A macrocyclic kinase inhibitor overcomes triple resistant mutations in EGFR-positive lung cancer.

Brigatinib-based therapy was effective against osimertinib-resistant EGFR C797S mutants and is undergoing clinical studies. However, tumor relapse suggests additional resistance mutations might emerge. Here, we first demonstrated the binding mode of brigatinib to the EGFR-T790M/C797S mutant by crystal structure analysis and predicted brigatinib-resistant mutations through a cell-based assay including N-ethyl-N-nitrosourea (ENU) mutagenesis. We found that clinically reported L718 and G796 compound mutations appeared, consistent with their proximity to the binding site of brigatinib, and brigatinib-resistant quadruple mutants such as EGFR-activating mutation/T790M/C797S/L718M were resistant to all the clinically available EGFR-TKIs. BI-4020, a fourth-generation EGFR inhibitor with a macrocyclic structure, overcomes the quadruple and major EGFR-activating mutants but not the minor mutants, such as L747P or S768I. Molecular dynamics simulation revealed the binding mode and affinity between BI-4020 and EGFR mutants. This study identified potential therapeutic strategies using the new-generation macrocyclic EGFR inhibitor to overcome the emerging ultimate resistance mutants.

MIG6 loss confers resistance to ALK/ROS1 inhibitors in NSCLC through EGFR activation by low-dose EGF.

Although tyrosine kinase inhibitor (TKI) therapy shows marked clinical efficacy in patients with anaplastic lymphoma kinase-positive (ALK+) and ROS proto-oncogene 1-positive (ROS1+) non-small cell lung cancer (NSCLC), most of these patients eventually relapse with acquired resistance. Therefore, genome-wide CRISPR/Cas9 knockout screening was performed using an ALK+ NSCLC cell line established from pleural effusion without ALK-TKI treatment. After 9 days of ALK-TKI therapy, sequencing analysis was performed, which identified several tumor suppressor genes, such as NF2 or MED12, and multiple candidate genes. Among them, this study focused on ERRFI1, which is known as MIG6 and negatively regulates EGFR signaling. Interestingly, MIG6 loss induced resistance to ALK-TKIs by treatment with quite a low dose of EGF, which is equivalent to plasma concentration, through the upregulation of MAPK and PI3K/AKT/mTOR pathways. Combination therapy with ALK-TKIs and anti-EGFR antibodies could overcome the acquired resistance in both in vivo and in vitro models. In addition, this verified that MIG6 loss induces resistance to ROS1-TKIs in ROS1+ cell lines. This study found a potentially novel factor that plays a role in ALK and ROS1-TKI resistance by activating the EGFR pathway with low-dose ligands.

Targeting Podoplanin for the Treatment of Osteosarcoma.

PURPOSE: Osteosarcoma, the most common bone malignancy in children, has a poor prognosis, especially when the tumor metastasizes to the lungs. Therefore, novel therapeutic strategies targeting both proliferation and metastasis of osteosarcoma are required. Podoplanin (PDPN) is expressed by various tumors and is associated with tumor-induced platelet activation via its interaction with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously found that PDPN contributed to osteosarcoma growth and metastasis through platelet activation; thus, in this study, we developed an anti-PDPN humanized antibody and evaluated its effect on osteosarcoma growth and metastasis. EXPERIMENTAL DESIGN: Nine osteosarcoma cell lines and two osteosarcoma patient-derived cells were collected, and we evaluated the efficacy of the anti-DPN-neutralizing antibody PG4D2 and the humanized anti-PDPN antibody AP201, which had IgG4 framework region. The antitumor and antimetastasis effect of PG4D2 and AP201 were examined in vitro and in vivo. In addition, growth signaling by the interaction between PDPN and CLEC-2 was analyzed using phospho-RTK (receptor tyrosine kinase) array, growth assay, or immunoblot analysis under the supression of RTKs by knockout and inhibitor treatment. RESULTS: We observed that PG4D2 treatment significantly suppressed tumor growth and pulmonary metastasis in osteosarcoma xenograft models highly expressing PDPN. The contribution of PDGFR activation by activated platelet releasates to osteosarcoma cell proliferation was confirmed, and the humanized antibody, AP201, suppressed in vivo osteosarcoma growth and metastasis without significant adverse events. CONCLUSIONS: Targeting PDPN with a neutralizing antibody against PDPN-CLEC-2 without antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is a novel therapeutic strategy for PDPN-positive osteosarcoma.

