Knockout Serum Replacement Promotes Cell Survival by Preventing BIM from Inducing Mitochondrial Cytochrome C Release.

Yuki Ishii,Michele Massimino,Donald L Durden,May Keu Nhiayi,Paolo Vigneri,Jonathan Cheng,Edison Tse,Jean Y J Wang

doi:10.1371/journal.pone.0140585

Abstract

Knockout serum replacement (KOSR) is a nutrient supplement commonly used to replace serum for culturing stem cells. We show here that KOSR has pro-survival activity in chronic myelogenous leukemia (CML) cells transformed by the BCR-ABL oncogene. Inhibitors of BCR-ABL tyrosine kinase kill CML cells by stimulating pro-apoptotic BIM and inhibiting anti-apoptotic BCL2, BCLxL and MCL1. We found that KOSR protects CML cells from killing by BCR-ABL inhibitors—imatinib, dasatinib and nilotinib. The protective effect of KOSR is reversible and not due to the selective outgrowth of drug-resistant clones. In KOSR-protected CML cells, imatinib still inhibited the BCR-ABL tyrosine kinase, reduced the phosphorylation of STAT, ERK and AKT, down-regulated BCL2, BCLxL, MCL1 and up-regulated BIM. However, these pro-apoptotic alterations failed to cause cytochrome c release from the mitochondria. With mitochondria isolated from KOSR-cultured CML cells, we showed that addition of recombinant BIM protein also failed to cause cytochrome c release. Besides the kinase inhibitors, KOSR could protect cells from menadione, an inducer of oxidative stress, but it did not protect cells from DNA damaging agents. Switching from serum to KOSR caused a transient increase in reactive oxygen species and AKT phosphorylation in CML cells that were protected by KOSR but not in those that were not protected by this nutrient supplement. Treatment of KOSR-cultured cells with the PH-domain inhibitor MK2206 blocked AKT phosphorylation, abrogated the formation of BIM-resistant mitochondria and stimulated cell death. These results show that KOSR has cell-context dependent pro-survival activity that is linked to AKT activation and the inhibition of BIM-induced cytochrome c release from the mitochondria.

Highlights

Of the recent advancements in cancer therapy, the most important has been the development of inhibitors that target specific oncogenic tyrosine kinases activated by mutations, translocations or over-expression in cancer cells
Induction of this tyrosine kinase inhibitors (TKIs)-resistance in chronic myelogenous leukemia (CML) K562 cells is linked to Knockout serum replacement (KOSR)-induced increase in reactive oxygen species (ROS), a transient increase in AKT phosphorylation, and the formation of mitochondria that are resistant to BIM-induced cytochrome c release
We found that media switch failed to induce ROS and p-AKT in LAMA-84 cells, and these cells were not protected from the cytotoxic effect of TKI by media switch

Summary

Introduction

Of the recent advancements in cancer therapy, the most important has been the development of inhibitors that target specific oncogenic tyrosine kinases activated by mutations, translocations or over-expression in cancer cells. The second category of TKI-resistance involves biological adaptation where cancer cells activate oncogene-independent mechanisms to survive and proliferate, and this mechanism of TKI-resistance underlies the persistence of CML stem cells [3]. Cancer cell addiction to oncogenic tyrosine kinases occurs when one or more of those kinases become the only activators of the mitogenic and survival pathways, e.g., RAS-MEK, PI3K-AKT, and JAK-STAT [4]. These pathways converge upon activation of the pro-survival BCL2-proteins and suppression of the pro-apoptotic BH3-proteins such as BIM [5]. These findings suggest that the BH3-induced MOMP is subjected to regulation beyond the mere increase in the relative abundance of BH3-containing proteins

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Conclusion