Leukemic Cell Expressing a Novel Kinase Fusion Protein NCOR1-LYN Exhibits High Sensitivity to Dasatinib and Rapamycin

[Background and aim of this study]

Kinase fusion protein is putative therapeutic target of hematological malignancies. Previously, we identified NCOR1-LYN fusion gene in which NCOR1 exon 34 was fused in-frame to LYN exon 7 in a pediatric patient with Ph-like acute lymphoblastic leukemia (ALL) treated according to Japan Childhood Leukemia Study Group ALL-02 protocol (Yano M, et al. Br J Haematol 2015). Because rearrangement of LYN is the recurrent genetic abnormality in high risk B-ALL, targeting therapy for LYN rearranged ALL could be attractive. Thus, in this study, we performed functional analysis of NCOR1-LYN fusion protein to get insight of biological property of this fusion protein to establish targeting therapy for LYN rearranged ALL.

[Materials and Methods]

Full length of NCOR1-LYN cDNA was cloned into pRetroX-Tight-Pur retroviral vector. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with this retroviral vector to establish Ba/F3 cells expressing NCOR1-LYN under doxycycline (DOX) dependent manner. Once Ba/F3 cells expressing NCOR1-LYN were established, cells were analyzed whether IL-3 independent growth was achieved. To determine whether the tyrosine residues of LYN (Y397 and Y508) in this fusion protein were critical for IL-3-independent proliferation, Ba/F3 cells expressing NCOR1-LYN mutants in which both tyrosine residues of LYN were replaced with phenylalanine (Y397F and Y508F) was established. In cytotoxic assay, proliferation of Ba/F3 cells expressing NCOR1-LYN was assessed under the media with tyrosine kinase inhibitors (TKI), such as imatinib and dasatinib. To determine changes of gene expression pattern in Ba/F3 cells expressing NCOR1-LYN, gene expression analysis was performed using Mouse Genome 430 2.0 Array.

[Results and discussions]

The expression of NCOR1-LYN in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3 cells expressing NCOR1-LYN could proliferate without IL-3 in contrast to Ba/F3 cells not expressing NCOR1-LYN couldn't, suggesting that NCOR1-LYN could induce IL-3 independent proliferation of Ba/F3 cells. Western blot analysis also revealed that constitutive tyrosine phosphorylation of NCOR1-LYN fusion protein was present, intriguing that constitutive phosphorylation of tyrosine residues in LYN was associated with IL-3 independent proliferation activity of NCOR1-LYN. Consistent with our hypothesis, Ba/F3 cells expressing NCOR1-LYN mutant (Y397F and Y508F), which didn't show constitutive phosphorylation of NCOR1-LYN, could not proliferate without IL-3.

Considering that TKIs could block constitutive phosphorylation of LYN, TKIs might block the IL-3 independent proliferation of Ba/F3 cells expressing NCOR1-LYN. Cytotoxic assay revealed that 1nM of dasatinib suppressed the proliferation of Ba/F3 cells expressing NCOR1-LYN completely (p<0.01), although imatinib didn't show any effect. Annexin V assay also determined that 96% of Ba/F3 cells treated with dasatinib (10nM, 48hrs) were Annexin positive. However, dasatinib didn't show any effect on Ba/F3 cells not expressing NCOR1-LYN. These findings suggest that dasatinib abolishes the proliferation activity of NCOR1-LYN selectively.

To determine other signaling pathways to be targeted in Ba/F3 cells expressing NCOR1-LYN, Gene Set Enrichment Analysis (GSEA) was performed. GSEA revealed that multiple signaling pathways including mTOR pathway, MYC, E2A and EZH2 related pathways. Because mTOR pathway was activated in Ba/F3 cells expressing NCOR1-LYN (NOM p-value<0.01), mTOR inhibitor might suppress the proliferation activity of NCOR1-LYN. Consistent with our hypothesis, cytotoxic assay revealed that rapamycin (20nM) could suppress the proliferation of Ba/F3 cells expressing NCOR1-LYN efficiently compared to Ba/F3 cells not expressing NCOR1-LYN.

[Conclusion]

Our in vitro study clearly demonstrated that dasatinib and rapamycin could be effective for the patient with B-ALL harboring LYN rearrangement.