Transcriptional Regulation of Peripheral Immune Cells as a Modulator of Melanoma Immunotherapy Resistance and Toxicity

黑色素瘤是美国第五大最常见的癌症，而早期- 阶段黑色素瘤是有利的，晚期疾病长期被认为是不可治愈的。免疫检查点抑制剂 （ICI）彻底改变了转移性黑色素瘤的治疗;然而，约40%的患者仍然没有反应。 约80%的ICI治疗患者发生免疫相关不良事件（irAE）。识别 迫切需要一种可靠的治疗前生物标志物，对患者进行分层，以获得最大疗效，同时将毒性降至最低。 临床任务，因为现有的肿瘤或肿瘤/免疫替代物有限。最近的研究表明，基线 宿主免疫力由影响T细胞活化和分化的特定调节网络控制， 包括CD 8 + T细胞（ICI的主要靶点）。积累的数据表明，调控基因组（ 由转录因子、增强子和其他元件（例如lncRNA）组成的调节基因组（“调节基因组”）控制转录因子的表达。 细胞毒性T细胞的转录重编程，在宿主抗肿瘤免疫应答中起关键作用。 我们和其他人已经报道，调节组是丰富的潜在遗传变异相关， 自身免疫风险，并且这种遗传易感性与ICI结果相关。基于这些有趣的 根据观察，我假设外周免疫细胞的基线“调节组”状态，由免疫调节蛋白决定。 遗传基因组和转录组因子相互作用，代表ICI抗性的新生物标志物， 对irAE的易感性。我假设这些转录调控网络（TRN）控制着T细胞的活性， 潜在地揭示生物学途径以降低毒性和增强ICI功效。对于F99阶段，我将 开发捕获治疗前外周CD 8 + T细胞（pre-pCD 8+）活性TRN的基因组方法， 使用ICI治疗的转移性黑色素瘤样本的ICI在转移性黑色素瘤中疗效的替代物 百时美施贵宝公司领导的一项大型临床试验的患者。我会识别转录信号 与pre-pCD 8+细胞中的ICI结果相关;单细胞RNA-seq将提供对免疫细胞 ICI背景下的亚型（目标1.1）。转座酶可降解染色质（ATAC）-seq的组合测定， RNA测序和全基因组测序，我将确定CD 8+特异性调节组TRN作为ICI的调节剂 晚期黑色素瘤患者的结局（目标1.2）。对于K 00阶段，我将扩展此假设， 探索治疗前和治疗后单细胞水平的变化，与特定免疫细胞的变化相关 使用黑色素瘤患者PBMC的ICI应答和irAE类型（目的2）。我还将建立模型， ICI相关的单细胞PBMC细胞类型和具有特异性肿瘤新表位的转录组特征， 治疗靶向使用这些生物标志物识别最有可能对ICI有反应的患者也可能指向 新的生物学途径，用于更有效和毒性更低的治疗，进一步改善ICI患者 黑色素瘤和其他潜在癌症的结果。

PROJECT SUMMARY Melanoma is the fifth most common cancer in the US and while the prognosis for early- stage melanoma is favorable, late-stage disease was long considered incurable. Immune checkpoint inhibitors (ICIs) have revolutionized metastatic melanoma treatment; nevertheless, ~40% of patients still do not respond to therapy and ~80% of ICI-treated patients experience immune-related adverse events (irAEs). Identifying reliable pre-treatment biomarkers stratifying patients for maximal efficacy while minimizing toxicity is an urgent clinical task, as existing tumor or tumor/immune surrogates are limited. Recent research suggests that baseline host immunity is controlled by specific regulatory networks impacting T-cell activation and differentiation, including CD8+ T cells (the primary targets of ICI). Accumulated data show that the regulatory genome (the “regulome”), composed of transcription factors, enhancers, and other elements (e.g. lncRNAs), controls the transcriptional reprogramming of cytotoxic T-cells, playing a critical role in host anti-tumor immune response. We and others have reported that the regulome is enriched by underlying genetic variation associated with autoimmune risk and that this genetic susceptibility associates with ICI outcomes. Based on these intriguing observations, I hypothesize that baseline “regulome” states of peripheral immune cells, determined by the interplay of inherited genomic and transcriptomic factors, represent novel biomarkers of ICI resistance and susceptibility to irAEs. I postulate that these transcriptional regulatory networks (TRNs) govern T cell activity, potentially revealing biological pathways to reduce toxicity and enhance ICI efficacy. For the F99 phase, I will develop a genomic approach capturing TRNs of pre-treatment peripheral CD8+ T-cell (pre-pCD8+) activity as surrogates of ICI efficacy in metastatic melanoma using samples from ICI-treated metastatic melanoma patients from a large clinical trial led by Bristol-Myers Squibb. I will identify transcriptional signatures associated with ICI outcomes in pre-pCD8+ cells; single-cell RNA-seq will offer insight into immune cell subtypes in the ICI context (Aim 1.1). Combining Assay for Transposase-Accessible Chromatin (ATAC)-seq, RNA seq, and whole-genome sequencing, I will determine CD8+-specific regulome TRNs as modulators of ICI outcomes in advanced melanoma patients (Aim 1.2). For the K00 phase, I will expand this hypothesis to explore pre- and post-treatment changes at the single-cell level, correlating alterations in specific immune cell types with ICI response and irAEs using melanoma patient PBMCs (Aim 2). I will also develop models linking ICI-associated single-cell PBMC cell types and transcriptomic signatures with specific tumor neo-epitopes for therapeutic targeting. Identifying patients most likely to respond to ICI using these biomarkers may also point to novel biological pathways for more efficacious and less toxic treatments, further improving ICI patient outcomes in melanoma and potentially other cancers.