Dissecting the Roles of p38β and p38γ in Cutaneous T Cell Lymphoma
Authors: Zhang Xu Hannah , Querfeld Christiane , Rosen Steven T 
Published: October 2019

Abstract
Cutaneous T cell lymphoma (CTCL) is a disfiguring, incurable cancer that develops from clonal expansion of T cells and most commonly presents on the skin. For patients with advanced disease, current therapies are inadequate, and outcome is poor. An incomplete understanding of CTCL molecular regulators has limited development of effective targeted therapies. One candidate molecular regulator is p38γ, a mitogen-activated protein kinase (MAPK) crucial for malignant T cell activity and growth in response to T cell receptor (TCR) signaling. We previously reported that p38β (Bliss-Moreau, JID, 2014) and p38γ (Zhang, JID, 2018) are promising therapeutic targets for CTCL. We examined both RNA-seq and microarray public databases for gene expression and found that both targets are elevated in CTCL cell lines and patient samples, compared to normal T cells. This is consistent with literature showing that in several organs of the healthy human immune system, although p38α and p38δ isoforms are highly expressed, p38β and p38γ are typically very low (Wang, JBC, 1997). We demonstrated that knockdown of p38γ, but not p38β, causes cell death in the human CTCL cell line Hut78. In addition, overexpression of either target causes proliferation in CTCL cells.
In this study, we dissected the roles of both p38β and p38γ in CTCL and delineated their downstream pathways. DLGH1 is an important scaffolding protein that directs T-cell signaling through the nuclear factor of activated T-cells (NFAT) pathway rather than through the NF-κB pathway (Round, Nat Immunol, 2007; Sabio, EMBO J, 2005). Our recent data indicate that knockdown of p38β reduced expression of DLGH1 at both the mRNA and protein level, but upregulated gene expression of CARMA1, which directs TCR signaling to the NF-kB pathway (Thome and Tschopp, Trends in Immunology, 2003). p38γ directly phosphorylates DLGH1 at ser158 in Hut78 cells (Sabio, EMBO J, 2005; Zhang, JID, 2018). We showed that inhibition of this phosphorylation reduced the role of both NFAT and NF-kB signaling in regulation of the immune response. Thus, our data indicate that DLGH1 is a common target of both p38β and p38γ isoforms. In addition, curcumin treatment causes induction of both p38γ and DLGH1 protein expression, but causes their reduction in p38β-knockdown Hut78 cells. This suggests an important role for p38β in TCR signaling via DLGH1 and its possible crosstalk with p38γ. We performed confocal microscopy of immunofluorescent p-p38 Tyr323 staining in p38β-knockdown Hut78 cells, which revealed a "ring-like" structure formed in the cytosol, in contrast to a polarized structure in wild-type Hut78 cells. This suggests that loss of p38β abolishes initiation of TCR complex formation in the plasma membrane of Hut78 cells. It is reported that TCR signaling can be modulated by the actin cytoskeleton (Yu, J Cell Sci, 2013); we further pursued its potential mechanisms and found that loss of p38β reduced F-actin expression in Hut78 cells. However, that p38β modulates with cytoskeletal structure in malignant T cells via altered actin expression remains further investigation.
In summary, DLGH1 is a common target of p38γ and p38β, which is involved in regulation of TCR signaling through either phosphorylation or downregulation of its protein expression by these two MAPK isoforms. Furthermore, DLGH1 provides a potential pathway for modulation of the T cell cytoskeleton by MAPKs and through which the downstream NFAT and NFkB pathways maybe directed. This opens new possibilities for the development of targeted therapies for CTCL.

Disclosures
Querfeld: Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees; Medivir: Membership on an entity's Board of Directors or advisory committees; Bioniz: Membership on an entity's Board of Directors or advisory committees; Kyowa: Membership on an entity's Board of Directors or advisory committees; Acelion: Membership on an entity's Board of Directors or advisory committees.