靶向miR-185新靶基因PAK6抑制耐药性白血病干细胞的研究

Chronic myeloid leukemia (CML) and BCR-ABL+ acute lymphoblastic leukemia (ALL) stem/progenitor cells are insensitive to tyrosine kinase inhibitor (TKI) monotherapies, as they do not exclusively rely on BCR-ABL activity for survival. This population rapidly generates therapy-resistant clones in vitro ¬and in vivo and is often responsible for disease relapse. Therefore, novel treatments that target new, distinct key molecular events active in leukemic stem cells (LSCs) are needed. MicroRNAs (miRNAs) are small molecules that regulate the gene expression network and are highly deregulated in many cancers. Using Illumina deep sequencing analysis, we have recently many differentially expressed miRNAs in pre-treatment CD34+ CML stem/progenitor cells from CML patients compared to healthy bone marrow controls; among those miR-185 was discovered to be one of the most highly deregulated miRNAs, with significant reduction in CD34+ stem/progenitor cells from TKI-nonresponders compared to TKI-responders. Importantly, restoration of miR-185 expression by lentiviral transduction in CD34+ TKI-nonresponder cells significantly impairs survival of these cells and sensitizes them to TKI treatment in vitro. Restored miR-185 expression in BCR-ABL+ blast crisis cells decreases leukemia burden and enhances survival in immunodeficient mice; these effects are enhanced by TKI, indicating that miR-185 acts as a tumor suppressor and is involved in regulating TKI response/resistance of CML stem/progenitor cells. Several miRNA target genes were further identified by integrating miRNA expression profiles with gene expression profiles from the same patient samples using strand-specific RNA-seq. Strikingly, PAK6, a serine/threonine-protein kinase, was found to be highly upregulated in CML stem/progenitor cells from IM-nonresponders compared to IM-responders. It was confirmed as a target gene of miR-185 by a luciferase reporter assay. PAK6 silencing reduced viability of CML CD34+ cells. Indeed, the use of a pre-clinically validated pan PAK inhibitor (PF-3758309) significantly inhibits CML stem/progenitor cell growth, and these effects can be enhanced by TKIs. Thus, PAK6, a novel target of miR-185, emerges as an attractive druggable target for complementary therapies to target drug-insensitive LSCs. These findings open a new avenue to investigate synergistic effects of drug combinations, to effectively eradicate long-term initiating LSCs from TKI-nonresponders in vitro and in vivo..In this proposal, we hypothesize that the combined suppression of BCR-ABL and miR-185-mediated PAK6 activities to specifically target drug-insensitive LSCs may present a novel therapeutic approach to overcome TKI-resistant CML/ALL. We will undertake the following studies: (1) Characterize the biological effects of genetic and pharmacological suppression of PAK6 in CML/ALL LSCs and their progenitors by lentiviral-mediated shRNA and the use of new PAK6 inhibitors in vitro; (2) Investigate the ability of new PAK6 inhibitors, alone or in combination with TKIs, to inhibit long-term leukemia initiating activity of LSCs and block leukemia development in vivo. This translational study will determine whether combination therapies designed to simultaneously target both BCR-ABL and PAK6 activities in primary CML/ALL LSCs, including dormant LSCs, may improve outcomes in patients.

BCR-ABL诱发慢性髓细胞白血病（CML）和部分急性淋巴细胞白血病（ALL），单独应用激酶抑制剂（TKI）不能治愈疾病。microRNA参与多种生理/病理过程。依据高通量数据提示，我们验证CML干/祖细胞中miR-185低表达，发挥抑癌基因功能；miR-185的新靶基因PAK6（p21(RAC1) activated kinase）高表达；PAK6沉默/PAK抑制剂与IM联合能较单一处理更有效地抑制IM耐受患者CD34+细胞。为研究靶向PAK6与TKI联合清除白血病干细胞的能力，将分离CML/ALL患者祖细胞、干细胞和静止期CD34+细胞，检测PAK6沉默/PAK6抑制剂和TKI单独或联合处理对细胞活性、周期、凋亡、集落生成和长期培养起始细胞的影响；使用异种移植模型评价这两类药物及其组合抑制白血病生成的能力和停药后的长期效果。将为靶向PAK6和TKI联合清除白血病干细胞提供实验基础。

慢性髓细胞白血病（Chronic myeloid leukemia, CML）是源自造血干细胞的恶性血液病。异常活化的酪氨酸蛋白激酶BCR-ABL是诱发疾病的重要因素，针对BCR-ABL的酪氨酸激酶抑制剂（TKI；比如伊马替尼，IM）能治愈部分患者，但药物耐受和复发仍是CML治疗的难点。microRNA（miRNAs）在多种生理和病理过程中发挥重要作用，但有关它在CML细胞中作用与机制的研究还较为有限。为研究miRNAs在调控白血病干细胞命运和药物耐受中的作用，我们获取临床治疗中IM敏感和IM耐受患者在未经药物治疗前的CD34+细胞与健康供体的正常CD34+细胞，并建立miRNA转录组数据。通过比较和鉴定，我们发现miR-185在CML患者CD34+细胞中表达较正常对照细胞显著下降，且miR-185在IM耐受患者CD34+细胞中的表达较IM敏感患者CD34+细胞显著下降。利用随机森林分布策略对临床数据进行机器学习显示miR-185可以预测患者的TKI反应。为研究miR-185在CML中的作用，使用慢病毒递送miR-185和对照载体至BV173细胞和患者CD34+细胞中。异种移植研究证实miR-185的恢复表达可以抑制白血病细胞的体内生长且增强这些细胞对TKI治疗的反应。体外干/祖细胞检测中也获得类似结果。利用组学和信息学数据，我们鉴定到PAK6是miR-185的重要底物。转录组数据显示患者CD34+细胞、特别是IM耐受患者CD34+细胞中氧化磷酸化和活性氧基因集获得富集，提示线粒体活性和活性氧途径受到扰动。PAK6抑制剂降低IM耐受患者细胞中的线粒体活性和活性氧产生，而TKI能显著增强这些效应；两者的共同处理较单一处理能更有效地抑制IM耐受患者细胞的生长并诱发凋亡。综上，研究表明miR-185可能成为预测患者TKI反应的生物标志物，miR-185在CML细胞中发挥抑癌基因功能，同时靶向miR-185下游的PAK6和BCR-ABL可能为克服CML治疗中的药物耐受提供新策略。