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Diefenbacher Lab

Experimental Immuno-Oncology

Lung cancer is the leading cause of cancer death globally, claiming 1.8 million lives in 2020. Projections show that lung cancer annual cases are expected to reach 3.8 million in 2050. This demonstrates how important it is to learn more about the development of this disease and to investigate novel therapeutic approaches.

Like all cancer researchers, we want to tear away the cloak of invisibility from tumor cells, we want to see what they do. To do this, we analyze the interaction between tumor and genetics as well as between tumor and environment: What processes take place, which proteins are involved, how are they regulated? Our visions are new, advanced models to read, control and stop or reverse these processes with the help of the immune system.

Lung cancer is the leading cause of cancer death globally, claiming 1.8 million lives in 2020. Projections show that lung cancer annual cases are expected to reach 3.8 million in 2050. This demonstrates how important it is to learn more about the development of this disease and to investigate novel therapeutic approaches.

Like all cancer researchers, we want to tear away the cloak of invisibility from tumor cells, we want to see what they do. To do this, we analyze the interaction between tumor and genetics as well as between tumor and environment: What processes take place, which proteins are involved, how are they regulated? Our visions are new, advanced models to read, control and stop or reverse these processes with the help of the immune system.

Target identification and validation

Our focus is the deregulation of protein turnover as a central driver in tumorigenesis, using state-of-the-art ex vivo and in vivo tools like CRISPR/Cas9 and patient derived Organoids.

One central mechanism that tumor cells alter during oncogenic transformation is the ubiquitin proteasome system (UPS), which is regulating virtually all biological processes, including, but not limited to DNA damage, senescence, cell cycle, DNA replication and the immune system. Despite the prominent involvement of the UPS in cancer, our understanding of how tumor cells alter the UPS system very early in transformation is rather limited.

The targeting of these common essential pathways and exploiting tumor intrinsic vulnerabilities holds the potential to develop preclinical models as well as personalized therapies not only for end- stage but also for early-stage patients.

Methods - CRISPR/Cas9

We utilize and combine novel techniques like CRISPR/Cas9 for tumor onset and develop isogenic transplant models. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before.

We employ these models to study the early consequences of oncogenic transformation in the lung, and how cells, in combination with expression of different tumor drivers, reshape their niche and respond to standard of care therapy. These new models will provide a foundation for understanding lung cancer formation in molecular detail, from onset to late-stage disease.

Picture left:

Large lesions represent lung tumours caused by somatic engineering of KrasG12D and TP53mutant by CRISPR in adult lung.
Green: Deubiquitylating enzyme overrepresented in tumours
Red: Marker for club cells

Methods - Organoids

Another focus of our Lab is to culture primary murine and human lung epithelial and primary tumor cells as spheroids or ‘so called’ organoids. In the last years, we have established a novel protocol which simplifies the culture conditions and enabled us to cultivate not only primary murine cells derived from the trachea and alveolar compartment, but also patient material derived primary organoids from resected material.

Apart from genetically tailored murine respiratory organoid models, we culture and propagate patient organoids, derived from resection material. Combining genetically modified organoids with patient organoids, together with the corresponding control organoids, allows us the testing of novel therapeutic strategies for the treatment of all lung cancer variations.

Methods - Adeno-associated viruses (AAV)

As vehicle to facilitate gene transfer into recipient animals or patient samples do we use adeno-associated viruses, or short AAVs. These non-integrating, non-pathologic vectors carry as a payload all genetic components for e.g. CRISPR mediated gene editing, including the homology repair template or sgRNA sequences. This allows us to ‘just’ clone our genotypes of interest, in the case of modelling human disease, or express shRNA or cDNA variants of disease-causing genes. We established a simplified production protocol, hence, allowing to perform all steps required form design to production on site, making it the go-to tool for gene delivery in our model systems and beyond.  

Scientists at Diefenbacher Lab

Bohnacker_Bianka_Portrait

Bianka Bohnacker

PhD Student
Chowdhury_Niti_Portrait

Niti Chowdhury

Master Student
Hartmann_Oliver_LHI_Portrait

Dr. Oliver Hartmann

Postdoc
Pahor_Nikolett_LHI_Portrait

Nikolett Pahor

PhD Student
Shukla_Kimaya_Portrait

Kimaya Shukla

Master Student
Portrait Diana Steinhart LHI

Diana Steinhart

Technical Assistant
Ünal_Ceren_Yagmur_Portrait

Yağmur Ceren Ünal

PhD Student
Von_Heyl_zu_Herrnsheim_Viktoria_Portrait

Viktoria von Heyl zu Herrnsheim

Master Student

Publications

2024, Scientific Article in Nature Communications

LungVis 1.0: An automatic AI-powered 3D imaging ecosystem unveils spatial profiling of nanoparticle delivery and acinar migration of lung macrophages.

Targeted (nano-)drug delivery is essential for treating respiratory diseases, which are often confined to distinct lung regions. However, spatio-temporal profiling of drugs or nanoparticles (NPs) and their interactions with lung macrophages remains unresolved. Here, we present LungVis 1.0, an AI-powered imaging ecosystem that integrates light sheet fluorescence microscopy with deep learning-based image analysis pipelines to map NP deposition and dosage holistically and quantitatively across bronchial and alveolar (acinar) regions in murine lungs for widely-used bulk-liquid and aerosol-based delivery methods. We demonstrate that bulk-liquid delivery results in patchy NP distribution with elevated bronchial doses, whereas aerosols achieve uniform deposition reaching distal alveoli. Furthermore, we reveal that lung tissue-resident macrophages (TRMs) are dynamic, actively patrolling and redistributing NPs within alveoli, contesting the conventional paradigm of TRMs as static entities. LungVis 1.0 provides an advanced framework for exploring pulmonary delivery dynamics and deepening insights into TRM-mediated lung immunity.

