Mps1 Inhibition Research Articles

HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos.

Human fertility is suboptimal, partly due to error-prone divisions in early cleavage-stages that result in aneuploidy. Most human pre-implantation are mosaics of euploid and aneuploid cells, however, mosaic embryos with a low proportion of aneuploid cells have a similar likelihood of developing to term as fully euploid embryos. How embryos manage aneuploidy during development is poorly understood. This knowledge is crucial for improving fertility treatments and reducing developmental defects. To explore these mechanisms, we established a new mouse model of chromosome mosaicism to study the fate of aneuploid cells during pre-implantation development. We previously used the Mps1 inhibitor reversine to generate aneuploidy in embryos. Here, we found that treatment with the more specific Mps1 inhibitor AZ3146 induced chromosome segregation defects in pre-implantation embryos, similar to reversine. However, AZ3146-treated embryos showed a higher developmental potential than reversine-treated embryos. Unlike reversine-treated embryos, AZ3146-treated embryos exhibited transient upregulation of Hypoxia Inducible-Factor-1A (HIF1A) and lacked p53 upregulation. Pre-implantation embryos develop in a hypoxic environment in vivo, and hypoxia exposure in vitro reduced DNA damage in response to Mps1 inhibition and increased the proportion of euploid cells in the mosaic epiblast. Inhibiting HIF1A in mosaic embryos also decreased the proportion of aneuploid cells in mosaic embryos. Our work illuminates potential strategies to improve the developmental potential of mosaic embryos.

Abstract 509: The MPS1/TTK inhibitor, NMS-01940153, synergizes with tisotumab vedotin in colorectal cancer

Abstract Background: MPS1 kinase plays a crucial role in maintaining genomic integrity by controlling the spindle assembly checkpoint (SAC). MPS1 is highly expressed in tumors of different origins and overexpression is associated with poor prognosis. By inhibiting MPS1, cancer cells are forced to enter anaphase before chromosomes are aligned, leading to mitotic catastrophe and apoptosis. NMS-01940153 (NMS-153) is a potent and selective MPS1 inhibitor, with antitumor activity in vitro and in vivo. NMS-153, under investigation in a clinical trial, shows early signs of clinical activity, an acceptable safety profile, with manageable and reversible neutropenia as the main adverse effect (EudraCT 2020-001002-26; Reig et al, Eur J Cancer 174S1, 2022). Microtubule targeting agents (MTAs), such as taxanes and vinca alkaloids, cause mitotic arrest and activate the SAC. Concomitant SAC disruption by MPS1 inhibition synergizes with MTAs, but the combination is limited by neutropenia in the clinic. Purpose and Methods: The goal is to show synergism between NMS-153 and MTA and to find a strategy to overcome the limiting neutropenia of this combination. For this, Tisotumab vedotin (TV), an anti-Tissue Factor (TF) with Monomethyl auristatine E (MMAE) payload, was used. TF is aberrantly expressed in a variety of tumors compared to normal tissue counterpart. In colorectal cancer (CRC), TF is overexpressed in patients with distinct molecular features including BRAF mutation, a colon cancer population that hardly responds to chemotherapy or BRAF inhibitors. At therapeutic doses, TV induces low rate of grade ≥3 neutropenia. The combination of NMS-153 and TV is expected to synergize and have an acceptable safety profile. LS411N CRC model expressing TF and harboring a BRAFV600E mutation was selected for testing NMS-153 and TV in combination. In vivo, NMS-153 was tested at 13 or 20 mpk every 4 days for 7 doses. TV was tested at 1 or 4 mpk weekly for 2 doses. The 2 therapeutic doses of both agents were tested in combination. Results: In vitro, synergism of NMS-153 with TV in LS411N cells was observed. In vivo, NMS-153 and TV as single agents were efficacious in LS411N xenograft with TGIs up to 53% and 89%, respectively. The median survival time (MST) for the vehicle group was 35.5 days. NMS 153 and TV single agents prolonged survival with MSTs up to 53 and 73.3 days, respectively. When combined, a dose-dependent therapeutic benefit was observed, with an MST of >137 days, and 6 out of 10 mice profiting of long-term tumor regressions at the highest doses tested. The combination benefit was statistically significantly different from single agents and more than additive. Treatments were well tolerated. Conclusions: The combination of NMS-153 with TV is synergistic in the LS411N CRC model. These data strongly suggest the potential for clinical efficacy of NMS-153 with TV and the combinability of NMS-153 with TV and other MMAE-based ADCs in different tumor contexts. Citation Format: Gemma Texido, Fabio Gasparri, Antonella Ciavolella, Laura Gianellini, Anders E. Pedersen, Alberto Ocaña, Riccardo Colombo, Domenico Roberti, Lisa Mahnke, Claudia Perrera. The MPS1/TTK inhibitor, NMS-01940153, synergizes with tisotumab vedotin in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 509.

