Interfering with protein-protein interactions

[Cell Cycle 7:11, 1569-1574; 1 June 2008]; ©2008 Landes Bioscience

Perspective

Interfering with protein-protein interactions Potential for cancer therapy Tomoyuki Tanaka and Terence H. Rabbitts* Leeds Institute of Molecular Medicine; Wellcome Trust Brenner Building; St. James’s University Hospital; Leeds, United Kingdom

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of genotype-specific therapeutics. Whilst enzymes are in principle amenable to blocking by small molecule inhibitors, other therapeutic routes are needed for a large number of cancer targets that work via protein-protein interaction. However, the current dogma argues for the intractability of protein-protein interaction as a small molecule drug target due to the large and often flat surfaces where two proteins bind to each other. Some successes have begun to appear, such as Nutlin-3 specifically binding to MDM2, inhibiting MDM2-p53 interaction and activating p53,3 and indeed the notion of the intractability of ablating protein interactions is being questioned.4 Recently, evidence has begun to accumulate that interference with protein interactions using antibody fragments5,6 can be a route to design of cancer drugs, including macrodrugs and small molecules. We will discuss here the possible utility of targeting the RAS-effector interactions for cancer treatment, as an exemplar of macromolecular (macrodrug) intracellular targeting.

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Genotype-specific cancer therapy promises to engender the era of personalised medicines in which rapid identification of tumour specific gene mutations coupled to rapid methods for efficacious drug identification will be applied. Aberrant signal transduction via protein-protein interactions is generally difficult to target with small molecules. However, macromolecules (macrodrugs) can be developed that interfere with protein-protein interactions by binding with high affinity and specificity to contact surfaces. Inhibitors of mutant RAS and its effector protein interactions affect cancer by attenuating aberrant RAS-dependent signal transduction and would be effective against mutant RAS in dividing cells of overt tumours and in putative cancer stem cells when they move into cell cycle. Results with an antibody fragment blocking effector binding to RAS, illustrates that this is sufficient to prevent cancer. While macrodrugs have inherent problems of bio-distribution and delivery to target cells in patients, their efficacy suggests that efforts to achieve the goal of clinical use should be pursued.

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Key words: protein-protein interaction, signal transduction, single domains, cancer therapy, RAS, intracellular antibody, intrabody

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Introduction

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Cancer arises because of somatic mutation in cells that lead to unrestricted growth and in vivo mobility of the afflicted cells. Currently, most cancer therapy involves generalised treatments (e.g., chemotherapy, surgery or radiotherapy), and few therapies are based on the targeting of the mutant molecules in cancer (i.e., DNA, RNA or protein). Genotype-specific therapeutics promise to have a major impact on cure rates and can potentially be applied on a personalised basis. Nonetheless, there have been few successes thus far, due partly to the fact that most mutations are not in enzymes per se and thus small molecules inhibitors are not readily applicable. An exception to this is Gleevec that targets the ABL kinase of BCR-ABL in Philadelphia-chromosome positive chronic myelogenous leukaemia1 and Kit (CD117)-positive gastrointestinal stromal tumours (GIST).2 The success of Gleevec nonetheless exemplifies the exciting promise *Correspondence to: Terence H. Rabbitts; Leeds Institute of Molecular Medicine; Section of Experimental Therapeutics; Wellcome Trust Brenner Building; St. James’s University Hospital; Leeds LS9 7TF United Kingdom; Tel.: +44.0.113.343.8518; Fax: +44.0.113.343.8601; Email: [email protected] Submitted: 03/29/08; Accepted: 04/02/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6061

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The Functions of RAS in Cancer

