WO2020165570A1 - Methods relating to disrupting the binding of af9 partner proteins to af9 and/or enl - Google Patents

Abstract

The present invention relates to inhibitorsof AF9 and/or ENL, or DOT1L. The invention relates to the use of AF9 and/or ENL inhibitors for use in a method of treating an individual having cancer. In particular, the invention relates to a method of treating an individual having cancer, wherein the cancer has a mutation in KRAS; and wherein the cancer is a non-haematological cancer.

Description

Methods relating to disrupting the binding of AF9 partner proteins to AF9 and/or

ENL

BACKGROUND TO THE INVENTION

Approximately a third of all human cancers are associated with mutant Ras proteins that increase Ras activity. Activating KRAS mutations are associated with pancreatic, colorectal and lung cancers. Owing to aspects of its biology, KRAS has generally been considered to be "undruggable" for many years. The discovery of so-called 'synthetic lethal' cellular targets whose inhibition can selectively kill cancer cells carrying oncogenic KRAS mutations is, therefore, of significant interest in cancer therapy.

KRAS (K-Ras, K-ras, Ki-ras) is a member of the highly homologous p21 Ras family of monomeric GTPases. Three Ras isoforms (HRAS, KRAS and NRAS) are expressed in all mammalian cells and function as molecular switches downstream of cell surface receptors, such as Epidermal Growth Factor Receptor (EGFR), to stimulate cell proliferation and cell survival (Quinlan and Settleman, Future Oncol. 2009, 5, 105-16). Mutations of Ras at the conserved codons 12, 13 or 61 (corresponding to amino acid residues G12, G13 or Q61) result in an impaired ability to hydrolyse GTP, either intrinsically, or in response to GTPase activating proteins (GAPs) (Prior et al., Cancer Res. 2012, 72, 2457-67). Oncogenic mutations of Ras at codons 12, 13 or 61, each resulting in constitutive activation of the protein, are found in ~16% of all human cancer cases. Amongst the three major Ras proteins, KRAS (K-ras) is the most frequently mutated isoform in leading causes of malignant-related death in a wide range of cancer types including colorectal (colon) and pancreatic cancer.

Despite a high degree of similarity, Ras isoforms display distinct codon-specific mutational profiles (Prior et al., 2012). KRAS is typically mutated at codon 12 or codon 13 and whilst mutations at both sites are activating, due to impaired GAP binding, the position of the mutation has functional and clinical relevance.

Metastatic colorectal cancer (mCRC) is one of the leading causes of cancer related death world-wide. Overall survival is relatively poor; first-line therapy for advanced colon cancer involves treatment with anti-EGFR monoclonal antibodies such as Cetuximab in combination with standard chemotherapy. Failure to respond to Cetuximab is common and a key determinant of this resistance is the presence of activating mutations in KRAS, which are present in approximately one third of CRC tumours. Consequently, mCRC tumours are routinely genotyped for KRAS status, to predict Cetuximab responsiveness and this therapy is restricted to patients with homozygous wild type KRAS allele.

KRAS mutations occur in over 90% of human pancreatic cancers, almost always at codon 12. The KRAS mutations occur relatively early in cancer development: while they are rare in early pancreatic intraepithelial neoplasm (PIN) lesions, they are present in the majority of advanced lesions and are near universal in frank pancreatic cancers. 5 year survival rates for patients with pancreatic cancer diagnoses are very poor: the disease is one of the most lethal of all neoplasms.

Directly targeting mutant KRAS has been largely unsuccessful as a cancer therapy. Significant efforts have been made to develop therapies targeting downstream elements of the RAS signalling pathways, including RAF, MEK, ERK and PI3K inhibitors, but none of these agents have yet been approved to treat KRAS mutant colon cancer, in particular. The difficulties with targeting mutant KRAS directly or via downstream effectors mean that there remains a need to identify new therapeutic approaches to the treatment of cancers that harbour KRAS mutations, in particular colon cancers that harbour KRAS mutations. In addition, several other types of cancer are characterised by relatively high frequencies of KRAS mutation, including pancreatic and non-small- cell lung cancer.

SUMMARY OF THE INVENTION

The present invention is based on identifying KRAS mutated cancers as being suitable for treatment with AF9 and/or ENL inhibitors or DOTH inhibitors. The present inventors have shown that in KRAS mutated cancer cell lines the AF9 and/or ENL gene(s) are important to cell viability.

Inhibiting the activity of the AF9/ENL proteins, for example by using inhibitors that block the binding of one or more AF9/ENL partner proteins (such as DOT1L and AF4) to AF9/ENL, has enhanced lethality in cells harbouring the KRAS mutation. The invention therefore provides a new therapeutic approach for the treatment of KRAS mutant cancers, inhibitors of AF9 and/or ENL or DOT1L that may be useful in the treatment of KRAS mutant cancer, new screening methods to identify agents that are useful in the treatment of KRAS mutated cancer and new methods of selecting and/or treating patients with cancers that harbour KRAS mutations.

Thus in a first aspect the invention provides inhibitors of AF9 and/or ENL, or DOT1L, for use in a method of treating an individual having cancer, wherein the cancer has a mutation in KRAS. In some embodiments the cancer may be a non-haematological cancer, e.g. a solid tumour. Cancers which may be treated include those that have a high incidence of Kras mutations including colon, lung, pancreas, peritoneum, biliary tract, small intestine, ovarian and endometrial cancers.

Suitable inhibitors of AF9 and/or ENL, or DOT1L, for use in the method of treatment include nucleic acids, antibodies, small molecules, peptides, or inducers of AF9 and/or ENL or DOT1L degradation. In some embodiments the inhibitor is a peptide that binds to AF9 and/or ENL, preferably the C terminus of AF9 and/or ENL, for example, a peptide comprising or consisting of the Ras002 amino acid sequence (SEQ ID NO: 1), or a functional fragment thereof; or a peptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the Ras-002 peptide; or a peptide comprising or consisting of the DOT1L Sites 2 and 3 peptide amino acid sequence (SEQ ID NO: 2), or a functional fragment thereof such as DOTlL-18-site 3, DOTlL-10-site 2, DOTlL-7-site 2, DOTlL-7-site 3; or a peptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the DOT1L Sites 2 and 3 peptide; or a peptide comprising or consisting of the DOT1L Sites 1 peptide amino acid sequence (SEQ ID NO: 3), or a functional fragment thereof; or a peptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the DOT1L Site 1 peptide. In some embodiments the inhibitors, including the peptide inhibitors, bind to the C-terminus of AF9 and/or ENL. Binding to this region of the AF9 and/or ENL proteins may be advantageous because binding to this region is believed to block binding of endogenous DOT1L and/or other AF9 and/or ENL partner proteins. Examples AF9 partner protein include DOT1L and AF4, whose binding to AF9/ENL could be blocked. It has been reported by Shen et al., (J Biol Chem 2013 288(42):30585-30596) that the interaction of AF9/ENL is mutually exclusive with AF4 and DOTH. It was demonstrated that both AF4 and DOTIL compete for the same binding site on the C-terminal of AF9. Blocking the binding of one the AF9 partner proteins to AF9 could result in blocking the binding of other AF9 partner proteins.

In some embodiments the inhibitor of AF9 and/or ENL is selected from any of the inhibitors provided in WO2014127191.

In some embodiments the inhibitor is an RNAi molecule or a siRNA molecule or a shRNA molecule or a sgRNA or gRNA designed to recruit an RNA-guided nuclease.

In some embodiments the inhibitor is an agent that inhibits DOTIL by binding to DOTIL and thereby reducing the enzymatic activity of DOTIL, or is an agent that binds to DOTIL and thereby prevents DOTIL from binding to AF9 and/or ENL. In some embodiments the DOTIL inhibitor is selected from any of the inhibitors in W02014100662A1.

As will be apparent to the skilled person, any of inhibitors and methods of treatment provided herein may be combined with additional anti-cancer therapies.

In some embodiments the method of treatment is utilised to treat cancers that have been identified as comprising a mutation in KRAS. In some embodiments the method may further include the step of determining whether the cancer has a mutation in KRAS.

