Molecular understanding of cytokine-Ras signals in leukemic bone marrow

白血病骨髓中细胞因子-Ras 信号的分子理解

基本信息

批准号：
9296107
负责人：
JEROEN ROOSE
金额：
$ 35.38万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9296107
关键词：
Acute T Cell Leukemia Address Adult Affect Basic Science Biochemical Biochemical Pathway Biochemistry Biological Biological Assay Bone Marrow Bone Marrow Cells Buffers CRISPR/Cas technology Calcium Cell Line Cells Characteristics Child Childhood Precursor T Lymphoblastic Leukemia Clinical Cytokine Receptors Development Diglycerides Disease Equilibrium FRAP1 gene Future GTP Binding Genetic Goals Growth Guanine Nucleotide Exchange Factors Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Hematopoietic Human Intervention KRAS2 gene Leukemic Cell Link Lymphocyte MEKs Malignant Neoplasms Measures Methods Mission Modeling Modernization Molecular Mus Mutation Myelogenous NF1 gene Nude Mice Oncogenic Outcome Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phospholipase C Phospholipases A Phosphotransferases Positioning Attribute Receptor Signaling Relapse Reporting Research Research Personnel Rest Role Sampling Signal Pathway Signal Transduction Signal Transduction Inhibitor Stem cells Structure Study models System T-Cell Leukemia T-Lymphocyte Testing Therapeutic Therapeutic Effect Transplantation United States National Institutes of Health base biochemical model chemotherapy cytokine cytotoxicity hyperactive Ras improved in vivo inhibitor/antagonist innovation insight interest molecular targeted therapies mouse model novel overexpression phospholipase C gamma pre-clinical preclinical trial public health relevance ras Guanine Nucleotide Exchange Factors relapse risk small hairpin RNA small molecule inhibitor targeted treatment tool treatment strategy

项目摘要

DESCRIPTION (provided by applicant): T cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer that affects children and adults. Modern chemotherapy has improved clinical outcome but relapse and non-specific cytotoxicity are still problematic. Targeted therapy with specific inhibitors is highly desired but a better understanding of the aberrant biochemical pathways and pivotal molecules herein is required to reach this goal. ~50% of patient T-ALL patients show aberrantly active Ras signals. Until recently, the molecular players were unknown. We uncovered that T-ALL have two major mechanisms of abnormal Ras signaling: via overexpression of the Ras exchange factor Rasgrp1 or via oncogenic mutations in Ras (like K-RasG12D). Rasgrp1 overexpression occurs in ~55% of all pediatric T-ALL patients, mutations in KRAS in ~10%. We uncovered that Rasgrp1 continuously activates Ras that this is somehow counterbalanced by RasGAPs, and that cytokine receptor stimulation tips the balance in favor of active Ras. In 2013 we also reported (i) that myeloid leukemic cells with mutations in KRAS require Rasgrp3 and (ii) that Rasgrp molecules are autoinhibited and require 2nd messengers produced by Phospholipase C(PLC) for activation. In summary, these findings imply that Rasgrp's are critical components of leukemogenic Ras signals, are positioned downstream of cytokine receptor signaling, depend on PLCfor their activation, and are counterbalanced by RasGAPs. The mechanism of cytokine-Ras signaling, the molecular roles of RasGAPs, Rasgrp's, and PLCherein, and the potential therapeutic effect of PLCinhibiton in T-ALL are all unknowns. We obtained novel mechanistic insights through the development of innovative tools. We optimized a novel Ras activation assay to measure flux in the Ras GDP/GTP cycle that suggests critical buffering by RasGAPs. We optimized a quantitative, high-throughput method of phospho-flow combined with barcoding and a novel pINDUCER system for Dox-indicible shRNA. We established growth characteristics of Rasgrp1 and K-RasG12D T-ALL transplanted into nude mice. We can analyze primary patient T-ALL transplanted into recipient mice. Lastly, we developed an entirely novel genetic mouse model with overexpression of Rasgrp1 in bone marrow cells that leads to T-ALL and will compare this model to a genetic K-RasG12D model. Our biochemical-, cell biological-, and in vivo- approaches will reveal molecular insights into this novel but uncharacterized cytokine receptor-Rasgrp signaling pathway that is counterbalanced by RasGAP Ras inactivators (Aim 1) and will establish the molecular role of PLCin Rasgrp-Ras-Ras effector activation (Aim 2). In Aim 3, we will explore PLCinhibition using pINDUCER or small molecule inhibitors in preclinical trials in the three above-mentioned mouse models. We anticipate that our studies will provide significant molecular insights into the basic science of leukemogenic signals but also provide translational insights in the therapeutic potential of PLCinhibition that could impact clinical therapy forT-ALL in the future.

