Molecular understanding of cytokine-Ras signals in leukemic bone marrow

 描述（由申请人提供）：T细胞急性淋巴细胞白血病（T-ALL）是一种侵袭性癌症，影响儿童和成人。 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。使用特异性抑制剂的靶向治疗是高度期望的，但是需要更好地理解本文中的异常生化途径和关键分子以达到该目标。 约50%的T-ALL患者显示异常活跃的Ras信号。直到最近，分子的参与者还是未知的。我们发现T-ALL有两种主要的异常Ras信号传导机制：通过Ras交换因子Rasgrp 1的过度表达或通过Ras中的致癌突变（如K-RasG 12 D）。 Rasgrp 1过表达发生在约55%的所有儿童T-ALL患者中，KRAS突变发生在约10%。我们发现Rasgrp 1持续激活Ras，这在某种程度上被RasGAP抵消，并且细胞因子受体刺激使平衡有利于活性Ras。在2013年，我们还报道了（i）KRAS突变的髓系白血病细胞需要Rasgrp 3和（ii）Rasgrp分子是自抑制的，需要磷脂酶C受体（PLC）产生的第二信使来激活。总之，这些发现意味着Rasgrp是致白血病Ras信号的关键组分，位于细胞因子受体信号传导的下游，依赖于PLC介导其活化，并且被RasGAP抵消。T-ALL中丝氨酸-Ras信号传导的机制、RasGAP、Rasgrp和PLC抑制剂的分子作用以及PLC抑制剂的潜在治疗作用都是未知的。 我们通过创新工具的开发获得了新的机械见解。我们优化了一种新的Ras活化测定法，以测量Ras GDP/GTP循环中的通量，这表明RasGAP具有关键缓冲作用。我们优化了磷酸流结合条形码的定量、高通量方法和一种新的Dox指示性shRNA的pINDUCER系统。我们建立了Rasgrp 1和K-RasG 12 D 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中的原发患者T-ALL。最后，我们开发了一种全新的遗传小鼠模型，在骨髓细胞中过表达Rasgrp 1，导致T-ALL，并将此模型与遗传K-RasG 12 D模型进行比较。 我们的生物化学、细胞生物学和体内方法将揭示对这种新型但未表征的细胞因子受体-Rasgrp信号传导途径的分子见解，该信号传导途径被RasGAP Ras失活剂（Aim 1）抵消，并将建立PLC抑制剂在Rasgrp-Ras-Ras效应物激活中的分子作用（Aim 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或小分子抑制剂的PLC抑制。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抑制剂的治疗潜力提供翻译见解，这可能会影响未来T-ALL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.