In another aspect provided herein is a method of screening for agents useful in the treatment of cancer, the method comprising: (a) contacting an AF9 and/or ENL protein with at least one candidate agent; and (b) selecting a candidate agent that inhibits the activity of the AF9 and/or ENL protein as being useful for the treatment of a cancer having a mutation in KRAS. The method may be performed by adding the candidate agent to a cell line that comprises one or more mutations in KRAS. The method may be performed by contacting AF9 and/or ENL protein with at least one candidate agent and the inhibitor of AF9 and/or ENL, or DOTIL such as Ras002, DOTILsites 1 and 3 or DOTIL sites 1 peptides; and selecting the candidate agent that disrupts the interaction between the inhibitors and AF9 and/or ENL. This method would select for a candidate agent that competes with Ras002 or DOTIL derived peptides to bind to AF9 and/or ENL. By identifying a candidate agent that binds to AF9 and/or ENL, the binding of endogenous DOTIL or AF4 or other proteins that bind at the same site, as detailed in Table 1, to AF9 would be disrupted and thus be useful for treatment of cancer.

In another aspect provided herein is a peptide comprising or consisting of the Ras002 amino acid sequence (SEQ ID NO: 1), or a functional fragment thereof, or a peptide having 70%, 75%, 80%, 85%, 90% ,95%, 96%, 97%, 98% or 99% identity to the Ras-002 peptide (SEQ ID NO:l); or a peptide comprising or consisting of the DOTIL Sites 2 and 3 peptide amino acid sequence (SEQ ID NO: 2), or a functional fragment thereof, or a peptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the DOTIL Sites 2 and 3 peptide; or a peptide comprising or consisting of the DOTIL Sites 1 peptide amino acid sequence (SEQ ID NO: 3), or a functional fragment thereof; or a peptide having 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the DOTIL Site 1 peptide

In some embodiments the peptide binds to AF9 and/or ENL, preferably the C-terminus of AF9 and/or ENL. Also provided herein are nucleic acid molecules comprising a nucleic acid sequence encoding any of the peptides provided herein, vectors comprising the nucleic acid sequences and cells containing the nucleic acids/vectors.

Also provided herein are binding moieties that bind to the peptides provided herein, for example antibodies or functional fragment thereof.

The peptides, nucleic acids, vectors or cells may be for use in medicine, particularly for use in the treatment of cancer. The novel peptides provided herein, particularly those that bind to AF9 and/or ENL, may be useful for treating leukaemia, particularly MLL-fusion protein associated leukaemia.

Also provided herein are pharmaceutical compositions comprising any of the inhibitors (for example peptides), nucleic acid molecules, vectors, or binding moieties together with a pharmaceutically acceptable carrier.

FIGURES

Figure 1: Analysis of PROTEINi probes identified in the screen. Each dot represents a PROTEINi sequence and the circle size is 1/CV of replicates. The large circles have less noise. The average number of hits was measured against Score (l/p_value). The hit threshold was p < 0.01. Ras-002 is circled in the figure.

Figure 2: Ras-002 demonstrates a consistent reduction in cell viability with oncogenic KRAS in follow-up studies. Normalised cell viability is shown on the y-axis. Reduced cell numbers indicate cell death. The shaded columns represent cells where oncogenic KRAS is induced with doxycycline.

In the Ras-002 chart the effect window is indicated.

Figure 3: Ras-002 binds to the crucial, C-terminal region of the proteins encoded by the MLLT3 and MLLT1 genes. Note the very high amino acid identity between MLLT3 and MLLT1 proteins (known as AF9 and ENL respectively) in this region.

Figure 4: Ras-002 binds to the crucial, C-terminal region of the proteins encoded by the MLLT3 and MLLT1 genes. Note the very high amino acid identity between MLLT3 and MLLT1 proteins (known as AF9 and ENL respectively) in this region.

Figure 5: The DOT1L peptide sequence engages C-terminal MLLT3 through a clear peptide binding groove. The structure shown in PDB 2mv7.

Figure 6: Ras-002 and DOTIL-peptide both show synthetic lethality in the presence of oncogenic KRAS, and this can be rescued by ectopic AF9 expression.

Figure 7: Fluorescence polarisation assay shows the binding of DOTIL-peptides to AF9 C- terminal. DOTlL-18-site 3 and DOTlL-10-site have a higher affinity for AF9 compared to DOTlL-7-site 2 and DOTlL-7-site 3 derived peptides. DETAILED DESCRIPTION OF THE INVENTION

The data provided herein indicates that the disrupting the activity of AF9 and/or ENL protein(s) may be useful in the treatment of cancers, particularly cancers having a mutation in KRAS.

The Ras-002 peptide provided herein binds to AF9 and ENL and kills cancer cells having one or more KRAS mutations. Thus the Ras-002 peptide, and agents that mimic the activity of Ras-002, may be useful in the treatment of cancers having one or more KRAS mutations. It is hypothesised that RAS002 competes for binding with one or more endogenous partner proteins (such as DOT1L) at the C terminal domain of AF9/ENL. It is known that AF9/ENL have a number of partner proteins (see for example those listed in Table 1 below which are known partner proteins of AF9) and preventing the binding of one or more of these proteins to AF9/ENL may function to inhibit the activity of AF9/ENL and thus be useful in the treatment of KRAS mutant cancers.

Table 1 - AF9 partner proteins

Inhibitors of AF9/ENL activity that may be employed in the methods of treatment, uses or assays provided herein include AF9/ENL binding agents (inhibitors) that bind to AF9 and/or ENL and block the protein-protein interactions between AF9 and/or ENL and one or more endogenous partner proteins e.g. such as DOTIL, thereby inhibiting the activity of the AF9 and/or ENL, for example by inhibiting the formation and/or activity of DOTIL: AF9/ENL complex. Inhibitors of AF9 and/or ENL activity may function by blocking binding of one or more endogenous partner proteins at the C terminal domain of AF9 and/or ENL (amino acids 498 to 568 of AF9).

Examples of AF9/ENL binding agents (inhibitors) include the Ras-002 peptide (SEQ ID NO:l) or analogues thereof, or the DOTIL peptides such as the DOTIL Sites 2 and 3 peptide- see SEQ ID NO:2, and the DOTIL Site 1 peptide SQISEKQRHCLELQISIVELEKSQRQ (SEQ ID NO: 3), which is sometimes referred to as DOTIL 628-653 sequence. Other DOTIL sites 2 and 3 peptides are listed in Table 2 and Table 3.

Other suitable AF9/ENL binding agents include those described in WO2014127191 (Al),

incorporated herein by reference in its entirety, which describes the protein:protein interaction (PPI) between DOT1L and AF9 and/or ENL as a promising therapeutic target for the treatment of MLL- fusion protein associated leukemia. The results in WO2014127191 indicated that disruption of AF9- DOT1L interaction abolishes M LL-AF9 leukemia transformation, without affecting the global level of FI3K79 methylation level.

In some embodiments provided herein, inhibiting the activity of AF9 and/or ENL may be

accomplished through blocking the region of AF9 and/or ENL that binds DOT1L with, for example, using an agent (e.g., a peptide, a small molecule, peptidomimetic, a cyclic peptide) that binds the AF9/ENL interaction site (e.g. amino acids 498 to 568 of AF9) thereby preventing DOT1L binding. In some embodiments, such an inhibitor has binding properties similar to the region of DOT1L corresponding to DOTIL 865LPISIPLSTV874. In some embodiments, such binding of the region of AF9 and/or ENL that binds with DOTIL furthermore results in prevention of aberrant H3K79 methylation in KRAS mutant cancers.

In certain embodiments, the present invention provides methods for screening drugs through identifying agents capable of, for example, binding to the C terminal domain of AF9 and/or ENL, and/or more specifically the interaction site for DOTIL (e.g., an agent having binding properties similar to the region of DOTIL corresponding to DOTIL 865LPISIPLSTV874).

