The tumor suppressor and transcription factor p53 plays critical roles in tumor prevention by orchestrating a wide variety of cellular responses, including damaged cell apoptosis, maintenance of genomic stability, inhibition of angiogenesis, and regulation of cell metabolism and tumor microenvironment. TP53 is one of the most commonly deregulated genes in cancer. The p53-ARF-MDM2 pathway is deregulated in 84% of glioblastoma (GBM) patients and 94% of GBM cell lines. Deregulated p53 pathway components have been implicated in GBM cell invasion, migration, proliferation, evasion of apoptosis, and cancer cell stemness. These pathway components are also regulated by various microRNAs and long non-coding RNAs. TP53 mutations in GBM are mostly point mutations that lead to a high expression of a gain of function (GOF) oncogenic variants of the p53 protein. These relatively understudied GOF p53 mutants promote GBM malignancy, possibly by acting as transcription factors on a set of genes other than those regulated by wild type p53. Their expression correlates with worse prognosis, highlighting their potential importance as markers and targets for GBM therapy. Understanding mutant p53 functions led to the development of novel approaches to restore p53 activity or promote mutant p53 degradation for future GBM therapies.

Glioblastoma (GBM) is the most common and most lethal primary malignant brain tumor. The receptor tyrosine kinase MET is frequently upregulated or overactivated in GBM. Although clinically applicable MET inhibitors have been developed, resistance to single modality anti-MET drugs frequently occurs, rendering these agents ineffective. We aimed to determine the mechanisms of MET inhibitor resistance in GBM and use the acquired information to develop novel therapeutic approaches to overcome resistance. We investigated two clinically applicable MET inhibitors: crizotinib, an ATP-competitive small molecule inhibitor of MET, and onartuzumab, a monovalent monoclonal antibody that binds to the extracellular domain of the MET receptor. We developed new MET inhibitor-resistant cells lines and animal models and used reverse phase protein arrays (RPPA) and functional assays to uncover the compensatory pathways in MET inhibitor-resistant GBM. We identified critical proteins that were altered in MET inhibitor-resistant GBM including mTOR, FGFR1, EGFR, STAT3, and COX-2. Simultaneous inhibition of MET and one of these upregulated proteins led to increased cell death and inhibition of cell proliferation in resistant cells compared with either agent alone. In addition, treatment of mice bearing MET-resistant orthotopic xenografts with COX-2 or FGFR pharmacological inhibitors in combination with MET inhibitor restored sensitivity to MET inhibition and significantly inhibited tumor growth. These data uncover the molecular basis of adaptive resistance to MET inhibitors and identify new FDA-approved multidrug therapeutic combinations that can overcome resistance.

We investigate mutations in trβ2, a splice variant of thrb, identifying changes in function, structure, and behavior in larval and adult zebrafish retinas. Two N-terminus CRISPR mutants were identified. The first is a 6BP+1 insertion deletion frameshift resulting in a truncated protein. The second is a 3BP in frame deletion with intact binding domains. ERG recordings of isolated cone signals showed that the 6BP+1 mutants did not respond to red wavelengths of light while the 3BP mutants did respond. 6BP+1 mutants lacked optomotor and optokinetic responses to red/black and green/black contrasts. Both larval and adult 6BP +1 mutants exhibit a loss of red-cone contribution to the ERG and an increase in UV-cone contribution. Transgenic reporters show loss of cone trβ2 activation in the 6BP+1 mutant but increase in the density of cones with active blue, green, and UV opsin genes. Antibody reactivity for red-cone LWS1 and LWS2 opsin was absent in the 6BP+1 mutant, as was reactivity for arrestin3a. Our results confirm a critical role for trβ2 in long-wavelength cone development.

We investigate a splice variant of thrb isolated in the retina, trβ2, identifying functional changes in larval and adult mutant zebrafish lacking trβ2. We constructed two CRISPR mutant zebrafish with mutations located in the N-terminus region. The first is a 6BP+1 insertion deletion frameshift resulting in a truncated protein. The second is a 3BP in frame deletion with intact binding domains. ERG recordings showed that the 6BP+1 mutants did not respond to red wavelengths of light while the 3BP mutants did respond. 6BP+1 mutants lacked optomotor and optokinetic responses to red/black and green/black contrasts. Adult 6BP+1 mutants exhibit a loss of red-cone contribution to the ERG, and an increase in green and UV contributions. Anatomical markers show loss of red-cones in the 6BP+1 mutant but increase in blue, green, and UV cone density. Our results confirm trβ2's role in retinal cone development. Author SummaryThere are four cone photoreceptors responsible for color vision in zebrafish: red, green, blue, and UV. The thyroid hormone receptor trβ2 is localized in the vertebrate retina. We know that it is necessary for the development of long-wavelength-sensitive cones (red), but here we investigate the functional alterations that accompany a loss of trβ2. Our work contributes to the ongoing investigations of retinal development and the involvement of thyroid hormone receptors. Confirming previous morphological findings, we see that the fish become red colorblind when trβ2 is knocked out, but the contributions of the other three cone types shift in response. Our work highlights the plasticity of the retinal circuit as we see changes in opsin peaks and cone sensitivity, increases in contributions of UV cones, and an attempt at a mosaic pattern in the adult retina all in the absence of trβ2 and red cones. We now have an increased understanding of mechanisms underlying retinal development ResultsCRISPR mutations are located in the N-terminus of trβ2. The two splice variants of the thrb gene, trβ1 and trβ2 differ only in the first exon [14]. To target trβ2, we designed gRNA targeting exon1 of trβ2 and injected into one-cell-stage eggs of tg(trβ2:tdTomato). We isolated