Genetic variability in NOTCH1: association with overweight/obesity and therapeutic implications in Chronic Lymphocytic Leukemia in elderlies.

Name: Estevão Carlos Silva Barcelos
Type: PhD thesis
Publication date: 03/09/2021
Advisor:

Namesort descending Role
Flavia Imbroisi Valle Errera (M) Co-advisor *
Sandra Lúcia Ventorin von Zeidler Advisor *

Examining board:

Namesort descending Role
Breno Valentim Nogueira Internal Examiner *
Carlos Magno da Costa Maranduba External Examiner *
Flavia de Paula (M/D) Internal Examiner *
Flavia Imbroisi Valle Errera (M) Co advisor *
MICHEL SATYA NASLAVSKY External Examiner *
Sandra Lúcia Ventorin von Zeidler Advisor *

Summary: NOTCH1 is critical for the maintenance of stem cells and adult tissues, and appears to play as a key molecule in aging and in a variety of chronic conditions, including obesity and cancer, especially Chronic Lymphocytic Leukemia (CLL). Recently, the NOTCH1 signaling pathway has been identified as fundamental in adipocyte homeostasis, in addition to being directly linked to the inflammatory process. Furthermore, constitutive activation of NOTCH1 signaling is associated with poor prognosis in some types of cancer such as CLL WHERE recurrent NOTCH1 mutations are responsible for stabilizing the signaling state. Given the involvement of the NOTCH1 gene in cell metabolism and development, the hypothesis of the present study is that genetic variations of NOTCH1 are associated with increased risk for overweight (overweight and obesity) and also alter the response of CLL cells to induced-stress. Therefore, as a first objective, we investigated whether genetic
variations in NOTCH1 are associated with overweight/obesity in a Brazilian elderly cohort. To assess the association of NOTCH1 polymorphisms and overweight/obesity, anthropometric, biochemical, medical history, and lifestyle data were collected. Sequencing data from 1.024 individuals (aged 59 to 99 years) were analyzed: 424 were overweight, 320 were obese, and 280 were normal weight. We
analyzed one hundred and sixty-one tag SNPs spanning the entire NOTCH1 gene and borders using the SNP tagging approach (minor allele frequency≥0.01 and pairwise linkage disequilibrium r2≥0.8). We observed the association of SNP rs9411207 with risk of overweight/obesity under the additive model, and the genotype distribution showed an increased frequency of homozygous TT (OR = 1.50, 95% CI: 1.20-1.88; P = 0.0002). The GAT haplotype constructed from this and other SNPs in high linkage disequilibrium (LD) was more frequent in overweight/obese subjects (P = 0.003). In silico analysis suggested that these SNPs likely affect the transcription of NOTCH1 and other genes. This is the first study reporting the association between NOTCH1 SNPs and risk of overweight/obesity. Considering the possibility of modulation of NOTCH1, additional population studies are needed to replicate these results and confirm the usefulness of these risk genotypes for new therapeutic strategies. A second objective was to evaluate whether the mutation in the NOTCH1 gene and the deregulation of the NOTCH1 pathway in CLL cases alter the response to curcumin. For this, we investigated the anti-CLL effect of curcumin and its ability to interfere in the integrated stress response (ISR) and in the NOTCH1 signaling pathway in primary CLL cells with mutation in the NOTCH1 gene and in the murine model of CLL Eμ-TCL1. Initially, gene expression data from cells with c.7544-7545 delCT mutation in NOTCH1 were analyzed against data from Wild-Type (WT) cells. In silico analysis of gene expression of CLL cells mutated in NOTCH1 showed an increase in the expression of genes involved in Endoplasmic Reticulum (ER) and ISR stress. Primary CLL cells were collected and cultured with curcumin, a natural compound that also targets ER stress, for further analysis by Western blotting and real-time PCR. After in vitro exposure to curcumin, stress-related mechanisms were observed in primary cells with mutated NOTCH1 compared to WT cells. This response was preceded by an early increase in Ca2+ in the cytoplasm of CLL cells mutated in NOTCH1, which can trigger and maintain ER stress. Furthermore, curcumin increased the apoptosis of CLL cells carrying the NOTCH1 mutation, regardless of allelic load, when compared to WT cells. NOTCH1 signaling also appears modulated after exposure to curcumin. In particular, CLL cells mutated in NOTCH1 showed a reduction in the active portion of NOTCH1 (ICN1) and in the antiapoptotic proteins of the BCL2 family (BCL2 and MCL1), and the combination of curcumin with venetoclax, an anti-BCL2, shown to have a synergistic effect on CLL cells. In addition, we used the murine model Eμ-TCL1 to evaluate treatment with curcumin. In vivo administration of curcumin in the Eμ-TCL1 model significantly reduced the percentage of CD5+/CD19+ leukemic cells infiltrating the liver, spleen and bone marrow, with concomitant inhibition of NOTCH1 signaling in leukemic cells in the bone marrow. Our results suggest that ER stress-induced ISR activation and NOTCH1 signaling inhibition converge to amplification of CLL cell death and provide a crucial target for CLL treatment.

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