Co-Targeting IL-6 and EGFRsignaling for the Treatment of Schwannomatosis and Associated Pain

摘要 神经鞘瘤病是一种以多发性非恶性神经鞘瘤生长为特征的遗传性疾病。 在脊椎和周围神经上。SWN患者绝大多数表现为顽固、虚弱的症状 慢性疼痛，严重到足以导致永久性残疾。SWN患者疼痛的病因尚不清楚， 治疗SWN和相关疼痛的新疗法的开发极其缓慢和低效。治疗 因为SWN仅限于侵入性手术，这会带来进一步神经损伤的重大风险。目前还没有药物 FDA批准用于阻止SWN肿瘤生长或缓解SWN相关疼痛。认识到世界上最大的 SWN研究面临的挑战是缺乏临床相关的模型，我们成功地建立了患者衍生的模型 SWN细胞株，并在原位坐骨神经和脊柱小鼠模型中生长，复制肿瘤- 诱导疼痛。使用这些新的患者来源的异种移植模型(PDX)，在初步研究中，我们发现： I)生长在周围神经的肿瘤病变导致巨噬细胞涌入背根神经节 (DRG)，ii)SWN肿瘤衍生的高迁移率族蛋白1(HMGB1)，一个关键的炎症启动者和 放大，调节关键巨噬细胞C-C基序趋化因子配体2(CCL2)的神经元表达 趋化因子，iii)肿瘤刺激的巨噬细胞产生高水平的促炎细胞因子， 白介素6(IL-6)，iv)IL-6中和抗体显著降低疼痛反应，但对 原位SWN PDX模型中的肿瘤生长，以及v)抗IL-6治疗激活EGFR信号，从而 可以补偿肿瘤的生长。基于这些令人兴奋的发现，我们假设：i)SWN派生 HMGB1刺激感觉神经元表达CCL2，从而将巨噬细胞招募到DRG，II) 巨噬细胞通过过度产生IL-6而引起疼痛反应，III)联合IL-6和EGFR阻断可以 在SWN模型中同时减轻疼痛和抑制肿瘤生长。在目标1中，使用原位SWN PDX 模型，我们将进行功能丧失实验，以评估肿瘤来源的HMGB1在 调节神经元中CCL2的表达。然后，我们将研究神经元(CCL2)-巨噬细胞(CCR2)轴是否 在巨噬细胞募集和疼痛反应中必不可少的，使用过继转移的骨髓来源 CCR2-/-小鼠的巨噬细胞。在目标2中，使用遗传沉默和药物抑制，我们将评估 肿瘤来源与巨噬细胞来源的IL-6信号在原位SWN疼痛反应中的作用 PDX模型；此外，我们还将确定IL-6中和抗体在降低疼痛反应方面的疗效。 在目标3中，我们将验证IL-6和EGFR联合阻断在延缓肿瘤方面有效的假设 原位SWN PDX模型中的生长和减轻疼痛反应。影响：如果成功，这项研究将I) 为肿瘤(HMGB1)-神经元(CCL2)的作用提供关键的细胞和分子机制见解- 巨噬细胞(IL-6)在SWN中引起疼痛的串扰，以及II)使用FDA批准的IL-6和EGFR抑制剂 目的：在减轻疼痛的同时控制肿瘤的生长。

ABSTRACT Schwannomatosis (SWN) is a genetic disorder characterized by multiple non-malignant schwannomas growing on the spine and peripheral nerves. Patients with SWN overwhelmingly present with intractable, debilitating chronic pain, severe enough to cause permanent disability. The etiology of pain in SWN is not clear and the development of novel treatments for SWN and related pain has been extremely slow and inefficient. Treatment for SWN is limited to invasive surgery, which carries significant risk of further nerve damage. No drug is currently FDA-approved to halt SWN tumor growth or ameliorate SWN-associated pain. Recognizing that one of the biggest challenges in SWN research is the lack of clinically-relevant models, we successfully established patient-derived SWN cell lines and grew them in the orthotopic sciatic nerve and spine mouse models that reproduce tumor- induced pain. Using these novel patient-derived xenograft models (PDX), in preliminary studies, we found that: i) tumor lesions that grew in the peripheral nerve cause an influx of macrophage into the dorsal root ganglia (DRG), ii) SWN tumor-derived Highly Mobility Group Box1 (HMGB1), a key inflammation initiator and amplifier, regulates neuronal expression of C-C motif chemokine ligand 2 (CCL2), the key macrophage chemokine, iii) tumor-primed macrophages produce elevated levels of the pro-inflammatory cytokine, Interleukin-6 (IL-6), iv) IL-6 neutralizing antibody significantly reduces pain response but had little efficacy on tumor growth in the orthotopic SWN PDX model, and v) anti-IL-6-treatment activates EGFR signaling, which can compensate for tumor growth. Based on these exciting discoveries, we hypothesize that: i) SWN-derived HMGB1 stimulates sensory neurons to express CCL2, which recruits macrophages into the DRG, ii) macrophages cause pain response via overproduction of IL-6, and iii) combined IL-6 and EGFR blockade can concurrently reduce pain and inhibit tumor growth in SWN models. In Aim 1, using orthotopic SWN PDX models, we will perform loss-of-function experiments to assess the functional role of tumor-derived HMGB1 in regulating CCL2 expression in neurons. Then, we will study if the neuron (CCL2)-macrophage (CCR2) axis is essential in macrophage recruitment and pain response, using the adoptive transfer of bone marrow-derived macrophage from Ccr2-/- mice. In Aim 2, using genetic silencing and pharmacologic inhibition, we will assess the contributions of tumor-derived vs. macrophage-derived IL-6 signaling on pain response in orthotopic SWN PDX models; moreover, we will determine the efficacy of IL-6 neutralizing antibody in reducing pain response. In Aim 3, we will test the hypothesis that combined IL-6 and EGFR blockade is effective in delaying tumor growth and reducing pain response in orthotopic SWN PDX models. Impact: If successful, this study will i) provide pivotal cellular and molecular mechanistic insights into the role of tumor (HMGB1)–neuron (CCL2)– macrophage (IL-6) crosstalk in causing pain in SWN, and ii) employ FDA-approved IL-6 and EGFR inhibitors to simultaneously alleviate pain and control tumor growth in SWN.