Reverse IMiD screen identifies ZBTB7A degrader with fetal hemoglobin induction effects and anti-tumor effects in colorectal carcinoma

Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular, Cellular, and Developmental Biology

First Advisor

Crews, Craig

Abstract

Zinc finger and BTB/POZ domain-containing 7A (ZBTB7A), also known as LRF or Pokemon, is a transcription factor involved in a wide range of biological processes, including repression of fetal hemoglobin, cell differentiation, and tumorigenesis. In terms of globin expression, ZBTB7A is a direct repressor of the fetal-like globin genes HBG1 and HBG2. Knockout or knockdown of ZBTB7A elevates fetal hemoglobin (HbF), a validated therapeutic strategy for sickle cell disease (SCD) and β-thalassemia, but direct pharmacologic inhibition of ZBTB7A has proven challenging. In colorectal carcinoma (CRC), ZBTB7A is upregulated in both CRC cell lines and tissue samples, where it functions as a driver of tumor proliferation. Evidence indicates that ZBTB7A expression correlates with the grade of dysplasia and may serve as a diagnostic marker, prognostic marker, and predictor of survival. Recently, derivatives of immunomodulatory (IMiD)-based drugs have been shown to induce degradation of transcription factors with C2H2 zinc finger DNA binding domains. Since ZBTB7A contains four tandem C2H2 zinc fingers, it represents a prime substrate for targeted protein degradation by IMiDs. In this study, we aimed to develop an IMiD-based degrader for ZBTB7A and validate its use to (1) promote induction of fetal hemoglobin and (2) slow the progression and proliferation of CRC. Using a dual-luciferase ZBTB7A reporter system in K562 cells, we screened 1,138 compounds with scaffolds derived from thalidomide, lenalidomide, and pomalidomide. Structure–activity relationship optimization of initial hits yielded MJB IL7973, a compound with improved selectivity and potency for ZBTB7A degradation, exhibiting a low nanomolar DC₅₀. In HUDEP-2 cells and primary human CD34+ stem and progenitor cells, MJB IL7973 degraded ZBTB7A in a dose-dependent manner, increased the proportion of HbF-positive cells, and elevated HbF protein levels without impairing erythroid cell proliferation or viability. In vivo, intraperitoneal administration of MJB IL7973 resulted in marked F-cell induction and enhanced HBG1/2 transcription in bone marrow cells. In CRC models, treatment of SW1116 and HT29 cell lines reduced cell viability and proliferation, while intraperitoneal treatment of mice bearing SW1116 xenografts significantly slowed tumor growth without evidence of toxicity. In conclusion, we identified MJB IL7973 as a novel IMiD-based degrader of ZBTB7A that induces HbF and inhibits CRC progression. These findings highlight the therapeutic potential of pharmacologically optimized LRF degraders for the treatment of SCD, β-thalassemia, CRC, and potentially other cancers.

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