Traf3: A potential new target to improve the effectiveness of cancer immunotherapy

By Haoyu Li

In recent years, cancer immunotherapy has developed rapidly. Immune checkpoint inhibitors activate the patient’s own immune system by targeting and suppressing immune checkpoints, significantly improving the prognosis of many cancer patients. However, most cancer patients are still resistant to immune checkpoint therapy. Among them, the lack or down-regulation of cancer cell antigen presenting molecules such as Major Histocompatibility Complex Class-I (MHC-I) is one of the main reasons for such resistances (Gao et al, 2016; Zaretsky et al, 2016; Rodig et al, 2018). Studies have predicted that more than 65% of cancer patients have MHC-I related defects (Garrido et al, 2016). Since MHC-I and the immunosuppressive signal protein PD-L1 are often regulated by common molecular signals, the up-regulation of MHC-I may also be accompanied by up-regulation of PD-L1 and suppress the immune response. Therefore, how to effectively increase MHC-I expression without increasing PD-L1 expression has become a major problem in cancer immunotherapy.

Recently, Professor Xiaole Liu from Harvard University/Dana-Farber Cancer Institute and Professor Myles Brown’s laboratory jointly published an article entitled ‘Therapeutically increasing MHC-I expression potentiates immune checkpoint blockade’ on Cancer Discovery (2021), which systematically explored MHC- The regulation mechanism of I and PD-L1 and data mining to find targeted drugs that can specifically increase the expression of MHC-I.

The author first applied the dual-label CRISPR screening technology to search for genes that can regulate MHC-I and PD-L1 in B16F10 melanoma cells with low baseline expression of MHC-I. The analysis of this high-throughput data revealed many negative regulatory genes specific for MHC-I, among which Traf3 is a gene encoding a key transduction protein in the TNF family signaling pathway. In vitro experiments further confirmed that under the stimulation of different interferons, Traf3 knockout significantly up-regulated the expression of MHC-I in B16F10 cells but did not up-regulate the expression of PD-L1. In addition, in multiple mouse and human cancer cell lines, Traf3/TRAF3 knockout also specifically increased the expression of MHC-I.

In order to explore the mechanism by which Traf3 specifically regulates the expression of MHC-I, the authors used RNA-seq, ATAC-seq, western-blot to find that Traf3 knockout can significantly increase the activity of the NF-kB pathway, thereby up-regulating the expression of MHC-I. Further experiments found that the IKK inhibitor of the NF-kB signaling pathway almost completely eliminated the up-regulation of MHC-I expression by Traf3 knockout, thus confirming that the regulatory effect of Traf3 on MHC-I is achieved through the NF-kB signaling pathway.

So, what is the impact of increased expression of MHC-I molecules on cancer immune response? The author found that Traf3 knockout promoted T cell-mediated tumor cell killing through in vitro cancer cell and immune cell co-culture experiments. At the same time, in vivo experiments in mice also show that Traf3 knockout greatly improves the efficacy of immune checkpoint inhibitors in the treatment of melanoma.

Another question stemming from the finding is whether Traf3 related to the clinical resistance of cancer patients to immunotherapy. By comparing the RNA-seq data of Traf3 knockout cells and control cells through differential expression analysis, the author compiled a set of differential gene maps of Traf3 knockout. By comparing the differential gene profile with the gene expression profile of cancer patients in the TCGA database, and the RNA-seq data of clinical samples of immune checkpoint inhibitors in public databases, the author found that the differential expression profile of Traf3 knockout in many cancer types is positively correlated with patient survival and the efficacy of immune checkpoint inhibitors. In addition, the authors isolated cancer cells with high or low expression of MHC-I from melanoma samples from the Dana-Farber Cancer Institute, and found that the differential expression profile of Traf3 knockout was also positively correlated with the expression level of MHC-I.

Can Traf3 therefore be targeted to improve the efficacy of patients with immune checkpoint inhibitors? Since TRAF3 is a transduction protein, there is currently no targeted drug for it. To answer the question, the author took a different approach, using data mining methods to find drugs that can mimic the Traf3 knockout effect. By comparing the differential gene expression profile of Traf3 knockout with a large number of drug-stimulated cell transcriptome data included in the GEO database, they found that SMAC mimetic (IAP inhibitor) stimulation can well mimic the effect of Traf3 knockout on gene transcription. Among the many SMAC mimetic, the author selected birinapant, which has undergone multiple phase I/II clinical trials, for the next step of verification. In the B16F10 model, birinapant can mimic the effect of Traf3 knockout, specifically increase the expression of MHC-I, thereby increasing the sensitivity of cancer cells to T cell killing and the response of mouse tumor models to immune checkpoint inhibitors. It has been shown that in some other human tumor cell lines, birinapant also specifically increased MHC-I expression. Interestingly, the authors found that birinapant did not increase MHC-I expression in a few other cell lines (including some cell lines with higher baseline NF-kB pathway activity); suggesting that in these cell lines, the expression of MHC-I may be regulated by other mechanisms.

In summary, by studying the regulatory mechanism of MHC-I, the study has discovered a way to enhance the effect of cancer immunotherapy by specifically enhancing cancer cell antigen presentation, which is expected to improve the therapeutic effect of immunotherapy in some tumor patients with low MHC-I expression levels.

References:

Gao J, Shi LZ, Zhao H, Chen J, Xiong L, He Q, et al (2016). Loss of IFN-γ Pathway Genes in Tumor Cells as a Mechanism of Resistance to Anti-CTLA-4 Therapy. Cell. Elsevier Inc.; 2016; 167:397-404.e9. 

Garrido F, Aptsiauri N, Doorduijn EM, Garcia Lora AM, van Hall T (2016). The urgent need to recover MHC class I in cancers for effective immunotherapy. Curr Opin Immunol. Elsevier Ltd; 2016; 39:44–51. 

Gu SS, Zhang W, Wang X, Jiang P, et al (2021). Therapeutically increasing MHC-I expression potentiates immune checkpoint blockade. Cancer Discov. 2021 Feb 15: candisc.0812.2020. doi: 10.1158/2159-8290.CD-20-0812. PMID: 33589424. 

Rodig SJ, Gusenleitner D, Jackson DG, Gjini E, Giobbie-Hurder A, Jin C, et al (2018). MHC proteins confer differential sensitivity to CTLA-4 and PD-1 blockade in untreated metastatic melanoma. Sci Transl Med. 2018 Jul 18;10(450): eaar3342. 

Zaretsky JM, Garcia-Diaz A, Shin DS, Escuin-Ordinas H, Hugo W, Hu-Lieskovan S, et al (2016). Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma. N Engl J Med. 2016; 375:819–29. 

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