The present invention is not limited to certain types or kinds of agents capable of binding the AF9/ENL interaction site for DOTIL. In some embodiments, the agent is a peptide capable of binding the MLL-fusion protein interaction site for DOTH (e.g., the AF9/ENL interaction site for DOTH). In some embodiments, the peptide comprises 865LPISIPLSTV874 of DOTH. In some embodiments, the agent is a peptidomimetic designed and synthesized to mimic a peptide capable of binding a region of AF9 or ENL known to interact with DOT1L (e.g., 865LPISIPLSTV874 of DOTIL).

In some embodiments, the agent is a cyclic peptide designed and synthesized to mimic a peptide capable of binding a region of AF9 or ENL known to interact with DOTI1L (e.g., 865LPISIPLSTV874 of DOTIL).

In some embodiments, the agent is a small molecule capable of binding to the AF9 and/or ENL interaction site for DOTIL.

In some embodiments provided herein it may be advantageous to utilise DOTIL inhibitors to reduce the amount of endogenous DOTIL available to bind to AF9 and/or ENL and thereby reduce the activity of AF9/ENL:DOTlL inhibitors include agents that bind to the DOTIL enzyme and inhibit the enzymatic activity thereof, or agents that bind to DOTIL and block DOTIL binding to AF9/ENL.

The development of a specific small molecule inhibitor of DOTIL, EPZ004777, which is a competitive inhibitor of the methyl donor S adenosyl-methionine provided proof of principle for the development of DOTIL inhibitors as targeted therapeutics for MLL-rearranged leukemia (see, e.g., Daigle SR, et al., (2011) Cancer Cell 20: 53-65). However, constitutive and conditional knockout studies of DOTIL, which is the only known H3K79 methyltransferase, have shown it is essential for embryonic development, prenatal and postnatal hematopoiesis and cardiac function. This universal and essential function of DOTIL in multiple cell types suggests that directly inhibiting DOTIL histone methyltransferase activity might be toxic. Consequently, development of therapeutic strategies allowing selective inhibition of DOTIL function is important and necessary.

Further compounds that interact with DOTIL and/or the DOTIL binding site on AF9/ENL may be identified by the use of high throughput screening strategies. For detecting compounds that interact with DOTIL and/or the DOTIL binding site on AF9/ENL those skilled in the art will be familiar with biophysical techniques such as surface plasmon resonance, thermal melt assays, fluorescence polarization assays, homogeneous time resolved fluorescence assays or other binding assays such as those provided on the KinomeScan platform (DiscoverX). Knowledge of the structure of DOTIL and/or the DOTIL binding site on AF9/ENL, for example the nucleic acid sequence, the amino acid sequence, the secondary, tertiary or quaternary structure of the proteins, e.g. the crystal structure of AF9/ENL, the location of particular functional domain(s) within the nucleic acid sequence, amino acid sequence or protein structure, the location of domain(s) that interact with a receptor or target of AF9/ENL, can be exploited in order to design putative AF9/ENL or DOTIL inhibitors for use in any aspect of the invention.

As discussed above, small molecule inhibitors could be identified using a range of assay technologies which monitor Protei Protein interactions. Examples are the NanoBit split reporter system, a Fluorescence Polarisation (FP) Assay, or an Enzyme-Linked Immunosorbent Assay (ELISA). Any small molecule capable of disrupting the interaction between Ras-002 or the DOT1L peptides and AF9 in such an assay might reasonably be expected to block the interaction between AF9 and its endogenous ligands in a cell. By extension from the synthetic lethal data provided herein, it is expected that such small molecules should be able to selectively kill cancer cells carrying oncogenic KRAS mutations.

Accordingly, the present invention extends to the use of agents or molecules found to interact with DOT1L (e.g. DOT1L inhibitors) and/or the DOT1L binding site on AF9/ENL (AF9/ENL inhibitors) including those identified using the screening methods disclosed herein and to derivatives thereof.

A person skilled in the art will also recognise that it will be possible to take small molecules that interact with DOT1L and/or the DOT1L binding site on AF9/ENL and modify them to include linker groups (e.g. flexible polyethylene glycol linkers) and a second protein-interacting moiety known to interact with the ubiquitin ligase system that is responsible for a large fraction of protein turnover in the cell. Suitable targets for the second protein-interacting moiety include VEIL, MDM2, cerebelon and c-IAP. Bifunctional compounds comprise BRD4 antagonists such as JQ1 and cerebelon ligands such as pomalidomide will be known by those skilled in the art to target BRD4 for ubiquitin/proteosome mediated destruction.

Peptides

Agents useful for treating cancer, including KRAS mutant solid tumours, includes the peptides provided herein for example peptides that bind to AF9/ENL and block DOT1L binding to AF9/ENL, e.g. Ras-002, including functional fragments thereof. Peptides may be also be generated that block the catalytic sites of the DOT1L enzyme. Peptide fragments can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods, general descriptions of which are broadly available.

The amino acid residues comprising the peptides described herein may be chemically modified. Examples of chemical modifications include those corresponding to post translational modifications for example phosphorylation, acetylation and deamidation. For example, the N or C terminal ends of the peptide may be modified to improve the stability, bioavailability and or affinity of the peptides. The peptides described herein may be modified by amino acid substitution, insertion or deletion. Amino acid substitution means that an amino acid residue is substituted for a replacement amino acid residue at the same position. Inserted amino acid residues may be inserted at any position and may be inserted such that some or all of the inserted amino acid residues are immediately adjacent to one another or may be inserted such that none of the inserted amino acid residues is immediately adjacent another inserted amino acid residue.

Inserted amino acids and replacement amino acids may be naturally occurring amino acids or may be non-naturally occurring amino acids and, for example, may contain a non-natural side chain, and/or be linked together via non-native peptide bonds. In some embodiment the peptides of the invention bind to the C terminus of AF9/ENL. Generally the amino acid modifications described above will not impair the ability of the peptide to bind AF9/ENL. In some embodiments, the amino acid modifications improve the ability of the peptide to bind AF9/ENL. Inhibitors of the invention may be conjugated to additional moieties such as carrier molecules. Carriers are compounds and substances that improve and/or prolong the delivery of an active ingredient to a subject in the context of a pharmaceutical composition. Carrier may serve to prolong the in vivo activity of a drug or slow the release of the drug in a subject, using controlled-release technologies. Carriers may also decrease drug metabolism in a subject and/or reduce the toxicity of the drug. Carrier can also be used to target the delivery of the drug to particular cells or tissues in a subject. Common carriers (both hydrophilic and hydrophobic carriers) include fat emulsions, lipids, PEGylated phospholids, PEGylated liposomes, PEGylated liposomes coated via a PEG spacer with a cyclic RGD peptide c(RGDDYK), liposomes and lipospheres, microspheres (including those made of biodegradable polymers or albumin), polymer matrices, biocompatible polymers, protein-DNA complexes, protein conjugates, erythrocytes, vesicles, nanoparticles, and side-chains for hydro carbon stapling. The aforementioned carriers can also be used to increase cell membrane permeability of the AF9 and/or ENL inhibitors of the invention. In addition to their use in the pharmaceutical compositions of the present invention, carriers may also be used in compositions for other uses, such as research uses in vitro (e.g., for delivery to cultured cells) and/or in vivo.

The use and delivery of therapeutic peptides for cancer treatment, such as those provided herein in described in detail in Cancer Treatment Using Peptides: Current Therapies and Future Prospects Journal of Amino Acids Volume 2012, Article ID 967347, 13 pages.

The peptides may also be employed in accordance with the present invention by expression of the peptides in vivo, i.e., via gene therapy approaches known in the art.

Nucleic acid inhibitors

Another class of inhibitors useful for treatment of KRAS mutated cancer includes nucleic acid inhibitors of AF9/ENL, or the complements thereof, which inhibit activity or function by down regulating production of active polypeptide. This can be monitored using conventional methods well known in the art, for example by screening using real time PCR as described in the examples.

