T-cell Acute Lymphoblastic Leukemia (T-ALL) accounts for about 15% of pediatric ALL and is characterized as a high-risk disease with frequent relapse, chemotherapy resistance, and a poorer prognosis. LIM domain only protein 2 (LMO2) is a regulator of hematopoiesis and an oncogene that is overexpressed in about 10% of T-ALL. The IKZF1 gene encodes a zinc finger protein called Ikaros which is a master regulator of lymphoid development and a tumor suppressor. In pediatric ALL, Ikaros alteration is considered independent prognostic marker for poor outcome. Pro oncogenic Casein Kinase II (CK2) is overexpressed in various malignancies including leukemia. CK2 directly phosphorylates Ikaros in vivo and inhibits its function as transcriptional regulator. Inhibition of CK2 restores Ikaros tumor suppressor function and results in anti-leukemic effect. Objective of this study is to understand the mechanisms of transcriptional regulation of LMO2 in T-cell ALL. Global chromatin immunoprecipitation (ChIP) coupled with the next-generation sequencing (ChIP-seq) studies in primary human ALL cells and in cell lines, demonstrated Ikaros occupancy of the promoter of LMO2 gene. We hypothesize that Ikaros negatively regulates expression of LMO2 at transcriptional level and CK2 impairs Ikaros mediated repression of LMO2 in T-cell ALL.
Results: qChIP in primary leukemia cells confirmed that Ikaros binds to the promoter region of LMO2. Using gain-of-function and loss-of-function experiments we dissected the role of Ikaros in regulation of LMO2 transcription in T-ALL. Ikaros silencing using shRNA transfection revealed increase in LMO2 expression as measured by qRT PCR. Conversely, overexpression of Ikaros was associated with strongly reduced transcription of LMO2. T-ALL cells that are derived from Ikaros-knockout mouse models express high level of LMO2. Retroviral transduction of these cells with Ikaros, resulted in significant reduction of LMO2 expression. Next we investigated how CK2 affects the regulatory functions of Ikaros towards LMO2. Molecular and pharmacological inhibition of CK2 resulted in reduced expression of LMO2 in primary human T-ALL. Ikaros binding at promoter of LMO2 was noted to be significantly increased following CK2 inhibition. This effect was not seen when cell were subjected to CK2 inhibition after Ikaros silencing. Further, we analyzed changes in the histone markers at the heterochromatin at the LMO2 promoter following Increased Ikaros binding such as reduced histone H3K9ac and H3K4me3 markers. This suggests that Ikaros regulates LMO2 transcription via chromatin remodeling.
Conclusion: This data reveals new regulatory mechanism for oncogene LMO2 in pediatric T-ALL. New evidence suggests that repression of LMO2 expression in T cell ALL via Ikaros can be potentiated using CK2 inhibitors. Findings provide the rationale for the use of CK2 inhibitors in T-ALL with LMO2 overexpression.
Citation Format: Chandrika S. Gowda, Yali Ding, Chunhua Song, Malika Kapadia, Kimberly J. Payne, Sinisa Dovat. Signaling pathways that regulate LMO2 oncogene expression in pediatric high risk T cell acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5533. doi:10.1158/1538-7445.AM2017-5533