Periodic Reporting for period 1 - SoMuKT (SOmatic MUtagenesis in Kidney Tubule is enhanced by specific metabolic pathways)
Reporting period: 2020-09-01 to 2022-08-31
Somatic genetic changes accumulate during a lifetime in every cell of the body and are a key factor promoting tumor initiation. Slowing down mutation accumulation could, in principle, reduce the risk of cancer. Unfortunately, our current knowledge about the processes and lifestyle factors that enhance mutation accumulation is very limited.
Latest research has shown that different tissues and cell types display different mutation rates, which depend on cell-type-specific exposure to mutagens and ability to repair DNA lesions. In addition to differences in the number of mutations, cell-type-specific DNA-damage and repair also determine differences in the quality of mutations and produce recognizable mutational patterns and profiles. These patterns represent the mark that specific mutational processes leave on the genome and can be exploited to understand cellular mechanism leading to mutation.
Having this in mind, we have studied single nucleotide variants (SNVs) and small insertions/deletions (indels) in normal cells derived from human kidney tubules. We have identified a subset of kidney tubule cells that showed a particularly interesting somatic mutation profile. Peculiar features included:
-high mutation burden
-direct correlation between mutation rate and transcription
-specific alterations in the DNA strand-bias of SNVs
Similar features have been observed in tumors with alterations of glutamine/ammonia metabolism. This branch of metabolism is involved in nucleotide synthesis and DNA repair. For these reasons, we hypothesize that similar alterations of glutamine/ammonia metabolism could be a mutational process occurring in normal kidney cells.
This project objectives are:
1) test whether metabolic alterations similar to those observed in tumors occur in normal kidney tubule cells as well
2) assess whether similar alterations are the underlying cause of excessive mutation in a subset of normal kidney tubule cells
3) verify whether hypoxia signaling might be the driver of metabolic rearrangement connected with mutagenesis.
Preliminary data in support of each point have been obtained.
Latest research has shown that different tissues and cell types display different mutation rates, which depend on cell-type-specific exposure to mutagens and ability to repair DNA lesions. In addition to differences in the number of mutations, cell-type-specific DNA-damage and repair also determine differences in the quality of mutations and produce recognizable mutational patterns and profiles. These patterns represent the mark that specific mutational processes leave on the genome and can be exploited to understand cellular mechanism leading to mutation.
Having this in mind, we have studied single nucleotide variants (SNVs) and small insertions/deletions (indels) in normal cells derived from human kidney tubules. We have identified a subset of kidney tubule cells that showed a particularly interesting somatic mutation profile. Peculiar features included:
-high mutation burden
-direct correlation between mutation rate and transcription
-specific alterations in the DNA strand-bias of SNVs
Similar features have been observed in tumors with alterations of glutamine/ammonia metabolism. This branch of metabolism is involved in nucleotide synthesis and DNA repair. For these reasons, we hypothesize that similar alterations of glutamine/ammonia metabolism could be a mutational process occurring in normal kidney cells.
This project objectives are:
1) test whether metabolic alterations similar to those observed in tumors occur in normal kidney tubule cells as well
2) assess whether similar alterations are the underlying cause of excessive mutation in a subset of normal kidney tubule cells
3) verify whether hypoxia signaling might be the driver of metabolic rearrangement connected with mutagenesis.
Preliminary data in support of each point have been obtained.
To reach the project objectives, we studied primary cells derived from human kidney tubules and clonally expanded in vitro. From each clone, we have obtained whole genome sequencing data, together with expression data of selected genes. For some clones we have obtained metabolomic data. To test our hypothesis on hypoxia signaling as underlying cause of alterations (objective 3), we have used a genetic model, i.e. kidney cells from individuals that carry an inherited pathogenic mutation in the VHL (Von Hippel Lindau) gene. The VHL gene is a tumor suppressor and master regulator of hypoxia signalling. Loss of VHL causes ectopic activation of hypoxia signaling and, according to our hypothesis, should enhance mutagenesis. Of note, the genetic loss of VHL is very often the first event in kidney tumorigenesis. The project has been carried out in collaboration with the Urological Research Institute and the Laboratory of Clinical Molecular Genetics of San Raffaele Hospital.
The project has achieved the following results:
1) Establishment of a method for detection of somatic mutations in single genomes from human kidneys avoiding the need for kidney biopsies and using, instead, non-invasive liquid biopsies (urine samples).
2) Establishment of a protocol for concomitant assessment of somatic mutations, gene expression and metabolite quantification in in vitro-expanded, clonal populations from human kidneys.
3) Detailed characterization of proliferating kidney cells found in human urines and able to grow in vitro. This characterization has been obtained by culturing cells from the urine of 6 healthy individuals in the age range (24-63) and 6 VHL patients (age range 39-56) and is focused on describing changes associated with aging and heterozygous loss of VHL.