Expression of AF9/ENL may be inhibited using anti-sense or RNAi technology. The use of these approaches to down-regulate gene expression is now well-established in the art. Suitable nucleic acid sequences for use in RNAi to inhibit AF9/ENL may be found in, for example, Xing et al. (Int J Mol Med. 2016 Aug; 38(2): 407-416), Zhao et al. (Mol Med Rep. 2015 Jul; 12(1): 960-966), Zheng et al. (Acta Pharmacol Sin. 2009 Dec; 30(12): 1625-1633) and Zhu et al. (Genet Mol Biol. 2012;35:538- 544). Methods for the design and evaluation of other suitable nucleic acid sequences for use in RNAi are known to those skilled in the art.

DNA or RNA guided nuclease systems as AF9/ENL inhibitors

DNA or RNA guided nuclease systems, such as the CRISPR/Cas9 genome editing system can also be used to inhibit AF9/ENL activity by editing the AF9/ENL gene to knock out AF9/ENL expression or reduce functional AF9/ENL protein. For example, a guide nucleic (e.g., a guide RNA that binds to an AF9/ENL nucleic acid is administered to or expressed in the cell, along with a CRISPR/Cas9 nuclease that results in targeted cleavage of the AF9/ENL gene sequence and introduces a mutation in the gene that inhibits the production of functional AF9/ENL, see, e.g., Jinek et al. Science 337, 816-821 (2012); Jiang et al., Nat. Biotechnol. 31, 233-239 (2013); Hou, Z. et al. Proc. Natl. Acad. Sci. USA 110, 15644-15649 (2013); Mali et al., Science 339, 823- 826 (2013); Jinek, M. et al. RNA-programmed genome editing in human cells.

The CRISPR/Cas system can alternatively be used like RNA interference, turning off the AF9/ENL gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the Cas protein to the promoter, sterically blocking RNA polymerases.

The AF9/ENL inhibitors may also be employed in accordance with the present invention by expression of the inhibitors in vivo, i.e., via gene therapy.

The present invention also encompasses: (i) polynucleotide sequences encoding AF9 and/or ENL inhibitors (for example the Ras002 or DOT1L peptides, or functional fragments or variants thereof) or polynucleotide sequences encoding DOT1L inhibitors of the present invention, (ii) vectors into which the polynucleotide sequences are inserted, (iii) host cells genetically engineered (transduced, transformed, or transfected) with the vectors, (iv) methods of culturing the host cells under conditions promoting production of the AF9 and/or ENL inhibitors encoded by the polynucleotide sequences.

A person skilled in the art would appreciate that the term "AF9/ENL inhibitor" as used in the context of any aspect of the present invention encompasses a molecule or agent that results in a decrease in the activity or function of AF9/ENL. The molecule or agent could act at the nucleic acid level or at the protein level of AF9/ENL. The decrease in the activity or function of AF9/ENL may be as a result of a reduction in the level of AF9/ENL. This reduction in the level of AF9/ENL may be due to e.g. reduced expression of AF9/ENL or degradation of AF9/ENL.

Methods of screening

In some aspects, the present invention is concerned with methods of screening candidate compounds to determine whether one or more candidate agents (putative AF9/ENL inhibitors) are likely to be useful for the treatment of KRAS mutated cancer, including solid tumours having one or more KRAS mutations. As described herein, there are a number of approaches that may be used for these methods of screening, either alone or in any combination or order.

In one approach, a method of screening may involve using cell lines to profile candidate agents to identify and select compounds that are synthetically lethal to cells with KRAS mutations: namely compounds that are cytotoxic to a high fraction of cells or cell lines that are KRAS mutant, but less toxic to cells or cell lines with homozygous wild-type KRAS alleles including normal cells. For example, a higher level of cytotoxicity is observed in the KRAS mutant cells than compared to the wildtype cells. Suitable methods of determining cytotoxicity are described herein. Suitably, the candidate agents show a greater lethal effect in KRAS mutant cells as compared to wildtype cells.

The present invention also includes methods of screening that employ AF9/ENL as a protein target for the screening of candidate compounds to find AF9/ENL inhibitors. Accordingly, methods of screening may be carried out for identifying candidate agents that are capable of inhibiting AF9/ENL for subsequent use of development as agents for the treatment of KRAS mutated cancer. Conveniently, this may be done in an assay buffer to help the components of the assay interact, and in a multiple well format to test a plurality of candidate agents. The activity of AF9/ENL can then be determined in the presence and absence of the one or more candidate compounds to determine whether a given candidate is an inhibitor of AF9/ENL.

By way of example, the candidate agent may be a known or putative AF9/ENL inhibitor, an antibody, a peptide, a nucleic acid molecule or an organic or inorganic compound, e.g. a small molecule with molecular weight of less than 100 Da.

Following identification of a candidate agent for further investigation, the agent in question may be tested to determine whether it is not lethal to normal cells or otherwise is suited to therapeutic use. Following these studies, the agent may be manufactured and/or used in the preparation of a medicament, pharmaceutical composition or dosage form.

Treatment of cancer

The present invention provides methods and medical uses for the treatment of cancer, particularly KRAS mutated cancer. A cancer may be identified as KRAS mutated by testing a sample of cancer cells from an individual, for example to determine whether the KRAS protein contains one or more mutations or to determine the expression of the KRAS gene to evaluate whether expression is increased compared to normal. It is known that KRAS mutations are present at high frequency in colon, lung, pancreas, peritoneum, biliary tract, small intestine, ovarian and endometrial cancers. Cancer types that may be treatable in accordance with the methods of the invention include colon, lung, pancreas, peritoneum, biliary tract, small intestine, ovarian and endometrial cancers. Cancer types that may be treatable in accordance with the methods of the invention include colon, lung, peritoneum, biliary tract, small intestine, ovarian and endometrial cancers. Other cancers with KRAS mutations include gastric, hepatocellular, breast, prostate, testicular, soft tissue and bladder cancers (COSMIC http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=KRAS). Other KRAS mutated cancers that are treatable in accordance with the present invention are likely to be found as further work is done to identify the prevalence of KRAS mutations, and to confirm that the presence of the KRAS mutation(s) is associated with AF9/ENL expression. Thus further cancer types that may be treatable in accordance with the methods of the invention include but are not limited to: adrenal gland, autonomic ganglia, biliary tract, bladder, bone, breast, CNS, cervix, endometrial, eye, fallopian tube, gastrointestinal tract, genital tract, haematopoietic and lymphoid tissue, kidney, large intestine, liver, lung, meninges, nervous system, oesophagus, ovary, pancreas, penis, peritoneum, pleura, prostate, salivary gland, skin, small intestine, soft tissue, stomach, testis, thymus, thyroid, upper aerodigestive tract, urinary tract, or vulva.

As discussed above, databases such as the COSMIC database, include further information on the cancer types that have been identified as containing KRAS activating mutations, including G12 and G13 mutations. Pharmaceutical compositions

According to a further aspect of the invention there is provided a pharmaceutical composition comprising an agent for use in the in the treatment of cancer combined with any pharmaceutically acceptable carrier, adjuvant or vehicle wherein the cancer is a KRAS mutated cancer.

Examples of pharmaceutically acceptable carriers are known to those skilled in the art and include but are not limited to preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms. The compositions may be in the form of, for example, tablets, capsules, powders, granules, elixirs, lozenges, suppositories, syrups and liquid preparations including suspensions and solutions.

The term "pharmaceutical composition" in the context of this invention means a composition comprising an active agent and comprising additionally one or more pharmaceutically acceptable carriers. The composition may further contain ingredients selected from, for example, diluents, adjuvants, excipients, vehicles, preserving agents, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavouring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispersing agents, depending on the nature of the mode of administration and dosage forms.

The pharmaceutical compositions of the invention may be administered orally in any orally acceptable dosage form including, but not limited to tablets, dragees, powders, elixirs, syrups, liquid preparations including suspensions, sprays, inhalants, tablets, lozenges, emulsions, solutions, cachets, granules and capsules. Such dosage forms are prepared according to techniques known in the art of pharmaceutical formulation. When in the form of sprays or inhalants the pharmaceutical compositions may be administered nasally. Suitable formulations for this purpose are known to those skilled in the art.

