3D layered co-culture model enhances Trastuzumab Deruxtecan sensitivity and reveals the combined effect with G007-LK in HER2-positive non-small cell lung cancer.

Human epidermal growth factor receptor 2 (HER2) aberrations are observed in various cancers. In non-small cell lung cancer, genetic alterations activating HER2, mostly exon 20 insertion mutations, occur in approximately 2-4% of cases. Trastuzumab deruxtecan (T-DXd), a HER2-targeted antibody-drug conjugate has been approved as the first HER2-targeted drug for HER2-mutant lung cancer. However, some cases are not responsive to T-DXd and the primary resistant mechanism remains unclear. In this study, we assessed sensitivity to T-DXd in JFCR-007, a patient-derived HER2-mutant lung cancer cell line. Although JFCR-007 was sensitive to HER2 tyrosine kinase inhibitors, it showed resistance to T-DXd in attachment or spheroid conditions. Accordingly, we established a three-dimensional (3D) layered co-culture model of JFCR-007, where it exhibited a lumen-like structure and became sensitive to T-DXd. In addition, an in-house inhibitor library screening revealed that G007-LK, a tankyrase inhibitor, was effective when combined with T-DXd. G007-LK increased the cytotoxicity of topoisomerase-I inhibitor, DXd, a payload of T-DXd and SN-38. This combined effect was also observed in H2170, an HER2-amplified lung cancer cell line. These results suggest that the proposed 3D co-culture system may help in evaluating the efficacy of T-DXd and may recapitulate the tumor microenvironment.

3D culture system containing gellan gum restores oncogene dependence in ROS1 rearrangements non-small cell lung cancer.

The ROS1 fusion gene has been identified in approximately 1% of non-small cell lung cancer (NSCLC) cases. Several clinical studies have highlighted ROS1 as a promising therapeutic target because crizotinib, a multi-targeted drug against ROS1, ALK, and the MET proto-oncogene, has elicited remarkable responses in ROS1-rearrangements NSCLC. However, acquired resistance mediated by ROS1 kinase domain mutations has been identified and a system to assess ROS1 inhibitors for these resistant mutations is necessary for the promotion of drug development. Publicly available NSCLC cell lines harboring the ROS1 fusion gene are limited to only HCC78 cells carrying SLC34A2-ROS1. This cell line exhibits resistance to ROS1 inhibitors through activation of the EGFR pathway, although the cells were established from ROS1-TKI naïve pleural effusion. Here, we demonstrate that 3D culture with gellan gum can restore the ROS1 oncogene dependence of HCC78 cells by upregulating the expression of the ROS1 fusion gene and reducing the activity of the EGFR pathway. Moreover, we established the HCC78xe3 cell line, a subclone of the HCC78 cell line, by repeated in vitro cultures from tumor xenografts and created xenograft tumors three times using in vitro cultured cells. This eventually enabled us to engraft and stably grow the cells in vivo, and subsequently evaluate various ROS1 inhibitors against HCC78xe3 cells by overexpressing crizotinib-resistant mutations in the ROS1 kinase domain including G2032R and D2033 N. We newly found that lorlatinib, a next generation ROS1/ALK inhibitor, remain the activity against D2033 N mutation. Furthermore, we demonstrated that HCC78xe3 cells expressing SLC34A2-ROS1 G2032R, and D2033 N, but not wild type (WT) cells, were resistant to crizotinib in vivo. Taken together, our data suggested that 3D cultures of HCC78 might reflect the features in patients and this new system will be a useful tool for evaluating ROS1 inhibitors.

Expression of Aggrus/podoplanin in bladder cancer and its role in pulmonary metastasis.

