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RESEARCH

© dkfz.de

Our research aims to explore the vulnerabilities and driver mutations of human cancer cells, particularly in rare cancers, to find new targets for the successful implementation of pathogenesis-based cancer therapies. To achieve this goal, we are currently pursuing the following research areas and projects:

IDENTIFICATION OF SECONDARY GENE DEPENDENCIES

Somatically acquired genetic alterations lead to complex changes in intracellular signaling pathways during malignant transformation, thereby rendering cancer cells dependent on other intact genes. Such secondary dependencies can provide insights into the functions of the altered genes and may be exploited therapeutically. To identify such vulnerabilities in cancer cells, our laboratory uses functional small- and large-scale screening methods to systematically inhibit genes and study their impact on cell survival and proliferation. Specifically, we use targeted and genome-wide CRISPR/Cas9 knockout or CRISPR interference (CRISPRi) screening and other multi-omics approaches to uncover specific dependencies in different malignancies.

For example, we performed genome-wide shRNA screens in acute myeloid leukemia (AML) cell lines, which did not have any immediately targetable genetic alterations, and identified the receptor tyrosine kinase RET as a vulnerability in multiple AML subtypes. AML cells exhibited activation of RET signaling via ARTN/GFRA3 and NRTN/GFRA2 ligand/co-receptor complexes, and mTORC1-mediated suppression of autophagy and subsequent stabilization of leukemogenic drivers such as mutant FLT3 were important RET effectors. Accordingly, genetic or pharmacological RET inhibition impaired the growth of FLT3-dependent AML cell lines. Our results suggest that RET-mTORC1 signaling promotes AML by suppressing autophagy, suggesting that targeting RET or, more generally, inhibiting leukemogenic drivers by autophagy induction is a therapeutic option for a relevant subset of AML patients.

Reference: Rudat, S. et al. RET-mediated autophagy suppression as targetable co-dependence in acute myeloid leukemia. Leukemia 373, 1–14, 2018

May-Grünwald-Giemsa-stained MOLM-14 AML cells. Left: non-targeting shRNA; right: cell differentiation after LIMK1 knockdown.
© dkfz.de

Using a similar shRNA screening strategy, we identified the intracellular kinase LIMK1 as a potential therapeutic target in AML. Briefly, we found that high LIMK1 expression significantly correlated with shorter survival of AML patients and was associated with FLT3 mutations, MLL rearrangements, and increased HOX gene expression. LIMK1 (and LIMK2) inhibition affected MLL-rearranged AML cell lines and patient-derived xenografts, and induced myeloid differentiation. In addition, we observed, among other effects, a reciprocal regulation between LIMK1/2 and CDK6, a kinase that we had previously shown to be involved in the differentiation blockade of MLL-altered AML. The addition of the CDK6 inhibitor palbociclib further enhanced the antiproliferative effect of LIMK inhibition. This study suggests that LIMK1/2 might be promising targets for AML therapy.

References: Jensen, P. et al. Requirement for LIM kinases in acute myeloid leukemia. Leukemia 1–13, 2020
Placke T, et al. Requirement for CDK6 in MLL-rearranged acute myeloid leukemia. Blood 124:13-23, 2014

Tumor growth on chicken chorio-allantoic membranes of two myxoid liposarcoma cell lines following treatment with the YAP inhibitor verteporfin.
© dkfz.de

By using shRNA screening, we also identified a new driver and vulnerability in myxoid liposarcoma (MLS). MLS is a malignant tumor of adipocytic origin and is driven by the FUS-DDIT3 fusion gene, which encodes an abnormal transcription factor. We discovered a novel requirement of YAP1 in this cancer type, and demonstrated overactive YAP1 signaling as a unifying feature of MLS development. Mechanistically, FUS-DDIT3 promotes YAP1 expression, nuclear localization, and transcriptional activity and is physically associated with YAP1 in the nucleus of MLS cells. Pharmacological inhibition of YAP1 activity impairs MLS cell growth in vitro and in vivo. Therefore, YAP1 might represent a novel target for therapeutic intervention. This project was carried out together with our long-time cooperation partners Wolfgang Hartmann (Translational Pathology, University Hospital Münster) and Stefan Fröhling (NCT Heidelberg and DKFZ).

Reference: Trautmann, M. et al. Requirement for YAP1 signaling in myxoid liposarcoma. EMBO Molecular Medicine e9889-15, 2019

Ongoing projects

Dividing chordoma cell. Green: phospho-histone H3; red: ß-tubulin; blue: DNA.
© dkfz.de

CHORDOMA
One main focus of the lab is the systematic interrogation of essential genes in chordoma. Chordomas are rare but devastating malignant tumors with limited therapy options and incomplete knowledge regarding the pathophysiology of the disease. We are using targeted and large-scale CRISPR screening in human chordoma cells to identify and characterize genes, signaling proteins, and pathways that are essential for the growth of chordomas and may be exploited for the development of novel therapeutic strategies.

Funding: This project is funded by the German Cancer Aid (Deutsche Krebshilfe).

