CRISPRi Screen Identifies INSTAR as a Novel Growth Suppressor in THP-1 Cells

Monocytes and macrophages are central to the innate immune response, acting as primary responders to pathogens. Their proliferation must be tightly controlled, as dysregulation can lead to diseases such as acute myeloid leukemia (AML), which comprises ~25% of pediatric leukemia cases. While most research in AML has focused on protein-coding genes, the disease's heterogeneity suggests involvement of additional regulatory elements. Long noncoding RNAs (lncRNAs) are highly cell-type specific and have emerged as promising biomarkers and regulators in cancer. However, fewer than 3% of validated lncRNAs have known functions. To uncover functional lncRNAs in monocyte biology, we performed a CRISPR interference (CRISPRi) dropout screen targeting over 2,000 lncRNAs in THP-1 monocytic cells. Sixteen candidate growth suppressors were identified, with INSTAR (Intergenic Nuclear Suppressor lncRNA Targeting Adjacent Regulator SFMBT2) emerging as the top hit. Functional validation revealed that INSTAR knockdown promotes monocyte proliferation. RNA-seq analysis demonstrated widespread transcriptional changes affecting genes involved in cell proliferation, development, and metabolism. Among neighboring genes within a 2Mb range of INSTAR, only SFMBT2 was significantly downregulated upon INSTAR knockdown. SFMBT2 encodes a paternally imprinted polycomb group (PcG) protein previously shown to suppress tumor metastasis in prostate cancer. Mechanistic studies suggest that INSTAR acts in cis to regulate SFMBT2 expression in the nucleus of THP-1 cells. This work highlights the power of high-throughput CRISPR screening for functional lncRNA discovery and underscores the importance of lncRNA loci in controlling monocyte proliferation. Our findings contribute to a deeper understanding of monocyte regulation with a future goal of informing novel therapeutic strategies for AML and other myeloid disorders. Overall design: For our candidate RNA-sequencing experiments we cloned the top 3 performing guides from our THP1 CRISPRi dropout screen (doi.org/10.1016/j.jbc.2025.110204) and created three negative controls cell lines, 3 CRISPRi-INSTAR cell lines, 3 CRISPRi-STARD7-AS1 cell lines, and 3 CRISPRi-SNHG17 cell lines. RNA samples were collected after 1 week of lentiviral infection and subsequent puromycin selection.