Targeting Epigenetic Crosstalk as a Therapeutic Strategy for EZH2-Aberrant Solid Tumors.

Mutations or aberrant upregulation of EZH2 occur frequently in human cancers, yet clinical benefits of EZH2 inhibitor (EZH2i) remain unsatisfactory and limited to certain hematological malignancies. We profile global posttranslational histone modification changes across a large panel of cancer cell lines with various sensitivities to EZH2i. We report here oncogenic transcriptional reprogramming mediated by MLL1's interaction with the p300/CBP complex, which directs H3K27me loss to reciprocal H3K27ac gain and restricts EZH2i response. Concurrent inhibition of H3K27me and H3K27ac results in transcriptional repression and MAPK pathway dependency in cancer subsets. In preclinical models encompassing a broad spectrum of EZH2-aberrant solid tumors, a combination of EZH2 and BRD4 inhibitors, or a triple-combination including MAPK inhibition display robust efficacy with very tolerable toxicity. Our results suggest an attractive precision treatment strategy for EZH2-aberrant tumors on the basis of tumor-intrinsic MLL1 expression and concurrent inhibition of epigenetic crosstalk and feedback MAPK activation.

Identification of benzo[b]thiophene-1,1-dioxide derivatives as novel PHGDH covalent inhibitors.

The increased de novo serine biosynthesis confers many advantages for tumorigenesis and metastasis. Phosphoglycerate dehydrogenase (PHGDH), a rate-limiting enzyme in serine biogenesis, exhibits hyperactivity across multiple tumors and emerges as a promising target for cancer treatment. Through screening our in-house compound library, we identified compound Stattic as a potent PHGDH inhibitor (IC50 = 1.98 ± 0.66 µM). Subsequent exploration in structural activity relationships led to the discovery of compound B12 that demonstrated the increased enzymatic inhibitory activity (IC50 = 0.29 ± 0.02 µM). Furthermore, B12 exhibited robust inhibitory effects on the proliferation of MDA-MB-468, NCI-H1975, HT1080 and PC9 cells that overexpress PHGDH. Additionally, using a [U-13C6]-glucose tracing assay, B12 was found to reduce the production of glucose-derived serine in MDA-MB-468 cells. Finally, mass spectrometry-based peptide profiling, mutagenesis experiment and molecular docking study collectively suggested that B12 formed a covalent bond with Cys421 of PHGDH.

Design, synthesis, and evaluation of thiazolecarboxamide derivatives as stimulator of interferon gene inhibitors.

Stimulator of interferon gene (STING) plays critical roles in the cytoplasmic DNA-sensing pathway and in the induction of inflammatory response. Aberrant cytoplasmic DNA accumulation and STING activation are implicated in numerous inflammatory and autoimmune diseases. Here, we reported the discovery of a series of thiazolecarboxamide-based STING inhibitors through a molecular planarity/symmetry disruption strategy. The privileged compound 15b significantly inhibited STING signaling and suppressed immune-inflammatory cytokine levels in both human and murine cells. In vivo experiments demonstrated 15b effectively ameliorated immune-inflammatory cytokines upregulation in MSA-2-stimulated and Trex1-D18N mice. Furthermore, compound 15b exhibited enhanced efficacy in suppressing interferon-stimulated gene 15 (ISG15), a critical positive feedback regulator of STING. Overall, compound 15b deserves further development for the treatment of STING-associated inflammatory and autoimmune diseases.

Discovery and optimization of 3-(indolin-5-yloxy)pyridin-2-amine derivatives as potent necroptosis inhibitors.

Necroptosis is a form of regulated necrotic cell death and has been confirmed to play pivotal roles in the pathogenesis of multiple autoimmune diseases such as rheumatoid arthritis (RA) and psoriasis. The development of necroptosis inhibitors may offer a promising therapeutic strategy for the treatment of these autoimmune diseases. Herein, starting from the in-house hit compound 1, we systematically performed structural optimization to discover potent necroptosis inhibitors with good pharmacokinetic profiles. The resulting compound 33 was a potent necroptosis inhibitor for both human I2.1 cells (IC50 < 0.2 nM) and murine Hepa1-6 cells (IC50 < 5 nM). Further target identification revealed that compound 33 was an inhibitor of receptor interacting protein kinase 1 (RIPK1) with favorable selectivity. In addition, compound 33 also exhibited favorable pharmacokinetic profiles (T1/2 = 1.32 h, AUC = 1157 ng·h/mL) in Sprague-Dawley rats. Molecular docking and molecular dynamics simulations confirmed that compound 33 could bind to RIPK1 with high affinity. In silico ADMET analysis demonstrated that compound 33 possesses good drug-likeness profiles. Collectively, compound 33 is a promising candidate for antinecroptotic drug discovery.