Maximizing Anticancer Response with MPS1 and CENPE Inhibition Alongside Apoptosis Induction.

Antimitotic compounds, targeting key spindle assembly checkpoint (SAC) components (e.g., MPS1, Aurora kinase B, PLK1, KLP1, CENPE), are potential alternatives to microtubule-targeting antimitotic agents (e.g., paclitaxel) to circumvent resistance and side effects associated with their use. They can be classified into mitotic blockers, causing SAC-induced mitotic arrest, or mitotic drivers, pushing cells through aberrant mitosis by overriding SAC. These drugs, although advancing to clinical trials, exhibit unsatisfactory cancer treatment outcomes as monotherapy, probably due to variable cell fate responses driven by cyclin B degradation and apoptosis signal accumulation networks. We investigated the impact of inhibiting anti-apoptotic signals with the BH3-mimetic navitoclax in lung cancer cells treated with the selective CENPE inhibitor GSK923295 (mitotic blocker) or the MPS1 inhibitor BAY1217389 (mitotic driver). Our aim was to steer treated cancer cells towards cell death. BH3-mimetics, in combination with both mitotic blockers and drivers, induced substantial cell death, mainly through apoptosis, in 2D and 3D cultures. Crucially, these synergistic concentrations were less toxic to non-tumor cells. This highlights the significance of combining BH3-mimetics with antimitotics, either blockers or drivers, which have reached the clinical trial phase, to enhance their effectiveness.

Age-dependent loss of cohesion protection in human oocytes

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a "cohesin bridge" between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

RZZ-Spindly and CENP-E form an integrated platform to recruit dynein to the kinetochore corona.

Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin-7) and dynein-dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona's building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we show that when corona assembly is prevented through MPS1 inhibition, CENP-E is absolutely required to retain RZZS at kinetochores. An RZZS phosphomimetic mutant bypasses this requirement, demonstrating the existence of a second receptor for polymeric RZZS. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

Design, synthesis and biological evaluation of a new class of 7H-pyrrolo[2,3-d]pyrimidine derivatives as Mps1 inhibitors for the treatment of breast cancer

Monopolar spindle kinase 1 (Mps1), a core component of the spindle assembly checkpoint (SAC), plays a crucial role in the transition of cells from mid-to late mitosis. As an attractive therapeutic target, inhibition of Mps1 induces cell cycle arrest and apoptosis in a variety of tumors, including breast cancer. However, early clinical development of Mps1 inhibitors remains unsatisfactory. Here, we designed and synthesized a new class of Mps1 inhibitors with 7H-pyrrolo[2,3-d]pyrimidine structure using a scaffold hopping approach. Structure–activity relationship (SAR) revealed that 12 is a potent Mps1 inhibitor (IC50 = 29 nM), which inhibited phosphorylation of Mps1 in vitro and in vivo. Treatment with 12 not only impeded proliferation of breast cancer cell lines, but also induced cell cycle arrest and apoptosis of MCF-7 and 4T1 cells. 12 suppressed tumor growth in vivo, and no obvious toxicities were observed. These results demonstrated the potential of Mps1 inhibitor 12 for the treatment of breast cancer.

