Page 205 - Haematologica May 2020

BCR/PI3K blockade upregulates CXCR4 in DLBCL
cell lines (DHL4, DHL6, LY7 [low NF-κB]; and TMD8 [high NF-κB]), were transduced with a vector encoding constitutively active (myristoylated) AKT1 (mAKT) or an empty vector control.29 Thereafter, GFP+-selected cells were treated with vehicle control or R406 and analyzed for CXCR4 expression. Following R406 treatment, all four BCR-dependent DLBCL cell lines infected with the control vector expressed increased CXCR4 (Figure 3A, top panel). In contrast, chemical SYK inhibition did not modulate CXCR4 expression in mAKT-expressing DLBCL cell lines with constitutive activation of AKT1 (Figure 3A, lower panel). These data confirmed the role of PI3K/AKT in
A
SYK-dependent modulation of CXCR4 expression.
Given these findings, we assessed the consequences of chemical pan-PI3K inhibition on CXCR4 expression in BCR-dependent DLBCL cell lines (Figure 3B) using the tool compound, LY294002. Like chemical SYK inhibition, pan- PI3K blockade with LY294002 increased CXCR4 transcript abundance (Figure 3B) and cell surface expression (Figure
3C).
We next examined the mechanism by which prolonged
SYK/PI3K inhibition induces CXCR4 expression in BCR- dependent DLBCLs. BCR signaling is known to promote CXCR4 internalization and inhibit SDF-1α induced
B
C
Figure 3. PI3K/AKT signaling regulates CXCR4 expression in BCR-dependent DLBCL cell lines. (A) BCR-dependent DLBCL cell lines, DHL4, DHL6, LY7 and TMD8, were retrovirally transduced with pMIG- mAKT1-IRES-GFP or pMIG-IRES-GFP vector, FACS- sorted for GFP expression, treated with 1 mM R406 or vehicle for 24 h (h), and analyzed for CXCR4 expression by flow cytometry. (B) BCR-dependent DLBCL cell lines were treated with 1 μM R406 (red), 10 mM LY294002 (blue) or vehicle for 24 h. Thereafter, CXCR4 expression was analyzed by qRT- PCR relative to PPIA. The P-values for vehicle versus R406 treated or vehicle versus LY294002 treated were determined with a one-sided Welch t-test. ***P<0.0001; **P<0.001; *P<0.01. Error bars represent the SD of three independent assays in a representative experiment. (C) Cell surface expression of CXCR4 was measured by flow cytome- try in DLBCL cell lines treated with vehicle (black), R406 (red) or LY294002 (blue) for 24 h.
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