Functional Analysis Of SEPT9-ABL1 Chimeric Fusion Gene Derived From T-Prolymphocytic Leukemia

ABL1 gene encoding a non-receptor tyrosine kinase has been involved as ABL1 chimeric genes in hematological malignancies. Their partner genes include BCR in chronic myeloid leukemia (CML), B-acute lymphoblastic leukemia (B-ALL), T-acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML), ETV6 in B-ALL, T-ALL, AML and myeloproliferative neoplasms (MPNs), RCSD1, SFPQ, ZMIZ1, FOXP1, SNX2, GAG in B-ALL, NUP214 and EML1 in T-ALL. The resultant chimeric gene products have been shown to associate with a cellular proliferation through constitutive activation of ABL1 signaling. In addition, they are the therapeutic targets for tyrosine kinase inhibitors (TKIs).

We identified a novel chimeric fusion gene SEPT9 (Septin9)-ABL1 in a case of T-prolymphocytic leukemia (T-PLL) using 5’- RACE PCR. It is generated by an in-frame fusion between SEPT9 exon 4 and ABL1 exon 2. The case was treated with imatinib and dasatinib, but the tumor burden did not reduce at all. The purpose of this study was to analyze the biological functions of the SEPT9-ABL1 gene product including TKIs sensitivity.

The expression analysis in the case using RT-PCR and Western blot revealed that there were at least 4 isoforms applying SEPT9a, SEPT9d, SEPT9e and SEPT9f as the fusion partners. SEPT9a-ABL1 was expressed most strongly among the SEPT9-ABL1 isoforms.

The SEPT9-ABL1 isoforms were retrovirally transduced into 32D cells, a murine interleukin3 (IL-3) dependent hematopoietic cell line. All of these isoforms caused 32D cells an IL-3 independent proliferation. Their proliferation rates were less than 32D cells with BCR-ABL1, and that of 32D cells with SEPT9d-ABL1 was subtle (day0, 1×10⁵ cells/ ml; day2, the densities of 32D cells with BCR-ABL1, SEPT9a-ABL1, SEPT9d-ABL1 SEPT9e-ABL1 and SEPT9f-ABL1 were 17.8×10⁵, 6.7×10⁵, 2.7×10⁵, 6.0×10⁵ and 6.1×10⁵ cells /ml, respectively). In consistent with the results of cellular proliferation, the immunocytological analysis revealed SEPT9a-ABL1, SEPT9e-ABL1 and SEPT9f-ABL1 proteins localized mainly in the cytoplasm.

When these cells were transplanted into C3H/HeJ mice (5×10⁶/ mouse). Mice with SEPT9e-ABL1- and SEPT9f-ABL1-infected cells died with the infiltration of transplanted cells into bone marrow, spleen and liver within 50 days. It was later than mice with BCR-ABL1 infected cells.

Half maximal inhibitory concentrations (IC₅₀) of imatinb in 32D cells infected with BCR-ABL1, SEPT9a-ABL1, SEPT9e-ABL1 and SEPT9f-ABL1 were 2.52μM, 0.96μM, and 2.08μM, respectively. They were higher than IC₅₀ with BCR-ABL1 (0.28μM)( SEPT9a-ABL1, 9.0 times, p<0.001; SEPT9e-ABL1, 3.4 times, p=0.011; SEPT9f-ABL1, 9.0 times, p=0.009). In addition, IC₅₀ of dasatinib in the above cells were 12.6nM, 15.8nM and 16.0nM, respectively. They were also higher than IC₅₀ with BCR-ABL1 (0.66nM) (SEPT9a-ABL1, 19.1 times, p=0.019; SEPT9e-ABL1, 23.9 times, p=0.016; SEPT9f-ABL1, 24.2 times, p<0.001)

These data demonstrated that SEPT9-ABL1 fusion gene provide autonomous cellular proliferation in vitro and in vivo, according to the isoforms which showed different cellular distribution. In addition, the sensitivity of 3 evaluated SEPT9-ABL1 isoforms to TKIs was significantly higher than BCR-ABL1, confirming the TKI- resistant character as seen in the case.