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Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells. Cancer Res; 75(16); 3340–54. ©2015 AACR.

The protein kinase MPS1 is a crucial component of the spindle assembly checkpoint signal and is aberrantly overexpressed in many human cancers. MPS1 is one of the top 25 genes overexpressed in tumors with chromosomal instability and aneuploidy. PTEN-deficient breast tumor cells are particularly dependent upon MPS1 for their survival, making it a target of significant interest in oncology. We report the discovery and optimization of potent and selective MPS1 inhibitors based on the 1H-pyrrolo[3,2-c]pyridine scaffold, guided by structure-based design and cellular characterization of MPS1 inhibition, leading to 65 (CCT251455). This potent and selective chemical tool stabilizes an inactive conformation of MPS1 with the activation loop ordered in a manner incompatible with ATP and substrate-peptide binding; it displays a favorable oral pharmacokinetic profile, shows dose-dependent inhibition of MPS1 in an HCT116 human tumor xenograft model, and is an attractive tool compound to elucidate further the therapeutic potential of MPS1 inhibition.

Genomic aberrations frequently target and activate the MAPK and PI3K pathways in human cancers and combined inhibition of these pathways is being explored widely in the clinic. In a panel of 45 human colorectal cancer cell lines we found the presence of a KRAS mutation resulted in reduced sensitivity to PI3K and MEK inhibitors when used as single treatments. In addition, prolonged exposure to the individual PI3K (GDC-0941) or MEK (GDC-0973) inhibitors resulted in resistance that was associated with acquisition of mutations of the respective pathway being targeted. Resistance to individual targeted agents is challenge in clinic and can be overcome by combining treatments and in our colorectal cancer panel combined MEK/PI3K inhibition exhibited potent synergy in the KRAS mutant lines. However, as with the single treatments prolonged exposure to the combination regime also resulted in resistance through acquisition of a mutation of the allosteric drug-binding site of MEK2 that abrogates binding of the inhibitor. The clinical development of small-molecule ERK inhibitors is of considerable interest as ERK signaling represents a key downstream effector of RAS mutations and ERK inhibitors may retain activity in cells resistant to RAF and MEK inhibitors. Consistent with the latter, the combination resistant line retained sensitivity to an ERK inhibitor (VTX-11e) that also rescued the synergistic interaction when combined with PI3K inhibition. However, prolonged exposure of this MEK/PI3K resistant line to the combination of VTX-11e/GDC0941 resulted in the acquisition of additional resistance to the ERK inhibitor component of the combination. Resistance to VTX-11e was a result of an ERK2 mutation, novel to protein kinases, in a conserved residue of the kinase domain P-loop. Recombinant wild-type and P-loop mutant ERK2 had similar Vmax and ATP Km values, but exhibited a 20-fold increase in IC50 for VTX-11e. Stable expression of the P-loop mutant ERK2 in parent cells resulted in resistance to VTX-11e. Development of an additional VTX-11e resistant line, in this case by prolonged exposure to a single treatment resulted in the acquisition of the same ERK2 mutation. The repeated development of resistance to the MEK/ERK inhibitor arm of the combination is intriguing as the parent cells were not particularly sensitive to treatment with MEK or ERK inhibitors alone, with a GI50 30-fold higher than BRAF mutant cell lines that are sensitive to these inhibitors. Our data show that dual combination of agents, at least for MEK and PI3K pathway inhibition, is insufficient to block the acquisition of resistance, an observation similar to that of clinical trials of BRAF and MEK inhibitors in melanoma. Finally we recommend sequencing of P-loop of the ERKs in clinical studies of ERK inhibitors to determine whether these mutations will occur in the clinic. Citation Format: Paul A. Clarke, Toby Roe, Kate Swabey, Craig McAndrew, Kathy Boxall, Isaac Westwood, Robert van Montfort, Bissan Al-Lazikhani, Paul Workman. Resistance to ERK inhibitors as a result of an acquired novel P-loop mutation of ERK2. [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 2843. doi:10.1158/1538-7445.AM2015-2843

Acquired resistance is the greatest challenge to the effectiveness of targeted anti-cancer therapies in the clinic. With several MPS1 inhibitors under pre-clinical development, we aimed to investigate how cancer cells will develop resistance against these inhibitors; therefore we modeled acquired resistance using a range of MPS1 inhibitors. We identified and characterized five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed that several MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. One mutation in particular, p.C604W, which is close to the gatekeeper residue, rendered MPS1 resistant to all the inhibitors we tested. However, we were able to design new compounds to specifically overcome this mutation, which in fact targeted the mutant with more potency than the wild-type MPS1 protein. Importantly, we show that these mutations are present in untreated cancer cell lines and primary tumour samples, and also pre-exist in normal lymphoblast and breast tissues. Furthermore, to confirm this is not specific to MPS1, we show that the EGFR p.T790M mutation is also pre-existing in cancer cell lines and normal tissue. Our data therefore suggest that mutations conferring resistance to targeted therapy are naturally occurring mutations in normal and cancer cells that are not introduced due to cancer cells being more mutagenic. Citation Format: Mark D. Gurden, Isaac Westwood, Amir Faisal, Sébastien Naud, Jack Cheung, Craig McAndrew, Amy Wood, Jessica Schmitt, Kathy Boxall, Grace Mak, Paul Workman, Rosemary Burke, Swen Hoelder, Julian Blagg, Rob Van Montfort, Spiros Linardopoulos. Naturally occurring mutations in the MPS1 gene predispose cells to kinase inhibitor drug-resistance. [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 5450. doi:10.1158/1538-7445.AM2015-5450

