Eriocalyxin B induces apoptosis of t(8;21) leukemia cells through NF-kappaB and MAPK signaling pathways and triggers degradation of AML1-ETO oncoprotein in a caspase-3-dependent manner

Diterpenoids isolated from Labiatae family herbs have strong antitumor activities with low toxicity. In this study, Eriocalyxin B (EriB), a diterpenoid extracted from Isodon eriocalyx, was tested on human leukemia/lymphoma cells and murine leukemia models. Acute myeloid leukemia cell line Kasumi-1 was most sensitive to EriB. Significant apoptosis was observed, concomitant with Bcl-2/Bcl-XL downregulation, mitochondrial instability and caspase-3 activation. AML1-ETO oncoprotein was degraded in parallel to caspase-3 activation. EriB-mediated apoptosis was associated with NF-kappaB inactivation by preventing NF-kappaB nuclear translocation and inducing IkappaBalpha cleavage, and disturbance of MAPK pathway by downregulating ERK1/2 phosphorylation and activating AP-1. Without affecting normal hematopoietic progenitor cells proliferation, EriB was effective on primary t(8;21) leukemia blasts and caused AML1-ETO degradation. In murine t(8;21) leukemia models, EriB remarkably prolonged the survival time or decreased the xenograft tumor size. Together, EriB might be a potential treatment for t(8;21) leukemia by targeting AML1-ETO oncoprotein and activating apoptosis pathways.     

Altered chromatin modifications in AML1/RUNX1 breakpoint regions involved in (8;21) translocation

The RUNX1/AML1 gene is the most frequent target for chromosomal translocation, and often identified as a site for reciprocal rearrangement of chromosomes 8 and 21 in patients with acute myelogenous leukemia. Virtually all chromosome translocations in leukemia show no consistent homologous sequences at the breakpoint regions. However, specific chromatin elements (DNase I and topoisomerase II cleavage) have been found at the breakpoints of some genes suggesting that structural motifs are determinant for the double strand DNA-breaks. We analyzed the chromatin organization at intron 5 of the RUNX1 gene where all the sequenced breakpoints involved in t(8;21) have been mapped. Using chromatin immunoprecipitation assays we show that chromatin organization at intron 5 of the RUNX1 gene is different in HL-60 and HeLa cells. Two distinct features mark the intron 5 in cells expressing RUNX1: a complete lack or significantly reduced levels of Histone H1 and enrichment of hyperacetylated histone H3. Strikingly, induction of DNA damage resulted in formation of t(8;21) in HL-60 but not in HeLa cells. Taken together, our results suggest that H1 depletion and/or histone H3 hyperacetylation may have a linkage with an increase susceptibility of specific chromosomal regions to undergo translocations.     

成人t(8;21)急性髓系白血病患者初诊Ki-67的表达特征及预后意义

目的:研究成人t(8; 21)急性髓系白血病(AML)初诊Ki-67抗原的表达特征及预后意义。方法:采集2012年7月至2019年2月本院57例成人初诊t(8; 21) AML患者的新鲜骨髓标本,采用流式细胞术(FCM)检测CD34和Ki-67抗原,分析Ki-67表达与患者初诊生物学特征、疗效及复发的关系。结果:全部患者中,CD34~+Ki-67~+细胞比例的中位值为30. 5%(范围:10. 0%～65. 8%);通过受试者工作特征(ROC)曲线确定CD34~+Ki-67~+细胞比例的最适分界阈值,CD34~+Ki-67~+细胞高比例与初诊c-KIT基因突变阳性及WT1转录本低水平均明显相关(P=0. 001; P=0. 042)。随访的36例患者中,CD34~+Ki-67~+高比例比低比例患者具有明显更高的1年累积复发(CIR)率(P=0. 035);此外,初诊WT1转录本低水平和微小残留病(MRD)高水平(2个疗程巩固治疗后RUNX1-RUNX1T1转录本水平下降3-log)均与更高的1年CIR率明显相关(P 0. 0001;P=0. 041),初诊c-KIT基因突变阳性和白细胞计数 10×109/L的患者分别有较高的1年CIR率趋势(P=0. 091; P=0. 054)。联合分组显示,MRD高水平同时CD34~+Ki-67~+细胞高比例的患者比其他患者具有明显更高的1年CIR率(P 0. 0001)。结论:初诊骨髓高比例的CD34~+Ki-67~+可能是成人t(8; 21) AML患者预后不良因素,MRD联合初诊CD34~+Ki-67~+细胞比例可能比单纯MRD更好地预测复发。     

Ring chromosome 8 and translocation t(8;21) in a patient with acute myeloblastic leukemia

Recurrent translocation t(8;21)(q22;q22) acute myeloid leukemia (AML) is often associated with secondary chromosome changes of which the clinical significance is not clear since they do not seem to impair the prognosis. Uncommon chromosome changes may lead to the identification of leukemogenetic factors associated with t(8;21) since the AML1/RUNX1-ETO fusion gene resulting from the translocation is thought to be unable alone to induce leukemia. We here report a patient with AML, t(8;21) and ring chromosome 8 resulting in partial chromosome 8 deletion. Another patient with partial 8q deletion has been previously reported. It is suggested that more attention be paid to the genes located in distal 8q in relation to leukemogenesis.     

