Alox-5 As a Potent Therapeutic Target on Overcoming TKI-Resistance in Chronic Myeloid Leukemia with T315I Mutation in Bcr-Abl

Background&Significance: Chronic myeloid leukemia(CML) is a malignant disease of a primitive haematological cell, characterised by inappropriate expansion of myeloid cells. Although the disease is readily controlled by Tyrosine kinase inhibitors, approximately one third of patients will eventually fail treatment. And we believed it corresponds to insensitive leukemia stem cells(LSCs) with unresponsive genes to the kinase inhibitors in CML and mutation of Bcr-Abl. 5-lipoxygenase gene(Alox5) was proved as a novel therapeutic target in cancer stem cells of CML. It encodes a member of the lipoxygenase gene family and plays a role in the synthesis of leukotrienes from arachidonic acid. Without Alox5, Bcr-Abl fails to induce CML in mice due to the impairments of the functions of LSCs. However, recent report suggest that Alox5 regulation may not be important for the development of CML in human. Interestingly, we found specific upregulation of Alox5 in CML patients with strongly positive expression of p210 in mRNA level, including the patients primary diagnosed as CML and who suffered in relapse for TKI resistance. Therefore, we characterized the function and regulation of Alox5 in TKI-resistant CML.

Results: Firstly, we verified the upregulation of Alox5 by real-time PCR on sorted human CML progenitor populations with strongly positive expression of Bcr-Abl(p210), but not on CML patients obtained remission after treatment of TKI. To evaluate the function and regulation of Alox5, we silenced Alox5 by siRNA and chemical inhibitior in human CML cell lines K562, its TKI-resistant cell lines K562R, murine CML cell lines BaF3^wild, and its TKI-resistant cell line BaF3^T315I. As a result, the apoptotic rate induced by Alox5 inhibition alone in K562 and BaF3^wild cells was lower than by TKI. Conversely, blockage of Alox5 in BaF3^T315I cells caused increasingly apoptotic rate, which was higher than imatinib treatment. To further study if Alox5 could play an important role in impairing leukemia stem cells in CML, we cultured LSCs(Lin-c-Kit+Sca-1+) derived from CML patients with Bcr-Abl-T315I mutation in the presence of Alox5 inhibitor or imatinib alone. For 24h treatment, obvious apoptosis was observed in cells cultured with Zileuton, a kind of Alox5 inhibitor, but not in cells cultured with imatinib. Therefore, apoptosis related genes were detected, significant downregulation of Bcl-2 was found compared to in LSCs without Alox5 knockouted. In addition, downregulation of Alox5 followed Bcr-Abl inhibition in CML, we hypothesised that Alox5 was a downstream of Bcr-Abl, genomic array was used to uncover the signaling pathway connected Alox5 with Bcr-Abl. The results shown us that PKC-β was significantly downregulated when Bcr-Abl was inactivated by TKI. Selectivly inhibited PKC-β could decreased Alox5 expression in mRNA level. As for this mechanism, we found p38/MAPK signaling pathway mediated regulation of Alox5 by PKC-β. Next, we evaluated the in vivo anti-CML effect of Alox5 inhibition with a xenograft mice model. Two weeks after the transplantation of human CML cells with mutant Bcr-Abl-T315I(n=12). NOD/SCID/IL2Rg-KO(KOG) mice were treated with normal saline, 10mg/kg Zileuton or imatinib intraperitoneally on everyday schedule. At first, we confirmed that Zileuton doesn't affect normal hematopoiesis(n=3), but eventually inhibite LSCs growth(n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ CML cells and LSCs were significantly reduced by Zileuton treatment in bone marrow((BM) of the recipient mice compared with normal saline-treated control mice and imatinib treated mice, indicating that Alox5 inhibition can inhibit the survival of CML-T315I mutant cells and LSCs in vivo.

Conclusion: Together, these results suggest that Alox5 would be a potent therapeutic target on overcoming TKI-resistance involved in Bcr-Abl-T315I mutation.