Soluble PD-L1 works as a decoy in lung cancer immunotherapy via alternative polyadenylation.

Immune checkpoint therapy targeting the PD-1/PD-L1 axis is a potentially novel development in anticancer therapy and has been applied to clinical medicine. However, there are still some problems, including a relatively low response rate, innate mechanisms of resistance against immune checkpoint blockades, and the absence of reliable biomarkers to predict responsiveness. In this study of in vitro and in vivo models, we demonstrate that PD-L1-vInt4, a splicing variant of PD-L1, plays a role as a decoy in anti-PD-L1 antibody treatment. First, we showed that PD-L1-vInt4 was detectable in clinical samples and that it was possible to visualize the secreting variants with IHC. By overexpressing the PD-L1-secreted splicing variant on MC38 cells, we observed that an immune-suppressing effect was not induced by their secretion alone. We then demonstrated that PD-L1-vInt4 secretion resisted anti-PD-L1 antibody treatment, compared with WT PD-L1, which was explicable by the PD-L1-vInt4's decoying of the anti-PD-L1 antibody. The decoying function of PD-L1 splicing variants may be one of the reasons for cancers being resistant to anti-PD-L1 therapy. Measuring serum PD-L1 levels might be helpful in deciding the therapeutic strategy.

Platelet-derived lysophosphatidic acid mediated LPAR1 activation as a therapeutic target for osteosarcoma metastasis.

Osteosarcoma is the most common primary malignant bone cancer, with high rates of pulmonary metastasis. Osteosarcoma patients with pulmonary metastasis have worse prognosis than those with localized disease, leading to dramatically reduced survival rates. Therefore, understanding the biological characteristics of metastatic osteosarcoma and the molecular mechanisms of invasion and metastasis of osteosarcoma cells will lead to the development of innovative therapeutic intervention for advanced osteosarcoma. Here, we identified that osteosarcoma cells commonly exhibit high platelet activation-inducing characteristics, and molecules released from activated platelets promote the invasiveness of osteosarcoma cells. Given that heat-denatured platelet releasate maintained the ability to promote osteosarcoma invasion, we focused on heat-tolerant molecules, such as lipid mediators in the platelet releasate. Osteosarcoma-induced platelet activation leads to abundant lysophosphatidic acid (LPA) release. Exposure to LPA or platelet releasate induced morphological changes and increased invasiveness of osteosarcoma cells. By analyzing publicly available transcriptome datasets and our in-house osteosarcoma patient-derived xenograft tumors, we found that LPA receptor 1 (LPAR1) is notably upregulated in osteosarcoma. LPAR1 gene KO in osteosarcoma cells abolished the platelet-mediated osteosarcoma invasion in vitro and the formation of early pulmonary metastatic foci in experimental pulmonary metastasis models. Of note, the pharmacological inhibition of LPAR1 by the orally available LPAR1 antagonist, ONO-7300243, prevented pulmonary metastasis of osteosarcoma in the mouse models. These results indicate that the LPA-LPAR1 axis is essential for the osteosarcoma invasion and metastasis, and targeting LPAR1 would be a promising therapeutic intervention for advanced osteosarcoma.

Novel knock-in mouse model for the evaluation of the therapeutic efficacy and toxicity of human podoplanin-targeting agents.

