Leukaemic cells expressing ETV6::FRK are sensitive to dasatinib in vivo

Abstract ETV6::Fyn‐related kinase (FRK), which is a Src family tyrosine‐kinase‐related fusion gene and firstly identified in our patient with paediatric high risk B cell precursor acute lymphoblastic leukaemia (B‐ALL), has no evidence of efficacy of tyrosine kinase inhibitor in vivo. We performed functional analysis of ETV6::FRK to establish molecular targeting therapy and determined that dasatinib could abrogate proliferation activity of ETV6::FRK through the repression of FRK‐STAT3/STAT5 pathway in vitro and significantly extended the survival time of the xenografted mice in vivo (p < 0.01). Our data support the potential of dasatinib as a therapeutic option for patients with B‐ALL harboring FRK rearrangements.


INTRODUCTION
Fyn-related kinase (FRK) is a member of the breast tumuor kinase family of tyrosine kinases, which are related to Src family tyrosine kinases. FRK was originally attributed a tumour-suppressive function; however, further functional characterization in diverse cancer types has revealed that it can also play oncogenic roles [1]. ETV6::FRK, which is chimeric fusion gene comprising the oncogenic transcription factor, ETV6 (ETS variant transcription factor 6, also known as TEL), and FRK, was first identified in acute myeloid leukaemia (AML) [2]. Expression of ETV6::FRK induces IL-3-independent growth in Ba/F3 cells, suggesting that this fusion is a critical pathogenic driver of leukaemogenesis [2]. Other FRK-related fusion genes, including CAPRIN1::FRK, PABPC1::FRK, and MAPK9::FRK, have been identified in ALK-negative anaplastic large cell lymphoma (ALCL) [3]. These findings suggest that kinase of the Src family, the tyrosine kinase inhibitor, dasatinib, has been proposed as a potentially effective treatment for targeting cells harboring FRK-related fusion genes; however, its efficacy has only been reported in vitro against cells carrying CAPRIN1::FRK [3]. Thus, targeting FRK-related fusion proteins with dasatinib warrants more extensive investigation. Herein, we first identified the ETV6::FRK fusion gene in a patient with high-risk B-cell precursor acute lymphoblastic leukaemia (B-ALL) and performed functional analysis of cells expressing ETV6::FRK to evaluate the efficacy of targeting FRK with dasatinib in vitro and in vivo.

Patient and methods
Our patient was 11-year-old boy with B-ALL who was resistant to induction chemotherapy. 46,XY,t(6;12)(q21;p13) karyotype ( Figure 1A). To search for the potential fusion gene, target capture RNA-Seq was performed using RNA isolated from the diagnostic leukaemic sample, as described previously [4]. Chimera gene-inducible cell lines were generated by retroviral transfection using the Retro-X Tet-On Advanced Inducible Expression System (Takara Bio, USA). Generation of retroviral supernatants by Plat-E cells, infection and transduction of the interleukin (IL)−3-dependent murine pro-B-cell line, Ba/F3 cells, was carried out as previously described [5]. Cell proliferation assay, cytotoxic assay and apoptosis assay were also performed as described previously [5].
To characterize the transcriptome profile of established cells, whole transcriptome sequencing (WTA) was conducted (Macrogen Japan, Waltham, MA, USA), as described previously [7]. All mouse studies were approved by institutional committees. An unpaired two-tailed Student's t-test was used to determine the significance of differences and p-values < 0.05 considered statistically significant. Kaplan-Meier curves for survival analyses were compared using the log-rank test.
Next, to investigate which pathways were affected by ETV6::FRK, we compared gene expression profiles from Ba/F3-ETV6::FRK cells with those of non-DOX-induced control cells using WTA and GSEA.
WTA revealed that, among 14238 genes analysed, expression of 394 and 121 were increased and decreased (log 2 fold change ≥ |2.0|; adjusted p < 0.05), respectively, in Ba/F3-ETV6::FRK cells compared with controls ( Figure 1G). The top 20 up-and down-regulated genes are listed in Table S1. GSEA of oncogenic gene sets (C6) from the GSEA Molecular Signatures Database [6] revealed that, compared with control cells, Ba/F3-ETV6::FRK cells were enriched for up-regulation of SNF5 target genes and down-regulation of RB target genes involved in cell cycle regulation ( Figure 1H, Table S2). Ba/F3-ETV6::FRK cells were also enriched for down-regulation of ALK target genes, consistent with previous reports that FRK and ALK regulatory networks share several proteins in common, including STAT3 [1,8]. Further, the observed enrichment for up-regulation of EGFR target genes may reflect that the EGFR signalling inhibition function of FRK is diminished in the context of ETV6::FRK, relative to wild-type FRK [9].
To investigate whether dasatinib can block constitutive phos- In this study, we analysed the functional consequences of the ETV6::FRK fusion, which we first identified in a patient with B-ALL.
The ETV6::FRK chimeric protein enhanced cell proliferation and constitutional tyrosine autophosphorylation of ETV6::FRK was critical for this proliferative activity. Although the downstream signalling pathways mediated by ETV6::FRK were not fully elucidated in previous studies [2], our findings reveal that ETV6::FRK is catalytically active in the FRK-STAT pathway, as evidenced by the observation of constitutive phosphorylation of STAT3, STAT1 and STAT5. These data are consistent with another report that the FRK fusion gene, CAPRIN1::FRK, identified in ALCL, induced phosphorylation of STAT3 in the Ba/F3 model [3].
Dasatinib is considered potentially effective for the treatment of cancers with FRK rearrangements; however, evidence of dasatinib use is sparse and limited to in vitro studies; preclinical data showed that dasatinib inhibits CAPRIN1::FRK-driven cell growth in vitro in ALK-negative ALCL [3]. Here, we clearly demonstrate that dasatinib is effective in inhibiting ETV6::FRK-driven cell growth in vitro and in vivo, as demonstrated using xenograft models. Our data support the hypothesis that dasatinib is effective for treatment of haematological malignancies harboring FRK-related fusion genes.
In summary, we report: (1) the first identification of the rare kinase-