Signal Transduction Events and the Regulation of Cell Growth

我们在2022财年的主要活动是与临床研究人员合作，开发适合其临床试验的新药效学（PD）分析，并在我们在2022财年开放的临床试验中实施这些PD分析。我们的具体目标是：(1)确定一种疗法是否达到了患者的目标；(2)在系统水平、肿瘤和肿瘤微环境中探究治疗对宿主的影响；(3)与临床研究人员合作，将新技术引入他们的项目，包括在内部、外部和国际上转让我们的技术；(4)与临床和基础转化研究人员合作，确定新的药物靶点和新的药物作用机制。我们在2022财年进行了60多项临床试验。我们专注于生物标志物分析的三个主要领域；(1)免疫PD，(2)罕见细胞非免疫PD（即循环肿瘤细胞和循环内皮细胞），以及(3)可在新鲜、冷冻或福尔马林固定石蜡包埋组织（FFPE）上进行基因表达的数字分析，包括FFPE肿瘤的数字空间分析，极大地促进了肿瘤样本中基因表达的评估。此外，我们还研究了免疫和非免疫靶向治疗对外周血免疫基因表达的全身影响。在我们合作的大多数临床试验中，我们通过多参数流式细胞术评估了免疫PD。我们已经在多个临床试验中发现并发表了与生存率的相关性。这些探索性数据为治疗对外周免疫的影响提供了见解，正如发表在Nature （Wherry等人）和PNAS （Ramalingam等人）上的那样，可以提供反映效应T细胞与肿瘤相互作用的有价值信息，并作为对检查点封锁反应的潜在血液指标，正如我们在2019年与包括博士在内的临床团队合作发表的那样。Karzai, Madan， Gulley和Dahut研究了抗pd - l1抗体durvalumab加PARP抑制剂olaparib治疗的去势抵抗性前列腺癌患者的免疫谱，证明了CD8+ T细胞再生的证据。2022年，我们对完成的durvalumab/olaparib临床试验的所有参与者进行了评估，目前正在准备发表。在我们的免疫表型研究中，我们专注于单核细胞、肿瘤相关巨噬细胞、髓源性抑制细胞、活化和调节性t细胞的多个群体和亚群，包括功能标记的表达。在2022财年，我们对多个HDAC抑制剂临床试验进行了免疫亚群分析。综上所述，这些数据为我们提供了先天免疫和适应性免疫在患者基线和治疗反应中的功能相互作用的观点，这反过来又为未来临床研究提供了联合治疗以增强抗肿瘤活性的建议。除了HDAC抑制剂外，我们还专注于药物类别，包括伴侣抑制剂和多激酶抑制剂cabozantinib。之前，我们开发了一种PD测定法来评估体内HDAC抑制剂的活性。NCI为我们的工作申请了专利，并于2016年发布。我们已经在多个临床试验中实施了这项技术，其中包括13个已发表的临床试验。2021年，我们与儿科早期临床试验网络（pediatric Early Phase Clinical Trials Network）合作，发表了HDAC抑制剂entinostat的首个儿科试验。我们还发表了一篇关于恩替诺他联合派姆单抗对转移性非小细胞肺癌外周免疫影响的论文。我们是ECOG-ACRIN癌症研究小组（ECOG-ACRIN Cancer Research Group）在FDA注册的依替诺他治疗激素受体阳性晚期乳腺癌的3期临床试验的国家实验室中心，我们的PD是唯一的综合生物标志物。该试验最近发表，我们的PD评估证实了恩替诺他治疗患者的靶抑制作用。我们正在帮助将安替司他带到日本，用于治疗女性乳腺癌。我们与Kyowa Kirin密切合作，完成了两项将恩替诺他单独治疗或与依西美坦联合治疗的临床试验。第一本于2022年出版，第二本正在审查中。总的来说，我们一直在与美国国立卫生研究院的15个分支机构、三家生物技术公司、阿斯利康的两项制药试验以及与学术界（约翰霍普金斯大学、克利夫兰诊所、达纳法伯、麻省理工学院）的四项试验进行临床试验，包括国内和国际的1期、2期和3期试验。在研究伴侣蛋白抑制剂时，我们之前发现Hsp40是一个新的抗癌靶点。NIH申请了该发明的专利，并于2018财年颁发了初始专利，并于2020年颁发了第二项专利。Drs。国家癌症研究所的Len Neckers和加州大学旧金山分校的Jason Gestwicki我们正在争取两项国防部奖励，包括进一步开发药物，以达到我们在实验室确定的目标。我们一直与Brigitte Widemann博士和她的团队合作分析nf1相关肿瘤的免疫浸润，包括外周血和nf1相关丛状神经纤维瘤、非典型神经纤维瘤、生物潜能不确定的非典型神经纤维瘤肿瘤和恶性周围神经鞘肿瘤的免疫分析。我是儿童肿瘤小组多机构卡博赞替尼试验的主要合作伙伴，我们收到了来自全国各地的样本。2022年，我们完成了这些样本的外周免疫分析，这些分析结果将被纳入描述试验结果的手稿中。在2022财年，我们继续与Anish Thomas博士就其小细胞肺癌（SCLC）临床试验的免疫分析和循环肿瘤细胞分析进行合作，包括Thomas博士于2022年在cancer cell上发表我们的循环肿瘤细胞结果。作为HSP90抑制剂工作的延续，我们还与Thomas博士合作进行了一项一流的HSP90结合分子与拓扑异构酶I抑制剂的临床试验，为此我们正在使用NanoString和液滴数字PCR进行数字基因表达。我们在Andrea Apolo博士的cabozantinib治疗转移性尿路上皮癌的2期试验中确定了cabozantinib对先天和适应性免疫细胞的影响（Lancet Oncol. 2020）。在2021财年和2022财年，我们与Apolo博士合作，继续确定cabozantinib与检查点抑制剂nivolumab和ipilimumab联合使用对全身免疫的影响，并正在准备进一步的分析。我们继续在循环内皮细胞方面的工作，并在Christine Alewine博士针对晚期胰腺腺癌患者的LMB-100 + nab -紫杉醇试验中确定了毛细血管渗漏综合征的严重程度与凋亡循环内皮细胞的增加之间的关联。2022年，我们正在与Alewine博士合作进行超级增强剂Minnelide用于晚期难治性胰腺腺鳞癌的II期试验。在2021年和2022年，我们一直与Jeremy Davis博士合作，使用NanoString GeoMx平台进行数字空间分析，分析与CDH1基因种系失活变异相关的遗传性弥漫性胃癌研究参与者的组织。

Our major activities in FY2022 were to work with clinical investigators to develop new pharmacodynamic (PD) assays tailored to their clinical trials and to implement these PD assays in the clinical trials we have open in FY2022. Our specific objectives are (1) to determine if a therapy hit its target in the patient; (2) interrogate the impact of the therapy on the host, both at the systemic level, and in the tumor and tumor microenvironment; (3) work with clinical investigators to introduce new technology into their programs, including transfer of our technology intramurally, extramurally and internationally; and (4) work in collaboration with clinical and basic translational investigators to identify