Tetrandrine synergizes with MAPK inhibitors in treating KRAS-mutant pancreatic ductal adenocarcinoma via collaboratively modulating the TRAIL-death receptor axis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies lacking effective therapies. KRAS mutations that occur in over 90% of PDAC are major oncogenic drivers of PDAC. The MAPK signaling pathway plays a central role in KRAS-driven oncogenic signaling. However, pharmacological inhibitors of the MAPK pathway are poorly responded in KRAS-mutant PDAC, raising a compelling need to understand the mechanism behind and to seek new therapeutic solutions. Herein, we perform a screen utilizing a library composed of 800 naturally-derived bioactive compounds to identify natural products that are able to sensitize KRAS-mutant PDAC cells to the MAPK inhibition. We discover that tetrandrine, a natural bisbenzylisoquinoline alkaloid, shows a synergistic effect with MAPK inhibitors in PDAC cells and xenograft models. Mechanistically, pharmacological inhibition of the MAPK pathway exhibits a double-edged impact on the TRAIL-death receptor axis, transcriptionally upregulating TRAIL yet downregulating its agonistic receptors DR4 and DR5, which may explain the limited therapeutic outcomes of MAPK inhibitors in KRAS-mutant PDAC. Of great interest, tetrandrine stabilizes DR4/DR5 protein via impairing ubiquitination-mediated protein degradation, thereby allowing a synergy with MAPK inhibition in inducing apoptosis in KRAS-mutant PDAC. Our findings identify a new combinatorial approach for treating KRAS-mutant PDAC and highlight the role of TRAIL-DR4/DR5 axis in dictating the therapeutic outcome in KRAS-mutant PDAC.

Development of a novel lactate dehydrogenase A inhibitor with potent antitumor activity and immune activation.

Lactate accumulation in the tumor microenvironment was shown to be closely related to tumor growth and immune escape, and suppression of lactate production by inhibiting lactate dehydrogenase A (LDHA) has been pursued as a potential novel antitumor strategy. However, only a few potent LDHA inhibitors have been developed and most of them did not show potent antitumor effects in vivo. To this end, we designed new LDHA inhibitors and obtained a novel potent LDHA inhibitor, ML-05. ML-05 inhibited cellular lactate production and tumor cell proliferation, which was associated with inhibition of ATP production and induction of reactive oxygen species and G1 phase arrest. In a mouse B16F10 melanoma model, intratumoral injection of ML-05 significantly reduced lactate production, inhibited tumor growth, and released antitumor immune response of T cell subsets (Th1 and GMZB+ CD8 T cells) in the tumor microenvironment. Moreover, ML-05 treatment combined with programmed cell death-1 Ab or stimulator of interferon genes protein (STING) could sensitize the antitumor activity in B16F10 melanoma model. Collectively, we developed a novel potent LDHA inhibitor, ML-05, that elicited profound antitumor activity when injected locally, and was associated with the activation of antitumor immunity. In addition, ML-05 could sensitize immunotherapies, which suggests great translational value.

LS-106, a novel EGFR inhibitor targeting C797S, exhibits antitumor activities both in vitro and in vivo.

