Graduation Date

Spring 5-9-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Cancer Research

First Advisor

Amarnath Natarajan

Abstract

Heterobifunctional compounds (HBCs) that recruit E3 ligases to induce targeted protein degradation have emerged as promising therapeutic agents for cancer. Current HBC screening strategies prioritize biochemical degradation assays over functional anti-cancer activity assays. This approach may miss compounds with modest target protein degradation yet significant biological activity via alternative mechanisms. To address this, we developed an integrated chemical-genetic screening platform using isogenic wild-type, target-knockout, and E3-ligase-knockout cell lines that simultaneously assesses viability-based anticancer efficacy, target dependency, E3-ligase dependency, and heterobifunctional mechanism within a single primary screening assay. We applied this platform to Screen 23 CRBN-based HBCs targeting MAP3K1, a dual-function kinase and E3 ubiquitin ligase whose dysregulation contributes to poor prognosis and disease progression across multiple tumor types, including breast, pancreas, and ovarian cancers, making degradation an attractive therapeutic strategy. This approach identified HBC-8, which exhibits potent growth-inhibitory activity and induces S-phase arrest and apoptosis in a MAP3K1- and CRBN-dependent manner, validating target engagement and a heterobifunctional mechanism. HBC-8 induces dose-dependent degradation of MAP3K1 and perturbs its downstream signaling. Large-scale PRISM viability screens across numerous cancer cell lines identified MAP3K1 and CRBN expression as dual biomarkers for sensitivity, while genome-wide CRISPR screens revealed genetic determinants of response. Quantitative proteomics distinguished direct degradation from broader cellular effects, revealing that HBC-8's anti-cancer activity extends beyond the removal of MAP3K1 to include the disruption of the ubiquitin-proteasome system and perturbation of the protein translation machinery. These findings suggest that therapeutic effects arise from both target degradation and dysregulation of cellular quality control pathways. This work establishes a practical alternative approach for rapidly identifying and characterizing therapeutically relevant HBCs whose anti-cancer activities depend on the presence of the targeted proteins.

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