Ceri M. Wiggins

ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL.

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim−/− fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild‐type counterparts. In viable cells, Bax associates with Bcl‐2, Bcl‐xL and Mcl‐1. Upon serum withdrawal, newly expressed BimEL associates with Bcl‐xL and Mcl‐1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro‐survival proteins by preventing Bim expression. However, we now show that even preformed BimEL/Mcl‐1 and BimEL/Bcl‐xL complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl‐1 is specific for BimEL and requires ERK1/2‐dependent phosphorylation of BimEL at Ser65. Finally, ERK1/2‐dependent dissociation of BimEL from Mcl‐1 and Bcl‐xL may play a role in regulating BimEL degradation, since mutations in the BimEL BH3 domain that disrupt binding to Mcl‐1 cause increased turnover of BimEL. These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.

Bim and the Pro-Survival Bcl-2 Proteins: Opposites Attract, ERK Repels

Bim (Bcl-2-interacting mediator of cell death) is a BH3-only protein (BOP), a pro-apoptotic member of the Bcl-2 protein family. The Bim mRNA undergoes alternate splicing to give rise to the short, long and extra long protein variants (BimS, BimL and BimEL). These proteins have distinct potency in promoting death and distinct modes of regulation conferred by their interaction with other proteins. Quite how Bim and other BOPs promote apoptosis has been the subject of some debate. Bim was isolated by it’s interaction with pro-survival proteins such as Bcl-2 and it has been suggested that this is key to the ability of Bim to induce apoptosis. However, an alternative model argues that some forms of Bim can bind directly to the pro-apoptotic Bax and Bak proteins to initiate apoptosis. A new study may finally put this debate to rest as it provides strong evidence to suggest that Bim and other BOPs act primarily by binding to pro-survival Bcl-2 proteins, thereby releasing Bax or Bak proteins to promote apoptosis. The importance of the interaction between Bim and the pro-survival Bcl-2 proteins is underlined by our demonstration that it is regulated by ERK1/2-dependent phosphorylation of BimEL. ERK1/2-dependent dissociation of BimEL from pro-survival proteins is the first step in a process by which the pro-survival ERK1/2 pathway promotes the destruction of this most abundant Bim splice variant. In this review we outline the significance of these new studies to our understanding of how BOPs such as Bim initiate apoptosis and how this process is regulated by growth factor-dependent signalling pathways.

Apoptosis and autophagy: BIM as a mediator of tumour cell death in response to oncogene-targeted therapeutics.

The BCL‐2 homology domain 3 (BH3)‐only protein, B‐cell lymphoma 2 interacting mediator of cell death (BIM) is a potent pro‐apoptotic protein belonging to the B‐cell lymphoma 2 protein family. In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal‐regulated kinase 1/2 and protein kinase B pathways. In tumour cells these oncogene‐regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival. In parallel, a new generation of targeted therapeutics has been developed, which show remarkable specificity and efficacy in tumour cells that are addicted to particular oncogenes. It is now apparent that the expression and activation of BIM is a common response to these new therapeutics. Indeed, BIM has emerged from this marriage of basic and applied biology as an important mediator of tumour cell death in response to such drugs. The induction of BIM alone may not be sufficient for significant tumour cell death, as BIM is more likely to act in concert with other BH3‐only proteins, or other death pathways, when new targeted therapeutics are used in combination with traditional chemotherapy agents. Here we discuss recent advances in understanding BIM regulation and review the role of BIM as a mediator of tumour cell death in response to novel oncogene‐targeted therapeutics.

BIMEL, an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation

BIM-extra long (BIMEL), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIMEL in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIMEL undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIMELΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIMEL is degraded by isolated 20S proteasomes but that this is prevented when BIMEL is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIMEL turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIMEL, causes Cdc25A accumulation but does not inhibit BIMEL turnover. These results provide new insights into the regulation of BIMEL by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

Abstract 700: Investigating KRAS synthetic lethal/co-dependency interactions using siRNA and CRISPR