With the wide clinical use of the third-generation epidermal growth factor receptor (EGFR) inhibitor osimertinib for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC), acquired resistance caused by EGFR C797S tertiary mutation has become a concern. Therefore, fourth-generation EGFR inhibitors that could overcome this mutation have gained increasing attention in recent years. Here, we identified LS-106 as a novel EGFR inhibitor against C797S mutation and evaluated its antitumor activity both in vitro and in vivo. In cell-free assay, LS-106 potently inhibited the kinase activities of EGFR19del/T790M/C797S and EGFRL858R/T790M/C797S with IC50 values of 2.4 nmol/L and 3.1 nmol/L, respectively, which was more potent than osimertinib. Meanwhile, LS-106 exhibited comparable kinase inhibitory effect to osimertinib on EGFRL858R/T790M and wild-type EGFR. Results from cellular experiments demonstrated that LS-106 potently blocked the phosphorylation of EGFR C797S triple mutations in the constructed BaF3 cells that highly expressed EGFR19del/T790M/C797S or EGFRL858R/T790M/C797S , and thus inhibited the proliferation of these cells. We also constructed tumor cells harboring EGFR19del/T790M/C797S (named PC-9-OR cells) using the CRISPR/Cas9 system and found that LS-106 markedly suppressed the activation of EGFR19del/T790M/C797S and the proliferation of PC-9-OR cells. Moreover, cells harboring EGFR19del/T790M/C797S underwent remarkable apoptosis upon LS-106 treatment. In vivo experiments further demonstrated that oral administration of LS-106 caused significant tumor regression in a PC-9-OR xenograft model, with a tumor growth inhibition rate (TGI) of 83.5% and 136.6% at doses of 30 and 60 mg/kg, respectively. Taken together, we identified LS-106 as a novel fourth-generation EGFR inhibitor against C797S mutation and confirmed its preclinical antitumor effects in C797S-triple-mutant tumor models.

Antitumor pharmacological research in the era of personalized medicine.

Anticancer drug discovery has yielded unprecedented progress in recent decades, resulting in the approval of innovative treatment options for patients and the successful implementation of personalized medicine in clinical practice. This remarkable progress has also reshaped the research scope of pharmacological research. This article, as a tribute to cancer research at Shanghai Institute of Materia Medica in celebration of the institute's 90th birthday, provides an overview of the conceptual revolution occurring in anticancer therapy, and summarizes our recent progress in the development of molecularly targeted therapeutics and exploration of new strategies in personalized medicine. With this review, we hope to provide a glimpse into how antitumor pharmacological researchers have embraced the new era of personalized medicine research and to propose a future path for anticancer drug discovery and pharmacological research.

SAF-248, a novel PI3Kδ-selective inhibitor, potently suppresses the growth of diffuse large B-cell lymphoma.

PI3Kδ is expressed predominately in leukocytes and overexpressed in B-cell-related malignances. PI3Kδ has been validated as a promising target for cancer therapy, and specific PI3Kδ inhibitors were approved for clinical practice. However, the substantial toxicity and relatively low efficacy as a monotherapy in diffuse large B-cell lymphoma (DLBCL) limit their clinical use. In this study, we described a novel PI3Kδ inhibitor SAF-248, which exhibited high selectivity for PI3Kδ (IC50 = 30.6 nM) over other PI3K isoforms at both molecular and cellular levels, while sparing most of the other human protein kinases in the kinome profiling. SAF-248 exhibited superior antiproliferative activity against 27 human lymphoma and leukemia cell lines compared with the approved PI3Kδ inhibitor idelalisib. In particular, SAF-248 potently inhibited the proliferation of a panel of seven DLBCL cell lines (with GI50 values < 1 µM in 5 DLBCL cell lines). We demonstrated that SAF-248 concentration-dependently blocked PI3K signaling followed by inducing G1 phase arrest and apoptosis in DLBCL KARPAS-422, Pfeiffer and TMD8 cells. Its activity against the DLBCL cells was negatively correlated to the protein level of PI3Kα. Oral administration of SAF-248 dose-dependently inhibited the growth of xenografts derived from Pfeiffer and TMD8 cells. Activation of mTORC1, MYC and JAK/STAT signaling was observed upon prolonged treatment and co-targeting these pathways would potentiate the activity of SAF-248. Taken together, SAF-248 is a promising selective PI3Kδ inhibitor for the treatment of DLBCL and rational drug combination would further improve its efficacy.

SAF-189s, a potent new-generation ROS1 inhibitor, is active against crizotinib-resistant ROS1 mutant-driven tumors.

