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Conventional CRISPR approaches for precision genome editing rely on the introduction of DNA double-strand breaks (DSB) and activation of homology-directed repair (HDR), which is inherently genotoxic and inefficient in somatic cells. The development of base editing (BE) systems that edit a target base without requiring generation of DSB or HDR offers an alternative. Here, we describe a novel BE system called Pin-point(TM) that recruits a DNA base-modifying enzyme through an RNA aptamer within the gRNA molecule. Pin-point is capable of efficiently modifying base pairs in the human genome with precision and low on-target indel formation. This system can potentially be applied for correcting pathogenic mutations, installing premature stop codons in pathological genes, and introducing other types of genetic changes for basic research and therapeutic development.

Studies to identify predictive biomarkers can be carried out in isogenic cancer cell lines, which enable interrogation of the effect of a specific mutation. We assessed the effects of four drugs, the PI3K-mammalian target of rapamycin inhibitor dactolisib, the PI3K inhibitor pictrelisib, and the MEK (MAPK/ERK Kinase) inhibitors PD 0325901 and selumetinib, in isogenic DLD1 parental, KRAS(+/-), KRAS(G13D/-), PIK3CA(+/-) and PIK3CA(E545K/-) colorectal carcinoma cell lines. Importantly, we found substantial differences in the growth of these cells and in their drug sensitivity depending on whether they were studied under 2D (standard tissue culture on plastic) or 3D (in vitro soft agar and in vivo xenograft) conditions. DLD1 KRAS(+/-) and DLD1 PIK3CA(+/-) cells were more sensitive to MEK inhibitors than parental, DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) cells under 2D conditions, whereas DLD1 KRAS(G13D/-) and DLD1 PIK3CA(E545K/-) xenografts were sensitive to 10 mg/kg daily ×14 PD 0325901 in vivo (p ≤ 0.02) but tumours derived from parental DLD1 cells were not. These findings indicate that KRAS and PIK3CA mutations can influence the response of DLD1 colorectal cancer cell lines to MEK and PI3K inhibitors, but that the effect is dependent on the experimental model used to assess drug sensitivity.

Cell panel screening of cancer lines grown in standard 2D conditions is a well-established component of the drug discovery toolbox. A sufficiently large cell line panel, reflecting the molecular heterogeneity within a cancer type/sub-type, is required to detect correlations with robust statistical power that aid biomarker identification and patient stratification. However, the heterogenous exposure of tumor cells to nutrients, oxygen and other physical or chemical stresses found in three-dimensional solid tumors also has a role to play in patient response and this is not well mimicked in 2D monolayer cell cultures. To meet the need for high-throughput cell-panel screening in more complex culture systems, we have developed 3D cell-based screens to more closely model the complex physiological environment found in tumors. We have characterised our OncoSignature panel of 300 cell lines for growth as spheroids to produce a 200 strong panel of spheroid forming lines. Using our high-throughput platform to compare drug activity in 2D and 3D cultures, we have identified clear differentials for some compounds in single agent and drug combination screens. For example, MEK and EGFR inhibitors have significantly higher activity in 3D cultures than 2D cultures, with a greater than 10-fold shift in EC50 values. These results were validated in 3D soft agar assays, with similar compound potencies being observed in the HTS spheroid assay and the lower throughput soft-agar assay. We have further extended our 3D screening into organoid cultures. Organoid cultures, with increased complexity in terms of structure and cell heterogeneity compared to spheroids, have historically been challenging to use in high-throughput screening. However, our initial results show encouragingly robust data, with compound activity that is congruent with the genetic characteristics of the organoids. Finally, we used our integrated screening platform to evaluate the power of CRISPR approaches to examine genetic dependencies in KRAS mutant cells in 2D, 3D spheroid and organoid cell cultures. Pooled, next-generation sequencing-linked CRISPRko allows an alternative and complementary genomics approach to arrayed cell panel screening, providing a powerful and robust target identification and validation strategy. Overall, Horizon’s 3D screening platforms will enable large scale interrogation of both drug and gene interactions in more complex cell culture systems. Citation Format: Tim M. Scales, Anett Rada-Kovacs, Helen N. Pemberton, Ceri M. Wiggins, Nicola J. McCarthy, Annette S. Little, David A. Sorrell. High-throughput cell line panel drug screening in organoids and 3D systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1927.

