AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs

AFF1 and AFF4 belong to the AFF （AF4/FMR2） family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b （P-TEFb） and ELL1/2, in super elongation complexes （SECs）. Both AFF1 and AFF4 regulate gene transcription through elongation and chromatln remodeling. However, their function in the osteogenic differentiation of mesenchymal stem cells （MSCs） is unknown. In this study, we show that small interfering RNA （siRNA）-mediated depletion of AFF1 in human MSCs leads to increased alkaline phosphatase （ALP） activity, enhanced mineralization and upregulated expression of osteogenic-related genes. On the contrary, depletion of AFF4 significantly inhibits the osteogenic potential of MSCs. In addition, we confirm that overexpression of AFF1 and AFF4 differentially affects osteogenic differentiation in vitro and MSC-mediated bone formation in vivo. Mechanistically, we find that AFFI regulates the expression of DKK1 via binding to its promoter region. Depletion of DKK1 in HA-AFFl-overexpressing MSCs abrogates the impairment of osteogenic differentiation. Moreover, we detect that AFF4 is enriched in the promoter region of ID1. AFF4 knockdown blunts the BRE luciferase activity, SP7 expression and ALP activity induced by BMP2 treatment. In conclusion, our data indicate that AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.AFF1 and AFF4 belong to the AFF （AF4/FMR2） family of proteins, which function as scaffolding proteins linking two different transcription elongation factors, positive elongation factor b （P-TEFb） and ELL1/2, in super elongation complexes （SECs）. Both AFFI and AFF4 regulate gene transcription through elongation and chromatln remodeling. However, their function in the osteogenic differentiation of mesenchymal stem cells （MSCs） is unknown. In this study, we show that small interfering RNA （siRNA）-mediated depletion of AFF1 in human MSCs leads to increased alkaline phosphatase （ALP） activity, enhanced mineralization and upregulated expression of osteogenic-related genes. On the contrary, depletion of AFF4 significantly inhibits the osteogenic potential of MSCs. In addition, we confirm that overexpression of AFF1 and AFF4 differentially affects osteogenic differentiation in vitro and MSC-mediated bone formation in vivo. Mechanistically, we find that AFFI regulates the expression of DKK1 via binding to its promoter region. Depletion of DKK1 in HA-AFFl-overexpressing MSCs abrogates the impairment of osteogenic differentiation. Moreover, we detect that AFF4 is enriched in the promoter region of ID1. AFF4 knockdown blunts the BRE luciferase activity, SP7 expression and ALP activity induced by BMP2 treatment. In conclusion, our data indicate that AFF1 and AFF4 differentially regulate the osteogenic differentiation of human MSCs.

急性B淋巴细胞白血病KMT2A::AFF1基因表达的临床特征分析

目的分析伴KMT2A::AFF1基因阳性急性B淋巴细胞白血病(B-ALL)的临床特点与患者预后及其预后风险因素的相关性。方法回顾性队列研究。纳入2011年4月1日至2022年7月31日上海市第一人民医院和海军军医大学附属第一医院收治的B-ALL病例167例,按照基因类型进行分组。KMT2A::AFF1基因阳性B-ALL病例22例作为实验组,BCR::ABL基因阳性B-ALL 54例作为对照组1和91例KMT2A::AFF1及BCR::ABL基因阴性B-ALL作为对照组2。实验组、对照组1和对照组2的首诊中位年龄分别为43.5(30.5,56)岁、43.5(34,55)岁和32(24,46)岁。三组中位白细胞计数分别为142.4(25.7,247.2)×109/L、37.6(15.7,102.2)×109/L和13.4(4.3,33.0)×109/L。三组骨髓异基因造血干细胞移植(allo-HSCT)率分别为45.5%、72.2%和72.5%。运用SPSS 26.0软件,采用非参数检验的秩和检验、卡方检验、Kaplan-Meier及Cox回归的统计学方法,分析比较试验组与对照组间患者的临床特征、化疗和预后的差异,分析实验组风险因素及allo-HSCT预后差异。结果实验组与对照组2的年龄差异有统计学意义(Z=-2.151,P=0.031);实验组白细胞计数明显高于对照组1组(Z=-2.363,P=0.018)和对照组2(Z=-4.886,P<0.001);实验组的allo-HSCT率低于对照组1(45.5%比72.2%,χ^(2)=4.890,P=0.027)和对照组2(45.5%比72.5%,χ^(2)=5.897,P=0.015)。三组一个疗程缓解率分别为60%(12/20)、83.3%(45/54)及76.7%(69/90),两个疗程化疗缓解率分别为25%(5/20)、7.4%(4/54)、12.2%(11/90),两个疗程以上或未缓解率分别为15%(3/20)、9.3%(5/54)、11.1%(10/90)。实验组较对照组1化疗效果差(Z=-1.979,P=0.048)。3组在性别、染色体是否为标危核型、初发时血红蛋白、血小板计数及骨髓原始细胞百分比差异无统计学意义。实验组总生存(OS)率低于对照组1和对照组2(23.9%比36.7%,χ^(2)=7.608,P=0.006和23.9%比44.8%,χ^(2)=6.442,P=0.011),3年无复发生存(RFS)也低于其他两组(14.0%比57.6%,χ^(2)=17.823,P<0.001和14.0%比48.2%,χ^(2)=16.432,P<0.001)。实验组allo-HSCT与未进行allo-HSCT的总OS率差异有统计学意义(45.0%比9.2%,χ^(2)=15.254,P<0.001)。单因素分析显示,年龄是实验组患者RFS的风险因素,allo-HSCT治疗是OS的保护因素。多因素分析显示,allo-HSCT为实验组OS的独立保护因素。结论伴KMT2A::AFF1基因阳性的B-ALL患者白细胞高,化疗较不敏感,预后差。年龄是其风险因素,allo-HSCT可以改善预后。

MLL-AF4(KMT2A/AFF1)阳性急性白血病系别转化三例报告并文献复习

在急性白血病治疗过程中系别转化罕见,在与系别转化相关的染色体畸变中,t(4;11)(q21;q23)与KMT2A/AFF1融合蛋白[原混合谱系白血病(mixed linage leukemia,MLL)基因MLL/AFF1或MLL/AF4]重排最为常见[1].研究表明,MLL重排急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)起源于一个有淋巴细胞分化潜能的前体细胞,从而使系别发生潜在的变化[2].细胞毒性治疗后患者的系别转化是由选择或基因重组引起的[2].博纳吐单抗是一种双特异性单克隆抗体,由一种抗CD19抗体和一种抗CD3抗体连接而成,对B细胞CD19和细胞毒性T细胞CD3均具有特异性.在与2个表位同时结合后,正常和肿瘤B细胞被宿主细胞毒性T细胞裂解.有研究报道,6例婴幼儿和4例成人急性B淋巴细胞白血病(B cell acute lymphoblastic leukemia,B-ALL)在CD19靶向治疗[嵌合抗原受体T细胞(chimeric anti-gen receptor T-cells,CAR-T)和博纳吐单抗]后转化为急性髓系白血病[1,3-9].有研究观察到CD19靶向免疫治疗后复发与获得髓样表型和B淋巴系抗原缺失有关,是一种新的复发机制,不同于以前注意到的孤立CD19表达丢失[3,10].可能是ALL患者接受靶向免疫治疗后经过髓系转化而产生的一种耐药和适应.