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

Abstract

We determined the molecular mechanisms by which the novel therapeutic GZ17-6.02 killed non-small cell lung cancer (NSCLC) cells. Erlotinib, afatinib, and osimertinib interacted with GZ17-6.02 to kill NSCLC cells expressing mutant EGFR proteins. GZ17-6.02 did not interact with any EGFR inhibitor to kill osimertinib-resistant cells. GZ17-6.02 interacted with the thymidylate synthase inhibitor pemetrexed to kill NSCLC cells expressing mutant ERBB1 proteins or mutant RAS proteins or cells that were resistant to EGFR inhibitors. The drugs interacted to activate ATM, the AMPK, and ULK1 and inactivate mTORC1, mTORC2, ERK1/2, AKT, eIF2α; and c-SRC. Knockdown of ATM or AMPKα₁ prevented ULK1 activation. The drugs interacted to cause autophagosome formation followed by flux, which was significantly reduced by knockdown of ATM, AMPKα₁, and eIF2α, or by expression of an activated mTOR protein. Knockdown of Beclin1, ATG5, or [BAX + BAK] partially though significantly reduced drug combination lethality as did expression of activated mTOR/AKT/MEK1 or over-expression of BCL-XL. Expression of dominant negative caspase 9 weakly reduced killing. The drug combination reduced the expression of HDAC2 and HDAC3, which correlated with lower PD-L1, IDO1, and ODC levels and increased MHCA expression. Collectively, our data support consideration of combining GZ17-6.02 and pemetrexed in osimertinib-resistant NSCLC.

Keywords: EGFR; ER stress; GZ17-6.02; NSCLC; autophagy; osimertinib; pemetrexed; resistance.

Figure 1

GZ17-6.02 interacts with ERBB1 inhibitors to kill NSCLC cells expressing mutant active forms of ERBB1. (A, B) H1650, wild-type sensitive, and afatinib-resistant (AR) H1975 and erlotinib-resistant (ER) HCC827 cells were treated with vehicle, erlotinib (500 nM), afatinib (500 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion (n = 3 ± SD). ^# p < 0.05 greater than GZ17-6.02 alone; ^¶ p < 0.05 less than the corresponding values in drug-sensitive cells. (C) H1650, wild-type sensitive, and afatinib-resistant (AR) H1975 and erlotinib-resistant (ER) HCC827 cells were treated with vehicle, osimertinib (100 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion (n = 3 ± SD). ^# p < 0.05 greater than GZ17-6.02 alone; ^¶ p < 0.05 less than the corresponding values in drug-sensitive cells.

Figure 2

GZ17-6.02 interacts with pemetrexed to kill NSCLC cells. (A) H1975 and H1650 cells [wild-type sensitive and osimertinib resistant (OR)] were treated with vehicle, erlotinib (500 nM), afatinib (500 nM), osimertinib (100 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion (n = 3 ± SD). ^# p < 0.05 greater than GZ17-6.02 alone; ^¶ p < 0.05 less than the corresponding values in drug-sensitive cells. (B) H1975 and H1650 cells [wild-type sensitive and osimertinib resistant (OR)] were treated with vehicle, pemetrexed (500 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion (n = 3 ± SD). ^# p < 0.05 greater than GZ17-6.02 alone; ^¶ p < 0.05 less than the corresponding values in drug-sensitive cells. (C) NSCLC cells were treated with vehicle, pemetrexed (500 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion (n = 3 ± SD). ^# p < 0.05 greater than GZ17-6.02 alone. The mutational status of K-/N-RAS or of ERBB1 is noted in each graph. * = mutated active

