Periodic Reporting for period 1 - MACHETE (Unraveling the Functional Complexity of Cancer Genomes through Chromosome Engineering)
Reporting period: 2023-01-01 to 2025-06-30
Cancer develops when changes in genes turn normal cells into tumors. One common type of genetic change is called a copy number alteration (CNA), where parts of DNA are either gained or lost. These changes can affect up to 30% of a cancer cell’s DNA and are often linked to worse outcomes for patients. However, we still don’t fully understand how specific CNAs contribute to cancer’s behavior, like spreading to other tissues or resisting treatment. This is because current methods can’t accurately replicate these complex DNA changes to understand how they change cancer cells. Unlike other types of mutations, CNAs can influence many genes at once and even alter the structure of the genome, which in some cases leads to the appearance of tumor promoting genes in circular DNA outside of chromosomes (also known as extra chromosomal DNA).
To better understand the role of CNAs in cancer, this grant uses a new genome engineering approach called MACHETE, which can create deletions, gains, and extra chromosomal DNA. Focusing on pancreatic cancer, a deadly type of tumor, we will engineer the most common CNAs to see how they help cancer evade the immune system, spread to other organs, or promote resistance to treatments. It will also explore whether the order in which these DNA changes occur affects how the cancer behaves—a question that hasn’t been studied before.
By combining MACHETE with in vivo models and state of the art molecular profiling techniques, this grant aims to uncover how CNAs drive pancreatic cancer. The findings from this proposal could lead to new treatments targeting these large-scale DNA changes. Importantly, the tools and ideas developed here could also be applied to other cancers and diseases with similar genetic alterations, potentially opening doors to new therapies.
Overall objectives:
-Identify how specific copy number alterations allow pancreas cancer cells to avoid the immune system.
-Study if the way in which oncogenes are amplified (inside or outside of chromosomes) changes the ability of pancreas cancer cells to invade distant organs and respond to therapies.
-Dissect if the order in which copy number alterations arise change the way pancreas cancers behave.
To better understand the role of CNAs in cancer, this grant uses a new genome engineering approach called MACHETE, which can create deletions, gains, and extra chromosomal DNA. Focusing on pancreatic cancer, a deadly type of tumor, we will engineer the most common CNAs to see how they help cancer evade the immune system, spread to other organs, or promote resistance to treatments. It will also explore whether the order in which these DNA changes occur affects how the cancer behaves—a question that hasn’t been studied before.
By combining MACHETE with in vivo models and state of the art molecular profiling techniques, this grant aims to uncover how CNAs drive pancreatic cancer. The findings from this proposal could lead to new treatments targeting these large-scale DNA changes. Importantly, the tools and ideas developed here could also be applied to other cancers and diseases with similar genetic alterations, potentially opening doors to new therapies.
Overall objectives:
-Identify how specific copy number alterations allow pancreas cancer cells to avoid the immune system.
-Study if the way in which oncogenes are amplified (inside or outside of chromosomes) changes the ability of pancreas cancer cells to invade distant organs and respond to therapies.
-Dissect if the order in which copy number alterations arise change the way pancreas cancers behave.
In these initial two years we have made substantial efforts to progress in completing the established objectives outlined in this grant, which will shed light into the role of copy number alterations in pancreas cancer.
Our work has been centered in establishing the group (recruitment of team members, purchase and optimization of essential equipment, approval of ethics protocols). Beyond this, we are setting up the methods and models to reach the project objectives.
Objective-specific achievements:
Objective 1. Identify how specific copy number alterations allow pancreas cancer cells to avoid the immune system.
-Establishment of the in vivo colony of pancreas cancer and neoantigen-specific CD8 T cell mouse models.
-Generation of the relevant mouse cell lines to study how deletions of specific clusters of immune genes alter pancreas cancer progression.
-Creation of novel neoantigen expressing vectors and showed their ability to elicit activation and cytotoxicity of matching CD8 T cells in vitro.
Objective 2. Study if the way in which oncogenes are amplified (inside or outside of chromosomes) changes the ability of pancreas cancer cells to invade distant organs and respond to therapies.
-Establishment of DNA FISH and confirmation that we can engineer de novo extra chromosomal DNA.
-Identification that engineered extra chromosomal DNA encoding Dhfr endows drug-naïve cells to resist methotrexate treatment.
-Redesign and optimisation of ecDNA engineering approach in pancreas cancer cells.
-Establishment of pancreas cancer with an extra chromosomal DNA reporter system.
Objective 3. Dissect if the order in which copy number alterations arise change the way pancreas cancers behave.
-Optimisation of MACHETE for creating large chromosomal deletions of PDAC tumor suppressor genes.
-Creation of allelic series of chromosome 18E2 and in vivo test of these alleles.
-Establishment of novel microcell mediated chromosome transfer protocol.
Our work has been centered in establishing the group (recruitment of team members, purchase and optimization of essential equipment, approval of ethics protocols). Beyond this, we are setting up the methods and models to reach the project objectives.
Objective-specific achievements:
Objective 1. Identify how specific copy number alterations allow pancreas cancer cells to avoid the immune system.
-Establishment of the in vivo colony of pancreas cancer and neoantigen-specific CD8 T cell mouse models.
-Generation of the relevant mouse cell lines to study how deletions of specific clusters of immune genes alter pancreas cancer progression.
-Creation of novel neoantigen expressing vectors and showed their ability to elicit activation and cytotoxicity of matching CD8 T cells in vitro.
Objective 2. Study if the way in which oncogenes are amplified (inside or outside of chromosomes) changes the ability of pancreas cancer cells to invade distant organs and respond to therapies.
-Establishment of DNA FISH and confirmation that we can engineer de novo extra chromosomal DNA.
-Identification that engineered extra chromosomal DNA encoding Dhfr endows drug-naïve cells to resist methotrexate treatment.
-Redesign and optimisation of ecDNA engineering approach in pancreas cancer cells.
-Establishment of pancreas cancer with an extra chromosomal DNA reporter system.
Objective 3. Dissect if the order in which copy number alterations arise change the way pancreas cancers behave.
-Optimisation of MACHETE for creating large chromosomal deletions of PDAC tumor suppressor genes.
-Creation of allelic series of chromosome 18E2 and in vivo test of these alleles.
-Establishment of novel microcell mediated chromosome transfer protocol.
We are developing novel mouse models of cancer, establishing methods to create extra chromosomal DNA, and a more efficient whole chromosome transfer technique. All of these approaches will have broad implications on the study of cancer genetics, immune surveillance of tumors, and cancer genome evolution.
We still need further research to substantiate these approaches and biological insights to the broader academic community. Despite this, we believe we are moving forward the state of the art at a technical and biological level by developing models and pursuing unique questions in cancer genetics.
We still need further research to substantiate these approaches and biological insights to the broader academic community. Despite this, we believe we are moving forward the state of the art at a technical and biological level by developing models and pursuing unique questions in cancer genetics.