR4 in DLBCL cell lines treated for 24 h with vehicle or 1 mM R406 (upper panel), or vehicle or 2 μM GS-9973 (lower panel) was measured by flow cytometry. Isotype-matched control in gray. (D) CXCR4 expression in primary DLBCL patient samples following SYK inhibition. Cryopreserved viable DLBCL tumor cell suspensions from newly diagnosed patients were thawed and treated with vehicle or 1 mM R406 for 24 h. RNA samples were prepared and CXCR4 expression was determined by qRT-PCR relative to PPIA. The P-values for vehicle versus R406 treatment were determined with a one-sided Welch t-test. **P<0.001; *P<0.01; #P<0.05. Error bars represent the SD of three independent assays in a representative experiment.
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