bluuebunny/crossref_metadata_2025 huggingface dataset:
Data from Impact of the Anticancer Drug NT157 on Tyrosine Kinase Signaling Networks
Authors: Su Shih-Ping , Flashner-Abramson Efrat , Klein Shoshana , Gal Mor , Lee Rachel S. , Wu Jianmin , Levitzki Alexander , Daly Roger J.
Published: March 2024

Abstract
The small-molecule drug NT157 has demonstrated promising efficacy in preclinical models of a number of different cancer types, reflecting activity against both cancer cells and the tumor microenvironment. Two known mechanisms of action are degradation of insulin receptor substrates (IRS)-1/2 and reduced Stat3 activation, although it is possible that others exist. To interrogate the effects of this drug on cell signaling pathways in an unbiased manner, we have undertaken mass spectrometry–based global tyrosine phosphorylation profiling of NT157-treated A375 melanoma cells. Bioinformatic analysis of the resulting dataset resolved 5 different clusters of tyrosine-phosphorylated peptides that differed in the directionality and timing of response to drug treatment over time. The receptor tyrosine kinase AXL exhibited a rapid decrease in phosphorylation in response to drug treatment, followed by proteasome-dependent degradation, identifying an additional potential target for NT157 action. However, NT157 treatment also resulted in increased activation of p38 MAPK α and γ, as well as the JNKs and specific Src family kinases. Importantly, cotreatment with the p38 MAPK inhibitor SB203580 attenuated the antiproliferative effect of NT157, while synergistic inhibition of cell proliferation was observed when NT157 was combined with a Src inhibitor. These findings provide novel insights into NT157 action on cancer cells and highlight how globally profiling the impact of a specific drug on cellular signaling networks can identify effective combination treatments. Mol Cancer Ther; 17(5); 931–42. ©2018 AACR.
Data from Genomic and Immunological Tumor Profiling Identifies Targetable Pathways and Extensive CD8+/PDL1+ Immune Infiltration in Inflammatory Breast Cancer Tumors
Authors: Hamm Christopher A. , Moran Diarmuid , Rao Kakuturu , Trusk Patricia B. , Pry Karen , Sausen Mark , Jones Siân , Velculescu Victor E. , Cristofanilli Massimo , Bacus Sarah
Published: March 2024

Abstract
Inflammatory breast cancer (IBC) is a rare and aggressive form of breast cancer that remains poorly understood at the molecular level. Comprehensive tumor profiling was performed to understand clinically actionable alterations in IBC. Targeted next-generation sequencing (NGS) and IHC were performed to identify activated pathways in IBC tumor tissues. siRNA studies examined the impact of IBC genomic variants in cellular models. IBC tumor tissues were further characterized for immune infiltration and immune checkpoint expression by IHC. Genomic analysis identified recurrent alterations in core biologic pathways, including activating and targetable variants in HER/PI3K/mTOR signaling. High rates of activating HER3 point mutations were discovered in IBC tumors. Cell line studies confirmed a role for mutant HER3 in IBC cell proliferation. Immunologic analysis revealed a subset of IBC tumors associated with high CD8+/PD-L1+ lymphocyte infiltration. Immune infiltration positively correlated with an NGS-based estimate of neoantigen exposure derived from the somatic mutation rate and mutant allele frequency, iScore. Additionally, DNA mismatch repair alterations, which may contribute to higher iScores, occurred at greater frequency in tumors with higher immune infiltration. Our study identifies genomic alterations that mechanistically contribute to oncogenic signaling in IBC and provides a genetic basis for the selection of clinically relevant targeted and combination therapeutic strategies. Furthermore, an NGS-based estimate of neoantigen exposure developed in this study (iScore) may be a useful biomarker to predict immune infiltration in IBC and other cancers. The iScore may be associated with greater levels of response to immunotherapies, such as PD-L1/PD-1–targeted therapies. Mol Cancer Ther; 15(7); 1746–56. ©2016 AACR.
Data from Circulating Tumor Cells with Stemness and Epithelial-to-Mesenchymal Transition Features Are Chemoresistant and Predictive of Poor Outcome in Metastatic Breast Cancer
Authors: Papadaki Maria A. , Stoupis Giannis , Theodoropoulos Panayiotis A. , Mavroudis Dimitris , Georgoulias Vassilis , Agelaki Sofia
Published: March 2024

