Overcoming Tyrosine Kinase Inhibitor Resistance to SEPT9-ABL1 Chimeric Fusion Protein Using CRM1 Inhibitors

ABL1 fusion genes, which have been reported in various hematological malignancies, are thought to contribute to leukemogenesis through aberrant ABL1 signaling and activating multiple downstream signals. Tyrosine kinase inhibitors (TKIs), such as imatinib and dasatinib, have been developed to inhibit the function of the most- characterized ABL1 fusion product, BCR-ABL1. Because TKIs target the ABL1 kinase domain, ABL1 fusion products are usually sensitive to TKIs both in vitro and in vivo .

We previously identified the chimeric fusion gene SEPT9 - ABL1 in a case of T-prolymphocytic leukemia that was unexpectedly resistant to imatinib and dasatinib without mutations in the domains derived from ABL1. We proved that SEPT9-ABL1 exhibited TKI resistance in vitro and in vivo, as seen in theclinical course (Leuk Res 2014). The purpose of this study was to reveal the mechanism underlying TKI resistance and to overcome it in a specific way.

SEPT9f-ABL1, which has the most typical characteristics among SEPT9 isoforms, was retrovirally transduced into interleukin-3 (IL-3)-dependent murine hematopoietic cell lines 32D and BaF3 cells to generate 32D/SEPT9-ABL1 and BaF3/SEPT9-ABL1 cells. 32D/BCR-ABL1 and BaF3/BCR-ABL1 cells were also generated as references. The 50% inhibitory concentration (IC₅₀) of imatinib was higher with SEPT9-ABL1 than with BCR-ABL1 (in 32D cells and BaF3 cells, respectively), confirming that SEPT9-ABL1 contributed to TKI resistance.

Because TP53 has been suggested to involved in TKI resistance, we first analyzed the TP53 expression and phosphorylation in these cells. Western blot revealed that TP53 was downregulated and less phosphorylated in the cells expressing SEPT9-ABL1 than in those with BCR-ABL1. These findings were consistent with the analysis using primary leukemic cells. Imatinib treatment reduced the TP53 expression in the cells expressing SEPT9-ABL1. When the cell pellets were fractioned, the TP53 expression in the cytoplasm was comparable between cells expressing SEPT9-ABL1 and those with BCR-ABL1 under imatinib treatment. However, the TP53 expression remained higher in the nucleus of the cells with BCR-ABL1 than in those with SEPT9-ABL1, suggesting that the decreased nuclear TP53 expression prevented the cells expressing SEPT9-ABL1 from undergoing apoptosis induced by imatinib.

Because the distribution of TP53 in the nucleus and the cytoplasm is controlled by chromosomal region maintenance 1 (CRM1), a nuclear export receptor, we next evaluated the effects of CRM1 inhibition. When the cells were cultured with the combination of imatinib (1 µM) and KPT-185 (1 µM), TP53 was markedly accumulated in the cells expressing SEPT9-ABL1; however, no such accumulation was noted when cells were cultured with imatinib alone, suggesting that KPT-185 treatment enhanced the effect of imatinib in the cells expressing SEPT9-ABL1 through the inhibition of TP53 export from the nucleus. This combination dramatically increased the apoptotic cells with SEPT9-ABL1. To evaluate CRM1 inhibition in vivo, we utilized a subcutaneous tumor model with BaF3/SEPT9-ABL1 cells. Treatment with imatinib 20 mg/kg plus another CRM1 inhibitor (KPT-330, 20 mg/kg) led to the significant regression of tumors composed of BaF3/SEPT9-ABL1 cells compared to imatinib alone (p=0.029).

In conclusion, decreased nuclear expression of TP53 is a possible mechanism underlying TKI resistance in cells harboring SEPT9-ABL1. CRM1 inhibition is an effective method of overcoming TKI resistance in cells with SEPT9-ABL1 through induction of the nuclear accumulation of TP53 and apoptosis.

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