Downregulation of activated leukemic oncogenes AML1-ETO and RUNX1(K83N) expression with RNA-interference

In the present study we have applied the siRNA approach for substantial reduction of AML1-ETO and RUNX1 (K83N) expression, which are frequently found in the leukemic cells. We have designed small hairpin RNAs (shRNA) for targeting AML1-ETO oncogene and a region close to the 5'-untranslated region of mRNA for the mutant RUNX1 (K83N) oncogene and expressed the shRNAs in lentiviral vectors. We report a stable reduction in expression of the oncogenes following the introduction of shRNAs into cells.     

Acute megakaryocytic leukaemia with acquired polysomy 21 and translocation t(1;21)

Cytogenetic study of a young child with acute megakaryocytic leukaemia (AML-M7) has shown a karyotype with 49-50 chromosomes with one and two acquired extra chromosomes 21. Fluorescence in situ hybridization detected a minor clone with translocation t(1;21) and loss of a part of chromosome band 1p36. Trisomy and polysomy 21 are not uncommon in AML-M7. A more systematic search for chromosome 21 rearrangements in AML-M7 using FISH techniques is proposed.     

Interchange trisomy 21 by t(1;21)(p22;q22)mat

Interchange trisomy 21 by t(1:21)(p22:q22)mat: Interchange trisomy 21 by t(1;21)(p22;q22)mat was identified in a sporadic patient with Down syndrome. With a 21q22 specific probe, we observed signals on both normal 21 chromosomes and on the der. We reviewed the 23 published reports of families with reciprocal translocations leading to viable offspring with interchange trisomy 21. The breakpoints in chromosome 21 were mainly located in 21q (19/24 instances, including the present report) and in 19/23 cases the other chromosome involved in the translocation was (pairs 1-12). The underlying 3:1 segregation occurred mainly in carrier mothers; only one patient presented a de novo imbalance and in another case the father was the carrier. In addition, there were 4 instances of concurrence with another unbalanced segregation (adjacent-1 or tertiary trisomy) and 3 families with recurrence of interchange trisomy 21. The mean age of 14 female carriers at birth of interchange trisomy 21 offspring (24.8 yr) was lower that the mean (28.3 yr) found in a larger sample of mothers of unbalanced offspring due to 3:1 segregation (mostly tertiary trisomics) and was not increased with respect to the general population average. Overall, these data agree with previous estimates regarding recurrence risk (9-15%) and abortion rate (about 28%) in female carriers ascertained through an interchange trisomic 21 child.     

Leukemogenic AML1-ETO fusion protein upregulates expression of connexin 43: the role in AML 1-ETO-induced growth arrest in leukemic cells

AML1-ETO, a fusion protein generated by the chromosomal translocation t(8;21), is frequently associated with acute myeloid leukemia (AML). In addition to blocking differentiation, AML1-ETO is also shown to induce growth arrest in AML cells, which is unfavorable for leukemogenesis harboring the t(8;21) translocation. However, its precise mechanism is still unclear. Here we provide the first demonstration that the conditional expression of AML1-ETO by the ecdysone-inducible system dramatically increases the expression of connexin 43 (CX43), together with growth arrest at G1 phase in leukemic U937 cells. We also show that the CX43 induction inhibits the proliferation of U937 cells at G1 phase, while the suppression of CX43 expression by small interfering RNA (siRNA) effectively overcomes the growth-inhibitory effect of AML1 -ETO in leukemic cells. Furthermore, either AML1-ETO or CX43 induction elevates cell-cycle negative regulator P27(kip1) protein by inhibiting its degradation, which is antagonized by siRNA against CX43. Taken together, our data indicate that CX43 plays a role in AML1-ETO-induced growth arrest possibly through the accumulation of P27(kip1) protein. The potential mutation or/and epigenetic alterations of CX43 and its related gene(s) deserve to be explored in AML1-ETO-positive AML patients.     

Complex chromosome abnormality mimicking t(8;21)in an acute myeloblastic leukemia

A patient with acute myeloblastic leukemia (AML) and karyotypic abnormality mimicking t(8;21) is reported. Because of the uncommon morphology of blasts for AML with t(8;21), and absence of AML1-ETO fusion in the RT-PCR analysis, fluorescence in situ hybridization (FISH) was applied to precise the abnormality. FISH revealed a complex rearrangement involving chromosomes 7, 21 and 8, and involvement of the AML1 gene without evidence of AML1-ETO fusion. This case of unusual AML illustrates the necessity of collecting all hematologic, cytogenetic, and eventual FISH and RT-PCR data to analyse genetic rearrangements in leukemic patients.