Treating post-transplant lymphoproliferative disorders (PTLD) remains a clinical challenge due to adverse effects such as graft rejection. mTOR inhibitors, including rapamycin (RAPA), have shown both anti-tumor and anti-rejection properties. As a monotherapy, however, RAPA has several well-described shortcomings including incomplete suppression of downstream signaling and feedback activation of upstream Akt, which can upregulate alternative proliferation pathways. We hypothesize that inhibiting the Akt axis upstream of RAPA provides additional, synergistic suppression of PTLD lymphoma growth.

Epstein-Barr virus (EBV)-positive B lymphoma cell lines, established from PTLD patients, were cultured in the presence or absence of small molecule inhibitors targeting the PI3K/Akt/mTOR pathway. Western blot analysis was conducted to validate the efficacy and specificity of these drugs. Cell proliferation after treatment with two-drug combinations was determined by MTS assay (n=3 per cell line). Finally, the effect of two-drug combinations on the growth of PTLD-derived B cell lymphoma xenografts in NOD-SCID mice (n=8 per group) was assessed in vivo.

Whereas RAPA alone did not suppress the phosphorylation of Akt, a combination of RAPA with either CAL101 (PI3K inhibitor) or MK2206 (Akt inhibitor) suppressed the phosphorylation of both Akt and S6K. Three PTLD cell lines (AB5, MF4, VB5) were sensitive to RAPA alone in a dose-dependent manner, with max inhibition of 64.6±1.6% at 100 nM of RAPA. Combining RAPA (100 nM) with up to 1 uM of either CAL101 or MK2206 shifted the proliferation curve down in a dose-dependent manner, with max inhibition of 78.3±1.6% and 77.9±2.0%, respectively (p<0.001). Furthermore, the additive effect of each of the second drugs on proliferation was synergistic with RAPA, as determined by IC50 isobologram analysis.

Mice injected with a PTLD cell line showed progressive, linear tumor growth. By week 7, control mice had tumors of 2240.8±620.7mm³, compared with RAPA-treated mice which had tumors of 440.6±111.1mm³ (p<0.005). Mice treated with RAPA in combination with either CAL101 or MK2206 had even smaller tumors (185.1±70.6mm³ (p<0.002) and 147.45±50.8mm³ (p<0.001), respectively).

Based on these results, dual-drug therapy of RAPA with either CAL101 or MK2206, both of which have entered clinical trials for other malignancies, is an attractive option for treatment of EBV+ PTLD.

CITATION INFORMATION: Sang A, Ivison G, Qu X, Esquivel C, Krams S, Martinez O. Inhibition of Multiple Nodes in the PI3K/Akt/mTOR Pathway Synergistically Suppresses Epstein-Barr Virus B Cell Lymphomas. Am J Transplant. 2017;17 (suppl 3).

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