In addition, we are developing Designed Ankyrin Repeat Proteins (DARPins) for targeting brachyury in chordoma. The embryonic transcription factor brachyury is highly expressed in nearly all chordomas, but not normal adult tissues, and is required for chordoma cell survival and proliferation. Thus, brachyury represents in principle an attractive therapeutic target, but transcription factors are difficult to inhibit with small molecules. In close collaboration with Prof. Andreas Plückthun (University of Zurich), we are developing brachyury-targeting DARPins, which is a new class of small-protein therapeutics derived from natural ankyrin repeat proteins that recognize and bind their target proteins by mimicking the functional principle of antibodies.

Funding: This project is funded by the Wilhelm Sander-Stiftung.

FUNCTIONAL CHARACTERIZATION OF CANCER ALTERATIONS

Most cancers are characterized by few mutations found in tumors with high frequency. These are often well characterized, which has enabled pathogenesis-oriented therapeutic strategies that have led to remarkable therapeutic success in selected cases. However, the majority of alterations are rare or even unique, and their contribution to tumorigenesis is largely unclear, but needs to be elucidated for the successful implementation of individualized diagnosis and treatment of cancer patients.

For the systematic analysis of the tumorigenic potential of unknown cancer mutations, we established a workflow for the generation of isogenic immortalized cell lines harboring the wildtype or mutant forms of candidate genes that are then analyzed in cancer-relevant phenotypic readouts, including colony formation, EGF independence, and anchorage-independent growth. Using this approach, we contributed to a number of collaborative projects, such as the discovery of FGFR1 as driver in multiple soft tissue sarcoma subtypes, supporting FGFR1 inhibition as therapeutic option in this challenging disease (1), the integrative genomic and transcriptomic analysis of leiomyosarcoma (2), the analysis of new NRG1 fusions in KRAS wild-type pancreatic cancer (3), and the functional analysis of novel gene fusions that were detected in patient RNA sequencing data by a novel fusion detection algorithm (4).

References:
1. Chudasama, P. et al. Targeting Fibroblast Growth Factor Receptor 1 for Treatment of Soft-Tissue Sarcoma. Clin Cancer Res 23, 962–973, 2017
2. Chudasama, P. et al. Integrative genomic and transcriptomic analysis of leiomyosarcoma. Nature Communications 9, 1–15, 2018
3. Heining, C. et al. NRG1 Fusions in KRAS Wild-Type Pancreatic Cancer. Cancer Discovery 8, 1087–1095 (2018).
4. Uhrig, S. et al. Accurate and efficient detection of gene fusions from RNA sequencing data. Genome Res 31, gr.257246.119, 2021

Ongoing projects

SYSTEMATIC FUNCTIONAL ANALYSIS OF RARE MUTATIONS IN CANCER

Using our established pipeline, we systematically investigate the oncogenic function of uncharacterized rare cancer mutations, which will improve the interpretation of cancer genomes and help investigators translate genetic information into clinical application.

MULTIDIMENIONAL CHARACTERIZATION OF SOFT-TISSUE SARCOMAS WITH FUS-TFCP2 OR EWSR1-TFCP2 FUSIONS

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma subtype consisting of malignant immature progenitor cells with myogenic differentiation and is thought to originate from skeletal muscle. Our collaborators of the NCT/DKTK MASTER program (Stefan Fröhling, Christoph Heilig) identified several RMS cases with FUS or EWSR1 fused to the transcription factor TFCP2. These FUS- or EWSR1-TFCP2-positive RMS were recognized in the recently updated WHO classification (2020), but their classification, pathogenesis, and optimal treatment are unclear. We are studying their clinical, histopathological, molecular, and functional characteristics. For example, we found that all tumors showed outlier expression of the receptor tyrosine kinase ALK, which was additionally affected by intragenic deletions and aberrant splicing resulting in the expression of oncogenic ALK variants.


Funding: This project is funded by the German Cancer Aid (Deutsche Krebshilfe).

Reference (bioRxiv preprint): Schöpf J., Uhrig S., Heilig C.E. et al. Multidimensional Characterization of Soft-Tissue Sarcomas with FUS-TFCP2 or EWSR1-TFCP2 Fusions. https://doi.org/10.1101/2023.02.19.528670

SINGLE-CELL SEQUENCING OF THE REGENERATING MOUSE LUNG

UMAP embedding of single-cell RNA sequencing data from mouse lungs.
© dkfz.de

Understanding the molecular and cellular processes involved in lung epithelial regeneration may fuel the development of new therapeutic approaches for lung diseases. We combined new mouse models that allow diphtheria toxin (DTA)-mediated depletion of specific epithelial cell types and GFP-labeling of dividing cells with single-cell transcriptomics to characterize the regeneration of the distal lung. We uncovered new cell types, some of which likely represent epithelial precursors, propose goblet cells as progenitor cells, and provide evidence that adventitial fibroblasts act as supporting cells in epithelial regeneration. We also found that DTA-expressing cells can persist in the lung, express specific inflammatory factors, and resemble a previously undescribed population in the lungs of COVID-19 patients. Our study provides a comprehensive single-cell atlas of the distal lung that characterizes early transcriptional and cellular responses to defined epithelial injury, encompassing proliferation, differentiation, and cell-to-cell interactions.


Funding: German Research Foundation (DFG) as part of the Collaborative Research Center SFB873.

Reference (bioRxiv preprint): Martins et al. Cell division tracing combined with single-cell transcriptomics reveals new cell types and differentiation paths in the regenerating mouse lung. https://doi.org/10.1101/2023.01.18.524384

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