RAS family proteins have pivotal roles as molecular switches to transmit a variety of extracellular signals to the nucleus controlling cell growth, differentiation, migration and survival7,8 via the interaction with a range of effectors molecules (Fig. 1). RAS mutations, impairing the GTPase activity and consequently causing accumulation of active GTP-bound RAS, are found in up to 30% of human cancers, being particularly prevalent in pancreatic, colon and lung adenocarcinoma (Table 1). In addition, secondary RAS-associated signalling aberrations occur by gene mutation of receptor tyrosine kinases (e.g., EGFR), RAS regulators (e.g., GAP) and RAS effectors (e.g., PI3Kα, BRAF) in many cancers,9-12 suggesting that the overall RAS-signal transduction network is involved in a high percentage of human cancer. Growing insight in RAS signal transduction reveals that more than ten proteins with distinct functions (including PI3K, RAF and RALGEF) have been identified as RAS effectors (Fig. 1), directly interacting with RAS to constitute different downstream signalling pathways (e.g., RAF-MEK-ERK, PI3K-AKT and RALGEF-RAL).7 The mutation of the RAS family members, and downstream effectors, are excellent examples of mutations that can be classified as genotype-specific targets for therapy. Furthermore, genetically manipulated mouse models have validated that oncogenic RAS functions are essential for early onset of tumours.13 In addition, continuous RAS activation is necessary for maintenance of tumour viability, and cancerous cells harbouring RAS

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Protein interactions and cancer therapy

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mutation can revert to normal phenotype by removing the mutation or suppressing the expression of RAS-dependent signal transduction. For instance, melanocytespecific inducible mutant HRAS mice develop melanoma upon application of inducer14 and tumours regress, via apoptosis, when inducer is withdrawn. In addition, it has been shown that directly interfering with RAS-effector interactions, using an antibody binding the RAS switch region, prevents tumourigenesis in mouse models.5 Tiam115 and Ralgds KO mice,16 respectively have resistance to DMBA carcinogen-induced skin tumourigenesis, which generally causes specific HRAS mutation. It has also been demonstrated recently that PI3KCA mutant mice (which specifically abolish RAS-PI3Kα interaction by mutation of RAS-binding domain (RBD), also have impaired lung tumourigenesis.17 Single or multiple RAS-dependent signal cascades may contribute oncogenesis and Figure 1. Diagrammatic representation of RAS-effector signalling pathways. Schematic diagram showmaintenance of tumours. ing a variety of RAS effector interactions and their downstream pathways. Plasma membrane-associated Despite many years of research on GTP-bound activated RAS can directly interact with multiple, different effectors (rounded rectangles) to RAS mutations and their effects in cancer, activate various signal cascades. These occur in different contexts, which in turn regulate different celattempts to develop drugs directed to lular functions, ranging from cell proliferation, migration, survival/death, differentiation, endocytosis and adhesion. RAS have proven ineffective (see below). It remains unclear which of the RAS signal cascades are most important for general tumourigenesis and begin the process to overt cancer, the cancer initiating cells (CIC). maintenance and therefore it still appears that the RAS protein itself For instance, in skin carcinoma, HRAS mutation is an initiating may be the best target for treatment of cancer with RAS mutation. event19 and in colon KRAS mutation predominates initially.20,21 Alternatively, the contribution of each RAS cascade may depend on Thus theoretically, anti-RAS therapies can target CIC, overt neoplathe cell type and cancer type and concurrent blockade of multiple sias and invasive stage cancers (Fig. 2). An additional wrinkle to RAS-cascades may be needed. These facts highlight the importance genotype-specific therapy options, is the possible existence of the of mutant RAS and RAS-effector interactions as targets for cancer cancer stem cell (CSC), which may not be susceptible to therapies, therapy. Another important issue is the putative cancer stem cell, either conventional or genotype-specific. Note that herein the CIC which is thought to be a largely quiescent cell with the stem-cell-like and the CSC are regarded as conceptually distinct cells, but mutaproperties of self renewal and the ability to produce cells which can tions in the CIC may confer stem cell-like properties, thus the CIC eventually comprise the overt tumour. The putative cancer stem cell becomes the CSC and they are therefore synonymous (Fig. 2). seems likely to evade conventional therapy since it is mostly quiesWhether the occurrence of RAS mutations invokes stem cell-like cent. Recently, it has been shown that the microRNA let-7 regulates properties on the primary transformed cell or if secondary mutations cancer stem cell stem-cell properties in breast cancer by silencing contribute, therapies to target the CIC/CSC will be difficult but HRAS indicating that HRAS plays a role in conferring stem-cell like valuable. The putative CSC is thought to be a quiescent cell in G0 characteristics on mutated cells.18 Therefore, genotype-specific thera- of the cell cycle and in this state, the constitutive signal transduction pies targeting mutant RAS offer alternative and possible universally of mutant RAS protein may not be important. However, at times more effective approaches to cancer therapy and cancer control. when the putative CSC undergoes proliferation to generate progenitors, the mutant RAS signalling would be active and thus become a Anti-RAS Therapeutic Opportunities target, remaining so through the stages to overt, metastatic cancers Unlike conventional chemotherapy and radiotherapy that mainly depicted in Figure 2. Further, oncogene addiction22,23 may prove target dividing cells in a non-specific manner, an attack on mutant a chink in the armour of RAS, since in this setting the cancer cell RAS proteins should offer robust genotype-specific therapies. RAS becomes reliant on the mutant oncogenic protein at the expense of is a GTP-binding protein located at the plasma membrane by post- normal cellular control processes. Thus targeting mutant RAS could translationally added lipid tails. The GTP-binding pocket is also produce an effective cytotoxic response, providing an opportunity a possible target for small molecule drugs, as is the farnesylation to capitalise therapeutically on mutations in cancer. In this scenario, process but neither has proved to be effective so far (see below). In genotype-based cancer therapy may require recurring administration addition, RAS mutations may well be found in the very first cells that of therapeutics and so effectively cancer may become a chronic disease 1570

