Most of Anaplastic Large Cell Lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK. NPM-ALK deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive due to heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or re-localization of NPM-ALK to the cytoplasm by NPM genetic knock-out or knock-down caused ERK1/2 increased phosphorylation and cell death through the engagement of an ATM/Chk2 and γH2AX mediated DNA damage response. Remarkably, human NPM-ALK amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A “drug holiday” where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

Non-small cell lung cancer (NSCLC) harboring chromosomal rearrangements of the anaplastic lymphoma kinase (ALK) gene is treated with ALK tyrosine kinase inhibitors (TKIs), but is successful for only a limited amount of time; most cases relapse due to the development of drug resistance. Here we show that a vaccine against ALK induced a strong and specific immune response that both prophylactically and therapeutically impaired the growth of ALK-positive lung tumors in mouse models. The ALK vaccine was efficacious also in combination with ALK TKI treatment and significantly delayed tumor relapses after TKI suspension. We found that lung tumors containing ALK rearrangements induced an immunosuppressive microenvironment, regulating the expression of PD-L1 on the surface of lung tumor cells. High PD-L1 expression reduced ALK vaccine efficacy, which could be restored by administration of anti-PD-1 immunotherapy. Thus, combinations of ALK vaccine with TKIs and immune checkpoint blockade therapies might represent a powerful strategy for the treatment of ALK-driven NSCLC.

<p>Supplementary Figure S1. ALK Tg mice develop lung multifocal adenocarcinomas similar to human ALK-rearranged NSCLC. Supplementary Figure S2. Therapeutic ALK vaccine impairs tumor growth of ALK-rearranged lung tumors. Supplementary Figure S3. Immunophenotype of EML4-ALK transgenic mice Supplementary Figure S4. Human ALK-rearranged NSCLC show a pattern of low expression of T cell markers. Supplementary Figure S5. PD-L1 expression is dependent on ALK kinase activity in ALK-rearranged NSCLC cells. Supplementary Figure S6. Schematic protocols of the in vivo treatment with anti-PD-1 blocking antibody. Supplementary Figure S7. ALK vaccine can be combined with ALK inhibitor TAE684. Supplementary Figure S8. Immunophenotype of intratumoral T cells in ALK vaccinated mice.</p>