Mutations in Ras family proteins are implicated in 33% of human cancers, but direct pharmacological inhibition of Ras mutants remains challenging. As an alternative to direct inhibition, we screened for sensitivities in Ras-mutant cells and discovered 249C as a Ras-mutant selective cytotoxic agent with nanomolar potency against a spectrum of Ras-mutant cancers. 249C binds to vacuolar (V)-ATPase with nanomolar affinity and inhibits its activity, preventing lysosomal acidification and inhibiting autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. Unexpectedly, potency of 249C varies with the identity of the Ras driver mutation, with the highest potency for KRASG13D and G12V both in vitro and in vivo, highlighting a mutant-specific dependence on macropinocytosis and lysosomal pH. Indeed, 249C potently inhibits tumor growth without adverse side effects in mouse xenografts of KRAS-driven lung and colon cancers. A comparison of isogenic SW48 xenografts with different KRAS mutations confirmed that KRASG13D/+ (followed by G12V/+) mutations are especially sensitive to 249C treatment. These data establish proof-of-concept for targeting V-ATPase in cancers driven by specific KRAS mutations such as KRASG13D and G12V.

Ras 家族蛋白的突变与 33% 的人类癌症有关，但直接药理学抑制 Ras 突变体仍然具有挑战性。作为直接抑制的替代方法，我们筛选了 Ras 突变细胞的敏感性，并发现 249C 作为 Ras 突变选择性细胞毒剂，对一系列 Ras 突变癌症具有纳摩尔效力。249C 以纳摩尔亲和力与液泡 (V)-ATP 酶结合并抑制其活性，防止溶酶体酸化并抑制多种 Ras 驱动的癌症赖以生存的自噬和巨胞饮途径。出乎意料的是，249C 的效力随 Ras 驱动突变的身份而变化，在体外和体内对KRAS G13D 和 G12V的效力最高，突显了对巨胞饮作用和溶酶体 pH 的突变体特异性依赖性。事实上，249C 能有效抑制KRAS驱动的肺癌和结肠癌小鼠异种移植物中的肿瘤生长，且没有不良副作用。具有不同KRAS突变的同基因 SW48 异种移植物的比较证实，KRAS G13D/+（随后是 G12V/+）突变对 249C 治疗特别敏感。这些数据确立了针对由特定KRAS突变（例如KRAS G13D 和 G12V ）驱动的癌症中靶向 V-ATP 酶的概念验证。