描述（由申请人提供）：T 细胞急性淋巴细胞白血病 (T-ALL) 是一种影响儿童和成人的侵袭性癌症，现代化疗已改善了临床结果，但复发和非特异性细胞毒性仍然是问题。非常需要，但要实现这一目标，从根本上需要更好地了解异常的生化途径和关键分子。直到最近，分子参与者才发现大约 50% 的 T-ALL 患者表现出异常活跃的 Ras 信号。我们发现 T-ALL 有两种异常 Ras 信号传导的主要机制：通过 Ras 交换因子 Rasgrp1 的过度表达或通过 Ras 的致癌突变（如 K-RasG12D），约 55% 的儿童 T-ALL 发生这种情况。所有患者中，约 10% 的 KRAS 发生突变，我们发现 Rasgrp1 持续激活 Ras，这在某种程度上被 RasGAP 和细胞因子抵消。受体刺激使平衡向有利于活性 Ras 的方向倾斜。2013 年，我们还报道了 (i) KRAS 突变的骨髓白血病细胞需要 Rasgrp3，以及 (ii) Rasgrp 分子具有自身抑制作用，需要由磷脂酶 C(PLC) 产生的第二信使。总之，这些发现表明 Rasgrp 是致白血病 Ras 信号的关键组成部分，位于下游。细胞因子受体信号传导依赖于 PLC 的激活，并通过 RasGAP 进行平衡。细胞因子-Ras 信号传导的机制、RasGAP、Rasgrp 和 PLC 的分子作用，以及 PLC 的潜在治疗作用。 T-ALL 中的抑制作用都是未知的。通过开发创新工具，我们获得了新颖的机制见解。我们优化了一种新颖的 Ras 激活测定。测量 Ras GDP/GTP 循环中的通量，这表明 RasGAP 的关键缓冲作用。我们优化了一种定量、高通量的磷酸化流方法，结合了条形码和一种用于 Dox 指示的 shRNA 的新型 pINDUCER 系统。我们建立了 Rasgrp1 的生长特征。和K-RasG12D T-ALL移植到裸鼠中我们可以分析移植到受体小鼠中的原发性患者T-ALL最后，我们开发了一种全新的基因小鼠模型，其过度表达。骨髓细胞中的 Rasgrp1 会导致 T-ALL，并将该模型与遗传 K-RasG12D 模型进行比较，我们的生化、细胞生物学和体内方法将揭示对这种新颖但未表征的细胞因子受体 Rasgrp 的分子见解。由 RasGAP Ras 灭活剂平衡的信号通路（目标 1），并将确立 PLC在 Rasgrp-Ras-Ras 效应子中的分子作用激活（目标 2）。在目标 3 中，我们将探索。在上述三种小鼠模型的临床前试验中使用 pINDUCER 或小分子抑制剂抑制 PLC——我们预计我们的研究将为白血病信号的基础科学提供重要的分子见解，同时也为 PLC 的治疗潜力提供转化见解。 - 抑制可能影响未来 T-ALL 的临床治疗。