Expression of the ETS Transcription Factor GABPα Is Correlated to BCR-ABL/ABL ratio in Human CML and Mediates Imatinib Sensitivity
Authors: Manukjan Georgi , Ripperger Tim , Santer Laura , von Neuhoff Nils , Ganser Arnold , Schambach Axel , Schlegelberger Brigitte , Steinemann Doris 
Published: October 2019

Abstract
Chronic myeloid leukemia (CML) is characterized by the reciprocal translocation t(9;22) leading to a constitutively active ABL kinase as disease driver. For tailored treatment, specific tyrosine kinase inhibitors (TKI) were developed. Still, drug resistance is frequently observed and mostly caused by point mutations in the ABL kinase domain. However, further mechanisms promoting disease progression and TKI resistance are discussed.
The GA-binding protein (GABP) consists of two distinct subunits, GABPα and GABPβ. GABPα belongs to the E26 transformation-specific (ETS) transcription factor family and bears the DNA-binding domain (DBD). GABPβ contains the transcriptional activation (TAD) and the nuclear localization signal (NLS) domains. In mice, GABP is known as an important regulator in myeloid and lymphoid differentiation. Recently, its influence on murine CML-like myeloproliferative disease (MPD) was shown in independent studies (Yu et al., 2012, Cell Stem Cell; Yang et al., 2013, PNAS). Knockout of GABPα or GABPβ in BCR-ABL-transfected hematopoietic stem cells leads to prolonged survival of recipient mice. Focusing on these findings in murine models, we aimed to investigate GABP’s role in human CML.
Initially, GABPA transcript expression was quantified in 70 untreated CML patients at time of diagnosis and displayed a positive and significant correlation to the BCR-ABL/ABL ratio, which is a rough estimate for the tumor cell burden in peripheral blood.
In subsequent in vitro experiments, effects on imatinib response were studied following stable knockdown and ectopic overexpression of GABPA as well as overexpression of a dominant-negative GABPβ TAD deletion mutant in the BCR-ABL+ cell lines K-562 and NALM-1. As observed by enhanced apoptosis and reduced proliferation, knockdown of GABPA in K-562 cells resulted in significantly elevated imatinib sensitivity at concentrations below the determined IC50 value. In accordance with this, ectopic overexpression of GABPA led to enhanced proliferation and clonogenic capacity in comparison to empty vector controls, thus indicating a protective effect against imatinib.
To study GABP’s capability to affect imatinib resistance, the GABPβ TAD deletion mutant was overexpressed in the TKI-resistant cell line NALM-1. This mutant construct lacks the transcriptional activation domain but is still capable of interacting with GABPα as confirmed by co-immunoprecipitation. Consequently, the result is a functionally impaired GABP transcription factor complex. Remarkably, overexpression of the deletion construct sensitizes NALM-1 cells to imatinib as observed in subsequent viability assays.
Furthermore, we analyzed expression of two putative GABP targets with known impact on murine MPD, i.e. protein kinase D2 (PRKD2) and Rho-GTPase RAS-related C3 botulinum substrate 2 (RAC2), in K-562 cells as well as in primary CML samples. Knockdown and overexpression of GABPA in K-562 cells led to accordingly reduced or enhanced expression of PRKD2 and RAC2, respectively. In line with this, PRKD2 expression is significantly elevated in CML patients with prominent GABPA expression.
In summary, our findings demonstrate that GABP plays a role in human CML and highlight the potential clinical relevance of high GABP expression possibly acting in TKI resistance mechanisms. It remains to be shown at which stage of BCR-ABL driven transformation GABP is involved and how GABP and its direct target PRKD2 are functionally embedded in BCR-ABL-triggered pro-proliferative and anti-apoptotic pathways. In this context, it is of interest whether GABP or PRKD2 may serve as alternative treatment targets for TKI-resistant CML.

Disclosures
No relevant conflicts of interest to declare.