Jon Roffey

Protein kinase C intervention-the state of play

Intervention in protein kinase C (PKC) has a chequered history, partly because of the poor selectivity of many inhibitors and partly a reflection of the sometimes antagonistic action of related PKC isoforms. Recent advances in targeting PKC isoforms have come from structural work on isolated kinase domains that have provided opportunities to drive selectivity through structure-based avenues. The promise of isoform selective inhibitors and the rationale for their development are discussed in the broader context of the PKC inhibitor arsenal.

Adenosine-binding motif mimicry and cellular effects of a thieno[2,3-d]pyrimidine-based chemical inhibitor of atypical protein kinase C isoenzymes.

The aPKC [atypical PKC (protein kinase C)] isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCι kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, CRT0066854 displaces a crucial Asn-Phe-Asp motif that is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors.

Regulation of polarized morphogenesis by protein kinase C iota in oncogenic epithelial spheroids

Protein kinase C iota (PKCι), a serine/threonine kinase required for cell polarity, proliferation and migration, is commonly up- or downregulated in cancer. PKCι is a human oncogene but whether this is related to its role in cell polarity and what repertoire of oncogenes acts in concert with PKCι is not known. We developed a panel of candidate oncogene expressing Madin-Darby canine kidney (MDCK) cells and demonstrated that H-Ras, ErbB2 and phosphatidylinositol 3-kinase transformation led to non-polar spheroid morphogenesis (dysplasia), whereas MDCK spheroids expressing c-Raf or v-Src were largely polarized. We show that small interfering RNA (siRNA)-targeting PKCι decreased the size of all spheroids tested and partially reversed the aberrant polarity phenotype in H-Ras and ErbB2 spheroids only. This indicates distinct requirements for PKCι and moreover that different thresholds of PKCι activity are required for these phenotypes. By manipulating PKCι function using mutant constructs, siRNA depletion or chemical inhibition, we have demonstrated that PKCι is required for polarization of parental MDCK epithelial cysts in a 3D matrix and that there is a threshold of PKCι activity above and below which, disorganized epithelial morphogenesis results. Furthermore, treatment with a novel PKCι inhibitor, CRT0066854, was able to restore polarized morphogenesis in the dysplastic H-Ras spheroids. These results show that tightly regulated PKCι is required for normal-polarized morphogenesis in mammalian cells and that H-Ras and ErbB2 cooperate with PKCι for loss of polarization and dysplasia. The identification of a PKCι inhibitor that can restore polarized morphogenesis has implications for the treatment of Ras and ErbB2 driven malignancies.

Addressing the Right Targets in Oncology: Challenges and Alternative Approaches

Translating existing and emerging knowledge of cancer biology into effective novel therapies remains a great challenge in drug discovery. A firm understanding of the target biology, confidence in the supporting preclinical research, and access to diverse chemical matter is required to lower attrition rates and prosecute targets effectively. Understanding past successes and failures will aid in refining this process to deliver further therapeutic benefit to patients. In this review, we suggest that early oncology drug discovery should focus on selection and prosecution of cancer targets with strong disease biology rather than on more chemically “druggable” targets with only modest disease-linkage. This approach offers higher potential benefit but also increases the need for innovative and alternative approaches. These include using different methods to validate novel targets and identify chemical matter, as well as raising the standards and our interpretation of the scientific literature. The combination of skills required for this emphasizes the need for broader early collaborations between academia and industry.

Abstract C126: Molecular profiling of the HES transcription factor-TLE co-repressor protein-protein interaction site.

