Preclinical evaluation of sorafenib in combination with rapamycin for the treatment of acute myeloid leukemia (AML)

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The mammalian target of rapamycin (mTOR) is a key regulator of cell growth and survival. Aberrant activation of AKT/mTOR signaling is observed in 50-70% of AML patients. On the other hand, the mitogen-activated protein kinase (MAPK) signaling cascade is constitutively activated in approximately 75% of AML cases, and simultaneous signaling through MAPK and AKT/mTOR may diminish the efficacy of therapies directed against receptor tyrosine kinase inhibitors (TKIs). Inhibiting both pathways was hypothesized to enhance antitumor effects by concurrently targeting parallel signaling pathways. In the current study, sorafenib, a multitargeted TKI previously shown to block MAPK signaling in AML cell lines (S. Hu et al, Proc AACR 2007), in combination with rapamycin, a specific mTOR inhibitor shown to inhibit p-AKT in AML (Z. Zeng, Blood 2007), was evaluated in three AML cell lines (MV4-11, U937 and THP-1) with constitutive activation of MAPK and AKT/mTOR signaling and different genetic alterations in tyrosine kinases. In a 72-hr MTT assay (2 independent experiments, 8 replicates each), rapamycin (0.001-10 µM) administered simultaneously with sorafenib (0.001-10 µM) at a non-fixed ratio resulted in synergistic effects with combination index (CI) values at ≥ ED50 of 0.41-0.81, 0.53-0.96, and 0.57-0.79 in MV4-11 (FLT-3 ITD), THP-1 (N-RAS mutation), and U937 cells, respectively. Rapamycin 10 nM enhanced the antiproliferative effects of sorafenib at most sorafenib concentrations in the 3 cell lines (P < 0.001). Compared to sorafenib 0.01 µM alone, rapamycin 10 nM had minimal effects on promoting cell cycle block in G0/G1 (% cells increased from 83 ± 15% to 88 ± 9%) and apoptosis (% cells annexin V positive increased from 15 ± 4% to 22 ± 6%) in MV4-11 cells; rapamycin had no influence on sorafenib-induced G0/G1 block and apoptosis in THP-1 and U937 cells. Using a multi-plex phospoprotein assay in MV4-11 cells, rapamycin 10 nM did not inhibit p-AKT, but inhibited p-p70S6K (70 ± 3% of control), p-MEK (52 ± 15% of control) and p-ERK (62 ± 13% of control); sorafenib 0.01 µM alone inhibited p-MEK (8.6 ± 15% of control), p-ERK (1.8 ± 0.38% of control) and p-p70S6K (52 ± 0.38 of control), but with negligible further inhibition by rapamycin. Investigations in AML blasts from patients are in progress. The results from this study indicate that rapamycin enhanced the anti-proliferative effects of sorafenib in AML cell lines, but with minimal to negligible enhancement of G0/G1 block, apoptosis and p-AKT inhibition. Future efforts will aim to evaluate additional drug combinations for more effective parallel inhibition of MAPK and AKT signaling.