NYMC Student Theses and Dissertations

Date of Award

5-19-2020

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Microbiology and Immunology

First Advisor

Dr. Raj Tiwari

Second Advisor

Dr. Jan Geliebter

Third Advisor

Dr. Paul Arnaboldi

Abstract

Thyroid cancer incidence is increasing at an alarming rate almost trebling every decade. 52,070 new cases of thyroid cancer (14,260 in men and 37,810 in women) were diagnosed in 2019 with an estimated death toll of 2170. Although most thyroid tumors are treatable and has good prognosis, anaplastic thyroid cancer (ATC) is extremely aggressive with a grim poor prognosis of 6-9 months post diagnosis. ATC is completely refractory to mainstream therapies. In our study, immunohistochemical analyses of ATC tissues confirmed a T cell inflamed “hot” tumor immune microenvironment (TIME) as evidenced by presence of CD3+ and CD8+ T cells. This kind of tumors are amenable to immune checkpoint blockade (ICB) therapy. This therapeutic avenue is unexplored in ATC. In this study, we explored the feasibility of a combination therapy of small molecule inhibitor and ICB in ATC. We used an in vitro model system representative of papillary (TPC-1, K1, BCPAP), anaplastic (8505C, T238, SW1736, HTh74), follicular (CGTH-W-1) thyroid cancer and Nthy-ori-3-1 as normal thyroid follicular cell. The cells were screened for expression of 29 immune checkpoint molecules by qRT PCR. We noted a higher expression of HVEM, BTLA, CD160 in ATC cell lines compared to the rest. Expression level of HVEM was more than 30-fold higher in ATC compared to the other cell lines on average. immunocytochemistry, western blot and flow cytometry analyses confirmed expression of these proteins in ATC. Additionally, HVEM had highest surface expression in ATC. HVEM is a member of TNFRSF which acts as a bidirectional switch by interacting with BTLA, CD160 and LIGHT in a cis or trans manner. Given xxiii the T cell inflamed hot TIME in ATC, expression of HVEM on tumor cells was suggestive of a possibility of complex crosstalk of HVEM with inflammatory cytokines. Increased transcription and solubilization of HVEM were observed in ATC cell lines in response to pro-inflammatory cytokines IL-8 and TNFα. Our study also indicates that HVEM is inducible by IFNγ as evidenced by more than 5-fold increase in HVEM transcription in response to the cytokine. Our study reports for the first time, a tumor intrinsic stress induced MAPK signaling transduced by HVEM upon interaction with soluble LIGHT. Induction of pJNK and p-c-Jun was indicative of increased proliferative potential of these cells triggered by this interaction. HVEM/LIGHT interaction also triggered nuclear translocation of NFB in ATC. Silencing HVEM in 8505C by CRISPRi highly dampened proliferative and invasive potential of the cells signifying possible tumor intrinsic function of HVEM in ATC. BRAFV600E is a common mutation in ATC which is largely responsible for remodeling of TIME and is a prominent candidate for targeted therapy. Unfortunately, emergence of resistance is extremely common. BRAFV600E inhibitor PLX4032 resistant ATC cell lines were generated in this study, and their immune phenotypes were profiled. PLX4032 resistant cells had activation of alternate signaling molecules rescuing BRAFV600E inhibition and had dramatically higher expression of HVEM, BTLA and CD160 compared to the sensitive phenotype. The surface expression of HVEM persisted in resistant cells after combination therapy with PLX4032 and trametinib. Altogether, our studies provide evidence for T cell inflamed TIME in ATC along with expression of immune checkpoint proteins HVEM, BTLA and CD160 in ATC. We also discovered active tumor intrinsic signaling transduced by HVEM/LIGHT interaction in ATC and the persistent xxiv expression of these immune checkpoint molecules in case of BRAFV600Ei resistant ATC. We propose that a combination of small molecule inhibitor targeting downstream effectors of MAPK pathway and antagonistic antibodies targeting HVEM/BTLA axis might provide a viable therapeutic avenue for ATC patients.

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