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H-RAS

Thyroid

6%

3%

3%

Breast

1%

5%

1%

Skin

4%

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7%

Melanoma

21%

2%

3%

HCC

11%

7%

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Biliary tract

2%

35%

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Lung

Adenocarcinoma

1%

22%

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Pancreas

Adenocarcinoma

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71%

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Lymphoid

10%

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AML

16%

5%

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MDS

8%

4%

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Multiple myeloma

26%

16%

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Seminoma

8%

7%

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Adenocarcinoma

1%

37%

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Glioblastoma

2%

1%

0%

Bladder

3%

4%

11%

SCC

Liver

Haematopoietic lymphoid

Testis Large intestine CNS Urinary tract

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4%

17%

Ovary

4%

16%

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Endometrium

0%

15%

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Prostate

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8%

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Stomach

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Cervix

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concept is being challenged4,27 and new types of inhibitors are proving effective in pre-clinical mouse models. Principle among these are antibodies which nature has provided as molecules ‘designed’ to have high affinity and specificity for protein surfaces. Antibody engineering has allowed the expression of antibody fragments intracellularly28 and we and other researchers have shown that single variable domains of antibodies are the more efficacious intracellular antibody fragments.29,30 There are also devised protocols for allow direct selection31-34 (and TT & THR, in preparation). Furthermore, we have shown by X-ray crystallography, that single domains do not require intra-domain disulphide bridges to function in cells, providing the basis for single domain libraries with stable domain scaffold for selection against any antigen.35 We have recently reported an anti-RAS single VH domain that specifically binds to active GTP-bound RAS with high affinity (an intra-fering antibody36) and neutralises oncogenic function in human cancerous cells with RAS mutation and also those harbouring additional gene alternations such as p53 mutation.5 Furthermore, the X-ray crystal structure of the RAS-single domain complex disclosed that antibody fragments can specifically recognise the conformational structure of activated GTP-bound RAS and inhibit RAS-effector interactions, consequently blocking aberrant RAS signal transduction.5 Interestingly, a recent mathematical model of RAS signalling shows that a reagent specifically binding and sequestering GTPbound RAS would impair mutated RAS-effector interactions more than wild type RAS-effector interactions.37 This suggests that the single domain would a more ideal inhibitor in cancer cells with RAS mutation than against normal cells. The single VH domain specifically binds to the switch I of the activated form of RAS (Fig. 3) and the structural study shows the precision with which the CDR regions of the VH forms hydrogen bonds with residues in the RAS protein. This defines the binding site and demonstrating why the single domain blocks RAS function since it covers the surface of RAS where most signaling effectors interact [RAF, RALGDS and PI3K (Fig. 3)]. Our recent data show that the anti-RAS VH can be expressed in developing mouse lung without detectable changes to the lung structure and function but with almost completely suppression of RAS-dependent lung tumour occurrence (TT & THR, unpublished). Thus, this single VH is a RAS-specific antagonist that is both more specific and effective in interfering with RAS-dependent cancer than are the available antiRAS blockers currently in clinical trials.