Abstract
Circulating tumor cells (CTCs) bearing phenotypes related to cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) have been identified in breast cancer; however, their clinical significance is not clear. In the current study, we investigated the prognostic relevance of single CSC+/partial-EMT+ CTCs in patients with metastatic breast cancer and the effect of first-line chemotherapy on their incidence. For this purpose, triple immunofluorescence against cytokeratin, ALDH1, and TWIST1 was performed in peripheral blood mononuclear cell (PBMC) cytospins from 130 patients before and after first-line chemotherapy. CSC+/partial-EMT+ CTCs were characterized as cells co-expressing cytokeratin, high levels of ALDH1, and nuclear TWIST1. CSC+/partial-EMT+ CTCs were evident in 27.7% of patients at baseline and were correlated to lung metastases (P = 0.010) and decreased progression-free survival [PFS; median 10.2 (8.9–11.6) vs. 13.5 (11.3–15.7) months; P = 0.024]. Their detection was an independent factor predicting for increased risk of relapse [multivariate analysis; HR (95% confidence interval (CI)): 1.785 (1.171–2.720); P = 0.007]. In HER-2–negative patients, CSC+/partial-EMT+ CTCs were additionally associated with reduced overall survival (OS) [median 39 (26.2–51.9) vs. 51 (15.7–86.4) months; P = 0.020] and increased risk of death [multivariate analysis; HR (95% CI): 2.228 (1.066–4.655); P = 0.033]. Chemotherapy resulted in a significant increase in the incidence of CSC+/partial-EMT+ CTCs (mean CTC% per patient: 59.4% post vs. 39.5% pre; P = 0.018), which was subsequently confirmed only in HER2-negative patients (P = 0.040) and in non-responders at the end of treatment (P = 0.020). In conclusion, CSC+/partial-EMT+ CTCs represent a chemoresistant subpopulation, which independently predicts for unfavorable outcome in metastatic breast cancer. Efficient targeting of these CTCs could potentially increase patient survival.
Data from 15α-methoxypuupehenol Induces Antitumor Effects In Vitro and In Vivo against Human Glioblastoma and Breast Cancer Models
Authors: Hilliard Tyvette S. , Miklossy Gabriella , Chock Christopher , Yue Peibin , Williams Philip , Turkson James
Published: March 2024

Abstract
Studies with 15α-methoxypuupehenol (15α-MP), obtained from the extracts of Hyrtios species, identified putative targets that are associated with its antitumor effects against human glioblastoma and breast cancer. In the human glioblastoma (U251MG) or breast cancer (MDA-MB-231) cells, treatment with 15α-MP repressed pY705Stat3, pErk1/2, pS147CyclinB1, pY507Alk (anaplastic lymphoma kinase), and pY478ezrin levels and induced pS10merlin, without inhibiting pJAK2 (Janus kinase) or pAkt induction. 15α-MP treatment induced loss of viability of breast cancer (MDA-MB-231, MDA-MB-468) and glioblastoma (U251MG) lines and glioblastoma patient–derived xenograft cells (G22) that harbor aberrantly active Stat3, with only moderate or little effect on the human breast cancer, MCF7, colorectal adenocarcinoma Caco-2, normal human lung fibroblast, WI-38, or normal mouse embryonic fibroblast (MEF Stat3fl/fl) lines that do not harbor constitutively active Stat3 or the Stat3-null (Stat3−/−) mouse astrocytes. 15α-MP–treated U251MG cells have severely impaired F-actin organization and altered morphology, including the cells rounding up, and undergo apoptosis, compared with a moderate, reversible morphology change observed for similarly treated mouse astrocytes. Treatment further inhibited U251MG or MDA-MB-231 cell proliferation, anchorage-independent growth, colony formation, and migration in vitro while only moderately or weakly affecting MCF7 cells or normal mouse astrocytes. Oral gavage delivery of 15α-MP inhibited the growth of U251MG subcutaneous tumor xenografts in mice, associated with apoptosis in the treated tumor tissues. Results together suggest that the modulation of Stat3, CyclinB1, Alk, ezrin, merlin, and Erk1/2 functions contributes to the antitumor effects of 15α-MP against glioblastoma and breast cancer progression. Mol Cancer Ther; 16(4); 601–13. ©2017 AACR.
Data from Suppression of SRC Signaling Is Effective in Reducing Synergy between Glioblastoma and Stromal Cells
Authors: Calgani Alessia , Vignaroli Giulia , Zamperini Claudio , Coniglio Federica , Festuccia Claudio , Di Cesare Ernesto , Gravina Giovanni Luca , Mattei Claudia , Vitale Flora , Schenone Silvia , Botta Maurizio , Angelucci Adriano
Published: March 2024

Abstract
Glioblastoma cells efficiently interact with and infiltrate the surrounding normal tissue, rendering surgical resection and adjuvant chemo/radiotherapy ineffective. New therapeutic targets, able to interfere with glioblastoma's capacity to synergize with normal brain tissue, are currently under investigation. The compound Si306, a pyrazolo[3,4-d]pyrimidine derivative, selected for its favorable activity against SRC, was tested in vitro and in vivo on glioblastoma cell lines. In vivo, combination treatment with Si306 and radiotherapy was strongly active in reducing U-87 xenograft growth with respect to control and single treatments. The histology revealed a significant difference in the stromal compartment of tumoral tissue derived from control or radiotherapy-treated samples with respect to Si306-treated samples, showing in the latter a reduced presence of collagen and α-SMA–positive cells. This effect was paralleled in vitro by the capacity of Si306 to interfere with myofibroblastic differentiation of normal fibroblasts induced by U-87 cells. In the presence of Si306, TGF-β released by U-87 cells, mainly in hypoxia, was ineffective in upregulating α-SMA and β-PDGFR in fibroblasts. Si306 efficiently reached the brain and significantly prolonged the survival of mice orthotopically injected with U-87 cells. Drugs that target SRC could represent an effective therapeutic strategy in glioblastoma, able to block positive paracrine loop with stromal cells based on the β-PDGFR axis and the formation of a tumor-promoting microenvironment. This approach could be important in combination with conventional treatments in the effort to reduce tumor resistance to therapy. Mol Cancer Ther; 15(7); 1535–44. ©2016 AACR.
Data from Combining Nonclinical Experiments with Translational PKPD Modeling to Differentiate Erlotinib and Gefitinib
Authors: Eigenmann Miro J. , Frances Nicolas , Hoffmann Gerhard , Lavé Thierry , Walz Antje-Christine
Published: March 2024