new drug targets and new drug mechanisms. We worked on more than 60 clinical trials in FY2022. We have focused on three main areas of biomarker analysis; (1) immune PD, (2) rare cell non-immune PD (i.e. circulating tumor cells and circulating endothelial cells), and (3) digital analyses of gene expression that can be performed on fresh, frozen, or formalin-fixed, paraffin-embedded tissue (FFPE), including digital spatial profiling of FFPE tumor, to greatly facilitate the assessment of gene expression in tumor samples. In addition, we examined systemic effects of immune- and non-immune targeted therapy on immune gene expression in peripheral blood. For the majority of the clinical trials on which we collaborate we are including immune PD as assessed by multiparameter flow cytometry. We have found and published correlations with survival in multiple clinical trials. These exploratory data have provided insight into the impact of therapy on peripheral immunity, which, as published in Nature (Wherry et al.) and PNAS (Ramalingam et al.), can provide valuable information reflective of the interaction of effector T cells and tumor and serve as a potential blood-based indicator of response to checkpoint blockade, as we published in 2019 in a collaboration with a clinical team including Drs. Karzai, Madan, Gulley and Dahut on immune profiling of castration-resistant prostate cancer patients treated with the anti-PD-L1 antibody durvalumab plus the PARP inhibitor olaparib, demonstrating evidence of CD8+ T cell reinvigoration. In 2022 we assessed all of the participants in the completed durvalumab/olaparib clinical trial, and this is currently being prepared for publication. In our immune phenotyping studies we focused on multiple populations and subpopulations of monocytes, tumor-associated macrophages, myeloid-derived suppressor cells, activated and regulatory T-cells, including expression of functional markers. In FY2022 we have performed immune subset analysis on multiple HDAC inhibitor clinical trials. Together these data have afforded us a view of the functional interplay of innate and adaptive immunity in patients at baseline and in response to treatment, which in turn, suggests combination therapies for future clinical studies to enhance antitumor activity. Drug classes we have focused on in addition to HDAC inhibitors include chaperone inhibitors, and the multikinase inhibitor cabozantinib. Previously we developed a PD assay for assessment of HDAC inhibitor activity in vivo. The NCI applied for a patent on our work, which issued in 2016. We have implemented this technology in multiple clinical trials, including 13 published clinical trials. In 2021 we published the first pediatric trial of the HDAC inhibitor entinostat in collaboration with the Pediatric Early Phase Clinical Trials Network. We also published a manuscript on the impact of entinostat plus pembrolizumab on peripheral immunity in metastatic non-small cell lung cancer. We are the National Laboratory Center of a Phase 3 ECOG-ACRIN Cancer Research Group FDA registration trial for entinostat in hormone receptor-positive advanced breast cancer, for which our PD is the only integrated biomarker. The trial was recently published and our PD assessment confirmed target inhibition in entinostat-treated patients. We are helping to bring entinostat to Japan