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Pedro Pablo Medina
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EP1.14 - Targeted Therapy (ID 204)
- Event: WCLC 2019
- Type: E-Poster Viewing in the Exhibit Hall
- Track: Targeted Therapy
- Presentations: 2
- Now Available
- Moderators:
- Coordinates: 9/08/2019, 08:00 - 18:00, Exhibit Hall
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EP1.14-25 - Development of New Lung Cancer Therapies Based on Gene-Editing Technologies (Now Available) (ID 821)
08:00 - 18:00 | Author(s): Pedro Pablo Medina
- Abstract
Background
Genome editing has enriched our understanding of mechanisms of the human pathology. Genome editing took a significant advance with the recent development of the CRISPR-Cas9 technology. CRISPR is an acronym for: Clustered Regularly Interspaced Short Palindromic Repeats and it is an adaptation of a prokaryotic functional system. It uses a single guide RNA to direct Cas9 activity to a specific part of the genome, therefore, this system can be used for gene editing and regulation.
Cancer is a genetic disease where some DNA-damaged cells begin to divide without stopping and spread into surrounding tissues. Interestingly, in some tumors there is a dependency of a single oncogenic activity (oncogene addiction). This phenomenon indicates that mutations in key oncogenes (driver mutation) are able to drive carcinogenesis and maintain the tumor phenotype. Suggestively, if we can prevent or disrupt these mutations, we can difficult carcinogenesis or damage an established tumoral phenotype
Method
We seek out for using Crispr-Cas9 technology to target driver mutations and evaluate its therapeutic and preventive value. To develop a proof of concept, we focused on KRAS gene which represents the most frequently mutated family across all cancer types. About one-third of human lung adenocarcinomas, the most prevalent form of lung cancer, carry KRAS mutations. Most of these mutations are located on codon 12; the mutations KRAS c.34G>T (G12C) and KRAS c.35G>A (G12D) are the most frequent and important ones.
Result
We designed specific targeting strategies using HiFiCas9 nuclease which induced KRAS G12C and G12D edition while leaving KRAS WT untouched.
Disruption of these KRAS mutations with the specific gRNA-guided-CRISPR-Cas9 decreases viability and proliferation of mutated cells such as H23 and A427. The generation of a transgenic mouse expressing Crispr-Cas9 designed to target these mutations will allow us to test the potential cancer-resistant phenotype.
Conclusion
Crispr-Cas9 can be engineered to specifically target single nucleotide oncogenic mutations of KRAS.
Edition of KRAS oncogenic mutations G12C and G12D led to a reduction in the viability of mutated lung adenocarcinoma cell lines.
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EP1.14-36 - Suicide Gene Therapy Directed by MicroRNA Activity (Now Available) (ID 290)
08:00 - 18:00 | Author(s): Pedro Pablo Medina
- Abstract
Background
Lung cancer is one of the most prevalent types of malignancies worldwide, accounting for 1.6 million deaths every year. Despite last efforts, the overall survival rate at five years after diagnosis is under 15%. Therefore, new approaches are needed to improve the current clinic.
Suicide gene therapy is an interesting technology which consists in expressing a toxic gene into tumor cells. The most studied suicide gene system is the Herpes Simplex Virus Thymidine Kinase (HSV-TK) that converts the non-toxic prodrug ganciclovir (GCV) into a guanosine analog, promoting cell apoptosis. However, there are problems regarding the expression of the toxic gene in non-tumor cells, which would cause unwanted cell death.
MicroRNAs (miRNAs) have been revealed as one of the most important families of gene expression regulators, acting at a posttranscriptional level by binding messenger RNAs (mRNAs) and blocking protein translation. Alterations in the expression levels of these miRNAs in carcinogenesis have been largely described in the literature, being some of them biomarkers in the diagnosis of cancer. The aim of this work is to improve the selectivity of the HSV-TK suicide gene therapy to target exclusively tumor cells, taking advantage of the alterations in the expression of miRNAs in lung cancer.
Method
We have constructed a plasmid that expresses the HSV-TK mRNA under the control of an artificial 3’-UTR containing binding sites for different members of the let-7 family, a miRNA family whose expression is frequently lost during lung cancer development. Thus, we could direct the expression of the toxic gene preferably in tumor cells where the levels of let-7 are low, allowing the expression of the HSV-TK.
To determine the ability to direct the expression of the suicide gene on this system, we used a lung cancer cell line and a non-tumor, lung tissue one. Both lung cancer and non-tumor cell lines were infected with lentiviruses containing the plasmid construction and then cell viability assays, competitive cell growth assays by immunofluorescence and apoptosis assays were performed to check for differences between the GCV-treated and untreated cells.
Result
Our preliminary results suggest that miRNA expression can selectively drive the expression of the HSV-TK in lung cancer cells due to their low let-7 expression values, resulting in a decrease of cell viability in lung cancer cells compared to non-tumor cells after treating them with GCV.
Ectopically overexpressing let-7 into the lung cancer cells blocked the cell viability decrease caused by the GCV, meaning that our artificial 3'-UTR with binding sites for let-7 is working.
Conclusion
According to our results, we have successfully directed the expression of a suicide gene to tumor cells using miRNA activity. Thanks to that, our HSV-TK/GCV suicide gene therapy model adds an extra layer of safety by preventing off-target cell death.
Differential miRNA expression between tumor and normal tissues could be used to direct the expression of a gene of our choice to the tumor tissue, which could be an interesting approach and a potential tool to treat this disease.
