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Ultra Sensitive Proteomics and CRISPR-CAS9 Gene Editing in PDX Models in VIVO Identify an Essential Function of ADAM10 for Acute Leukemias
Irmela Jeremias1, Jan Philipp Schmid1, Ehsan Bahrami1, Vindi Jurinovic1, Martin Becker1, Diana Amend1, Katharina Hunt1, Ruppert Öllinger2, Roland Rad2, Matthias Mann3, Binje Vick1, Tobias Herold1, Ashok Kumar Jayavelu3
1 Research Unit Apoptosis in Hematopoietic Stem Cells, Helmholtz Munich (HMGU), Munich, Germany;
2 Institute of Molecular Oncology and Functional Genomics, Technische Universität München, Munich, Germany;
3 Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Munich, Germany
Backgound: Tumor-microenvironment interactions are essential for the maintenance of leukemia, and their disruption represents an attractive therapeutic strategy. We developed a CRISPR-Cas9 screening approach for functional analysis of surface molecules in patient-derived xenograft (PDX) models of acute leukemias (AL) in vivo.
Aim: We aimed to identify leukemia surface molecules required for tumor growth in vivo.
Methods: Ultra sensitive proteomics was performed comparing cycling with resting PDX ALL cells. For molecular insights in PDX cells in vivo, a split version of Cas9 fused to a fluorochrome was stably expressed in PDX models of patients with ALL or AML. An sgRNA library of surface molecules was lentivirally transduced and PDX cells transplanted into NSG mice. After in vivo growth, recovered cells were analyzed for sgRNA depletion using next generation sequencing and data analysis using the MAGeCK algorithm. Hits were functionally validated by competitive in vivo assays. PDX cells with ADAM10 Knockout (KO) or treated with different ADAM10 inhibitors were characterized for in vivo engraftment, frequency of leukemic stem cells using limiting dilution transplantation assay (LDTA) or for sensitivity towards routine chemotherapy by in vivo competitive chemotherapy trials. To determine the role of single ADAM10 protein domains, rescue assays were performed in PDX cells in vivo.
Results: A customized CRISPR-Cas9 screen was performed targeting roughly 100 different surface molecules in two AL PDX samples in vivo and candidate hits validated using a competitive molecular in vivo approach testing the PDX cells with and without KO in the same mouse. These experiments confirmed essential functions of two well-described candidates CXCR4 and ITGB1. ADAM10 was depleted in both PDX models. In vivo competitive experiments affirmed its essential role in 6 PDX models of individual patients with either acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML). This indicates a broad ADAM10 dependency of AL PDX cells, regardless of the underlying oncogenic-driver mutations and chromosomal aberrations. Treating PDX cells with ADAM10 inhibitor significantly reduced the engraftment capacity into the bone marrow (BM), emphasizing the importance of ADAM10 during early engraftment. KO of ADAM10 reduced the frequency of leukemia stem cells (LSCs) and ADAM10 KO PDX samples of both lineages showed increased sensitivity towards routine chemotherapy treatments. Reconstitution of ADAM10 KO PDX cells with a recombinant wildtype variant in vivo rescued the phenotype, while an enzymatic domain lacking variant did not, highlighting the importance of ADAM10’s sheddase function.
Conclusion: We established a CRISPR-Cas9 drop-out screening pipeline in PDX models in vivo as a technology to decipher patient-specific tumor dependencies. Our data revealed a yet unknown role of ADAM10 to maintain patient leukemic cells in vivo. Targeting the interaction between leukemia cells and their tumor microenvironment by inhibition of ADAM10 represents an attractive future therapeutic strategy for the treatment of acute leukemias.
The work was funded by Consolidator Grant (CoG) #681524 from the European Research Council (ERC).
Speakers
Irmela Jeremias