DIRAS3 disrupts K-RAS clustering and signaling, enhancing autophagy and response to autophagy inhibition

突变的KRAS从多个位点驱动人类癌症，包括胰腺导管腺癌（PDAC） 低级别浆液性卵巢癌（LGSOC）尽管RAS突变在不同癌症中普遍存在， 有效的RAS靶向治疗仍然是一个挑战。KRAS单体形成同二聚体和纳米簇， 细胞膜以优化信号传导并有效转化细胞。需要KRAS的二聚化， RAS驱动的转型和癌症生长。破坏突变RAS二聚体和簇的药物可以阻断 致癌活性最近的证据表明，抑制RAS信号转导诱导自噬，并增强自噬。 抗自噬治疗的反应尽管经过四十年的努力， 突变KRAS驱动的癌症的治疗仍在进行中。我们的实验室发现了一种 一种新的内源性生理性RAS抑制剂DIRAS 3，一种26 KDa的GT酶，具有50-60%的同源性 与经典的RAS家族成员，但具有独特的34个氨基酸的N-末端延伸， 功能与RAS一样，DIRAS 3在C末端CAAX位点被异戊烯化，以高亲和力结合GTP，表现出高亲和力。 GT3活性弱，其生物学功能需要膜结合。DIRAS 3下调 在包括PDAC和LGSOC在内的许多癌症中，DIRAS 3的再表达阻断了癌细胞的增殖， 增殖，抑制运动性，并且重要的是，通过多种机制诱导自噬。最近我们 发现DIRAS 3和来自其α5结构域的DIRAS 3衍生的钉合肽直接与突变型KRAS相互作用， 减少KRAS二聚化和纳米簇化，并抑制KRAS信号传导。完整的DIRAS 3和 DIRAS 3衍生的钉合肽诱导自噬并增强自噬的促凋亡活性 PDAC和LGSOC细胞中的抑制剂。在这个建议中，我们将研究DIRAS 3对KRAS依赖性细胞的影响。 MEF细胞中的细胞生长、迁移和效应物信号传导以及具有突变体的基因工程小鼠模型 KRAS，以及KRAS驱动的PDAC和LGSOC，更好地定义了DIRAS 3抑制 KRAS（目标1）。我们将研究DIRAS 3在KRAS驱动的PDAC中诱导自噬的机制 和LGSOC（目标2）。最后，我们将测试DIRAS 3或DIRAS 3衍生的钉合肽在细胞中的表达能力。 与自噬抑制剂（CQ/DC 661）组合以增强PDAC中的细胞凋亡和生长抑制， LGSOC（Aim3）.这些研究不仅将为探索新的治疗策略奠定基础， KRAS突变型癌症，而且有助于从根本上理解DIRAS 3， 作为肿瘤抑制因子，抑制突变型KRAS活性并诱导自噬。

Mutant KRAS drives human cancers from several sites, including pancreatic ductal adenocarcinoma (PDAC) and low-grade serous ovarian cancer (LGSOC). Despite the prevalence of RAS mutations in different cancers, effective RAS-targeted treatment remains a challenge. KRAS monomers form homodimers and nanoclusters in the cell membrane to optimize signaling and to transform cells efficiently. Dimerization of KRAS is required for RAS-driven transformation and cancer growth. Agents that disrupt mutant RAS dimers and clusters can block oncogenic activity. Recent evidence indicates that inhibition of RAS signaling induces autophagy and enhances the response to anti-autophagic therapy. Despite four decades of effort, development of effective strategies for the treatment for mutant KRAS-driven cancers remains a work in progress. Our laboratory has discovered a novel endogenous physiological RAS inhibitor designated DIRAS3, a 26 KDa GTPase sharing 50-60% homology with classical RAS family members, but with a distinctive 34 amino acid N-terminal extension that reverses RAS function. Like RAS, DIRAS3 is prenylated at the C-terminal CAAX site, binds GTP with high affinity, exhibits weak GTPase activity, and requires membrane association for its biological function. DIRAS3 is downregulated in a number of cancers including PDAC and LGSOC, and re-expression of DIRAS3 blocks cancer cell proliferation, inhibits motility, and, importantly, induces autophagy by multiple mechanisms. Recently we have found that DIRAS3 and a DIRAS3-derived stapled peptide from its α5 domain interact directly with mutant KRAS, reducing KRAS dimerization and nanoclustering, and inhibiting KRAS signaling. Both intact DIRAS3 and DIRAS3-derived stapled peptide induce autophagy and potentiate the pro-apoptotic activity of autophagy inhibitors in PDAC and LGSOC cells. In this proposal, we will study the effect of DIRAS3 on KRAS-dependent cell growth, migration and effector signaling in MEF cells and genetically engineered mouse model with mutant KRAS, as well as in KRAS-driven PDAC and LGSOC, better defining the mechanism by which DIRAS3 inhibits KRAS (Aim 1). We will investigate the mechanisms by which DIRAS3 induces autophagy in KRAS-driven PDAC and LGSOC (Aim 2). Finally, we will test the ability of DIRAS3 or a DIRAS3-derived stapled peptide in combination with autophagy inhibitors (CQ/DC661) to enhance apoptosis and growth inhibition in PDAC and LGSOC (Aim3). These studies will not only lay the groundwork for exploring new therapeutic strategies targeting KRAS-mutant cancers, but also contribute to a fundamental understanding of the mechanisms by which DIRAS3, as a tumor suppressor, inhibits mutant KRAS activity and induces autophagy.