Podoplanin is a key molecule for enhancing tumor-induced platelet aggregation. Podoplanin interacts with CLEC-2 on platelets via PLatelet Aggregation-inducing domains (PLAGs). Among our generated antibodies, those targeting the fourth PLAG domain (PLAG4) strongly suppress podoplanin-CLEC-2 binding and podoplanin-expressing tumor growth and metastasis. We previously performed a single-dose toxicity study of PLAG4-targeting anti-podoplanin-neutralizing antibodies and found no acute toxicity in cynomolgus monkeys. To confirm the therapeutic efficacy and toxicity of podoplanin-targeting antibodies, a syngeneic mouse model that enables repeated dose toxicity tests is needed. Replacement of mouse PLAG1-PLAG4 domains with human homologous domains drastically decreased the platelet-aggregating activity. Therefore, we searched the critical domain of the platelet-aggregating activity in mouse podoplanin and found that the mouse PLAG4 domain played a critical role in platelet aggregation, similar to the human PLAG4 domain. Human/mouse chimeric podoplanin, in which a limited region containing mouse PLAG4 was replaced with human homologous region, exhibited a similar platelet-aggregating activity to wild-type mouse podoplanin. Thus, we generated knock-in mice with human/mouse chimeric podoplanin expression (PdpnKI/KI mice). Our previously established PLAG4-targeting antibodies could suppress human/mouse chimeric podoplanin-mediated platelet aggregation and tumor growth in PdpnKI/KI mice. Repeated treatment of PdpnKI/KI mice with antibody-dependent cell-mediated cytotoxicity activity-possessing PG4D2 antibody did not result in toxicity or changes in hematological and biochemical parameters. Our results suggest that anti-podoplanin-neutralizing antibodies could be used safely as novel anti-tumor agents. Our generated PdpnKI/KI mice are useful for investigating the efficacy and toxicity of human podoplanin-targeting drugs.

Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer.

ALK gene rearrangement was observed in 3%-5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI-resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey (Macaca fascicularis).

Hematogenous metastases are enhanced by platelet aggregation induced by tumor cell-platelet interaction. Podoplanin is a key molecule to enhance the platelet aggregation and interacts with C-type lectin-like receptor 2 (CLEC-2) on platelet via PLAG domains. Our previous reports have shown that blocking podoplanin binding to platelets by neutralizing antibody specific to PLAG4 domain strongly reduces hematogenous metastasis. However, podoplanin is expressed in a variety of normal tissues such as lymphatic vessels and the question remains whether treatment of tumors with anti-podoplanin neutralizing antibodies would be toxic. Monkeys are the most suitable species for that purpose. PLAG3 and PLAG4 domains had high homology among various monkey species and human. PLAG domain deleted mutants were indicated that monkey PLAG4 domain played a more crucial role in podoplanin-induced platelet aggregation than did the PLAG3 domain as in human. Moreover, newly established neutralizing antibodies (1F6, 2F7, and 3F4) targeting the monkey PLAG4 domain blocked interaction between monkey podoplanin and CLEC-2. Especially, the 2F7 neutralizing antibody strongly suppressed platelet aggregation and pulmonary metastasis. Furthermore, inhibiting podoplanin function with 2F7 neutralizing antibody exhibited no acute toxicity in cynomolgus monkeys. Our results suggested that targeting podoplanin with specific neutralizing antibodies may be an effective anticancer treatment.

Podoplanin: An emerging cancer biomarker and therapeutic target.

Podoplanin (PDPN) is a transmembrane receptor glycoprotein that is upregulated on transformed cells, cancer associated fibroblasts and inflammatory macrophages that contribute to cancer progression. In particular, PDPN increases tumor cell clonal capacity, epithelial mesenchymal transition, migration, invasion, metastasis and inflammation. Antibodies, CAR-T cells, biologics and synthetic compounds that target PDPN can inhibit cancer progression and septic inflammation in preclinical models. This review describes recent advances in how PDPN may be used as a biomarker and therapeutic target for many types of cancer, including glioma, squamous cell carcinoma, mesothelioma and melanoma.

Platelet-activating factor podoplanin: from discovery to drug development.

