Multilevel Molecular Profiling to Dissect Resistance to Tyrosine Kinase Inhibitors in TEL/ABL Positive Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemias (ALLs) bearing TEL/ABL (ETV6/ABL1) fusion gene are generally rare but can be identified within the Ph-like (BCR/ABL-like) ALLs - a poor risk ALL subtype characterized by the presence of various molecular lesions activating kinase signaling. Identification of these druggable lesions brought the possibility to improve the outcome of Ph-like ALL by incorporation of targeted therapeuticals – e.g. tyrosine kinase inhibitors (TKI) – into the current treatment strategies. In vitro studies proved the efficiency of TKI to inhibit TEL/ABL kinase activity justifying TKI use in clinical practice. However, there is a lack of knowledge on potential development of TKI resistance and its mechanism in TEL/ABL-positive ALL.

To address this issue we aimed to establish TKI resistant TEL/ABL-positive cell line and study the mechanism of acquired resistance. After a long-term exposure to gradually growing doses of imatinib mesylate we induced a stable pan-TKI resistance not resulting from the most common mechanisms described in BCR/ABL ALL such as ABL kinase domain mutations, fusion gene amplification or enhanced fusion gene expression/activity.

To elucidate the molecular basis of the resistance we performed multilevel molecular profiling. Using high density whole genome SNP array we identified a single acquired copy number aberration in the resistant cell line - a 60 kb intragenic deletion in KDM6A gene encoding lysine-specific histone demethylase. However, this deletion did not result in expression of the predicted aberrant protein. Using whole exome sequencing we identified 25 non-synonymous single nucleotide variants (SNVs) within gene coding regions to be gained by the resistant cell line. Only half of these variants (12/25) affected genes transcribed in the studied cell line and were thus subjected to further verification by Sanger sequencing. Finally, 5 of the 12 SNVs were confirmed to be stably present and expressed in the resistant cell line. All the affected genes – AEBP1, GNB1, KMT2C, RREB1, STAM2 – are involved in important biological processes and have not been associated with TKI resistance yet.

Gene expression profiling of multiple replicates of parental sensitive and induced resistant cell line identified a gene expression pattern associated with TKI resistance consisting of more than 500 genes with significantly changed expression. Further analysis of this pattern revealed broad changes in many unrelated biological pathways and processes, hence providing a limited potential to specifically focus the study direction.

We further employed two independent methods to study the resistant cell line proteome.

Using 2-dimensional electrophoresis of the whole cell protein lysates we detected a single differentially expressed protein spot present in the resistant compared to parental sensitive cell line. Strikingly, mass spectrometry identified this protein as a transducin beta chain, product of the aforementioned GNB1 gene, one of the five beta subunits of human G-proteins playing an important role as signal transmitters.

Using Size-Exclusion Chromatography-Microsphere-based Affinity Proteomic array (SEC-MAP; Wu, MCP 2009) we analyzed the total expression of more than 300 proteins by flow cytometry. We have found 39 proteins to be differentially expressed in resistant compared to parental sensitive cell line. Interestingly, several distinctive changes indicate enhanced B-cell receptor signaling in the resistant cell line. This mechanism was already implicated as a potential strategy of resistant cells for evading TKI induced apoptosis in BCR/ABL-positive leukemia (Klein, Cell Cycle 2004).

In conclusion we have successfully used several high-throughput technologies to study genomic, transcriptomic and proteomic changes associated with acquired TKI resistance in TEL/ABL-positive ALL model and identified dysregulation of G-protein and BCR-signaling as its potential causes.

Support: IGA MZ NT/13170-4; GAUK 694414; GAUK 596912; UNCE 204012; RVO-FNM64203; BIOCEV –(CZ.1.05/1.1.00/02.0109) from the ERDF