Platelet aggregation-inducing factor Aggrus, also known as podoplanin, is associated with tumor malignancy by promoting hematogenous metastasis. Aggrus overexpression has been reported in some tumor tissues including lung, esophagus, head and neck and brain. We here found the frequent upregulation of aggrus mRNA in urinary bladder cancers using cancer tissue panels from various organs. Immunohistochemical analysis confirmed Aggrus protein expression in urinary bladder cancers and suggested a positive correlation between Aggrus expression and metastatic tendency in bladder cancers. Endogenous expression of Aggrus protein on the cell surface was found in the mouse bladder cancer MBT-2 cell line and human bladder cancer SCaBER cell lines. Knockdown of Aggrus expression in MBT-2 cells decreased their ability to induce platelet aggregation and form pulmonary metastasis in syngeneic mouse models. Knockdown of Aggrus expression in the human bladder cancer SCaBER cells also attenuated their ability to induce platelet aggregation and form pulmonary metastasis in mice. Moreover, pulmonary metastasis of SCaBER cells was prevented by prior administration of our generated anti-Aggrus neutralizing monoclonal antibodies by attenuating their retention in lung. These results indicate that Aggrus plays an important role in bladder cancer metastasis. Thus, anti-Aggrus neutralizing antibodies would be useful for the prevention of hematogenous metastasis of Aggrus-positive bladder cancer.

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 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.

Acquired resistance to BRAF inhibitors is mediated by BRAF splicing variants in BRAF V600E mutation-positive colorectal neuroendocrine carcinoma.

Neuroendocrine carcinomas (NECs), a poorly differentiated subtype of neuroendocrine neoplasms, are aggressive and have a poor prognosis. Colorectal neuroendocrine carcinomas (CRC-NECs) are observed in about 0.6% of all patients with CRC. Interestingly, patients with CRC-NECs show higher frequencies of BRAF mutation than typical CRC. BRAF V600E mutation-positive CRC-NECs were shown to be sensitive to BRAF inhibitors and now are treated by BRAF inhibitors. Similar to the other BRAF V600E mutated cancers, resistances against BRAF inhibitors have been observed, but the resistance mechanisms are still unclear. In this study, we established BRAF V600E mutated CRC-NEC cell line directly from surgical specimens and experimentally obtained BRAF inhibitor dabrafenib resistant cell lines. The resistant cells are revealed to express at least three types of BRAF splicing variants harboring V600E-mutation, and contribute to RAF/MEK/ERK pathway activation. In these cells, MEK and ERK inhibitors but not dabrafenib significantly suppressed cell growth and survival. Thus, in BRAF V600E mutation-positive CRC-NECs, BRAF splicing variants activate the RAF/MEK/ERK pathway and contribute to acquire BRAF inhibitor resistance. Hence, MEK or ERK are potential therapeutic targets to overcome BRAF resistance.

Prevention of hematogenous metastasis by neutralizing mice and its chimeric anti-Aggrus/podoplanin antibodies.

The platelet aggregation-inducing factor, Aggrus (also known as podoplanin), is reported to contribute to cancer metastasis by mediating cancer cell-platelet interaction. Aggrus has been shown to be upregulated in many different types of cancers. Thus, not only the functional inhibition of Aggrus, but also its application as a cancer-specific antigen has therapeutic potential. Among a series of anti-Aggrus mAb established previously, no mouse anti-human Aggrus mAb exists that possesses the ability to neutralize platelet aggregation. For precise preclinical examinations of mouse and monkey models, the establishment of Aggrus-neutralizing mouse mAb and their chimeric Abs is needed. In this study, we established two mouse anti-human Aggrus mAb, P2-0 and HAG-3. A precise analysis of their epitopes revealed that P2-0 recognized the conformation near the bioactive O-glycosylation site at the Thr(52) residue. In contrast, HAG-3 recognized the amino-terminus side at a short distance from the conformation recognized by P2-0. We observed that only P2-0 attenuated Aggrus-induced platelet aggregation and Aggrus binding to its platelet receptor, that is, the C-type lectin-like receptor-2. Consistent with these data, only P2-0 prevented the experimental metastasis of human Aggrus-overexpressing CHO cells. Subsequently, we cloned the complementary determining region of P2-0 and produced the murine/human chimeric P2-0 antibody. This chimeric antibody maintained its inhibitory activity of Aggrus-induced platelet aggregation and experimental metastasis. Thus, P2-0 and its chimeric antibody are expected to aid the development of preclinical and clinical examinations of Aggrus-targeted cancer therapy.

