AZD6244 (ARRY-142886), a potent and highly selective MEK1/2 inhibitor, demonstrates anti-HIF properties, and increases the therapeutic response in a lung tumor xenograft model when combined with radiotherapy

A244 Introduction: AZD6244 is a potent and selective, uncompetitive MEK1/2 inhibitor that is highly active in both in vitro and in vivo tumor models and is currently being evaluated as monotherapy in Phase II clinical trials. The primary objective of this study was to determine whether AZD6244 and radiotherapy can be used in combination to greater therapeutic effect than either modality alone, and to examine the impact of MEK1/2 inhibition on the molecular responses to hypoxia in a human lung tumor xenograft model (Calu-6; mutant KRAS).
 Methods: The impact of scheduling AZD6244 treatment (before, after or concomitantly) in combination with radiotherapy was investigated in vitro using clonogenic assays. Western immunoblotting was performed to assess the effect of AZD6244 and radiation on the biomarker (phospho-ERK), and on HIF-1α and its downstream targets under hypoxic (1% O 2 ) conditions. HIF-1α is only detectable under hypoxic conditions and is a useful marker of hypoxia. The effect of drug on HIF-specific transactivation was assessed by a hypoxia-response element (HRE)-luciferase reporter assay, using a Calu-6 (dual clone: DC) cell line stably expressing a firefly luciferase gene under the control of a hypoxia-driven promoter.
 In vivo , short-term pharmacodynamic studies were performed in Calu-6 DC xenografts with oral dosing of AZD6244 (25 mg/kg BID); tumors were harvested 4 h after a single dose of AZD6244 or following five days of dosing. Subsequently, a course of 10-day treatment with AZD6244 (25 mg/kg BID) was combined with fractionated radiotherapy (5 x 2 Gy) during the first or last five days of drug therapy. Tumors were allowed to reach three times their original pre-treatment tumor volume.
 Results: Clonogenic assays demonstrated that the neo-adjuvant schedule (48h AZD6244 treatment before irradiation) was more effective than sequential (drug after irradiation) or concomitant treatment in vitro . AZD6244 (≥ 2 nM) reduced phospho-ERK expression in the Calu-6 human lung cancer cell line in vitro and within 4 h of one dose of AZD6244 (25 mg/kg) in vivo . Radiation alone had no effect on ERK activation in vitro . AZD6244 ablated HIF-1 transactivation in vitro and in vivo (5 days). Furthermore, in vitro there was a reduction in HIF-1α protein expression following 6-24 h treatment with 200 nM AZD6244 under hypoxic conditions, as well as a reduction in GLUT-1 expression (after 24 h).
 AZD6244 resulted in tumor growth stasis from the first day of treatment in the Calu-6 tumor xenograft model. Combination treatment with 10-day AZD6244 and fractionated radiotherapy resulted in a significantly enhanced tumor growth delay compared to either modality alone. This enhancement was independent of whether radiotherapy was administered during the first or last five days of AZD6244 treatment.
 Conclusions: In summary, a regime combining AZD6244 and radiotherapy in the Calu-6 xenograft model demonstrates significant therapeutic benefit compared to single therapy treatment. Collectively, these data support the clinical development of AZD6244 in combination with radiotherapy and indicate a novel role for AZD6244 in inhibiting the tumor hypoxia response.