GZ17-6.02 Interacts With [MEK1/2 and B-RAF Inhibitors] to Kill Melanoma Cells

Abstract

We defined the lethal interaction between the novel therapeutic GZ17-6.02 and the standard of care combination of the MEK1/2 inhibitor trametinib and the B-RAF inhibitor dabrafenib in PDX isolates of cutaneous melanoma expressing a mutant B-RAF V600E protein. GZ17-6.02 interacted with trametinib/dabrafenib in an additive fashion to kill melanoma cells. Regardless of prior vemurafenib resistance, the drugs when combined interacted to prolong ATM S1981/AMPK T172 and eIF2α S51 phosphorylation and prolong the reduced phosphorylation of JAK2 Y1007, STAT3 Y705 and STAT5 Y694. In vemurafenib-resistant cells GZ17-6.02 caused a prolonged reduction in mTORC1 S2448, mTORC2 S2481 and ULK1 S757 phosphorylation; regardless of vemurafenib resistance, GZ17-6.02 caused a prolonged elevation in CD95 and FAS-L expression. Knock down of eIF2α, Beclin1, ATG5, ATM, AMPKα, CD95 or FADD significantly reduced the ability of GZ17-6.02 to kill as a single agent or when combined with the kinase inhibitors. Expression of activated mTOR, activated STAT3, activated MEK1 or activated AKT significantly reduced the ability of GZ17-6.02 to kill as a single agent or when combined with kinase inhibitors; protective effects that were significantly less pronounced in cells treated with trametinib/dabrafenib. Regardless of vemurafenib resistance, the drugs alone or in combination all reduced the expression of PD-L1 and increased the levels of MHCA, which was linked to degradation of multiple HDAC proteins. Our findings support the use of GZ17-6.02 in combination with trametinib/dabrafenib in the treatment of melanomas expressing mutant B-RAF V600E proteins.

Keywords: B-Raf mutation V600E; GZ17-6.02; HDAC; autophagy; er stress.

Figure 1

GZ17-6.02 interacts with [trametinib + dabrafenib] to kill cutaneous melanoma cells expressing B-RAF V600E. (A) Melanoma cells were treated with vehicle control, isovanillin (37.2 µM), harmine (4.5 µM), curcumin (2 µM) or GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)] for 24h. Cells were isolated and viability determined by trypan blue exclusion (n = 3 +/- SD). ^#p < 0.05 greater than vehicle control; ^$p < 0.05 greater than value in cells treated with curcumin alone. (B) Melanoma cells were treated with vehicle control, [isovanillin (37.2 µM) + harmine (4.5 µM)], curcumin (2 µM) or GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)] for 24h. Cells were isolated and viability determined by trypan blue exclusion (n = 3 +/- SD). ^#p < 0.05 greater than vehicle control; ^$p < 0.05 greater than value in cells treated with curcumin alone. (C) Melanoma cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (Tram, 2 µM) + dabrafenib (dab, 2 µM)] or the drugs in combination for 12h. Cells were isolated and viability determined by trypan blue exclusion (n = 3 +/- SD). ^#p < 0.05 greater than vehicle control; ^$p < 0.05 greater than value in cells treated with GZ17-6.02 alone.

Figure 2

GZ17-6.02 and [trametinib + dabrafenib] interact to increase endoplasmic reticulum stress signaling and inactivate STAT transcriptionfactors. Melanoma cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (2 µM) + dabrafenib (2 µM)] or the drugs in combination for 4h (the three bars per protein from left to right are trametinib/dabrafenib, GZ17-6.02 and the drugs combined). Cells were fixed in place and in-cell immunostaining performed to determine the expression and the phosphorylation of the indicated proteins (n = 3 +/-SD) *p < 0.05 less than vehicle control; £ p < 0.05 less than either drug as a single agent; ^#p < 0.05 greater than vehicle control; ^$p < 0.05 greater than either drug as a single agent.

