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Molecular and therapeutic implications of NOTCH1 signaling in pediatric T-cell acute lymphoblastic leukemia.

机译：儿科T细胞急性淋巴细胞白血病中NOTCH1信号传导的分子和治疗意义。

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摘要

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 15% of pediatric ALL cases and is associated with early relapse and inferior outcome. The poorer prognosis of T-ALL compared to B-precursor ALL may in part reflect the lack of unique features on which to base therapy. NOTCH1 mutations are of particular interest since these were reported in 37--71% of T-ALLs. The prognostic value of NOTCH1 mutations remains controversial as both favorable and unfavorable associations were reported, whereas in other studies, there were no associations between NOTCH1 mutations and treatment outcome. We explored the impact of mutations in NOTCH1, FBW7 and PTEN on prognosis and downstream signaling in pediatric T-cell acute lymphoblastic leukemia. We identified a high frequency of mutations in NOTCH1 (16 patients), FBW7 (5 patients) and PTEN (26 patients) in a well defined cohort of 47 pediatric T-ALL specimens. Our results suggest (1) multiple factors should be considered with attempting to identify molecular-based prognostic factors for pediatric T-ALL and (2) that, depending on the NOTCH1 signaling status, modifications in the types or dosing of standard chemotherapy drugs for T-ALL, or combinations of agents capable of targeting NOTCH1, AKT and/or mTOR with standard chemotherapy agents may be warranted.;To determine the contribution of genetic alteration to the development of relapse in T-ALL, we assessed the frequency of mutations in NOTCH1 alone or in combination with mutations in FBW7 and PTEN at the time of diagnosis and relapse in 11 paired clinical T-ALL specimens. We observed that the 7 patients harboring mutations in NOTCH1 and/or PTEN at some stage in their disease had a longer remission period (13 months vs. 5.5 months), and were typically diagnosed at an early age (120 months vs. 132 months). In these 7 patients, nearly 70% of relapse appeared to be associated with the emergence of a new leukemic clone, an assumption made by the presence of a new mutation or loss of a mutation at relapse. Using real-time PCR techniques with specific hybridization probes, we were able to determine that the leukemic clone for one patient was present at the time of diagnosis, but at a very low expression level. This suggests that the clone responsible for relapse was resistant to the initial chemotherapy treatment. For another patient, the relapse clone could not be detected at diagnosis, suggesting that it was induced following chemotherapy. This study strongly warrants future studies with a larger patient cohort to systematically identify specific hallmarks of relapse.;NOTCH1 is a potentially attractive therapeutic target for T-ALL since constitutively activating effects of mutant NOTCH1 can be abolished with gamma-secretase inhibitors (GSIs). Because of possible effects of GSIs on other cellular targets in addition to NOTCH1, we explored shRNA knockdown of NOTCH1 to identify novel NOTCH1-regulated genes that may serve as prognostic indicators or therapeutic targets in T-ALL. To characterize genotypic changes accompanying NOTCH1 knockdown, we performed microarray analysis with