Haspin Kinase Research Articles

A protein phosphatase 1 specific phosphatase targeting peptide (PhosTAP) to identify the PP1 phosphatome

Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates ~80% of all ser/thr phosphorylation sites. Here, we developed a phosphatase targeting peptide (PhosTAP) that binds all PP1 isoforms and does so with a stronger affinity than any other known PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for Phosphorylation Targeting Chimera (PhosTAC)-type recruitment in in vitro and cellular experiments, as well as in phosphoproteomics experiments to identify PP1-specific substrates and phosphosites. The latter is especially important to further our understanding of cellular signaling, as the identification of substrates and especially phosphosites that are targeted by specific phosphatases lags behind that of their kinase counterparts. Using PhosTAP-based proteomics, we show that, counter to our current understanding, many PP1 regulators are also substrates, that the number of residues between regulator PP1-binding and phosphosites vary significantly, and that PP1 counteracts the activities of mitotic kinases. Finally, we also found that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Together, we show that PP1-specific PhosTAPs are a powerful tool for +studying PP1 activity in vitro and in cells.

The Natural HASPIN Inhibitor Coumestrol Suppresses Intestinal Polyp Development, Cachexia, and Hypogonadism in a Mouse Model of Familial Adenomatous Polyposis (ApcMin/+).

(1) Background: HASPIN kinase is involved in regulating spindle function and chromosome segregation, as well as phosphorylating histone H3 at Thr3 in mitotic cells. Several HASPIN inhibitors suppress cancer cell proliferation. It was recently reported that coumestrol from bean sprouts inhibits HASPIN, and a cultivation method for bean sprouts containing large amounts of coumestrol has been established. Here, we showed the effects of bean sprout ingestion on intestinal polyp development, cachexia, and hypogonadism in a mouse model of familial adenomatous polyposis (ApcMin/+). (2) Methods: ApcMin/+ mice were randomized into control and treatment groups. Mice in the control group were given the standard diet, while those in the treatment group were given the same standard diet with the addition of 15% bean sprouts. Treatments were commenced at 7 weeks old and analyses were performed at 12 weeks old. (3) Results: ingesting bean sprouts suppressed the development of intestinal polyps, cachexia, and hypogonadism, and also increased serum levels of testosterone in male wild-type and ApcMin/+ mice. (4) Conclusions: ingesting bean sprouts helps prevent cancer and increases serum levels of testosterone in a mouse model. These results are expected to be applicable to humans.

Aurora B and Aurora C pools at two chromosomal regions collaboratively maintain chromosome alignment and prevent aneuploidy at the second meiotic division in mammalian oocytes.

Correct chromosome segregation is essential to preserve genetic integrity. The two protein kinases, Aurora B and its meiotic homolog Aurora C, regulate attachments between chromosomal kinetochores and microtubules, thereby contributing to the accuracy of the chromosome segregation process. Here we performed a detailed examination of the localization and activity of Aurora B/C kinases, their partner Incenp and the kinetochore target Hec1, during the second meiotic division in mouse oocytes. We found that a majority of Aurora B and C changed their localization from the outer kinetochore region of chromosomes at prometaphase II to an inner central region localized between sister centromeres at metaphase II. Depletion of the Aurora B/C pool at the inner central region using the haspin kinase inhibitor 5-iodotubercidin resulted in chromosome misalignments at the metaphase II stage. To further understand the role of the Aurora B/C pool at the central region, we examined the behaviour of single chromatids, that lack a central Aurora B/C pool but retain Aurora B/C at the outer kinetochores. We found that kinetochore-microtubule attachments at single chromatids were corrected at both prometaphase II and metaphase II stages, but that single chromatids compared to paired chromatids were more prone to misalignments following treatment of oocytes with the Aurora B/C inhibitory drugs AZD1152 and GSK1070916. We conclude that the Aurora B/C pool at the inner central region stabilizes chromosome alignment during metaphase II arrest, while Aurora B/C localized at the kinetochore assist in re-establishing chromosome positioning at the metaphase plate if alignment is lost. Collaboratively these two pools prevent missegregation and aneuploidy at the second meiotic division in mammalian oocytes.

