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Juan Carlos Alvarez Álvarez Pérez



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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: 1
    • 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  |  Presenting Author(s): Juan Carlos Alvarez Álvarez Pérez

      • Abstract
      • Slides

      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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    MA17 - Molecular Mechanisms and Therapies (ID 143)

    • Event: WCLC 2019
    • Type: Mini Oral Session
    • Track: Biology
    • Presentations: 1
    • Now Available
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      MA17.06 - Plakophilin 1 Enhances MYC Expression, Promoting Squamous Cell Lung Cancer (Now Available) (ID 823)

      15:45 - 17:15  |  Author(s): Juan Carlos Alvarez Álvarez Pérez

      • Abstract
      • Presentation
      • Slides

      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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