【作者】 张路；

【导师】 陈崇；

【作者基本信息】 四川大学 ， 生物与医药， 2023， 硕士

【摘要】 急性早幼粒细胞白血病(Acute promyelocytic leukemia,APL)是急性髓系白血病(acute myeloid leukemia,AML)的一种亚型,其发病机制与染色体易位形成的融合基因密切相关。大多数APL患者都具有复杂的、隐蔽的或变异的易位,这些易位通常涉及视黄酸受体α(RARA)。由t(15;17)(q24.1;q21.2)易位形成的PML-RARA 是最典型的APL融合基因,此类APL患者对含有全反式维甲酸(ATRA)和三氧化二砷(ATO)的分化诱导疗法敏感,具有良好的临床疗效。然而,还有小部分的 APL 以 ZBTB16、NPM1、NUMA、STAT5B、FIPlLl、PRKARlA、BCOR、OBFC2A、TBLR1、GTF2I、IRF2BP2、FNDC3B、TFGA、NUP98、TNRC18、STAT3、SNX15和TFG的非典型重排为特征1-3。RARA融合伴侣基因在形态学和临床特征中起着重要作用4,部分变体具有与经典APL相似的形态学和免疫表型特征。替代融合类型表现出对ATRA的不同敏感性5。对变异APL的研究不仅可以深入了解APL的发病机制,还有助于为患者选择合适的治疗方案。某些变异对全反式维甲酸、三氧化二砷和蒽环类药物表现出抗性,因此识别和研究这些非典型的隐性易位和变异易位具有重要的临床意义。在对临床上一例APL患者研究中,我们发现了一个新的t(14;17)变异易位。患者因发热和皮肤瘀斑入院,诊断时发现其外周血出现白细胞减少和凝血病变,这是急性早幼粒细胞白血病的典型表现。在患者骨髓(BM)中,91.5%的细胞为超颗粒早幼粒细胞,免疫表型为cMPO+、CD13+、CD33+、CD56+、CD45+low、CD117+low、CD123+low、CD64+low、CD34-和 HLA-DR-。对患者骨髓细胞进行RNA测序(RNA-seq)分析,发现与正常BM细胞相比,患者中一些与APL相关的经典基因明显上调。因此,该患者与经典APL具有相似的表型和转录组分子特征。为了明确患者中可能存在的融合基因,对患者的BM细胞进行荧光原位杂交(FISH)试验,以检测PML和RARA。但没有检测到PML-RARA融合或任何已知的其他变异融合基因。细胞的核型分析结果显示14和17号染色体之间发生了易位。为验证是否存在新的融合基因,我们通过对患者的BM细胞RNA-seq测序数据进行融合基因分析及Sanger测序验证,发现STRN3的3号外显子和RARA的3号外显子之间发生易位,形成了STRN3-RARA融合基因。因此,我们鉴定发现了一个全新的APL融合基因—STRN3-RARA,该融合此前未曾被报道。为深入探究STRN3-RARA融合基因的功能,我们构建STRN3-RARA(SR)慢病毒载体,将其转导到U937细胞中建立了过表达SR的细胞模型。药物实验结果显示SR对ATRA敏感,而对ATO不敏感。SR细胞的免疫荧光分析显示,SR主要位于细胞核中,与PML-RARA和其他报道的变异融合蛋白相似6。这些结果表明SR的位置和药物反应与PML-RARA和其他变体融合蛋白类似。深入分析pMIG和SR过表达细胞的转录组数据,观察到一些此前被报道与APL相关的信号通路在SR过表达组中显著正富集,而髓系分化相关通路则在SR过表达组中下调,说明SR能够抑制分化,并增强了 APL相关基因的表达。根据已报道的PML-RARA研究结果,分析比较STRN3-RARA与其差异表达基因,二者上调和下调的基因存在显著重叠。为进一步我们验证STRN3-RARA是否与PML-RARA一致,能够直接结合基因并调节其表达水平。于是我们进行SR的CUT&Tag测序验证STRN3-RARA是否直接结合基因。结果显示与PML-RARA 一样,STRN3-RARA融合也能直接结合基因。SR结合位点位于基因启动子和远端基因间区。此外,与pMIG相比,SR过表达组中上调的JAGI、SLCIA4、DDIT4和MMP9等APL特征基因和下调的FGR、CEBPE、CXCR4和CCL5等分化相关基因,都显著直接与SR结合。这表明STRN3-RARA融合基因也像PML-RARA 一样7,是通过直接结合发挥抑制和激活功能。除了STRN3-RARA融合外,在该APL患者的BM细胞中还检测到UTX的移码突变。UTX(亦称为KDM6A)是一种位于Xp11.3的组蛋白H3(H3K27)上赖氨酸27的去甲基化酶,是人类实体瘤和白血病中常见的突变肿瘤抑制因子8。二代测序结果显示,移码突变c.3372 del(p.Phe1109LeufsTer11)位于UTX的Jmjc结构域,而该结构域负责UTX的去甲基酶活性,因而该移码突变将导致UTX功能的丧失。此APL患者同时存在UTX缺失和STRN3-RARA融合,暗示二者的合作对于APL发展可能是必要的。基于此我们猜测UTX突变联合SR促进了 APL。利用sgRNA对U937细胞的UTX进行敲除,进而分析pMIG、sgUTX、SR和sgUTX;SR的RNA-seq数据。发现相较pMIG,APL患者中显著高表达的基因在SR和sgUTX;SR中明显上调,但在sgUTX中没有上调;反之亦然,APL患者中显著低表达的基因在SR和sgUTX;SR中明显下调,但在sgUTX中没有下调。这表明SR与UTX突变的协同作用导致APL相关上调基因的上调和下调基因的下调。然后分析pMIG、sgUTX、SR和sgUTX;SR各自特异表达的基因及其通路富集情况,发现SR调控APL通路相关基因,sgUTX调控一般血癌相关基因,而sgUTX与SR协同促进了 APL的产生。此外,我们构建的小鼠模型显示,Utx敲除和SR过表达会导致髓系偏位、肝脾肿大和白血病,这也再次印证了我们的假设。与APL的其他RARA融合变体一致,伴有STRN3-RARA融合的APL细胞对RARA治疗不太敏感,患者在对RARA治疗的初始反应后很快复发。测序数据分析的结果显示,TNFa通路在sgUTX;SR中显著上调。药物筛选证实,FDA(Food and Drug Administration)批准的能够抑制TNFa产生的药物——千金藤素,既可以抑制过表达STRN3-RARA的U937细胞生长,也可以抑制患者复发的APL细胞生长。综上所述,本研究发现了一个新的APL融合基因STRN3-RARA,并通过体内外功能学实验证明STRN3-RARA融合基因协同UTX突变促进APL的形成,结合转录组和CUT&Tag测序深入探究UTX缺失和STRN3-RARA融合协同促进APL形成的潜在机制。鉴定了STRN3-RARA融合的新的治疗药物。为STRN3-RARA癌蛋白的临床特征、药物反应和潜在机制提供了理论基础,为变异APL患者的诊断和治疗提供了新的思路。更多还原

【Abstract】 Acute promyelocytic leukemia（APL）is a subtype of acute myeloid leukemia（AML）whose pathogenesis is closely linked to the formation of fusion genes due to chromosomal translocations.Most patients with APL harbor complex,cryptic,or variant translocations,which typically involve the retinoic acid receptor alpha（RARA）.PML-RARA,formed by t（15;17）（q24.1;q21.2）translocations,is the most typical APL fusion gene,and such APL patients are sensitive to differentiation induction therapy containing all-trans retinoic acid（ATRA）and arsenic trioxide（ATO）with a good clinical outcome.However,a small proportion of APL is characterized by