The ROS1 fusion kinase is an attractive antitumor target. Though with significant clinical efficacy, the well-known first-generation ROS1 inhibitor (ROS1i) crizotinib inevitably developed acquired resistance due to secondary point mutations in the ROS1 kinase. Novel ROS1is effective against mutations conferring secondary crizotinib resistance, especially G2032R, are urgently needed. In the present study, we evaluated the antitumor efficacy of SAF-189s, the new-generation ROS1/ALK inhibitor, against ROS1 fusion wild-type and crizotinib-resistant mutants. We showed that SAF-189s potently inhibited ROS1 kinase and its known acquired clinically resistant mutants, including the highly resistant G2032R mutant. SAF-189s displayed subnanomolar to nanomolar IC50 values against ROS1 wild-type and mutant kinase activity and a selectivity vs. other 288 protein kinases tested. SAF-189s blocked cellular ROS1 signaling, and in turn potently inhibited the cell proliferation in HCC78 cells and BaF3 cells expressing ROS1 fusion wild-type and resistance mutants. In nude mice bearing BaF3/CD74-ROS1 or BaF3/CD74-ROS1G2032R xenografts, oral administration of SAF-189s dose dependently suppressed the growth of both ROS1 wild-type- and G2032R mutant-driven tumors. In a patient-derived xenograft model of SDC4-ROS1 fusion NSCLC, oral administration of SAF-189s (20 mg/kg every day) induced tumor regression and exhibited notable prolonged and durable efficacy. In addition, SAF-189s was more potent than crizotinib and comparable to lorlatinib, the most advanced ROS1i known against the ROS1G2032R. Collectively, these results suggest the promising potential of SAF-189s for the treatment of patients with the ROS1 fusion G2032R mutation who relapse on crizotinib. It is now recruiting both crizotinib-relapsed and naive ROS1-positive NSCLC patients in a multicenter phase II trial (ClinicalTrials.gov Identifier: NCT04237805).

A novel tricyclic BTK inhibitor suppresses B cell responses and osteoclastic bone erosion in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by joint leukocyte infiltration, synovial inflammation and bone damage result from osteoclastogenesis. Bruton's tyrosine kinase (BTK) is a key regulator of B cell receptor (BCR) and Fc gamma receptor (FcγR) signaling involved in the pathobiology of RA and other autoimmune disorders. SOMCL-17-016 is a potent and selective tricyclic BTK inhibitor, structurally distinct from other known BTK inhibitors. In present study we investigated the therapeutic efficacy of SOMCL-17-016 in a mouse collagen-induced arthritis (CIA) model and underlying mechanisms. CIA mice were administered SOMCL-17-016 (6.25, 12.5, 25 mg·kg-1·d-1, ig), or ibrutinib (25 mg·kg-1·d-1, ig) or acalabrutinib (25 mg·kg-1·d-1, ig) for 15 days. We showed that oral administration of SOMCL-17-016 dose-dependently ameliorated arthritis severity and bone damage in CIA mice; it displayed a higher in vivo efficacy than ibrutinib and acalabrutinib at the corresponding dosage. We found that SOMCL-17-016 administration dose-dependently inhibited anti-IgM-induced proliferation and activation of B cells from CIA mice, and significantly decreased anti-IgM/anti-CD40-stimulated RANKL expression in memory B cells from RA patients. In RANKL/M-CSF-stimulated RAW264.7 cells, SOMCL-17-016 prevented osteoclast differentiation and abolished RANK-BTK-PLCγ2-NFATc1 signaling. In summary, this study demonstrates that SOMCL-17-016 presents distinguished therapeutic effects in the CIA model. SOMCL-17-016 exerts a dual inhibition of B cell function and osteoclastogenesis, suggesting that it to be a promising drug candidate for RA treatment.