4) Generation of somatic mutation catalogues (n=19) of normal kidney genomes obtained from the urine of 3 healthy individuals and 1 patient with VHL disease.
5) Analyses of additional somatic mutation catalogues, obtained from 4 cysts and 7 ccRCC,from the kidney of the same patient with VHL disease.
6) Evidence of increased mutagenesis in normal kidney cells of an individual carrying a germline heterozygous pathogenic mutation in VHL.
7) Evidence, based on mutational profiles, that metabolic changes downstream hypoxia signalling are a cell-type specific cause of mutagenesis in kidney tubule epithelia.
Results have been disseminated both within the host institution and at international events. This work has been presented multiple times at internal congresses and retreats within the host institution (San Raffaele Hospital); at the “14th International VHL Medical/Research Symposium”, organized by the “VHL alliance”, which gathers researchers, clinicians and patients with VHL disease; at the Congress of the European Society of Human Genetics (ESHG) 2022; at the FASEB congress on Polycystic Kidney Disease in July 2022.
The project has achieved the following results:
1) Establishment of a method for detection of somatic mutations in single genomes from human kidneys avoiding the need for kidney biopsies and using, instead, non-invasive liquid biopsies (urine samples).
2) Establishment of a protocol for concomitant assessment of somatic mutations, gene expression and metabolite quantification in in vitro-expanded, clonal populations from human kidneys.
3) Detailed characterization of proliferating kidney cells found in human urines and able to grow in vitro. This characterization has been obtained by culturing cells from the urine of 6 healthy individuals in the age range (24-63) and 6 VHL patients (age range 39-56) and is focused on describing changes associated with aging and heterozygous loss of VHL.
4) Generation of somatic mutation catalogues (n=19) of normal kidney genomes obtained from the urine of 3 healthy individuals and 1 patient with VHL disease.
5) Analyses of additional somatic mutation catalogues, obtained from 4 cysts and 7 ccRCC,from the kidney of the same patient with VHL disease.
6) Evidence of increased mutagenesis in normal kidney cells of an individual carrying a germline heterozygous pathogenic mutation in VHL.
7) Evidence, based on mutational profiles, that metabolic changes downstream hypoxia signalling are a cell-type specific cause of mutagenesis in kidney tubule epithelia.
Results have been disseminated both within the host institution and at international events. This work has been presented multiple times at internal congresses and retreats within the host institution (San Raffaele Hospital); at the “14th International VHL Medical/Research Symposium”, organized by the “VHL alliance”, which gathers researchers, clinicians and patients with VHL disease; at the Congress of the European Society of Human Genetics (ESHG) 2022; at the FASEB congress on Polycystic Kidney Disease in July 2022.
The project has validated the use of kidney cells derived from the urine as a representative model to study patho-physiological processes occurring in the kidney during a lifetime. This method avoids the need of collecting invasive kidney biopsies and substantially enhances the number of individuals that can be tested.
A protocol for concomitant collection of genomic, transriptomic and metabolomic data from clonal cell populations has been developed (figure).
Ultimate goal was the characterization of a specific mutagenic process that occurs in the kidney of normally-aging individuals. This process induces excessive accumulation of mutations, specifically in transcribed genes, thereby enhancing the risk of tumor initiation. Our idea that this process is initiated by hypoxia and is based on alterations of cellular metabolism reveals possible strategies for preventing excessive mutagenesis and limiting cancer risk. One possible intervention could be at the level of the diet. In fact, a diet that exasperates kidney workload might result in micro-ischemic episodes in kidney proximal tubules and finally enhance mutagenesis. Adequate knowledge about lifestyle factors that increase cancer risk is a very important goal for society and might have an impact on citizens, patients with genetic predisposition to kidney cancer, clinicians and policymakers.
A protocol for concomitant collection of genomic, transriptomic and metabolomic data from clonal cell populations has been developed (figure).
Ultimate goal was the characterization of a specific mutagenic process that occurs in the kidney of normally-aging individuals. This process induces excessive accumulation of mutations, specifically in transcribed genes, thereby enhancing the risk of tumor initiation. Our idea that this process is initiated by hypoxia and is based on alterations of cellular metabolism reveals possible strategies for preventing excessive mutagenesis and limiting cancer risk. One possible intervention could be at the level of the diet. In fact, a diet that exasperates kidney workload might result in micro-ischemic episodes in kidney proximal tubules and finally enhance mutagenesis. Adequate knowledge about lifestyle factors that increase cancer risk is a very important goal for society and might have an impact on citizens, patients with genetic predisposition to kidney cancer, clinicians and policymakers.