Cancer is a disease with a major socio-economic impact and the second leading cause of death in the European Union. Pancreatic ductal adenocarcinoma (PDAC), which is driven by oncogenic KRAS, is almost universally fatal and the 4th leading cause of cancer death in the western...
Cancer is a disease with a major socio-economic impact and the second leading cause of death in the European Union. Pancreatic ductal adenocarcinoma (PDAC), which is driven by oncogenic KRAS, is almost universally fatal and the 4th leading cause of cancer death in the western world. The incidence of PDAC is constantly rising and despite modern treatment regimens like multimodal radio-chemotherapy and targeted therapies, the prognosis of PDAC patients did not change significantly in the last 30 years with less than 8% of patients surviving 5 years. Accordingly, the annual number of deaths equals the number of newly diagnosed cancers despite maximal treatment. This contrasts other solid tumor entities such as breast and colon cancer, where death rates have decreased >30% due to technical innovations and novel insights into biological function. Due to the increasing incidence and the lack of efficient therapies, PDAC is predicted to become the second leading cause of cancer death in the next decade. Therefore, PDAC poses a major challenge in Europe and novel therapeutic approaches are urgently needed.
The overarching goal of this ERC funded research project is to gain a comprehensive mechanistic understanding of PDAC and its microenvironment, to investigate why it resists conventional and targeted therapies and to pave the way to novel individualized treatment strategies for this deadly disease.
Though 5-year mortality rates have remained stagnant for more than 30 years, recent advances provide reason for true optimism to achieve our goal. Breakthroughs in various fields of cancer research, such as genomic analysis and next-generation sequencing, high-throughput drug and genetic screening and computational approaches are providing a wealth of resources that hold the promise of advancing treatment strategies for PDAC patients. Thus, the field is primed for major advances as never before.
What has so far been lacking to take full advantage of these developments is a means of modeling and manipulating the developing tumor entity and its microenvironment in a realistic manner on a genome-wide scale in vivo. Access to these aspects of the disease is vital for identifying and assessing candidate therapeutic targets and mechanisms of resistance, as well as revealing basic principles of tumor biology. Therefore, there is an urgent need for a next generation of improved pre-clinical model systems, which will pave the way to novel therapies.
We generated such models by combining two recombination system, Flp/frt and Cre/loxP (termed dual-recombinase system - DRS). These novel models permit spatial and temporal control of gene expression and provide unparalleled access to the native biology of cancer cells and their hosting stroma, and rigorous genetic validation of candidate therapeutic targets.
In the first 2.5 years of funding, we (I) engineered and further improved our pre-clinical PDAC models and generated triple-recombination systems that allow for the first time intersectional genetic manipulation of PDAC; (II) performed cell-autonomous and non-autonomous targeting of autochthonous tumors thereby identifying novel therapeutic targets; (III) uncovered hallmarks of human catastrophic as well as multistep carcinogenesis providing a mechanistic understanding of PDAC heterogeneity; (IV) showed that immune cell function in the tumor microenvironment strictly depend on the molecular makeup of the primary tumor providing the rational for individualized immunomodulatory therapies; and (V) uncovered molecular mechanisms of primary (intrinsic) and secondary (acquired) resistance towards targeted therapies.
By the application of cutting edge genetic engineering and interfering technologies we were able to address long-standing biological questions in a rigorous manner that could not be addressed before. The project thus opened already new the horizons for the functional understanding of pancreatic cancer biology with a strong impact on cli
In the first 2.5 years of funding, we performed the following work and achieved the following major goals and results of work packages (WP) 1-3
WP 1 Development and refinement of highly versatile PDAC models
We have extensively validated our novel dual recombinase system (DRS) in vivo and demonstrated that it is a versatile and unique tool to sequentially manipulate pancreatic cancer and the host on a genome wide scale in vivo. To further improve the model, we generated a DRS using a novel knock-in line to overcome limitations of transgenic lines, such as loss of transgene activity over time.