Figure 3

GZ17-6.02 and [trametinib + dabrafenib] interact to inactivate ERK1/2 and p70 S6K. Melanoma cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (2 µM) + dabrafenib (2 µM)] or the drugs in combination for 4h. (the three bars per protein from left to right are trametinib/dabrafenib, GZ17-6.02 and the drugs combined). Cells were fixed in place and in-cell immunostaining performed to determine the expression and the phosphorylation of the indicated proteins (n = 3 +/-SD) *p < 0.05 less than vehicle control; ^£p < 0.05 less than either drug as a single agent; ^#p < 0.05 greater than vehicle control; ^$p < 0.05 greater than either drug as a single agent.

Figure 4

GZ17-6.02 and [trametinib + dabrafenib] can interact to inactivate c-SRC and JAK2 and increase expression of protein serine/threonine phosphatase 1. (A–C) Melanoma cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (t, 2 µM) + dabrafenib (d, 2 µM)] or the drugs in combination for 4h. Cells were fixed in place and in-cell immunostaining performed to determine the expression and the phosphorylation of the indicated proteins (n = 3 +/-SD) *p < 0.05 less than vehicle control; ^£p < 0.05 less than either drug as a single agent; ^#p < 0.05 greater than vehicle control; ^$p < 0.05 greater than either drug as a single agent.

Figure 5

Exposure to [GZ17-6.02 + trametinib + dabrafenib] maintains elevated endoplasmic reticulum stress signaling and inactivation of STAT transcription factors. Melanoma cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (2 µM) + dabrafenib (2 µM)] or the drugs in combination for 8h. (the three bars per protein from left to right are trametinib/dabrafenib, GZ17-6.02 and the drugs combined). Cells were fixed in place and in-cell immunostaining performed to determine the expression and the phosphorylation of the indicated proteins (n = 3 +/-SD) *p < 0.05 less than vehicle control; ^£p < 0.05 less than either drug as a single agent; ^#p < 0.05 greater than vehicle control; ^$p < 0.05 greater than either drug as a single agent.

Figure 6

Exposure to [GZ17-6.02 + trametinib + dabrafenib] has the potential to increase the expression of ATG5 or reduce MCL1 and BCL-XL levels. Melanoma cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (2 µM) + dabrafenib (2 µM)] or the drugs in combination for 8h. (the three bars per protein from left to right are trametinib/dabrafenib, GZ17-6.02 and the drugs combined). Cells were fixed in place and in-cell immunostaining performed to determine the expression and the phosphorylation of the indicated proteins (n = 3 +/-SD) *p < 0.05 less than vehicle control; ^£p < 0.05 less than either drug as a single agent; ^#p < 0.05 greater than vehicle control; ^$p < 0.05 greater than either drug as a single agent.

Figure 7

Enhanced tumor cell killing by [GZ17-6.02 + trametinib + dabrafenib] requires autophagosome formation and CD95 death receptor signaling. Cells were transfected with a scrambled siRNA (siSCR) or with validated siRNA molecules to knock down the expression of the indicated proteins. Twenty-four h later, cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (t, 2 µM) + dabrafenib (d, 2 µM)] or the drugs in combination for 24h. Cells were isolated and viability determined by trypan blue exclusion (n = 3 +/- SD). *p < 0.05 less than corresponding value in siSCR transfected cells.

Figure 8

Enhanced tumor cell killing by [GZ17-6.02 + trametinib + dabrafenib] requires mitochondrial dysfunction. Cells were transfected with an empty vector plasmid (CMV) or with plasmids to express the indicated proteins. Twenty-four h later, cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (t, 2 µM) + dabrafenib (d, 2 µM)] or the drugs in combination for 24h. Cells were isolated and viability determined by trypan blue exclusion (n = 3 +/- SD). *p < 0.05 less than corresponding value in siSCR transfected cells.