atypical rearrangements with ZBTB16,NPM1,NUMA,STAT5B,FIP1L1,PRKAR1A,BCOR,OBFC2A,TBLR1,GTF2I,IRF2BP2,FNDC3B,TFGA,NUP98,TNRC18,STAT3,SNX15,and TFG^1-3.RARA fusion partner plays an important role in morphological and clinical features⁴,and some variants have similar morphologic and immunophenotypic features as classic APL.Alternative fusion types exhibit different sensitivities to ATRA⁵.The study of variant APL not only provides insight into the pathogenesis of APL but also helps to select appropriate treatment options for patients.Certain variants are resistant to treatment with all-trans retinoic acid,arsenic trioxide,and anthracyclines.Therefore identification and study of these atypical recessive and variant translocations are of clinical importance.In a clinical study of a patient with APL,we identified a new t（14;17）variant translocation.The patient was admitted to the hospital with fever and skin petechiae and was diagnosed with leukopenia and coagulopathy in the peripheral blood,which are typical of acute promyelocytic leukemia.In the patient’s bone marrow（BM）,91.5%of cells were hypergranular promyelocyte,and the immunophenotype was c MPO+,CD13+,CD33+,CD56+,CD45+low,CD117+low,CD123+low,CD64+low,CD34-and HLA-DR-.RNA sequencing（RNA-seq）analysis of the patient’s bone marrow cells revealed that some classical genes associated with APL were significantly upregulated in the patient compared to normal BM cells.Thus,this patient has similar phenotypic and transcriptomic molecular features to classical APL.To clarify the possible presence of fusion genes in the patient,fluorescence in situ hybridization（FISH）tests were performed on the patient’s BM cells to detect PML and RARA.But no PML-RARA fusion or any other known variant fusion gene was detected.Karyotype analysis of the cells showed a translocation between chromosomes 14 and 17.To verify the existence of the new fusion gene,we performed fusion gene analysis by RNA-seq sequencing data of BM cells from patient’s and Sanger sequencing,and found that a translocation occurred between exon 3 of STRN3 and exon 3 of RARA,forming a STRN3-RARA fusion gene.Thus,we identified a novel APL fusion gene,the STRN3-RARA,which has not been previously reported.To further explore the function of the STRN3-RARA fusion gene,we constructed the STRN3-RARA（SR）lentiviral vector and transduced it into U937 cells to establish a cell model overexpressing SR.The results of drug assays showed that SR was sensitive to ATRA but not to ATO.Immunofluorescence analysis of SR cells showed that SR was mainly located in the nucleus,similar to PML-RARA and other reported variant fusion proteins⁶.These results suggest that the location and drug response of SR is similar to that of PML-RARA and other variants of fusion proteins.In-depth analysis of transcriptome data from p MIG and SR overexpressing cells observed that some signaling pathways previously reported to be associated with APL were significantly positively enriched in the SR overexpressing group,whereas myeloid differentiation-related pathways were downregulated in the SR overexpressing group,suggesting that SR inhibits differentiation and enhances the expression of APL-related genes.According to the reported results of the PML-RARA study,analysis comparing STRN3-RARA with its differentially expressed genes revealed a significant overlap of up-and down-regulated genes between the two.To further we verified whether STRN3-RARA,consistent with PML-RARA,can directly bind genes and regulate their expression levels.We