Microsecond-timescale MD simulation of EGFR minor mutation predicts the structural flexibility of EGFR kinase core that reflects EGFR inhibitor sensitivity.

Approximately 15-30% of patients with lung cancer harbor mutations in the EGFR gene. Major EGFR mutations (>90% of EGFR-mutated lung cancer) are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs). Many uncommon EGFR mutations have been identified, but little is known regarding their characteristics, activation, and sensitivity to various EGFR-TKIs, including allosteric inhibitors. We encountered a case harboring an EGFR-L747P mutation, originally misdiagnosed with EGFR-del19 mutation using a routine diagnostic EGFR mutation test, which was resistant to EGFR-TKI gefitinib. Using this minor mutation and common EGFR-activating mutations, we performed the binding free energy calculations and microsecond-timescale molecular dynamic (MD) simulations, revealing that the L747P mutation considerably stabilizes the active conformation through a salt-bridge formation between K745 and E762. We further revealed why several EGFR inhibitors, including the allosteric inhibitor, were ineffective. Our computational structural analysis strategy would be beneficial for future drug development targeting the EGFR minor mutations.

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.

PRMT5, a novel TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB activation.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-alpha-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of kappaB kinase (IKK) and nuclear factor-kappaB (NF-kappaB), leading to induction of several NF-kappaB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-alpha, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-alpha-mediated NF-kappaB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKbeta, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-kappaB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments

Overcoming resistance by ALK compound mutation (I1171S + G1269A) after sequential treatment of multiple ALK inhibitors in non-small cell lung cancer.

BACKGROUND: Anaplastic lymphoma kinase (ALK) fusion genes are found in 3%-5% of non-small cell lung cancers (NSCLCs). ALK inhibitors show a very high response rate to ALK-positive NSCLCs. However, the emergence of acquired resistance is inevitable. In this study, we investigated the drugs for overcoming resistance especially compound mutations after sequential treatment with crizotinib, alectinib, and lorlatinib. METHOD: Next-generation sequencing (NGS) and Sanger sequencing were performed on a liver biopsy tissue obtained from a clinical case. Ba/F3 cells in which mutant EML4-ALK were overexpressed were prepared, and cell viability assay and immunoblotting were performed to check the sensitivity of five independent ALK inhibitors. RESULTS: I1171S + G1269A double mutation was identified by NGS and Sanger sequencing on a liver biopsy tissue from a patient who relapsed on lorlatinib treatment. Ceritinib and brigatinib-but not other ALK inhibitors-were active against the compound mutations in the cell line model. CONCLUSIONS: With the sequential ALK inhibitors treatment, cancer cells accumulate new mutations in addition to mutations acquired previously. The identified compound mutation (I1171S + G1269A) was found to be sensitive to ceritinib and brigatinib, and indeed the patient's tumor partially responded to ceritinib. KEY POINTS: ALK compound mutation was found in a clinical sample that was resistant to lorlatinib after sequential ALK-tyrosine kinase inhibitor (TKI) treatment. Ceritinib and brigatinib are potential overcoming drugs against ALK I1171S + G1269A double mutation.

Casein kinase 2-interacting protein-1, a novel Akt pleckstrin homology domain-interacting protein, down-regulates PI3K/Akt signaling and suppresses tumor growth in vivo.

