Cotton, C. | Search bioRxiv

bioRxiv2024

Upregulation of a nonsense mediated decay (NMD) insensitive CFTR mRNA isoform has therapeutic potential for the treatment of 3 Prime CFTR PTC variants

BackgroundNonsense or Premature Termination Codon (PTC) mutations of the CFTR gene are pathogenic and found in [~]10% of North American people with cystic fibrosis. PTC mutations induce Nonsense-Mediated mRNA Decay (NMD), leading to a substantial ([~]80-90%) reduction in full-length mRNA. This reduction is a key contributor to PTC mutation-related pathology. Various approaches to evade NMD and preserve the impacted mRNA transcript have been explored but have not progressed to clinical development, leaving NMD a significant hurdle for PTC readthrough therapy. MethodsAntisense oligonucleotides (ASOs) were tiled across intron 22 splice donor (SD) and acceptor (SA) sites of the CFTR gene. Immortalized airway cells were treated with SD and SA ASOs, and those yielding the greatest increase in a CFTR NMD-insensitive mRNA isoform (e22 trunc mRNA) were tested in combinations. Top SD/SA ASO pairs were assessed for their impact on e22 trunc mRNA via ddPCR, e22 trunc protein via western blot, and CFTR-mediated chloride (Cl-) transport via transepithelial electrophysiological measurements in immortalized and primary human bronchial epithelial (hBE) cell cultures. ResultsWe demonstrate that e22 trunc mRNA generates a truncated CFTR protein whose Cl- transport function can be enhanced with elexacaftor/tezacaftor/ivacaftor (ETI) treatment. ASO and ETI treatment in combination restore [~]20% and 25% of wild-type CFTR Cl- transport function in immortalized epithelial and primary hBE cells homozygous for CFTR W1282X, respectively. ConclusionsThis study lays the groundwork for advancing ASO-mediated upregulation of e22 trunc mRNA and protein as a therapeutic approach for cystic fibrosis caused by 3-terminal CFTR PTC mutations.

genomics↗

bioRxiv2019

β-HPV 8E6 Dysregulates the Hippo Signaling Pathway and Induces Aneuploidy

Beta genus human papillomaviruses ({beta}-HPVs) are associated with cutaneous squamous cell carcinomas (cSCCs) in a subset of immunocompromised patients. Although {beta}-HPVs are not necessary for tumor maintenance, they are hypothesized to destabilize the genome in the early stages of cancer development. Supporting this idea, {beta}-HPVs 8E6 protein attenuates p53 accumulation after failed cytokinesis. This paper identifies the mechanism of this abatement. We show {beta}-HPV 8E6 dysregulates the Hippo signaling pathway (HP). It increases pro-proliferative gene expression, enhances TEAD activity and promotes cell growth. {beta}-HPV 8E6 also reduces LATS activation and p53-mediated apoptosis following unsuccessful division of mitotic cells. These phenotypes are dependent on {beta}-HPV 8E6 binding and destabilizing a cellular histone acetyltransferase, p300. Despite circumventing apoptosis, {beta}-HPV 8E6 caused increased senescence after unsuccessful cytokinesis. We linked this lack of growth to the viral proteins inability to prevent cytoplasmic sequestration of the HP transcription factor, YAP. We also show that increased telomerase reverse transcriptase activity (a common alteration in cSCCs) acts synergistically with {beta}-HPV 8E6 to promote cellular proliferation after abortive cytokinesis. While {beta}-HPV 8E6 promoted aneuploidy on its own, this genome destabilization is amplified in cells that do not divide after mitosis. Although our group and others have previously described inhibition of DNA repair, to the best of our knowledge this marks the first time that a {beta}-HPV protein has been connected to chromosome level changes in the cellular genome. This represents a substantial escalation in the known genome destabilizing properties likely to occur during a {beta}-HPV infection.\n\nIMPORTANCEThere is mounting evidence that {beta}-HPVs contribute to cSCCs development in immunocompromised populations. They may also augment UVs mutagenic potential, increasing cancer risk in the general population. We demonstrate that {beta}-HPV 8E6 dysregulates the Hippo signaling pathway (HP). HP regulates cell growth and apoptosis in response to a myriad of stimuli, including failed cytokinesis. {beta}-HPV 8E6 attenuates phosphorylation of the HP kinase, LATS, decreasing some but not all downstream signaling events. This allows binucleated cells to avoid apoptosis, however they succumb to senescence. We show that {beta}-HPV 8E6 synergizes with a common cSCC mutation (telomerase activation) to avoid both apoptosis and senescence. We did not find any telomerase immortalized {beta}-HPV 8E6 expressing cells that were not aneuploid after aberrant cytokinesis. This represents a substantial escalation in {beta}-HPV E6s known mutagenic potential.

cancer biology↗