Regulation of AML1-ETO via the 3'UTR in t(8;21) leukemia

Daniel Johnson
Appointment Period: 2016-2018, Grant Year: [30,31]

My primary research project is to examine the regulation of the AML1-ETO (AE) fusion gene transcript via its 3’UTR in t(8;21) acute myeloid leukemia (AML). The t(8;21)(q22;q22) translocation is one of the most common AML-associated recurrent chromosomal abnormalities and is observed in 8-15% of all AML patients. This translocation results in the formation of a stable fusion protein, AE, which functions as part of an aberrant regulatory complex that dysregulates the transcription of genes involved in normal blood cell function and differentiation; this dysregulation results in the blocking of differentiation at an immature blast stage, a critical step in AML development. Recently, single-cell qPCR data from our lab has suggested that a subset of low-expressing AE cells can differentiate to the granulocyte and monocyte lineages. Based on this and other observations, we hypothesize that stabilization of AE expression is an important step in t(8;21) eukemogenesis, which may be mediated by alterations in regulatory factors associated with the AE 3’UTR.

During my time on the training grant, we have observed that AE transcripts in patient leukemic blast cells preferentially use a proximal alternative poly-adenylation site, resulting in a 3’UTR which is 3.7kb in length, whereas the lower AE expressing granulocytes and monocytes use a distal 5.2kb 3’UTR. We have also determined that knock-down of cleavage and poly-adenylation factor 1 (CPSF1) increases distal 5.2kb 3’UTR usage and results in lower AE expression and cell proliferation in the t(8;21)+ Kasumi-1 cell line. Through mRNA decay and dual-luciferase assays I have shown that the 3.7kb proximal 3’UTR fragment is significantly more stable than the distal 5.2kb 3’UTR.

Therefore, I have begun searching for the specific factors which are responsible for regulating the stability of both the 3.7 & 5.2kb AE 3’UTR fragments. Several experiments using DICER1 siRNA or shRNA knockdown in t(8;21)+ Kasumi-1 and SKNO1 cells suggests that micro-RNAs are involved in the regulation of AE 3’UTR stability. Furthermore, using luciferase assays with 600bp fragments spanning the AE 3’UTR, I have determined the relative stability contributed by cis-elements within small portions of the AE 3’UTR. My current and future efforts are to identify the specific micro-RNAs and RNA binding proteins that target cis-elements found in the most relevant parts of the AE 3’UTR. Regulation of the 3’UTR to control the post-transcriptional regulation of transcription factors and oncofusion proteins, like AE, is a nearly untouched area in the hematopoietic field. Therefore, I anticipate that my training will soon result in the prospective publication of this work and may eventually lead to novel insights into ways to inhibit the leukemogenic potential of the t(8;21) AE fusion.

My training has also contributed to research on additional cancer-related projects. I have made an AML mouse model as part of a collaboration testing a novel nano-particle based dendritic cell vaccine immunotherapy. Currently, I am testing the nano-particles’ ability to induce immune responses in my AML mouse model. Working with others that I’ve met through the training grant has also allowed me to learn how to analyze DNA and RNA sequencing data from The Cancer Genome Atlas as well as how to do the necessary statistics, which directly contributed to the below publication, investigating the roles of LZTS2 and PTEN mutation in prostate cancer.

PUBLICATIONS (resulting from this training)

Yu EJ*, Hooker E*, Johnson DT*, Kwak MK, Zou K, Luong R, He Y, Sun Z. (2017) LZTS2 and PTEN Collaboratively Regulate ß-catenin in Prostatic Tumorigenesis. PLoS One. 12(3): e0174357. PMID: 28323888. PMCID: PMC5360334.

Petrie KL, Palmer ND, Johnson DT, Medina SJ, Yan SJ, Li V, Burmeister AR, Meyer JR. Destabilizing mutations encode nongenetic variation that drives evolutionary innovation. (2018) Science. 359(6383):1542-1545. doi: 10.1126/science.aar1954. PMID: 29599247