Abstract
We quantitatively compare the efficacy of two approved EGFR tyrosine kinase inhibitors, erlotinib and gefitinib, based on in vivo and in vitro data and show how a modeling approach can be used to scale from animal to humans. Gefitinib shows a higher tumor uptake in cancer patients, and we explored the potential impact on pharmacologic and antitumor activity in in vitro and in xenograft mice. Tumor growth inhibition was monitored, and the pharmacokinetics (PK) in plasma and tumor, as well as temporal changes of phospho-Erk (pErk) signals were examined in patient-derived tumor xenograft mice. These data were integrated in a translational PKPD model, allowing us to project an efficacious human dose, which we retrospectively compared with prescribed doses for cancer patients. In vitro experiments showed that cell-cycle arrest was similar for erlotinib and gefitinib. Similar pERK biomarker responses were obtained despite a 6.6-fold higher total tumor exposure for gefitinib. The PKPD model revealed a 3.7-fold higher in vivo potency for gefitinib, which did not translate into a lower anticipated efficacious dose in humans. The model-based dose prediction matched the recommended clinical doses well. These results suggest that despite having lower total tumor-to-plasma ratios, active drug exposure at target site is higher for erlotinib. Considering the PK properties, this translates in a 50% lower recommended daily dose of erlotinib in cancer patients. In summary, total exposure at target site is not suitable to rank compounds, and an integrated modeling and experimental approach can assess efficacy more accurately. Mol Cancer Ther; 15(12); 3110–9. ©2016 AACR.
Data from Cotargeting of MEK and PDGFR/STAT3 Pathways to Treat Pancreatic Ductal Adenocarcinoma
Authors: Sahu Nisebita , Chan Emily , Chu Felix , Pham Thinh , Koeppen Hartmut , Forrest William , Merchant Mark , Settleman Jeff
Published: March 2024

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal human diseases and remains largely refractory to available drug treatments. Insufficient targeting of the known oncogenic drivers and activation of compensatory feedback loops and inability to prevent metastatic spread contribute to poor prognosis for this disease. The KRAS-driven MEK pathway is mutationally activated in most pancreatic cancers and is an important target for therapeutics. Using a two-dimensional monolayer culture system as well as three-dimensional spheroid culture system, we conducted a screen of a large panel of anticancer agents and found that MAP2K (MEK) inhibitors were most effective in targeting PDAC spheroids in comparison with monolayer cultures. Combination treatment with an MEK inhibitor and the multikinase inhibitor ponatinib was effective in targeting pancreatic cancer cells both in monolayer and spheroids by effectively blocking signaling via the PDGFRα and MEK kinases, while also preventing the activation of STAT3- and S6-mediated compensatory feedback loops in cancer cells. Furthermore, using xenograft models, we demonstrate that cotreatment with a MEK inhibitor and ponatinib causes significant tumor regression. PDAC patient samples also provided evidence of increased STAT3 activation in PDAC tumors and MAPK1 (ERK) activation in liver metastases, implicating STAT3 and ERK as key drivers in primary tumors and metastases, respectively. These results reveal a combination drug treatment strategy that may be effective in pancreatic cancer. Mol Cancer Ther; 16(9); 1729–38. ©2017 AACR.
Data from Alkylating Agent–Induced NRF2 Blocks Endoplasmic Reticulum Stress–Mediated Apoptosis via Control of Glutathione Pools and Protein Thiol Homeostasis
Authors: Zanotto-Filho Alfeu , Masamsetti V. Pragathi , Loranc Eva , Tonapi Sonal S. , Gorthi Aparna , Bernard Xavier , Gonçalves Rosângela Mayer , Moreira José C.F. , Chen Yidong , Bishop Alexander J.R.
Published: March 2024

Abstract
Alkylating agents are a commonly used cytotoxic class of anticancer drugs. Understanding the mechanisms whereby cells respond to these drugs is key to identify means to improve therapy while reducing toxicity. By integrating genome-wide gene expression profiling, protein analysis, and functional cell validation, we herein demonstrated a direct relationship between NRF2 and Endoplasmic Reticulum (ER) stress pathways in response to alkylating agents, which is coordinated by the availability of glutathione (GSH) pools. GSH is essential for both drug detoxification and protein thiol homeostasis within the ER, thus inhibiting ER stress induction and promoting survival, an effect independent of its antioxidant role. NRF2 accumulation induced by alkylating agents resulted in increased GSH synthesis via GCLC/GCLM enzyme, and interfering with this NRF2 response by either NRF2 knockdown or GCLC/GCLM inhibition with buthionine sulfoximine caused accumulation of damaged proteins within the ER, leading to PERK-dependent apoptosis. Conversely, upregulation of NRF2, through KEAP1 depletion or NRF2-myc overexpression, or increasing GSH levels with N-acetylcysteine or glutathione-ethyl-ester, decreased ER stress and abrogated alkylating agents–induced cell death. Based on these results, we identified a subset of lung and head-and-neck carcinomas with mutations in either KEAP1 or NRF2/NFE2L2 genes that correlate with NRF2 target overexpression and poor survival. In KEAP1-mutant cancer cells, NRF2 knockdown and GSH depletion increased cell sensitivity via ER stress induction in a mechanism specific to alkylating drugs. Overall, we show that the NRF2-GSH influence on ER homeostasis implicates defects in NRF2-GSH or ER stress machineries as affecting alkylating therapy toxicity. Mol Cancer Ther; 15(12); 3000–14. ©2016 AACR.
Data from EGFR-Mediated Reactivation of MAPK Signaling Induces Acquired Resistance to GSK2118436 in BRAF V600E–Mutant NSCLC Cell Lines
Authors: Kim Sung-Moo , Kim Hwan , Jang Kang Won , Kim Min Hwan , Sohn Jinyoung , Yun Mi Ran , Kang Han Na , Kang Chan Woo , Kim Hye Ryun , Lim Sun Min , Moon Yong Wha , Kim Joo Hang , Paik Soonmyung , Cho Byoung Chul
Published: March 2024

