Overcoming BRAF and MEK Inhibitors Resistance in Advanced Melanoma

 描述（由申请人提供）： BRAF抑制剂（BRAFi）已成为BRAFV 600 E突变的关键治疗之一，BRAFV 600 E突变占皮肤黑色素瘤的60%。虽然反应率很高，反应可能很快，但复发是不可避免的，通常在6个月内发生。MEK抑制剂（MEKi）的加入可将反应持续时间从6个月增加到9个月，并将1年生存率提高到40%。然而，复发总是发生，没有有效的补救治疗。另一种方法是使用检查点抑制剂的免疫疗法。Ipilimumab（抗CTLA 4抗体）和抗程序性死亡1（抗PD 1）抗体（Nivolumab和Pembrolizumab）显示缓解持续时间延长（99周）和总生存期延长（63%一年生存率）。然而，这些药物的反应时间通常较长，并且当发生快速疾病进展时，特别是在BRAF失败后，患者可能无法完成计划的治疗过程，如伊匹单抗所报告的那样。目前，关于BRAFi失败或BRAFi和MEKi失败患者后抗PD 1和抗PD-L1的抗肿瘤活性的已发表数据很少。在本申请中，我们计划靶向治疗这组疾病进展迅速且预后极差的患者。我们的初步数据表明，这些耐药细胞对精氨酸剥夺非常敏感，并且将精氨酸降解为瓜氨酸的聚乙二醇化精氨酸脱亚胺酶（ADI-PEG 20）在体内使这些耐药肿瘤减少>80%。我们计划研究潜在的机制，并将其转化为临床试验。我们以前的工作和其他人已经表明，某些黑色素瘤不表达精氨酸琥珀酸合成酶（ASS），尿素循环中的关键酶，以合成精氨酸。这些肿瘤依赖外源性精氨酸生存。ADI-PEG 20抑制肿瘤生长的毒性最小，因为正常细胞可以摄取瓜氨酸生成精氨酸。然而，我们发现自噬确实发生，这可以减弱抗肿瘤作用，并且可以发展ASS的重新表达。这两种机制可以解释这种治疗所见的耐药性。重要的是，我们已经发现cMyc是ASS再表达的正调控因子。有趣的是，我们发现BRAF抗性（BR）和双重抗性[对BRAFi和MEKi都具有抗性（BMR）]细胞由于AMPK 1-α和Atg 5的减少而不能容易地进行自噬。这使得这些耐药细胞对ADI-PEG 20治疗非常敏感。此外，由于cMyc的减少，BR和BMR细胞都不能再表达ASS基因，因此它们不能逃避ADI-PEG 20处理的凋亡效应。我们计划研究cMyc、AMPK 1-α和Atg 5是如何下调的。重要的是，我们计划通过使用肿瘤外植体和原代培养物检查从BR和BRM患者中获得的肿瘤样本的AMPK 1-α、Atg 5和ASS表达以及ADI-PEG 20敏感性，研究我们实验室结果的临床相关性，如目的2所述。在临床上，BR和BMR患者通常进展非常迅速，单独的ADI-PEG 20可能不足以控制肿瘤。在目标3中，我们计划研究与ADI-PEG 20的哪种组合产生最佳抗肿瘤功效。基于我们的初步数据，我们计划在BR肿瘤中将联合收割机ADI-PEG 20与MEKi结合，在BMR肿瘤中将ADI-PEG 20与Hsp-90抑制剂结合。我们的最终目标是应用这种组合来治疗这组预后极差的患者。免疫检查点抑制剂已成为治疗黑色素瘤的主要成分，尚未探索ADI-PEG 20对PD-1和PD-L1表达的影响。我们的初步数据表明，ADI-PEG 20可以上调BR和BMR细胞中的PD-L1，这可能增强抗PD-1和抗PD-L1抗体的抗肿瘤作用。我们计划在ADI-PEG 20治疗前后检查肿瘤外植体、BR和BMR细胞的PD-1/PD-L1表达。此外，我们还将检查这些患者在ADI-PEG 20治疗前后淋巴细胞的PD-1表达。因此，未来使用这两种药物的联合治疗可以合理地制定治疗ASS（-）黑色素瘤患者。

 DESCRIPTION (provided by applicant): BRAF inhibitor (BRAFi) has emerged as one of the key treatments for BRAFV600E mutation which accounts for 60% of cutaneous melanomas. While the response rate is high and responses can be rapid, relapse is inevitable and often occurs within 6 months. The addition of MEK inhibitor (MEKi) could increase the duration of response from 6 to 9 months and increase one year survival to 40%. However, relapse invariably occurs and there is no effective salvage treatment available. Another approach is the use of immunotherapy with checkpoint inhibitors. Ipilimumab (anti-CTLA 4 antibody) and anti-programmed death 1 (anti-PD1) antibody (Nivolumab and Pembrolizumab) showed an increase in duration of response (99 weeks) and increased overall survival (63% one year survival rate). However, the time to response is often long for these agents, and when rapid disease progression occurs, especially after BRAF failure, patients may not be able to complete the planned treatment course, as has been reported for ipilimumab. Currently, there is a paucity of published data on the antitumor activity of anti-PD1 and anti-PD-L1 after BRAFi failure or BRAFi and MEKi failure patients. In this application, we plan to target to treat this group of patients which has rapid progressive disease and extremely poor prognosis. Our