Clec12a is required for the pathogenesis of NUP98::NSD1 AML

NUP98-NSD1 is one of the most recurring nucleoporin 98 (NUP98) fusions in acute myeloid leukemia (AML). NUP98-NSD1 AML is associated with adverse outcomes and poor response to conventional treatments. However, limited studies have been done to identify new potential targets to develop better treatment approaches. The C-type lectin domain family 12, member A (CLEC12A) is a cell surface receptor that is differentially expressed in leukemic stem cells (LSCs) compared to healthy hematopoietic stem cells (HSCs). We demonstrated a strong overexpression of CLEC12A in both NUP98-NSD1 patients and NUP98-NSD1 transformed murine cells. To understand the role of Clec12a in NUP98-NSD1 AML, we depleted Clec12a expression in NUP98-NSD1+NRASG12D immortalized cells using 3 different sgRNAs targeting 3 different exons. We observed an efficient gene editing in all target sites. NUP98-NSD1+NRASG12D/Clec12a knockout cells showed higher apoptosis and lower colony numbers in vitro compared to NUP98-NSD1+NRASG12D/Clec12a wildtype cells. Importantly, the deletion of Clec12a significantly reduced the leukemic engraftment and prolonged the survival of the NUP98-NSD1+NRASG12D murine model. Our data suggest to further explore CLEC12A as a potential target for the treatment of NUP98-NSD1 AML. To deplete the expression of Clec12a from NUP98-NSD1+ NRASG12D immortalized murine bone marrow cells, we designed 3 distinct sgRNAs against Clec12a. We cloned these sgRNAs into the Cas9-expressing lentiviral vector L40C-CRISPR.EFS.dTomato, and the dTomato reporter was used to select sgRNA transduced cells. A sgRNA against the lacz gene was used as transduction control. For gene expression profiling, we used the Clec12a T44 sgRNA and the lacZ control. Following transduction, single-cell sorting was performed in triplicate. Once the cells expanded, total RNA was isolated and sent for sequencing.