Aberrant Notch signalling has been implicated in a variety of tumor types, where it is known to play a role in tumorigenisis and angiogenisis. In particular, recent evidence suggests that the Notch pathway is activated by somatic mutations in a significant proportion of human T cell acute lymphoblastic leukaemias (T-ALLs). Therefore targeting of the Notch pathway represents an attractive avenue for anti-cancer drug discovery. However, apart from the potential use of gamma-secratase inhibitors, there are very few known tractable intervention points along this predominantly protein-protein interaction mediated pathway. The structural characteristics of protein-protein contact surfaces present a huge challenge for small molecule drug discovery as these contact surfaces are generally larger than those involved in protein-small molecule interactions and often lack the grooves and pockets on the protein surface that small molecules efficiently bind to. Recent discoveries in our laboratories indicate that the interaction of the HES transcription factor with the TLE co-repressor could provide a tractable point for small molecule inhibitors that would inhibit the Notch signalling pathway. The interaction between HES/TLE is dependent on a highly conserved contiguous tetra-peptide WRPW motif present in the C-terminus of HES family members which binds to the WD domain of TLE. The binding site on TLE is a potentially druggable hydrophobic recess located at the central pore of a 7-bladed β-propeller structure, and the WRPW motif sits on top of the recess contacting all 7 blades of the -propeller. We have established and validated biochemical fluorescence polarisation (FP) and GST-WRPW pull down assays to identify compounds that disrupt HES/TLE binding in vitro and a GAL4-WRPW reporter luciferase assay was developed as an effective way of monitoring the WRPW dependent interactions between HES and TLE proteins in a cell culture model. Using these assays a small library of peptide derivatives, based around the highly conserved WRPW motif, was screened. SAR from the peptide array in conjunction with structural information from a WRPW peptide bound to TLE1 WD domain was used to construct a 3D pharmacophore model which was applied as a search query for in silico virtual screening. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C126.

Abstract 1356: Cell-based screening to identify repressors of wild type and mutated telomerase reverse transcriptase gene promoter activity

Cellular immortality is a near-universal phenotype of cancer cells with the central role played by the multicomponent telomerase complex firmly established. The reactivation of telomerase is found in approximately 85% of all cancers. Furthermore, frequent activating non-coding mutations in the hTERT promoter region have been identified in multiple tumour types. A considerable body of evidence supports the concept of targeting the signal transduction pathways that contribute to telomerase overexpression in cancer cells. We have exploited our knowledge in this area to develop cell-based luciferase reporter gene assays to measure hTERT and hTR gene promoter activity. To this end we undertook high throughput screening in the A2780 human ovarian cancer cell line against both hTR and hTERT promoters. Secondary specificity and cytotoxicity assays reduced the initial hits to a single chemical series of dual inhibitors which strongly suppress both hTR and hTERT promoter activity without primary cytotoxicity, as predicted of telomerase inhibitors. Through a process of Hit to Lead we have further developed the chemical series. Lead compounds are active across a panel of cell lines from distinct histological origins, reduce endogenous hTERT mRNA levels and decrease telomerase catalytic activity at nM concentrations. Furthermore, these compounds repress both wild type and mutant C228T and C250T hTERT promoter activity. In specificity assays no off-target activity has been identified in an expanded promoter screen against a panel of 15 off-target promoter reporters. Citation Format: Alan Bilsland, Yu Liu, Sharon Burns, David Jenkinson, Jon Roffey, W. Nicol Keith. Cell-based screening to identify repressors of wild type and mutated telomerase reverse transcriptase gene promoter activity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1356.

