p38 MAPK and NF-κB Survival Pathways As Targets for Action of Rituximab In Vivo in a Xenographic Model,

Treatment of B-NHL cell lines with rituximab has been shown to inhibit the p38 MAPK and NF-κB pathways, resulting in chemosensitization to drug-induced apoptosis (Vega et al., 2004 Oncogene). Chemosensitization was a direct consequence of the inhibition of the Bcl-2 / Bcl-xL anti-apoptotic proteins. It has recently shown for us (Vega, et al BMC-Cancer 2015), that activation and expression of p38 MAPK correlate with a poor prognosis in patients with DLBCL-NHL treated with CHOP alone. However, cellular signaling by rituximab in tissue from patients with B-NHL has not been described, and therefore we have examined in an in vivo model that carries a tumor xenograft for validation. Balb/c nu/nu mice were inoculated s.c. with the B-NHL Raji cell line; one group of mice was left untreated, and another group was treated with rituximab (30 mg/kg) at days 5 and 10 after implantation with 8x10⁶ Raji cells. We monitored the tumor growth, and the animals sacrificed at day 30, the tumors collected for further analysis. Treatment with rituximab resulted in significant growth inhibition of tumor cells compared to the control. The IHC examination of the tissue tumors for the expression of phospho and nonphospho-total-p38 MAPK, NF-κB (phospho p65), and Bcl-xL revealed an overexpression of phospho-p38 MAPK, Bcl-xL, and NF-κB with a strong nuclear localization of phospho-p65. In contrast, tumor tissues derived from rituximab treated mice demonstrated a significant inhibition of phospho-p38 MAPK, Bcl-xL, NF-κB, and a reduction of nuclear localization of phospho-p65. We also analyzed the expression of CD20 in tumors and its correlation with the expression of phospho-p38 MAPK. Furthermore, we inoculated a group of mice with Ramos cells (8X10⁶), and two weeks post-incubation the tumors were collected, and their expression of phospho-p38 MAPK and NF-κB analyzed. A section of the tumor was re-inoculated into another group of mice, and we repeated this procedure three more times. The re-inoculated tumors showed an increased phospho-p38 MAPK and NF-κB expression. These findings established for the first time, in a preclinical model, the rituximab-mediated inhibition of cell survival pathways mediated by p38 MAPK and NF-κB. Moreover, the study also corroborates the role of Bcl-xL expression in resistance and its inhibition by rituximab. It also allows establishing that p38 MAPK activation correlates with tumor malignancy in a tumor re-transplantation model. Overall, this study suggests that the survival pathways of p38 MAPK and NF-κB are targets for rituximab-mediated effects and suggests these proteins can use for intervention in cases of resistance to rituximab and p38 MAPK as well as a prognostic factor.

Vega MI, et al Rituximab inhibits p38 MAPK activity in 2F7 B NHL and decreases IL-10 transcription: pivotal role of p38 MAPK in drug resistance. Oncogene. 2004;23(20):3530-40

Vega GG, et al. P38 MAPK expression and activation predicts failure of response to CHOP in patients with Diffuse Large B-Cell Lymphoma. BMC Cancer. 2015;15:722.