Molecular biomarkers in melanoma susceptibility and therapy response
Author: Yang, Muyi
Date: 2021-12-21
Location: J3:06 Ulf von Euler, BioClinicum, Karolinska University Hospital Solna
Time: 09.00
Department: Inst för onkologi-patologi / Dept of Oncology-Pathology
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Thesis (5.686Mb)
Abstract
Melanoma accounts for the majority of the mortality from all types of skin cancers. Five to ten percent of the cases are familial melanoma, yet more than 50% of the melanoma families cannot be explained by established susceptibility genes. By understanding the genetics behind unexplained families may shed lights on melanoma etiology and in turn provide these susceptible individuals with opportunities of more precise medical care. Over the last decade, treatment options for melanoma patients have expanded with the introduction of targeted therapies and immunotherapies. For the melanoma patients with advanced disease whose tumors harbor BRAF mutations, targeted therapy (TT) of BRAF-inhibitors (BRAFi) in combination with MEK-inhibitors have shown promising results with improved clinical outcome. However, acquired resistance is common among these patients. Immunotherapy with immune checkpoint inhibitors (ICIs) have also been proven successful in melanoma. Yet a subset of patients shows intrinsic or acquired resistance and benefits little from ICIs. There is an urgent need of predictive biomarkers to identify patients who will benefit from a durable response to TT or ICIs.
In this thesis, we first describe a targeted sequencing project in which we performed massive parallel sequencing of a selected 120 genes in the germline DNA of 92 melanoma patients with family history or multiple primary melanomas. We reported a rare nonsense variant in BRCA2 and showed moderately increased risk for melanoma according to our case-control analysis. Moreover, we identified another rare variant in the BRIP1gene that segregated with the disease in one family, but the variant did not seem to alter the expression or subcellular location of BRIP protein.
In paper II, we genotyped the MC1R gene for 103 melanoma patients receiving ICIs and compared the characteristics and prognosis of those carrying ≥1 R alleles and those not. Our data showed that patients with ≥1 R alleles had more favorable progression free survival (PFS) after ICIs, but no significant difference in overall survival (OS). Multivariate analyses suggested that the superior PFS in “≥1 R alleles” group was attributed to more than the previously anticipated R allele-associated pigmentation phenotype. This study was the first to assess the impact of germline MC1R variants on the efficacy of ICIs in melanoma.
In paper III, we performed whole-exome sequencing on multiple cases from a melanoma-prone family and identified a germline, heterozygous, frameshift variant in DENND5A gene. We were able to report two additional rare variants in two separate melanoma-prone families segregating with the disease. Our follow-up functional study discovered that DENND5A was enriched in pigmented tissue. Impaired DENND5A function led to mis-trafficking of melanosomal cargo protein and thereafter reduction of melanin content in vitro and in zebrafish. Altogether, paper III uncovered a novel biological function of DENND5A in melanoma and linked its loss-of-function variant to melanoma susceptibility.
In paper IV, we revisited our previously published quantitative proteome profiling on vemurafenib-induced BRAFi-resistant subline A375VR4 and its parental A375 cell line, and identified the down-regulation of RAB7B in A375VR4. We showed that silencing of RAB7B in A375 cells led to reduced sensitivity towards BRAFi in 3D spheroids and to enhanced cell motility in vitro and in zebrafish. We performed RAB7B-immunoprecipitated mass spectrometry (IP-MS) and identified tyrosine kinase SRC as a novel effector of RAB7B. Silencing RAB7B in A375 spheroids led to up-regulated kinase activity reflected by increase of phospho-SRC (Tyr416), which might have contributed to the BRAFi resistance and enhanced cell motility.
In this thesis, we first describe a targeted sequencing project in which we performed massive parallel sequencing of a selected 120 genes in the germline DNA of 92 melanoma patients with family history or multiple primary melanomas. We reported a rare nonsense variant in BRCA2 and showed moderately increased risk for melanoma according to our case-control analysis. Moreover, we identified another rare variant in the BRIP1gene that segregated with the disease in one family, but the variant did not seem to alter the expression or subcellular location of BRIP protein.
In paper II, we genotyped the MC1R gene for 103 melanoma patients receiving ICIs and compared the characteristics and prognosis of those carrying ≥1 R alleles and those not. Our data showed that patients with ≥1 R alleles had more favorable progression free survival (PFS) after ICIs, but no significant difference in overall survival (OS). Multivariate analyses suggested that the superior PFS in “≥1 R alleles” group was attributed to more than the previously anticipated R allele-associated pigmentation phenotype. This study was the first to assess the impact of germline MC1R variants on the efficacy of ICIs in melanoma.
In paper III, we performed whole-exome sequencing on multiple cases from a melanoma-prone family and identified a germline, heterozygous, frameshift variant in DENND5A gene. We were able to report two additional rare variants in two separate melanoma-prone families segregating with the disease. Our follow-up functional study discovered that DENND5A was enriched in pigmented tissue. Impaired DENND5A function led to mis-trafficking of melanosomal cargo protein and thereafter reduction of melanin content in vitro and in zebrafish. Altogether, paper III uncovered a novel biological function of DENND5A in melanoma and linked its loss-of-function variant to melanoma susceptibility.