Inhibition of Mps1 kinase enhances taxanes efficacy in castration resistant prostate cancer

Androgen ablation therapy is the standard of care for newly diagnosed prostate cancer (PC) patients. PC that relapsed after hormonal therapy, referred to as castration-resistant PC (CRPC), often presents with metastasis (mCRPC) and is the major cause of disease lethality. The few available therapies for mCRPC include the Taxanes Docetaxel (DTX) and Cabazitaxel (CBZ). Alas, clinical success of Taxanes in mCRPC is limited by high intrinsic and acquired resistance. Therefore, it remains essential to develop rationally designed treatments for managing therapy-resistant mCRPC disease. The major effect of Taxanes on microtubule hyper-polymerization is a prolonged mitotic block due to activation of the Spindle Assembly Checkpoint (SAC). Taxane-sensitive cells eventually inactivate SAC and exit mitosis by mitotic catastrophe, resulting in genome instability and blockade of proliferation. Resistant cells remain in mitotic block, and, upon drug decay, resume mitosis and proliferation, underlying one resistance mechanism. In our study we explored the possibility of forced mitotic exit to elevate Taxane efficacy. Inactivation of the SAC component, mitotic checkpoint kinase Mps1/TTK with a small molecule inhibitor (Msp1i), potentiated efficacy of Taxanes treatment in both 2D cell culture and 3D prostasphere settings. Mechanistically, Mps1 inhibition forced mitotic catastrophe in cells blocked in mitosis by Taxanes. Androgen receptor (AR), the main driver of PC, is often mutated or truncated in mCRPC. Remarkably, Mps1i significantly potentiated CBZ cytotoxicity regardless of AR status, in both AR-WT and in AR-truncated CRPC cells. Overall, our data demonstrate that forced mitotic exit by Mps1 inhibition potentiates Taxanes efficacy. Given that several Mps1i’s are currently in different stages of clinical trials, our results point to Mps1 as a new therapeutic target to potentiate efficacy of Taxanes in mCRPC patients.

MPS1 inhibition primes immunogenicity of KRAS-LKB1 mutant lung cancer.

KRAS-LKB1 (KL) mutant lung cancers silence STING owing to intrinsic mitochondrial dysfunction, resulting in Tcell exclusion and resistance to programmed cell death (ligand) 1 (PD-[L]1) blockade. Here we discover that KL cells also minimize intracellular accumulation of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) to further avoid downstream STING and STAT1 activation. An unbiased screen to co-opt this vulnerability reveals that transient MPS1 inhibition (MPS1i) potently re-engages this pathway in KL cells via micronuclei generation. This effect is markedly amplified by epigenetic de-repression of STING and only requires pulse MPS1i treatment, creating a therapeutic window compared with non-dividing cells. A single course of decitabine treatment followed by pulse MPS1i therapy restores Tcell infiltration invivo, enhances anti-PD-1 efficacy, and results in a durable response without evidence of significant toxicity.

A STING operation to expose KRAS and STK11 co-mutated lung cancers.

KRAS and STK11 (LKB1) co-mutated (KL) tumors define an immunologically cold and anti-PD-(L)1-refractory non-small-cell lung cancer (NSCLC) subset. In this issue of Cancer Cell, Kitajima etal. outline a strategy to unleash innate immunity in KL tumors by utilizing epigenetic de-repression of STING and pulsed inhibition of spindle assembly checkpoint kinase MPS1.

Development of the First Covalent Monopolar Spindle Kinase 1 (MPS1/TTK) Inhibitor.

Monopolar spindle kinase 1 (MPS1/TTK) is a key element of the mitotic checkpoint and clinically evaluated as a target in the treatment of aggressive tumors such as triple-negative breast cancer. While long drug-target residence times have been suggested to be beneficial in the context of therapeutic MPS1 inhibition, no irreversible inhibitors have been reported. Here we present the design and characterization of the first irreversible covalent MPS1 inhibitor, RMS-07, targeting a poorly conserved cysteine in the kinase's hinge region. RMS-07 shows potent MPS1 inhibitory activity and selectivity against all protein kinases with an equivalent cysteine but also in a broader kinase panel. We demonstrate potent cellular target engagement and pronounced activity against various cancer cell lines. The covalent binding mode was validated by mass spectrometry and an X-ray crystal structure. This proof of MPS1 covalent ligandability may open new avenues for the design of MPS1-specific chemical probes or drugs.

Targeting Mps1 in combination with paclitaxel inhibits osteosarcoma progression by modulating spindle assembly checkpoint and Akt/mTOR signaling.

Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents and is characterized by early metastasis and frequent recurrence, which greatly affects patient prognosis and survival rates. However, the treatment of OS, its recurrence and subsequent metastasis is now at a clinical bottleneck. To explore new OS chemotherapeutic targets, investigate new therapeutic strategies and improve patient prognosis and survival rates, the roles of paclitaxel (PTX) and monopolar spindle kinase 1 (Mps1) in OS were investigated using in vivo and in vitro models. Mps1 expression was upregulated in OS samples and associated with patient survival times. Moreover, spindle assembly checkpoint (SAC) activation and upregulation of Akt/mTOR signaling were both positively associated with OS progression. PTX treatment significantly inhibited Mps1 expression, as well as migration of OS cells both in vitro. In addition, the combination of Mps1 knockdown and PTX treatment inhibited OS progression in vivo. Mps1 overexpression inhibited the expression of SAC markers and upregulated Akt and mTOR expression, while Mps1 knockdown had the opposite effect. Cells subjected to combined Mps1 knockdown and PTX treatment exhibited activation of SAC and inhibition of Akt/mTOR signaling compared with Mps1 knockdown or PTX treatment alone. Based on these observations, Mps1 inhibition combined with PTX treatment may represent a potentially effective strategy for the treatment of OS.

Abstract 980: Preclinical evaluation of mps1 inhibition in uterine leiomyosarcoma

Abstract Background: Uterine leiomyosarcoma (Ut-LMS) is the commonest malignant mesenchymal tumor arising from the uterus. It is aggressive and the prognosis is dismal. To-date, no specific oncogenic drivers have been identified. Conventional adjuvant therapy and immunotherapy are ineffective and treatment largely remains surgical. MPS1 kinase is vital in spindle assembly checkpoint which safeguards the faithful segregation of chromosomes. Its inactivation leads to mitotic defects and induces mitotic catastrophe. Compare with normal tissue, MPS1 has shown to be overexpressed in Ut-LMS. Method: The Ut-LMS cell lines SK-UT-1 and SKN were treated with a MPS1 inhibitor. RT-qPCR was performed to analyze mRNA expression levels of Spindle Assembly Checkpoint-related genes. Cells were seeded in 96-well plates and treated with MPS1 inhibitor for 4 days. Sulforhodamine B stain was used to labelled viable cells. IC50 of different cell lines was determined by SRB assay. Cell cycle analysis was done after 2 days treatment by propidium iodide staining. Apoptosis study was done after 3 days treatment by AnnexinV/PI apoptotic assay. Flow cytometry was used to analyze PI assay and AnnexinV/PI apoptotic assay. Immunofluorescence staining of centromere, α-tubulin and DAPI was used to label kinetochore, mitotic spindles and DNA. Cells with chromosome missegregation were counted in treated and non-treated samples. Ut-LMS cell lines SKN was injected subcutaneously in Balb/c nude mice and MPS1 inhibitor was administrated through oral gavage daily, tumor weight was record and tumor size was estimated by caliper. Results: The Spindle Assembly Checkpoint-related genes MPS1, CDC20, MAD2L1, BUB1, BUB1B, and KNL1 were found to be overexpressed. SRB assays had shown that there was growth inhibition of both cell lines in a concentration-dependent manner with IC50 values ranged between 40-65 nM. An increase in aneuploidy cells was demonstrated by PI assay, which suggested that MPS1 inhibitor disrupted cell cycle and induced aneuploidy. The inhibitor also induced apoptosis as demonstrated an increase in AnnexinV+/PI+ cells. Chromosome missegregation was demonstrated in double-thymidine block synchronized anaphase cells with immunofluorescence staining. Micronuclei were also identified in tetraploid cells. In a preliminary in vivo study, MPS1 inhibitor was shown to reduce tumor growth in xenograft models. Conclusion: Our preclinical results indicated that MPS1 inhibitor was an effective treatment in Ut-LMS as a single agent by causing chromosome missegregation, mitotic catastrophe, genomic instability and cell death. Further evaluation of its use in combination with other pharmacological agents is in progress. Citation Format: Ho Shing Wong, Hok Yeung H. Lee, Ka Yu Tse, Tak Wah Mak, Philip PC Ip. Preclinical evaluation of mps1 inhibition in uterine leiomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 980.

Mps1 controls spindle assembly, SAC, and DNA repair in the first cleavage of mouse early embryos.