Abstract
Although treatment of BRAF V600E–mutant non–small cell lung cancer (NSCLCV600E) with GSK2118436 has shown an encouraging efficacy, most patients develop resistance. To investigate the mechanisms of acquired resistance to GSK2118436 in NSCLCV600E, we established GSK2118436-resistant (GSR) cells by exposing MV522 NSCLCV600E to increasing GSK2118436 concentrations. GSR cells displayed activated EGFR–RAS–CRAF signaling with upregulated EGFR ligands and sustained activation of ERK1/2, but not MEK1/2, in the presence of GSK2118436. Treatment of GSR cells with GSK2118436 enhanced EGFR-mediated RAS activity, leading to the formation of BRAF–CRAF dimers and transactivation of CRAF. Interestingly, sustained activation of ERK1/2 was partly dependent on receptor-interacting protein kinase-2 (RIP2) activity, but not on MEK1/2 activity. Combined BRAF and EGFR inhibition blocked reactivation of ERK signaling and improved efficacy in vitro and in vivo. Our findings support the evaluation of combined BRAF and EGFR inhibition in NSCLCV600E with acquired resistance to BRAF inhibitors. Mol Cancer Ther; 15(7); 1627–36. ©2016 AACR.
Data from Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity
Authors: Wengner Antje M. , Siemeister Gerhard , Koppitz Marcus , Schulze Volker , Kosemund Dirk , Klar Ulrich , Stoeckigt Detlef , Neuhaus Roland , Lienau Philip , Bader Benjamin , Prechtl Stefan , Raschke Marian , Frisk Anna-Lena , von Ahsen Oliver , Michels Martin , Kreft Bertolt , von Nussbaum Franz , Brands Michael , Mumberg Dominik , Ziegelbauer Karl
Published: March 2024

Abstract
Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis (“mitotic breakthrough”), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583–92. ©2016 AACR.
Data from β-Catenin Inhibitor BC2059 Is Efficacious as Monotherapy or in Combination with Proteasome Inhibitor Bortezomib in Multiple Myeloma
Authors: Savvidou Ioanna , Khong Tiffany , Cuddihy Andrew , McLean Catriona , Horrigan Stephen , Spencer Andrew
Published: March 2024

Abstract
Currently available treatment options are unlikely to be curative for the majority of multiple myeloma patients, emphasizing a continuing role for the introduction of investigational agents that can overcome drug resistance. The canonical Wnt/β-catenin signaling pathway, essential for self-renewal, growth, and survival, has been found to be dysregulated in multiple myeloma, particularly in advanced stages of disease. This provides the rationale for evaluating the novel β-catenin inhibitor BC2059 as monotherapy and in combination with proteasome inhibitors in vitro and in vivo. Here, we show nuclear localization of β-catenin in human myeloma cell lines (HMCL), consistent with activation of the canonical Wnt pathway. BC2059 attenuates β-catenin levels, in both the cytoplasm and the nucleus, reducing the transcriptional activity of the TCF4/LEF complex and the expression of its target gene axin 2. Treatment of HMCL with BC2059 inhibits proliferation and induces apoptosis in a dose-dependent manner. This is also observed in HMCL–stromal cell cocultures, mitigating the protective effect afforded by the stroma. Similarly, BC2059 induces apoptosis in primary multiple myeloma samples in vitro, causing minimal apoptosis on healthy peripheral blood mononuclear cells. Furthermore, it synergizes with the proteasome inhibitor bortezomib both in HMCL and primary multiple myeloma samples. Finally, in xenograft models of human myelomatosis, BC2059 delays tumor growth and prolongs survival with minor on-target side effects. Collectively, these results demonstrate the efficacy of targeting the Wnt/β-catenin pathway with BC2059 both in vitro and in vivo, at clinically achievable doses. These findings support further clinical evaluation of BC2059 for the treatment of multiple myeloma. Mol Cancer Ther; 16(9); 1765–78. ©2017 AACR.
Data from Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody–Drug Conjugate Targeting 5T4
Authors: Harper Jay , Lloyd Christopher , Dimasi Nazzareno , Toader Dorin , Marwood Rose , Lewis Leeanne , Bannister David , Jovanovic Jelena , Fleming Ryan , D'Hooge Francois , Mao Shenlan , Marrero Allison M. , Korade Martin , Strout Patrick , Xu Linda , Chen Cui , Wetzel Leslie , Breen Shannon , van Vlerken-Ysla Lilian , Jalla Sanjoo , Rebelatto Marlon , Zhong Haihong , Hurt Elaine M. , Hinrichs Mary Jane , Huang Keven , Howard Philip W. , Tice David A. , Hollingsworth Robert E. , Herbst Ronald , Kamal Adeela
Published: March 2024

