Abstract：
BACKGROUND: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive.
METHODS: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate.
RESULTS: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation.
CONCLUSIONS: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.
METHODS: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate.
RESULTS: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation.
CONCLUSIONS: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones.
摘要：
背景：急性髓性白血病（AML）是一种造血系统癌症，其特征是髓系未分化细胞的过度增殖。虽然大多数AML疗法都集中在肿瘤减积上，全反式维甲酸(ATRA)诱导AML亚型急性早幼粒细胞白血病(APL)中性粒细胞分化.巨自噬已在各种癌症的背景下进行了广泛的研究，并且在AML中经常失调，在AML中它可能具有环境依赖性的促或抗白血病作用。相反,伴侣介导的自噬(CMA)对疾病病理生理学的影响仍在探索中,其在AML中的作用仍然难以捉摸.
方法：我们利用人类AML原发样本和数据库来分析CMA基因的表达和活性。此外，我们使用ATRA敏感(NB4)和耐药(NB4-R1)细胞进一步剖析了CMA在ATRA介导的中性粒细胞分化中的潜在功能.NB4-R1细胞的独特之处在于它们确实对视黄酸有转录反应，但除非通过添加cAMP触发成熟，否则不会单独响应类视黄醇信号而成熟。
结果：这里，我们报告说，CMA相关的mRNA转录本在未成熟的造血细胞中的表达明显高于中性粒细胞。对比巨自噬基因表达模式。因此，在ATRA诱导的APL细胞分化过程中mCherry-KFERQCMA报道分子的溶酶体降解减少。另一方面,使用NB4-R1细胞，我们发现巨自噬通量使ATRA抗性NB4-R1细胞在ATRA治疗后分化，但溶酶体相关膜蛋白2A（LAMP-2A）和热休克蛋白家族A（Hsp70）成员8（HSPA8）的关联减少，这是中性粒细胞完全成熟所必需的。因此，HSPA8的消耗减弱了CMA活性并促进了APL细胞分化。相比之下,通过LAMP-2A的异位表达维持高CMA活性阻碍了APL分化。
结论：总体而言，我们的研究结果表明,APL中性粒细胞分化需要CMA失活,该途径主要依赖于HSPA8,并可能由其他共同伴侣辅助.
方法：我们利用人类AML原发样本和数据库来分析CMA基因的表达和活性。此外，我们使用ATRA敏感(NB4)和耐药(NB4-R1)细胞进一步剖析了CMA在ATRA介导的中性粒细胞分化中的潜在功能.NB4-R1细胞的独特之处在于它们确实对视黄酸有转录反应，但除非通过添加cAMP触发成熟，否则不会单独响应类视黄醇信号而成熟。
结果：这里，我们报告说，CMA相关的mRNA转录本在未成熟的造血细胞中的表达明显高于中性粒细胞。对比巨自噬基因表达模式。因此，在ATRA诱导的APL细胞分化过程中mCherry-KFERQCMA报道分子的溶酶体降解减少。另一方面,使用NB4-R1细胞，我们发现巨自噬通量使ATRA抗性NB4-R1细胞在ATRA治疗后分化，但溶酶体相关膜蛋白2A（LAMP-2A）和热休克蛋白家族A（Hsp70）成员8（HSPA8）的关联减少，这是中性粒细胞完全成熟所必需的。因此，HSPA8的消耗减弱了CMA活性并促进了APL细胞分化。相比之下,通过LAMP-2A的异位表达维持高CMA活性阻碍了APL分化。
结论：总体而言，我们的研究结果表明,APL中性粒细胞分化需要CMA失活,该途径主要依赖于HSPA8,并可能由其他共同伴侣辅助.
