Deregulation of the PI3K/Akt Signal Transduction Pathway Is Associated with the Development of Chemotherapy Resistance and Can be Effectively Targeted to Improve Chemoresponsiveness in Burkitt Lymphoma Pre-Clinical Models

Multi-agent chemotherapy regimens have significantly improved long-term survival of pediatric Burkitt Lymphoma (BL) to over 80%. However, patients with refractory or relapsed disease have a dismal prognosis, stressing the need to identify mechanisms of therapeutic resistance and develop novel therapeutic approaches. Recent data demonstrated tonic B-cell receptor (BCR) activation of PI3K in BL (Srinivisan et al, Cell 2009); and implicated PI3K, in coordination with MYC, in Burkitt lymphomagenesis (Sander et al, Cancer Cell 2012). In order to investigate mechanisms of resistance, our laboratory generated several resistant BL cell lines. We exposed Raji cells to escalating doses of rituximab +/- human serum and generated several BL rituximab-resistant (Raji 7R and Raji 8RH) (RRCL) or therapy (rituximab-chemotherapy) resistant (Raji 2R and Raji 4RH) (TRCL) cell lines. We then screened for aberrant activation of signal transduction pathways using western blot, phospho-flow cytometry and phosphoproteomics to define pathways associated with the development of resistance. While total Akt expression was similar between all cell lines tested, Raji 2R and Raji 4RH cells had increased phosphorylation of Akt at Ser473 and Thr308, indicating Akt activation, when compared to Raji, Raji 7R and Raji 8RH by western blot and phospho-flow cytometry. Phosphoproteomic analysis comparing Raji and Raji 4RH identified an increase of at least 2 fold in phosphorylation of 315 proteins in Raji 4RH cells, including multiple direct targets of AKT (e.g. GSK3B, WEE1, FOXO1 and PRAS40). Altered phosphorylation of proteins downstream of Akt (e.g. BAD, 4EBP1, GSK3B and ERK) was also detected by western blot in Raji 2R and Raji 4RH compared to Raji, Raji 7R and Raji 8RH. These findings suggest that activation of PI3K/Akt may play a role in the development of chemoresistance noted in our cell line model. KEGG analysis of phosphoproteome and gene expression comparison of Raji to Raji 4RH cells identified the BCR pathway as one of the top pathways altered. Additionally, the microRNA (miR) 17-92 has been noted to increase Akt activation through its inhibitory effects on PTEN, a key regulator of Akt activation. A recent copy number analysis indicated that a recurrent gain in 13q, containing the MIR17HG locus, was also associated with higher mir17 RNA levels and early relapse in pediatric BL patients (Schiffman et al, Br J Haematol 2011). An analysis of miR expression in Raji 2R and Raji 4RH compared to Raji noted a 1.93 and 3.77 fold increase in miR17 respectively. These findings suggest possible mechanisms for increased PI3K/Akt activation in our chemoresistant cell lines. In vitro exposure to MK-2206, a pan-Akt inhibitor, resulted in dose- and time-dependent decreases in viability of TRCLs (IC50 at 72 hours: Raji 2R = 2.6µM; Raji 4RH = 3.2µM) and to a slightly lesser degree Raji and RRCL (Raji= 4.4µM; Raji 7R = 4.0µM; Raji 8RH = 5.2µM) (TRCL vs. Raji/RRCL, p<0.05), by alamarBlu assay. Cell cycle analysis following exposure to MK2206 identified G1 cell cycle arrest in Raji/RRCL, but G2/M cell cycle arrest was observed in TRCL, suggesting differing mechanisms of anti-proliferative effect between sensitive and resistant cells. The combination of MK2206 and either doxorubicin or dexamethasone resulted in synergistic decreases in cell viability at a variety of dose combinations in both sensitive and resistant cells, as well as in BL Ramos and Daudi cells, using the Chou-Talalay method to measure synergy. In Raji and Ramos, the combination of MK2206 and doxorubicin also resulted in a higher degree of PARP cleavage compared to either agent alone, though no PARP cleavage was noted in Raji 2R and Raji 4RH cells likely related to impaired apoptotic potential previously noted in these cell lines. An siRNA knockdown of Akt in Raji cells resulted in a decrease in viable cells compared to controls following exposure to doxorubicin. Similar effects on cell proliferation, cell cycle and synergy were seen with upstream inhibition of PI3K by the PI3Kδ inhibitor idelalisib. Our data suggests that constitutive activation of the PI3K/Akt pathway, through activation of the BCR pathway or over-expression of miR17, is associated with the development of resistance in BL cell lines, and inhibition of PI3K/Akt can sensitize BL cells to the effects of some chemotherapeutic agents. Targeting the PI3K/Akt signaling pathway may be a clinically relevant approach in pediatric BL.