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Table 1 RAS somatic mutations in human cancers*

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*Source from COSMIC (Catalogue of Somatic Mutation in Cancer) database (Sanger centre, UK, http://www. sanger.ac.uk/genetics/CGP/cosmic/).

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requiring prolonged treatment application. While the presentation and relapse characteristics of many human tumours does not fit a simplistic cyclic model of tumour occurrence, a complicating factor is drug resistance which results in treatment failure. This may of course be an issue with repeat use of any therapeutic regimen.

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“Intra-fering” Antibody Fragments as Anti-RAS Therapeutic Exemplars

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Mutant RAS proteins work by signalling to the nucleus independently of extra-cellular stimuli. This process occurs through plasma-membrane located RAS and is mediated by protein-protein interactions (Fig. 1). Over the last decade, a variety of approaches for anti-RAS therapy have been attempted, including farnesyltransferase inhibitors (FTIs),24 antisense oligonucleotides and RNA interference (RNAi).25 FTIs can inhibit membrane localisation of RAS family proteins by preventing from their post-translational modification and block downstream RAS signalling. However, recently it has become clear that FTIs only partly target RAS and mainly HRAS rather than KRAS which is most often mutated in human cancer (Table 1). In addition, the FTIs lack specificity for RAS. One new approach is the possible option of blocking the interaction of RAS with its effectors (e.g., RAF, PI3K) to cause a cessation of signal transduction. Unfortunately, this has been considered “undruggable”26 for several considerations, mainly because proteinprotein interaction surfaces are often large, molecularly flat and specified by widely spaced residues in each interaction surface. This www.landesbioscience.com

Interference of Protein-Protein Interactions in Cancer: The Future Protein-protein interactions play pivotal and executive roles in various cellular functions, including in cancerous cells. While they are crucially important as drug targets, the interfaces of protein interactions are very difficult to target with small-molecule compounds because of thermodynamic obstacles encountered by small-molecules in binding a relatively large and featureless interface. Single domain antibody fragments can bind to protein surfaces with high affinity and therefore suitably characterised single domains have the potential to interfere with specific protein-protein interaction. The anti-RAS single domain described 5 has implications for future biotherapy of cancers with RAS mutations, but methods need to be developed for introducing the anti-RAS single domain into cancer cells to convert

