Kit-mediated autophagy suppression driven by a viral oncoprotein emerges as a crucial survival mechanism in Merkel cell carcinoma

The KIT/c-KIT proto-oncogene is frequently over-expressed in Merkel cell carcinoma (MCC), an aggressive skin cancer commonly caused by Merkel cell polyomavirus (MCPyV). Here, we demonstrated that truncated MCPyV-encoded large T-antigen (LT) suppressed macroautophagy/autophagy by stabilizing and sequestering KIT in the paranuclear compartment via binding VPS39. KIT engaged with phosphorylated BECN1, thereby enhancing its association with BCL2 while diminishing its interaction with the PIK3C3 complex. This process ultimately resulted in the suppression of autophagy. Depletion of KIT triggered both autophagy and apoptosis, and decreased LT expression. Conversely, blocking autophagy in KIT-depleted cells restored LT levels and rescued apoptosis. Additionally, stimulating autophagy efficiently increased cell death and inhibited tumor growth of MCC xenografts in mice. These insights into the interplay between MCPyV LT and autophagy regulation reveal important mechanisms by which viral oncoproteins are essential for MCC cell viability. Thus, autophagy-inducing agents represent a therapeutic strategy in advanced MCPyV-associated MCC. <b>Abbreviation</b>: 3-MA, 3-methyladenine; AL, autolysosome; AP, autophagosome; Baf-A1, bafilomycin A₁; BARA, β-α repeated autophagy specific domain; BH3, BCL2 homology 3 domain; CCD, coiled-coil domain; CHX, cycloheximide; Co-IP, co-immunoprecipitation; CQ, chloroquine; CTR, control; DAPI, 4′,6-diamidino-2-phenylindole; EBSS, Earle’s balanced salt solution; ECD, evolutionarily conserved domain; EEE, three-tyrosine phosphomimetic mutations Y229E Y233E Y352E; ER, endoplasmic reticulum; FFF, three-tyrosine non-phosphomimetic mutations; FFPE, formalin-fixed paraffin-embedded; FL, full-length; GIST, gastrointestinal stromal tumor; IB, immunoblotting; IHC, immunohistochemistry; KIT-HEK293, KIT stably expressing HEK293 cells; KRT20/CK20, keratin 20; LT, large T-antigen; LT339, MCPyV truncated LT antigen; LTco, codon-optimized MCPyV LT antigen; MCC, Merkel cell carcinoma; MCPyV⁻, MCPyV-negative; MCPyV, Merkel cell polyomavirus; MCPyV⁺, MCPyV-positive; PARP1, poly(ADP-ribose) polymerase 1; PCI, pan-caspase inhibitor; PI, propidium iodide; PtdIns3K, class III phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; RB1, RB transcriptional corepressor 1; RTKs, receptor tyrosine kinases; KITLG/SCF, KIT ligand; sT, small T-antigen; sTco, codon-optimized MCPyV sT antigen; T-B, Tat-BECN1; T-S, Tat-scrambled; TEM, transmission electron microscopy.

KIT/c-KIT原癌基因（KIT/c-KIT proto-oncogene）在默克尔细胞癌（Merkel cell carcinoma, MCC）中常呈过度表达状态。默克尔细胞癌是一种侵袭性皮肤癌，其发病通常与默克尔细胞多瘤病毒（Merkel cell polyomavirus, MCPyV）感染相关。本研究证实，截短型MCPyV编码的大T抗原（large T-antigen, LT）可通过结合VPS39，将KIT稳定并隔离于核旁区室，从而抑制巨自噬/自噬（macroautophagy/autophagy）。KIT可与磷酸化的BECN1结合，进而增强其与BCL2的相互作用，同时削弱其与PIK3C3复合物的结合，最终导致自噬受到抑制。敲除KIT可同时触发自噬与凋亡，并降低LT的表达水平。反之，在KIT敲除细胞中阻断自噬，可恢复LT的表达水平并挽救凋亡现象。此外，激活自噬可有效促进细胞死亡，并抑制小鼠体内MCC移植瘤的生长。上述关于MCPyV LT与自噬调控之间相互作用的研究结果，揭示了病毒癌蛋白对MCC细胞存活至关重要的关键机制。因此，诱导自噬的药物可作为晚期MCPyV相关性MCC的治疗策略。

缩写对照表：3-MA：3-甲基腺嘌呤（3-methyladenine）；AL：自噬溶酶体（autolysosome）；AP：自噬体（autophagosome）；Baf-A1：巴弗洛霉素A₁（bafilomycin A₁）；BARA：β-α重复自噬特异性结构域（β-α repeated autophagy specific domain）；BH3：BCL2同源3结构域（BCL2 homology 3 domain）；CCD：卷曲螺旋结构域（coiled-coil domain）；CHX：环己酰亚胺（cycloheximide）；Co-IP：免疫共沉淀（co-immunoprecipitation）；CQ：氯喹（chloroquine）；CTR：对照组（control）；DAPI：4′,6-二脒基-2-苯基吲哚（4′,6-diamidino-2-phenylindole）；EBSS：伊格尔氏平衡盐溶液（Earle’s balanced salt solution）；ECD：进化保守结构域（evolutionarily conserved domain）；EEE：三酪氨酸磷酸模拟突变Y229E、Y233E、Y352E（three-tyrosine phosphomimetic mutations Y229E Y233E Y352E）；ER：内质网（endoplasmic reticulum）；FFF：三酪氨酸非磷酸模拟突变（three-tyrosine non-phosphomimetic mutations）；FFPE：福尔马林固定石蜡包埋（formalin-fixed paraffin-embedded）；FL：全长（full-length）；GIST：胃肠道间质瘤（gastrointestinal stromal tumor）；IB：免疫印迹（immunoblotting）；IHC：免疫组化（immunohistochemistry）；KIT-HEK293：稳定表达KIT的HEK293细胞（KIT stably expressing HEK293 cells）；KRT20/CK20：角蛋白20（keratin 20）；LT：大T抗原（large T-antigen）；LT339：MCPyV截短型LT抗原（MCPyV truncated LT antigen）；LTco：密码子优化型MCPyV LT抗原（codon-optimized MCPyV LT antigen）；MCC：默克尔细胞癌（Merkel cell carcinoma）；MCPyV⁻：MCPyV阴性（MCPyV-negative）；MCPyV：默克尔细胞多瘤病毒（Merkel cell polyomavirus）；MCPyV⁺：MCPyV阳性（MCPyV-positive）；PARP1：多聚ADP核糖聚合酶1（poly(ADP-ribose) polymerase 1）；PCI：泛半胱天冬酶抑制剂（pan-caspase inhibitor）；PI：碘化丙啶（propidium iodide）；PtdIns3K：Ⅲ型磷脂酰肌醇3-激酶（class III phosphatidylinositol 3-kinase）；PtdIns3P：磷脂酰肌醇-3-磷酸（phosphatidylinositol-3-phosphate）；RB1：RB转录共抑制因子1（RB transcriptional corepressor 1）；RTKs：受体酪氨酸激酶（receptor tyrosine kinases）；KITLG/SCF：KIT配体（KIT ligand）；sT：小T抗原（small T-antigen）；sTco：密码子优化型MCPyV sT抗原（codon-optimized MCPyV sT antigen）；T-B：Tat-BECN1；T-S：Tat-scrambled；TEM：透射电子显微镜（transmission electron microscopy）。