Agilent Whole Genome oligonucleotide microarrays and microRNA (miR) HumanV2 arrays. The microarray identified Rictor, a key component to in the mTOR2 complex, as a novel downstream target of NOTCH1 signaling. Upon NOTCH1 inhibition, an increase in the expression of Rictor was observed, both at the transcript and protein levels. Initial computational analysis of the Rictor promoter suggests that NOTCH1 may regulate its expression directly (via RBPJkappa) or indirectly (via HES1). The miR array identified 20 miRs in J.N1KD 2-4 and J.N1KD 2-7 cells with altered expression compared to J.ntc greater than 1.5-fold (p0.05) and ranging from 3-to10-fold. miRs hsa-Let-7e, hsa-miR-125a-5p and hsa-miR-99b, reportedly derived from a polycistronic transcript, were decreased 10-fold accompanying NOTCH1 knockdown. Using miR qPCR, we confirmed decreased levels of hsa-miR-125a-5p and hsa-miR-99b in the J.N1KD 2-4 and J.N1KD 2-7 sublines. In conclusion, we have developed novel T-ALL cell line models to study the impact of decreased NOTCH1 levels and activity independent of GSI treatment. Our results implicate NOTCH1 in regulating levels of Rictor and hsa-miR-125a-5p, and suggest that caution may be warranted in targeting NOTCH1 with GSIs in the therapy of T-ALL, reflecting the potential promotion of cell survival via the upregulation of Rictor. The downstream effect of regulating hsa-miR-125a-5p has yet to be determined. (Abstract shortened by UMI.)
机译：T细胞急性淋巴细胞白血病（T-ALL）占小儿ALL病例的15％，与早期复发和不良预后相关。与B前体ALL相比，T-ALL的预后较差可能部分反映了缺乏基础疗法的独特特征。 NOTCH1突变特别令人感兴趣，因为在37-71％的T-ALL中报告了这些突变。由于已经报道了有利和不利的关联，因此NOTCH1突变的预后价值仍然存在争议，而在其他研究中，NOTCH1突变与治疗结果之间没有关联。我们探讨了NOTCH1，FBW7和PTEN突变对小儿T细胞急性淋巴细胞白血病预后和下游信号的影响。我们在一组明确定义的47例儿科T-ALL标本中确定了NOTCH1（16例患者），FBW7（5例患者）和PTEN（26例患者）的高频率突变。我们的结果表明（1）在尝试确定儿童T-ALL的基于分子的预后因素时应考虑多种因素，（2）根据NOTCH1信号状态，对T的标准化疗药物的类型或剂量进行修改-ALL或能够将靶向NOTCH1，AKT和/或mTOR的药物与标准化疗药物组合使用是有必要的。为了确定基因改变对T-ALL复发发展的贡献，我们评估了T-ALL的突变频率在11对配对的临床T-ALL标本中，NOTCH1单独或与FBW7和PTEN突变一起诊断并复发时。我们观察到，在疾病的某个阶段具有NOTCH1和/或PTEN突变的7位患者的缓解期较长（13个月对5.5个月），通常被诊断为较早（120个月对132个月）。 。在这7例患者中，近70％的复发似乎与新的白血病克隆的出现有关，这是由新突变的存在或复发时突变的丧失所引起的。使用具有特定杂交探针的实时PCR技术，我们能够确定在诊断时存在一名患者的白血病克隆，但其表达水平非常低。这表明负责复发的克隆对最初的化学疗法治疗有抗性。对于另一名患者，在诊断时无法检测到复发克隆，表明该克隆是在化疗后诱导的。这项研究有力地保证了将来有一个更大的患者队列来系统地确定复发的特征。NOTCH1是T-ALL潜在的有吸引力的治疗靶标，因为可以用γ-分泌酶抑制剂（GSI）消除突变型NOTCH1的组成型激活作用。由于GSI除NOTCH1以外还可能对其他细胞靶标产生影响，因此我们研究了NOTCH1的shRNA敲除，以鉴定可作为T-ALL预后指标或治疗靶标的新型NOTCH1调控基因。为了表征伴随NOTCH1敲除的基因型变化，我们使用安捷伦全基因组寡核苷酸微阵列和microRNA（miR）HumanV2阵列进行了微阵列分析。微阵列将Rictor（mTOR2复合物中的关键成分）鉴定为NOTCH1信号传导的新型下游靶标。在NOTCH1抑制后，在转录本和蛋白质水平上都观察到Rictor表达的增加。 Rictor启动子的初步计算分析表明，NOTCH1可以直接（通过RBPJkappa）或间接（通过HES1）调节其表达。 miR阵列在J.N1KD 2-4和J.N1KD 2-7细胞中鉴定出20个miR，与J.ntc相比，表达改变的程度大于1.5倍（p <0.05），范围是3至10倍。据报道，源自多顺反子转录本的miRs hsa-Let-7e，hsa-miR-125a-5p和hsa-miR-99b随NOTCH1敲低而降低了10倍。使用miR qPCR，我们确认了J.N1KD 2-4和J.N1KD 2-7子系中hsa-miR-125a-5p和hsa-miR-99b的水平降低。总之，我们已经开发出新颖的T-ALL细胞系模型来研究降低的NOTCH1水平和活性的影响，而与GSI治疗无关。我们的结果表明，NOTCH1调节Rictor和hsa-miR-125a-5p的水平，并建议在T-ALL治疗中以GSI靶向NOTCH1可能需要谨慎，反映出通过Rictor的上调可能促进细胞存活。调节hsa-miR-125a-5p的下游效应尚未确定。 （摘要由UMI缩短。）

著录项

  • 作者

    Larson Gedman, Amanda.;

  • 作者单位

    Wayne State University.;

  • 授予单位 Wayne State University.;
  • 学科 Biology Molecular.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2010
  • 页码 171 p.
  • 总页数 171
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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