Targeting p38 gamma signaling to advance Cutaneous T Cell Lymphoma Therapy

皮肤 T 细胞淋巴瘤 (CTCL) 是一种毁容且无法治愈的癌症。对于病情晚期的患者来说， 目前的治疗方法不足，效果不佳。对 CTCL 分子的了解不完全 监管机构限制了有效靶向治疗的开发。一种候选调节因子是 p38γ，该基因 它的表达在 CTCL 细胞系和患者样本中选择性增加，但在健康 T 细胞中则不然。我们 证明 p38γ 的抑制或沉默会抑制增殖并诱导 CTCL 细胞死亡。 NF-κB 该通路在 CTCL 中具有组成型活性，提供与 p38γ 互补的 T 细胞信号通路，并且可以 被组蛋白脱乙酰酶抑制剂 (HDACi) 抑制。 HDACi是目前临床上最有效的 经批准的针对 CTCL 的细胞毒性化合物，与 p38γ 组合时表现出协同杀伤作用 抑制。我们的目标是结合使用以下方法来了解和利用 CTCL 中的 p38γ 通路： 分子、化学和遗传学方法。我们的首要目标是确定 p38γ 的机制 抑制会诱导 CTCL 中的细胞死亡。我们将定义参与 p38γ 抑制诱导的激酶级联 CTCL细胞杀伤并识别p38γ信号传导的磷酸化靶点；使用合成致死性 RNAi 筛选 鉴定在 p38γ 抑制存在的情况下，在耗尽时导致细胞死亡的信号传导成分；和 确定 p38γ 和互补途径（包括 HDAC）联合抑制的程度， 产生协同治疗作用。我们将验证已识别的蛋白质影响下游的能力 使用 CTCL 细胞系异种移植物和患者来源的异种移植物进行体外和体内信号传导和细胞反应 (PDX) 模型。我们的第二个目标是开发新型 p38γ 抑制剂以用于潜在的治疗应用。使用 通过高通量筛选和分子建模，我们鉴定出了多激酶抑制剂 F7（也称为 PIK75），并表明它是一种 ATP 竞争性 p38γ 抑制剂，对 CTCL 细胞具有纳摩尔级细胞毒性功效。 为了开发更具选择性的 p38γ 抑制剂，我们将结合基于配体和结构的计算方法 与有机合成；使用F7作为支架分子，我们将鉴定F7类似物并将F7衍生化为 与其他激酶相比，对 p38γ 的结合亲和力更高。此外，我们将使用基于 CRISPR 的筛选来识别 用于开发变构下一代疗法的新功能域和非保守位点。我们会 合成各种类似物并在体外验证 CTCL 细胞毒性和 p38γ 特异性激酶抑制的命中 在体内，使用 CTCL 异种移植和 PDX 模型。我们预计该提案的成功完成将 产生有关 p38γ 信号传导的独特生物学和临床相关性的机制信息 CTCL 中的互补途径。重要的是，验证特定 p38γ 抑制剂在 CTCL 中的功效 动物模型将与 CTCL 治疗直接相关。

Cutaneous T cell lymphoma (CTCL) is a disfiguring, incurable cancer. For patients with advanced disease, current therapies are inadequate, and outcome is poor. An incomplete understanding of CTCL molecular regulators has limited development of effective targeted therapies. One candidate regulator is p38γ, the gene expression of which is selectively increased in CTCL cell lines and patient samples, but not healthy T cells. We demonstrate that inhibition or silencing of p38γ inhibits proliferation and induces CTCL cell death. The NF-κB pathway is constitutively active in CTCL, provides a complementary T cell signaling pathway to p38γ, and can be inhibited by histone deacetylase inhibitors (HDACi). HDACi, which are currently the most effective clinically approved cytotoxic compounds against CTCL, demonstrate synergistic killing when combined with p38γ Inhibition. Our objective is to understand and exploit the p38γ pathway in CTCL, using a combination of molecular, chemical, and genetic approaches. Our first Aim is to determine the mechanisms by which p38γ inhibition induces cell death in CTCL. We will define the kinase cascade involved in p38γ inhibition-induced CTCL cell killing and identify phosphorylation targets of p38γ signaling; use a synthetic lethal RNAi screen to identify signaling components that cause cell death upon depletion in the presence of p38γ inhibition; and determine the extent to which combined inhibition of p38γ and complementary pathways, including HDACs, induce synergistic therapeutic effects. We will validate identified proteins for the ability to affect downstream signaling and cellular responses in vitro and in vivo, using CTCL cell line xenograft and patient-derived xenograft (PDX) models. Our second Aim is to develop novel p38γ inhibitors for potential therapeutic application. Using high throughput screening and molecular modeling, we identified the multi-kinase inhibitor F7 (also known as PIK75), and showed it is an ATP-competitive p38γ inhibitor with nanomolar cytotoxic efficacy against CTCL cells. To develop a more selective p38γ inhibitor, we will combine ligand- and structure-based computational methods with organic synthesis; using F7 as a scaffold molecule, we will identify F7 analogs and derivatize F7 to have higher a binding affinity for p38γ than other kinases. In addition, we will use CRISPR-based screening to identify novel functional domains and non-conserved sites for developing allosteric next-generation therapeutics. We will synthesize the various analogs and validate hits for CTCL cytotoxicity and p38γ-specific kinase inhibition in vitro and in vivo, using CTCL xenograft and PDX models. We expect that successful completion of this proposal will yield mechanistic information about the unique biological and clinical relevance of p38γ signaling and complementary pathways in CTCL. Importantly, validation of a specific p38γ inhibitor with efficacy in CTCL animal models will have immediate relevance for CTCL therapy.