for the treatment of women with breast cancer. Working closely with Kyowa Kirin, we completed two clinical trials of entinostat as monotherapy or in combination with exemestane. The first was published in 2022 and the second is under review. Overall, we have been working on clinical trials with 15 Branches of the NIH, three biotech companies and on two pharma trials with AstraZeneca, as well as four trials with academia (Johns Hopkins, Cleveland Clinic, Dana Farber, MIT), together encompassing Phase 1, Phase 2, and Phase 3 trials, both nationally and internationally. Working on chaperone inhibitors we previously identified Hsp40 as a new anticancer target. NIH filed for patent on this invention and the initial patent issued in FY2018 and a second patent issued in 2020. With Drs. Len Neckers of NCI and Jason Gestwicki of UCSF we are working on two DOD awards that include further development of drugs designed to hit the target we identified in my lab. We have been collaborating with Dr. Brigitte Widemann and her team on analysis of the immune infiltrate in NF1-associated tumors, including immune analysis of peripheral blood and tumor of NF1-associated plexiform neurofibromas, atypical neurofibromas, atypical neurofibromatous neoplasms of uncertain biologic potential, and malignant peripheral nerve sheath tumors. I am a key collaborator on a Childen's Oncology Group multi-institutional trial of cabozantinib from which we received samples from all over the country. In 2022 we completed analysis of peripheral immunity on these samples, and these analyses are being included in the manuscript describing the trial results. In FY2022 we have continued our collaborations with Dr. Anish Thomas on immune profiling and circulating tumor cell analyses of his small cell lung cancer (SCLC) clinical trials, including publishing of our circulating tumor cell results by Dr. Thomas in Cancer Cell in 2022. As a continuation of our work on HSP90 inhibitors we also collaborated with Dr. Thomas on a first-in-class clinical trial of an HSP90-binding molecule linked to a topoisomerase I inhibitor, for which we are doing digital gene expression using NanoString and droplet digital PCR. We identified the impact of cabozantinib on innate and adaptive immune cells in Dr. Andrea Apolo's Phase 2 trial of cabozantinib in metastatic urothelial carcinoma (Lancet Oncol. 2020). In FY2021 and 2022 in collaboration with Dr. Apolo we have continued to define the the impact of cabozantinib on systemic immunity in combination with the checkpoint inhibitors nivolumab and ipilimumab, and further analyses are in preparation. We have continued our work on circulating endothelial cells and identified an association of the severity of capillary leak syndrome with increases in apoptotic circulating endothelial cells in Dr. Christine Alewine's LMB-100 plus Nab-Paclitaxel trial of patients with advanced pancreatic adenocarcinoma and in 2022 we are working with Dr, Alewine on a phase II trial of the super-enhancer Minnelide in advanced refractory adenosquamous carcinoma of the pancreas. With Dr. Jeremy Davis in 2021 and 2022 we have been working on analysis of tissue from participants in his study of hereditary diffuse gastric cancer associated with germline inactivating variants in the CDH1 gene using the NanoString GeoMx platform for digital spatial profiling.