Abstract LB-052: Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification

Protein kinases are attractive targets for pharmacological intervention through their frequent disease associated aberration of cellular signalling networks. In this context an understanding of the myriad of substrates on which a protein kinase operates can help to reveal potential pharmacodynamic and patient stratification biomarkers. Kinase Identification of Proximal Substrates (KIPS) utilises an innovative chemical genetics approach to identify novel proximal biomarkers of kinase target response. This approach relies on the generation of a desensitized mutant protein that retains its kinase function, but loses sensitivity to small-molecule mediated inhibition. Comparative phospho-proteomics is performed on immuno-complexes of tagged wild type and mutant protein kinases in the presence and absence of inhibitor to isolate specific phospho-proteomic changes in proximal substrates. To exemplify the KIPS approach we have focused on the identification of novel substrates of the atypical protein kinase C isoform, PKCι. Through structure based design we identified two acidic amino acid residues in the PKCι nucleotide binding site that are crucial for efficacy of a previously characterized PKCι inhibitor, CRT0066854. Upon mutation to non-acidic alanine residues the PKCι protein is rendered insensitive to compound-mediated inhibition, allowing a comparison of wild type and mutant responses to PKCι inhibition to be made. Using HCT116 cells, we identified Myosin X as a putative substrate for PKCι, and demonstrated its specificity to the PKCι signalling axis through alanine-scanning peptide arrays and generation of phospho-specific antibodies. We show that the key acidic amino acids utilised in KIPS are highly conserved across the AGC and CAMK kinase super families. Furthermore, extensive compound SAR, diverse kinome profiling and structural biology have identified a tool box of inhibitor compounds with a broad range of activities across these kinase families leading to broader applicability of KIPS to enable the identification of individual kinase specific substrates and response across approximately 20% of the human kinome. Citation Format: jon roffey, Andrew Turnbull, Christian Dillon, Susan Boyd, Philippe Riou, Mark Linch, Peter Parker, Sven Kjaer, Neil McDonald. Kinase identification of proximal substrates (KIPS): A novel chemical genetics approach for kinase substrate identification. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-052. doi:10.1158/1538-7445.AM2015-LB-052

Abstract LB-125: The identification of FOXA1/ERα pathway modulators for the treatment of hormone-based therapy resistant breast cancer

Estrogen receptor alpha (ERα) is expressed in ∼75% of breast cancers where it binds and regulates specific target genes which drive cancer cell proliferation. Hormone-based treatments such as selective estrogen receptor modulators (SERMs) and aromatase inhibitors prevent estrogen signalling through the ERα, but in many cases resistance to these therapies develops. FOXA1 is a pioneer factor for ERα that is required to facilitate ERα binding to its target genes and its expression is considered to be a defining gene that characterizes ERα positive luminal breast cancers. Here we show that FOXA1 is an essential gene in ERα positive, tamoxifen resistant and aromatase-resistant breast cancer cell lines suggesting inhibitors that target the FOXA1/ERα pathway could have broad utility in these settings. To this end we performed a cell-based screen to identify novel small molecule regulators of FOXA1/ERα pathway activity. Our novel cell-based assay allowed us to screen over 180, 000 discrete compounds in a high through-put manner. Counter screens and phenotypic profiling in FOXA1 positive and negative breast cancer cell lines were used to remove non-specific compounds and direct ERα modulators. Second batches of confirmed actives were profiled through the screening cascade and by using ChIP assays we have observed a reduction in FOXA1 binding at the chromatin that is commensurate with a similar loss of ERα binding, demonstrating for the first time pharmacological modulation of FOXA1/ERα occupancy at the chromatin level. Citation Format: jon roffey, Aurore Lejeune, Michelle Barnard, Julie Stock, Ai Ching Wong, Jenny McKelvie, Kelly Holmes, Jason Carroll, Stephen Myatt. The identification of FOXA1/ERα pathway modulators for the treatment of hormone-based therapy resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-125. doi:10.1158/1538-7445.AM2015-LB-125

Abstract LB-99: Identification and characterization of small molecule thieno[3,2-d]pyrimidine inhibitors of Protein Kinase C iota (PKCι)

