??PK mediated melanoma stem cell oncolysis through non-redundant programmed cell

描述（由申请人提供）：黑色素瘤是第六常见的癌症，约占所有新诊断恶性肿瘤的5%。这种高发病率和死亡率的癌症在很大程度上对现有疗法具有抗性。这至少部分是由于癌症干细胞（CSC）越来越多地被认为是复发性和治疗抗性疾病的主要来源。溶瘤病毒是一种很有前途的新治疗药物，具有靶向CSC的潜力。早期的临床试验表明，病毒治疗耐受性良好，但其疗效受到细胞溶解活性差、肿瘤穿透不足和在静止肿瘤细胞中复制失败的限制。目前的努力寻求改善复制，但基于诱导程序性细胞死亡（PCD）的病毒治疗策略是不可用的。单纯疱疹病毒（HSV）是一种特别有吸引力的溶瘤病毒，因为它具有广泛的宿主谱，其基因组不整合到宿主基因组中，从而排除插入诱变，抗病毒药物可用于防止不利的病毒复制，并且病毒逃避免疫，允许再治疗。我们最近发现HSV-2基因ICP 10 PK（delta PK）中缺失的HSV-2突变体诱导黑色素瘤细胞死亡。黑色素瘤细胞中激活的Ras信号传导途径提供选择性δ PK复制，但溶瘤作用由多个非冗余PCD途径介导。这些途径包括钙蛋白酶和胱天蛋白酶活性增加，上调表观遗传学沉默基因H11（在黑色素瘤中具有肿瘤抑制样功能），增强自噬相关蛋白Beclin 1的表达，以及激活炎性胱天蛋白酶-1（焦亡）。Delta PK是一种特别有前途的病毒疗法，因为它能刺激Th 1细胞，Th 1细胞能克服抑制性黑色素瘤环境，并且在人体中耐受性良好。拟议的研究旨在更好地阐明δ PK诱导/激活的死亡途径之间的关系，并确定其是否可以杀死黑色素瘤干细胞。具体目的是：（i）阐明自噬和焦亡对δ PK诱导的黑素瘤肿瘤溶解的潜在贡献。重点将是这些替代死亡途径和既定的蛋白酶途径之间的串扰和（ii）表征δ PK的潜力，感染和杀死黑色素瘤干细胞在体外和体内。工作假设是，自噬和焦亡增强了由δ PK诱导的PCD，并且δ PK通过这些途径中的一个或多个感染并杀死黑素瘤干细胞。

DESCRIPTION (provided by applicant): Melanoma is the sixth most common cancer and accounts for approximately ~5% of all newly diagnosed malignancies. This high morbidity and mortality cancer is largely resistant to available therapies. This is due at least in part, to cancer stem cells (CSC) that are increasingly recognized as the primary source of recurrent and therapeutically resistant disease. Oncolytic viruses are a promising new therapeutic that has the potential to target CSC. Early clinical trials indicate that virotherapy is tolerated well but its efficacy is limited by poor cytolytic activity, insufficient tumor penetration and failures to replicate in quiescent tumor cells. Current efforts seek to improve replication but virotherapy strategies based on the induction of programmed cell death (PCD) are not available. Herpes simplex virus (HSV) is a particularly attractive oncolytic virus because it has a broad host spectrum, its genome does not integrate into the host genome thereby precluding insertion mutagenesis, antiviral drugs are available to safeguard against unfavorable virus replication and the virus evades immunity, allowing re-treatment. The proposed studies follow on our recent findings that an HSV-2 mutant deleted in the HSV-2 gene ICP10PK (delta PK) induces melanoma cell death. The activated Ras signaling pathways in melanoma cells provide for selective delta PK replication but oncolysis is mediated by multiple non-redundant PCD pathways. These pathways include increased calpain and caspase activities, upregulation of the epigenetically silenced gene H11 that has a tumor suppressor-like function in melanoma, enhanced expression of the autophagy-related protein Beclin 1, and activation of the inflammatory caspase-1 (pyroptosis). Delta PK is a particularly promising virotherapy because it elicits Th1 cells, which override the suppressive melanoma milieu and it is tolerated well in humans. The proposed studies seek to better elucidate the relationship between the death pathways induced/activated by delta PK and determine whether it can kill melanoma stem cells. The specific aims are: (i) To elucidate the potential contributions of autophagy and pyroptosis to delta PK induced melanoma oncolysis. Focus will be on the cross-talk between these alternate death pathways and established protease pathways and (ii) To characterize delta PK's potential to infect and kill melanoma stem cells both in vitro and in vivo. The working hypothesis is that PCD induction by delta PK is augmented by autophagy and pyroptosis and delta PK infects and kills melanoma stem cells through one or more of these pathways.