Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity

Abstract

BackgroundThe success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein ‘cytokine inducible SH2-containing protein’ (CISH) in NK cells to evaluate the impact on their functions and antitumor properties.MethodsTo further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions.ResultsIn Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions.ConclusionThis study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

CISH Expression Is Associated with Metastasis-Free Interval in Triple-Negative Breast Cancer and Refines the Prognostic Value of PDL1 Expression

“…Thus, the CISH-up/PDL1-up patients, who displayed an 81% 5-year MFI that deserves to be improved, might benefit from a dual blockade of CISH and PDL1. Indeed, several studies highlighted the inhibition of CISH as a method of unleashing the NK cell [ 18 , 19 , 40 , 41 ] and T cell [ 15 , 16 , 17 ] antitumor response, leading to increase of IFNγ production, cytotoxicity, and PDL1 expression. These in vivo data highlighted the potential of inhibiting the CISH intracellular immune checkpoint in combination with ICIs to overcome ICI resistance.…”

“…CISH deletion in NK cells increases ex-vivo proliferation, functions and signaling activation of several signaling pathways such as cytokines and Natural cytotoxicity receptors (NCR). In vivo CISH absence favors NK cell numbers to the tumor burden, optimizes their killing properties and limits NK cell exhaustion leading to primary breast cancer growth in addition to superior control of spontaneous tumor metastasis [ 19 ]. These data suggested possibilities for immunotherapies directed at blocking CISH function and their potential for improving the efficacy of ICIs in combination.…”

Applications of CRISPR Epigenome Editors in Tumor Immunology and Autoimmunity

“…This intervention resulted in a higher frequency of CD107a + TNF-α double-positive cells and reduced expression of the inhibitory receptor TIGIT, underscoring the potential of CRISPR-mediated modulation in augmenting the antitumor activity of primary human cells. 137 Another noteworthy approach involves the development of a CAR-T model known as RB-340-1, where an anti-HER2 scFv is combined with CD28 and CD3ζ costimulatory domains. To enhance its efficacy, TEV protease and a PD-1 sgRNA targeting the TSS region of the PDCD1 gene were introduced to these CAR-T cells.…”

“…Through CRISPRi targeting of the cytokine-inducible SH-2 containing protein ( CISH ) gene, researchers enhanced the antitumor response of the NK-92 cell line and primary human NK cells. This intervention resulted in a higher frequency of CD107a + TNF-α double-positive cells and reduced expression of the inhibitory receptor TIGIT, underscoring the potential of CRISPR-mediated modulation in augmenting the antitumor activity of primary human cells …”

Focusing on NK cells and ADCC: A promising immunotherapy approach in targeted therapy for HER2-positive breast cancer

“…CISH is a key negative regulator of IL-15 signaling in NK cells, and it plays a key role in the regulation of human NK cell metabolic activity and thereby modulates anti-tumor activity ( 85 ). CISH deletion favors the accumulation of NK cells in primary breast cancer, thus optimizing NK cell-killing properties, and decreases T cell immune receptors with immunoglobulin and ITIM domain (TIGIT) immune checkpoint receptor expression ( 90 ). The activation of STAT3 helps tumor cells to evade NK cell-mediated immune surveillance, which is associated with high expression of tumor-promoting cytokines and growth factors, such as IL-10, TGF-β, and vascular endothelial growth factor-A ( 91 ).…”