Abstract 7056: The impact of aberrant ZMIZ1 protein expression in pediatric acute lymphoblastic leukemia

Abstract While the outcomes for pediatric acute lymphoblastic leukemia (ALL) have improved dramatically over the past 30 years, up to 20% of patients relapse and their prognosis is poor. Collaborators at St. Jude’s Research Hospital discovered that mutations in ZMIZ1 are highly enriched at relapse in a subset of patients. Subsequent studies showed that the transcript level is correlated to event-free survival. Thus, our objective is to study the function of ZMIZ1 in cancer survival under the selective pressures of chemotherapy. To accomplish this, we generated a panel of B-ALL cell lines overexpressing either wild-type (WT), or two different mutant forms of ZMIZ1 found in patients (A291-A305 deletion (Del), or A297T substitution (Sub)) and compared them to an empty vector (EV) control. We performed reverse transcription quantitative PCR (RT qPCR) to confirm mRNA expression, western blots to determine expression of ZMIZ1, as well as cell viability, apoptosis, and proliferation assays to assess functional impact. While full length transcripts were confirmed in all three lines, full length protein was only detected in the WT overexpressing line. All lines expressing the mutant forms of ZMIZ1 demonstrated a lower molecular weight product, suggesting post-translational modification. Phenotypically, we observed profound resistance to prednisolone, a cornerstone of ALL therapy, upon WT (IC50 >1e-1mM) expression compared to EV (IC50=3e-4mM), while cell lines expressing the Del or Sub showed no difference in drug sensitivity (IC50s=3e-4mM). No differences were seen with other commonly used chemotherapies. Since these experiments were done in cell lines with two WT alleles, we then used CRISPR-cas9 to knock out endogenous ZMIZ1. Allele specific sequencing for individual clones was confirmed using IDT’s rhAmpSeqTM CRISPR kit. ZMIZ1 complete knockouts (KO) showed no detectable protein expression and had increased sensitivity to prednisolone (IC50=3e-5mM) compared to the Rosa control (IC50=5.7e-5mM) in both cell viability and apoptosis assays. Subsequently, we reintroduced ZMIZ1 (WT and mutants) into KO cells and again only WT had detectable full length protein expression, but now all three lines showed the lower molecular weight product. Resistance to prednisolone was restored completely upon rescue with WT ZMIZ1 (IC50>1e-1mM) compared to the complete KO cells and Rosa. In contrast, mutant cell lines now showed modest resistance to prednisolone (IC50s 4.9e-5mM Del; 5.6e-5mM Sub) compared to KO. Collectively our results indicate a complex relationship between the products of WT and mutant alleles mediated in part by a post-translationally modified species involved in glucocorticoid resistance. A more thorough understanding of how mutations in ZMIZ1 affect the regulation and drug resistance is vital in determining the clinical significance and evaluating ZMIZ1 as a potential therapeutic target in patients. Citation Format: Caroline S. Greenstone, Jessica Krugman, Danielle W. Cohen, John Easton, Heather Mulder, Ying Shao, Xiaotu Ma, Nikki A. Evensen, William L. Carroll. The impact of aberrant ZMIZ1 protein expression in pediatric acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7056.