Molecular understanding of cytokine-Ras signals in leukemic bone marrow

 描述(申请人提供)：T细胞急性淋巴细胞白血病(T-ALL)是一种侵袭性癌症，影响儿童和成人。现代化疗已经改善了临床结果，但复发和非特异性细胞毒性仍然存在问题。使用特定的抑制剂进行靶向治疗是非常必要的，但要达到这一目标，需要对异常的生化途径和关键分子有更好的了解。约50%的T-ALL患者RAS信号异常活跃。直到最近，分子玩家还不为人所知。我们发现T-ALL有两个主要的RAS信号异常机制：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)。在所有儿童T-ALL患者中，RASGRP1过表达的比例约为55%，KRAS突变的比例约为10%。我们发现，RASGRP1不断激活RAS，这在某种程度上被RasGAP平衡，细胞因子受体刺激使平衡有利于活跃的RAS。2013年，我们也报道了(I)KRAS突变的髓系白血病细胞需要Rasgrp3和(Ii)Rasgrp分子是自我抑制的，需要磷脂酶C(PLC)产生的第二信使才能激活。综上所述，这些发现表明Rasgrp是导致白血病的RAS信号的关键成分，位于细胞因子受体信号的下游，依赖于PLC的激活，并被RasGAP抵消。细胞因子-RAS信号转导机制、RasGAP、Rasgrp‘s和PLC的分子作用，以及PLC抑制剂对T-ALL的潜在治疗作用都是未知的。通过开发创新的工具，我们获得了新的机械学见解。我们优化了一种新的RAS激活实验来测量RAS GDP/GTP循环中的通量，这表明RasGAP具有关键的缓冲作用。我们优化了一种结合条形码的定量、高通量的磷酸流动方法和一种新的Dox标记shRNA的pINDUCER系统。我们建立了RASGRP1和K-RasG12D T-ALL移植到裸鼠体内的生长特性。我们可以分析移植到受体小鼠体内的原发患者T-ALL。最后，我们开发了一种全新的遗传小鼠模型，该模型在骨髓细胞中过表达RASGRP1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这一新的但尚未确定的细胞因子受体-Rasgrp信号通路的分子洞察力，该信号通路被RasGAP RAS失活剂(AIM 1)抵消，并将建立PLC在Rasgrp-RAS-RAS效应器激活(AIM 2)中的分子作用。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基础科学提供重要的分子见解，也将为PLC抑制的治疗潜力提供翻译见解，这可能会影响未来的临床治疗。

 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.