Abstract 646: CRISPR/Cas9 screening to identify mechanisms of azacitidine failure in myelodysplastic syndrome

Abstract

Abstract Myelodysplastic syndrome (MDS) is a heterogenous myeloid lineage malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of secondary transformation to acute myeloid leukemia (sAML). The mainstay of treatment in patients with higher-risk MDS are the hypomethylating agents (HMAs) azacitidine and decitabine, which extend overall survival by approximately 12 to 18 months. Unfortunately, disease relapse and HMA resistance are inevitable and carry a very poor prognosis. Genomic sequencing of large MDS cohorts has led to the identification of recurrent genetic abnormalities in MDS, some of which predict sensitivity to HMA therapy. For example, abnormalities in TET2, but not ASXL1, are associated with a favorable response to the HMAs. However, it is unknown why some patients with otherwise similar clinical features have variable responses to HMA therapy. We hypothesize that there are common cellular mechanisms promoting resistance to HMA therapy and that these can be therapeutically targeted to enhance treatment responses. To uncover cellular processes promoting HMA treatment failure we performed duplicate genome-wide CRISPR/Cas9 screen in the human MDS-L cell line using resistance to azacitidine as a positive selection readout. MDS-L was established from bone marrow mononuclear cells in a 52-year-old male patient with high-risk MDS. We transduced the Brunello sgRNA CRISPR library into Cas9 expressing MDS-L cells and subjected the cells to azacitidine treatment for 7 days. Cells surviving azacitidine treatment were then expanded and harvested for genomic DNA. High throughput sequencing of the barcoded DNA produced raw reads that were analyzed using the PinAPL-Py web-based software. sgRNAs appearing in duplicate with over 10-fold enrichment compared to the control library were considered significant. We identified 125 candidate genes involved in HMA resistance from the CRISPR-Cas9 screen. Pathway analysis revealed a significant enrichment of genes involved in lipid metabolism, DNA methylation, and response to small molecule catabolism. Future experiments to validate these candidates as well as test whether they contribute in vivo to HMA resistance are underway. We conclude genome-wide CRISPR-Cas9 screens for drug-resistance in the MDS-L model are feasible and may uncover future therapeutic targets to augment HMA therapy or utilize upon HMA failure. Citation Format: Michael Schieber, John D. Crispino. CRISPR/Cas9 screening to identify mechanisms of azacitidine failure in myelodysplastic syndrome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 646.