Pediatric acute myeloid leukemia (AML) is often refractory to chemotherapy and remains a devastating disease with poor outcomes. Consequently, there is an urgent need to characterize molecular drivers of pediatric AML with the goal of developing more effective molecular therapies. Our group recently identified somatic tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) as a subtype-defining genomic alteration in pediatric AML that is associated with poor outcomes, high relapse rate, and measurable residual disease (MRD) positivity. However, how these alterations drive leukemogenesis is yet to be investigated. Wild-type UBTF regulates rRNA transcription, ribosome biogenesis, and nucleolar formation. Our recent work suggests that UBTF-TD retains these functions. However, we found that expression of UBTF-TD in cord blood CD34+ cells, but not wild-type UBTF, leads to their increased proliferation, self-renewal, and CD117/CD33 surface marker expression, suggesting a potential gain of function as well. Furthermore, we also observed increased expression of HOXB locus genes, PRDM16, MEIS1, and NKX2-3 in the CD34+ UBTF-TD overexpression model relative to our WT control. These expression patterns are similar to what we see in patients, collectively implicating UBTF-TD driving leukemic phenotypes. Based on our localization studies of UBTF-TD to DNA, we hypothesized that UBTF-TD protein may gain the ability to interact with genomic loci that characterize UBTF-TD AML, like the HOXB locus. We therefore wanted to test if UBTF-TD localized to the HOXB cluster and if this lead direct transcriptional upregulation. To test this, we expressed HA-tagged UBTF-TD in cord-blood CD34+ cells and mapped UBTF-TD genomic occupancy using CUT&RUN. We found that UBTF-TD localized to the promoters and gene bodies of key genes, including PRDM16, MEIS1, NKX2-3, and the HOXB cluster. These sites were accompanied by H3K4me3 and H3K27ac active marks without the H3K27me3 repressive mark. To validate these as bone-fide UBTF-TD-occupied regions, we utilized a expressed FKBP12F36V-HA-UBTF-TD fusion in CD34+ cells. This degradation tag (dTAG) system allows for rapid degradation of UBTF-TD protein upon dTAG-13 treatment. We treated our FKBP12F36V-HA-UBTF-TD expressing cord blood CD34+ cells with 1uM of dTAG-13 or DMSO. We found that a 3-day treatment with dTAG-13 led to downregulation of HOXB gene locus expression, but loss of CD117 expression or cell growth required prolonged treatment. Furthermore, our CUT&RUN data showed that dTAG-13 treatment resulted in loss of UBTF-TD protein at the HOXB gene cluster. Collectively, these data show that UBTF-TD interacts with genomic loci at the HOXB gene cluster to directly upregulate their expression. This loss of HOX gene expression leads then leads to loss of a leukemic phenotype. Our data highlights a potential mechanism for UBTF-TD leukemogenesis and sets a foundation where inhibiting the UBTF-TD molecular signature may be a viable therapeutic option.

Citation Format: Juan M Barajas, Masayuki Umeda, Ryan Hiltenbrand, Reethu Krishnan, Tamara Westover, Michael P Walsh, Jing Ma, Sherif Abdelhamed, Jeffery M Klco. UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A45.