The pharmaceutical compositions of the invention may be administered by injection and may be in the form of a sterile liquid preparation for injection, including liposome preparations.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Compositions comprising agents disclosed herein for the treatment of cancer, particularly the treatment of KRAS mutated cancer may be used in the methods described herein in combination with standard chemotherapeutic regimes or in conjunction with radiotherapy. Examples of other chemotherapeutic agents include Amsacrine (Amsidine), Bleomycin, Busulfan, Capecitabine (Xeloda), Carboplatin, Carmustine (BCNU), Chlorambucil (Leukeran), Cisplatin, Cladribine (Leustat), Clofarabine (Evoltra), Crisantaspase (Erwinase), Cyclophosphamide, Cytarabine (ARA-C), Dacarbazine (DTIC), Dactinomycin (Actinomycin D), Daunorubicin, Docetaxel (Taxotere), Doxorubicin, Epirubicin, Etoposide (Vepesid, VP-16), Fludarabine (Fludara), Fluorouracil (5-FU), Gemcitabine (Gemzar), Flydroxyurea (Hydroxycarbamide, Hydrea), Idarubicin (Zavedos), Ifosfamide (Mitoxana), Irinotecan (CPT-11, Campto), Leucovorin (folinic acid), Liposomal doxorubicin (Caelyx, Myocet), Liposomal daunorubicin (DaunoXome^®), Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin (Eloxatin), Paclitaxel (Taxol), Pemetrexed (Alimta), Pentostatin (Nipent), Procarbazine, Raltitrexed (Tomudex^®), Streptozocin (Zanosar^®), Tegafur-uracil (Uftoral), Temozolomide (Temodal), Teniposide (Vumon), Thiotepa, Tioguanine (6-TG) (Lanvis), Topotecan (Hycamtin), Treosulfan, Vinblastine (Velbe), Vincristine (Oncovin), Vindesine (Eldisine) and Vinorelbine (Navelbine).

Administration in-vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.

In general, a suitable dose of the active compound is in the range of about 100 pg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound, and so the actual weight to be used is increased proportionately.

The active agents disclosed herein for the treatment of KRAS mutated cancer according to the present invention are preferably for administration to an individual in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams & WAF9/ENLins. A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.

A number of different strategies for the targeting and delivery of therapeutic peptides to cancer cells in-vivo are discussed in Marqus S et al; Journal of Biomedical Science201724:21.

The present invention also encompasses: (i) polynucleotide sequences encoding AF9 and/or ENL inhibitors (for example polynucleotides encoding the Ras002 or DOT1L peptides, or functional fragments or variants thereof) or DOT1L inhibitors of the present invention, (ii) vectors into which the polynucleotide sequences are inserted, (iii) host cells genetically engineered (transduced, transformed, or transfected) with the vectors, (iv) methods of culturing the host cells under conditions promoting production of the AF9 and/or ENL inhibitors encoded by the polynucleotide sequences. As described herein, targeting the DOT1L binding site on AF9/ENL may be useful in the treatment of non-haematological malignancies, e.g. solid tumors. In some embodiments these solid tumours may comprise one or more KRAS mutations.

Cancer-driving KRAS mutations are colloquially termed activating mutations and increase the fraction of protein present in its signalling-promoting GTP-bound form. These mutations typically occur at codons 12 or 13 in human KRAS (but at lower frequency elsewhere in the protein, including codons 59 or 61) and impair the GTP-ase activity of the molecule.

Such activating mutations are well documented and include those described in Siena et al., 2009, J NCI, 101:1308-1324. The K-Ras mutation may comprise one or more activating mutations in codon 12, 13, 59 and/or 61.

The presence of a K-Ras mutation in a cancer can be determined by molecular diagnostic testing methods that include, but are not restricted to, mutation-specific PCR-amplification methods, tandem PCR amplification and DNA-sequencing methods (including Sanger-based, pyrosequencing- based and mass-spectrometry-based methods), whole genome sequencing, or proteomic-based technologies (including Western blotting, immuno-histochemistry and protein mass-spectrometry). Examples of such cancers are diverse, with K-Ras mutations being present in a significant proportion of many cancer types, including but not limited to colon, lung, pancreas, prostate, endometrium, ovarian, liver, thyroid, biliary tract, stomach and ovary.

Thus the present invention may be useful in the treatment of these cancers or any other cancer that is identified as KRAS mutated. The present invention may be useful in the treatment of KRAS mutated cancers in humans, or in other mammalian species.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. Flowever various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

EXAMPLES

Three million PROTEINi^® probes were used to scan for all targets that can selectively kill mutant KRAS cells. These probes are derived from DNA-encoded, protein-fragment expression libraries that can be screened in high throughput in phenotypic assays (such as described in WO 2013/116903); often dubbed 'Protein-interference' (PROTEINi^®).

A cell line with Doxycycline-inducible KRAS^G12V (HeLa Flp-in™ T-Rex™ KRAS^G12V) was constructed using the Flp-ln™ T-REx™ Core Kit (Invitrogen Cat#650001) and cells were expanded and infected with lentivirus encoding for the PROTEINi probes. Cells were infected at a multiplicity of infection of 0.3 in order to obtain cells carrying only one PROTEINi insert on average. Cells were subsequently selected on puromycin to remove uninfected cells and were propagated and expanded for an additional 5 days to remove all PROTEINi's that are toxic to HeLa cells without active Ras. Cells were then split into 6 replicates; 3 were treated with Doxycycline to induce KRAS^G12V expression and 3 were mock treated. Cells were then expanded for an additional 5 days, cell pellets were collected and genomic DNA was extracted. DNA samples were then subsequently used to amplify PROTEINi coding sequences using constant flanking primer sites, analogous to (Shalem et. al, Science 2014) and samples were submitted to NGS sequencing using an lllumina NextSeq platform. Obtained reads were counted and mapped against the library reference. This produced a dataset of read counts for every insert identified in the screen. Read counts were then normalised by total read count for each sample and then compared across KRAS^G12V induced and uninduced (reference set) conditions. Sequences with less than 10 reads in the reference samples were removed from the analysis. We then calculated the average coefficient of variation (CV) and p-value (using a student's t-test) for each PROTEINi insert and ranked the hits based on these metrics. The results of this analysis are shown in Figure 1. A total of 20 hits were selected for further follow-up experiments.

On re-testing (>3 biological repeats) it was found that the PROTEINi probe "Ras-002" (the second- ranked hit from the screen, shown in Figure 1) gave the strongest and most consistent response. Ras-002 demonstrated an increased ability to kill cancer cells carrying an activating KRASG12V mutation compared to WT KRAS FleLa cells. It was associated with approximately 50% depletion of KRAS^G12V cells (against control cells) in 72 hours (Figure 2).

In order to discover the cellular target of Ras-002, and thus the mechanism through which it shows synthetic lethality with oncogenic KRAS, a yeast two hybrid system was employed. This system conceptually relies on the activation of a downstream reporter gene by binding of a transcription factor to an upstream activating sequence. The transcription factor is split into two fragments: one called the DNA binding domain and the other known as the activating domain. When the two fragments of the transcription factor are physically proximal, the transcription factor is functional and therefore can activate transcription of the reporter gene.

A third party contractor, Flybrigenics Services of Paris, France, was used to conduct the yeast two hybrid screening. The most common screening approach is to carry out a survival screen. In this system, the yeast is genetically engineered to lack the biosynthesis of a specific nutrient, on which the yeast depends on entirely for its survival. The yeast is then transformed with the "bait", which in our case is the PROTEINi fused to the binding domain of GAL4 and a human cDNA library "prey" fused to the activating domain of GAL4. Once cells are transformed with these plasmids, yeast cells are transferred to medium lacking the given nutrient and only cells where the two fragments of GAL4 are found in proximity (due to the interaction of the fusions) will survive.

