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（由适用提供）：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略性癌症。现代化学疗法改善了临床结局，但是继电器和非特异性细胞毒性仍然有问题。具有特定抑制剂的靶向疗法是高度期望的，但需要更好地理解此处的异常生化途径和关键分子才能实现此目标。约50％的患者T-ALL患者表现出异常活跃的RAS信号。直到最近，分子玩家还不知道。我们发现T-All具有两个主要的RAS信号传导的主要机制：通过RAS交换因子RasGRP1的过表达或通过RAS中的致癌突变（例如K-Rasg12d）。 RASGRP1过表达发生在所有儿科T-ALL患者中约55％，KRAS中的突变约为10％。我们发现RASGRP1不断激活Ras，这是Rasgaps以某种方式平衡的，并且细胞因子受体刺激使平衡取决于有利于活性RA的平衡。 2013年，我们还报道了（i），KRAS中具有突变的髓样白血病细胞需要RasgRP3和（ii）（ii），RasGRP分子被自抑制，并且需要由磷脂酶C（PLC）产生的第二个使者进行活化。总而言之，这些发现表明RASGRP是白血病RAS信号的关键组成部分，将细胞因子受体信号的下游定位，取决于PLC的激活，并由Rasgaps平衡。细胞因子-RAS信号传导的机制，rasgaps，rasgrp和plchere的分子作用以及plc的潜在治疗作用在T-ALL中都是未知的。我们通过开发创新工具获得了新颖的机械见解。我们优化了一种新型的RAS激活测定法，以测量RAS GDP/GTP循环中的通量，该循环表明Rasgaps的临界缓冲。我们优化了一种磷酸流的定量，高通量方法，结合了条形码和用于DOX引人注目的SHRNA的新型Pinducer系统。 We established growth characteristics of Rasgrp1 and K-RasG12D T-ALL transplanted into nude mice.我们可以分析原发性患者T-HALL移植到受体小鼠中。最后，我们开发了一种完全新颖的遗传小鼠模型，在骨髓细胞中用RASGRP1过表达，这将导致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信号传导途径的分子见解，而Rasgap ras Inactivator则（AIM 1）平衡了（AIM 1），并将建立PLCGRP-RAS-RAS-RAS-RAS-RAS-RAS-RAS-RAS-RAS-RAS-RAS-RAS-RAS-RAS-RAS RAS RAS效应的分子作用（AIM 1）。在AIM 3中，我们将探索 plc在三种上述小鼠模型中使用pinducer或小分子抑制剂的抑制作用。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，但也为PLC抑制作用的治疗潜力提供了转化见解，这可能会影响临床治疗的未来。