Genetic perturbations mediated by CRISPR-Cas9 are transforming the drug discovery process. In the hunt for novel drug targets, researchers finally have a screening tool with sufficient penetrance and power to identify new interactions, followed by a rapid gene editing technology to streamline validation approaches. At Horizon Discovery, we have now initiated more than 250 CRISPR-Cas9 knockout (KO) screens, including a significant effort to identify novel synthetic lethal interactions in colon and lung cancer genotypes. These screens have identified a reassuring number of known essential interactions, such as MDM2 and PPMID in TP53 wild type cancer cell lines. These results provide confidence in the ability of these screens to also discover novel targets that should make it through the validation process. Indeed, we have discovered novel targets for selective ablation of TP53 mutant colon cancers, several of which are strongly supported by the literature. In FBXW7 mutant colon cancers, CRISPR-Cas9 KO screens identified a set of potential novel druggable targets in addition to MCL1 - a known substrate of FBXW7, which showed a strong dependency in FBXW7 mutant lines. An important step in triaging primary hits ahead of extensive validation work has been secondary screens that target functional domains with high density sgRNA tiling. They allow more targets to be examined than would be feasible by any arrayed approach. Data from such screens, which test tens to hundreds of guides in unison, not only increases hit calling power, but can also yield information regarding important domains and mechanism of action. This was evident in the FBXW7 secondary screen data, where guides targeting the key functional BH domains of MCL1 proved to be the most effective. This information can be used to prioritise individual targets for further validation, using techniques that include an inducible CRISPR-Cas9 system to assess the effect of individual guides in an arrayed format on cell proliferation and survival, and CRISPRi. These data exemplify how CRISPR-Cas9 can enable the discovery of a new, more robust generation of drug and antibody targets. Citation Format: Ceri M. Wiggins, David Walter, Paul Russell, Clare Sheridan, Chantelle Hudson, Joanne Yarker, Carlos le Sage, Nicola McCarthy, Jonathan D. Moore. CRISPR-Cas9: A tool for rapid target discovery and validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2912.

The pro-apoptotic protein BIM(EL) is phosphorylated by ERK1/2 and this targets the protein for poly-ubiquitination and degradation by the proteasome as a survival mechanism. To define in greater detail the sequence determinants required for BIM(EL) turnover we have compared various BIM splice variants and truncation mutants. Of the naturally occurring splice variants BIMbeta1, which lacks the C-terminal hydrophobic domain, the BH3 domain and is cytosolic, exhibited the fastest turnover rate. Indeed, neither the C-terminus, the BH3 domain nor the DLC1 binding region was required for poly-ubiquitination and turnover of BIM. However, we demonstrate that a region consisting of the ERK1/2 docking domain, ERK1/2 phosphorylation sites and either of the two potential ubiquitin-acceptor lysine residues is sufficient to allow poly-ubiquitination and turnover of BIM. In the process we demonstrate that the C-terminal hydrophobic domain, previously suggested to be important in membrane localisation, is as important as the BH3 domain for BIM to induce cell death; similarly, the pro-death BH3-domain can also confer correct mitochondrial localisation in the absence of the C-terminus. These results refine the minimal sequence for ERK1/2-driven degradation and further define the functional importance of key regions within BIM(EL), highlighting the complexity of this pro-apoptotic protein.

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 protein's 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.

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.

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 (Bim(S), Bim(L) and Bim(EL)). 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 Bim(EL). ERK1/2-dependent dissociation of Bim(EL) 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.

BIM-extra long (BIM(EL)), 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 BIM(EL) 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 BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, 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 BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

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.

Bim(EL) the most abundant Bim splice variant, is subject to ERK1/2-catalysed phosphorylation, which targets it for ubiquitination and proteasome-dependent destruction. In contrast, inactivation of ERK1/2, following withdrawal of survival factors, promotes stabilization of Bim(EL). It has been proposed that the RING finger protein Cbl binds to Bim(EL) and serves as its E3 ubiquitin ligase. However, this is controversial since most Cbl substrates are tyrosine phosphoproteins and yet Bim(EL) is targeted for destruction by ERK1/2-catalysed serine phosphorylation. Consequently, a role for Cbl could suggest a second pathway for Bim(EL) turnover, regulated by direct tyrosine phosphorylation, or could suggest that Bim(EL) is a coincidence detector, requiring phosphorylation by ERK1/2 and a tyrosine kinase. Here we show that degradation of Bim(EL) does not involve its tyrosine phosphorylation; indeed, Bim(EL) is not a tyrosine phosphoprotein. Furthermore, Bim(EL) fails to interact with Cbl and growth factor-stimulated, ERK1/2-dependent Bim(EL) turnover proceeds normally in Cbl-containing or Cbl-/- fibroblasts. These results indicate that Cbl is not required for ERK1/2-dependent Bim(EL) turnover in fibroblasts and epithelial cells and any role it has in other cell types is likely to be indirect.

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-x(L) and Mcl-1. Upon serum withdrawal, newly expressed Bim(EL) associates with Bcl-x(L) 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 Bim(EL)/Mcl-1 and Bim(EL)/Bcl-x(L) complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for Bim(EL) and requires ERK1/2-dependent phosphorylation of Bim(EL) at Ser(65). Finally, ERK1/2-dependent dissociation of Bim(EL) from Mcl-1 and Bcl-x(L) may play a role in regulating Bim(EL) degradation, since mutations in the Bim(EL) BH3 domain that disrupt binding to Mcl-1 cause increased turnover of Bim(EL). These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway.

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