A short-list of 5 high confidence interactions, based on sequences found to be in frame, in the sense orientation and not occurring frequently across multiple Y2H screens, was identified. The prey genes encoded by these clones included MLLT3 and MLLT1. Of specific interest were the 48 clones encoding 10 different regions of MLLT3, and the 2 clones encoding MLLT1. MLLT3 and MLLT1 are closely-related genes, both components of the super elongation complex. MLLT3 encodes for the AF9 protein while MLLT1 encodes for the ENL protein. In both cases, based on the clones emerging from Y2H screening, Ras-002 interacts with the C-terminal region of MLLT3 and MLLT1. In addition to exploring the role of Ras-002 and AF9/ENL in KRAS mutant tumours, as both proteins are known to play a key role as oncogenic drivers in leukemia, this paved the path for a leukemia model. Both proteins are common fusion partners of MLL in acute leukemias. Moreover, the C- terminal region of the proteins to which Ras-002 binds to is the same region which is translocated in pro-leukemic fusion transgenes. This adds significant weight to the hypothesis that Ras-002 binds to the crucial, oncogenic region of a fusion transgene that is commonly present in lymphoid and myeloid leukemias. Therefore, the Ras-002 and AF9/ENL interaction in leukemias is a second model being explored. This is shown in Figure 3.

Interestingly, Ras-002 shows significant amino acid identity to DOT1L, a histone methyltransferase, which has been implicated in the development of MLL-rearranged leukemias, and which is a known binding partner for the C-terminal region of AF9. Mistargeting of DOT1L in the MLL-MLLT3 (also known as MLL-AF9) fusion transgene causes aberrant FI3K79 methylation at homeobox genes, and subsequent transcriptional dysregulation. As such, DOTIL's essential role in leukemic transformation has made it an attractive therapeutic target. Therefore, one possible explanation for Ras-002's mechanism of action is that it competes for M LLT3 binding with endogenous client proteins, such as DOT1L, and that oncogenic KRAS signalling relies upon some aspect of MLLT3::DOTlL-dependent histone methylation for its function. Further this data suggests that peptides derived from the AF9 binding domain of DOT1L should phenocopy the effects of Ras-002. This is shown in a simplified cartoon form in Figure 4.

The NMR structure is available for the MLLT3::DOTlL peptide complex (PDB: 2mv7) (Kuntimaddi et al., Cell Reports 11, 808-820, May 5, 2015). The C-terminal of AF9 is partly unfolded in the absence of peptide ligand. There is a clear peptide groove, and within this grove, two residues (D544, D546) are essential for peptide binding. Additional binding energy is from hydrophobic interactions with the groove floor (Figure 5).

Target validation experiments were carried out to a) confirm the Ras002 phenotype and validate the phenotype of DOT1L derived peptides in the presence of KRAS and b) identify if this expression changes upon overexpression of AF9. These experiments were carried out as stated below:

Using lentiviral transduction the PROTEINi^® was expressed in FleLa Flpln-TRex cells at a MOI of 0.3 to generate a stable cell line. A PROTEINi^® control, which contains the same nucleotide sequence as the PROTEINi^® but randomized so it is non-functional, was expressed in the same manner to generate a scramble control cell line. The FleLa Flpln-TRex cells can be induced to express KRAS mutant upon addition of doxycycline. Cells not induced with doxycycline are referred to as wild type (WT) cells. Upon expression of the PROTEINi^® and PROTEINi^® scramble, each of the two cell populations were plated into 96 well at 5000 cells/well and a 1:2 serial dilution of cell number carried out to 156 cells/well. Each condition was plated in triplicate. Following six hours of plating to allow the cells to adhere, 200 ng/ml doxcycyline was added to half the population of each of the two cell lines to induce KRASG12V mutant expression - the second half did not express KRAS mutant and therefore was used as a control. Following five days of treatment, cell proliferation was measured in the two populations using a Sulforhodamine B (SRB) assay. To perform the SRB assay, cells were fixed in 1% TCA for 30 min at 4 °C. Cells were then washed with water three times. The SRB reagent was then added to the cells for 30 minutes at room temperature. Following 30 minutes, cells were washed with 1% acetic acid three times and left to air-dry overnight. Once dry, the cells were re-suspended in 10 mM Tris ph8.0 media. Absorbance was measured using a ClarioStar plate reader at 510 nm wavelength.

The results of these studies are shown in Figure 6. The data show that

(i) Expression of Ras-002 (amino acid sequence

'CTSRAGRRGRNSTVALTLTSPSGILSPSLFSQSSVQAVKTNISIPVRL' (SEQ ID NO. 1)) and DOT1L Sites 2 and 3 peptide(amino acid sequence 'TSLPISIPLSTVQPNKLPVSIPLASVVL' SEQ ID NO.2) expression, phenocopy each other and reduce cell viability with oncogenic KRAS compared to scrambled controls

(ii) this effect is blunted by over-expression of AF9, which acts as a buffer to absorb ectopically-expressed Ras-002 and DOT1L peptides.

To further demonstrate and elucidate the interaction between AF9 and DOT1L derived peptides a Nanobit split reporter assay system was employed. These experiments were carried out detailed below.

AF9 / DOT1L interaction assay using NanoBit (Promega)

Cloning of pBiT-LgC-AF9 plasmid

AF9 C-terminal region (498 to 568 aa) was PCR amplified from a gene synthesised full length AF9 ORF, and cloned into pBiTl.l-C[TK/LgBiT] (Promega) to generate the pBiT-LgC-AF9 using the Bglll and EcoRI sites. Primers were designed to insert a translation initiation region upstream of the AF9 sequence (AF9_DN_F_gra: 5'- ACGTGGATCCATGGGCGAGTGCGACAAGGCCTACCTGG-3' (SEQ ID NO: 4) and AF9_DN_R_gra: 5'- ACGTCTCGAGTCAGCTGGTGCCGCTTGTTTCC-3' (SEQ ID NO:5)), and ligations were carried out using T4 DNA ligase (NEB) following manufacturer's recommendations.

Cloning of pBiT-SmN-DOTlL

Wild type DOT1L region encompassing amino acids 862 to 890 and indicated mutants (in Table 2) were cloned into pBiT2.1-N[TK/SmBiT] (Promega) using Xhol and Bglll sites. In summary, oligonucleotides corresponding to DOT1L sense and anti-sense region were synthesised (SIGMA), 5' ends phosphorylated using T4 PNK (NEB) according to manufacturer's instructions (1 uM oligonucleotide, 0.5 mM ATP, 10U of PNK enzyme in a total volume of 50 uL with T4 PNK buffer) for 60 minutes at 37°C, followed by inactivation of enzyme at 65°C for 20 minutes. Double strand DNA were formed by mixing 10 uL of each corresponding DNA oligonucleotide phosphorylation reactions followed by boiling at 95°C for 3 minutes and allowed to slowly cool to ambient temperature. Ligations were carried out using T4 DNA ligase (NEB) following manufacturer's recommendations.

NanoBit experiment

FIEK-293FT cells (Invitrogen) were seeded onto six well plates (Corning) at a total of 5E+05 per well in 2 mL total of DMEM supplemented with 10% FBS (Flyclone). Cells were transfected 24 hours after plating using jetPRIME reagent (Polyplus-transfection). In brief, 1 ug of each plasmid DNA expressing indicated proteins was mixed with 200 uL of jetPRIM E buffer, followed by 8uL of jetPRIM E reagent. The mixture was mixed by 10 second vortexing followed by brief centrifugation to bring contents down. Transfection mixture was added to corresponding well after a 10 minute incubation at ambient temperature. Cells were returned to incubator for further 24 hours. Analysis of the NanoBit activity was carried out using the NanoGlo reagent (Promega). Media was removed from each well and replaced by 250 uL of fresh media equilibrated at ambient temperature. To each well 250 uL of NanoGlo reagent of ambient temperature equilibrated was added, prepared according to manufacturer's instructions. Plates were mixed vigorously in a plate shaker for 10 minutes, then 100 uL of each well was transferred into a white opaque 96 well assay plate in triplicate (Corning). Luminescence was read on a ClarioStar plate reader (BMG LABTECFI), and data analysed using Prism 7 (GraphPad).

The results for a series of threonine amino acid 873 mutants are shown in Table 2. The data shows that replacement of threonine-873 with aliphatic amino acids significantly increases the binding of the mutated DOT1L peptides to AF-9. Replacement of threonine-873 by isoleucine, leucine and tryptophan in particular increase the binding of the DOT1L derived peptide with AF-9.

