Abstract 2920: Excess of cytoplasmic NPM-ALK driven oncogenic signaling is toxic and promotes cellular apoptosis and drug dependency

Abstract

Abstract Most of Anaplastic Large Cell Lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein nucleophosmin (NPM)-ALK. NPM provides an oligomerization domain that causes the spontaneous dimerization and ligand-independent activation of the ALK. NPM-ALK deregulated kinase activity drives several pathways involved in cellular proliferation and survival and sustains the process of malignant transformation. NPM-ALK is localized both in the cytoplasm and the nucleus, but the role of NPM-ALK cytoplasmic or nuclear fractions on ALCL phenotype maintenance is undetermined. We found that in all ALK+ ALCL cell lines and in primary ALCL NPM-ALK was equally distributed between cytoplasm and nucleus, but only the cytoplasmic portion, formed by NPM-ALK homodimers, was kinetically active and had transforming properties. The nuclear portion, formed by NPM/NPM-ALK heterodimers, was inactive. Thus, about 50% of the NPM-ALK fusion is sequestered and inactivated in the nucleus of ALCL cells by WT NPM1. Indeed, relocalization of NPM-ALK entirely to the cytoplasm in NPM-/- cells or its overexpression in several ALK+ ALCL cell lines resulted in cell death through activation of the ERK1/2 pathway and of γ-H2AX, a key player of the DNA damage response pathway. Thus, excessive oncogenic ALK signaling induces a cell stress leading to apoptosis and a balanced amount of ALK activation is necessary for optimal ALCL growth. Interestingly, we selected three human NPM-ALK positive cell lines resistant to the dual ALK/EGFR inhibitor AP26113. Resistance was due to genomic NPM-ALK amplification that caused overexpression of NPM-ALK and consequently hyperactivation of downstream ALK-dependent pathways. These cell lines were not only resistant, but also drug-dependent, meaning that addition of a low amount of AP26113 was required for cellular growth and to keep “normal” NPM-ALK signalling levels. Indeed, upon drug withdrawal, NPM-ALK and its downstream pathways were hyperactivated. The viability of all three drug-addicted cell lines dramatically dropped, confirming that an excess of NPM-ALK signaling was detrimental for cell growth. In two resistant cell lines out of three a new population recovered in the absence of the drug and was re-sensitized to AP26113. Notably, in these new drug-sensitive populations NPM-ALK overexpression was abrogated. All together, these findings support the idea that deregulation of NPM-ALK localization might be an attractive therapeutic option. Moreover, for a subset of patients that may develop drug dependency as well as drug resistance as a consequence of NPM-ALK overexpression, a discontinuous tyrosine kinase inhibitor therapeutic schedule may be more effective than a continuous one, as it is usually proposed. Citation Format: Monica Ceccon, Maria Elena Boggio Merlo, Luca Mologni, Lydia Varesio, Teresa Poggio, Matteo Menotti, Silvia Bombelli, Roberta Rigolio, Andrea Manazza, Chiara Ambrogio, Giovanni Giudici, Cesare Casati, Mara Compagno, Suzanne Turner, Carlo Gambacorti Passerini, Roberto Chiarle, Claudia Voena. Excess of cytoplasmic NPM-ALK driven oncogenic signaling is toxic and promotes cellular apoptosis and drug dependency. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2920. doi:10.1158/1538-7445.AM2015-2920