Thesis Format
Monograph
Degree
Master of Science
Program
Microbiology and Immunology
Collaborative Specialization
Developmental Biology
Supervisor
DeKoter, Rodney P.
Abstract
B cell acute lymphoblastic leukemia (B-ALL) involves the cooperation between primary and secondary mutations. Using the Mb1-CreΔPB mouse model, we previously showed that PU.1 and Spi-B deletion leads to B-ALL. Whole-exome sequencing of leukemias revealed secondary mutations in the transcription factor Ikzf3. H195Y, corresponding to H196Y in humans, occurs within the DNA binding domain. We hypothesized that expression of H195/6Y IKZF3 alters gene expression in precursor B cells. RT-qPCR revealed H195/6Y Ikzf3-transduced cells had increased expression of Igll1, which encodes λ5—a component of the pre-B cell receptor. Interactions between IKZF3 and Igll1 were measured by co-transfecting cells with wild-type or H195/6Y Ikzf3 and the Igll1 promoter fused to firefly luciferase. H195/6Y Ikzf3-transfected cells had increased Igll1-driven expression of firefly luciferase, and mutating IKZF3 binding sites within the Igll1 promoter abolished this effect. Collectively, these results suggest that H195/6Y IKZF3 has altered activity and transcriptionally activates Igll1 through direct interactions.
Summary for Lay Audience
B cell acute lymphoblastic leukemia (B-ALL) is a common type of cancer observed in children that arises in early developing B cells. Like all cancers, B-ALL develops through the acquisition and cooperation of initial and later occurring secondary mutations. Using a mouse model of B-ALL, we showed that deletion of Spi1 and Spib, two important B cell development genes, leads to B-ALL. Further analysis of leukemias in these mice revealed secondary mutations in Ikzf3, a gene encoding a transcription factor. Transcription factors regulate the expression of a wide variety of genes by directly interacting with DNA binding sites via a DNA binding domain. Within Ikzf3, we identified the H195/6Y mutation, occurring in the DNA binding domain. Based on this, we hypothesized that H195/6Y functions to alter gene expression in early developing B cells. We compared gene expression between cells expressing unmutated and H195/6Y IKZF3 and found that cells expressing H195/6Y IKZF3 had increased expression of Igll1—a gene encoding a component of the pre-B cell receptor. To determine whether this increase was due to direct interactions between H195/6Y IKZF3 and Igll1, we fused Igll1 to a measurable marker and added both H195/6Y Ikzf3 and the fused gene into B cells. We found that these cells had increased expression of the marker, suggesting that H195/6Y IKZF3 directly interacted with Igll1, driving marker expression. In further support of this, mutating DNA binding sites within Igll1 abolished this effect, indicating that H195/6Y IKZF3 was unable to interact with these sites to drive marker expression. Collectively, our results suggest that the H195/6Y mutation alters IKZF3 function, leading to increased expression of Igll1.
Recommended Citation
Rysan, Heidi, "Role of Ikzf3 as a Driver Mutation in B Cell Acute Lymphoblastic Leukemia" (2024). Electronic Thesis and Dissertation Repository. 10203.
https://ir.lib.uwo.ca/etd/10203