Table 2: Relative luminescence for T873 DOT1L mutants

Previous work by Shen et al., (J Biol Chem 2013 288(42):30585-30596) demonstrated that a mutation of threonine to alanine at position 837 decreased the binding affinity compared to the wild-type DOT1L peptide. The data reported in Table 2 shows that replacement of threonine to isoleucine, leucine or tryptophan increased the binding affinity of the DOT1L mutants compared to the wild-type DOT1L peptide.

Cloning of AF9 C-terminal (490-568) construct into expression plasmid

A synthetic plasmid containing a DNA sequence encoding polyhistidine tag, GB1 solubility domain (B1 domain of Streptococcal protein G), TEV (Tobacco Etch virus) protease cleavage site and the C- terminal region of AF9 (residues 470 to 568) was synthesised by Thermo GeneArt services. This construct was further subcloned into Ncol and Xhol sites in pEt28a protein expression plasmid (Novagen). This was carried out by digesting both plasmids with Ncol and Xhol enzymes (NEB) for 2h at 37°C, followed by ligation of gel purified insert of synthetic plasmid to the backbone of pEt28a, using the T4 DNA ligase (NEB) and following manufacturer's protocol. E. coli Stellar^® (Takara) cells were transformed with the ligation mixture and screened for correct final plasmid by DNA sequencing. The final plasmid will be called from here forward pMPl-AF9. Expression of the AF9 C-terminal (490-568) protein

Chemocompetent E. coli BL21 (DE3) (NEB) bacteria were transformed with the pMPl-AF9 plasmid and grown overnight on LB agar containing 30 ug/mL of kanamycin antibiotic for selection. A single colony was then selected and used to inoculate 50 mL LB media supplemented with 30 ug/ mL kanamycin. It was then incubated overnight at 37°C with shaking (200 rpm). Overnight bacterial culture was then added (1:100, v/v) to fresh LB media supplemented with 30 ug/ mL kanamycin. Bacterial culture was incubated at 37°C sharking at 200 rpm until it reached optical density (600 nm) between 0.6-0.8. Protein expression was induced by addition of Isopropyl b-D-l- thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Protein expression was carried out by incubation for 16 -20h at 16°C with shaking (180 rpm). Cells were harvested by centrifugation at 4,000 x g for 15 min at 4°C, and proceeded with purification.

Purification of the AF9 C-terminal (490-568) protein

Cell pellet was resuspended (3:1, v/w) in lysis buffer (20 mM HEPES pH 7.5, 500 mM NaCI, 5% glycerol, 20 mM imidazole, 5 mM beta-mercaptoethanol, ImM PMSF, lx protease inhibitor cocktail EDTA-free (Roche)). Lysis of cells was carried out using a refrigerated FPG12800 homogeniser (Homogenising Systems Ltd) at 15,000 Psi. Cell lysate was then supplemented with Triton X-100 up to the final concentration of 0.5% and incubated at 4°C for lh. Extract was cleared by centrifugation at 25000 x g at 4C for 30 minutes, and soluble fraction pooled for further purification.

The protein was initially purified using Immobilised Metal Affinity Chromatography (IMAC) in an AKTA Start (GE Healthcare). Cleared cell lysate was loaded onto a 5 mL HisTrap HP column (GE Healthcare) previously equilibrated with 10 column volumes of the buffer consisting of 20 mM HEPES pH 7.5, 500 mM NaCI, 5% glycerol, 5 mM beta-mercaptoethanol and 20 mM imidazole. The column was then washed with 150 mL of 20 mM HEPES pH 7.5, 500 mM NaCI, 5% glycerol, 5 mM beta-mercaptoethanol and 100 mM imidazole. AF9 was eluted from the column by increasing the imidazole concentration to 300 mM. Fractions of the eluent were analysed by SDS-PAGE, and fractions containing AF9 were pooled and concentrated using a 5 kDa MWCO centrifugal concentrator (GE Healthcare) and quantified.

The protein was further purified using Size Exclusion Chromatography. HiLoad 16/600 Superdex 200 pg column (GE Healthcare) was equilibrated with 1 column volume of 20 mM HEPES pH 7.5, 250 mM NaCI and 1 mM TCEP. Samples containing 10 mg of AF9 were loaded onto the column and resolved using 1.5 column volume of the same buffer, with corresponding fractions collected and analysed by SDS-PAGE. Fractions containing AF9 were pooled, concentrated using a 5 kDa MWCO centrifugal concentrator (GE Healthcare), quantified and stored at -80°C.

Fluorescence polarisation (FP) assay

All peptides used for the development of the FP assay were designed by the inventors and purchased from Designer Biosciences (Cambridge, UK). Seven to eighteen amino-acid long DOT1L derived tracer peptides were singly labelled with tetramethylrhodamine (TAMRA) either through their amino end or side chain of their N-terminal lysine residue. An unrelated fifteen amino-acid long control peptide was singly labelled with tetramethylrhodamine (TAMRA) via its amino terminal. Peptides were analysed by HPLC and MS for quality assessment prior to fluorescence polarisation experiments. AF9 C-terminal protein (490-568) was purified as described in previous paragraph.

In the saturation binding experiments, 2 nM TAMRA-labelled tracer peptides were mixed in triplicate with increasing concentrations of AF9 protein (0.5 nM to 9.2 uM) and incubated for 15 min at room temperature. Final reaction buffer was composed of 20 mM H EPES pH 7.5, 150 mM NaCI, 0.5 mM imidazole, 5% glycerol and 2 mM DTT. Total sample volume was 20 uL. All FP measurements were performed on a Clariostar plate reader (BMG Labtech) in low volume 384-well black microplates. FP values (in milli-polarization units) were calculated according to the following equation, using MARS software (BMG Labtech):

The saturation binding of AF9 and DOTH derived peptides is shown in Figure 7.

Table 3: Binding affinity of DOT1L derived peptides

Fluorescence polarisation experiment demonstrated specific interaction between the C-terminal (490-568) domain of AF9 and peptides that mimic the AF9-binding motif of DOT1L. AF9 binds DOT1L- derived peptides with affinity positively correlated to their size. DOTlL-18-site 3 and DOTlL-10-site 2 have a higher binding affinity for AF9 compared to DOTlL-7-site 2 and DOTlL-7-site 3.

Homogenous time-resolved FRET (HTRF) assay

A set of probe peptides mimicking AF9-binding motifs of DOT1L and AF4 was designed to build a HTRF assay with a suitable assay window. All peptides used for the development of the HTRF assay were purchased from GenScript (Leiden, Netherlands). Probe peptides were N-terminally labelled with biotin. Peptides were analysed by HPLC and MS for quality check prior to HTRF experiments.

AF9 C-terminal protein (490-568) was purified as described in previous paragraph.

HTRF donor and acceptor were Streptavidin-Tb cryptate (CisBio, #610SATLF) and MAb Anti 6HIS-d2 (CisBio, #61HISDLF), respectively. Working solutions of HTRF labelling reagents were prepared in a dedicated Terbium detection buffer (CisBio).

Optimal assay conditions were identified by matrix cross-titration of peptides (0 to 500 nM) and AF9 protein (0 to 250 nM), in a buffer consisting of 20 mM HEPES pH 7.5, 150 mM NaCI, 1 mM DTT and 5% glycerol. Components of reaction were mixed in low volume 384-well microplates and incubated for 15 min at room temperature. HTRF labelling reagents were added at lx and 2x concentration, as recommended by the manufacturer. Samples were incubated for lh at room temperature prior to data collection, in a total sample volume of 20 uL. All HTRF measurements were performed on a Clariostar plate reader (BMG Labtech). HTRF ratio values were calculated according to the following equation using MARS software (BMG Labtech):

HTRF ratio= R_665nJ Fl_620nm * 10000

Maximal signal-to-background ratios were calculated using samples without AF9 as background.

All tested peptides showed binding to AF9 in the HTRF experiment, but signal magnitude varied between them. The experiment allowed to identify optimal probes for the HTRF assay.