Discovery of a New Dihydropyrimidinone Derivative as a Potential Haspin Kinase Inhibitor: Structure‐Guided Insights, In‐Vitro and Molecular Dynamics‐Based Validation

AbstractStructure‐based virtual screening is efficient method for finding novel scaffolds/molecules for biological targets. Due to the accessibility of the Haspin crystal structure and its prevalence in various diseases; we sought exploring novel Haspin kinase inhibitors. We used the commercially available Chembridge library for virtual screening which had nearly 7,00,000 compounds. We employed variety of computational tools such as molecular docking, molecular dynamics simulations, in silico pharmacokinetics analysis to identify novel inhibitors targeting Haspin kinase. Based on chemical features, docking score, and MMGB/SA binding strength, 10 ligands were chosen. On in vitro determination, one chemical entity was found to inhibit Haspin's enzyme function at 72 % at 10 μM. Compound 16503395 had higher selectivity over Haspin kinase activity than the other kinases. The IC50 value for the compound 16503395 that inhibits Haspin kinase significantly was determined and was found to be 2.6 μM. After subjecting to molecular dynamics simulation analysis for compound 16503395 Haspin protein complex, we found that the Haspin kinase binding site amino acid unique residues Gly608, Glu492, Lys511, Asn654, Asp687, Ala503, Leu656 and Phe605 play important role in binding kinetics. We claim 5‐(hydroxymethyl)‐N‐[cis‐3‐(6‐oxo‐2‐pyrrolidin‐1‐yl‐1,6‐dihydropyrimidin‐4‐yl)cyclobutyl]‐2‐furamide (16503395) as a selective and potential Haspin kinase inhibitor.

Function and inhibition of Haspin kinase: targeting multiple cancer therapies by antimitosis.

Haploid germ cell-specific nuclear protein kinase (Haspin) is a serine/threonine kinase as an atypical kinase, which is structurally distinct from conventional protein kinases. Functionally, Haspin is involved in important cell cycle progression, particularly in critical mitosis regulating centromeric sister chromatid cohesion during prophase and prometaphase, and subsequently ensuring proper chromosome alignment during metaphase and the normal chromosome segregation during anaphase. However, increasing evidence has demonstrated that Haspin is significantly upregulated in a variety of cancer cells in addition to normal proliferating somatic cells. Its knockdown or small molecule inhibition could prevent cancer cell growth and induce apoptosis by disrupting the regular mitotic progression. Given the specificity of its expressed tissues or cells and the uniqueness of its current known substrate, Haspin can be a promising target against cancer. Consequently, selective synthetic and natural inhibitors of Haspin have been widely developed to determine their inhibitory power for various cancer cells in vivo and in vitro. Here our perspective includes a comprehensive review of the roles and structure of Haspin, its relatively potent and selective inhibitors and Haspin's preliminary studies in a variety of cancers.

Tip60 acetylation of histone H3K4 temporally controls chromosome passenger complex localization.

The Chromosome Passenger Complex (CPC) generates chromosome autonomous signals that regulate mitotic events critical for genome stability. Tip60 is a lysine acetyltransferase that is a tumor suppressor and is targeted for proteasomal degradation by oncogenic papilloma viruses. Mitotic regulation requires the localization of the CPC to inner centromeres, which is driven by the Haspin kinase phosphorylating histone H3 on threonine 3 (H3T3ph). Here we describe how Tip60 acetylates histone H3 at lysine 4 (H3K4ac) to block both the H3T3ph writer and the reader to ensure that this mitotic signaling cannot begin before prophase. Specifically, H3K4ac inhibits Haspin phosphorylation of H3T3 and prevents binding of the Survivin subunit to H3T3ph. Tip60 acetylates H3K4 during S/G2 at centromeres. Inhibition of Tip60 allows the CPC to bind centromeres in G2 cells, and targeting of Tip60 to centromeres prevents CPC localization in mitosis. The H3K4ac mark is removed in prophase by HDAC3 to initiate the CPC localization cascade. Together, our results suggest that Tip60 and HDAC3 temporally control H3K4 acetylation to precisely time the targeting of the CPC to inner centromeres.

Haspin participates in AURKB recruitment to centromeres and contributes to chromosome congression in male mouse meiosis.

