Fig. 6

Induction of mitochondrial stress via ONC213 supports intrinsic apoptosis activation via venetoclax. (A) CRISPR knockdown (KD) of Mcl-1 and non-target control (NTC) was generated using THP-1 cells. Representative western blots of whole cell lysates probed with the indicated antibodies are shown in the left panel. The fold changes for the Mcl-1 densitometry measurements, normalized to β-actin and then compared to NTC, are indicated. Cells were treated with vehicle control or venetoclax (VEN) for 24 h. Flow cytometry results of annexin V/PI staining are show in the right panel. *** p < 0.001 compared to NTC treated with VEN. (B) THP-1 RFP (red fluorescent protein) and Mcl-1 OE (overexpression) whole cell lysates were subjected to western blot analysis. The fold changes for the Mcl-1 densitometry measurements, normalized to β-actin and then compared to RFP, are indicated. Cells were treated with vehicle control, VEN, ONC, or in combination for 24 h. Flow cytometry results of annexin V/PI staining are show in the right panel. *** p < 0.001 compared to RFP cells under the same treatment conditions. (C) THP-1 NTC and Bak/Bax KD whole cell lysates were subjected to western blot analysis. The fold changes for the Bax and Bak densitometry measurements, normalized to β-actin and then compared to NTC, are indicated. THP-1 NTC and Bak/Bax KD cells were cultured in either glucose or galactose containing media for 48 h and then treated with VEN, ONC213, or the combination of ONC + VEN for 24–48 h. Then the cells were stained with annexin V/PI and analyzed via flow cytometry. *** p < 0.001 and ns not significant compared to NTC cells under the same treatment conditions. (D) MV4-11 RFP and Mcl-1 OE whole cell lysates were subjected to western blot analysis. The fold changes for the Mcl-1 densitometry measurements, normalized to β-actin and then compared to RFP, are indicated. Cells were treated with vehicle control, VEN, ONC, or in combination for 24 h. Flow cytometry results of annexin V/PI staining are show in the right panel. *** p < 0.001 compared to RFP cells under the same treatment conditions. (E) MV4-11 NTC and Bak/Bax KD whole cell lysates were subjected to western blot analysis. The fold changes for the Bax and Bak densitometry measurements, normalized to β-actin and then compared to NTC, are indicated. MV4-11 NTC and Bak/Bax KD cells were cultured in glucose containing media for 48 h and then treated with VEN, ONC, or the combination of ONC + VEN for 24 h. Then the cells were stained with annexin V/PI and analyzed via flow cytometry. *** p < 0.001 compared to NTC cells under the same treatment conditions. (F) THP-1 cells and primary AML patient samples AML#213 and AML#214 were treated with VEN, ONC, or VEN + ONC for 48 h. The cells were then stained with annexin V/PI and subjected to flow cytometry analyses. Combination Index (CI) values were calculated using CompuSyn software to determine synergy. CI < 1.0, CI = 1.0, and CI > 1.0 indicate synergistic, additive, and antagonistic effects, respectively. ** p < 0.01 and *** p < 0.001 compared to control and single drug treatments. (G) Proposed mechanism of action of ONC + VEN. ONC213 targets oxidative phosphorylation via inhibition of α-KGDH which subsequently induces mitochondrial stress pathways (p-eIF2-α) and reduces levels of Mcl-1. Mcl-1 is a known contributor to resistance to venetoclax and reduction of Mcl-1 activates the intrinsic apoptosis pathway and contributes to reduction of oxidative phosphorylation, enhancing the antileukemic activity of venetoclax against AML