Overcoming BRAF and MEK Inhibitors Resistance in Advanced Melanoma

 描述(由申请人提供)： BRAF抑制剂(BRAFi)已成为治疗BRAFV600E突变的关键药物之一，该突变占皮肤黑色素瘤的60%。虽然应答率高，反应可能很快，但复发是不可避免的，通常发生在6个月内。加入MEK抑制剂(Meki)可使缓解时间从6个月延长到9个月，一年生存率提高到40%。然而，复发总是会发生的，而且没有有效的抢救治疗可用。另一种方法是使用带有检查点抑制剂的免疫疗法。Ipilimumab(抗CTLA4抗体)和抗程序性死亡1(抗PD1)抗体(nivolumab和pembrolizumab)的有效时间延长(99周)，总生存率提高(63%一年生存率)。然而，这些药物的起效时间往往很长，当疾病快速发展时，特别是在BRAF失败后，患者可能无法完成计划的疗程，正如已报道的ipilimumab。目前，有关抗PD1和抗PD-L1在BRAFi衰竭或BRAFi和Meki衰竭患者中的抗肿瘤活性的文献报道较少。在这一应用中，我们计划针对这一组病情进展迅速、预后极差的患者进行治疗。我们的初步数据显示，这些耐药细胞对精氨酸剥夺和聚乙二醇化精氨酸脱亚胺酶(ADI-PEG20)非常敏感，在这些耐药肿瘤中，聚乙二醇化精氨酸脱亚胺酶(ADI-PEG20)将精氨酸降解为瓜氨酸，体内产生的精氨酸减少了80%。我们计划研究其潜在机制(S)，并将其转化为临床试验。我们以前工作和其他研究表明，某些黑色素瘤不表达精氨酸琥珀酸合成酶(ASS)，精氨酸琥珀酸合成酶是尿素循环中合成精氨酸的关键酶。这些肿瘤依靠外源精氨酸生存。Adi-PEG20抑制肿瘤生长，毒性最小，因为正常细胞可以摄取瓜氨酸来制造精氨酸。然而，我们发现确实会发生自噬，这会削弱ASS的抗肿瘤作用，并可能导致ASS的重新表达。这两种机制可以解释这种治疗中出现的耐药性。重要的是，我们发现cMyc是ASS重新表达的正调控因子。有趣的是，我们已经发现，由于AMPK1-α和ATG5的减少，抗BRAF(BR)和双重抗性(BMR)的细胞都不能很容易地进行自噬。这使得这些耐药细胞对ADI-PEG20治疗非常敏感。此外，由于cMyc的减少，BR和BMR细胞都不能重新表达ASS基因，因此它们无法逃脱ADI-PEG20的凋亡效应。我们计划研究cMyc、AMPK1-α和ATG5是如何下调的。重要的是，我们计划通过检测从BR和BRM患者获取的肿瘤标本中AMPK1-α、ATG5和ASS的表达，以及使用肿瘤外植体和原代培养检测ADI-PEG20敏感性来研究我们的实验室结果的临床相关性。临床上，BR和BMR患者通常进展非常迅速，单靠ADI-PEG20可能不足以控制肿瘤。在目标3中，我们计划研究ADI-PEG20的哪种组合产生最好的抗肿瘤效果。基于我们的初步数据，我们计划将ADI-PEG20与Meki联合用于BR肿瘤，将ADI-PEG20与HSP-90抑制剂联合用于BMR肿瘤。我们的最终目标是应用这种组合来治疗这群预后极差的患者。免疫检查点抑制剂已经成为治疗黑色素瘤的主要成分，而ADI-PEG20对PD-1和PD-L1表达的影响尚未被探索。我们的初步研究结果表明，ADI-PEG20可以上调BR和BMR细胞中PD-L1的表达，这可能增强了抗PD-1和抗PD-L1抗体的抗肿瘤作用。我们计划在ADI-PEG20治疗前后检测肿瘤外植体、BR和BMR细胞中PD-1/PD-L1的表达。此外，我们还将检测这些患者在ADI-PEG20治疗前后淋巴细胞PD-1的表达。因此，未来使用这两种药物的联合治疗可以合理地用于治疗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.