Tumor cell-induced platelet aggregation facilitates hematogenous metastasis by promoting tumor embolization, preventing immunological assaults and shear stress, and the platelet-releasing growth factors support tumor growth and invasion. Podoplanin, also known as Aggrus, is a type I transmembrane mucin-like glycoprotein and is expressed on wide range of tumor cells. Podoplanin has a role in platelet aggregation and metastasis formation through the binding to its platelet receptor, C-type lectin-like receptor 2 (CLEC-2). The podoplanin research was originally started from the cloning of highly metastatic NL-17 subclone from mouse colon 26 cancer cell line and from the establishment of 8F11 monoclonal antibody (mAb) that could neutralize NL-17-induced platelet aggregation and hematogenous metastasis. Later on, podoplanin was identified as the antigen of 8F11 mAb, and its ectopic expression brought to cells the platelet-aggregating abilities and hematogenous metastasis phenotypes. From the 8F11 mAb recognition epitopes, podoplanin is found to contain tandemly repeated, highly conserved motifs, designated platelet aggregation-stimulating (PLAG) domains. Series of analyses using the cells expressing the mutants and the established neutralizing anti-podoplanin mAbs uncovered that both PLAG3 and PLAG4 domains are associated with the CLEC-2 binding. The neutralizing mAbs targeting PLAG3 or PLAG4 could suppress podoplanin-induced platelet aggregation and hematogenous metastasis through inhibiting the podoplanin-CLEC-2 binding. Therefore, these domains are certainly functional in podoplanin-mediated metastasis through its platelet-aggregating activity. This review summarizes the platelet functions in metastasis formation, the role of platelet aggregation-inducing factor podoplanin in pathological and physiological situations, and the possibility to develop podoplanin-targeting drugs in the future.

Podoplanin enhances lung cancer cell growth in vivo by inducing platelet aggregation.

Podoplanin/Aggrus, known as a platelet aggregation-inducing factor, is frequently overexpressed in lung squamous cell carcinomas (LSCC) and glioblastomas among other tumours, and its expression has been reported to be correlated with poor prognosis. However, the contribution of podoplanin to malignant progression has been elusive. Here we demonstrate that in podoplanin-positive LSCC cells, their growth was abrogated by podoplanin knockout in vivo but not in vitro. Conversely, ectopic expression of podoplanin promoted cell growth in vivo and facilitated intratumoral platelet activation. Consistently, LSCC cells evoked podoplanin-mediated platelet aggregation (PMPA), and the releasates from platelets during PMPA promoted the growth of LSCC cells in vitro. Phospho-receptor-tyrosine-kinase array analysis revealed that epidermal growth factor receptor (EGFR) phosphorylation of LSCC cells was responsible for the growth promotion induced by platelet releasates. Treatment with an antiplatelet agent or podoplanin-neutralizing antibody depressed the growth of an LSCC tumour xenograft via suppression of EGFR phosphorylation. These results suggested that podoplanin in LSCC enhanced cell growth by inducing PMPA in vivo and contributed to malignant progression.

A critical role of platelet TGF-ß release in podoplanin-mediated tumour invasion and metastasis.

The tumour microenvironment is critical for various characteristics of tumour malignancies. Platelets, as part of the tumour microenvironment, are associated with metastasis formation via increasing the rate of tumour embolus formation in microvasculature. However, the mechanisms underlying the ability of tumour cells to acquire invasiveness and extravasate into target organs at the site of embolization remain unclear. In this study, we reported that platelet aggregation-inducing factor podoplanin expressed on tumour cell surfaces were found to not only promote the formation of tumour-platelet aggregates via interaction with platelets, but also induced the epithelial-mesenchymal transition (EMT) of tumour cells by enhancing transforming growth factor-ß (TGF-ß) release from platelets. In vitro and in vivo analyses revealed that podoplanin-mediated EMT resulted in increased invasiveness and extravasation of tumour cells. Treatment of mice with a TGF-ß-neutralizing antibody statistically suppressed podoplanin-mediated distant metastasis in vivo, suggesting that podoplanin promoted haematogenous metastasis in part by releasing TGF-ß from platelets that was essential for EMT of tumour cells. Therefore, our findings suggested that blocking the TGF-ß signalling pathway might be a promising strategy for suppressing podoplanin-mediated haematogenous metastasis in vivo.

Nuclear envelope expansion is crucial for proper chromosomal segregation during a closed mitosis.

