Rituximab-Induced CD20-Mediated Signals and Suppression of PI3K-AKT Pathway Cooperates to Inhibit B-Cell Lymphoma Growth By Down-Regulation of Myc.

Rituximab, anti-CD20 monoclonal antibody, shows anti-lymphoma effects by either activating host immune mechanism against lymphoma cells such as ADCC and CDC or suppressing signal(s) important for growth of B-cell lymphoma. Accumulated evidence shows de-regulated PI3K-AKT pathway is important therapeutic target in B-cell lymphoma. Although studies suggested suppression of PI3K-AKT pathway is possibly critical in rituximab-induced anti-lymphoma signals, details of molecular mechanisms covering CD20-mediated signals and PI3K-AKT pathway are still unknown.

We analyzed signals in B-cell lymphoma cell line, SD07, which had bi-allelic deletions of both CD20(MS4A1) and PTEN gene. Briefly, SD07 was established from a patient with diffuse large B-cell lymphoma(DLBCL)who showed refractory CD20-negative relapse(Eur.J. Haematol. 2012).

We transiently restored protein expression of either or both of CD20 and PTEN by electroporation of expression plasmid having cDNA of those genes in SD07. In this experiment, expression of both proteins were clearly detected by western blot until 72 hours after transfection. CD20 protein was also detectable on cell surface of SD07 cells by flow cytometric(FCM) analysis at comparable levels of those of Daudi cells after transfection on CD20 cDNA.

Our previous study showed expression of both c-myc RNA and protein, as judged by FCM, was induced by transient transfection of CD20 cDNA in SD07 cells. Western blot analysis also showed significant increase of myc protein 24hrs after transfection (vector=13.7±0.2, CD20=14.5±0.4, P<0.05, arbitrary density unit) and the increase was not affected by the addition of rituximab(20µg/ml)in culture. In contrast transfection of PTEN cDNA produced significant decrease of myc protein (PTEN=11.2±0.1, P<0.01). Myc protein levels was comparable between cells transfected with CD20 plus PTEN and PTEN alone. However in the presence of rituximab, myc protein expression was markedly downregulated in SD07 cells at both 24 hours(vector=13.7±0.2,CD20+PTEN=8.3±0.1,P=0.0008)and 48hours(vector=12.4±0.3 vs CD20+PTEN=11.2±0.1,P=0.04).

24 hours after transfection, de-phosphorylation and down-regulation of retinoblastoma(RB) protein expression was evident only in SD07 cells transfected with both CD20 and PTEN, but not with either alone.

Rituximab also augmented down-regulation of RB protein in SD07 cells transfected with both CD20 and PTEN.

After transfection, in vitro cell growth of SD07, as evaluated by MTT, was as follows;vector=4.6±0.3, CD20=4.8±0.3(P=NS), PTEN=4.1±0.2(P<0.05), CD20+PTEN=3.6±0.2(P<0.01), CD20+PTEN+rituximab=2.7±0.2(P<0.01),OD570nm, days3)

72 hours after transfection, by FCM, SD07 transfected with both CD20 and PTEN showed significant increase of subG1 DNA content potion compared with cells transfected with either alone(vector=2.5±0.2%,CD20=5.3±0.2%,PTEN=8.8 ±0.5%,CD20+PTEN= 16.1±0.6, P<0.01). Furthermore, addition of rituximab

significantly increase of cells in subG1 in SD07 with transfected with both CD20 and PTEN(18.6±0.5%,P<0.01), suggesting suppression of PI3K-AKT is important for mediateing rituximab-induced apoptotis.

Accordance with proliferative effect of CD20 in

SD07, transfection of CD20 alone produced significant increase of phosphorylation of AktSer473(vector=11.2±2.3, CD20=13.3±2.3,pAkt/total Akt,P<0.05). Co-transfection of PTEN with CD20 suppress the CD20-induced phosphorylation of Akt in SD07 cells. (CD20+PTEN=12.8±2.3,P=NS).

In summary, although data is limited in a cell line, it clearly shows rituximab-induced CD20-mediated signals and suppression of PI3K-AKT pathway by tumor suppressor PTEN cooperatively inhibits growth and induce apoptosis of DLBCL cell line, SD07. Myc protein is critical molecules in this particular cooperative pathways. The present result partly agree studies which showed that inhibition of PI3K-AKT signaling and down-regulation of myc is important in PTEN-induced cytotoxicity in GCB(germinal center B cell)type DLBCL. In addition, we show here myc is not only for PTEN, but also involved in rituximab-induced anti-lymphoma effect. The present result provide a rationale for combination of rituximab and PI3K inhibitor in the treatment of DLBCL. In this context, myc is a candidate of the therapeutic target in rituximab-based immunochemotherapy for B cell lymphoma.