The serine/threonine kinase Akt plays a central role in cell survival and proliferation. Its activation is linked to tumorigenesis in several human cancers. Although many Akt substrates have been elucidated, the Akt-binding proteins that regulate Akt function remain unclear. We report herein having identified casein kinase 2-interacting protein-1 (CKIP-1) as an Akt pleckstrin homology (PH) domain-binding protein with Akt inhibitory function. CKIP-1 formed a complex with each Akt isoform (Akt1, Akt2, and Akt3) via its NH2 terminus. Dimerization of CKIP-1 via its leucine zipper (LZ) motif at the COOH terminus was found to be associated with Akt inactivation because deletion of the LZ motif eliminated Akt inhibitory function, although it could still bind to Akt. Expression of the NH2 terminus-deleted CKIP-1 mutant containing the LZ motif, but lacking Akt-binding ability, induced Akt phosphorylation and activation by sequestering the ability of endogenous CKIP-1 to bind to Akt. Stable CKIP-1 expression caused Akt inactivation and cell growth inhibition in vitro. In addition, the growth of stable CKIP-1 transfectants xenografted into nude mice was slower than that of mock transfectants. These results indicate that CKIP-1, a novel Akt PH domain-interacting protein, would be a candidate of tumor suppressor with an Akt inhibitory function.

Prediction of ALK mutations mediating ALK-TKIs resistance and drug re-purposing to overcome the resistance.

BACKGROUND: Alectinib has shown a greater efficacy to ALK-rearranged non-small-cell lung cancers in first-line setting; however, most patients relapse due to acquired resistance, such as secondary mutations in ALK including I1171N and G1202R. Although ceritinib or lorlatinib was shown to be effective to these resistant mutants, further resistance often emerges due to ALK-compound mutations in relapse patients following the use of ceritinib or lorlatinib. However, the drug for overcoming resistance has not been established yet. METHODS: We established lorlatinib-resistant cells harboring ALK-I1171N or -G1202R compound mutations by performing ENU mutagenesis screening or using an in vivo mouse model. We performed drug screening to overcome the lorlatinib-resistant ALK-compound mutations. To evaluate these resistances in silico, we developed a modified computational molecular dynamic simulation (MP-CAFEE). FINDINGS: We identified 14 lorlatinib-resistant ALK-compound mutants, including several mutants that were recently discovered in lorlatinib-resistant patients. Some of these compound mutants were found to be sensitive to early generation ALK-TKIs and several BCR-ABL inhibitors. Using our original computational simulation, we succeeded in demonstrating a clear linear correlation between binding free energy and in vitro experimental IC50 value of several ALK-TKIs to single- or compound-mutated EML4-ALK expressing Ba/F3 cells and in recapitulating the tendency of the binding affinity reduction by double mutations found in this study. Computational simulation revealed that ALK-L1256F single mutant conferred resistance to lorlatinib but increased the sensitivity to alectinib. INTERPRETATION: We discovered lorlatinib-resistant multiple ALK-compound mutations and an L1256F single mutation as well as the potential therapeutic strategies for these ALK mutations. Our original computational simulation to calculate the binding affinity may be applicable for predicting resistant mutations and for overcoming drug resistance in silico. FUND: This work was mainly supported by MEXT/JSPS KAKENHI Grants and AMED Grants.

The new-generation selective ROS1/NTRK inhibitor DS-6051b overcomes crizotinib resistant ROS1-G2032R mutation in preclinical models.

ROS1 gene rearrangement was observed in around 1-2 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. Crizotinib, an ALK/ROS1/MET inhibitor, is highly effective against ROS1-rearranged lung cancer and is used in clinic. However, crizotinib resistance is an emerging issue, and several resistance mechanisms, such as secondary kinase-domain mutations (e.g., ROS1-G2032R) have been identified in crizotinib-refractory patients. Here we characterize a new selective ROS1/NTRK inhibitor, DS-6051b, in preclinical models of ROS1- or NTRK-rearranged cancers. DS-6051b induces dramatic growth inhibition of both wild type and G2032R mutant ROS1-rearranged cancers or NTRK-rearranged cancers in vitro and in vivo. Here we report that DS-6051b is effective in treating ROS1- or NTRK-rearranged cancer in preclinical models, including crizotinib-resistant ROS1 positive cancer with secondary kinase domain mutations especially G2032R mutation which is highly resistant to crizotinib as well as lorlatinib and entrectinib, next generation ROS1 inhibitors.