In order generate experimental PDAC cohorts more quickly, we successfully developed novel non-genetic strategies to deliver Flp recombinase to the pancreas via plasmid based electroporation strategies as well as viral AAV based gene transfer methods for somatic gene targeting and CRISPR/Cas9 based gene engineering of the pancreas in vivo. Thereby, we achieved a major goal of the project, which is important for all other WPs.
To exploit the full potential of the DRS based PDAC model, we are in the process of systematically re-evaluating existing Cre driver lines for their capability of targeting specific subpopulations in the tumor microenvironment of PDAC. Such an unbiased, careful and rigorous re-evaluation is urgently needed because it is increasingly recognized that published data regarding fibroblast specificity of particular Cre drivers are not always reproducible. For example, we and others observed that a particular Fsp1-Cre line (S100a4-Cre (1Egn) recombines myeloid and dendritic cells. The comparative re-evaluation of lines with a potential stroma cell specific recombination pattern will generate a resource, which is of fundamental importance to investigate the contribution of different stromal cell subpopulations for the initiation, progression and maintenance of PDAC. This resource is urgently needed and demanded by the cancer research field, because of the conflicting results of stroma depletion strategies in mice and men. So far, we have analysed the recombination pattern of 15 individual stroma Cre driver lines and performed a subtyping of fibroblasts according to their recombination pattern.
To target specific cell subtypes, which are not precisely characterized by a single marker, but only by marker combinations, we validated the Dre/ROX recombination system for engineering of triple-recombinase systems (TRS) to specifically manipulate fibroblast cell subtypes or activated stellate cells, which are â€œuntargetableâ€ at the moment. We have tested various Dre versions for their ability to recombine ROX sites specifically and efficiently, and are in the process of generating the respective knock-in lines.
WP 2 Therapeutic targeting of PanIN progression and PDAC maintenance
The DRS PDAC models developed here can be used to address therapeutic questions of high clinical relevance for PDAC patients. We applied these models to genetic studies of autochthonous tumors and the microenvironment and inactivated tumor cell-autonomous and non-autonomous pathways/cells during PanIN progression and in established PDAC. We used the DRS models to 1) mechanistically uncover hallmarks of human catastrophic as well as multistep carcinogenesis, which provided us with a functional understanding of PDAC heterogeneit, which has important clinical implications and explains contect specificity (see below); 2) investigate candidate therapeutic targets in the epithelial compartment of PanINs and PDAC functionally, 3) perform synthetic lethal CRISPR/Cas-based genetic loss of function screen to identify genes which induce cell death in combination; these candidates are beeing validated at the moment. The analysis of tumor associated cells of the tumor microenvironment for PanIN progression, PDAC maintenance and metastasis formation is ongoing. Preliminary data show that the molecular makeup of the primary PDAC dictates if a cell in the tumor mic
Our new dual-recombinase-system (DRS) based PDAC models provide novel and unique opportunities to model, manipulate, investigate and understand diverse aspects of malignant tumors. This includes rigorous mechanistic analysis of i) genetic alterations in catastrophic & multi-step carcinogenesis and tumor heterogeneity, ii) tumor cell subpopulations such as cancer stem cells, iii) the tumor microenvironment, iv) immune cell subpopulations, v) the metastatic niche of host organs, vi) therapeutic targets and vii) resistance mechanisms. The next-generation models of human PDAC, which we developed in the project, are highly flexible and of ground breaking importance to the field, because they open the door to address fundamental biological and therapeutic questions at the genetic level in established tumors in vivo. Therefore, they set a new global standard for basic cancer research and drug target discovery and validation. In addition, they are of great clinical relevance because they contribute directly to the development of novel therapeutic strategies for this deadly disease. The application of cutting-edge genetic engineering and screening technologies allows us to address questions that could not be addressed before. Thereby, we open new horizons for the mechanistic understanding of pancreatic cancer biology. We expect that the results of our project until the end of the funding period will reveal several new strategies for individualized cancer therapies in PDAC patients, by targeting the tumor and its microenvironment. This will radically impact clinical management and prognosis of PDAC patients.