then performed CUT&Tag sequencing of SR to verify whether STRN3-RARA directly binds genes.The results showed that like PML-RARA,STRN3-RARA fusion can also bind genes directly.SR binding site is located in the gene promoter and distal intergenic region.In addition,APL signature genes such as JAG1,SLC1A4,DDIT4,and MMP9,which are upregulated in SR,and differentiation-related genes such as FGR,CEBPE,CXCR4,and CCL5,which are downregulated in SR,were significantly and directly bound to SR compared to p MIG.This suggests that STRN3-RARA fusion genes also exert repressive and activating functions through direct binding,like PML-RARA⁷.In addition to the STRN3-RARA fusion,a shift mutation in UTX was detected in the BM cells of this APL patient.UTX（also known as KDM6A）is a demethylase of lysine 27 on histone H3（H3K27）located at Xp11.3 and is a common mutated tumor suppressor in human solid tumors and leukemia⁸.The next-generation sequencing showed that the shift mutation c.3372 del（p.Phe1109Leufs Ter11）is located in the Jmjc structural domain of UTX,which is responsible for the demethylase activity of UTX,and thus the shift mutation would result in loss of UTX function.The presence of both UTX deletion and STRN3-RARA fusion in this APL patient implies that cooperation between the two may be necessary for APL development.Based on this we speculate that the UTX mutation combined with SR promotes APL.UTX was knocked down using sg RNA in U937 cells,and then RNA-seq data of p MIG,sg UTX,SR and sg UTX;SR were analyzed.Genes significantly highly expressed in APL patients were found to be significantly up-regulated in SR and sg UTX;SR but not in sg UTX compared to p MIG,and vice versa,genes significantly under-expressed in APL patients were significantly down-regulated in SR and sg UTX;SR but not in sg UTX.This suggests that the synergistic effect of SR and UTX mutation leads to upregulation of APL-related upregulated genes and downregulation of downregulated genes.Then,we analyzed the genes specifically expressed by each of p MIG,sg UTX,SR and sg UTX;SR and their pathway enrichment,and found that SR regulated APL pathway-related genes,sg UTX regulated general blood cancer-related genes,and sg UTX synergistically promoted APL production with SR.In addition,we constructed mouse models showing that Utx knockout and SR overexpression drives myeloid bias,hepatosplenomegaly,and leukemia.Consistent with other RARA fusion variants of APL,APL cells with STRN3-RARA fusion were less sensitive to RARA treatment and patients relapsed soon after the initial response to RARA treatment.The results of sequencing data analysis showed that the TNFa pathway was significantly upregulated in sg UTX;SR.Drug screening verified that the TNFa inhibitor cepharanthine,an FDA-approved drug,could inhibit both U937 cells transduced with STRN3-RARA and also the relapsed APL cells from the patient.In summary,this study identified a novel APL fusion gene,STRN3-RARA,and demonstrated that the STRN3-RARA fusion gene synergizes with UTX mutation to promote APL formation by vitro functional assays,and combined with transcriptome and CUT&Tag sequencing to explore the potential mechanisms by which UTX deletion and STRN3-RARA fusion synergize to promote APL formation.Novel therapeutic agents for STRN3-RARA fusion were identified.It provides a theoretical basis for clinical characterization,drug response and potential mechanisms of STRN3-RARA oncoprotein,and provides new ideas for diagnosis and treatment of patients with variant APL.更多还原