High ratio of T790M to EGFR activating mutations correlate with the osimertinib response in non-small-cell lung cancer.

OBJECTIVES: Osimertinib is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that can overcome resistance due to the Thr790Met (T790M) mutation. However, osimertinib occasionally shows limited efficacy in a small population of patients. We investigated the correlation between the ratio of T790M to EGFR activating mutation and the response to osimertinib. MATERIALS AND METHODS: Between April 2016 and April 2017, 44 patients started osimertinib therapy at the Cancer Institute Hospital of the Japanese Foundation for Cancer Research. We performed EGFR mutation analysis of cytological samples from 33 patients using droplet digital PCR. We calculated the ratio of T790M to EGFR activating mutations and correlated it with the systemic response to osimertinib. RESULTS: In tumors from the 33 patients, the average ratio of T790M to EGFR activating mutations was 0.420. Twenty-one of the 33 patients had tumors with a T790M ratio of ≥0.4. The osimertinib response rate was significantly higher (92.3%) in patients with a T790M ratio of ≥0.4 than in those with a T790M ratio of <0.4 (52.6%; p = 0.0237). We examined the correlation between the T790M ratio and the tumor reduction rate and obtained a coefficient of r = 0.417 (p = 0.0175). In patients with a T790M ratio of ≥0.4, the median progression-free survival was 355 days, which was longer, but not significant, than that in patients with a T790M ratio of <0.4 (median: 264 days). In patients with a T790M ratio of ≥0.4, the median treatment duration from first-line therapy onward was 931 days, which was significantly longer than that in patients with a T790M ratio of <0.4 (median, 567.5 days) (p = 0.044). CONCLUSION: The T790M ratio to EGFR activating mutation in tumor may correlate with the response to osimertinib, and patients with a higher T790M ratio have a longer treatment history.

Mechanisms of Resistance to NTRK Inhibitors and Therapeutic Strategies in NTRK1-Rearranged Cancers.

Neurotrophic receptor tyrosine kinase 1 (NTRK1) gene rearrangement leads to constitutive activation of NTRK1, which induces high-transforming ability. NTRK-rearranged cancers have been identified in several cancer types, such as glioblastoma, non-small cell lung cancer, and colorectal cancer. Although there are currently no clinically approved inhibitors that target NTRK1, several tyrosine kinase inhibitors (TKI), such as entrectinib and LOXO-101, are in clinical trials. The purpose of this study was to identify potential mechanisms of resistance to NTRK inhibitors and find potential therapeutic strategies to overcome the resistance. We examined the sensitivity of TPM3-NTRK1-transformed Ba/F3 cells and TPM3-NTRK1-harboring KM12 cells to multiple NTRK inhibitors. Acquired NTRK inhibitor-resistant mutations were screened by N-ethyl-N-nitrosourea mutagenesis with Ba/F3-TPM3-NTRK1 cells or by the establishment of NTRK-TKI-resistant cells from KM12 cells continuously treated with NTRK-TKIs. We identified multiple novel NTRK-TKI resistance mutations in the NTRK1 kinase domain, including G595R, and insulin growth factor receptor type 1 (IGF1R) bypass pathway-mediated resistance. After identifying the resistance mechanisms, we performed drug screening with small-molecule inhibitors to overcome the resistance. As a result, we found that ponatinib and nintedanib effectively inhibited the survival of TPM3-NTRK1-G667C but not G595R mutants, both of which showed resistance to entrectinib or larotrectinib (LOXO-101). Furthermore, cabozantinib with an IGF1R inhibitor such as OSI-906 could overcome bypass pathway-mediated resistance. We developed a comprehensive model of acquired resistance to NTRK inhibitors in cancer with NTRK1 rearrangement and identified cabozantinib as a therapeutic strategy to overcome the resistance. Mol Cancer Ther; 16(10); 2130-43. ©2017 AACR.

Blocking telomerase by dietary polyphenols is a major mechanism for limiting the growth of human cancer cells in vitro and in vivo.