Chromosome segregation requires that centromeres properly attach to spindle microtubules. This essential step regulates the accuracy of cell division and must therefore be precisely regulated. One of the main centromeric regulatory signaling pathways is the haspin-H3T3ph-chromosomal passenger complex (CPC) cascade, which is responsible for the recruitment of the CPC to the centromeres. During mitosis, the haspin kinase phosphorylates histone H3 at threonine 3 (H3T3ph), an essential epigenetic mark that recruits the CPC, in which the catalytic component is Aurora B kinase (AURKB). However, the centromeric haspin-H3T3ph-CPC pathway remains largely uncharacterized in mammalian male meiosis. We have analyzed haspin functions by either its chemical inhibition with LDN-192960 in cultured spermatocytes, or the ablation of the Haspin gene in Haspin-/- mice. Our studies suggest that haspin kinase activity is required for proper chromosome congression both during meiotic divisions and for the recruitment of Aurora B and kinesin MCAK (also known as KIF2C) to meiotic centromeres. However, the absence of H3T3ph histone mark does not alter borealin (or CDCA8) and SGO2 centromeric localization. These results add new and relevant information regarding the regulation of the haspin-H3T3ph-CPC pathway and centromere function during meiosis.

Design and biological evaluation of substituted 5,7-dihydro-6H-indolo[2,3-c]quinolin-6-one as novel selective Haspin inhibitors

A library of substituted indolo[2,3-c]quinolone-6-ones was developed as simplified Lamellarin isosters. Synthesis was achieved from indole after a four-step pathway sequence involving iodination, a Suzuki-Miyaura cross-coupling reaction, and a reduction/lactamization sequence. The inhibitory activity of the 22 novel derivatives was assessed on Haspin kinase. Two of them possessed an IC50 of 1 and 2 nM with selectivity towards a panel of 10 other kinases including the parent kinases DYRK1A and CLK1. The most selective compound exerted additionally a very interesting cell effect on the osteosarcoma U-2 OS cell line.

Ginsenoside Rg1 suppresses cancer cell proliferation through perturbing mitotic progression

BackgroundAlthough the tumor-suppressive effects of ginsenosides in cell cycle have been well established, their pharmacological properties in mitosis have not been clarified yet. The chromosomal instability resulting from dysregulated mitotic processes is usually increased in cancer. In this study, we aimed to investigate the anticancer effects of ginsenoside Rg1 on mitotic progression in cancer. Materials and methodsCancer cells were treated with ginsenoside Rg1 and their morphology and intensity of different protein were analyzed using immunofluorescence microscopy. The level of proteins in chromosomes was compared through chromosomal fractionation and Western blot analyses. The location and intensity of proteins in the chromosome were confirmed through immunostaining of mitotic chromosome after spreading. The colony formation assays were conducted using various cancer cell lines. ResultsGinsenoside Rg1 reduced cancer cell proliferation in some cancers through inducing mitotic arrest. Mechanistically, it inhibits the phosphorylation of histone H3 Thr3 (H3T3ph) mediated by Haspin kinase and concomitant recruitment of chromosomal passenger complex (CPC) to the centromere. Depletion of Aurora B at the centromere led to abnormal centromere integrity and spindle dynamics, thereby causing mitotic defects, such as increase in the width of the metaphase plate and spindle instability, resulting in delayed mitotic progression and cancer cell proliferation. ConclusionGinsenoside Rg1 reduces the level of Aurora B at the centromere via perturbing Haspin kinase activity and concurrent H3T3ph. Therefore, ginsenoside Rg1 suppresses cancer cell proliferation through impeding mitotic processes, such as chromosome alignment and spindle dynamics, upon depletion of Aurora B from the centromere.

The C‐terminus of VRK1 Governs Interactions with Nucleosomes to Influence Histone H3 Threonine 3 (H3T3) Phosphorylation

Vaccinia-related kinase 1 (VRK1) is a highly abundant nuclear kinase that has numerous functions during mitosis, including the phosphorylation of the histone H3 tail at threonine 3 (H3T3ph). This post-translational modification, attributed to both VRK1 and the haspin kinase, is crucial for ensuring appropriate chromosome segregation. Despite the importance of VRK1 to cell cycle regulation, little is known about the biochemical basis of its activity on nucleosomes. Because placement of histone phosphorylation during mitosis is tightly spatiotemporally regulated, we sought to determine the chromatin template itself serves as a regulatory mechanism for VRK1 activity. In this work, we characterize the interaction between VRK1 and nucleosomes and determine how nucleosome binding affects nucleosome-directed kinase activity at H3T3. We find that VRK1 directly and stably interacts with nucleosomes with an apparent Kd of ~200 nM. VRK1 binds nucleosomes via a C-terminal alternating arginine motif, mutations or truncations of which are found in rare cases of genetic spinal muscular atrophy. Mutation or truncation of this motif leads to a 4-50 fold reduction in nucleosome affinity that is correlated with mutation position and severity. C-terminal arginine mutants of VRK1 show reduced capacity for histone H3T3 phosphorylation, suggesting that stable interaction with nucleosomes is required for nucleosome-directed kinase activity at H3T3. The nucleosome acidic patch, a negatively charged region at the dimer interface of H2A/H2B, is a hot spot for arginine-mediated nucleosome interactions. Neutralization of the acidic patch impairs VRK1 binding but surprisingly increases the activity of VRK1 at H3T3. In contrast, the presence of extended lengths of linker DNA promotes both nucleosome binding and phosphorylation of H3T3. Cryo-EM single particle analysis of VRK1-bound nucleosome particles demonstrates both acidic patch binding and heterogenous DNA binding that concentrates around the nucleosome linker DNA. This evidence suggests that VRK1 adopts at least two alternative binding modes on the nucleosome (acidic patch-governed vs. linker DNA-governed) that either promote or inhibit nucleosome-directed kinase activity based on the position and distance of VRK1 from the H3 tail. We suggest a model wherein dynamic access to the nucleosome acidic patch regulates the activity of VRK1 towards H3T3 during mitosis.