Abstract
Antibody–drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576–87. ©2017 AACR.
Data from Development and Application of a Novel Model System to Study “Active” and “Passive” Tumor Targeting
Authors: Mukherjee Amarnath , Kumar Binod , Hatano Koji , Russell Luisa M. , Trock Bruce J. , Searson Peter C. , Meeker Alan K. , Pomper Martin G. , Lupold Shawn E.
Published: March 2024

Abstract
Macromolecular reagents can be targeted to tumors through active and passive mechanisms. “Active” targeting involves moieties, such as receptor ligands, to direct tumor cell binding, whereas “passive” targeting relies on long reagent circulating half-life, abnormal tumor vasculature, and poor lymphatic drainage for tumor entrapment. Here, we sought to study the impact of reagent circulating half-life on “active” and “passive” tumor uptake. The humanized prostate-specific membrane antigen (PSMA)-targeting antibody HuJ591 was used as the “active” targeting agent. HuJ591 was labeled with a Near Infrared (NIR) dye and its circulating half-life was modified by conjugation to high-molecular-weight Polyethylene Glycol (PEG). PEGylation did not negatively impact PSMA-binding specificity. “Active” and “passive” tumor targeting of intravenously injected antibody conjugates were then quantified by NIR fluorescent imaging of immunocompromised mice bearing bilateral isogenic PSMA-positive and PSMA-negative human tumor xenografts. Two isogenic tumor pairs were applied, PC3 ± PSMA (PC3-PIP/PC3-Flu) or LMD-MDA-MB-231 ± PSMA (LMD-PSMA/LMD). This study provided a unique model system to simultaneously observe “active” and “passive” tumor targeting within a single animal. “Passive” targeting was observed in all PSMA-negative tumors, and was not enhanced by increased HuJ591 size or extended circulating half-life. Interestingly, “active” targeting was only successful in some situations. Both PSMA-positive tumor models could be actively targeted with J591-IR800 and J591-PEG10K. However, the larger J591-PEG30K enhanced “active” targeting in the PC-3 tumor models, but inhibited “active” targeting the LMD-MDA-MB-231 tumor model. Successful “active” targeting was associated with higher PSMA expression. These results support the potential for “active” targeting to enhance overall macromolecular reagent uptake within tumors. Mol Cancer Ther; 15(10); 2541–50. ©2016 AACR.
Data from An HRE-Binding Py-Im Polyamide Impairs Hypoxic Signaling in Tumors
Authors: Szablowski Jerzy O. , Raskatov Jevgenij A. , Dervan Peter B.
Published: March 2024

Abstract
Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ fibrosis, age-related macular degeneration, and cancer. Hypoxia-inducible factor-1 (HIF1), a transcription factor central to the hypoxic gene expression, mediates multiple processes including neovascularization, cancer metastasis, and cell survival. Pyrrole-imidazole polyamide 1 has been shown to inhibit HIF1-mediated gene expression in cell culture but its activity in vivo was unknown. This study reports activity of polyamide 1 in subcutaneous tumors capable of mounting a hypoxic response and showing neovascularization. We show that 1 distributes into subcutaneous tumor xenografts and normal tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation of new tumor blood vessels, and suppresses tumor growth. Tumors treated with 1 show no increase in HIF1α and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased apoptosis in HIF1α-positive regions and the increased proximity of necrotic regions to vasculature. Overall, these results show that a molecule designed to block the transcriptional activity of HIF1 has potent antitumor activity in vivo, consistent with partial inhibition of the tumor hypoxic response. Mol Cancer Ther; 15(4); 608–17. ©2015 AACR.
Data from Combinatorial Drug Screening Identifies Ewing Sarcoma–specific Sensitivities
Authors: Radic-Sarikas Branka , Tsafou Kalliopi P. , Emdal Kristina B. , Papamarkou Theodore , Huber Kilian V. M. , Mutz Cornelia , Toretsky Jeffrey A. , Bennett Keiryn L. , Olsen Jesper V. , Brunak Søren , Kovar Heinrich , Superti-Furga Giulio
Published: March 2024

Abstract
Improvements in survival for Ewing sarcoma pediatric and adolescent patients have been modest over the past 20 years. Combinations of anticancer agents endure as an option to overcome resistance to single treatments caused by compensatory pathways. Moreover, combinations are thought to lessen any associated adverse side effects through reduced dosing, which is particularly important in childhood tumors. Using a parallel phenotypic combinatorial screening approach of cells derived from three pediatric tumor types, we identified Ewing sarcoma–specific interactions of a diverse set of targeted agents including approved drugs. We were able to retrieve highly synergistic drug combinations specific for Ewing sarcoma and identified signaling processes important for Ewing sarcoma cell proliferation determined by EWS-FLI1. We generated a molecular target profile of PKC412, a multikinase inhibitor with strong synergistic propensity in Ewing sarcoma, revealing its targets in critical Ewing sarcoma signaling routes. Using a multilevel experimental approach including quantitative phosphoproteomics, we analyzed the molecular rationale behind the disease-specific synergistic effect of simultaneous application of PKC412 and IGF1R inhibitors. The mechanism of the drug synergy between these inhibitors is different from the sum of the mechanisms of the single agents. The combination effectively inhibited pathway crosstalk and averted feedback loop repression, in EWS-FLI1–dependent manner. Mol Cancer Ther; 16(1); 88–101. ©2016 AACR.
Data from Afatinib against Esophageal or Head-and-Neck Squamous Cell Carcinoma: Significance of Activating Oncogenic HER4 Mutations in HNSCC
Authors: Nakamura Yu , Togashi Yosuke , Nakahara Hirokazu , Tomida Shuta , Banno Eri , Terashima Masato , Hayashi Hidetoshi , de Velasco Marco A. , Sakai Kazuko , Fujita Yoshihiko , Okegawa Takatsugu , Nutahara Kikuo , Hamada Suguru , Nishio Kazuto
Published: March 2024