Monopolar spindle-1 (Mps1) is a critical interphase regulator that also involves into the spindle assembly checkpoint for the cell cycle control in both mitosis and meiosis. However, the functions of Mps1 during mouse early embryo development is still unclear. In this study, we reported the important roles of Mps1 in the first cleavage of mouse embryos. Our data indicated that the loss of Mps1 activity caused precocious cleavage of zygotes to 2-cell embryos; however, prolonged culture disturbed the early embryo development to the blastocyst. We found that the spindle organization was disrupted after Mps1 inhibition, and the chromosomes were misaligned in the first cleavage. Moreover, the kinetochore-microtubule attachment was lost and Aurora B failed to accumulate to the kinetochores, indicating that the spindle assembly checkpoint (SAC) was activated. Furthermore, the inhibition of Mps1 activity resulted in an increase of DNA damage, which further induced oxidative stress, showing with positive γ-H2A.X signal and increased reactive oxygen species level. Ultimately, irreparable DNA damage and oxidative stress-activated apoptosis and autophagy, which was confirmed by the positive Annexin-V signal and increased autophagosomes. Taken together, our data indicated that Mps1 played important roles in the control of SAC and DNA repair during mouse early embryo development.

Abstract 608: The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML)

Abstract Background: AML is a genetically heterogenous disease with poor prognosis and new treatments are needed. MPS1 is the main kinase of the spindle assembly checkpoint (SAC), critical for segregation of chromatids during mitosis. A hallmark of cancer cells is chromosomal instability caused by deregulated cell cycle checkpoints and SAC dysfunction. MPS1 inhibition has been investigated in several solid tumors but not yet in AML. Here, we demonstrate the efficacy of the small molecule MPS1 inhibitor (MPS1i) S81694 in AML cell lines, primary cells and mouse models. Materials and Methods: Cell viability, cell stress and apoptosis were assessed by MTS assays, DiOC2(3) or annexin-V modifications and PI incorporation after exposure to S81694. Cell-cycle and polyploidy were determined by cytofluorometry and FISH. Phosphorylation of MPS1 was detected in synchronized cells by immunofluorescence detecting phosphorylated Thr33/Ser37 residues. Protein modifications were studied by WB. Time-lapse microscopy was used to determine mitosis duration using cells stained with the SiR-DNA live cell probe. Bone marrow cells from patients were obtained after informed consent and incubated on a MSC feeder or in methylcellulose for colony formation assays. All animal studies were performed in accordance with the animal ethics committee's guidelines. NSG mice were sub-lethally-irradiated and injected with AML-NS8 or AML-PS patient derived xenografts. Mice were treated intravenously with S81694 or with vehicle. Results: IC50 for viability after S81694 exposure alone was <1000nM in a panel of 8 AML cell lines. Three cell lines had IC50 >1000nM: the NPM1cmut cell line OCI-AML3, OCI-AML3-TP53mut and K562. In the most sensitive cell lines, including OCI-AML2, S81694 led to inhibition of MPS1 auto-phosphorylation, induced significant cell stress and apoptosis as detected by mitochondrial membrane potential loss, phosphatidylserine exposure and PI incorporation. In these cell lines, the cell cycle was strongly affected by treatment with S81694 showing aberrant 2n/4n ploidy distribution due to SAC abrogation as well as polyploidy (8n and 16n). S81694 exposure triggered mitotic exit as shown by cyclin B1 downregulation and significantly accelerated mitosis. For treated OCI-AML2 and OCI-AML3 cells we observed induction of gamma-H2AX (ser139), p53 upregulation and downstream caspase-3 and PARP cleavage indicating that S81694 induces p53 dependent apoptosis. Furthermore, MPS1i induced downregulation of anti-apoptotic proteins MCL-1 and BCLXL. Finally, for AML patient derived blast cells cells we observed apoptosis after exposure to S81694 and significant reduction of colony number. In two AML PDX models, intravenous administration of S81694 was shown to improve significantly median survival time, as compared to control, by 28 to 41 days and by 33 to 46 days respectively. Conclusion: The MPS1i S81694 shows significant preclinical activity in vitro and in vivo in AML. Combinations with different drugs active in AML are ongoing. Citation Format: Anna Kaci, Emilie Adicéam, Etienne De Braekeleer, Marine Garrido, Jeannig Berrou, Mélanie Dupont, Hanane Djamai, Virginie Eclache, Baruchel André, Claude Gardin, Hervé Dombret, Mike Burbridge, Thorsten Braun. The MPS1 inhibitor S81694 is active in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 608.

