Targeting ALK and WIP1 : neuroblastoma precision medicine under development

Abstract

Neuroblastoma is the childhood solid tumor accountable for the largest number of deaths, calling for improved treatment. Some genetic alterations are considered crucial in driving initiation and progression of neuroblastoma. These are usually associated with poor prognosis and are viewed as potential therapeutic targets. Anaplastic lymphoma kinase (ALK) is one of the best-established disease drivers in neuroblastoma, harboring oncogenic mutations or amplifications in about 10-15 % of all cases. In 2013, results from the first phase I study of an ALK inhibitor in neuroblastoma, crizotinib, were announced showing disappointing clinical response. Improved later-generation ALK inhibitors have since become available while biological and clinical understanding of ALK mutations in neuroblastoma has increased.

In Paper I, a patient with a metastatic high-risk neuroblastoma suffered extreme toxicity due to an underlying condition, Fanconi anemia, prohibiting further conventional treatment. Whole genome sequencing of tumor material revealed a novel ALK variant, ALK-I1171T, which was identified as a potent gain-of-function mutant resistant to crizotinib, but sensitive to later-generation ALK inhibitors including ceritinib. It was shown that ceritinib was equally effective as crizotinib in a panel of ALK-driven neuroblastoma cell lines. As a result, the child could be offered ceritinib in monotherapy, achieving complete metastatic remission and enabling resection of the primary tumor, and long-term survival.

In Paper II, activation of ALK as well as tropomyosin-related kinase A (TRKA) was observed in the absence of corresponding genomic alterations in an infant with metastasized and treatment-refractory neuroblastoma. A novel germline mutation of the ALK ligand ALKAL-2 was revealed by whole genome sequencing, which in cell culture and drosophila experiments was shown to be functional. Since an inhibitor targeting both ALK and TRKA was available in entrectinib, this drug was tested in neuroblastoma cell lines. Subsequently entrectinib treatment could be offered to the patient, producing a prompt improvement of clinical status and a gradual decline of catecholamine markers and metastases over the course of several years.

The majority of high-risk neuroblastomas contain a gain of chromosome 17q, a feature that correlates with aggressive disease and poor prognosis. The gene PPM1D is situated within the gained region and encodes the phosphatase WIP1, an inhibitor of p53 and a negative regulator of DNA damage response. WIP1 is often overexpressed in neuroblastoma, and overexpression in transgenic mice predisposes to tumor formation. In Paper III we showed that WIP1 expression correlates to 17q gain in neuroblastoma and medulloblastoma cell lines, which are also highly dependent on WIP1. Knockdown of WIP1 delayed tumor growth. Among different small molecule WIP1 inhibitors evaluated, SL-176 was demonstrated to be effective in all tested cell lines regardless of p53 mutational status. Pharmacologic inhibition of WIP1 with SL-176 in xenograft-bearing mice curbed tumor growth.

To achieve an improved cytotoxic effect, a drug combination screening with the WIP1 inhibitor SL-176 was conducted in Paper IV, identifying combination with epigenetic modification by inhibition of the histone demethylase JMJD3 as the most synergistic strategy. Combination of SL-176 and the JMJD3 inhibitor GSK-J4 showed strong synergism in a panel of neuroblastoma cell lines with regard to cell viability, WIP1 downstream targets, cell cycle arrest and apoptosis. Pathway analysis of differentially expressed genes, as identified by RNA sequencing, revealed enrichment of genes involved in pathways associated with DNA damage response.

While ALK inhibitors are already available to a subset of neuroblastoma patients, WIP1 is a promising therapeutic target where much work remains before patients may potentially benefit. It is our conviction that in the future, targeted therapy will be available to all high-risk neuro-blastoma patients. This thesis represents a few small steps on the road to accomplish that goal.