Cancer

Interference identifies immune modulators

A broad in vivo screen of the effects of specific gene inhibition on the antitumour activity of immune cells in mice bearing melanomas has revealed potential targets for cancer therapy. See Article p.52

Therapies designed to boost the immune system's response to tumours hold great promise for overcoming drug resistance in cancer. Advanced solid tumours inevitably develop resistance against currently available cytotoxic or molecularly targeted therapies, but durable responses have been observed following some immunotherapeutic treatments, leading to speculation that a cure for some patients could be possible. On page 52 of this issue, Zhou et al.1 use RNA-interference technology to identify genes that can be targeted to enhance the robustness and proliferation of immune cells called CD8+ T cells in mice bearing melanomas.

Two existing targets for cancer immunotherapy are the receptor molecules CTLA-4 and PD-1, which are expressed on the surface of T cells and transmit signals that dampen the cells' immune activity. Antibodies that bind these receptors, and thereby relieve the immune inhibition, have emerged as a powerful treatment for patients with advanced melanoma2,3, and the anti-CTLA-4 antibody ipilimumab was the first immunotherapy shown to significantly improve the overall survival of these patients4. However, patient responses to tumour immunotherapy are highly variable, and it is unclear why some tumours respond and others do not. Thus, to improve the efficiency of such treatments further, a deeper understanding and better mechanistic characterization of antitumour immune responses are needed.

The discovery of RNA interference (RNAi) — the process by which small RNA molecules specifically inhibit gene expression by binding to and inducing the cleavage of messenger RNAs — has revolutionized loss-of-function genetic studies. The experimental application of RNAi, using collections of short-interfering RNA (siRNA) or short hairpin RNA (shRNA) molecules, means that screens of gene function can be conducted in almost every biological system. For example, shRNA screens have been used successfully to dissect tumour-suppressor gene networks, to identify modulators of drug resistance and to pinpoint vulnerabilities in cancer cells5,6,7,8. Furthermore, in vitro shRNA screening was recently applied to identify genes that regulate the differentiation of T cells into T-helper 1 and 2 subsets9. However, RNAi-based functional genetic screens are commonly performed in vitro, and as such do not take into account the effects of tumour microenvironment on cancer growth or immune-cell function.

Zhou and colleagues have taken shRNA screening in immune cells to the next level by building on advances in stable shRNA technology and in vivo shRNA screening5,6,7,8,10. The authors compiled two thematically focused libraries of shRNA molecules (Fig. 1). The first comprised 1,275 shRNAs targeting 255 genes whose expression is associated with T-cell exhaustion or anergy — states of functional inactivity that arise in cancer. The second contained 6,535 shRNAs targeting 1,307 genes encoding kinase and phosphatase enzymes involved in cell-signalling pathways. These shRNAs were then delivered by lentiviruses into activated mouse CD8+ T cells carrying a specific T-cell receptor (OT-1); those cells that stably expressed shRNA molecules were then implanted into mice harbouring aggressive melanomas that expressed a model-antigen protein (Ova), which can activate the OT-1 T-cell receptor.

Figure 1: Screening for T-cell modulators.
figure1

Zhou et al.1 compiled libraries of short hairpin RNA (shRNA) molecules designed to specifically inhibit the expression of genes expressed by dysfunctional (anergic or exhausted) CD8+ T cells or of genes encoding cell-signalling enzymes (phosphatases and kinases). The authors then infected CD8+ T cells with these shRNAs and implanted the cells into mice bearing melanomas. Seven days later, they isolated T cells from the spleens and tumours of the mice and compared the relative representation of shRNA molecules in the two tissues. Those shRNA molecules found to be enriched in the tumour were postulated to be involved in facilitating T-cell survival or proliferation within the tumours and were selected for further analysis.

Seven days later, the OT-1 T cells were purified from the tumours and spleens of the mice and analysed to identify shRNA molecules that were substantially more highly represented in tumoural than in splenic T cells — the implication being that the genes targeted by these shRNAs are involved in mediating survival or proliferation of T cells in tumours. Top-scoring genes were then screened again using 15 different shRNAs targeting each candidate gene. In addition to several shRNAs targeting genes with known functions in T cells, the authors identified shRNAs against Ppp2r2d, a regulatory subunit of the phosphatase enzymes of the PP2A family, as strongly enriched in tumours.

In further experiments, the researchers found that shRNA-induced inhibition of Ppp2r2d expression increased the survival of CD8+ T cells within the tumour and resulted in increased intratumoural CD8+ T-cell proliferation. Most importantly, systemic delivery of melanoma-targeted CD8+ T cells expressing an anti-Ppp2r2d shRNA resulted in increased death of melanoma cells, a significantly reduced tumour burden over time and prolonged survival of tumour-bearing mice. Strikingly, Ppp2r2d suppression not only improved the antitumour activity of CD8+ T cells, but also increased that of another class of immune cell, CD4+ T cells, thus suggesting a broad applicability of Ppp2r2d as a therapeutic target in T cells.

Zhou and colleagues' paper breaks ground on several levels. Their study sets a new standard in how the function of immune cells can be genetically dissected by RNAi screening in vivo. On the basis of the authors' data, similar screens in genetically engineered mouse models of different tumour types seem feasible and should be given high priority. Furthermore, functional screens specifically aimed at identifying modulators of CD4+ T-cell function should be pursued.

The report also suggests an exciting potential for targeting Ppp2r2d in T cells for cancer therapy, assuming that the increased antitumour T-cell function observed following Ppp2r2d inhibition can be validated in other models — ideally ones that do not depend on model antigens and cell transplantation. It will also be interesting to explore the use of Ppp2r2d inhibition in conjunction with other immunotherapies. For example, Zhou et al. showed that shRNA-mediated suppression of Ppp2r2d did not reduce the expression of the inhibitory receptors PD-1 or LAG-3 on tumour-infiltrating T cells, so the effect of combining Ppp2r2d inhibitors with PD-1 or LAG-3 blockers should be investigated.

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Correspondence to Lars Zender.

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Zender, L. Interference identifies immune modulators. Nature 506, 39–40 (2014). https://doi.org/10.1038/nature13050

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