Role of p38γ - NFATc4 - IL17A Pathway As a Potential Therapeutic Target in Cutaneous T Cell Lymphoma
Authors: Zhang Xu Hannah , Ngo Vu N. , Sandoval Natalie , Cui Qi , Shi Yanhong , Zain Jasmine M. , Querfeld Christiane , Guo Chao , Wu Xiwei , Rosen Steven T. 
Published: October 2019

Abstract
Cutaneous T cell lymphoma (CTCL) is an incurable skin homing T cell malignancy. We have previously reported p38 as therapeutic targets for CTCL.1 However, the mechanism underlying p38 signaling is not completely understood. To further investigate p38 and its downstream signaling components, we examined public database of gene expression and found that p38γ is overexpressed in CTCL as compared to normal T cells. In addition, p38γ has negligible expression in normal lymphoid tissues, with the exception of high level expressed in smooth and cardiac muscle cells. We have demonstrated that p38γ over-expression increases cell proliferation and knockdown of p38γ causes Hut78 cell death. p38γ plays an important role in inflammation-associated tumorigenesis3 and inhibition of its activity has emerged as a strategy to treat a spectrum of cancers.4
The transcription factor, NFATc4, downstream of p38γ, is also significantly up-regulated in CTCL cells by microarray analysis, and it is at non-detectable level in normal T cells.1We have demonstrated that shRNA-mediated knockdown of p38γ reduced NFATc4 mRNA levels in Hut78 cells, and that inhibition of NFATc4 by siRNA reduces the proliferation of CTCL cells.
We also found that the cytokine IL17A functions downstream of p38γ and NFATc4, as knockdown of either p38γ or NFATc4 significantly reduced IL17A mRNA levels in Hut78 cells. This result suggests that IL17A is a target for transcriptionally activated NFATc4. Previously we have shown that IL17A rescues Hut78 cells from apoptosis induced by combined inhibition of NFAT and NFkB (treated with curcumin and Ly2228820). This implicates IL17A as a key mediator for CTCL survival. Therefore, we propose a novel p38γ - NFATc4 - IL17A signaling pathway in malignant T cells that promotes the survival of CTCL which provides potential therapeutic target against this disease.
To further define the role of p38 and identify targets that increase the antitumor efficacy of p38 inhibition, we performed a synthetic lethal RNA interference (RNAi) screen in Hut78 cells treated with 10 µM of the p38 MAPK inhibitor Ly2228820. We transduced control and Ly2228820-treated Hut78 cells with a pooled retroviral RNAi library consisting of 4290 shRNAs that targeted more than 1000 genes involved in human cancers. If a shRNA from the library is not toxic to the control cells, but causes cell death in Ly2228820-treated cells, the gene targeted by this shRNA would be identified by the screen as synthetically lethal to p38 inhibition. Among many hits identified from the screen, we selected UCHL5 for further analysis. UCHL5encodes a deubiquitin enzyme that cleaves K48-linked polyubiquitin chains and plays an important role in the regulation of protein stability. Interestingly, combination of Ly2228820 and b-AP15, a small molecule inhibitor of UCHL5, significantly reduced the protein levels of NFATc4 isoform but not other NFAT isoforms. NFATc4 protein levels are known to be regulated by ubiquitin-proteasome pathway.2 Our finding thus suggests UCHL5 as a potential new regulator that stabilizes NFATc4 protein. Further studies are needed to confirm this prediction. More importantly, combination of Ly2228820 and b-AP15 enhanced apoptosis in CTCL cell lines (HH and Hut78) and primary Sézary cells, but was not toxic in normal PBMC cells.
In summary, our findings suggest that the p38γ - NFATc4 - IL17A signaling pathway plays an important role in the survival of CTCL. In addition, improving the efficacy of targeting this pathway via p38 may also benefit from combined inhibition of UCHL5, a potentially important regulator of NFATc4 that needs further characterization.
Reference:
1 Bliss-Moreau M, Coarfa C, Gunaratne PH, Guitart J, Krett NL, Rosen ST (2015). Identification of p38beta as a therapeutic target for the treatment of Sezary syndrome. The Journal of investigative dermatology135:599-608.
2 Fan Y, Xie P, Zhang T, Zhang H, Gu D, She M et al (2008). Regulation of the stability and transcriptional activity of NFATc4 by ubiquitination. FEBS letters582:4008-4014.
3 Qi X, Yin N, Ma S, Lepp A, Tang J, Jing W et al (2015). p38gamma MAPK Is a Therapeutic Target for Triple-Negative Breast Cancer by Stimulation of Cancer Stem-Like Cell Expansion. Stem cells33:2738-2747.
4 Yin N, Qi X, Tsai S, Lu Y, Basir Z, Oshima K et al (2015). p38gamma MAPK is required for inflammation-associated colon tumorigenesis. Oncogene.

Disclosures
Querfeld: Actelion: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.