A forward genetic screen reveals novel independent regulators of ULBP1, an activating ligand for natural killer cells

Abstract

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.DOI: http://dx.doi.org/10.7554/eLife.08474.001

Immunosurveillance and immunotherapy of tumors by innate immune cells

“…The RNA-binding protein RBM4 supports ULBP1 expression by facilitating proper splicing of the first two exons of the primary transcript [62]. …”

Dysregulated cellular functions and cell stress pathways provide critical cues for activating and targeting natural killer cells to transformed and infected cells

“…The ATF4 mutant versions of each cell line failed to respond to these stressors, proving the role of ATF4. Finally, ChIP‐seq experiments showed that ATF4 binds directly to the ULBP1 promoter, and luciferase reporter experiments showed that ATF4 transactivates the ULBP1 promoter directly . Taken together, these results suggested that tumor cells are subject to varying levels of stress that activates the ISR and the ATF4 transcription factor, resulting in significant induction of ULBP1 expression.…”

“…These findings suggested that ATF4 is constitutively activated to varying extents in different cell lines, and that it induces ULBP1 expression even when no purposeful stress was applied to the cells. When wildtype cells were further exposed to agents that impart ER stress or result in amino acid starvation, ULBP1 expression increased dramatically in all three cell lines, by 5‐10‐fold . The ATF4 mutant versions of each cell line failed to respond to these stressors, proving the role of ATF4.…”

“…To identify the pathways that support ULBP1 expression, a library of HAP‐1 cells that had been infected with the retroviral promoter trap vector to knock out genes at random was selected for cells that expressed ULBP1 poorly at the cell surface. High‐throughput sequencing of the selected cells, in comparison to unselected cells, revealed a number of genes with retroviral insertions that were enriched in the selected population, including the ATF4 gene …”

“…The screen in HAP‐1 cells for cells with low expression of ULBP1 revealed a second regulator of interest: RBM4 . RBM4 regulates RNA processing and (in a different context) translation of preformed mRNAs .…”