Inhibition of Phosphatidylinositol 3-Kinase Is Effective to Suppress the Growth of CD20-Negative Refractory Diffuse Large B-Cell Lymphoma Cells

Abstract 2421

CD20 is not only a therapeutic target but also its expression has prognostic importance in B cell lymphoma. Decreased CD20 expression is often associated with refractory phenotypes, especially in lymphoma treated with therapy including rituximab, an anti-CD20 antibody.

To address the molecular mechanism(s) of down-regulation of its expression and to find an alternative therapeutic target(s) in resistant lymphoma, we analyzed a CD20-negative B-cell lymphoma cell line (SD07). SD07 was established from lymphoma cells without expressing CD20, which appeared in pleural effusion in a patient with diffuse large B-cell lymphoma (DLBCL), after 2-years of repeated anti-lymphoma therapy including rituximab, While initial diagnosis was CD20 positive activated B cell-like DLBCL (ABCDLBCL). SD07 selectively lacks CD20 protein expression, but not other B cell antigens, such as CD19, CD21 and CD22 by flowcytometry(FCM). In SCID mice, subcutaneously transplanted SD07 developed tumors which are positive for B cell antigen such as CD79a, but not CD20 (L26) and EBER. Array CGH revealed 0.7 Mb region with copy number loss less than −1.0 of log₂ ratios on chromosome 11q12 in SD07. Within this region, 30 kb segment, which showed further loss (less than −2.0),contained two genes, MS4A1(CD20) and MS4A5. Southern blot analysis using CD20 exon 1 as a probe showed homozygous deletion of CD20 gene in SD07. As expected, incubation with rituximab in culture failed to suppress the cell growth of SD07 up to 20 μg/ml(cell No. rituximab/control=0.98±0.04, P=NS). This suggests deletion of CD20 gene with genomic copy number loss in 11q12 produced the loss of CD20 expression and resulted in resistant to rituximab. It is currently studied whether the loss of CD20 expression is directly involved in the tumorigenicity of SD07.

Array CGH also showed several genomic regions with copy number loss which are possibly involved in refractory phenotype of SD07. Those includes 10q23 (PTEN), 12q23 (APAF1) and 16q21 (NFATC3). Immunoblot analysis showed the absence of PTEN protein expression and constitutive AKT Ser473 phosphorylation in SD07. SDO7 also showed constitutive phosphorylation of both SykTyr525/526 and Btk Tyr223,however, those did not differ much in those in three germinal center B-like(GCB) cell lines, two Burkitt lymphoma cell lines, Daudi and N8, and a DLBCL cell line, TK, derived from follicular lymphoma. In SD07, somatic mutations of ITAM of either CD79A or CD79B was not detected. This suggests inactivation of PTEN, rather than chronic active BCR signaling, is likely to promote constitutive PI3K-AKT signaling in SD07.

Nuclear NF-kB DNA binding by EMSA, SD07 showed constitutively higher binding signals of NF-kB, compared with either Daudi, N8 or TK (SD07=3.6±0.1,GCB cell lines(Daudi,N8,TK)=1.2±0.2, p<0.01, arbitrary density units). Supershift analysis showed increased NF-kB DNA binding in SD07 mainly consist of p50 and c-Rel. By immunoblot, SD07 showed steady-state increased accumulation of p50 protein, but not p65, in nuclear fraction, compared with either in Duadi,N8 or TK. Protein expression of BClxL, a NF-kB target gene, increased in SD07 (SD07=1.9±0.1, GCB cell lines=0.7±0.2, p<0.01, signal intensity, SI).

Those suggest that oncogenic PI3K-AKT activation and/or constitutive activation of NF-kB pathway contribute pro-survival signaling in SD07 and those are possible therapeutic target in SD07. Incubation with LY294002(LY), selective PI3K inhibitor, at more than 0.1μM for 4 days, dose-dependently inhibited the cell growth of SD07(10μMLY/DMSO=0.35±0.01, P<0.01), without significant effects on cell viability. FCM analysis with PI showed that incubation with LY produced G1 accumulation and decrease of cells in the S (LY/DMSO,G1=1.5±0.1,S=0.2±0.1,G2M=0.4±0.1, P<0.05).

10 μM LY inhibited both AKT Ser473 phosphorylation (LY/DMSO=0.18±0.01,SI,P<0.01) and protein expression of BClxL (LY/DMSO=0.43±0.02,P<0.05) in SD07. LY increased p27 protein expression (LY/DMSO=1.83±0.04, P<0.05), without affecting NF-kB DNA binding in SD07. It may suggest PI3K are required for expression of some NF-kB target gene without suppressing nuclear NF-kB DNA binding.

In summary, although a limited in a cell line, we clarify a novel molecular mechanism of acquired loss of expression of CD20 in DLBCL.

In addition, we show that de-regulated PI3K-AKT pathway is a possible therapeutic target for CD20-negative refractory ABCDLBCL.