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2024, Scientific Article in Oncogene

USP10 drives cancer stemness and enables super-competitor signalling in colorectal cancer.

The contribution of deubiquitylating enzymes (DUBs) to β-Catenin stabilization in intestinal stem cells and colorectal cancer (CRC) is poorly understood. Here, and by using an unbiassed screen, we discovered that the DUB USP10 stabilizes β-Catenin specifically in APC-truncated CRC in vitro and in vivo. Mechanistic studies, including in vitro binding together with computational modelling, revealed that USP10 binding to β-Catenin is mediated via the unstructured N-terminus of USP10 and is outcompeted by intact APC, favouring β-catenin degradation. However, in APC-truncated cancer cells USP10 binds to β-catenin, increasing its stability which is critical for maintaining an undifferentiated tumour identity. Elimination of USP10 reduces the expression of WNT and stem cell signatures and induces the expression of differentiation genes. Remarkably, silencing of USP10 in murine and patient-derived CRC organoids established that it is essential for NOTUM signalling and the APC super competitor-phenotype, reducing tumorigenic properties of APC-truncated CRC. These findings are clinically relevant as patient-derived organoids are highly dependent on USP10, and abundance of USP10 correlates with poorer prognosis of CRC patients. Our findings reveal, therefore, a role for USP10 in CRC cell identity, stemness, and tumorigenic growth by stabilising β-Catenin, leading to aberrant WNT signalling and degradation resistant tumours. Thus, USP10 emerges as a unique therapeutic target in APC truncated CRC.

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2024, Scientific Article in Molecular Carcinogenesis

Metabolism-focused CRISPR screen unveils mitochondrial pyruvate carrier 1 as a critical driver for PARP inhibitor resistance in lung cancer.

Homologous recombination (HR) and poly ADP-ribosylation are partially redundant pathways for the repair of DNA damage in normal and cancer cells. In cell lines that are deficient in HR, inhibition of poly (ADP-ribose) polymerase (poly (ADP-ribose) polymerase [PARP]1/2) is a proven target with several PARP inhibitors (PARPis) currently in clinical use. Resistance to PARPi often develops, usually involving genetic alterations in DNA repair signaling cascades, but also metabolic rewiring particularly in HR-proficient cells. We surmised that alterations in metabolic pathways by cancer drugs such as Olaparib might be involved in the development of resistance to drug therapy. To test this hypothesis, we conducted a metabolism-focused clustered regularly interspaced short palindromic repeats knockout screen to identify genes that undergo alterations during the treatment of tumor cells with PARPis. Of about 3000 genes in the screen, our data revealed that mitochondrial pyruvate carrier 1 (MPC1) is an essential factor in desensitizing nonsmall cell lung cancer (NSCLC) lung cancer lines to PARP inhibition. In contrast to NSCLC lung cancer cells, triple-negative breast cancer cells do not exhibit such desensitization following MPC1 loss and reprogram the tricarboxylic acid cycle and oxidative phosphorylation pathways to overcome PARPi treatment. Our findings unveil a previously unknown synergistic response between MPC1 loss and PARP inhibition in lung cancer cells.

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2024, Scientific Article in Apoptosis

ARTS and small-molecule ARTS mimetics upregulate p53 levels by promoting the degradation of XIAP.

Mutations resulting in decreased activity of p53 tumor suppressor protein promote tumorigenesis. P53 protein levels are tightly regulated through the Ubiquitin Proteasome System (UPS). Several E3 ligases were shown to regulate p53 stability, including MDM2. Here we report that the ubiquitin E3 ligase XIAP (X-linked Inhibitors of Apoptosis) is a direct ligase for p53 and describe a novel approach for modulating the levels of p53 by targeting the XIAP pathway. Using in vivo (live-cell) and in vitro (cell-free reconstituted system) ubiquitylation assays, we show that the XIAP-antagonist ARTS regulates the levels of p53 by promoting the degradation of XIAP. XIAP directly binds and ubiquitylates p53. In apoptotic cells, ARTS inhibits the ubiquitylation of p53 by antagonizing XIAP. XIAP knockout MEFs express higher p53 protein levels compared to wild-type MEFs. Computational screen for small molecules with high affinity to the ARTS-binding site within XIAP identified a small-molecule ARTS-mimetic, B3. This compound stimulates apoptosis in a wide range of cancer cells but not normal PBMC (Peripheral Blood Mononuclear Cells). Like ARTS, the B3 compound binds to XIAP and promotes its degradation via the UPS. B3 binding to XIAP stabilizes p53 by disrupting its interaction with XIAP. These results reveal a novel mechanism by which ARTS and p53 regulate each other through an amplification loop to promote apoptosis. Finally, these data suggest that targeting the ARTS binding pocket in XIAP can be used to increase p53 levels as a new strategy for developing anti-cancer therapeutics.

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2024, Scientific Article in Gut (eGut)

Targeting MYC effector functions in pancreatic cancer by inhibiting the ATPase RUVBL1/2.

OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.

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Contact

Diefenbacher_Markus_LHI_Portrait

Prof. Dr. Markus Diefenbacher

Group Leader