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MA17 - Molecular Mechanisms and Therapies (ID 143)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:Eloisa Jantus-Lewintre, Hongbin Ji
- Coordinates: 9/09/2019, 15:45 - 17:15, Melbourne (1991)
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MA17.06 - Plakophilin 1 Enhances MYC Expression, Promoting Squamous Cell Lung Cancer (Now Available) (ID 823)
15:45 - 17:15 | Presenting Author(s): Pedro Pablo Medina
- Abstract
- Presentation
Background
Plakophilin 1 (PKP1) is a member of the arm-repeat (armadillo) and plakophilin gene families, being an important component of the desmosome. Although desmosomes loss-of-function has been associated with increased cell migration and pro-oncogenic activity, we have observed consistent PKP1 overexpression in patient samples of squamous cell lung cancer (SqCLC) in comparison with lung adenocarcinoma (LUAD) and non-tumoral controls from two datasets achieved by our group, and also from three additional independent datasets.
Method
In order to explore this paradox, we developed in vitro and in vivo PKP1 gain/loss functional models in SqCLC cell lines and also we challenged our hypothesis in some LUAD cell lines.
Result
Greater cell dissemination but reduced cell proliferation was observed in CRISPR-Cas9 induced, PKP1-knockout clones. Furthermore, PKP1 expression promoted cell proliferation, cell survival, and in vivo xenograft engraftment.
Interestingly, we demonstrated through several functional experiments (chromatin immunoprecipitation, RNA immunoprecipitation, direct mutagenesis combined with luciferase assays, Western blot, qPCR... among others), and in 7 cell lines from different lung cancer subtypes (5 SqCLC and 2 LUAD cell lines), and different contexts (with and without PKP1 basal expression in order to set up gain and loss expression assays), that these pro-oncogenic activities were mediated by the functional direct relationship between PKP1 and the oncogene MYC. Specifically, PKP1 enhances MYC translation, and MYC increases PKP1 transcription, linking both proteins in a positive feedforward loop.
Conclusion
These observations provide a new molecular mechanism of cancer development, revealing PKP1 as a novel oncogene in SqCLC, and as an effective post-transcriptional regulator of MYC, which has been described as overexpressed in around 70% of NSCLC tumors.
Moreover, PKP1 unveiled as a valuable diagnostic biomarker and a potential therapeutic target for SqCLC. Importantly, PKP1 inhibition may open up the possibility of indirectly targeting MYC, not only in NSCLC (where, as mentioned before, is frequently overexpressed), but also in other tumors. This is of particular interest, because MYC is an oncogene that is dysregulated in most human cancers and is acknowledged as a “most wanted” target for cancer therapy.
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P1.03 - Biology (ID 161)
- Event: WCLC 2019
- Type: Poster Viewing in the Exhibit Hall
- Track: Biology
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2019, 09:45 - 18:00, Exhibit Hall
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P1.03-20 - Exploring Driver Mutations in Non-Coding RNAs in Lung Adenocarcinoma (ID 1686)
09:45 - 18:00 | Author(s): Pedro Pablo Medina
- Abstract
Background
Lung adenocarcinoma (LUAD) is the most frequent subtype of lung cancer, which is the leading cause of cancer death worldwide. Unraveling the molecular mechanisms of LUAD is crucial for identifying novel biomarkers and molecular targets for the diagnosis, prognosis and treatment of LUAD. One of the main molecular mechanisms responsible for LUAD and other cancers is the accumulation of driver somatic mutations, which undergo positive selection. However, driver mutations only represent a small proportion of the total mutations in a tumor and identifying true drivers can be challenging, especially in non-coding regions.
Non-coding RNAs are RNA molecules that do not code for protein. Increasing evidence suggests that many non-coding RNAs, especially microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play critical roles in most, if not all hallmarks of cancer. However, very little is known about the driver mutations that affect non-coding RNAs in cancer and, in particular, in lung adenocarcinoma (LUAD).
Method
We performed targeted next-generation sequencing on genomic DNA from 70 LUAD primary tumors, focusing on 1470 miRNAs and 913 lncRNAs. We developed a novel bioinformatics pipeline to identify somatic mutations by combining three state-of-the-art mutation calling tools, and then we prioritized the high-confidence somatic mutations based on various published functional impact metrics specifically designed for miRNAs and for lncRNAs. In addition, we further validated our pipeline and our results using whole-genome sequencing data from 59 LUAD primary tumors from The Cancer Genome Atlas (TCGA).
Result
We identified 193 miRNA mutations in our cohort of 70 LUAD primary tumors. These included 16 mutations affecting seed regions and 48 mutations affecting mature miRNAs. Using miRNA target prediction tools, we identified one somatic mutation that affected the seed of a well-known miRNA and that significantly altered the predicted targets of the miRNA. In lncRNAs, we found 2004 mutations in out cohort, out of which 565 passed preliminary filters based on the predicted functional impact. Of those mutations, 91 were recurrent in more than one patient, and some affected well-known lncRNAs such as HOTAIR and PVT1. The results from our internal cohort differed greatly from those obtained from TCGA patients. Future work will focus on refining the mutation prioritization pipeline and experimentally validating the most significant results.
Conclusion
Our novel pipeline may aid in the identification of novel driver mutations in non-coding RNAs, which may reveal novel biomarkers and molecular targets for the diagnosis, prognosis and treatment of LUAD.
