Casitas B-lineage lymphoma (CBL) proteins belong to a family of E3 ubiquitin ligases with oncogenic potential. In human T cells, they work as important gatekeeper components for T-cell receptor (TCR) activation and subsequent activation-induced cell death (AICD). AICD is a process of apoptosis that is triggered by the death receptor/ligand pair: FAS/FAS-ligand (FASL). Lack of FASL expression blocks the AICD apoptosis cascade even in FAS-high cells. Previously, we and others documented low FAS expression in the majority of cases of cutaneous T-cell lymphoma (CTCL). In keeping with its role as a tumor suppressor gene, we found that FAS was silenced by methylation of its promoter and that it can be upregulated by DNA demethylation. We also showed that FASL is generally not expressed by CTCL cells. In contrast to normal T cells, CTCL cells do not upregulate FASL in response to TCR engagement. While investigating this abnormality, we found that over- expressed c-CBL protein plays a key role in inhibiting FASL expression and contributing to resistance to AICD. We showed that c-CBL is over-expressed in most cases of CTCL compared to normal T cells. When c-CBL is reduced by siRNA treatment, TCR signaling cascades are restored, FASL expression is upregulated and CTCL cells undergo massive apoptosis as long as FAS expression is adequate. When FAS expression is inadequate, demethylating agents such as methotrexate can increase FAS and render cells sensitive to apoptosis. This groundbreaking work provided important insights into the correction of apoptotic defects in CTCL and was published in the Journal of Investigative Dermatology (135: 861-8, 2015)  and highlighted by an accompanying editorial comment. Our findings suggest that over-expressed c-CBL plays a key role in the pathogenesis of CTCL by blocking AICD. Therefore, it is critical to extend our research to elucidate the mechanisms underlying c-CBL over-expression. Our preliminary data suggest that c-CBL is transcriptionally regulated. CTCL lines and malignant T cells from CTCL patients have shown karyotypic abnormalities involving chromosome 11 which contains the c-CBL gene. Based on our preliminary findings, we hypothesize that c-CBL over-expression in CTCL is caused by structural abnormalities and/or epigenetic dysregulation of the c-CBL gene. To test this hypothesis, we will conduct experiments incorporated into two specific aims: 1) To determine whether structural abnormalities of the c-CBL gene result in c-CBL protein over-expression in CTCL; and 2) To determine whether epigenetic dysregualtion of the c-CBL gene results in c-CBL protein over- expression in CTCL. This project is a novel investigation of c-CBL regulatory mechanisms in the context of deficient FASL expression and associated apoptotic blockade in CTCL. These studies have strong translational significance. In addition to their potential for novel diagnostic assays, they will allow new prevention and treatment approaches targeting c-CBL for restoration of apoptosis in CTCL.
In this application, we follow-up on our discovery that the E3 ubiquitin ligase, c-CBL, is over-expressed in cutaneous T-cell lymphoma (CTCL), and that it's inhibition induces upregulation of FAS-ligand and apoptosis of CTCL cells. The purpose of this preclinical study is to perform in vitro and ex vivo experiments to determine the underlying genetic and/or epigenetic mechanisms responsible for this over-expression of c-CBL. Successful completion of our aims should lead to an improved understanding of CTCL pathogenesis and new approaches to its treatment as well as novel therapies for other chronic skin diseases involving cutaneous T- cell infiltrates.
|Zhao, Lei; Okhovat, Jean-Phillip; Hong, Eric K et al. (2018) Preclinical Studies Support Combined Inhibition of BET Family Proteins and Histone Deacetylases as Epigenetic Therapy for Cutaneous T-Cell Lymphoma. Neoplasia 21:82-92|
|Salva, Katrin A; Kim, Youn H; Rahbar, Ziba et al. (2018) Epigenetically Enhanced PDT Induces Significantly Higher Levels of Multiple Extrinsic Pathway Apoptotic Factors than Standard PDT, Resulting in Greater Extrinsic and Overall Apoptosis of Cutaneous T-cell Lymphoma. Photochem Photobiol 94:1058-1065|
|Salva, Katrin A; Reeder, Margo J; Lloyd, Rita et al. (2017) c-CBL E3 Ubiquitin Ligase Expression Increases Across the Spectrum of Benign and Malignant T-Cell Skin Diseases. Am J Dermatopathol 39:731-737|
|Nihal, Minakshi; Wood, Gary S (2016) c-CBL regulates melanoma proliferation, migration, invasion and the FAK-SRC-GRB2 nexus. Oncotarget 7:53869-53880|