That which does not kill me makes me stronger; combining ERK1/2 pathway inhibitors and BH3 mimetics to kill tumour cells and prevent acquired resistance

Abstract

Oncogenic mutations in RAS or BRAF can drive the inappropriate activation of the ERK1/2. In many cases, tumour cells adapt to become addicted to this deregulated ERK1/2 signalling for their proliferation, providing a therapeutic window for tumour-selective growth inhibition. As a result, inhibition of ERK1/2 signalling by BRAF or MEK1/2 inhibitors is an attractive therapeutic strategy. Indeed, the first BRAF inhibitor, vemurafenib, has now been approved for clinical use, while clinical evaluation of MEK1/2 inhibitors is at an advanced stage. Despite this progress, it is apparent that tumour cells adapt quickly to these new targeted agents so that tumours with acquired resistance can emerge within 6-9 months of primary treatment. One of the major reasons for this is that tumour cells typically respond to BRAF or MEK1/2 inhibitors by undergoing a G1 cell cycle arrest rather than dying. Indeed, although inhibition of ERK1/2 invariably increases the expression of pro-apoptotic BCL2 family proteins, tumour cells undergo minimal apoptosis. This cytostatic response may simply provide the cell with the opportunity to adapt and acquire resistance. Here we discuss recent studies that demonstrate that combination of BRAF or MEK1/2 inhibitors with inhibitors of pro-survival BCL2 proteins is synthetic lethal for ERK1/2-addicted tumour cells. This combination effectively transforms the cytostatic response of BRAF and MEK1/2 inhibitors into a striking apoptotic cell death response. This not only augments the primary efficacy of BRAF and MEK1/2 inhibitors but delays the onset of acquired resistance to these agents, validating their combination in the clinic.

Linked articles: This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8.

Keywords: BCL2; BRAF; ERK1/2; MEK1/2; RAS; acquired resistance; apoptosis; cancer; targeted therapeutics.

Figure 1

ERK1/2-mediated regulation of the G1-S phase transition. Nuclear ERK1/2 phosphorylates and stabilizes/activates members of ETS and AP1 transcription factor families, which can induce transcription of CCND1 (cyclin D1). The transcription factor MYC is stabilized by ERK1/2-mediated phosphorylation, and MYC can up-regulate the expression of cell cycle regulators such as cyclin D2 CCND2 (cyclin D2) and CDC25A. CCND1 and CCND2 bind to and activate CDK4 and CDK6 (CDK4/6). Phosphorylation of RB by CDK4/6 frees the E2F transcription factors from RB-mediated repression, allowing E2F-induced transcription of genes such as CCNE (cyclin E), CCNA (cyclin A) and MYC. Newly synthesized CCNE binds and activates CDK2, which can also phosphorylate RB in a feedforward loop. p27^KIP1, an endogenous inhibitor of CDK2, is down-regulated and inactivated during cell cycle entry by a variety of mechanisms mediated by cyclin E-CDK2, ERK1/2 and RSK. CDK2, initially in complex with CCNE and later with CCNA, and the E2F transcription factors regulate many target factors to drive progression into, and through, S phase. ERK1/2 signalling is frequently hyperactivated in tumour cells as a result of mutations in RTKs, RAS or BRAF (shown, yellow star), providing validation for selective inhibitors of mutant BRAF (e.g. vemurafenib) or MEK1/2 (e.g. selumetinib).

Figure 2

ERK1/2-mediated regulation of the BCL2 protein family. ERK1/2 activation by RTK signalling, mutant RAS or mutant BRAF (shown, yellow star) has pleiotropic effects on the expression and/or activity of BCL2 family members. The BH3-only protein BIM is phosphorylated by ERK1/2 on at least three sites, marking BIM for ubiquitination and subsequent degradation by the 26S proteasome. ERK1/2 also negatively regulates the expression and/or activity of BMF through incompletely understood mechanisms. Activation of RSK by ERK1/2 promotes BAD phosphorylation, which creates a 14-3-3 binding site and sequesters BAD away from the mitochondria. MCL1 stability is subject to reciprocal regulation by the actions of ERK1/2 and GSK3. Phosphorylation of MCL1 by ERK1/2 stabilizes MCL1, whereas GSK3-mediated phosphorylation promotes MCL1 degradation. In the nucleus, ERK1/2 influences the transcription of BCL2 family members. Activation of ERK1/2-dependent RSK and MSK1/2 activates CREB, which promotes transcription of the pro-survival genes BCL2, BCL-X_L and MCL1. ELK1 activation by ERK1/2 may also augment MCL1 transcription. ERK1/2 promotes the degradation of FOXO3, thereby inhibiting FOXO3-dependent transcription of pro-apoptotic BIM and PUMA. In contrast to other pro-apoptotic BH3-only proteins, ERK1/2 signalling induces the expression of NOXA mRNA and protein. Thus, with the exception of NOXA, tumour cell ERK1/2 signalling typically promotes the expression of pro-survival factors, and represses the expression and/or activity pro-apoptotic BCL2 family members.

Figure 3

Selumetinib and ABT-263 synergize to inhibit pro-survival BCL2 family proteins and activate BAX in ERK1/2-addicted tumour cell lines. Treatment of tumour cells with a MEK1/2 inhibitor such as selumetinib invariably induces strong expression of BH3-only proteins such as BIM and BMF (A, top). PUMA expression may also be induced. These BH3-only proteins then bind to pro-survival factors such as BCL-X_L and MCL1 (A, bottom). Despite this, little cell death occurs with MEK1/2 inhibition alone, likely due to residual pro-survival activity, including BCL-X_L and MCL1 (A, bottom). Addition of the BH3 mimetic ABT-263 (red triangle; B, top) causes a redistribution of selumetinib-induced BIM and BMF from ABT-263 sensitive BCL-X_L to ABT-263-resistant MCL1 (B, bottom). Thus, although ABT-263 cannot directly target MCL1, its combination with selumetinib results in indirect inhibition of MCL1 and consequently greater inhibition of pro-survivals (C, top). In addition, whereas selumetinib only resulted in partial displacement of BAX from BCL-X_L, ABT-263 efficiently disrupted this interaction. BAX could then potentially be directly activated by any residual BIM freed up from BCL-X_L (C, top). Activated BAX can subsequently oligomerize and insert into the outer mitochondrial membrane, resulting in MOMP (C, bottom). This allows cytochrome c release from the intermembrane space, followed by apoptosome formation, caspase activation and consequent apoptosis. (Reproduced with permission from Sale and Cook, 2013, The Biochemical Journal, 450, 285-294 © the Biochemical Society)