Graduation Date

Spring 5-9-2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Amarnath Natarajan

Abstract

Activation of NFκB pathway has been implicated in several malignancies and plays a role in many key processes including tumor initiation and progression. The NFκB pathway is activated when TNFα in the tumor microenvironment binds to its receptor, eventually leading to the phosphorylation of the kinase IKKβ. Once active, IKKβ phosphorylates IκBα, a protein that functions to sequester NFκB in the cytosol of resting cells. The phosphorylation of IκBα leads to its degradation in the proteasome and allows NFκB to translocate to the nucleus where it can drive gene transcription. The sulfhydryl groups on solvent-exposed cysteine (Cys) residues of the key NFκB pathway proteins IKKβ and p65 serve as critical targets for NFκB pathway inhibition through covalent modification. The Natarajan lab previously reported an isatin-derived spirocyclic α-methylene-γ-butyrolactone analog 19, that covalently binds to IKKβ and p65 resulting in NFκB pathway inhibition. Analog 19 also showed synergistic tumor growth inhibition with cisplatin in an orthotopic ovarian cancer model without inducing any overt toxicity. To determine the proteome wide targets of analog 19, we conducted pull-down MS with alkyne-tagged 19 treated lysates. This identified >100 proteins as potential targets of analog 19. Based on these results we hypothesized that dimers of analog 19 will crosslink proteins in the IKK complex and inhibit NFκB pathway. Synthesis and evaluation of the dimers showed that dimers n3 and n7 were more potent than analog 19 in inhibiting nuclear translocation of NFkB and the linker length contributed to their efficacy. The molecular basis for this was attributed to the formation of irreversible covalent bonds between the surface exposed cysteine residues of p65 and IKKβ and other proteins within the IKK complex resulting in higher molecular weight protein complexes.

Analyses of the pull-down MS data also revealed that analog 19 binds to a number of proteins involved in protein processing in the ER and protein folding. This suggested that the dimers could induce the unfolded protein response (UPR). Indeed, the dimers induced UPR and activated the PERK/eIF2α/CHOP arm of the UPR leading to robust apoptotic cell death in the cancer cells when compared to immortalized non-transformed cells. The dimers were ~3-9 -fold more potent than the analog 19 in inhibiting cancer cell growth. The potency was modulated by the linker length. We also showed that like analog 19 the Michael acceptor in the dimers is critical for activity against cancer cells. In summary studies presented in this dissertation demonstrate that dimers of the isatin-derived spirocyclic NFκB inhibitor analog 19, potently inhibits cancer cell growth and induce apoptosis by crosslinking NFκB pathway proteins and inducing UPR.

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