隐丹参酮靶向STAT3/PKR复合物介导的自噬通路诱导慢性粒细胞白血病干细胞分化的功能研究

Chronic myelogenous leukemia (CML) results from malignant transformation of a hematopoietic stem cell by the BCR/ABL oncogene. Imatinib mesylate (IM) induces clinical remissions in chronic-phase CML patients but IM resistance remains a problem. Primitive, quiescent CML leukemia stem cells (LSC) are resistant to apoptosis following IM treatment despite effective inhibition of BCR/ABL kinase activity.Since IM fails to eradicate CML at the stem cell level, strategies targeting LSC are required to achieve cure for CML patients. Autophagy is a dynamic process of protein degradation, which is essential for cell survival, differentiation, development and homeostasis. Accumulating evidence indicates autophagy plays an essential role in the regulation of differentiation of cancer stem cells (CSC), suggesting autophagy could be a promising therapeutic target for CSC. PKR, a eukaryotic translation initiation factor 2α (eIF2α) kinase, is known to act as a potent inducer of autophagy. During viral infection and stravation, PKR increased the phosphorylation of eIF2α, induing translational arrest which contributes to the induction of autophagy. Recent studies indicated that STAT3 interacted with PKR via the SH2 domian, forming a complex negativaly regulates autophagy. The STAT3/PKR complex provides a cross-regulation of differentiation and autophagy in the setting of cell stress, providing insights into their reciprocal roles in carcinogenesis. Disruption of the STAT3/PKR complex would facilitate the autophagy induction, which would contribute to the differentiation of cancer stem cell. Cryptotanshinone (CPT) is a major active component of Salvia miltiorrhiza. Recent studies indicated that CPT exhibited obvious antitumor activity in a diverse set of cancer cells. CPT could inhibit the activity of STAT3 by binding to the SH2 domain. Our studies demonstrated that CPT induced cell cycle arrest and apoptosis in multidrug resistant human CML cells. In addition, we found that CPT could inhibit the growth of primary CML CD34+ LSC cells, without obvious effect on the normal CD34+ progenitor cells. Thus, we plan to determine the CPT function on cell differentiation and autophagy of CML LSC, and clarify its specific mechanism. Our study would investigate the effect of CPT on differentiation of CML LSC cell, determine the role of autophagy and STAT3/PKR complex, and study the mechanism by which CPT regulates this complex. We would determine the effect of CPT on the dissociation of STAT3 and PKR, confirming that whether CPT increased the PKR dissociation through competitive combination to STAT3. The clarification of STAT3/PKR complex may provide new biomarkers for diagnosis and drug screening targeting CML LSC. Furthermore, our studies would confirm the anticancer effect of CPT targeting the LSCs, and provide candidate agent for CML therapy.

白血病干细胞是导致慢性粒细胞白血病(CML)耐药和复发的重要原因之一，开发靶向白血病干细胞的药物将有利于CML的治疗。本课题拟以中药单体隐丹参酮为研究对象，深入研究隐丹参酮诱导CML白血病干细胞分化的功能和机制。在细胞和动物水平研究隐丹参酮对白血病干细胞分化和成瘤能力的影响，并明确隐丹参酮对伊马替尼治疗的"增效减毒"功能；以STAT3/PKR复合物介导的自噬通路为切入点，研究自噬在隐丹参酮诱导干细胞分化中的功能；分析隐丹参酮对STAT3/PKR复合物的影响，明确隐丹参酮靶向STAT3/PKR复合物激活干细胞自噬的功能；研究隐丹参酮对STAT3、PKR结合能力的影响，阐明隐丹参酮发挥功能的分子机制。本研究将明确隐丹参酮在白血病干细胞分化中的功能和机制，为其在CML治疗中的应用奠定基础，STAT3/PKR复合物在白血病干细胞自噬和分化中功能的阐明为靶向干细胞药物的开发提供了新的筛选靶点。

伊马替尼是靶向Bcl/Abl基因的特异性分子抑制剂，目前已成为初治慢性粒细胞白血病（CML）患者的临床一线用药，但日益严重的耐药问题限制了其进一步的临床应用。寻找新的有效药物和干预手段，提高和改善伊马替尼的化疗敏感性和临床疗效，将有有利于耐药CML患者的治疗。.白血病干细胞聚集于CML患者的骨髓微环境中，伊马替尼等化疗药物无法清除其形成的药物病灶，是导致CML患者耐药和复发的重要原因之一。本项目在前期工作的基础上，以白血病干细胞和中药活性化合物隐丹参酮为切入点，对隐丹参酮体内外抗白血病的功能进行了系统研究。明确了CPT可通过抑制STAT3通路的活性，激活caspase依赖的凋亡途径，引起细胞G0/G1的细胞周期阻滞，发挥抗白血病功能；证实了CPT对伊马替尼体内外药效的影响，其作用机制不依赖于CML细胞中P-糖蛋白的表达抑制和胞内药物浓度的提高，而与Bcr-abl信号通路活性及其STAT3、eIF4E、mTOTR等下游靶点的活性降低有关，两者联合的体内抑瘤率可达60.2%。该研究明确了隐丹参酮与伊马替尼在治疗白血病中的“增效减毒”功能，阐明了两者协同的分子机制。.自噬调节与白血病干细胞的分化密切相关，本项目以STAT3/PKR通路介导的自噬为切入点，对白血病干细胞分化过程中，自噬相关基因的表达情况进行了研究。结果表明，低水平的自噬是维持白血病干细胞“干性”的重要因素，诱导白血病干细胞向巨核细胞和红系分化后可提高ATG4、Beclin1和LC3的表达水平，激活细胞自噬；抑制细胞自噬，可降低CD41和CD11b等成熟细胞标记物，阻断PMA对白血病干细胞的诱导效果；隐丹参酮可抑制STAT3通路的活性，加速STAT3/PKR复合物在胞内的解离，提高PKR的磷酸化水平，激活白血病干细胞的自噬，诱导其分化。.本项目的实施明确了STAT3/PKR介导的自噬通路在白血病干细胞中的功能，为靶向干细胞药物的开发提供了新的筛选靶点；隐丹参酮体内外抗白血病功能的明确，为耐药CML患者提供了新的化疗手段和策略，也为其进一步药物开发奠定了基础。