Figure 3

Resistance to ERBB1 inhibitors is associated with a reduced ability to form autophagosomes. (A) H1975 [wild-type sensitive and afatinib-resistant (AR)] were transfected to express LC3-GFP-RFP and subsequently treated with vehicle, pemetrexed (500 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 4 h and 8 h. The number of intense staining GFP+ and RFP+ punctae was determined randomly in at least 50 cells, and the mean number of punctae per cell was determined (n = 3 ± SD). ^# p < 0.05 greater than GZ17-6.02 value; ^¶ p < 0.05 greater than the corresponding values after 4 h; ~p < 0.05 less than the corresponding values in wild-type sensitive cells. (B) Afatinib-resistant H1975 cells were transfected with siRNA molecules to knock down protein levels or with plasmids to express activated forms of mTOR or STAT3 and then subsequently treated with vehicle or [pemetrexed (500 nM) + GZ17-6.02 (2 μM curcumin final)] in combination for 4 h and 8 h. The number of intense staining GFP+ and RFP+ punctae were determined randomly in at least 50 cells, and the mean number of punctae per cell was determined (n = 3 ± SD). ^¶ p < 0.05 greater than the corresponding values after 4 h; *p < 0.05 less than the corresponding values in siSCR/CMV-transfected cells. (C) Osimertinib-resistant H1975 cells were transfected with siRNA molecules to knock down protein levels or with plasmids to express activated forms of mTOR or STAT3 and then subsequently treated with vehicle or [pemetrexed (500 nM) + GZ17-6.02 (2 μM curcumin final)] in combination for 4 h and 8 h. The number of intense staining GFP+ and RFP+ punctae was determined randomly in at least 50 cells and the mean number of punctae per cell determined (n = 3 ± SD). ^¶ p < 0.05 greater than the corresponding values after 4 h; ~~p < 0.05 less than the corresponding values in afatinib-resistant H1975 cells; *p < 0.05 less than the corresponding values in siSCR/CMV-transfected cells.

Figure 4

GZ17-6.02 and pemetrexed interact to alter cell signaling, increase autophagosome formation, and kill via toxic autophagy A549 NSCLC cells that express a mutant K-RAS protein. (A) A549 cells were treated with vehicle control, GZ17-6.02 (2 μM final curcumin), pemetrexed (500 nM), or the drugs combined for 6 h. Cells were fixed in place and immunostaining was performed to determine protein expression and phosphorylation (n = 3 ± SD) *p < 0.05 less than vehicle; **p < 0.05 less than GZ17-6.02 alone; ^# p < 0.05 greater than vehicle control; ^## p < 0.05 greater than GZ17-6.02 alone. (B) A549 cells were transfected with siRNA molecules to knock down protein levels or with plasmids to express activated forms of mTOR or STAT3 and then subsequently treated with vehicle or [pemetrexed (500 nM) + GZ17-6.02 (2 μM curcumin final)] in combination for 4 h and 8 h. The number of intense staining GFP+ and RFP+ punctae were determined randomly in at least 50 cells, and the mean number of punctae per cell was determined (n = 3 ± SD). ^¶ p < 0.05 greater than the corresponding values after 4 h; *p < 0.05 less than the corresponding values in siSCR/CMV-transfected cells. (C) A549 cells were transfected to knock down Beclin1 or ATG5 expression. Subsequently cells were treated with vehicle, pemetrexed (500 nM), GZ17-6.02 (2 μM curcumin final), or the drugs in combination for 24 h. Cell viability was determined by trypan blue exclusion (n = 3 ± SD). *p < 0.05 less than the corresponding values in siSCR cells.

Figure 5

The killing of osimertinib-resistant NSCLC cells requires [BAX + BAK] and autophagosome formation and is significantly reduced by expression of activated AKT, activated mTOR or activated MEK1. Afatinib-resistant and osimertinib-resistant H1975 cells were transfected with siRNA molecules to knock down protein expression or with plasmids to express regulatory proteins. Subsequently, cells were treated with vehicle or [pemetrexed (500 nM) + GZ17-6.02 (2 μM curcumin final)] in combination for 24 h. Cell viability was determined by trypan blue exclusion (n = 3 ± SD). *p < 0.05 less than corresponding siSCR/CMV value; **p < 0.05 less than the corresponding values in afatinib-resistant cells; ^¶ p < 0.05 less than the corresponding values in all other conditions; ^§ p < 0.05 greater than the corresponding values in all other manipulated conditions.