Here, we screened a 10,371 library of diverse molecules using a drug-sensitive fission yeast strain to identify compounds which cause defects in chromosome segregation during mitosis. We identified a phosphorium-ylide-based compound Cutin-1 which inhibits nuclear envelope expansion and nuclear elongation during the closed mitosis of fission yeast, and showed that its target is the ß-subunit of fatty acid synthase. A point mutation in the dehydratase domain of Fas1 conferred in vivo and in vitro resistance to Cutin-1. Time-lapse photomicrography showed that the bulk of the chromosomes were only transiently separated during mitosis, and nucleoli separation was defective. Subsequently sister chromatids re-associated leading to chromosomal mis-segregation. These segregation defects were reduced when the nuclear volume was increased and were increased when the nuclear volume was reduced. We propose that there needs to be sufficient nuclear volume to allow the nuclear elongation necessary during a closed mitosis to take place for proper chromosome segregation, and that inhibition of fatty acid synthase compromises nuclear elongation and leads to defects in chromosomal segregation.

Targeting a novel domain in podoplanin for inhibiting platelet-mediated tumor metastasis.

Podoplanin/Aggrus is a sialoglycoprotein expressed in various cancers. We previously identified podoplanin as a key factor in tumor-induced platelet aggregation. Podoplanin-mediated platelet aggregation enhances tumor growth and metastasis by secreting growth factors and by forming tumor emboli in the microvasculature. Thus, precise analysis of the mechanisms of podoplanin-mediated platelet aggregation is critical for developing anti-tumor therapies. Here we report the discovery of a novel platelet aggregation-inducing domain, PLAG4 (81-EDLPT-85). PLAG4 has high homology to the previously reported PLAG3 and contributes to the binding of its platelet receptor CLEC-2. Mutant analyses indicated that PLAG4 exhibits a predominant platelet-aggregating function relative to PLAG3 and that conserved Glu81/Asp82/Thr85 residues in PLAG4 are indispensable for CLEC-2 binding. By establishing anti-PLAG4-neutralizing monoclonal antibodies, we confirmed its role in CLEC-2 binding, platelet aggregation, and tumor emboli formation. Our results suggest the requirement of simultaneous inhibition of PLAG3/4 for complete suppression of podoplanin-mediated tumor growth and metastasis.

Platelets promote osteosarcoma cell growth through activation of the platelet-derived growth factor receptor-Akt signaling axis.

The interactions of tumor cells with platelets contribute to the progression of tumor malignancy, and the expression levels of platelet aggregation-inducing factors positively correlate with the metastatic potential of osteosarcoma cells. However, it is unclear how tumor-platelet interaction contributes to the proliferation of osteosarcomas. We report here that osteosarcoma-platelet interactions induce the release of platelet-derived growth factor (PDGF) from platelets, which promotes the proliferation of osteosarcomas. Co-culture of platelets with MG63 or HOS osteosarcoma cells, which could induce platelet aggregation, enhanced the proliferation of each cell line in vitro. Analysis of phospho-antibody arrays revealed that co-culture of MG63 cells with platelets induced the phosphorylation of platelet derived growth factor receptor (PDGFR) and Akt. The addition of supernatants of osteosarcoma-platelet reactants also increased the growth of MG63 and HOS cells as well as the level of phosphorylated-PDGFR and -Akt. Sunitinib or LY294002, but not erlotinib, significantly inhibited the platelet-induced proliferation of osteosarcoma cells, indicating that PDGF released from platelets plays an important role in the proliferation of osteosarcomas by activating the PDGFR and then Akt. Our results suggest that inhibitors that specifically target osteosarcoma-platelet interactions may eradicate osteosarcomas.

A chemical biology strategy to analyze rheostat-like protein kinase-dependent regulation.

Protein kinases may function more like variable rheostats rather than two-state switches. However, we lack approaches to properly analyze this aspect of kinase-dependent regulation. To address this, we develop a strategy in which a kinase inhibitor is identified using genetics-based screens, kinase mutations that confer resistance are characterized, and dose-dependent responses of isogenic drug-sensitive and resistant cells to inhibitor treatments are compared. This approach has the advantage that function of wild-type kinase, rather than mutants, is examined. To develop this approach, we focus on Ark1, the fission yeast member of the conserved Aurora kinase family. Applying this approach reveals that proper chromosome compaction in fission yeast needs high Ark1 activity, while other processes depend on significantly lower activity levels. Our strategy is general and can be used to examine the functions of other molecular rheostats.

Analyzing fission yeast multidrug resistance mechanisms to develop a genetically tractable model system for chemical biology.