Abstract
The prognosis for patients with advanced esophageal or head-and-neck squamous cell carcinoma (ESCC or HNSCC) remains poor, and the identification of additional oncogenes and their inhibitors is needed. In this study, we evaluated the sensitivities of several ESCC and HNSCC cell lines to HER inhibitors (cetuximab, erlotinib, and afatinib) in vitro and found two cell lines that were hypersensitive to afatinib. Sequence analyses for the afatinib-targeted HER family genes in the two cell lines revealed that one cell line had a previously reported activating EGFR L861Q mutation, whereas the other had an HER4 G1109C mutation of unknown function. No amplification of HER family genes was found in either of the two cell lines. The phosphorylation level of HER4 was elevated in the HER4 G1109C mutation-overexpressed HEK293 cell line, and the mutation had a transforming potential and exhibited tumorigenicity in an NIH3T3 cell line, indicating that this HER4 mutation was an activating oncogenic mutation. Afatinib dramatically reduced the phosphorylation level of EGFR or HER4 and induced apoptosis in the two cell lines. In vivo, tumor growth was also dramatically decreased by afatinib. In a database, the frequencies of HER family gene mutations in ESCC or HNSCC ranged from 0% to 5%. In particular, HER4 mutations have been found relatively frequently in HNSCC. Considering the addiction of cancer cells to activating oncogenic EGFR or HER4 mutations for proliferation, HNSCC or ESCC with such oncogenic mutations might be suitable for targeted therapy with afatinib. Mol Cancer Ther; 15(8); 1988–97. ©2016 AACR.
Data from Development of a Novel Antibody–Drug Conjugate for the Potential Treatment of Ovarian, Lung, and Renal Cell Carcinoma Expressing TIM-1
Authors: Thomas Lawrence J. , Vitale Laura , O'Neill Thomas , Dolnick Ree Y. , Wallace Paul K. , Minderman Hans , Gergel Lauren E. , Forsberg Eric M. , Boyer James M. , Storey James R. , Pilsmaker Catherine D. , Hammond Russell A. , Widger Jenifer , Sundarapandiyan Karuna , Crocker Andrea , Marsh Henry C. , Keler Tibor
Published: March 2024

Abstract
T-cell immunoglobulin and mucin domain 1 (TIM-1) is a type I transmembrane protein that was originally described as kidney injury molecule 1 (KIM-1) due to its elevated expression in kidney and urine after renal injury. TIM-1 expression is also upregulated in several human cancers, most notably in renal and ovarian carcinomas, but has very restricted expression in healthy tissues, thus representing a promising target for antibody-mediated therapy. To this end, we have developed a fully human monoclonal IgG1 antibody specific for the extracellular domain of TIM-1. This antibody was shown to bind purified recombinant chimeric TIM-1-Fc protein and TIM-1 expressed on a variety of transformed cell lines, including Caki-1 (human renal clear cell carcinoma), IGROV-1 (human ovarian adenocarcinoma), and A549 (human lung carcinoma). Internalization studies using confocal microscopy revealed the antibody was rapidly internalized by cells in vitro, and internalization was confirmed by quantitative imaging flow cytometry. An antibody–drug conjugate (ADC) was produced with the anti-TIM-1 antibody covalently linked to the potent cytotoxin, monomethyl auristatin E (MMAE), and designated CDX-014. The ADC was shown to exhibit in vitro cytostatic or cytotoxic activity against a variety of TIM-1–expressing cell lines, but not on TIM-1–negative cell lines. Using the Caki-1, IGROV-1, and A549 xenograft mouse models, CDX-014 showed significant antitumor activity in a clinically relevant dose range. Safety evaluation in nonhuman primates has demonstrated a good profile and led to the initiation of clinical studies of CDX-014 in renal cell carcinoma and potentially other TIM-1–expressing tumors. Mol Cancer Ther; 15(12); 2946–54. ©2016 AACR.
Data from Linalool-Incorporated Nanoparticles as a Novel Anticancer Agent for Epithelial Ovarian Carcinoma
Authors: Han Hee Dong , Cho Young-Jae , Cho Sung Keun , Byeon Yeongseon , Jeon Hat Nim , Kim Hye-Sun , Kim Byoung-Gie , Bae Duk-Soo , Lopez-Berestein Gabriel , Sood Anil K. , Shin Byung Cheol , Park Yeong-Min , Lee Jeong-Won
Published: March 2024

