Discoidin Domain Receptor Tyrosine Kinase 2 in bladder cancer progression

项目总结 即使采用免疫检查点阻断疗法(ICBT)，大多数转移性膀胱癌(BC)患者也会死亡 他们的疾病。在之前的资助期间，我们研究了CD24，一种转移驱动因素和预后标记物。 我们最有翻译意义的发现是Discoidin结构域受体诱导CD24的表达 酪氨酸激酶2(DDR2)，一种胶原蛋白激活的受体，可用于治疗。在人类BC中，DDR2 表达与患者预后不良、侵袭性和ICBT不敏感的基底鳞状细胞有关 肿瘤亚型(Bss)，转化生长因子β信号，免疫抑制(M2)巨噬细胞(Mφ)和富含成纤维细胞， 肿瘤微环境。这些结果提出了一种耐人寻味的可能性，即DDR2是两者的关键驱动因素 转移与ICBT耐药。为了探索这种可能性，我们开发了一种基于shRNA的功能筛选 同基因小鼠BC模型探测DDR2和其他选定的可用药靶点在BC临床中的评估 对它们与不列颠哥伦比亚省常见的αPD-1的协同作用进行试验。我们发现了最有力的肿瘤减少量 耗尽DDR2，并用酪氨酸激酶抑制剂达沙替尼概括这一点。这一结果令人信服。 因为它定义了一种与CD24PD-1的新的协同联合疗法，与α靶向不同，这种疗法是可操作的 在近期的患者中。为了开始阐明DDR2可能用于推动肿瘤转移和 ICBT耐药性，我们用RNA-seq分析了DDR2沉默的小鼠BC肿瘤。我们鉴定了69个基因 被DDR2耗尽下调了2倍，并用这些来构建DDR2效应器签名(DES)。低 手术或αPD-L1治疗的患者的肿瘤DES评分与更好的预后相关，支持 进展中的DES基因与αPD-L1反应值得注意的是，MMP10或S100A2各自单独表达 分层结果与手术和αPD-L1治疗中的DES评分几乎一样，它们之间存在关联 在Mφ、转化生长因子β和成纤维细胞生物学中。这些发现和缺乏与T细胞衰竭相关的基因 DES，引导我们提出我们的创新的指导性假说，即癌细胞DDR2建立了BSS和 利用MMP10和S100A2激活M2 Mφ和肿瘤相关成纤维细胞(CAF)促进转移 和αPD-(L)1抗性。具体的目标是检验这一假设，目的是开发新的有效性 转移性BC的治疗。AIM 1将使用完全和有条件的DDR2缺失小鼠暴露在化学物质中 致癌物验证基本尿路上皮细胞中DDR2表达是BSS肿瘤所必需的假设 发展和进步。AIM 2将治疗DDR2、MMP10和S100A2改变的小鼠肿瘤 在带有成纤维细胞和Mφ缺陷的基因工程小鼠中生长，并带有αPD-1，以测试 肿瘤细胞DDR2驱动MMP10和S100A2及其在M-φ和成纤维细胞中作用的假说 有助于转移和αPD-1耐药。因为即使用DDR2+PD-1阻断，也不是所有的肿瘤都能治愈， 目标3将检验这样一种假设，即抑制DPP4，一种我们发现的一种肽酶和冠状病毒受体 潜在的耐药驱动因素DDR2+PD-1阻断，可提高此类治疗的疗效。

PROJECT SUMMARY Even with immune checkpoint blockade therapy (ICBT), most patients with metastatic bladder cancer (BC) die of their disease. In the previous funding period, we studied CD24, a metastasis driver and prognostic marker. Our most translationally significant finding is induction of CD24 expression by Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), a collagen-activated receptor that is therapeutically actionable. In human BC, DDR2 expression was associated with poor patient prognosis, the aggressive and ICBT insensitive basal squamous tumor subtype (BSS), TGFβ signaling, immunosuppressive (M2) macrophages (Mφ) and a fibroblast-enriched, tumor microenvironment. These results raised the intriguing possibility that DDR2 is a lynchpin driver of both metastasis and ICBT resistance. To explore this possibility, we developed an shRNA based functional screen in syngeneic murine BC models to probe DDR2 and other selected “druggable” targets evaluated in BC clinical trials for their synergism with αPD-1, a common ICBT in BC. We found the most robust tumor reduction with depletion of DDR2 and this was recapitulated with the Tyr kinase inhibitor, dasatinib. This result was compelling because it defined a novel synergistic combination therapy with αPD-1 that, unlike CD24 targeting, is actionable in the near term in patients. To begin elucidating the mechanisms DDR2 may use to drive tumor metastasis and ICBT resistance, we profiled DDR2-silenced murine BC tumors with RNA-seq. We identified 69 genes downregulated >2 fold by DDR2 depletion and used these to construct a DDR2 Effector Signature (DES). Low tumor DES scores of surgically or αPD-L1 treated patients associated with better outcome, supporting a role for DES genes in progression and αPD-L1 response. Remarkably, MMP10 or S100A2 expression each alone stratified outcome nearly as well as the DES score in surgery and αPD-L1 treatment and they were implicated in Mφ, TGFβ and fibroblast biology. These findings and the lack of genes associated with T-cell exhaustion in DES, lead us to propose our innovative Guiding Hypothesis that cancer cell DDR2 establishes the BSS and uses MMP10 and S100A2 to engage M2 Mφ and cancer-associated fibroblasts (CAFs) to promote metastasis and αPD-(L)1 resistance. Specific Aims test this hypothesis with the Objective to develop new effective treatments for metastatic BC. Aim 1 will use full and conditional DDR2-null mice exposed to chemical carcinogens to test the hypothesis that DDR2 expression in basal urothelial cells is necessary for BSS tumor development and progression. Aim 2 will treat murine tumors with alterations in DDR2, MMP10 and S100A2 expression grown in genetically engineered mice with fibroblast and Mφ defects, with αPD-1, to test the hypothesis that cancer cell DDR2 drives MMP10 and S100A2 and via their actions in Mφ and fibroblasts contribute to metastasis and αPD-1 resistance. Since even with DDR2+PD-1 blockade, not all tumors are cured, Aim 3 will test the hypothesis that inhibition DPP4, a peptidase and Coronavirus receptor we discovered as a potential driver of resistance to DDR2+PD-1 blockade, can improve effectiveness of such therapy.