Chemical inhibitors can help analyze dynamic cellular processes, particularly when probes are active in genetically tractable model systems. Although fission yeast has served as an important model system, which shares more cellular processes (e.g., RNAi) with humans than budding yeast, its use for chemical biology has been limited by its multidrug resistance (MDR) response. Using genomics and genetics approaches, we identified the key transcription factors and drug-efflux transporters responsible for fission yeast MDR and designed strains sensitive to a wide-range of chemical inhibitors, including commonly used probes. We used this strain, along with acute chemical inhibition and high-resolution imaging, to examine metaphase spindle organization in a "closed" mitosis. Together, our findings suggest that our fission yeast strains will allow the use of several inhibitors as probes, discovery of new inhibitors, and analysis of drug action.

The chromosomal association of condensin II is regulated by a noncatalytic function of PP2A.

Mitotic chromosomal assembly in vertebrates is regulated by condensin I and condensin II, which work cooperatively but have different chromosomal localization profiles and make distinct mechanistic contributions to this process. We show here that protein phosphatase 2A (PP2A), which interacts with condensin II but not condensin I, plays an essential role in targeting condensin II to chromosomes. Unexpectedly, our data indicate that PP2A acts as a recruiter protein rather than a catalytic enzyme to target condensin II to chromosomes. This recruiting activity of PP2A was inhibited by okadaic acid, but not by fostriecin, even though both molecules strongly inhibited the catalytic activity of PP2A. Additionally, we found that the chromokinesin KIF4a is also targeted to chromosomes via the noncatalytic activity of PP2A. Thus, our studies reveal a previously unknown contribution of PP2A to chromosome assembly.

Epstein-Barr virus BGLF4 kinase induces premature chromosome condensation through activation of condensin and topoisomerase II.

Previous studies of Epstein-Barr virus (EBV) replication focused mainly on the viral and cellular factors involved in replication compartment assembly and controlling the cell cycle. However, little is known about how EBV reorganizes nuclear architecture and the chromatin territories. In EBV-positive nasopharyngeal carcinoma NA cells or Akata cells, we noticed that cellular chromatin becomes highly condensed upon EBV reactivation. In searching for the possible mechanisms involved, we found that transient expression of EBV BGLF4 kinase induces unscheduled chromosome condensation, nuclear lamina disassembly, and stress fiber rearrangements, independently of cellular DNA replication and Cdc2 activity. BGLF4 interacts with condensin complexes, the major components in mitotic chromosome assembly, and induces condensin phosphorylation at Cdc2 consensus motifs. BGLF4 also stimulates the decatenation activity of topoisomerase II, suggesting that it may induce chromosome condensation through condensin and topoisomerase II activation. The ability to induce chromosome condensation is conserved in another gammaherpesvirus kinase, murine herpesvirus 68 ORF36. Together, these findings suggest a novel mechanism by which gammaherpesvirus kinases may induce multiple premature mitotic events to provide more extrachromosomal space for viral DNA replication and successful egress of nucleocapsid from the nucleus.

Analysis of the role of Aurora B on the chromosomal targeting of condensin I.

During mitosis, chromosome condensation takes place, which entails the conversion of interphase chromatin into compacted mitotic chromosomes. Condensin I is a five-subunit protein complex that plays a central role in this process. Condensin I is targeted to chromosomes in a mitosis-specific manner, which is regulated by phosphorylation by mitotic kinases. Phosphorylation of histone H3 at serine 10 (Ser10) occurs during mitosis and its physiological role is a longstanding question. We examined the function of Aurora B, a kinase that phosphorylates Ser10, in the chromosomal binding of condensin I and mitotic chromosome condensation, using an in vitro system derived from Xenopus egg extract. Aurora B depletion from a mitotic egg extract resulted in the loss of H3 phosphorylation, accompanied with a 50% reduction of chromosomal targeting of condensin I. Alternatively, a portion of condensin I was bound to sperm chromatin, and chromosome-like structures were assembled when okadaic acid (OA) was supplemented in an interphase extract that lacks Cdc2 activity. However, chromosomal targeting of condensin I was abolished when Aurora B was depleted from the OA-treated interphase extract. From these results, it is suggested that Aurora B-dependent and Cdc2-independent pathways of the chromosomal targeting of condensin I are present.