探討藉由誘發鐵死亡增加Molm13-XR和MV4-11-XR血癌細胞株對於cabozantinib之敏感性的可行性

Abstract

Acute myeloid leukemia (AML)患者中，約有三分之一的病人帶有FLT3突變，針對FLT3突變臨床上可以使用酪胺酸激酶抑制劑(tyrosine kinase inhibitor, TKI)進行治療，cabozantinib (CBZ)是目前FDA批准治療腎細胞癌 (renal cell carcinoma)、甲狀腺髓質癌 (medullary thyroid carcinoma)的酪胺酸激酶抑制劑。在本篇研究中所使用的細胞株Molm13-XR和MV4-11-XR，是經逐漸上升濃度的cabozantinib長時間培養後，產生抗藥性的AML細胞株。 由於鐵依賴型細胞死亡 (ferroptosis)，是因為細胞內氧化壓力過高，導致脂質過氧化物過度累積，而使造成細胞死亡，再將兩株抗藥性細胞株Molm13-XR和MV4-11-XR的RNA定序資料運用Metascape分析後發現，XR細胞較其母珠細胞中抗氧化能力提升，因此我們假設使用不同類型的ferroptosis誘導劑抑制XR細胞抗氧化系統，促使細胞發生ferroptosis，可以增加XR對於cabozantinib的敏感性，期能找出最能增加誘發Molm13-XR和MV4-11-XR細胞對cabozantinib之敏感性的組合。最後我們發現在Molm13-XR細胞中的ferroptosis誘導劑erastin和RSL3能誘發較高比例的細胞發生脂質氧化 (lipid peroxidation)，在MV4-11-XR則是RSL3能誘發較高比例的細胞發生脂質氧化 (lipid peroxidation)。在合併使用ferroptosis誘導劑與cabozantinib時，Molm13-XR細胞中erastin合併cabozantinib的組合具有能提升細胞對cabozantinib的敏感性，在使用2.7 µM erastin後，cabozantinib的IC50由原本0.6 µM下降至0.2 µM；而在MV4-11-XR細胞中則是RSL3合併cabozantinib能提升細胞對cabozantinib的敏感性。在使用0.05 µM RSL3後，cabozantinib的IC50由原本3.2 µM下降至1.1 µM。上述的結果說明，使用ferroptosis誘導劑後會使XR細胞對於cabozantinib更敏感。

Mutations of FLT3 can be observed in about one-third of AML patients. There are many specific targeted therapies for AML patients with FLT3 mutations, such as midostaurin and sunitinib. They are multikinase inhibitors. Cabozantinib (CBZ) is a tyrosine kinase inhibitor, which was FDA-approved to treat renal cell carcinoma and medullary thyroid carcinoma patients. Previous study in our laboratory discovered that cabozantinib was selectively cytotoxic to AML cell line harboring FLT3 mutations. We established the cabozantinib resistant cell line Molm13-XR and MV4-11-XR by long-term culture with gradually increasing concentrations of cabozantinib. Due to ferroptosis is a kind of programmed cell death which is induced by accumulate ROS (reactive oxygen species) and loss of lipid peroxide repair mechanism. We analyzed the RNA sequencing data of parental (P) and XR cells. Metascape and analysis showed that compared with parental cell, XR cells had enrichment in antioxidant pathway. Therefore, we hypothesized that inhibiting the antioxidant system in XR cells using different types of ferroptosis inducers could promote ferroptosis and increase the sensitivity of XR cells to cabozantinib. In this research, we wanted to identify the most effective combination that would enhance the sensitivity of Molm13-XR and MV4-11-XR cells to cabozantinib. We found that the ferroptosis inducers erastin and RSL3 could induce higher levels of lipid peroxidation in Molm13-XR cells, while RSL3 induced higher levels of lipid peroxidation in MV4-11-XR cells. The combination of erastin and cabozantinib showed synergistic effect which can enhance effective of cabozantinib in Molm13-XR cells. After treatment with 2.7 µM erastin, the IC50 of cabozantinib decreased from 0.6 µM to 0.2 µM. In MV4-11-XR cells, the combination of RSL3 and cabozantinib also had synergistic effect. After treatment with 0.05 µM RSL3, the IC50 of cabozantinib decreased from 3.2 µM to 1.1 µM. These results demonstrate that ferroptosis inducers increase the sensitivity of XR cells to cabozantinib.