Constitutive expression of I?B? promotes tumor growth and immunotherapy resistance in melanoma [mouse RNA-seq]

Background: I?B?, a rather unknown co-regulator of NF-?B, is mostly inducibly expressed and can either activate or repress a specific subset of NF-?B target genes. While its role as a transcriptional regulator of various cytokines and chemokines in immune cells has been revealed, I?B?'s function in solid cancer remains unclear. Methods: We investigated I?B? expression in melanoma, and assessed its impact on target gene expression, tumor growth, and response to immunotherapy in melanoma cell lines, mouse models, and patient samples. Results: Unlike in other cell types, I?B? protein was found to be constitutively expressed in a subfraction of melanoma cell lines, and around 35% of melanoma cases. This atypical expression pattern of I?B? did not correlate with its mRNA levels or known driver mutations, but instead seemed to result from changes in its post-transcriptional or post-translational regulation. Deleting constitutively expressed I?B? abrogated the activity and chromatin association of STAT3 and p65, leading to reduced expression of the pro-proliferative cytokines IL-1ß and IL-6 in melanoma cells. Consequently, loss of tumor-derived I?B? suppressed self-sustained melanoma cell growth both in vitro and in vivo. Additionally, constitutive I?B? expression suppressed the induction of the chemokines CXCL9, CXCL10, and CCL5, which impaired the recruitment of NK and CD8+ T-cells to the tumor, causing resistance to a-PD-1 immunotherapy in mice. Furthermore, the expression of tumor-derived I?B? also correlated with the absence of CD8+ T-cells in human melanoma samples and progressive disease during immunotherapy. Conclusion: We propose that tumor-derived I?B? could serve as a new therapeutic target and prognostic marker that characterizes melanoma with high tumor cell proliferation, cytotoxic T- and NK-cell exclusion, and unfavorable immunotherapy responses. Targeting I?B? expression might open up a new therapy option to re-establish the recruitment of cytotoxic cells, thereby resensitizing for immunotherapy. Overall design: To investigate the functional impact of constitutive I?B? expression in melanoma, we used CRISR-Cas9 to knock out NFKBIZ (I?B?) in the I?B?-expressing D4M-3A melanoma cells