Animal and epidemiological studies reveal that consuming food and beverages rich in polyphenols (e.g., catechins, flavones, and antocyanines) is associated with a lower incidence of cancer, and several molecular mechanisms have been proposed for explaining this effect. However, because most of these mechanisms were observed only under specific and nonphysiological conditions, and in most cases, with practically irrelevant concentrations, there is still no clear-cut or universal explanation for the major events that underlie the anticancer effects of polyphenols. In this study we present clear in vitro and in vivo evidence that the inhibition of the cancer-associated enzyme telomerase is a key mechanism involved in cancer inhibition by epigallocatechin gallate (EGCG), a major tea polyphenol. We demonstrate that EGCG and other selected polyphenols undergo structural rearrangements at physiologically permissible conditions that result in remarkably increased telomerase inhibition. In nude mice models bearing both telomerase-dependent and -independent xenograft tumors cloned from a single human cancer progeny, only the telomerase-dependent tumors responded to prolonged oral administration of EGCG. Thus, EGCG and likely other structurally related dietary polyphenols seem to act as prodrug-like molecules that, once ingested and distributed, undergo structural changes that favor potent activity against telomerase.

Predominant suppression of apoptosome by inhibitor of apoptosis protein in non-small cell lung cancer H460 cells: therapeutic effect of a novel polyarginine-conjugated Smac peptide.

The inhibitor of apoptosis proteins (IAPs) plays a central role in repressing caspase-mediated cell death. However, little is known about the actual role of endogenously expressed IAPs in cancer cells. We found that the cytochrome c/apoptotic protease-activating factor-1 (apoptosome)-dependent caspase activation is deficient in human non-small cell lung cancer (NSCLC) NCI-H460 cells. This dysfunctional apoptosome activity was not correlated with any decrease of apoptosome component factors, but it was linked to an increased X-linked inhibitor of apoptosis protein (XIAP). In H460 cells, the overexpressed XIAP, but not c-IAP1, bound to the processed form of caspase-9 and suppressed the activation of downstream effector caspases. Moreover, the defect in apoptosome activity in H460 cells was dramatically restored by the IAP-targeting SmacN7 peptide, which disrupted XIAP-caspase-9 binding, indicating an essential role of the IAP in the apoptosome inhibition. However, the SmacN7 did not show any striking effect on the apoptosome activity of normal lung fibroblast cells, although these cells also expressed modest amounts of IAP. To explore the therapeutic approach, we additionally developed SmacN7(R)8, a newly designed cell permeable peptide. The SmacN7(R)8 selectively reversed the apoptosis resistance of H460 cells, and when in combination with chemotherapy, regressed the tumor growth in vivo with little toxicity to the mice. Our results indicate that IAP-dependent suppression of apoptosome predominantly occurs in IAP-overexpressing tumor, and the IAP-targeting Smac peptide is an effective molecule to increase tumor cell death induced by chemotherapy in vitro and in vivo.

Telomere shortening and growth inhibition of human cancer cells by novel synthetic telomerase inhibitors MST-312, MST-295, and MST-1991.

Epidemiological studies suggest potent anticancer effects of tea catechins. Previously, we have reported (I. Naasani et aL, Biochem. Biophys. Res. Commun., 249: 391-396, 1998) that epigallocatechin gallate (EGCG), a major tea catechin, strongly and directly inhibits telomerase, a ribonucleoprotein that maintains telomeres and has been implicated in tumorigenesis. Here, we describe newly synthesized compounds MST-312, MST-295, and MST-199, as more effective telomerase inhibitors than EGCG. Continuous treatment of human monoblastoid leukemia U937 cells with a nontoxic dose of each drug caused progressive telomere shortening and eventual reduction of growth rate accompanied by induction of the senescence-associated beta-galactosidase activity. Particularly, in the case of MST-312, the effective dose required for the telomere shortening was 1-2 microM, which was 15- to 20-fold lower than that of EGCG. These compounds may provide a novel chemotherapeutic strategy for the treatment of cancers.