Fig 1.
Inhibition of VCP leads to accumulation of ubiquitylated proteins and activates the unfolded protein response (UPR) in human acute myeloid leukemia (AML) cells.
Immunoblot showing poly-ubiquitylated proteins in THP-1 cells (A) and in a primary human AML long-term culture (FFM05) (B) after treatment with the VCP inhibitors CB-5083 or NMS-873 or the proteasome inhibitor MG-132. (C) Immunoblot showing expression of UPR-associated proteins in THP-1 and MV4-11 cells after treatment with 200 or 500 nM CB-5083 overnight.
Fig 2.
Dynamics of the ubiquitin-modified proteome in AML cells after VCP inhibition.
(A) SILAC ratio distribution of all quantified ubiquitylation sites in MV4-11 and THP-1 cells. (B) Scatter plot showing SILAC ratios of quantified ubiquitylation sites in MV4-11 and THP-1 cells after treatment with the VCP inhibitor CB-5083.
Fig 3.
Inhibition of VCP induces apoptosis in AML cells.
(A) MV4-11 cells were treated with 200 nM or 1 μM CB-5083 overnight. Cells that were left untreated or treated with 500 mM H2O2 served as control. Induction of apoptosis was assessed by dual staining with Annexin V and 7-AAD. Relative fluorescence of Annexin V and 7-AAD is shown for all conditions. (B) Quantification of the data.
Fig 4.
VCP inhibition reduces AML cell growth and survival.
(A) AML cell lines THP-1, MV4-11, and KG-1 as well as the primary AML long term culture FFM05 were treated with different concentrations of CB-5083 and NMS-873. Cell count was measured after 72 hours and IC50 values for all cell lines were estimated. Additionally, IC50 values for CB-5083 were determined for CD34-positive cells from donors (B) and for the human bone marrow stromal cell line HS-5 (C). (D) Summary of all determined IC50 values.
Fig 5.
CB-5083 and cytarabine or venetoclax synergistically decrease AML cell survival.
(A-D) THP-1 and MV4-11 cell survival was assessed in a 96-well format with increasing concentrations of CB-5083 and cytarabine or venetoclax. Following a 72-hour incubation, CellTiter-Blue Reagent was added and fluorescence/cell viability was assessed. Cell viability data was used as input for the SynergyFinder software to estimate synergy δ scores. Synergy landscape maps display δ scores for the indicated cells lines and drug combinations.
Fig 6.
CB-5083-resistant MV4-11 cells exhibit features of permanent endoplasmic reticulum (ER) stress but no apoptosis.
(A) Immunoblot showing induction of ER stress in inhibitor-resistant cells after a few months of continuous treatment with 200 and 500 nM CB-5083. (B and C) Flow cytometry analysis using Annexin V and 7-AAD staining showed decreased apoptosis in CB-5083-resistant cell lines. (D and E) IC50 values for CB-5083 in the indicated cell lines upon withdrawal and re-treatment with CB-5083. (F) Analysis of the coding sequence of VCP identified a variant (c.1591C>A p.I531L) in the cell line treated with 1 μM CB-5083.
Table 1.
Potential CB-5082 resistance mutations characterized in this and previous studies.