SETDB1 suppresses interferon responses and NK cell-mediated immunosurveillance specifically in monocytic AML: CRISPR/Cas9 screens on cSAM leukemia cells

Monocytic acute myeloid leukemia (AML) responds poorly to current treatments, including venetoclax-based therapy. We conducted in vivo and in vitro CRISPR/Cas9 library screenings using a mouse monocytic AML model, and identified SETDB1 and its binding partners (ATF7IP and TRIM33) as crucial tumor promoters in vivo. The growth-inhibitory effect of Setdb1 depletion in vivo was mainly dependent on NK cell-mediated cytotoxicity. Mechanistically, SETDB1 depletion upregulated interferon-stimulated genes and NKG2D ligands through demethylation of histone H3 Lys9 at the monocyte-specific enhancer regions, thereby enhancing their immunogenicity to NK cells and intrinsic apoptosis. Importantly, these effects were not observed in non-monocytic leukemia cells. We also identified the expression of MNDA and its murine counterpart Ifi203 as biomarkers to predict the sensitivity of each AML to SETDB1 depletion. Our study highlights the critical and selective role of SETDB1 in monocytic AML and underscores its potential as a therapeutic target for current unmet needs. To identify epigenetic regulators that play essential roles in vivo, we performed CRISPR/Cas9 library screens using mouse AML cells transformed by SETBP1 and ASXL1 mutations (cSAM: combined expression of SETBP1 and ASXL1 Mutations12) and a pooled single guide RNA (sgRNA) library targeting 640 epigenetic genes (3 sgRNAs per gene). The amplification and virus production were performed by the Broad institute using standard protocols (https://portals.broadinstitute.org/gpp/public/resources/protocols). Viruses were tittered and optimal virus concentrations allowing for 10 - 20% infection were used. 1E07 cSAM cells were transduced with the sgRNA library. After 24 hours, puromycin (1 µg/ml) was added for selection. After 2 days, 20 million cells were frozen at −80 °C for genomic DNA extraction and deep-sequencing. The remaining cells were transplanted into sublethally irradiated (5.25 Gy) C57BL/6 mice (2 or 5E06 cells per mouse) or cultured in 6-well plates (2E06 cells per well). For the in vivo transplantation assay, bone marrow cells were harvested from the moribund leukemic mice, and GFP+ leukemia cells were sorted using BD FACSAriaIII (BD biosciences). For the in vitro culture, cSAM cells were passaged every 2 or 3 days, over 14-day incubation period. DNA was extracted from each sample using the Blood and Cell Culture DNA Maxi kit (QIAGEN) according to the manufacturer’s instructions.