Cdc42对范可尼贫血骨髓龛中造血干细胞的调控及其机理

Fanconi anemia（FA) is a rare inherited diseases associated with bone marrow failure (BMF), variable congenital/developmental abnormalities, and cancer susceptibility.The two most important clinical hallmarks of FA are BMF and progression to leukemia caused by hematopoietic stem cell (HSC) depletion and malignant transformation. Rho GTPase, Cdc42 acts as an intracellular signal transducer in response to a variety of extracellular stimuli. It is uniquely required for HSC retention and maintenance in the BM. Based on the preliminary results, the project will employ two in vivo systems: Murine Notch-eGFP/Fanca-/- model and Human FA xenograft model,to test an innovative hypothesis that Cdc42 may not only influence HSC renewal but also affect HSC residence in the BM niche, and that targeting Cdc42 could vacant BM niche otherwise occupied by FA mutant HSC or leukemic stem cells (LSCs) and allow wild-type or gene-corrected HSC to engraft. The goal of the project is to address two critical issues in the FA hematopoietic stem cell transplantation (HSCT) and gene therapy field: (1) the mechanism of FA HSC loss and (2) the urgent need for developing innovative therapy to improve FA HSCT. Especially we will investigate the mechanistic link between Cdc42-regulated cell polarity and FA HSC renewal and engraftment defects. We will also optimize CASIN regimen for effectively vacating recipient niche and engrafting donor HSCs; examine CASIN-mediated niche opening and donor HSC lodging in Fanca-/- mice by 2-photon confocal microscopy; and develop CASIN-based non-myeloablative preconditioning regimen for gene-corrected FA HSC engraftment. Scientifically, the proposed study has the potential to improve mechanistic understanding of HSC renewal and engraftment in general. Clinically, the project has the promise of developing innovative therapeutic regimens for stem cell and gene therapies in FA and other BM failure diseases, clinical settings in which stem cell numbers critically limit success and recipient patients are hypersensitive to pre-conditioning toxicity. In addition, the insights gained through these studies can be utilized to develop Cdc42 inhibitors with higher efficacy and less toxicity, for leukemic stem cell targeting and for HSC polarity.

骨髓衰竭是范可尼贫血（FA）典型的临床表现，是造血干细胞（HSC）从骨髓龛逐渐流失的反映。Cdc42是调控骨髓龛中HSC的关键因子。本项目以已有的FA HSC cdc42相关实验结果为基础，以已建立的Notch-eGFP/Fanca-/-小鼠和人性化的FA NSGS异种移植小鼠为模型:①揭示HSC极性状态对其不对称分裂模式的影响，比较极性、非极性FA HSC移植后的造血重建功能，阐明cdc42活性下降引起了FA HSC极性缺失，减少了不对称分裂模式而导致自我更新功能和造血重建功能减退的机理；②探讨cdc42活性抑制剂CASIN是否通过增加FA骨髓龛开放率而提高了正常或FA基因修复的HSC在 FA受体中的植入效果，明确CASIN有效动员FA HSC的机理。研究结果不仅有助于阐明FA骨髓衰竭的病理生理学分子基础，而且有助于形成以CASIN为基础的用于FA基因修复HSC移植的非清髓预处理新方案。

范可尼贫血（Fanconi anemia, FA）是一种由基因变异引发的遗传性贫血，临床上常见多发性先天畸形和骨髓衰竭等，易发展为白血病。其骨髓衰竭和白血病的发生是造血干细胞（HSCs）的衰竭和恶性转化导致的。骨髓龛是HSCs的支持环境，骨髓间充质基质细胞（MSCs）是骨髓龛的主要成份，对HSCs的维持至关重要。Cdc42是Rho GTP酶的一种，在调解细胞的极性形态和HSCs的分化中起很重要的作用。我们通过采用两种小鼠模型，研究了FA MSCs的造血支持功能。发现Fanca-/-和Fancc-/-小鼠的MSCs体外培养时增殖能力减弱且易于衰老。进一步研究表明， FA MSCs中的Cdc42活性降低。体内与体外实验均证明，与Fanca-/-和Fancc-/-的MSCs共培养后的WT HSCs 与WT MSCs共培养后的WT HSCs相比自我更新能力降低。FA缺陷基因修复可以恢复Cdc42的活性和改善FA MSCs的造血支持功能；活性持续的Cdc42突变体Cdc42F28L的强制表达可以明显改善FA MSCs的造血支持功能；采用非经典Wnt5a提高Cdc42的活性亦可以改善FA MSCs的造血支持功能。机理研究表明，FA蛋白通过调整Wnt5a的表达来协调Cdc42的活性，从而影响了FA HSCs自我更新功能和造血重建功能，因此，提高Cdc42活性有助于FA的干细胞治疗。另外，我们发现蛋白精氨酸甲基转移酶（PRMT5）活性降低与原癌基因K-rasG12D引起的白血病发生相关，对FA HSCs进行强化PRMT5表达可减缓受体小鼠的白血病发生；还发现免疫受体Trem1表达升高协同FA基因变异引起的DNA损伤反应降低，对Fanca缺陷的小鼠白血病前期小鼠造血干祖细胞扩充起到推动作用。这些结果为防治FA发展为白血病提供了病理生理学的理论依据。