Fig. 7: Inhibition of AKT restores sensitivity of venetoclax-resistant cells to venetoclax. | Leukemia

Fig. 7: Inhibition of AKT restores sensitivity of venetoclax-resistant cells to venetoclax.

From: Superoxide-mediated phosphorylation and stabilization of Mcl-1 by AKT underlie venetoclax resistance in hematologic malignancies

Fig. 7

A Western blot showing S473pAKT, AKT, T163pMcl-1, Mcl-1, Tubulin and/or β-Actin levels of VEN-S and VEN-R MOLM14, OCI-AML2, and OCI-Ly1 cells, as well as inherently VEN-R Su-DHL4 cells. B Western blot showing T163pMcl-1, Mcl-1, pro and cleaved caspase-3, β-Actin levels in VEN-R MOLM14 or VEN-R OCI-AML2 cells treated with capivasertib (1 µM/VEN-R MOLM14, 5 µM/VEN-R OCI-AML2) and/or VEN (0.1 µM/VEN-R MOLM14, 10 µM/VEN-R OCI-AML2) for 24 h. N = 3. C Heatmap of BH3-profiling showing percentage of cytochrome c loss from VEN-R MOLM14 cells following treatment with capivasertib (2 µM) or DMSO for 4 h, and subsequent exposure to different BH3 peptide or mimetic concentrations to indicate specific increase in Bcl-2 dependence. N = 3. D Graph showing cell viability assay of VEN-R MOLM14 cells treated with capivasertib (1–5 µM) and/or VEN (0.02–0.075 μM) for 48 h. Cell viability was assessed using CTG assay and data were normalized to untreated control cells. N = 3. Sidak’s multiple comparisons test was used. E Heatmap below showing synergy score based on Highest Single Agent (HSA) synergy model, calculated using SynergyFinder. F Graph showing a 7-day chase assay of VEN-R MOLM14 live cell count in fold change following treatment with DMSO, capivasertib (2 μM), VEN (0.1 μM), or combination. Day 3—DMSO vs combo P < 0.0001, Capi vs combo P < 0.02, VEN vs combo P < 0.001; Day 5—DMSO vs combo P < 0.0001, Capi vs combo P < 0.001, VEN vs combo P < 0.002; Day 7—DMSO vs combo P < 0.0001, Capi vs combo P < 0.0001, VEN vs combo P < 0.001. Tukey’s multiple comparisons test was used. G Cell viability assay of VEN-R OCI-AML2 (N = 3), VEN-R OCI-Ly1 (N = 3), Su-DHL4 (N = 4) cells treated with capivasertib and/or VEN for 48 h. Cell viability was assessed using the CTG assay and data were normalized to untreated control cells. Concentrations used for capivasertib (5 µM/VEN-R OCI-AML2, 0.5 µM/OCI-Ly1 VEN-R, 2 µM/Su-DHL4) and/or VEN (10 µM/VEN-R OCI-AML2, 1 µM/OCI-Ly1 VEN-R, 0.5 µM/Su-DHL4). Sidak’s multiple comparisons test was used. H Cell viability of CLL patient cells following ex vivo treatment with capivasertib (2 µM) and/or VEN (0.005 μM) for 24 h, measured via DAPI+/Annexin V−. N = 10. Sidak’s multiple comparisons test was used. I Western blot showing Mcl-1, S9pGSK3β, GSK3β, GAPDH levels in CLL patient cells following ex vivo treatment with capivasertib (2 µM) for 6 h. Representative blots shown for 2 CLL patient samples. Densitometric analyses of Mcl-1/β-Actin levels in CLL patient samples treated with capivasertib (2 µM) for 6 h, normalized to DMSO control. N = 5. Paired t-test was used. J Tumor burden of mice displayed by %hCD45 + hCD33 + VEN-R MOLM14 cells in blood sample harvested from mice that has been treated in vivo with vehicle, capivasertib, VEN or combo. N = 4 per study arm. Day 15 is the treatment initiation day. K Probability of survival following treatment with vehicle, capivasertib, VEN or combo in mice harboring VEN-R MOLM14 cells. N = 4 per study arm.

Back to article page