WIP1 phosphatase as a potential therapeutic target in neuroblastoma

Abstract

The wild-type p53-induced phosphatase 1 (WIP1) is a serine/threonine phosphatase that negatively regulates multiple proteins involved in DNA damage response including p53, CHK2, Histone H2AX, and ATM, and it has been shown to be overexpressed or amplified in human cancers including breast and ovarian cancers. We examined WIP1 mRNA levels across multiple tumor types and found the highest levels in breast cancer, leukemia, medulloblastoma and neuroblastoma. Neuroblastoma is an exclusively TP53 wild type tumor at diagnosis and inhibition of p53 is required for tumorigenesis. Neuroblastomas in particular have previously been shown to have 17q amplification, harboring the WIP1 (PPM1D) gene and associated with poor clinical outcome. We therefore sought to determine whether inhibiting WIP1 with a selective antagonist, GSK2830371, can attenuate neuroblastoma cell growth through reactivation of p53 mediated tumor suppression. Neuroblastoma cell lines with wild-type TP53 alleles were highly sensitive to GSK2830371 treatment, while cell lines with mutant TP53 were resistant to GSK2830371. The majority of tested neuroblastoma cell lines with copy number gains of the PPM1D locus were also TP53 wild-type and sensitive to GSK2830371A; in contrast cell lines with no copy gain of PPM1D were mixed in their sensitivity to WIP1 inhibition, with the primary determinant being TP53 mutational status. Since WIP1 is involved in the cellular response to DNA damage and drugs used in neuroblastoma treatment induce apoptosis through DNA damage, we sought to determine whether GSK2830371 could act synergistically with standard of care chemotherapeutics. Treatment of wild-type TP53 neuroblastoma cell lines with both GSK2830371 and either doxorubicin or carboplatin resulted in enhanced cell death, mediated through caspase 3/7 induction, as compared to either agent alone. Our data suggests that WIP1 inhibition represents a novel therapeutic approach to neuroblastoma that could be integrated with current chemotherapeutic approaches.

Fig 1. WIP1 is frequently overexpressed in neuroblastomas.

A, Survey of PPM1D (WIP1) mRNA expression from >25K microarray expression profiles across multiple human tumors, compared to composite of all normal tissues (detailed in S1 Fig.). B, PPM1D (204566_at) is frequently overexpressed in neuroblastoma and medulloblastoma among brain and CNS malignancies. Here, overexpression measured in tumor tissues is determined above the indicated threshold (see Materials and Methods).

Fig 2. WIP1 expression correlates with neuroblastoma disease stage.

A, Analysis of a large patient cohort (88 patients) with annotated clinical data and long term follow up (Versteeg-88-Mas5.0) demonstrates higher PPM1D levels in stage 4 metastatic subgroup (# = p < 0.01, # # = p < 0.005, by Student T-test). B, Long-term survival was also significantly (Kaplan-Meier analysis p = 0.005) different for high (n = 50) versus low (n = 38) expression of PPM1D.

Fig 3. GSK2830371 selectively inhibits WIP1 among related phosphatases.

A, Release of free phosphate was measured after incubation of each recombinant enzyme with the generic phosphopeptide, RRA(pT)VA. (* p < 0.01). B, GSK2830371 inhibits the dephosphorylation of WIP1 substrates. Release of free phosphate was measured after exposure of PPM1D (WIP1) to increasing concentrations of GSK2830371, followed by a reaction with phosphopeptide substrates, H2AX (Ser-139) and p53 (Ser-15). Error bars represent SD.

Fig 4. Neuroblastoma cell lines with wild-type TP53 and functional p53 response are sensitive to GSK2830371.

A, As a measure of p53 functional response, six neuroblastoma cell lines with different sensitivities to GSK2830371 (indicated by IC₅₀ value in a proliferation assay) and TP53 mutational status were exposed to ionizing radiation and allowed to recover for 1 hour, after which RT-PCR was carried out to measure changes in PPM1D and P21 transcript levels. Data represent fold change relative to untreated cells (mean ± SD). B, Anti-proliferative effect of GSK2830371 in a seven day cell proliferation assay. Data represent mean ± SD. Copy number for PPM1D and MYCN is represented as green for gain of >6 copies; yellow for 2–6 copies. WIP1 protein level was determined by western blotting of whole cell lysates from a panel of neuroblastoma cell lines. C. GSEA enrichment plot showing a TP53 gene signature that is significantly enriched in the sensitive group (S) relative to the resistant group (R) of neuroblastoma cell lines (see Table 1). Additional TP53 genesets tested by GSEA and their respective numbers of genes (n), normalized enrichment scores (NES) and FDR q values are shown.

Fig 5. GSK2830371 inhibits WIP1 signaling differentially in TP53 wild-type vs. mutant neuroblastoma cell lines.

TP53 wild-type (IMR-32) and mutant (SK-N-AS) cells were treated with indicated concentrations of GSK2830371 or DMSO for six hours. Cell lysates were analyzed by western blotting with antibodies to the indicated total and phospho-proteins. In both cell lines, GSK2830371 causes a concentration-dependent decrease in WIP1 protein and increases in WIP1 phospho-substrates, CHK2 (T68), H2AX (S139), and ATM (S1981). However, total and phospho-p53 (S15), and p53 transcriptional targets, p21 and PUMA, are only increased in the TP53 wild-type cells.

Fig 6. GSK2830371 has synergistic anti-proliferative activity with chemotherapeutic agents in neuroblastoma cells.

CHP-134 and IMR-32 cells were exposed to 1 μM GSK2830371 for one hour prior to addition of doxorubicin (4 nM proliferation; 1 nM caspase) or carboplatin (1 μM proliferation; 10 μM caspase). CellTiter-Glo (A) was added to plates at 72 hours and caspase-Glo 3/7 (B) was added to separate duplicate plates at 24 or 48 hours post-drug addition. Data represent mean ± SD. *p<0.05 vs DMSO control, ^#p<0.05 vs both single agents, ^$p<0.05 vs doxorubicin or carboplatin alone, using t-test, (2 tailed, unequal variance).