In paper IV, we revisited our previously published quantitative proteome profiling on vemurafenib-induced BRAFi-resistant subline A375VR4 and its parental A375 cell line, and identified the down-regulation of RAB7B in A375VR4. We showed that silencing of RAB7B in A375 cells led to reduced sensitivity towards BRAFi in 3D spheroids and to enhanced cell motility in vitro and in zebrafish. We performed RAB7B-immunoprecipitated mass spectrometry (IP-MS) and identified tyrosine kinase SRC as a novel effector of RAB7B. Silencing RAB7B in A375 spheroids led to up-regulated kinase activity reflected by increase of phospho-SRC (Tyr416), which might have contributed to the BRAFi resistance and enhanced cell motility.
List of papers:
I. Rainer Tuominen, Pär G Engström, Hildur Helgadottir, Hanna Eriksson, Per Unneberg, Sanela Kjellqvist, Muyi Yang, Diana Lindén, Daniel Edsgärd, Johan Hansson, Veronica Höiom. The role of germline alterations in the DNA damage response genes BRIP1 and BRCA2 in melanoma susceptibility. Genes Chromosomes Cancer. 2016;55(7):601-1.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Muyi Yang, Marie Jalsenius, Fernanda Costa Svedman, Vitali Grozman, Jasmine Dahlberg, Suzanne Egyházi Brage, Veronica Höiom, Hildur Helgadottir. Germline MC1R mutation status and efficacy of immune checkpoint inhibitor in patients with advanced melanoma. [Manuscript]
III. Muyi Yang, Per Johnsson, Lars Bräutigam, Xiaohong R. Yang, Kim Thrane, Jiwei Gao, Nicholas P. Tobin, Yitian Zhou, Rong Yu, Noemi Nagy, Pär G. Engström, Rainer Tuominen, Hanna Eriksson, Joakim Lundeberg, Margaret A. Tucker, Alisa M. Goldstein, Suzanne Egyházi Brage, Jian Zhao, Yihai Cao, Veronica Höiom. Novel loss-of-function mutation in DENND5A impedes melanosomal cargo transport and predisposes to familial cutaneous melanoma. [Accepted]
Fulltext (DOI)
Pubmed
IV. Muyi Yang, Jiwei Gao, Lars Bräutigam, Yafeng Zhu, Xingkang He, Alireza Azimi, Xiaofang Cao, Yihai Cao, Veronica Höiom, Suzanne Egyházi Brage. Silencing of RAB7B increases migratory capacity and decreases BRAF inhibitor sensitivity in melanoma cells. [Manuscript]
I. Rainer Tuominen, Pär G Engström, Hildur Helgadottir, Hanna Eriksson, Per Unneberg, Sanela Kjellqvist, Muyi Yang, Diana Lindén, Daniel Edsgärd, Johan Hansson, Veronica Höiom. The role of germline alterations in the DNA damage response genes BRIP1 and BRCA2 in melanoma susceptibility. Genes Chromosomes Cancer. 2016;55(7):601-1.
Fulltext (DOI)
Pubmed
View record in Web of Science®
II. Muyi Yang, Marie Jalsenius, Fernanda Costa Svedman, Vitali Grozman, Jasmine Dahlberg, Suzanne Egyházi Brage, Veronica Höiom, Hildur Helgadottir. Germline MC1R mutation status and efficacy of immune checkpoint inhibitor in patients with advanced melanoma. [Manuscript]
III. Muyi Yang, Per Johnsson, Lars Bräutigam, Xiaohong R. Yang, Kim Thrane, Jiwei Gao, Nicholas P. Tobin, Yitian Zhou, Rong Yu, Noemi Nagy, Pär G. Engström, Rainer Tuominen, Hanna Eriksson, Joakim Lundeberg, Margaret A. Tucker, Alisa M. Goldstein, Suzanne Egyházi Brage, Jian Zhao, Yihai Cao, Veronica Höiom. Novel loss-of-function mutation in DENND5A impedes melanosomal cargo transport and predisposes to familial cutaneous melanoma. [Accepted]
Fulltext (DOI)
Pubmed
IV. Muyi Yang, Jiwei Gao, Lars Bräutigam, Yafeng Zhu, Xingkang He, Alireza Azimi, Xiaofang Cao, Yihai Cao, Veronica Höiom, Suzanne Egyházi Brage. Silencing of RAB7B increases migratory capacity and decreases BRAF inhibitor sensitivity in melanoma cells. [Manuscript]
Institution: Karolinska Institutet
Supervisor: Höiom, Veronica
Co-supervisor: Egyházi Brage, Suzanne; Hansson, Johan; Zhao, Jian
Issue date: 2021-11-30
Rights:
Publication year: 2021
ISBN: 978-91-8016-407-8
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