DDX6 undergoes phase separation to modulate metabolic plasticity and drug resistance in AML [CRISPR Screen]

Chemoresistance in acute myeloid leukemia (AML) is a major cause of poor prognosis and frequent relapse among patients. Under stress conditions such as chemotherapy or radiotherapy, processing bodies (PBs) and stress granules (SGs) can sequester essential mRNAs and proteins to modulate leukemia cell survival. However, the precise role(s) of PBs and SGs in AML pathogenesis and chemotherapy response remains unclear. To systematically identify PB/SG-associated genes that are critical for AML survival, we selected 101 genes encoding RNA-binding proteins that are localized within PBs and SGs, and constructed a customized CRISPR library targeting these genes for both in vitro and in vivo CRISPR screening with AML cell line (Mono-Mac-6) and patient-derived xenograft (PDX) cells. Overall design: In the pilot experiment, the infection efficiency of 2,084 gRNAs was maintained at 0.2–0.3. Subsequently, the CRISPR library (containing 2,084 gRNAs) was introduced into Cas9-expressing Mono-mac-6 AML monoclonal cells and patient-derived xenograft (PDX) AML cells via lentiviral transduction, ensuring an infection efficiency of 0.2–0.3. Transduced AML cells were then selected with blasticidin (10 µg/mL) and puromycin (Mono-mac-6: 0.5 µg/mL; PDX-1: 16 µg/mL) for 3–5 days to obtain positively transduced populations. Cells collected after selection were designated as day 0 (baseline) samples. For the in vitro screening, both Mono-mac-6 and PDX cells were used (three biological replicates, 2 × 106 cells per replicate). For the in vivo screening, PDX cells were used (six biological replicates, 2 × 106 cells per replicate). For the in vivo CRISPR screen, PDX cells were injected into immunodeficient mice. After 28 days, bone marrow cells were harvested, and human CD45? PDX cells were isolated using magnetic bead sorting as endpoint samples for sequencing (six replicates, 2 × 106 cells per replicate). For the in vitro CRISPR screen, both Mono-mac-6 and PDX cells were continuously cultured for 28 days (maintaining =10,000-fold total cell expansion), after which cells were collected as endpoint samples for sequencing (three replicates per group, 2 × 106 cells per replicate).