Discovery of a novel third-generation EGFR inhibitor and identification of a potential combination strategy to overcome resistance.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients with activating EGFR mutations initially respond to first-generation EGFR inhibitors; however, the efficacy of these drugs is limited by acquired resistance driven by the EGFR T790M mutation. The discovery of third-generation EGFR inhibitors overcoming EGFR T790M and their new resistance mechanisms have attracted much attention. METHODS: We examined the antitumor activities and potential resistance mechanism of a novel EGFR third-generation inhibitor in vitro and in vivo using ELISA, SRB assay, immunoblotting, flow cytometric analysis, kinase array, qRT-PCR and tumor xenograft models. The clinical effect on a patient was evaluated by computed tomography scan. RESULTS: We identified compound ASK120067 as a novel inhibitor of EGFR T790M, with selectivity over EGFR WT. ASK120067 exhibited potent anti-proliferation activity in tumor cells harboring EGFR T790M (NCI-H1975) and sensitizing mutations (PC-9 and HCC827) while showed moderate or weak inhibition in cells expressing EGFR WT. Oral administration of ASK120067 induced tumor regression in NSCLC xenograft models and in a PDX model harboring EGFR T790M. The treatment of one patient with advanced EGFR T790M-positive NSCLC was described as proof of principle. Moreover, we found that hyperphosphorylation of Ack1 and the subsequent activation of antiapoptotic signaling via the AKT pathway contributed to ASK120067 resistance. Concomitant targeting of EGFR and Ack1 effectively overrode the acquired resistance of ASK120067 both in vitro and in vivo. CONCLUSIONS: Our results idenfity ASK120067 as a promising third-generation EGFR inhibitor and reveal for the first time that Ack1 activation as a novel resistance mechanism to EGFR inhibitors that guide to potential combination strategy.

Identification and Therapeutic Intervention of Coactivated Anaplastic Lymphoma Kinase, Fibroblast Growth Factor Receptor 2, and Ephrin Type-A Receptor 5 Kinases in Hepatocellular Carcinoma.

Though kinase inhibitors have been heavily investigated in the clinic to combat advanced hepatocellular carcinoma (HCC), clinical outcomes have been disappointing overall, which may be due to the absence of kinase-addicted subsets in HCC patients. Recently, strategies that simultaneously inhibit multiple kinases are increasingly appreciated in HCC treatment, yet they are challenged by the dynamic nature of the kinase networks. This study aims to identify clustered kinases that may cooperate to drive the malignant growth of HCC. We show that anaplastic lymphoma kinase, fibroblast growth factor receptor 2, and ephrin type-A receptor 5 are the essential kinases that assemble into a functional cluster to sustain the viability of HCC cells through downstream protein kinase B-dependent, extracellular signal-regulated kinase-dependent, and p38-dependent signaling pathways. Their coactivation is associated with poor prognosis for overall survival in about 13% of HCC patients. Moreover, their activities are tightly regulated by heat shock protein 90 (Hsp90). Thereby Combined kinase inhibition or targeting of heat shock protein 90 led to significant therapeutic responses both in vitro and in vivo. Conclusion: Our findings established a paradigm that highlights the cooperation of anaplastic lymphoma kinase, fibroblast growth factor receptor 2, and ephrin type-A receptor 5 kinases in governing the growth advantage of HCC cells, which might offer a conceptual "combined therapeutic target" for diagnosis and subsequent intervention in a subgroup of HCC patients.

EPHA2 feedback activation limits the response to PDEδ inhibition in KRAS-dependent cancer cells.

KRAS is one of the most important proto-oncogenes. Its mutations occur in almost all tumor types, and KRAS mutant cancer is still lack of effective therapy. Prenyl-binding protein phosphodiesterase-δ (PDEδ) is required for the plasma membrane association and subsequent activation of KRAS oncogenic signaling. Recently, targeting PDEδ has provided new promise for KRAS mutant tumors. However, the therapeutic potential of PDEδ inhibition remains obscure. In this study, we explored how PDEδ inhibition was responded in KRAS mutant cancer cells, and identified KRAS mutant subset responsive to PDEδ inhibition. We first performed siRNA screen of KRAS growth dependency of a small panel of human cancer lines, and identified a subset of KRAS mutant cancer cells that were highly dependent on KRAS signaling. Among these cells, only a fraction of KRAS-dependent cells responded to PDEδ depletion, though KRAS plasma membrane association was effectively impaired. We revealed that the persistent RAF/MEK/ERK signaling seemed responsible for the lack of response to PDEδ depletion. A kinase array further identified that the feedback activation of EPH receptor A2 (EPHA2) accounted for the compensatory activation of RAF/MEK/ERK signaling in these cells. Simultaneous inhibition of EPHA2 and PDEδ led to the growth inhibition of KRAS mutant cancer cells. Together, this study gains a better understanding of PDEδ-targeted therapeutic strategy and suggests the combined inhibition of EPHA2 and PDEδ as a potential therapy for KRAS mutant cancer.