Figure 9

Over-expression of GRP78 or HSP90, to a greater extent than HSP70, reduces drug lethality in melanoma cells. (A) and (B) Melanoma cells were transfected with an empty vector plasmid (CMV) or with plasmids to express the chaperones GRP78, HSP70 or HSP90. Twenty-four h later, cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (t, 2 µM) + dabrafenib (d, 2 µM)] or the drugs in combination for 24h. Cells were isolated and viability determined by trypan blue exclusion (n = 3 +/- SD). *p < 0.05 less than corresponding value in CMV transfected cells; £ p > 0.05 comparing d/t/602 value to 602 alone value in CMV cells. (C) and (D) Cells were transfected with a scrambled siRNA or with a validated siRNA molecule to knock down the expression of eIF2α. Cells were transfected with an empty vector plasmid (CMV) or with a plasmid to express activated STAT3. Twenty-four h later, cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (t, 2 µM) + dabrafenib (d, 2 µM)] or the drugs in combination for 24h. Cells were isolated and viability determined by trypan blue exclusion (n = 3 +/- SD). *p < 0.05 less than corresponding value in transfected cells; ^£p > 0.05 comparing d/t/602 value to 602 alone value in their corresponding transfected cells; ^¶p < 0.05 less than 602 single agent value in CMV transfected cells.

Figure 10

Exposure of cells to [GZ17-6.02 + trametinib + dabrafenib] reduces the expression of multiple HDAC proteins. Melanoma cells were treated with vehicle control or [GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)] + trametinib (t, 2 µM) + dabrafenib (d, 2 µM)] for 6h. Cells were fixed in place and in-cell immunostaining performed to determine the expression of HDACs1-11 and total ERK2 as a loading control. (n = 3 +/-SD) *p < 0.05 less than vehicle control value. ^#Greater than vehicle control.

Figure 11

Genetic manipulation of HDAC expression results in reduced expression of PD-L1, ODC and IDO1 and elevated levels of MHCA. (A) Melanoma cells were treated with vehicle control, GZ17-6.02 [curcumin (2 µM) + isovanillin (37.2 µM) + harmine (4.5 µM)], [trametinib (T, 2 µM) + dabrafenib (D, 2 µM)] or the drugs in combination for 6h. Cells were fixed in place and in-cell immunostaining performed to determine the expression of the indicated proteins and total ERK2 as a loading control. Bar graphs are corrected for the total loading of ERK2. (n = 3 +/-SD) *p < 0.05 less than vehicle control; ^#p < 0.05 greater than vehicle control. (B) Melanoma cells were transfected with an scrambled siRNA (siSCR) or with siRNA molecules to knock down the expression of HDAC proteins, as presented in the Figure. Twenty-four h later, cells were fixed in place and in-cell immuno-staining performed to define the expression of PD-L1, MHCA, ODC, IDO1 and the total expression of ERK2 as a loading control. The values presented in the bar graphs are corrected for total ERK2 levels under each condition. A representative study in triplicate from three independent experiments. (+/-SD) ^#p < 0.05 greater than siSCR value; *p < 0.05 less than siSCR value. ^$Less than individual knock down of an HDAC protein.

Figure 12

The molecular mechanisms by which GZ17-6.02 interacts with [trametinib + dabrafenib] to kill cutaneous melanoma cells that express B-RAF V600E. In MEL2 and MEL4 cells GZ17-6.02 initially interacted after a 4h exposure with [trametinib + dabrafenib] to cause inactivation of STAT3, p70 S6K and ERK1/2. In MEL4 cells they also combined to inactivate JAK2, STAT5, eIF2α, NFκB and reduced HSP70 levels. In MEL2 cells they also combined to inactivate c-SRC. Eight h after exposure the drugs interacted to maintain phosphorylation of the AMPK and eIF2α, and to decrease the phosphorylation of MEK1/2 and STAT3. The levels of CD95 in both isolates remained elevated at the 8h time-point. Molecular studies demonstrated that cells lacking ATM-AMPK signaling, lacking the ability to form autophagosomes or activate CD95 death receptor signaling were less able to undergo cell death processes. Over-expression of BCL-XL or dominant negative caspase 9, expression of activated STAT3 or knock down of eIF2α was more effective at suppressing cell killing than over-expression of FLIP-s or activated forms of mTOR, AKT or MEK1.