Table 4. HTRF signal of different peptides

AF4

27- 53 L S P L R D T P P P Q S L M V K I T L D L L S R I P Q mer

AF4

19- 11 P P Q S L M V K I T L D L L S R I P Q mer

Hybrid

A

DOT1L 8 L S P L R D T V Q P N K L P V S I P L

19- mer

Hybrid

B

DOT1L 10 L S P L R D T P P P Q S L P V S I P L

19- mer

Synthe

tic

DOT1L 2.5 G S G G G G S G G G G S L P V S I P L

19- mer Table 5. Peptides used in the HTRF assay

Claims

Claims

1. An inhibitor of AF9 and/or ENL, or DOTH, for use in a method of treating an individual having cancer, wherein the cancer has a mutation in KRAS.

2. The inhibitor of AF9 and/or ENL, or DOT1L, for use in a method of treatment according to claim 1, wherein the cancer is a non-haematological cancer.

3. The inhibitor of AF9 and/or ENL, or DOT1L, for use in a method of treatment according to claim 1 or claim 2, wherein the cancer is selected from colon, lung, pancreas, peritoneum, biliary tract, small intestine, ovarian and endometrial cancers.

4. The inhibitor of AF9 and/or ENL, or DOT1L, for use in a method of treatment according to any one of the preceding claims, wherein the inhibitor is a nucleic acid, an antibody, a small molecule, a peptide, or an inducer of AF9 and/or ENL, or DOT1L, degradation.

5. The inhibitor of AF9 and/or ENL, or DOT1L, for use in a method of treatment according to any one of the preceding claims, wherein the inhibitor is a peptide selected from the group consisting of:

a peptide comprising or consisting of the Ras002 amino acid sequence (SEQ ID NO: 1), or a functional fragment thereof;

a peptide having 70%, 75%, 80%, 85%, 90% or 95% identity to the Ras-002 peptide;

a peptide comprising or consisting of the DOT1L Sites 2 and 3 peptide amino acid sequence (SEQ ID NO: 2), or a functional fragment thereof;

a peptide having 70%, 75%, 80%, 85%, 90% 95%, 96%, 97%, 98% or 99% identity to the DOT1L Sites 2 and 3 peptide;

a peptide comprising or consisting of the DOT1L Site 1 peptide amino acid sequence (SEQ ID NO: 3), or a functional fragment thereof;

a peptide having 70%, 75%, 80%, 85%, 90% or 95% identity to the DOT1L Site 1 peptide; and wherein the peptide binds to AF9 and/or ENL, preferably the C terminus of AF9 and/or ENL.

6. The inhibitor of AF9 and/or ENL, or DOT1L, for use in a method of treatment according to any one of the preceding claims wherein the inhibitor is an AF9 and/or ENL inhibitor that binds to the C terminal domain of AF9 and/or ENL and blocks binding of endogenous DOT1L or AF4.

7. The inhibitor of AF9 and/or ENL, or DOT1L for use in a method of treatment according to any one of the preceding claims, where in the inhibitor an AF9 and/or ENL inhibitor selected from any of the inhibitors in WQ2014127191.

8. The inhibitor of AF9 and/or ENL, or DOTH, for use in a method of treatment according to claim 4, wherein the nucleic acid is an RNAi molecule or a siRNA molecule or a shRNA molecule or a sgRNA or gRNA designed to recruit an RNA-guided nuclease.

9. The inhibitor of AF9 and/or ENL, or DOT1L, for use in a method of treatment according to any one of the preceding claims wherein the inhibitor is a DOT1L inhibitor that inhibits DOT1L enzymatic activity.

10. The inhibitor of AF9 and/or ENL, or DOT1L, for use in a method of treatment according to any one of the preceding claims wherein the inhibitor is a DOT1L inhibitor selected from any of the inhibitors in W02014100662A1.

11. The inhibitor of AF9 and/or ENL, or DOT1L, for use in a method of treatment according to any one of the preceding claims, wherein treatment with the AF9 and/or ENL inhibitor or DOT1L inhibitor is combined with a further anti-cancer therapy.

12. The inhibitor of AF9 and/or ENL for use in a method of treatment according to any one of the preceding claims, wherein the method comprises the step of determining whether the cancer has a mutation in KRAS.

13. A method of treating an individual having a KRAS mutated cancer, the method comprising administering to the individual a therapeutically effective amount of an AF9 and/or ENL or DOT1L inhibitor.

14. A method of screening for agents useful in the treatment of cancer, the method comprising:

(a) contacting an AF9 and/or ENL protein with at least one candidate agent; and

(b) selecting a candidate agent that inhibits the activity of the AF9 and/or ENL protein as being useful for the treatment of a cancer having a mutation in KRAS.

15. A method according to claim 14, wherein the contacting step a) further comprises contacting the AF9 and/or ENL protein with a peptide a as defined in claim 5; and selecting the candidate agent that disrupts the interaction between the peptide and AF9 and/or ENL.

16. The method according to claim 14 wherein the candidate agent is added to a cell line that comprises one or more mutations in KRAS.

17. A method of screening for agents useful in the treatment of a cancer having a KRAS mutation, the method employing first and second cell lines, wherein the first cell line has a KRAS mutation and the second cell line is wildtype for KRAS, the method comprising: (a) contacting the first and second cell lines with at least one candidate agent;

(b) determining the amount of cell death in the first and second cell lines; and

(c) selecting a candidate agent which is synthetically lethal in the first cell line;

wherein the candidate agent is a known or putative AF9 and/or ENL inhibitor or DOTH inhibitor.

18. A method of screening for agents useful in the treatment of KRAS mutated cancer, the method comprising:

(a) contacting an AF9 and/or ENL, or DOT1L, with at least one candidate agent;

(b) determining an effect of the at least one candidate agent on an activity or stability of the AF9 and/or ENL, or DOT1L; and

(c) selecting a candidate agent that inhibits the activity of the AF9 and/or ENL, or DOT1L, as being useful for the treatment of the KRAS mutated cancer.

19. A method according to claim 18 wherein the method comprises a competitive binding assay and step (a) comprises contacting the AF9 and/or ENL, or DOT1L with a peptide as defined in claim 5.

20. A peptide comprising or consisting of the Ras002 amino acid sequence (SEQ ID NO: 1), or a functional fragment thereof, or a peptide having 70%, 75%, 80%, 85%, 90% or 95% identity to the Ras-002 peptide (SEQ ID NO:l); or a peptide comprising or consisting of the DOT1L Sites 2 and 3 peptide amino acid sequence (SEQ ID NO: 2), or a functional fragment thereof, or a peptide having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the DOT1L Sites 2 and 3 peptide; or a peptide comprising or consisting of the DOT1L Sites 1 peptide amino acid sequence (SEQ ID NO: 3), or a functional fragment thereof; or a peptide having 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the DOT1L Site 1 peptide.

21. The peptide according to claim 20 wherein the peptide binds to AF9 and/or ENL, preferably the C-terminus of AF9 and/or ENL.

22. A nucleic acid molecule comprising a nucleic acid sequence encoding the peptide as defined in claim 20 or claim 21.

23. A vector comprising a nucleic acid sequence as defined in claim 22.

24. A cell comprising a nucleic acid or vector as defined in claim 22 or claim 23.

25. A binding moiety that binds the polypeptide of claim 20 or claim 21.

26. The binding moiety of claim 25, wherein the binding moiety is an antibody or functional fragment thereof.

27. A peptide as defined in claim 20 or 21, a nucleic acid molecule as defined in claim 22, a vector as defined in claim 23, a cell as defined in claim 24 or a binding moiety as defined in claims 25 or 26 for use in medicine.

28. The peptide, nucleic acid, vector or cell for use as defined in claim 27 for use in treating or preventing cancer, preferably a non-haemotological cancer having a mutation in KRAS.

29. A pharmaceutical composition comprising a peptide as defined in claim 20 or claim 21, a nucleic acid molecule as defined in claim 22, a vector as defined in claim 23, a cell as defined in claim 24 or a binding moiety as defined in claims 25 or 26 together with a pharmaceutically acceptable carrier.