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Exploiting tumour cell dependency on pro-survival BCL2 proteins with BH3 mimetics


Type

Thesis

Change log

Authors

Minihane, Emma-Jayne 

Abstract

The RAS-RAF-MEK1/2-ERK1/2 signalling pathway is one of the most frequently deregulated pathways in human cancer, with mutations in KRAS and BRAF commonly occurring in cancers of the pancreas, skin, and colon, among others. Indeed, BRAF and MEK1/2 inhibitors are approved for the treatment of BRAF-mutant malignant melanoma and are improving patient outcomes. However, the development of acquired resistance to these agents is inevitable, highlighting the need for strategies to enhance their primary efficacy in the clinic. Inhibition of ERK1/2 signalling increases the abundance of pro-apoptotic BH3-only proteins such as BIM and BMF. However, despite this, responses to ERK1/2 pathway inhibitors are typically cytostatic, reflecting the residual activity of pro-survival BCL2 proteins. In this thesis, we determined that melanoma cells had a high MCL1:BCL-XL ratio, and as a result were biased towards MCL1 due to low expression of BCL-XL. Consequently, melanoma cells were uniquely dependent on MCL1 to restrain the pro-apoptotic BH3-only proteins induced by ERK1/2 pathway inhibition. The BH3 mimetic AZD5991, a selective MCL1 inhibitor, combined with ERK1/2 pathway inhibitors to drive striking synergistic apoptosis in vitro and robust tumour regressions in vivo. CRISPR/Cas9 gene editing of BIM and BMF in two melanoma cell lines showed that the cell death was substantially BIM/BMF-dependent, and required BAK and BAX to execute. In contrast to melanoma, colorectal cancer (CRC) cells exhibit increased expression of BCL-XL, with a low MCL1:BCL-XL ratio. The combination of MEK1/2 inhibitors with pan-BH3 mimetics that target BCL2, BCL-w and BCL-XL (such as ABT-263) has previously been explored in CRC cells. However, results in this thesis demonstrated that BCL-XL was the critical pro-survival protein in CRC cells responsible for buffering the pro-apoptotic effects of ERK1/2 pathway inhibition. CRC cell lines were completely refractory to BCL2 inhibition when combined with the MEK1/2 inhibitor selumetinib. MEK1/2 inhibitors primed CRC cells for rapid BAX-dependent apoptosis upon BCL-XL antagonism. BH3 mimetics combined with the ERK1/2 inhibitors SCH772984 or AZ6197 in a model of acquired resistance to BRAF and/or MEK1/2 inhibitors in A375 melanoma cells to drive striking apoptosis in vitro and in vivo. Combined BRAF and MCL1 inhibition delayed the onset of acquired resistance to BRAF inhibitor monotherapy in melanoma cells. In SW620 (CRC) cells, combined MEK1/2 and BCL-XL inhibition caused substantial cell death and did not result in the outgrowth of resistant clones; this combination possibly induced senescence in the residual “persister” cells. Finally, in addition to apoptosis, an alternate temporally-distinct cell death modality occurred in CRC cells treated with combined MEK1/2 and BCL-XL inhibition, and this was amplified when caspases were inhibited. This caspase-independent cell death was subsequently determined to be RIPK1-dependent necroptosis, which was reversed by inhibition of RIPK1 by necrostatin-1. BCL-XL may directly inhibit necroptosis induction at the level of the necrosome through the binding of the effector protein MLKL, which was displaced upon BCL-XL inhibition.

BH3 mimetics are effective as single agents for the treatment of haematological malignancies, but their application in solid tumours needs to be refined. The results presented here demonstrate that BH3 mimetics harness the apoptotic potential of ERK1/2 pathway inhibitors to drive striking, synergistic cell death in melanoma and CRC cells. The ratio of pro-survival proteins could represent a potential clinical biomarker, and be informative in the clinic to drive BH3 mimetic combination choices.

Description

Date

2020-02-28

Advisors

Cook, Simon

Keywords

Apoptosis, BH3 mimetic, Cancer, ERK, BRAF, Melanoma, Colorectal cancer, MEK1/2 inhibitors, Necroptosis

Qualification

Awarding Institution

University of Cambridge
Sponsorship
Cambridge International Trust scholarship AstraZeneca