preliminary data showed that these resistant cells are very sensitive to arginine deprivation and pegylated arginine deiminase (ADI-PEG20) which degrades arginine to citrulline produced >80% decrease in these resistant tumors in vivo. We plan to investigate the underlying mechanism(s) and translate this into clinical trial. Our previou work and others have shown that certain melanomas do not express argininosuccinate synthetase (ASS), a key enzyme in the urea cycle, to synthesize arginine. These tumors depend on exogenous arginine to survive. ADI-PEG20 inhibits tumor growth with minimal toxicity since normal cells can take up citrulline to make arginine. However, we have found that autophagy does occur which can blunt the antitumor effect and re-expression of ASS can develop. These two mechanisms can account for drug resistance seen with this treatment. Importantly, we have found that cMyc is a positive regulator for re- expression of ASS. Interestingly, we have found that both BRAF resistant (BR) and dual resistant [resistant to both BRAFi and MEKi (BMR)] cells cannot readily undergo autophagy due to decrease in AMPK1-α and Atg5. This makes these resistant cells exquisitely sensitive to ADI-PEG20 treatment. In addition, both BR and BMR cells are unable to re-express ASS gene due to decrease in cMyc, and hence they cannot evade the apoptotic effect by ADI-PEG20 treatment. We plan to investigate how cMyc, AMPK1-α, and Atg5 are down regulated. Importantly, we plan to investigate the clinical relevance of our laboratory findings by examining tumor samples obtained from BR and BRM patients for AMPK1-α, Atg5 and ASS expression and for ADI-PEG20 sensitivity using tumor explant and primary culture as outlined in aim 2. Clinically, BR and BMR patients usually progress very rapidly and ADI-PEG20 alone may not be sufficient to control the tumor. In aim 3, we plan to investigate which combination with ADI-PEG20 yields the best antitumor efficacy. Based on our preliminary data, we plan to combine ADI-PEG20 with MEKi in BR tumors and ADI-PEG20 with Hsp-90 inhibitors in BMR tumors. Our ultimate goal is to apply this combination to treat this group of extremely poor prognosis patients. Immune checkpoint inhibitors have become a major component for the treatment of melanoma, and the effect of ADI- PEG20 on PD-1 and PD-L1 expression has not been explored. Our preliminary data showed that ADI-PEG20 can upregulate PD-L1 in BR and BMR cells, which may potentiate the antitumor effect of anti-PD-1 and anti- PD-L1 antibody. We plan to examine tumor explants, BR, and BMR cells for PD-1/PD-L1 expression before and after ADI-PEG20 treatment. Furthermore, we will also examine PD-1 expression from lymphocytes in these patients before and after treatment with ADI-PEG20. Thus, future combinatorial treatment using both agents can be rationally formulated to treat ASS (-) melanoma patients.