Abstract
Although cytotoxic chemotherapy is widely used against epithelial ovarian cancer (EOC), adverse side effects and emergence of resistance can limit its utility. Therefore, new drugs with systemic delivery platforms are urgently needed for this disease. In this study, we developed linalool-incorporated nanoparticles (LIN-NP) as a novel anticancer agent. We prepared LIN-NPs by the self-assembly water-in-oil-in-water (w/o/w) emulsion method. LIN-NP–mediated cytotoxicity and apoptosis was assessed in EOC cells, and the role of reactive oxygen species (ROS) generation as the mechanism of action was evaluated. In addition, therapeutic efficacy of LIN-NP was assessed in cell lines and patient-derived xenograft (PDX) models for EOC. LIN-NPs had significant cytotoxicity and apoptotic activity against EOC cells, including A2780, HeyA8, and SKOV3ip1. LIN-NP treatment increased apoptosis in EOC cells through ROS generation and a subsequent decrease in mitochondrial membrane potential and increase in caspase-3 levels. In addition, 100 mg/kg LIN-NPs significantly decreased tumor weight in the HeyA8 (P < 0.001) and SKOV3ip1 (P = 0.006) in vivo models. Although treatment with 50 mg/kg LIN-NP did not decrease tumor weight compared with the control group, combination treatment with paclitaxel significantly decreased tumor weight compared with paclitaxel alone in SKOV3ip1 xenografts (P = 0.004) and the patient-derived xenograft model (P = 0.020). We have developed LIN-NPs that induce ROS generation as a novel anticancer agent for EOC. These findings have broad applications for cancer therapy. Mol Cancer Ther; 15(4); 618–27. ©2016 AACR.
Data from Dual Carfilzomib and Doxorubicin–Loaded Liposomal Nanoparticles for Synergistic Efficacy in Multiple Myeloma
Authors: Ashley Jonathan D. , Quinlan Charissa J. , Schroeder Valerie A. , Suckow Mark A. , Pizzuti Vincenzo J. , Kiziltepe Tanyel , Bilgicer Basar
Published: March 2024

Abstract
Here, we report the synthesis and evaluation of dual drug–loaded nanoparticles as an effective means to deliver carfilzomib and doxorubicin to multiple myeloma tumor cells at their optimal synergistic ratio. First, various molar ratios of carfilzomib to doxorubicin were screened against multiple myeloma cell lines to determine the molar ratio that elicited the greatest synergy using the Chou–Talalay method. The therapeutic agents were then incorporated into liposomes at the optimal synergistic ratio of 1:1 to yield dual drug–loaded nanoparticles with a narrow size range of 115 nm and high reproducibility. Our results demonstrated that the dual drug–loaded liposomes exhibited synergy in vitro and were more efficacious in inhibiting tumor growth in vivo than a combination of free drugs, while at the same time reducing systemic toxicity. Taken together, this study presents the synthesis and preclinical evaluation of dual drug–loaded liposomes containing carfilzomib and doxorubicin for enhanced therapeutic efficacy to improve patient outcome in multiple myeloma. Mol Cancer Ther; 15(7); 1452–9. ©2016 AACR.
Data from Estrogen Receptor β Is a Novel Target in Acute Myeloid Leukemia
Authors: Rota Sarah-Grace , Roma Alessia , Dude Iulia , Ma Christina , Stevens Robert , MacEachern Janet , Graczyk Joanna , Espiritu Shaundrei Mabriel G. , Rao Praveen N. , Minden Mark D. , Kreinin Elena , Hess David A. , Doxey Andrew C. , Spagnuolo Paul A.
Published: March 2024

Abstract
Acute myeloid leukemia (AML) is a devastating disease characterized by poor patient outcome and suboptimal chemotherapeutics. Here, a high-throughput screen identified diosmetin, a citrus flavonoid, with anti-AML activity. Diosmetin imparted selective toxicity against leukemia and leukemia stem cells in vitro and in vivo with no effect on normal hematopoietic stem cells. Mechanistically, we demonstrated that diosmetin targets estrogen receptor (ER) β. ERβ expression conferred cell sensitivity, as patient-derived AML cells with high levels of ERβ were sensitive, whereas cells with low ERβ were insensitive to diosmetin. Knockdown of ERβ confirmed resistance, whereas overexpression enhanced sensitivity to diosmetin, which was demonstrated to be mediated by reactive oxygen species signaling. In summary, these studies highlight targeting of ERβ with diosmetin as a potential novel therapeutic strategy for the treatment of AML. Mol Cancer Ther; 16(11); 2618–26. ©2017 AACR.
Data from Resistance to RET-Inhibition in RET-Rearranged NSCLC Is Mediated By Reactivation of RAS/MAPK Signaling
Authors: Nelson-Taylor Sarah K. , Le Anh T. , Yoo Minjae , Schubert Laura , Mishall Katie M. , Doak Andrea , Varella-Garcia Marileila , Tan Aik-Choon , Doebele Robert C.
Published: March 2024