Inhibition of mitotic kinase Mps1 promotes cell death in neuroblastoma

Neuroblastoma is the most common paediatric cancer type. Patients diagnosed with high-risk neuroblastoma have poor prognosis and occasionally tumours relapse. As a result, novel treatment strategies are needed for relapse and refractory neuroblastoma patients. Here, we found that high expression of Mps1 kinase (mitotic kinase Monopolar Spindle 1) was associated with relapse-free neuroblastoma patient outcomes and poor overall survival. Silencing and inhibition of Mps1 in neuroblastoma or PDX-derived cells promoted cell apoptosis via the caspase-dependent mitochondrial apoptotic pathway. The mechanism of cell death upon Mps1 inhibition was dependent on the polyploidization/aneuploidization of the cells before undergoing mitotic catastrophe. Furthermore, tumour growth retardation was confirmed in a xenograft mouse model after Mps1-inhibitor treatment. Altogether, these results suggest that Mps1 expression and inhibition can be considered as a novel prognostic marker as well as a therapeutic strategy for the treatment of high-risk neuroblastoma patients.

Chromosomal instability upregulates interferon in acute myeloid leukemia.

Chromosome instability (CIN) generates genetic and karyotypic diversity that is common in hematological malignancies. Low to moderate levels of CIN are well tolerated and can promote cancer proliferation. However, high levels of CIN are lethal. Thus, CIN may serve both as a prognostic factor to predict clinical outcome and as a predictive biomarker. A retrospective study was performed to evaluate CIN in acute myeloid leukemia (AML). Chromosome mis-segregation frequency was correlated with clinical outcome in bone marrow core biopsy specimens from 17 AML cases. Additionally, we induced chromosome segregation errors in AML cell lines with AZ3146, an inhibitor of the Mps1 mitotic checkpoint kinase, to quantify the phenotypic effects of high CIN. We observed a broad distribution of chromosome mis-segregation frequency in AML bone marrow core specimens. High CIN correlated with complex karyotype in AML, as expected, although there was no clear survival effect. In addition to CIN, experimentally inducing chromosome segregation errors by Mps1 inhibition in AML cell lines causes DNA damage, micronuclei formation, and upregulation of interferon stimulated genes. High levels of CIN appear to be immunostimulatory, suggesting an opportunity to combine mitotic checkpoint inhibitors with immunotherapy in treatment of AML.

Targeting Insulin-Like Growth Factor 1 Receptor Delays M-Phase Progression and Synergizes with Aurora B Inhibition to Suppress Cell Proliferation.

The insulin-like growth factor 1 receptor (IGF1R) is a receptor-type tyrosine kinase that transduces signals related to cell proliferation, differentiation, and survival. IGF1R expression is often misregulated in tumor cells, but the relevance of this for cancer progression remains unclear. Here, we examined the impact of IGF1R inhibition on cell division. We found that siRNA-mediated knockdown of IGF1R from HeLa S3 cells leads to M-phase delays. Although IGF1R depletion causes partial exclusion of FoxM1 from the nucleus, quantitative real-time PCR revealed that the transcription of M-phase regulators is not affected by decreased levels of IGF1R. Moreover, a similar delay in M phase was observed following 2 h of incubation with the IGF1R inhibitors OSI-906 and NVP-ADW742. These results suggest that the M-phase delay observed in IGF1R-compromised cells is not caused by altered expression of mitotic regulators. Live-cell imaging revealed that both prolonged prometaphase and prolonged metaphase underlie the delay and this can be abrogated by the inhibition of Mps1 with AZ3146, suggesting activation of the Spindle Assembly Checkpoint when IGF1R is inhibited. Furthermore, incubation with the Aurora B inhibitor ZM447439 potentiated the IGF1R inhibitor-induced suppression of cell proliferation, opening up new possibilities for more effective cancer chemotherapy.

High Proliferation Rate and a Compromised Spindle Assembly Checkpoint Confers Sensitivity to the MPS1 Inhibitor BOS172722 in Triple-Negative Breast Cancers.

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In in vivo pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant in vivo efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel.

