The Acquirement Of Rituximab Resistance Is Associated With De-Regulation Of The PI3K/Akt/mTOR Signaling Pathway Leading To Chemotherapy Resistance In Burkitt Lymphoma Pre-Clinical Models

Treatment with multi-agent chemotherapy regimens has significantly improved survival in pediatric Burkitt Lymphoma (BL) leading to long-term survival in over 80% of cases. The incorporation of rituximab in the treatment of pediatric B-cell non-Hodgkin lymphoma (B-NHL) has been slower than in adults, but may improve clinical outcomes in high risk pediatric BL patients when combined with chemotherapy regimens. On the other hand, BL patients with primary refractory or relapsed disease have a dismal prognosis, stressing the need to identify the mechanism(s) resulting in chemotherapy/rituximab resistance and to develop novel therapeutic approaches. To this end, we exposed a BL cell line (Raji) to escalating doses of rituximab with or without human serum and generated/characterized several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or rituximab-chemotherapy resistant (Raji 2R and Raji 4RH) (RCRCL) cell lines. Subsequently, we screened for aberrant activation of signal transduction pathways between RSCL (Raji), RRCL (Raji 7R and Raji 8RH), or RCRCL (Raji 2R and Raji 4RH) in an attempt to define what pathways were associated with resistance to both rituximab and chemotherapy agents. This was accomplished by the analysis of phosphorylation patterns on key-regulatory members of pre-defined signal transduction pathways using Western blotting, phospho-flow cytometry studies and phosphoproteomics. Effects on cell viability or cell cycle distribution of RRCL or RCRCL following pharmacological inhibition of key-regulatory pathways identified was then performed using the alamar blue reduction assay or flow cytometry respectively. While total Akt (Protein kinase B) expression was similar between all the types of BL cell lines tested, we found that RCRCL (Raji 2R and Raji 4RH) had an increase in basal phosphorylation levels of Akt at the Ser473 and Thr308 phosphorylation sites when compared to RSCL (Raji cells) or RRCL (Raji 7R and Raji 8RH). These findings were confirmed by phospho-flow cytometry studies. Phosphoproteomic analysis comparing Raji (RSCL) against Raji 4RH (RCRCL) cells identified an increase of at least 2 fold in the phosphorylation of 315 proteins in RCRCL including several direct targets of AKT such as GSK3B, WEE1, FOXO1 and PRAS40. Altered phosphorylation of multiple Akt/mTOR downstream proteins (BAD, 4EBP1, GSK3B and ERK) was detected by western blot in RCRCLs (Raji 2R and Raji 4RH) compared to RSCL and RRCLs. In vitro exposure of RCRCL (Raji 2R and Raji 4RH) to escalating doses of MK-2206, a selective Akt inhibitor, or idelalisib, a selective PI3 kinase delta inhibitor, resulted in a dose- and time-dependent decrease in cell viability of RCRCL and to a lesser degree RRCL and RSCL (RCRCL vs. RRCL/RSCL, p<0.05). The IC₅₀ of MK-2206 was lower in RCRCL (Raji-2R = 2.6µM and Raji-4RH = 3.2µM) than in RSCL (Raji= 4.4µM) or RRCL (Raji 7R = 4.0µM and Raji 8RH = 5.2µM). Similarly, the IC₅₀ for idelalisib was lower in RCRCL (Raji-2R = 61µM and Raji-4RH = 149µM) than in RSCL (Raji= 341µM) or RRCL (Raji 7R = 195µM and Raji 8RH = 318µM). In addition, PI3K/Akt/m-TOR inhibition with either MK-2206 or idelalisib induced cell cycle arrest in G1 phase in RSCL/RRCL, but G2/M cell cycle arrest was observed in RCRCL. In BL cells pre-treated with idelalisib (10µM and 50µM) for 24 hours prior to exposure to doxorubicin (1µM, 10µM or 20µM) for 48 hours, the RCRCL Raji 2R exhibited an increased sensitivity to doxorubicin compared to non-idelalisib exposed controls (untreated vs. idelalisib 10µM vs. idelalisib 50µM: doxorubicin 10µM=78% vs. 69% vs. 56%, p<0.05; doxorubicin 20µM=61% vs. 49% vs. 43%, p<0.05). Raji and Raji 7R cells pre-treated with idelalisib did not exhibit an increase in doxorubicin sensitivity. Together our data suggest that constitutive phosphorylation/activation of the PI3K/Akt signal transduction pathway is associated with the development of resistance and may play a role in shared resistant pathways that lead to the acquirement of chemotherapy resistance observed in some rituximab-resistant cell lines. Additionally, inhibition of the PI3K/Akt/mTOR pathway may partially re-sensitize chemotherapy resistant cells to the cytotoxic effects of chemotherapeutic agents. Targeting the PI3K/Akt/mTOR signaling pathway may be clinically relevant in some patients with relapsed/refractory BL. (Research supported by a grant from Hyundai Hope on Wheels and a St. Baldrick’s Foundation Scholar Award)