Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Kenneth L. Wright, Ph.D.

Committee Member

Shari Pilon-Thomas, Ph.D.

Committee Member

Bijal Shah, M.S.

Committee Member

Dennis Adeegbe, Ph.D.

Keywords

Non-Hodgkin’s Lymphoma, Antibody Dependent Cellular Cytotoxicity, PCI-34051, HDAC8 Inhibitors, Pan-HDAC Inhibitors, Apoptosis

Abstract

This study demonstrates for the first time that HDAC8 function is critical for MCL survival, and abrogating its activity in human primary NK cells does not interfere with NK IgG antibody directed ADCC therapies ex vivo. Human NK cells, isolated from healthy donors, are highly resistant to HDAC8 inhibitor treatment with PCI-34051. Even at the highest concentration, 20uM, no toxicity was observed. Conversely, MCL cell lines representative of the aggressive MCL subtype, classical MCL, were especially sensitive to PCI-34051, an HDAC8 selective inhibitor, treatment. Blocking HDAC8 activity and/or abrogating expression through shRNA silencing induced significant DNA damage, hyperacetylation of SMC3, and apoptosis. Furthermore, DNA damage was an indicator of sensitivity. Drug washout experiments indicated that PCI-34051 can be dosed twice at half the original dose, distributed across 2-sequential days, to achieve the same effect as a single high dose.

Stimulating human NK cells with IL-2, IL-12, and IL-15 or IL-18, leads to a significant upregulation of HDAC8. It was hypothesized that HDAC8 may play a role in NK activity. However, blocking HDAC8 activity did not dampen NK survival, proliferation, cytotoxic responses, cytokine secretion, or NK phenotype. Interestingly, PCI-34051 increased the abundance of IFNγ+ NK cells, possibly indicating enhanced activity. There was insufficient evidence that PCI-34051 sensitizes MCL cell lines to NK cytotoxicity. Rather, PCI-34051 may induce a NK memory-like phenotype, indicated by enrichment of IFNγ+ NK cells.

The focus of this study was to identify a novel HDAC protein critical for MCL survival and to evaluate if inhibiting activity interferes with IgG antibody therapies dependent on NK ADCC for therapeutic efficacy. NK cell cytolytic function is critical for anti-tumor response and is dependent on the expression of sensors that detect aberrancies. NK cells are capable of sensing abnormally low levels of HLA class I molecules through a plethora of KIR receptors. They are capable of detecting stress signals on the surface of tumor cells through their NKG2D receptor and elicit a robust cytotoxic response. Additionally, the low-affinity Fc binding receptor CD16 is imperative for antibody therapies dependent on NK ADCC for clinical efficacy. Thorough investigation was performed to determine if blocking HDAC8 in NK cells caused any attributes not favorable for NK anti-tumor responses. PCI-34051 did not alter NK anti-tumor phenotype. The results presented in this dissertation indicate that NK cells can robustly induce cytotoxic responses during treatment with the HDAC8 selective inhibitor, PCI-34051, in combination with IgG-targeting therapies.

MCL cell lines representing the highly aggressive SOX11+ MCL subtype, conventional MCL, had the highest sensitivity to HDAC8 inhibition, indicating a potential therapeutic option for treating this patient population.

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