Data_Sheet_1_GZ17-6.02 and Doxorubicin Interact to Kill Sarcoma Cells via Autophagy and Death Receptor Signaling.PDF

GZ17-6.02 (602) is presently under phase I clinical evaluation (NCT03775525). We defined the mechanisms by which it interacted with a standard of care therapeutic doxorubicin to kill sarcoma cells. Doxorubicin and 602 interacted to rapidly activate ATM and c-MET, inactivate mTOR, AKT, and p70 S6K, enhance the expression of Beclin1 and reduce the levels of K-RAS and N-RAS. This was followed later by the drugs interacting to reduce expression of MCL-1, BCL-XL, and HDAC6. Knock down of ATM prevented the drugs alone or in combination inactivating mTOR or activating ULK1. Knock down of c-MET significantly enhanced [doxorubicin + 602] lethality. Knock down of ATM and to a greater extent ULK1, Beclin1, or ATG5 significantly reduced killing by 602 alone or when combined with doxorubicin. Expression of an activated mTOR mutant suppressed killing, autophagosome formation and prevented autophagic flux. In the absence of Beclin1, knock down of CD95, or FADD, or over-expression of c-FLIP-s or BCL-XL abolished tumor cell killing. We conclude that 602 and doxorubicin interact to increase autophagosome formation and autophagic flux as well as causing elevated death receptor signaling resulting in mitochondrial dysfunction and tumor cell death.

GZ17-6.02（代号602）目前正处于I期临床评估阶段（临床试验编号NCT03775525）。本研究明确了其与临床标准治疗药物多柔比星（doxorubicin）协同杀伤肉瘤细胞的作用机制。多柔比星与602可快速激活ATM与c-MET，抑制mTOR、AKT及p70 S6K的活性，上调Beclin1的表达并降低K-RAS与N-RAS的蛋白水平。后续两药还可协同下调MCL-1、BCL-XL及HDAC6的表达。敲低ATM可阻断两药单独或联合使用时对mTOR的抑制作用，以及对ULK1的激活。敲低c-MET则可显著增强[多柔比星+602]的细胞杀伤活性。敲低ATM，以及更显著地敲低ULK1、Beclin1或ATG5，可显著减弱602单独使用或与多柔比星联合使用时的细胞杀伤效果。过表达激活型mTOR突变体可抑制细胞杀伤、自噬体形成并阻断自噬流。在Beclin1缺失的情况下，敲低CD95、FADD，或过表达c-FLIP-s与BCL-XL，可完全消除肿瘤细胞的杀伤效应。本研究表明，602与多柔比星协同作用可上调自噬体形成与自噬流，同时增强死亡受体信号通路活化，最终导致线粒体功能障碍与肿瘤细胞死亡。