Sorafenib demonstrated in vitro activity in a panel of molecularly heterogeneous AML cell lines and primary childhood AML samples at a clinically achievable concentration of 10 μM. When given simultaneously with cytarabine, synergistic to additive inhibition of cell viability was observed. In an AML xenograft model, administration of sorafenib (10 μM) plus cytarabine prolonged survival compared with cytarabine alone.
Although sorafenib appears to be more active in leukemias with the FLT3-ITD abnormality, antileukemic activity has been reported in several patients with AML and wild-type FLT3 (15
). Our study shows the in vitro antileukemic activity of sorafenib in 10 genetically heterogeneous AML cell lines as well as in 25 primary AML blast samples in which activity was observed in both FLT3-ITD and wild-type FLT3 AML cells. Sorafenib alone also exhibited a different spectrum of resistance compared with cytarabine alone, indicating that sorafenib may be active in some cells with de novo resistance to cytarabine. Sorafenib alone effectively inhibited ERK signaling in AML cell lines, consistent with previous studies (12
). Also, sorafenib alone inhibited AKT phosphorylation in one cell line, which may represent an additional mechanism of sorafenib-induced apoptosis in which AKT-mediated negative regulation of proapoptotic BAD is suppressed (11
). Treatment with sorafenib alone also inhibited phosphorylation of p70S6K, a downstream effector of both ERK and AKT signaling that is involved in transcription and translation of genes that regulate cell growth, proliferation, and survival. The precise mechanism of action of sorafenib in AML is unknown but likely involves inhibition of RTKs and downstream signaling pathways, the latter, which is supported by the results of our study.
Because sorafenib previously demonstrated modest single-agent activity in relapsed or refractory AML (15
), we evaluated its activity in combination with cytarabine, one of the most widely used chemotherapeutic agents for AML. In our study, sorafenib and cytarabine had a synergistic to additive activity in seven AML cell lines, and inhibition of cell viability was greater after treatment with sorafenib plus cytarabine in 13 of 15 childhood primary blast samples compared with either agent alone. In MV4-11 AML cells and BaF3 cells with a FLT3-ITD mutation, administration of the kinase inhibitor lestaurtinib simultaneously with or immediately following treatment with cytarabine produced cytotoxicity in a synergistic fashion, in contrast to the antagonist effect observed when lestaurtinib was administered before cytarabine (21
). In MV4-11 cells, we observed a synergistic interaction when sorafenib was administered before cytarabine, although stronger synergy was observed when cytarabine was administered before sorafenib. Our data suggest that the sequence of sorafenib and cytarabine administration may not be a critical factor to consider in FLT3-ITD AML. Our data showed that in wild-type FLT3 U937 AML tumor-bearing NSG mice, sorafenib did not prolong survival compared with untreated controls, which is generally consistent with the limited single-agent activity observed in phase I trials (15
). However, we observed that sorafenib administered simultaneously with cytarabine statistically significantly prolonged median survival compared with cytarabine alone in this AML xenograft model, findings that are consistent with the results of a recent phase II study in 51 adults with previously untreated AML receiving sorafenib simultaneously in combination with cytarabine and idarubicin during the first week of induction therapy. Overall, 38 (75%) of 51 patients achieved a complete response (44
), and the response rate was higher in patients with FLT3-ITD AML (14 of 15, 92%), but 24 (66%) of 36 of patients with wild-type FLT3 achieved a complete response, demonstrating the potential utility of sorafenib in combination with chemotherapy in some patients with FLT3 wild-type disease.
The twice-daily administration schedule of sorafenib plus cytarabine was more active than sorafenib given once daily plus cytarabine. Pharmacokinetic studies in NSG mice demonstrated that the time sorafenib plasma concentrations remained above 10 μM was longer with twice-daily vs once-daily administration (17 vs 8 hours), and exposure with the former schedule was more similar to human steady-state exposure with minimal fluctuations from peak to trough with an approximate average concentration of 10 μM. These pharmacokinetic studies provide a preliminary benchmark for effective sorafenib concentrations in patients receiving sorafenib in combination with cytarabine, particularly in wild-type FLT3.
Our study identified a potential mechanism underlying the interaction between sorafenib and cytarabine. Previous studies have demonstrated that protein kinase inhibitors can inhibit nucleoside transport into cells (45
), indicating that caution should be taken when combining a kinase inhibitor with a nucleoside analogue. However, our study shows that sorafenib did not decrease the initial uptake of cytarabine in AML cell lines but increased the cellular accumulation of cytarabine triphosphate, the active moiety of cytarabine, by up to three- to fivefold in cells in which synergistic drug effects were observed with combination treatment compared with cells treated with cytarabine alone. Because cytarabine-sensitive cells accumulate higher intracellular concentrations of cytarabine triphosphate than resistant cells (39
), it is plausible that enhanced accumulation of cytarabine triphosphate by sorafenib contributes to the synergistic to additive activity observed with the drug combination. The precise mechanism of this intracellular pharmacokinetic interaction is unknown but may involve enhanced formation of cytarabine to its phosphorylated metabolites or reduced cellular efflux. Members of the ABCC efflux transporter family have been shown to transport a range of base, nucleoside, and nucleotide analogs (47
), and cytarabine has recently been shown to be a substrate for ABCC10 (MRP7) and ABCC11 (MRP8) (42
). We determined that whereas ABCC11 was not expressed, ABCC10 protein was expressed in four of 10 AML cell lines studied. In addition to ABCC10, other ABCC family members may efflux cytarabine from AML cells, and studies are currently under way to investigate this mechanism. In U937 cells, the combination of sorafenib plus cytarabine resulted in additive drug effects in vitro, and survival was prolonged in a U937 xenograft model. Because sorafenib treatment did not increase the cellular retention of cytarabine triphosphate in U937 cells with high basal accumulation of the active metabolite, it is likely that other mechanisms are contributing to the antileukemic activity of the combination therapy. Recently, it was shown that the mitogen-activated protein kinase pathway was activated in NB4 and HL-60 AML cells treated with cytarabine (49
). A selective MEK inhibitor, AZD624 (AstraZeneca Pharmaceuticals), effectively blocked cytarabine-induced MEK/ERK activation and enhanced growth arrest and apoptosis in both the NB4 and HL-60 AML cell lines. Future investigations are needed to characterize the role of sorafenib in the inhibition of activated signaling pathways in drug-resistant leukemic cells.
Our study is not without several limitations. We evaluated different sequences of sorafenib and cytarabine combination treatment in one AML cell line with an FLT3-ITD mutation. The superiority of one sequence over the others in cells with wild-type FLT3 is unknown and may differ from that of AML cells with the FLT3-ITD mutation. Also, we suggest a target effective sorafenib plasma concentration of 10 μM in AML patients based on in vitro data in 10 molecularly heterogeneous AML cell lines (one with FLT3-ITD) and 25 primary blast samples (four with FLT3-ITD), and in vivo data in one AML xenograft with wild-type FLT3. Based on these data, it cannot be determined if the requirements for optimal sorafenib plasma exposure and/or sequence of drug administration with cytarabine (eg, simultaneous vs sequential administration) are different for FLT3-ITD and wild-type FLT3 AML. Current preclinical investigations are aimed to define these parameters in mouse models of AML.
In conclusion, sorafenib has antileukemic activity in molecularly heterogeneous AML in vitro and in vivo. Sorafenib enhances the antitumor activity of cytarabine in vitro and in vivo when administered simultaneously in combination. Our study provides the foundation for the future clinical evaluation of sorafenib in combination with cytarabine-based regimens in frontline and relapsed/refractory childhood AML.