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this powerful macrodrug into a clinically useable entity. New technologies are appearing that can either allow delivery of protein or vectors able to express proteins in cells. Cell-penetrating peptides38,39 could be linked to the macrodrug polypeptides (such as antibody fragments) for in-cell delivery. Alternatively proteins or vectors specifically expressing the macrodrug could be delivered using nanocarriers, which in turn could be targeted using receptor ligands, antibodies or antibody fragments to bind vesicles to tumour-associated surface proteins. A range of nanocarrier options are available40,41 and the medium future prospect is for tumourtype specific nanocarrier development to deliver inhibitory molecules. An alternative future approach will be development of antibody fragment mimetics, in which the binding site of the antibody is imitated by a peptide or Figure 2. The RAS mutations, cancer stem cells and therapeutic stage options. A model for progression of a peptide mimic that can bind with the cancer and the therapeutic potential at various stages is depicted. Normal differentiation (shown on the left same specificity as the original antibody hand side) occurs from a tissue stem cell giving rise to progenitor cells and finally to differentiated cells of fragment and with sufficient affinity the tissue. Any of these cells can be subject mutation to derive the cancer initiating cell (CIC), which is the cell with primary mutation, occurring as the “initiating” step. The initial mutation may confer stem cell-like to knock-down the relevant protein- properties on such cells. The CIC may actually be synonymous with the putative cancer stem cell (CSC) with protein binding. The prospect for such self-renewing properties and can produce cancer progenitor cells that undergo further mutation to form overt new inhibitors of protein interactions cancer and eventually metastatic, invasive disease. Aberrant RAS-dependent signal transduction, occurring is very promising especially, when the as a consequence of RAS mutation, may be involved in almost all steps from the CIC/CSC to metastatic, X-ray crystal or NMR structure of overt tumour cells cells. Thus anti-RAS therapeutics can be applicable to all these stages of cancer. While the CIC/CSC may be quiescent, and presumably signal transduction is minimal, it would constitute a target as the antibody fragment bound to its soon as it starts to cycle. It should be noted that recent work also calls into question the linear, mutation-based target can be obtained. This provides aetiology of disseminated disease.43 detailed molecular information about the target-antibody fragment interaction surface on which to build, using in silico methods, chemicals References that both look like the binding site and have similar contacts with 1. Druker BJ, Talpaz M, Resta DJ, Peng B, Buchdunger E, Ford JM, Lydon NB, Kantarjian H, Capdeville R, Ohno-Jones S, Sawyers CL. Efficacy and safety of a specific inhibitor of the the target molecule. Application of medicinal chemistry to lead BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med 2001; 344:1031-7. compounds will provide a spectrum of small molecules that can 2. Sawyers CL. Imatinib GIST keeps finding new indications: successful treatment of dermatofibrosarcoma protuberans by targeted inhibition of the platelet-derived growth factor initially be tested in mouse preclinical modes. Finally, idiopathic receptor. J Clin Oncol 2002; 20:3568-9. changes in individual cancers42 could be a target for macrodrugs or 3. Vassilev LT. Small-molecule antagonists of p53-MDM2 binding: research tools and potenmacrodrug mimetics and this would bring the concept of persontial therapeutics. Cell Cycle 2004; 3:419-21. alised medicine into reality. Achievement of these objectives will 4. Wells JA, McClendon CL. Reaching for high-hanging fruit in drug discovery at proteinprotein interfaces. Nature 2007; 450:1001-9. require focus on technology development in the next decade, as faster 5. Tanaka T, Williams RL, Rabbitts TH. Tumour prevention by a single antibody domain methods for isolation of macrodrug inhibitors of protein-protein inhibiting binding of signal transduction molecules to activated RAS. EMBO J 2007; 26:3250-9. interactions are needed and streamlining of procedures to convert 6. Nam CH, Lobato MN, Appert A, Drynan LF, Tanaka T, Rabbitts TH. An antibody inhibithese into small molecule inhibitors. tor of the LMO2 protein complex blocks its normal and tumourigenic functions. Oncogene Acknowledgements

This work was supported by the Medical Research Council and by the National Foundation For Cancer Research.

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Figure 3. Structural comparison between RAS-PI3Kγ and RAS-anti-RAS VH complexes. The binding site of the anti-RAS single VH domain in the RAS switch region coincides with the binding sites of the RAS effectors molecules such as PI3K. The structure of the HRAS (G12V) protein complexed with the anti-RAS VH (left, PDB, 2UZI)5 and RAS (G12V) or with RAS-binding domain (RBD) of PI3Kγ (right, PDB, 1HE8)44 is shown in ribbon form. HRAS (G12V), VH and PI3Kγ-RBD are shown in green, cyan and orange respectively. RAS switch I and II regions are shown in red and blue, GTP and a Mg2+ ion in RAS are shown in yellow. Insets: magnification of the interaction region between RAS and anti-RAS VH (top inset) and between RAS and PI3Kγ (bottom inset). Amino acid residues involved in the interface are shown in stick configuration. Specific residues of RAS switch I and II are shown in red and blue respectively.

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