No molecularly targeted therapy has yet been identified for KRAS mutant cancers. As oncogenic mutations reduce RAS enzymatic activity, classic small molecule approaches are ineffective, hence most work has focussed on drugging RAS-effector pathways. Multiple inhibitors of MEK, RAF and PI3K have been identified but toxicity issues and pathway adaptation have stymied their success against KRAS-driven cancers. An alternative approach is to exploit “non-oncogene addiction” by identifying targets with synthetic lethal or co-dependence interactions with KRAS. A number of siRNA and shRNA screens have identified targets that exhibit differential dependencies between KRAS mutant and KRAS wild-type tumours, but there is poor overlap between the different published studies. This discordance may arise from (1) the noise inherent in using cell line panels differing in much more than their KRAS mutant/wild-type status and (2) the use of RNA interference methodologies driving incomplete knockdown and associated with substantial off-target effects. Next generation screens that exploit both isogenic cell lines and cell line panels, and use a combination of knockdown and knock-out (i.e. CRISPR/Cas9-sgRNA) methodologies, may be better suited for identifying novel targets that withstand validation. However, if we are to detect co-dependence as well as synthetic lethal interactions, screens must be performed under conditions where mutant KRAS alleles are essential for growth. A library of siRNAs targeting proposed KRAS synthetic lethal targets was assembled and screened under conditions where proliferation is dependent on KRAS status. DLD1 cells harbour an activating KRASG13D mutation dispensable for proliferation in 2D, but essential for proliferation under 3D (soft agar) conditions. Knockdown of several targets including KRAS itself, PLK1, TBK1, BCL-XL & RAF1 proved more anti-proliferative under 3D conditions. This screen was extended to a panel of KRAS-mutant colon lines, with varying levels of KRAS sensitivity, where we found the requirement for RAF1 highly correlated with the requirement for KRAS. With the advent of CRISPR we are now able to design sgRNA libraries capable of probing the effect of ‘knocking out’ rather than ‘knocking down’ targets, providing a potentially superior alternative to RNA interference. Data from mouse models indicates RAF1 is required for the initiation of lung cancer by oncogenic KRAS. Although we found good correlation between sensitivity to KRAS and RAF1 depletion, we were unable to unambiguously validate RAF1 as a target in human lung cancer cells using RNA interference methodologies. However, using CRISPR-Cas9, we found complete loss of RAF1 expression was anti-proliferative in A549 cells, supporting the concept of targeting RAF1 in a KRAS mutant lung cancers. These results demonstrate that a more penetrant sgRNA based screening approach may identify novel KRAS synthetic lethal or co-dependent interactions. Citation Format: Simon F. Scrace, Elpida Tsonou, Paul Russell, Julie A. Wickenden, Steffen Lawo, Tim M. Scales, Ceri M. Wiggins, Jonathan D. Moore. Investigating KRAS synthetic lethal/co-dependency interactions using siRNA and CRISPR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2015-700

Abstract A25: Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer

Mutations in tumour suppressors and un-druggable oncogenes dominate the landscape of cancer driver genes. Only a minority of colon cancers have mutations in druggable cancer drivers, such as PIK3CA. Conversely, mutations in tumour suppressors such as APC and TP53 are frequent, as are mutations in the notoriously difficult to drug KRAS target. There is an urgent need for new therapeutics to target tumours driven by these mutations: immune checkpoint approaches are likely to only prove effective in the fraction of patients whose tumours bear high mutation loads, which is colon cancer may be restricted to the minority of mismatch repair deficient cancers. The concepts of non-oncogene addiction and synthetic lethality provide a conceptual framework for finding therapeutic targets in these cancers. We have used arrayed siRNA and pooled CRISPR-Cas9 libraries to screen a panel of isogenic and non-isogenic colon cancer cell lines under conditions designed to increase the cells dependency on oncogenic pathways. This panel contains cell lines with mutations in TP53, APC, KRAS, PIK3CA and/or FBXW7 for which we are aiming to identify co-dependencies that consistently segregate with genotype. Our screens have identified a number of novel targets for which reduced expression imposes specific fitness defects on cells with mutant PIK3CA, mutant TP53 or mutant FBXW7. Few of these targets were identified by both siRNA and CRISPR-Cas9 approaches. Furthermore, isogenic pairs of cell lines have not proved helpful for identifying synthetic lethal targets in the KRAS or PIK3CA genotypes. However, we believe the increased penetrance of the CRIPSR-Cas9 approach has uncovered novel candidate synthetic lethal genes that were not found by RNA interference. Although our current screens are not saturating, we have confirmed several previously reported genetic interactions including the requirements for expression of MDM2 in TP53 wild-type cancers and HK2 in colon cancer lines with KRAS mutations. The screens in the FBXW7 genotype were particularly interesting: in addition to identifying a potential synthetic lethal interaction with a target for which a drug has recently entered the clinic, we also found that FBXW7 itself seemed to be required in many of the colon cancers with FBXW7 mutations. This suggests that FBXW7 may sometimes act as an oncogene, rather than as a tumour suppressor in cancer initiation or progression. Finally, we have attempted to validate some of the candidate synthetic lethal genes identified by RNA interference via ultra-deep CRISPR-CAS9 pooled screening, extending the approach of the Vakoc lab (Nature Biotech. 33, 661-667, 2015). We can confirm this method can highlight functional domains of particular significance to a protein9s function, but note that many-fold drop outs of sgRNAs can also occur where they target multiple sites in the genome, presumably via a DNA damage response. Citation Format: Jonathan D. Moore, Chantelle Hudson, Paul Russell, Gaganpreet Tiwana, David Walter, Ceri M. Wiggins, Joanne Yarker. Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A25.