Synthesis and evaluation of C3 substituted chalcone-based derivatives of 7-azaindole as protein kinase inhibitors.

Chalcones are a group of naturally occurring or synthetic compounds which possess a wide range of biological activities. In this paper, a series of twenty-three 7-azaindole-chalcone hybrids (5a-w) were synthesized and evaluated as potential protein kinase inhibitors. Analyses of structure-activity relationships revealed that some of these compounds exhibit significant activity against Haspin kinase, with compounds 5f and 5q exhibiting IC50 values of 0.47 and 0.41µM, respectively. Furthermore, 5f also inhibits cyclin-dependent kinase 9 (CDK9/CyclinT) in a micromolar potency (IC50 =2.26µM). This novel dual-target inhibitor is a promising lead for the development of chemopreventive/chemotherapeutic agents.

Haspin kinase modulates nuclear architecture and Polycomb-dependent gene silencing.

Haspin, a highly conserved kinase in eukaryotes, has been shown to be responsible for phosphorylation of histone H3 at threonine 3 (H3T3ph) during mitosis, in mammals and yeast. Here we report that haspin is the kinase that phosphorylates H3T3 in Drosophila melanogaster and it is involved in sister chromatid cohesion during mitosis. Our data reveal that haspin also phosphorylates H3T3 in interphase. H3T3ph localizes in broad silenced domains at heterochromatin and lamin-enriched euchromatic regions. Loss of haspin compromises insulator activity in enhancer-blocking assays and triggers a decrease in nuclear size that is accompanied by changes in nuclear envelope morphology. We show that haspin is a suppressor of position-effect variegation involved in heterochromatin organization. Our results also demonstrate that haspin is necessary for pairing-sensitive silencing and it is required for robust Polycomb-dependent homeotic gene silencing. Haspin associates with the cohesin complex in interphase, mediates Pds5 binding to chromatin and cooperates with Pds5-cohesin to modify Polycomb-dependent homeotic transformations. Therefore, this study uncovers an unanticipated role for haspin kinase in genome organization of interphase cells and demonstrates that haspin is required for homeotic gene regulation.

Haspin regulates Ras localization to promote Cdc24-driven mitotic depolarization

Cell polarization is of paramount importance for proliferation, differentiation, development, and it is altered during carcinogenesis. Polarization is a reversible process controlled by positive and negative feedback loops. How polarized factors are redistributed is not fully understood and is the focus of this work. In Saccharomyces cerevisiae, mutants defective in haspin kinase exhibit stably polarized landmarks and are sensitive to mitotic delays. Here, we report a new critical role for haspin in polarisome dispersion; failure to redistribute polarity factors, in turn, leads to nuclear segregation defects and cell lethality. We identified a mitotic role for GTP-Ras in regulating the local activation of the Cdc42 GTPase, resulting in its dispersal from the bud tip to a homogeneous distribution over the plasma membrane. GTP-Ras2 physically interacts with Cdc24 regulateing its mitotic distribution. Haspin is shown to promote a mitotic shift from a bud tip-favored to a homogenous PM fusion of Ras-containing vesicles. In absence of haspin, active Ras is not redistributed from the bud tip; Cdc24 remains hyperpolarized promoting the activity of Cdc42 at the bud tip, and the polarisome fails to disperse leading to erroneously positioned mitotic spindle, defective nuclear segregation, and cell death after mitotic delays. These findings describe new functions for key factors that modulate cell polarization and mitotic events, critical processes involved in development and tumorigenesis.

