Immunomodulatory agents (IMiDs) and cereblon E3 ligase modulators (CELMoDs) are a cornerstone of Multiple Myeloma (MM) treatment. They bind to the cereblon (CRBN) component of the CRL4 E3 ubiquitin ligase complex and designate a new set of substrates for proteasomal degradation. Most patients initially respond well to IMiD/CELMoD therapy but over time will become resistant. Understanding resistance mechanisms will enable the development of new targeted treatment strategies to overcome this pressing clinical challenge. Acquired IMiD/CELMoD resistant human MM cell lines were generated by treating IMiD/CELMoD sensitive MM1s and H929 cells with lenalidomide (Len), pomalidomide (Pom) or iberdomide (Iber) at ~10x GI50 concentration for ~12 weeks until resistance was achieved. Cell lines were characterized by whole exome sequencing, RNA-Seq and proteomics. All resistant lines had reduced CRBN expression and some had CRBN mutations, reflecting patient data. A genome-wide loss-of-function CRISPR screen (Brunello library) was carried out in Iber-resistant MM1s. Gene effect scores were calculated with the Chronos algorithm and compared to parental MM1s using data from the Broad Institute DepMap portal. Potential new dependencies in the resistant setting (defined as gene effect score ←1 in resistant cells and >-0.5 in parental cells) were identified in 47 genes including SETD2, a histone 3 lysine 36 methyltransferase (H3K36me2 → H3K36me3). The specific SETD2 inhibitor EPZ-719 reduced viability to a greater extent in the IMiD/CELMoD resistant lines compared to their controls in a 14-day trypan blue exclusion assay. This was most pronounced in the Pom- and Iber-resistant H929 lines. For example in Iber-resistant H929 compared to its control line the viability with EPZ-719 was 13% vs 66% at 1µM, 5% vs 46% at 5µM and 2% vs 45% at 10µM, p<0.0001 (% viability compared to DMSO control at day 14). To investigate whether this novel dependency was related to reduced CRBN expression a CRBN knockout MM1s cell line was generated using CRISPR/Cas9. These cells showed a markedly greater reduction in viability with EPZ-719 compared to control MM1s (viability 20% vs 32% at 1µM, 8% vs 25% at 5µM and 2% vs 18% at 10µM, p<0.001). A reduction in H3K36me3 was seen on immunoblotting after EPZ-719 incubation across all cell lines suggesting an on-target effect. ChIP-Seq and RNA-Seq experiments are underway to identify downstream mechanisms. In conclusion, SETD2 inhibition is more active in IMiD/CELMoD-resistant cell lines compared to sensitive counterparts. Rapid clinical translation of these findings would be possible as a SETD2 inhibitor is currently in a phase 1 trial which includes MM patients. Previous pre-clinical data suggests the t(4;14) MM subgroup may be more sensitive to SETD2 inhibition but our data highlights a novel vulnerability in the IMiD/CELMoD resistant setting that should be explored further. Citation Format: Sarah Anne Bird, Marco Licciardello, Yakinthi Chrisochoidou, James Smith, Amy Barber, Jack Cheung, Yura Grabovska, Shannon Martin, Fernando Sialana, Harvey Che, Habib Bouguenina, Benjamin Bellenie, Brian Walker, Paul Clarke, Charlotte Pawlyn. SETD2 is a novel and druggable dependency in IMiD/CELMoD resistant multiple myeloma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3879.

Abstract 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

Abstract 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

Mitosis is controlled by multiple protein kinases, many of which are abnormally expressed in human cancers. Nek2, Nek6, Nek7, and Nek9 are NIMA-related kinases essential for proper mitotic progression. We determined the atomic structure of Nek7 and discovered an autoinhibited conformation that suggests a regulatory mechanism not previously described in kinases. Additionally, Nek2 adopts the same conformation when bound to a drug-like molecule. In both structures, a tyrosine side chain points into the active site, interacts with the activation loop, and blocks the alphaC helix. Tyrosine mutants of Nek7 and the related kinase Nek6 are constitutively active. The activity of Nek6 and Nek7, but not the tyrosine mutant, is increased by interaction with the Nek9 noncatalytic C-terminal domain, suggesting a mechanism in which the tyrosine is released from its autoinhibitory position. The autoinhibitory conformation is common to three Neks and provides a potential target for selective kinase inhibitors.

3994 Inhibition of the molecular chaperone Hsp90 is a highly attractive anticancer strategy because of its potential to provide combinatorial blockade of multiple oncogenic pathways and simultaneous antagonism of all of the malignant hallmarks of cancer. Although the geldanamycin analogue 17AAG shows promise in early clinical trials, the natural product inhibitors have liabilities that would best be overcome by identifying synthetic small molecule agents. We previously described the discovery by high throughput screening of an early lead compound designated CCT018159 (Aherne et al Proc. AACR 44 Abstract #4002). We now report the biological properties of a series of closely related analogues of this diarylpyrazole lead structure. Improved activity and potency has been observed against the yeast and human recombinant Hsp90 ATPase activity using a malachite green colorimetric assay for inorganic phosphate. Anticancer activity in cell culture has been demonstrated in a range of tumour types, including HCT116 colon carcinoma as well as various melanoma cell lines. The molecular signature of Hsp90 inhibition, defined in details by proteomic and gene expression microarray analysis, has been confirmed using western blotting and ELISA for elevation of Hsp70 and Aha1 and depletion of C-Raf-1, ErbB2 and phospho-ERK1/2, together with other client proteins and co-chaperones. Cell-line dependent G1 and G2M arrest has been demonstrated by dual-stain Hoechst-propidium iodide flow cytometry and apoptosis has been confirmed by sub-G1 peak, morphology, PARP and caspage cleavage. Activity is retained in a P-glycoprotein positive cell line made resistant to doxorubicin and also in cells made resistant to platinum agents. Pharmacokinetic properties are promising, with glucuromide conjugation of the resorcinol ring being a major clearance mechanism. In summary, this series of compounds shows significant promise for development as Hsp90 inhibitors with potential for broad spectrum anticancer activity. Supported by Cancer Research UK, the Wellcome Trust and Vernalis .