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We describe a novel series of small molecule ATP-competitive inhibitors of the atypical class of Protein Kinase C (PKC). The PKC family of serine/threonine kinases are divided into four structurally and functionally distinct sub-types: classic (PKCα, PKCβ, PKCγ); novel (PKCδ, PKCϵ, PKCη, PKCθ); atypical (PKCι,PKCζ); and PKNs (PKN1, PKN2, PKN3). The PKCs have been defined as key regulators in a multitude of signal transduction pathways that impinge on diverse cellular processes such as proliferation, differentiation, and survival. The regulation and functions of the atypical PKCs are distinct from the classic and novel PKCs.Both atypical isoforms have been implicated in various models of cancer, and PKCι in particular has been described as a potential oncogene. Its abundance is frequently increased, primarily through PKCι gene amplification, in many epithelial tumours including: subsets of squamous non-small cell lung carcinoma, serous ovarian carcinoma, and squamous esophageal carcinoma. It has been widely demonstrated that deregulation of PKCι signalling leads to unconstrained cell growth, increased migratory and invasive behaviour, and aberrant cellular polarity, which is a hallmark of aggressive cancers. Together, these data make a strong case for the inhibition of PKCι as a novel therapeutic strategy. We have discovered a series of ATP-competitive thieno[3,2-d]pyrimidine- based PKCι inhibitors from a high throughput screen. Using structure-based design we have optimized the series and demonstrated potent and selective inhibition of PKCι in biochemical and cellular models. The biochemical, structural, and cellular characterization of these compounds will be described herein. Citation Format: Christelle Soudy, Emma Stanway, Bhavisha Patel, Caroline Barton, Michelle Barnard, Leon Pang, Paul Owen, Andrew Turnbull, Bruce A. Ruggeri, Bruce D. Dorsey, Mark A. Ator, Greg R. Ott, Mark Linch, Philippe Riou, Peter J. Parker, Sven Kjaer, Christian Dillon, Neil Q. McDonald, Jon Roffey. Identification and characterization of small molecule thieno[3,2-d]pyrimidine inhibitors of Protein Kinase C iota (PKCι). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-99. doi:10.1158/1538-7445.AM2014-LB-99

Abstract A280: Small molecule inhibition of atypical protein kinase C iota reveals a functional basis for oncogenesis.

Background: Atypical protein kinase C iota (PKCι) is a serine/threonine kinase that has been implicated in a variety of cellular processes including polarity, migration, proliferation and survival. PKCι is amplified in many cancers correlating with poor prognosis, however the upstream activating inputs and downstream effectors are not well defined. Experimental Procedures: 3D MDCK epithelial cell spheroids were grown in Matrigel and used to assess polarized morphogenesis and growth. PKCι was manipulated by inhibition with CRT0066854, or transfection with siRNA or cDNA variants. A PKCι binding interface was identified by X-ray crystallography and interrogated by mass spectrometry of wildtype or binding mutant immunocomplexes. Results: To identify potential inputs into PKCι we assessed a panel of transformed MDCK cells; p110α, Src, Ras, ErbB2 and Raf. Parental MDCK cells form polarized cysts in Matrigel, however Ras, ErbB2 and p110α MDCK cells formed dysplastic spheroids. In the case of Ras and ErbB2, but not p110α MDCK spheroids, normal polarized morphogenesis could be rescued by siRNA-PKCι or partial PKCι inhibition with decreased spheroid size seen for all transformed cells lines. An effector binding site (PKCι RIPR motif) identified from the crystal structure of the kinase and characterized as a recruitment site for select targets including LLGL2 and Myosin X, was found to be critical for PKCι to confer polarized morphogenesis. Interestingly this binding motif shows a low frequency of mutation in a subset of human tumors. Conclusions: Downstream of Ras and ErbB2, PKCι activity is enhanced with a resulting dysplasia and loss of polarization, that can be rescued by PKCι inhibition. Mutation of the PKCι-RIPR effector binding motif that is found in cancer, prevents interaction with LLGL2 and its expression also elicits growth as non-polarized, dysplastic epithelial spheroids. This indicates that PKCι may promote cancer, not only by amplification, but by upstream oncogenic inputs and somatic mutation. The oncogene dependent PKCι hyperactivation is pro-proliferative in 3D culture, providing compelling evidence that PKCι is a good target for Ras mutant tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A280. Citation Format: Mark Linch, Marta Sanz-Garcia, Svend Kjaer, Erika Soriano, Philippe Riou, Philip Knowles, Christian Dillon, Jon Roffey, Neil McDonald, Peter Parker. Small molecule inhibition of atypical protein kinase C iota reveals a functional basis for oncogenesis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A280.