The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. Surveillance mechanisms, the so-called checkpoint pathways, monitor passage through mitosis at several stages. One of the best characterised is the spindle assembly checkpoint that prevents anaphase onset until the appropriate tension and attachment across kinetochores is achieved. One of the first components of the spindle assembly checkpoint signal, identified by a genetic screen in budding yeast, was MPS1 (monopolar spindle 1; also known as TTK). MPS1 gene was shown to encode an essential dual-specificity kinase conserved from yeast to humans. MPS1 activity peaks at the G2/M transition and is enhanced upon activation of the spindle assembly checkpoint with nocodazole. We and others, have identified the autophosphorylation of T676 in the activation loop of MPS1 and shown that this is essential for MPS1 function. MPS1 has been found aberrantly overexpressed in a wide range of human tumours including breast, lung, oesophagus, and prostate. MPS1 is required for the establishment and maintenance of the spindle assembly checkpoint during mitosis. Aneuploid tumour cells possess a compromised spindle checkpoint to allow onset of anaphase and cell division. We have shown that depletion of MPS1 by siRNA induces cell death selectively in PTEN-deficient breast cancer cell lines. We have developed biochemical and cellular assays for MPS1 activity and a high throughput screening of our compound library delivered multiple hit series. We have previously reported the discovery of CCT251455 as a selective and orally bioavailable MPS1 inhibitor that inhibits the growth of a panel of human tumour cell lines, abrogates nocodazole-induced mitotic arrest and reduces the time spent in mitosis. Medicinal chemistry in combination with X-ray crystallography led to the discovery of CCT271850, a novel inhibitor of MPS1 kinase activity. CCT271850 selectively inhibits MPS1 kinase activity with an IC50 of 0.004 μM, inhibits autophosphorylation of MPS1 in cells with an IC50 of 0.07 μM and reduces the growth of a panel of human tumour cell lines, particularly PTEN-deficient cell lines. Tumour cells treated with CCT271850 contain aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment. CCT271850 is orally bioavailable (F = 68%) and shows modulation of biomarkers in vivo. Citation Format: Amir Faisal, Paolo Innocenti, Isaac Westwood, Sebastan Naud, Jessica Schmitt, Angela Hayes, Grace Mak, Mark Gurden, Vassilios Bavetsias, Jack Cheung, Hannah Woodward, Peter Sheldrake, Butrus Atrash, Rosemary Burke, Ross Baker, Craig McAndrew, Martin Rowlands, Melanie Valenti, Paul Workman, Suzanne Eccles, Florence Raynaud, Rob vanMontfort, Swen Hoelder, Julian Blagg, Spiros Linardopoulos. CCT271850, a novel, selective, highly potent and orally bioavailable Mps1 kinase inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3242. doi:10.1158/1538-7445.AM2013-3242

Monopolar spindle 1 (Mps1, also known as TTK) is a dual-specificity, cell cycle-regulated kinase required for the establishment and maintenance of the spindle assembly checkpoint during mitosis. Aneuploid tumour cells possess a weak spindle checkpoint to allow onset of anaphase and cell division. Our hypothesis is that a complete inhibition of an already weakened mitotic checkpoint of cancer cells will cause gross chromosomal abnormalities leading to aneuploid cell death. We have shown that depletion of Mps1 by siRNA induces cell death selectively in aneuploid and PTEN-deficient cancer cell lines. We have demonstrated that Mps1 depletion inhibits MAD2 localisation to the kinetochores. We have developed biochemical and cellular assays for Mps1 activity and a high throughput screening of our Institute's compound library delivered multiple hit series. Medicinal chemistry in combination with X-ray crystallography led to the development of CCT251455, a small molecule inhibitor of Mps1 kinase activity. CCT251455 selectively inhibits Mps1 kinase with an IC50 of 0.003 μM, inhibits growth of a panel of human tumour cell lines with GI50 between 0.06 - 1 μM and is particularly potent in PTEN-deficient cell lines. Cells treated with CCT251455 abrogate nocodazole-induced mitotic arrest and reduce the time spent in mitosis. Mps1-inhibited cells contain aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment. CCT251455 is orally bioavailable (F = 82%) and shows modulation of biomarkers in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1817. doi:1538-7445.AM2012-1817

Monopolar spindle 1 (MPS1, also known as TTK) is a dual-specificity, cell cycle-regulated kinase required for the establishment and maintenance of the spindle assembly checkpoint during mitosis. Meta-analysis on expression array datasets showed increased MPS1 expression strongly correlated with time to relapse from breast cancer and this was significantly associated with breast tumors of a basal-like phenotype. However, the issue of whether inhibition of MPS1 in human tumor cells compromises their viability has not yet been fully addressed. We show here, using an RNA interference (RNAi) approach, that inhibition of MPS1 can indeed reduce the viability of human tumor cells and that this effect is selective; aneuploid tumor cell lines are sensitive to MPS1 inhibition whereas non-cancerous cells remained resistant. To identify chemical inhibitors of MPS1, a high-throughput screen of a library of small molecules using the Homogeneous Time-Resolved Fluorescence (HTRF) assay was performed. This strategy successfully identified a hit compound that selectively reduces viability in human breast tumor cell lines. These results further validate MPS1 as a therapeutic target and identify a proof-of-principle small molecule inhibitor. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A239.