Analysis of CD20-Mediated Signals in Refractory Diffuse Large B-Cell Lymphoma Cells.
Authors: Nakamaki Tsuyoshi , Fukuchi Kunihiko , Nakashima Hidetoshi , Ariizumi Hirotsugu , Maeda Takashi , Saito Bungo , Yanagisawa Kouji , Tomoyasu Shigeru 
Published: October 2019

Abstract
Abstract 2684
Loss of CD20 expression isimportant not only because of the loss of a therapeutic target, but because it is often associated with a poor prognosis of the patient with DLBCL at relapse. In newly diagnosed cases of DLBCL, reduced expression of CD20 at diagnosis is also associated with poor prognosis. CD20-mediated signaling pathway(s) and how rituximab affect the phenotype trough CD20 in refractory B-cell lymphoma remains to be studied.
We analyzed CD20-mediated signals in refractory DLBCL using a CD20-negative DLBCL cell line, SD07 as a model. SD07 was established from a patient with DLBCL at CD20-negative relapse after receiving immuno-chemotherapy that included rituximab. We transiently transfected expression vector(pIRES2-GFP) carrying CD20 cDNA into SD07 cells. CD20 gene was successfully re-expressed in SD07 cells. By immunoblot, CD20 protein was detectable in SD07 cells for 72 hours after transfection. By flowcytometry (FCM), expression levels of cell surface CD20 protein was even more than those in Daudi B-cell lymphoma cells, which is sensitive to growth inhibition of rituximab. Expression of CD20 gene in SD07 produced increased viable cell number through 6 days' culture compared with those in transfected with vector alone(SD07=4.9±0.1(d1), 4.8±0.2(d3), 4.5±0.1(d6) and SD07CD20=5.6±0.3(d1), 5.7±0.1(d3), 5.9±0.4(d6), x105cells/ml, P<0.05). Increase of number of cells transfected CD20 gene was accompanied with rapid increase of expression of c-myc mRNA analyzed by RT-PCR. c-myc mRNA was detectable 24hrs and remained to be detected up to 6days after transfection, while those was undetectable in cells tranfected with vector alone. By FCM, increase of c-myc protein was also detected 24 hours after transfection (SD07=5.9±0.2, SD07CD20=8.2±0.4, mean signal intensity(MSI), p<0.01). Incubation with rituximab (20μg/ml), unexpectedly, even stimulated the increase of viable cell number of CD20-tranfected SD07 cells (SD07CD20=5.7±0.1(d1), 5.9±0.3(d3), 6.1±0.2(d6), x105cells/ml). c-myc protein of CD20-transfected SD07 remained to be up-regulated in the presence of rituximab (SD07CD20=7.6±0.4, MSI). Accelerated phosphphorylation of retinoblastoma protein was observed in CD20-transfected SD07 cells. By immunoblot, expression levels of either of p-Src(Tyr416), p-Akt(Ser473), Bcl2 or BclxL protein was not affected by the transfection of CD20 into SD07 cells for 6 days' culture in the presence or absence of rituximab. Those results suggest that growth-promoting effect are involved in CD20-mediated signals in some types of B-cell lymphoma.
We hypothesized that oncogenic activation of PI3K-AKT pathway might be involved in the lack of rituximab-induced anti-lymphoma effects in CD20-tranfected SD07 cells in culture, because SD07 shows a homozygous deletion of PTEN and constitutive Akt Ser473 phosphorylation. Rituximab showed significant growth inhibition on CD20-transfected SD07 cells in combination with suboptimal concentration of various chemotherapeutic drugs only in the presence of LY294002 (LY, 10μM), a selective PI3K inhibitor, while rituximab without LY rather stimulated cell growth of those cells(medium=85±0.1%, cisplatin(1μg/ml)=71±0.3%, doxorubicin(100ng/ml)=78±0.2%, bendamustine(10μM)=71±0.1%, etoposide(2μM)= 61±0.3%, Ara-C(10μM)=70±0.5%, % of cell number of CD20-transfected SD07 incubated without LY at day 3, P<0.01) Moreover, FCM analysis of cells stained with PI showed that rituximab significantly increased of subG1 DNA content portion in CD20-transfected SD07 cells in the presence of both LY and chemotherapeutic drugs (medium=5.1±0.8%, cisplatin=6.7±0.2%, doxorubicin =7.6±0.1%, bendamustine=9.0±0.2%, etoposide=9.0±0.2%, Ara-C=11.3±0.2%). Those suggest that deregulated PI3K-AKT pathway may contribute, at least in part, to defects of growth-inhibitory/pro-apoptotic effect of rituximab on SD07 cells.
In summary, although a limited in a DLBCL cell line, we showed expression of CD20 is involved in cell growth and cell cycle progression accompanied with rapid induction of c-myc in refractory DLBCL cells. In chemo-refractory lymphoma cells, accumulating genetic alteration(s) of signal transduction pathways downstream of CD20 might impair rituximab-induced anti-lymphoma effects. CD20-mediated signaling pathways are possible therapeutic target(s) to overcome refractory B- cell lymphoma.

Disclosures:
No relevant conflicts of interest to declare.