骨髓基质细胞分泌小分子物质促进白血病细胞谷胱甘肽代谢并抗凋亡和耐药的研究及白血病治疗新方案的探讨

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries, and there is distinct increasing incidence trend of CLLL in Eastern countries. The interaction between CLL cells and the bone marrow stromal environment is thought to play a major role in promoting the leukemia cell survival and drug resistance. Here we study a novel biochemical mechanism by which the bone marrow stromal cells exert a profound influence on the redox status of primary CLL cells and enhance their ability to sustain oxidative stress and drug treatment. Fresh leukemia cells isolated from the peripheral blood of CLL patients exhibited two major redox alterations when they were cultured alone: a significant decrease in cellular glutathione (GSH) and an increase in basal ROS levels. However, we found that when cultured in the presence of bone marrow stromal cells, CLL cells restored their redox balance with an increased synthesis of GSH, a decrease in spontaneous apoptosis, and an improved cell survival. Further study showed that CLL cells were under intrinsic ROS stress and highly dependent on GSH for survival, and that the bone marrow stromal cells secreted low molecular weight molecule to promote GSH synthesis in CLL cells. Cysteine is a limiting substrate for GSH synthesis and is chemically unstable. Cells normally obtain cysteine by uptaking the more stable and abundant precursor cystine from the tissue environment and convert it to cysteine intracellularly. Based on our preliminary data and literature report, we hypothesize that bone marrow stromal cells effectively take up cystine and reduce it to cysteine for secretion into the tissue microenvironment to be taken up by CLL cells for GSH synthesis. The elevated GSH in CLL cells in the presence of bone marrow stromal cells could significantly protect the leukemia cells from stress-induced apoptosis, and render them resistant to standard therapeutic agents such as fludarabine and oxaliplatin. Importantly, we propose to test the significance of disabling of this protective mechanism by depletion of cellular GSH using a pharmacological approach to potently sensitize CLL cells to drug treatment, and effectively enhance the cytotoxic action of fludarabine and oxaliplatin against CLL in the presence of stromal cells. We expect to reveal a key biochemical mechanism of leukemia-stromal cells interaction, and identifies a new therapeutic strategy to overcome drug resistance in vivo.

慢性淋巴细胞白血病（CLL）至今仍然是不可治愈的恶性血液系统疾病。现有的化疗方案虽然可以达到缓解的目的,抗药导致复发是目前主要的临床问题，克服CLL抗药性已经成为治疗CLL研究的热点和难点。而在CLL抗药性研究中，骨髓基质细胞与CLL细胞相互作用以尚不明确的机制，极大影响了CLL细胞的存活和对药物的反应。申请人通过共同培养原代CLL细胞与骨髓基质细胞体外模拟骨髓微环境，首次提出了一种全新的生化机制：骨髓基质细胞通过摄取细胞外充足的胱氨酸，以此合成并释放半胱氨酸，供给CLL细胞谷胱甘肽（GSH）合成从而促进其存活和抗药性。为证实该假设，本课题将研究胱氨酸在骨髓基质细胞与CLL细胞共培养中的代谢通量，以及参与CLL细胞存活和耐药形成。在此机制基础上，本项目将通过白血病小鼠模型研究相关方案的体内药效。该课题能够进一步揭示CLL的抗药机制，对于设计新的治疗方案，提高CLL临床治疗水平具有重要意。

慢性淋巴细胞白血病 （Chronic lymphocytic leukemia，CLL）是一种成熟淋巴细胞恶性增生性疾病。CLL细胞抵抗传统药物诱发凋亡导致病情复发是目前主要的临床问题，克服CLL细胞耐药性是新治疗方案研发的热点和难点。骨髓微环境是CLL细胞逃避常规化疗的避难所。研究表明骨髓基质细胞与CLL细胞相互作用，对CLL细胞的存活和耐药起了重要作用，躲避于骨髓中的耐药CLL细胞成为白血病复发的根源。我们通过共同培养原代CLL细胞与骨髓基质细胞体外模拟骨髓微环境，研究骨髓基质细胞对CLL细胞抵抗传统化疗药物氟达拉滨和奥沙利铂，以及抗肿瘤潜力药物组蛋白去乙酰化酶抑制剂SAHA诱发凋亡的保护作用和机制，并在机制研究的基础上设计新的治疗药物和方案阻断骨髓基质细胞与CLL细胞之间的相互作用。我们发现，CLL细胞依赖胞内抗氧化物谷胱甘肽（glutatione，GSH）对抗内源性和SAHA等药物诱导的氧自由基（ROS）损伤。细胞合成GSH受限速酶催化亚基GCLC表达和必须底物半胱氨酸（cysteine）浓度的影响。我们的机制研究发现，一方面，骨髓基质细胞通过高表达转运蛋白Xc-摄取细胞外充足的氧化态胱氨酸（cystine），以此合成并释放还原态cysteine，供给CLL细胞合成GSH的必须底物；另一方面，SAHA通过上调CLL细胞中GCLC表达，与骨髓基质细胞共同为CLL细胞提供GSH合成的必须条件，造成CLL细胞中GSH大量合成，从而抵抗自身和SAHA诱发的自由基损伤。在此机制研究的基础上，我们将GSH清除剂PEITC与SAHA联合使用，一方面，PEITC与CLL细胞中高浓度GSH结合并迅速排出细胞外；另一方面，PEITC通过去谷胱甘肽化降低抗凋亡蛋白Mcl1稳定性促使其降解，从而极大提高SAHA对共培养CLL细胞的杀伤作用。在体内实验中，我们进一步验证了SAHA与PEITC联合用药对延长白血病小鼠生存期的影响。本课题首次揭示CLL细胞与骨髓微环境通过小分子物质相互作用并导致耐药的机制，解释了抗肿瘤潜力药物SAHA在临床试验中对治疗CLL病人效果不理想的重要原因，并证明PEITC与SAHA联合使用可以切断CLL细胞与骨髓基质细胞的相互作用，极大提高SAHA的抗白血病效果。