TAZ role in lung cancer: resistance to BET inhibitors and functional interaction with lncRNA

Despite extensive research and introduction of innovative therapy, lung cancer prognosis remains poor, with a five years survival of only 17%. The success of pharmacological treatment is often impaired by drug resistance. Thus, the characterization of response mechanisms to anti-cancer compounds and of the molecular mechanisms supporting lung cancer aggressiveness are crucial for patient’s management. In the first part of this thesis, we characterized the molecular mechanism behind resistance of lung cancer cells to the Inhibitors of the Bromodomain and Extraterminal domain containing Proteins (BETi). Through a CRISPR/Cas9 screening we identified three Hippo Pathway members, LATS2, TAOK1 and NF2 as genes implicated in susceptibility to BETi. These genes confer sensitivity to BETi inhibiting TAZ activity. Conversely, TAZ overexpression increases resistance to BETi. We also displayed that BETi downregulate both YAP, TAZ and TEADs expression in several cancer cell lines, implying a novel BETi-dependent cytotoxic mechanism. In the second part of this work, we attempted to characterize the crosstalk between the TAZ gene and its cognate antisense long-non coding RNA (lncRNA) TAZ-AS202 in lung tumorigenesis. As for TAZ downregulation, TAZ-AS202 silencing impairs NSCLC cells proliferation, migration and invasion, suggesting a pro-tumorigenic function for this lncRNA during lung tumorigenesis. TAZ-AS202 regulates TAZ target genes without altering TAZ expression or localization. This finding implies an uncovered functional cooperation between TAZ and TAZ-AS202. Moreover, we found that the EPH-ephrin signaling receptor EPHB2 is a downstream effector affected by both TAZ and TAZ-AS202 silencing. EPHB2 downregulation significantly attenuates cells proliferation, migration and invasion, suggesting that, at least in part, TAZ-AS202 and TAZ pro-oncogenic activity depends on EPH-ephrin signaling final deregulation. Finally, we started to dissect the mechanism underlying the TAZ-AS202 regulatory activity on EPHB2 in lung cancer, which may involve the existence of an intermediate transcription factor and is the object of our ongoing research.

Whole Exome Sequencing widens the genetic landscape of Hereditary Optic Neuropathies

Hereditary optic neuropathies (HON) are a genetic cause of visual impairment characterized by degeneration of retinal ganglion cells. The majority of HON are caused by pathogenic variants in mtDNA genes and in gene OPA1. However, several other genes can cause optic atrophy and can only be identified by high throughput genetic analysis. Whole Exome Sequencing (WES) is becoming the primary choice in rare disease molecular diagnosis, being both cost effective and informative. We performed WES on a cohort of 106 cases, of which 74 isolated ON patients (ON) and 32 syndromic ON patients (sON). The total diagnostic yield amounts to 27%, slightly higher for syndromic ON (31%) than for isolated ON (26%). The majority of genes found are related to mitochondrial function and already reported for harbouring HON pathogenic variants: ACO2, AFG3L2, C19orf12, DNAJC30, FDXR, MECR, MTFMT, NDUFAF2, NDUFB11, NDUFV2, OPA1, PDSS1, SDHA, SSBP1, and WFS1. Among these OPA1, ACO2, and WFS1 were confirmed as the most relevant genetic causes of ON. Moreover, several genes were identified, especially in sON patients, with direct impairment of non-mitochondrial molecular pathways: from autophagy and ubiquitin system (LYST, SNF8, WDR45, UCHL1), to neural cells development and function (KIF1A, GFAP, EPHB2, CACNA1A, CACNA1F), but also vitamin metabolism (SLC52A2, BTD), cilia structure (USH2A), and nuclear pore shuttling (NUTF2). Functional validation on yeast model was performed for pathogenic variants detected in MECR, MTFMT, SDHA, and UCHL1 genes. For SDHA and UCHL1 also muscle biopsy and fibroblast cell lines from patients were analysed, pointing to possible pathogenic mechanisms that will be investigated in further studies. In conclusion, WES proved to be an efficient tool when applied to our ON cohort, for both common disease-genes identification and novel genes discovery. It is therefore recommended to consider WES in ON molecular diagnostic pipeline, as for other rare genetic diseases.