Synthesis and evaluation of 7-azaindole derivatives bearing benzocycloalkanone motifs as protein kinase inhibitors

Protein kinases are important drug targets, especially in the area of oncology. This paper reports the synthesis and biological evaluation of new 7-azaindole derivatives bearing benzocycloalkanone motifs as potential protein kinase inhibitors. Four compounds 8g, 8h, 8i, and 8l were discovered to inhibit cyclin-dependent kinase 9 (CDK9/CyclinT) and/or Haspin kinase in the micromolar to nanomolar range. 8l was identified as the most potent Haspin inhibitor (IC50 = 14 nM), while 8g and 8h acted as dual inhibitors of CDK9/CyclinT and Haspin. These novel compounds constitute a promising starting point for the discovery of dual protein kinase inhibitors that have potential to be developed as anticancer agents, since both CDK9/CyclinT and Haspin are considered to be drug targets in oncology.

Asymmetric assembly of centromeres epigenetically regulates stem cell fate.

Centromeres are epigenetically defined by CENP-A-containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. We show that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. Our work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis.

Inhibition of Haspin Kinase Promotes Cell-Intrinsic and Extrinsic Antitumor Activity.

Patients with melanoma resistant to RAF/MEK inhibitors (RMi) are frequently resistant to other therapies, such as immune checkpoint inhibitors (ICI), and individuals succumb to their disease. New drugs that control tumor growth and favorably modulate the immune environment are therefore needed. We report that the small-molecule CX-6258 has potent activity against both RMi-sensitive (RMS) and -resistant (RMR) melanoma cell lines. Haspin kinase (HASPIN) was identified as a target of CX-6258. HASPIN inhibition resulted in reduced proliferation, frequent formation of micronuclei, recruitment of cGAS, and activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway. In murine models, CX-6258 induced a potent cGAS-dependent type-I IFN response in tumor cells, increased IFNγ-producing CD8+ T cells, and reduced Treg frequency in vivo. HASPIN was more strongly expressed in malignant compared with healthy tissue and its inhibition by CX-6258 had minimal toxicity in ex vivo-expanded human tumor-infiltrating lymphocytes (TIL), proliferating TILs, and in vitro differentiated neurons, suggesting a potential therapeutic index for anticancer therapy. Furthermore, the activity of CX-6258 was validated in several Ewing sarcoma and multiple myeloma cell lines. Thus, HASPIN inhibition may overcome drug resistance in melanoma, modulate the immune environment, and target a vulnerability in different cancer lineages. SIGNIFICANCE: HASPIN inhibition by CX-6258 is a novel and potent strategy for RAF/MEK inhibitor-resistant melanoma and potentially other tumor types. HASPIN inhibition has direct antitumor activity and induces a favorable immune microenvironment.

PRMT6-mediated H3R2me2a guides Aurora B to chromosome arms for proper chromosome segregation

The kinase Aurora B forms the chromosomal passenger complex (CPC) together with Borealin, INCENP, and Survivin to mediate chromosome condensation, the correction of erroneous spindle-kinetochore attachments, and cytokinesis. Phosphorylation of histone H3 Thr3 by Haspin kinase and of histone H2A Thr120 by Bub1 concentrates the CPC at the centromere. However, how the CPC is recruited to chromosome arms upon mitotic entry is unknown. Here, we show that asymmetric dimethylation at Arg2 on histone H3 (H3R2me2a) by protein arginine methyltransferase 6 (PRMT6) recruits the CPC to chromosome arms and facilitates histone H3S10 phosphorylation by Aurora B for chromosome condensation. Furthermore, in vitro assays show that Aurora B preferentially binds to the H3 peptide containing H3R2me2a and phosphorylates H3S10. Our findings indicate that the long-awaited key histone mark for CPC recruitment onto mitotic chromosomes is H3R2me2a, which is indispensable for maintaining appropriate CPC levels in dynamic translocation throughout mitosis.

HASPIN kinase inhibitor CHR-6494 suppresses intestinal polyp development, cachexia, and hypogonadism in Apcmin/+ mice.

