In Vivo CRISPR-Cas9 Screens in PDX Models Reveals ADAM10 As Novel Therapeutic Target in Acute Leukemia

Acute leukemias require more accurate and effective treatments, especially upon disease relapse. In search for novel therapeutic targets for acute leukemias, we established a pipeline for CRISPR-Cas9 mediated functional genomic screens which harbor the ability to elegantly increase our knowledge about vulnerabilities and gene dependencies. For a highly patient-related setting, we performed CRISPR knockout (KO) dropout screens in patient-derived xenograft (PDX) models in vivo, combining the advantages of studying an individual patient´s tumor cell in the physiologic in vivo bone marrow microenvironment.

Serially transplantable PDX models were lentivirally transduced to stably express Cas9. A customized CRISPR-Cas9 library targeting about 100 genes addressing surface molecules was designed, cloned and transduced into two PDX models of acute lymphoblastic leukemia (ALL). Enriched PDX ALL cells were transplanted into NSG mice and grown until advanced disease stage. Input versus end stage cells were subjected to next generation sequencing, followed by data analysis using MAGeCK algorithm. Data analysis revealed commonly depleted as well as sample-specific depleted genes between the two PDX models tested, and CXCR4 and ITGB1 were the top commonly depleted genes from the screens.

For target validation, single sgRNA were cloned into the knockout vector; concomitant expression of recombinant fluorochromes allowed competitive growth assays in PDX models in vivo, comparing cells with KO of interest versus control KO in the same animal. In vivo competitive assay showed that both PDX models clearly depended on both CXCR4 and ITGB1, validating an essential function for both genes in the two PDX ALL models.

Of note, disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) was among the list of dropout genes in the screens. ADAM10 is known for its role in the central nervous system and considered as a therapeutic target in Alzheimer disease, but poorly studied in the context of leukemia. In competitive validation assays in vivo, ADAM10 KO population showed a clear growth disadvantage compared to control KO cells in a number of PDX models of ALL, but also acute myeloid leukemia (AML); in some PDX models, ADAM10 KO cells were completely abrogated, indicating that ADAM10 plays an essential role for different types of acute leukemias and represents a yet unknown vulnerability.

To better characterize the role of ADAM10 for the clinical situation, we performed further in vivo assays with PDX models. Re-expression of ADAM10 in ADAM10 KO PDX cells could partially rescue the phenotype in an in vivo competitive reconstitution assay, unequivocally proving ADAM10 essentiality in ALL cells. Interestingly, a similar rescue assay expressing a ADAM10 variant lacking the disintegrin domain resulted in the same phenotypical compensation, highlighting essentiality of the enzymatic but not adhesion domain of ADAM10 in tumor engraftment and growth in BM. Important for translating the molecular insights into clinical use, PDX cells treated with an ADAM10 chemical inhibitor ex vivo, showed reduced tumor engraftment capacity compared to the vehicle treated cells, suggesting a role for ADAM10 in tumor-niche interactions and homing to the bone marrow. Further, we performed limiting dilution transplantation assays to determine stem cell frequencies; ADAM10 loss resulted in reduced stemness and a reduced number of leukemia-initiating cells compared to control KO cells, indicating that ADAM10 is essential also in leukemia stem cells. Of clinical relevance, ADAM10 KO significantly sensitized leukemic cells towards treatment of mice with the routine chemotherapeutic drug Cyclophosphamide in vivo, suggesting a putative synergistic effect when addressing ADAM10 as a therapeutic target.

In summary, our data revealed ADAM10 as attractive novel vulnerability in acute leukemias with essential function for the tumor-niche interaction, leukemia stem cells and anti-leukemia treatment. ADAM10 might be addressed as therapeutic target to treat acute leukemias in the future.