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Uncovering the anti-apoptotic mechanism of mutated ALK in neuroblastoma

Nathan F Moore

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National Institutes of Health (NIH)
Neuroblastoma (NB) is a common childhood cancer that accounts for 15% of all pediatric oncology deaths. The anaplastic lymphoma receptor tyrosine kinase, ALK is an important therapeutic target in NB. ALKF1174L is considered the most aggressive of all ALK mutations in NB, possessing higher transforming potential and segregating with the other frequently occurring molecular aberration, MYCN amplification. We generated a transgenic mouse model that develops ALKF1174L/MYCN-expressing NB tumors with a shorter latency and a more aggressive phenotype than MYCN-only tumors. Moreover, ALKF1174L/MYCN tumors demonstrate increased activation of PI3K/AKT and MAPK signaling pathways as well as decreased apoptosis, accompanied by activation of antiapoptotic BCL2 family members. The central hypothesis of this project is that mutated ALK promotes tumor cell survival by disrupting BCL2 family activity via specific downstream signaling pathways, and that the addition of one or more agents that inhibit antiapoptotic BCL2 proteins would complement the cytotoxicity of ALK inhibitors. In preliminary studies, we show that overexpression of ALKF1174L leads to marked inhibition of apoptosis in NB cells, increased expression of several antiapoptotic proteins, including MCL1, and decreased expression of proapoptotic BIM. Moreover, ALKF1174L-positive cells were 10-fold more sensitive to the MCL1 inhibitor TW-37 compared with ABT-737 and ABT-263, compounds that primarily inhibit BCL2 and BCLXL, suggesting that MCL1 is critical to regulating apoptosis in these cells. Finally, ALKF1174L-mediated activation of AKT was associated with decreased FOXO3 activity, a transcription factor that promotes a proapoptotic BCL2 phenotype. Together, these results suggest that ALKF1174L inhibits apoptosis in NB cells through activation of AKT and/or inhibition of FOXO3, driving aberrant regulation of MCL1 and possibly other members of the BCL2 family. In Aim 1, I will identify the BCL2 family members that are essential for the promotion of survival in ALKF1174L-expressing NB cells through BH3 profiling and determine whether these proteins confer dependency on ALK-mutant cells. I will also determine whether combined inhibition of the identified antiapoptotic BCL2 family members and ALK produces synergistic antitumor activity in ALKF1174L/MYCN-expressing NB models, and whether this targeted treatment can augment the cytotoxic effects of standard chemotherapy. In Aim 2, I will determine the mechanism(s) by which ALKF1174L differentially regulates BCL2 family members, specifically focusing on the signaling pathways that are active in the ALKF1174L/MYCN transgenic tumor model. I will elucidate the role of PI3K/AKT- and/or mTORC2/SGK1- mediated inhibition of FOXO3 and whether BCL2 family members are regulated directly through AKT activation to promote the survival of ALKF1174L-positive cells. I will also assess the role of the MAPK/ERK pathway in mutant ALK-mediated regulation of the BCL2 family. Together, these aims are designed to clarify the antiapoptotic role of mutant ALK in NB pathogenesis and its potential as a therapeutic target.

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