HASPIN has been identified as a nuclear Ser/Thr kinase specifically expressed in haploid germ cells. HASPIN kinase inhibitors were recently isolated, and their antitumor activity reported. Colorectal cancer occurs with high incidence worldwide. In this study, we examined whether HASPIN inhibitor CHR-6494 suppresses cancer progression in ApcMin/+ mice, a familial colon tumor disease model. Mice were treated by intraperitoneal injection of CHR-6494 for 50 days. Following the treatment period, intestinal polyps were counted and testosterone and spermatogenesis levels were observed. Intraperitoneal administration of CHR-6494 significantly inhibited intestinal polyp development and recovered body weight in ApcMin/+ mice. Although spermatogenesis was inhibited with increasing age in ApcMin/+ mice, CHR-6494 significantly improved blood testosterone levels and spermatogenesis. Our results suggest that HASPIN inhibitors may be useful as anti-cancer agents and for the treatment of hypogonadism in colorectal cancer patients.

Abstract B132: Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy

Abstract Oncogene-targeted therapies are initially effective in most patients with BRAF-mutated melanoma. However, patients frequently develop resistance during therapy. At the time of therapy resistance, they are also less likely to respond to immune checkpoint inhibitors (ICI) with response rates ranging from 0-12%, compared to 40-60% when used in the first line. This may be in part be due to rapid tumor growth that outpaces pharmacodynamics of ICI. New drugs that directly control tumor growth and effectively enhance ICI activity are therefore needed. We report that the small molecule CX-6258 has potent activity against both RAF/MEK inhibitor (RMS) and resistant (RMR) BRAF-mutant melanoma cell lines. CX-6258 is annotated as a pan-PIM kinase inhibitor, with an EC50 corrected for growth rate (GR50) of ~200 nM; in contrast, other PIM inhibitors had no in vitro activity, suggesting that PIMs may not be the true target in melanoma - we therefore sought to systematically study the targets of this compound. Using an unbiased kinase profiling approach, we identified Haspin Kinase (HASPIN) as the key target of CX-6258 with an IC50~10nM. Haspin kinase (Haspin) is a poorly understood mitotic kinase, whose only well-defined function is phosphorylation of Histon H3 at Thr3 (pH3T3). Treatment with CX-6258 resulted in reduced proliferation, reduced phosphorylation of pH3T3, frequent formation of micronuclei (MN), recruitment of cGAS and activation of the cGAS-STING-pathway. By using CRISPR/CAS9 knock outs of PIM kinase we show that these targets are not required for CX-6258 function. Furthermore, siRNA mediated knock-down of HASPIN phenocopied CX-6258. In murine models, CX-6258 induces a potent cGAS dependent type-I-interferon response in tumor cells, increases IFNγ-producing CD8+ T-cells, reduces Tregs, and enhances responses to ICIs in vivo. HASPIN is more strongly expressed in malignant compared to healthy tissue and its inhibition by CX-6258 has minimal toxicity in ex vivo expanded human tumor-infiltrating-lymphocytes (TILs) and in vitro differentiated human neurons, suggesting a potential therapeutic index for anti-cancer therapy. CX-6258 also shows activity in several Ewing sarcoma and multiple myeloma cell lines with. In summary, HASPIN inhibition may overcome drug resistance in melanoma, synergize with ICIs and target a vulnerability in different cancer lineages. Citation Format: Johannes C. Melms, Sreeram Vallabhaneni, Caitlin E. Mills, Kai Wucherpfennig, Peter K. Sorger, Benjamin Izar. Inhibition of Haspin kinase to promote cell-intrinsic and extrinsic anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B132. doi:10.1158/1535-7163.TARG-19-B132

Topoisomerase II SUMOylation activates a metaphase checkpoint via Haspin and Aurora B kinases.

Topoisomerase II (Topo II) is essential for mitosis since it resolves sister chromatid catenations. Topo II dysfunction promotes aneuploidy and drives cancer. To protect from aneuploidy, cells possess mechanisms to delay anaphase onset when Topo II is perturbed, providing additional time for decatenation. Molecular insight into this checkpoint is lacking. Here we present evidence that catalytic inhibition of Topo II, which activates the checkpoint, leads to SUMOylation of the Topo II C-terminal domain (CTD). This modification triggers mobilization of Aurora B kinase from inner centromeres to kinetochore proximal centromeres and the core of chromosome arms. Aurora B recruitment accompanies histone H3 threonine-3 phosphorylation and requires Haspin kinase. Strikingly, activation of the checkpoint depends both on Haspin and Aurora B. Moreover, mutation of the conserved CTD SUMOylation sites perturbs Aurora B recruitment and checkpoint activation. The data indicate that SUMOylated Topo II recruits Aurora B to ectopic sites, constituting the molecular trigger of the metaphase checkpoint when Topo II is catalytically inhibited.