GZ17-6.02 and Doxorubicin Interact to Kill Sarcoma Cells via Autophagy and Death Receptor Signaling

Abstract

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 Interacts With [MEK1/2 and B-RAF Inhibitors] to Kill Melanoma Cells

“…We next determined a pathway analysis to determine the mechanisms of cell killing. Prior studies in other cell types have shown GZ17-6.02 killing through mechanisms using the death receptor CD95 and autophagosome formation (10)(11)(12). We also previously demonstrated using other agents that activation of ATM can lead to activation of the AMPK followed by mTOR inactivation, ULK1 activation, ATG13 S318 phosphorylation and autophagosome formation (22).…”

“…Specific multiple independent siRNAs to knock down the expression of CD95, FADD, Beclin1, ATG5 and eIF2a, and scramble control, were purchased from Qiagen (Hilden Germany). Control studies were presented showing on-target specificity of our siRNAs, primary antibodies and our phosphospecific antibodies to detect both total protein levels and phosphorylated levels of proteins (10)(11)(12)(19)(20)(21)(22)(23)(24).…”

“…All bench-side Methods used in this manuscript have been performed and described in the peer-reviewed references (10)(11)(12)(19)(20)(21)(22)(23)(24). All cell lines were cultured at 37°C (5% (v/v CO2) in vitro using RPMI supplemented with dialyzed 5% (v/v) fetal calf serum and 1% (v/v) Non-essential amino acids.…”

“…The novel cancer therapeutic GZ17-6.02 undergoing phase I evaluation in solid tumor patients. GZ17-6.02 is comprised of three natural chemicals; curcumin (10%); isovanillin (77%); and harmine (13%) (10)(11)(12). The most widely studied of these three compounds is curcumin; turmeric, the spice most associated with Indian cuisine is comprised~95% of curcumin and curcuminoid derivatives.…”

GZ17-6.02 and Pemetrexed Interact to Kill Osimertinib-Resistant NSCLC Cells That Express Mutant ERBB1 Proteins

“…Studies have shown that while harmine has anti-proliferative effects in tumor cells, the compound appears to lack any anti-proliferative biologic effects in non-transformed cells. We have previously shown that GZ17-6.02 interacted with 5-fluorouracil (5FU) to kill GI tumor cells, with doxorubicin to kill sarcoma cells and with [trametinib + dabrafenib] to kill cutaneous melanoma cells expressing B-RAF V600E ( 1 , 2 ). Our new studies were performed to determine whether GZ17-6.02 could kill non-small cell lung cancer (NSCLC) cells expressing mutant activated forms of the EGF receptor (ERBB1).…”