Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in neuroblastoma, a devastating pediatric cancer of the sympathetic nervous system. Germline and somatically acquired ALK aberrations induce increased autophosphorylation, constitutive ALK activation and increased downstream signaling. Thus, ALK is a tractable therapeutic target in neuroblastoma, likely to be susceptible to both small-molecule tyrosine kinase inhibitors and therapeutic antibodies–as has been shown for other receptor tyrosine kinases in malignancies such as breast and lung cancer. Small-molecule inhibitors of ALK are currently being studied in the clinic, but common ALK mutations in neuroblastoma appear to show de novo insensitivity, arguing that complementary therapeutic approaches must be developed. We therefore hypothesized that antibody targeting of ALK may be a relevant strategy for the majority of neuroblastoma patients likely to have ALK-positive tumors. We show here that an antagonistic ALK antibody inhibits cell growth and induces in vitro antibody-dependent cellular cytotoxicity of human neuroblastoma-derived cell lines. Cytotoxicity was induced in cell lines harboring either wild type or mutated forms of ALK. Treatment of neuroblastoma cells with the dual Met/ALK inhibitor crizotinib sensitized cells to antibody-induced growth inhibition by promoting cell surface accumulation of ALK and thus increasing the accessibility of antigen for antibody binding. These data support the concept of ALK-targeted immunotherapy as a highly promising therapeutic strategy for neuroblastomas with mutated or wild-type ALK.

Autophagy is an evolutionarily conserved survival pathway in eukaryote and is frequently upregulated in cancer cells after chemotherapy or targeted therapy. Thus induction of autophagy has emerged as a drug resistance mechanism. In this study, we found that crizotinib induced a high level of autophagy in lung cancer cells through inhibition of STAT3. Ectopic expression of wild-type or constitutive activated STAT3 significantly suppressed the effect of crizotinib on autophagy. Interestingly, crizotinibmediated inhibition of STAT3 is in a step-wise manner. Firstly it inhibited cytoplasmic STAT3, which leads to the phosphorylation of EIF2A, then inhibited nuclear STAT3, which leads to the downregulation of BCL-2. Cell death induced by crizotinib was greatly enhanced after the inhibition of autophagy by the pharmacological inhibitors or shRNAs against Beclin-1. Moreover, the autophagy inhibitor HCQ significantly augmented the anti-tumor effect of crizotinib in a mouse xenograft model. In conclusion, crizotinib can induce cytoprotective autophagy by suppression of STAT3 in lung cancer cells. Thus, autophagy inhibition represents a promising approach to improve the efficacy of crizotinib in the treatment of targeted lung cancer patients.

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Patients with epidermal growth factor receptor (EGFR) exon 21 L858R substitution benefit less from standard EGFR tyrosine kinase inhibitor (TKI) treatment, and whether anti-angiogenic therapy was beneficial to the EGFR L858R subpopulation was inconclusive. A retrospective study was conducted to investigate the survival benefit and the target characteristics of the anti-angiogenic agent in the EGFR L858R patients in our center, comparing those treated with or without anti-angiogenic therapy (cohort A and cohort B). At the median follow-up time of 31.0 months vs 32.7 months (cohort A vs. B) respectively, Cohort A (n = 58) had a significantly prolonged median OS compared to Cohort B (n = 101) (60.0 months vs.37.0 months, HR 0.51, p = 0.016). Anti-angiogenic therapy significantly prolonged the OS in patients with liver metastases (NA vs.26.0 months, HR 0.17, p = 0.023) comparing to patients without liver metastases (60.0 months vs.37.0 months, HR 0.63, p = 0.129). For brain metastatic patients, anti-angiogenic treatment tended to improve median OS with (65.0 months vs.35.0 months, HR 0.29, p = 0.068) or without brain radiotherapy (73.0 months vs.29.0 months, HR 0.24, p = 0.171). The grade 3 or more adverse events were manageable and consistent with previous studies. Patients with EGFR L858R mutation treated with anti-angiogenic therapy in their course of treatment had a significantly prolonged OS compared to those who had never received an anti-angiogenic agent. Patients with liver metastases might benefit more from anti-angiogenic therapy than those without.