Author Correction: Targeting Hsp90 with FS-108 circumvents gefitinib resistance in EGFR mutant non-small cell lung cancer cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CUDC-907 displays potent antitumor activity against human pancreatic adenocarcinoma in vitro and in vivo through inhibition of HDAC6 to downregulate c-Myc expression.

Pancreatic adenocarcinoma is a highly malignant cancer that often involves a deregulation of c-Myc. It has been shown that c-Myc plays a pivotal role in the regulation of a variety of physiological processes and is involved in early neoplastic development, resulting in poor progression. Hence, suppression of c-Myc overexpression is a potential strategy for pancreatic cancer therapy. CUDC-907 is a novel dual-acting inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC). It has shown potential efficiency in patients with lymphoma, multiple myeloma, or thyroid cancer, as well as in solid tumors with c-Myc alterations, but the evidence is lacking for how CUDC-907 regulates c-Myc. In this study, we investigated the effect of CUDC-907 on human pancreatic cancer cells in vitro and in vivo. Our results showed that CUDC-907 potently inhibited the proliferation of 9 pancreatic cancer cell lines in vitro with IC50 values ranging from 6.7 to 54.5 nM. Furthermore, we revealed the antitumor mechanism of CUDC-907 in Aspc-1, PANC-1, and Capan-1 pancreatic cancer cells: it suppressed the HDAC6 subunit, thus downregulating c-Myc protein levels, which was a mode of action distinct from the existing mechanisms. Consistently, the extraordinary antitumor activity of CUDC-907 accompanied by downregulation of c-Myc and Ki67 expression in tumor tissue was observed in a human pancreatic cancer Aspc-1 xenograft nude mouse model in vivo. Our results suggest that CUDC-907 can be a valuable therapeutic option for treating pancreatic adenocarcinoma.

Polymeric immunoglobulin receptor promotes tumor growth in hepatocellular carcinoma.

Deregulation of the immune system is believed to contribute to cancer malignancy, which has led to recent therapeutic breakthroughs facilitating antitumor immunity. In a malignant setting, immunoglobulin receptors, which are fundamental components of the human immune system, fulfill paradoxical roles in cancer pathogenesis. This study describes a previously unrecognized pro-oncogenic function of polymeric immunoglobulin receptor (pIgR) in the promotion of cell transformation and proliferation. Mechanistically, pIgR overexpression is associated with YES proto-oncogene 1, Src family tyrosine kinase (Yes) activation, which is required for pIgR-induced oncogenic growth. Specifically, pIgR activates the Yes-DNAX-activating protein of 12 kDa-spleen tyrosine kinase-Rac1/CDC42-MEK (extracellular signal-regulated kinase kinase)/ERK (extracellular signal-regulated kinase) cascade in an immunoreceptor tyrosine-based activating motif (ITAM)-dependent manner to promote cell transformation and tumor growth, although pIgR itself does not contain an ITAM sequence. Additionally, the combination of pIgR and phosphorylated Yes (p-Yes) levels serves as a prognostic biomarker for hepatitis B surface antigen-positive and early-stage hepatocellular carcinoma (HCC) patients. Moreover, pharmacological targeting of MEK/ERK or Yes represents a therapeutic option for the subgroup of patients with pIgR/p-Yes-positive HCC based on our results with both cancer cell-line-based xenografts and primary patient-derived xenografts. CONCLUSION: Our findings reveal the molecular mechanism by which pIgR promotes cancer malignancy, suggest the clinical potential of targeting this pathway in HCC, and provide new insight into the oncogenic role of immunoglobulin receptors. (Hepatology 2017;65:1948-1962).

Discovery and structure-activity-relationship study of novel imidazole cyclopropyl amine analogues for mutant isocitric dehydrogenase 1 (IDH1) inhibitors.

The discovery and optimization of imidazole cyclopropyl amime analogues as mutant IDH1 inhibitors via structure-based rational design were reported. The optimal compounds demonstrated both potent inhibition in IDH1R132H enzymatic activity and 2HG production in IDH1 mutant HT1080 cell line, moderate liver microsome stability and PK properties.