Development of a collection of cell lines from a genetically engineered mouse model of DICER1 syndrome-associated sarcoma for therapeutic assessment

Background: DICER1 syndrome, driven by loss-of-function germline variants in the DICER1 gene, predisposes children and young adults to rare cancers in multiple organs such as the brain, thyroid, lung, kidney, and gynecologic tract. Most of these tumors are sarcomas, which share common histologic and molecular features regardless of their anatomic origins. A second hit impairing DICER1 RNase IIIb occurs in nearly all these cancers. We have recently developed two genetically engineered mouse models (GEMMs) of DICER1 syndrome-associated sarcomas, which fully recapitulates the human counterpart genetically and histologically. Here, we aim to develop a panel of cell lines from the GEMMs of DICER1 syndrome-associated sarcomas and utilize them for developing novel therapeutic strategies. Methods: Mouse DICER1 syndrome-associated sarcoma cell lines are derived from tumors growing in Hic1 CreERT2 ;Dicer1 fl/fl-D1693N ;(Rosa26) LSL-tdTomato (HDT) mice. Whole-exome sequencing was performed on HDT cell lines. IVADo regimen (ifosfamide/cyclophosphamide, vincristine, actinomycin-D, doxorubicin) and CDK4/6 inhibitor were selected to treat the cells. MTT assays were performed to assess cell viability. Metaphase chromosome analysis was used to detect chromosomal abnormalities. Results: We validated that HDT tumors predominantly express the Dicer1 IIIb -mutant transcript and most tumors were quadriphasic (epithelial, undifferentiated blastema, stromal cells, and rhabdomyoblastic cells) with no to variable amounts of anaplastic sarcoma components. From these murine HDT tumors, we have successfully derived five HDT cell lines (HDT298, HDT340, HDT366, HDT546, and HDT1003 cell lines). Some of these cell lines have aberrant p53 expression (or p53 mutation) and Kras mutation, confirming their resemblance to human DICER1-sarcoma. Furthermore, we tested the response of HDT cell lines to the chemotherapeutic agents of the IVADo regimen and found that they are sensitive to vincristine, actinomycin-D, and doxorubicin. Next, we test potential therapeutic agents on these cell lines. Considering that the KRAS-mutant lung cancer cells have been reported to be hypersensitive to CDK4/6 inhibitor, we tested palbociclib sensitivity and found that only one out of two Kras-mutant cell lines was sensitive to palbociclib. Additionally, we identified that these HDT cell lines have an average chromosome number of 73, indicating the presence of chromosomal instability (CIN). Supporting this, our GEMM DICER1 sarcoma cohort has nearly ubiquitous CIN. We are currently investigating the underlying mechanism and testing whether it can be exploited for therapeutic targeting. Relevance: Our DICER1-sarcoma GEMM and the derived tumor cell lines empower us to study the biology of DICER1 syndrome-associated sarcoma and inform future development of therapeutic strategies.