Deletion of APC7 or APC16 Allows Proliferation of Human Cells without the Spindle Assembly Checkpoint

SummaryThe multisubunit ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) is essential for mitosis by promoting timely degradation of cyclin B1. APC/C is tightly regulated by the spindle assembly checkpoint (SAC), which involves MPS1 and MAD2-dependent temporal inhibition of APC/C. We analyzed the contribution of the APC/C subunits APC7 and APC16 to APC/C composition and function in human cells. APC16 is required for APC7 assembly into APC/C, whereas APC16 assembles independently of APC7. APC7 and APC16 knockout cells display no major defects in mitotic progression, cyclin B1 degradation, or SAC response, but APC/C lacking these two subunits shows reduced ubiquitylation activity in vitro. Strikingly, deletion of APC7 or APC16 is sufficient to provide synthetic viability to MAD2 deletion. ΔAPC7ΔMAD2 cells display accelerated mitosis and require SAC-independent MPS1 function for genome stability. These findings reveal that the composition of APC/C critically influences the importance of the SAC in humans.

Abstract 4950: MPS1 as a novel target in endocrine- and palbociclib-resistant estrogen receptor positive breast cancer

Abstract Background: Despite treatment, many estrogen receptor positive (ER+) breast cancer (BC) patients, relapse with de novo or acquired endocrine resistant disease. Using a kinome siRNA library screen, we identified MPS1, which is required for recruitment of the spindle assembly checkpoint complex, as strongly associated with resistance to both endocrine therapy and CDK4/6 inhibitor palbociclib, a contemporary first-line combination for advanced ER+ BC. Until now, the target population for MPS1 inhibitors has been triple negative BC. Here we show for the first time, potential efficacy of MPS1 inhibitors in endocrine resistant BC models. Methods: A panel of ER+ BC cell lines adapted to estrogen independent growth (LTED) or after additional resistance to palbociclib (palboR), was subjected to a siRNA kinome screen targeting 709 genes. Kinases causative of resistance were identified using Z-scores. Cell viability upon MPS1 inhibition (MPS1i) with BOS172722 was assessed 2D and 3D, and the class effect confirmed with other compounds targeting MPS1. Impact of MPS1i on ER-mediated transcription was assessed using luciferase reporter assays. Effect of MPS1i on chromosomal alignment and time spent in mitosis was established by time lapse and confocal microscopy. Cell cycle and DNA content were analysed by FACS. Apoptosis was assessed using PARP cleavage. Efficacy of BOS172722 in a xenograft model of LTED was evaluated. Results: Kinome knockdown identified MPS1 as the top common hit in LTED and palboR cell lines. Increased MPS1 was evident in MCF7-LTED at the transcript and protein level. Notably, BOS172722 significantly reduced viability of the majority of LTED and palboR cell lines tested (IC50 between 25-100nM), an effect shown to be class specific using a second MPS1i. Upon MPS1i, cells demonstrated shorter time in mitosis, aberration of cell cycle, increased mitotic errors, culminating in apoptosis. Of note, as little as 48hr exposure to BOS172722 reduced colony formation over 3 weeks suggesting early accumulation of errors is sufficient to provide a long term anti-proliferative effect. To evaluate the clinical relevance of MPS1 in ER+ BC treated with endocrine therapy, we interrogated publicly available datasets from patients treated with neoajuvant AI therapy. In the anastrozole cohort, on-treatment MPS1 expression was significantly (p<0.0001) associated with poor response to anastrozole based on a 2-week residual Ki67 score <10%. In the letrozole cohort, increased on-treatment expression of MPS1 (p=0.0118) was associated with poor response based on tumor shrinkage <50%. Finally, in a xenograft model of AI relapse, BOS172722 caused significant tumor shrinkage, compared to vehicle. Conclusion: For the first time, we show that MPS1 inhibitors are capable of inducing mitotic aberrations and apoptosis in ER+ BC resistant to endocrine therapy and palbociclib providing a new therapeutic strategy. Citation Format: Joanna Nikitorowicz-Buniak, Sunil Pancholi, Nikiana Simigdala, Ricardo Ribas, Spiros Linardopoulos, Mitch Dowsett, Stephen Johnston, Lesley-Ann Martin. MPS1 as a novel target in endocrine- and palbociclib-resistant estrogen receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4950.