Whole-Genome CRISPR Screen Reveals the Mechanism of Relapse in Patient-Derived Cells Representing High-Risk Paediatric ALL
Authors: Szoltysek Katarzyna , Blair Helen , Nakjang Sirintra , Tirtakusuma Ricky , Singh Mankaran , Nelson Ryan , McNeill Hesta , Dodd Catherine , Beckett Melanie , McKenzie Lynsey , Law Edward , Vormoor Josef , Pal Deepali , Heidenreich Olaf 
Published: November 2019

Acute lymphoblastic leukemia (ALL) is the most common type of childhood leukaemia. Recently improved risk stratification resulted in therapy optimization and extended survival for the majority of cases. Unfortunately, there is still a significant number of patients either relapsing or not responding to treatment with response to glucocorticoids being one of the most important prognostic indicators of treatment outcome.
In order to investigate the mechanism of dexamethasone resistance, we performed genome-wide CRISPR screens in patient derived xenotransplant (PDX) material from t(17;19)-positive ALL. Primary material was obtained from the patient at the presentation and at relapse stage of disease and corresponding PDX samples were generated in immunocompromised NSG mice. PDX cells were lentivirally transduced with the CRISPR knockout pooled 'Brunello' library and then subjected to dexamethasone pressure both ex vivo and in vivo. For the in vivo screen, CRISPR-modified cells were intrafemorally injected into immunodeficient NSG mice followed by either 7.5mg/kg dexamethasone or vehicle treatment. In parallel, PDXs were co-cultured with mesenchymal and endothelial-like human stromal cells generated from human bone marrow-derived iPSCs.
Data analysis performed with the MAGeCKFlute software identified the glucocorticoid receptor gene NR3C1 as a main driver of chemoresistance-mediated relapse in this high-risk ALL. Notably, a homozygous deletion of NR3C1 was present in the relapse PDX sample. Furthermore, we identified that loss of the NR3C1 gene in those cells was associated with an inferior engraftment potential in the absence of dexamethasone.
Interestingly, the whole-genome CRISPR screen in the relapse sample identified BCL2 and several genes associated with the mTOR pathway as crucial for leukaemic propagation. Knockout of NR3C1 in the diagnostic PDX also established dexamethasone resistance and further enhanced the already significant sensitivity towards mTOR inhibitors. To explore a potential synergism between BCL2 and mTOR inhibition, we assessed the effect of the BCL2 inhibitor ABT-199 and several mTOR inhibitors in both presentation and relapse PDX samples. PDX samples were co-cultured with MSCs and treated with drug combinations in a matrix format for 96 hrs followed by high-throughput fluorescence microscopy-based analysis. These experiments revealed substantial synergism of ABT-199 and mTOR inhibitors associated with increased cell death and prolonged growth inhibition in both presentation and relapse samples.
In conclusion, our studies (i) demonstrate that genome-wide CRISPR screens are feasible in PDX material both ex vivo and in vivo, (ii) provide an explanation for the relative rarity of NR3C1 mutations in relapsed material and (iii) identify drug combinations effective in both diagnostic and relapse PDX for further preclinical evaluation.

Disclosures
Vormoor: Abbvie (uncompensated): Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding; AstraZeneca: Research Funding.