Abstract
Oncogenic rearrangements in RET are present in 1%–2% of lung adenocarcinoma patients. Ponatinib is a multi-kinase inhibitor with low-nanomolar potency against the RET kinase domain. Here, we demonstrate that ponatinib exhibits potent antiproliferative activity in RET fusion–positive LC-2/ad lung adenocarcinoma cells and inhibits phosphorylation of the RET fusion protein and signaling through ERK1/2 and AKT. Using distinct dose escalation strategies, two ponatinib-resistant LC-2/ad cell lines, PR1 and PR2, were derived. PR1 and PR2 cell lines retained expression, but not phosphorylation of the RET fusion and lacked evidence of a resistance mutation in the RET kinase domain. Both resistant lines retained activation of the MAPK pathway. Next-generation RNA sequencing revealed an oncogenic NRAS p.Q61K mutation in the PR1 cell. PR1 cell proliferation was preferentially sensitive to siRNA knockdown of NRAS compared with knockdown of RET, more sensitive to MEK inhibition than the parental line, and NRAS dependence was maintained in the absence of chronic RET inhibition. Expression of NRAS p.Q61K in RET fusion expressing TPC1 cells conferred resistance to ponatinib. PR2 cells exhibited increased expression of EGFR and AXL. EGFR inhibition decreased cell proliferation and phosphorylation of ERK1/2 and AKT in PR2 cells, but not LC-2/ad cells. Although AXL inhibition enhanced PR2 sensitivity to afatinib, it was unable to decrease cell proliferation by itself. Thus, EGFR and AXL cooperatively rescued signaling from RET inhibition in the PR2 cells. Collectively, these findings demonstrate that resistance to ponatinib in RET-rearranged lung adenocarcinoma is mediated by bypass signaling mechanisms that result in restored RAS/MAPK activation. Mol Cancer Ther; 16(8); 1623–33. ©2017 AACR.
Data from Cell Panel Profiling Reveals Conserved Therapeutic Clusters and Differentiates the Mechanism of Action of Different PI3K/mTOR, Aurora Kinase and EZH2 Inhibitors
Authors: Uitdehaag Joost C.M. , de Roos Jeroen A.D.M. , Prinsen Martine B.W. , Willemsen-Seegers Nicole , de Vetter Judith R.F. , Dylus Jelle , van Doornmalen Antoon M. , Kooijman Jeffrey , Sawa Masaaki , van Gerwen Suzanne J.C. , de Man Jos , Buijsman Rogier C. , Zaman Guido J.R.
Published: March 2024

Abstract
Cancer cell line panels are important tools to characterize the in vitro activity of new investigational drugs. Here, we present the inhibition profiles of 122 anticancer agents in proliferation assays with 44 or 66 genetically characterized cancer cell lines from diverse tumor tissues (Oncolines). The library includes 29 cytotoxics, 68 kinase inhibitors, and 11 epigenetic modulators. For 38 compounds this is the first comparative profiling in a cell line panel. By strictly maintaining optimized assay protocols, biological variation was kept to a minimum. Replicate profiles of 16 agents over three years show a high average Pearson correlation of 0.8 using IC50 values and 0.9 using GI50 values. Good correlations were observed with other panels. Curve fitting appears a large source of variation. Hierarchical clustering revealed 44 basic clusters, of which 26 contain compounds with common mechanisms of action, of which 9 were not reported before, including TTK, BET and two clusters of EZH2 inhibitors. To investigate unexpected clusterings, sets of BTK, Aurora and PI3K inhibitors were profiled in biochemical enzyme activity assays and surface plasmon resonance binding assays. The BTK inhibitor ibrutinib clusters with EGFR inhibitors, because it cross-reacts with EGFR. Aurora kinase inhibitors separate into two clusters, related to Aurora A or pan-Aurora selectivity. Similarly, 12 inhibitors in the PI3K/AKT/mTOR pathway separated into different clusters, reflecting biochemical selectivity (pan-PI3K, PI3Kβγδ-isoform selective or mTOR-selective). Of these, only allosteric mTOR inhibitors preferentially targeted PTEN-mutated cell lines. This shows that cell line profiling is an excellent tool for the unbiased classification of antiproliferative compounds. Mol Cancer Ther; 15(12); 3097–109. ©2016 AACR.
Data from An Antibody-Tumor Necrosis Factor Fusion Protein that Synergizes with Oxaliplatin for Treatment of Colorectal Cancer
Authors: Bajic Davor , Chester Kerry , Neri Dario
Published: March 2024

Abstract
We have cloned and characterized a novel fusion protein (Sm3E-TNF), consisting of the mAb, S 6m3E, in single-chain Fv fragment format, fused to murine TNF. The protein, which was expressed in mammalian cells and purified as a noncovalent stable homotrimer, bound to the cognate carcinoembryonic antigen (CEA) and retained TNF activity. A quantitative biodistribution experiment, performed in immunocompetent mice with CT26 colon carcinomas transfected with human CEA, revealed that Sm3E-TNF was able to preferentially accumulate in the tumors with excellent selectivity (tumor:blood ratio = 56:1, 24 hours after intravenous administration). The fusion protein mediated a rapid hemorrhagic necrosis of a large portion of the tumor mass, but a rim survived and eventually regrew. Surprisingly, the combination of Sm3E-TNF with 5-fluorouracil led to a reduction of therapeutic activity, while a combination with oxaliplatin led to a prolonged stabilization, with complete tumor eradication in 40% of treated mice. These therapy results were confirmed in a second immunocompetent mouse model of colorectal cancer (CEA-transfected C51 tumors) and provide a rationale for the possible clinical use of oxaliplatin in combination with fully human antibody-TNF fusions.
Data from Characterization of 7A5: A Human CD137 (4-1BB) Receptor Binding Monoclonal Antibody with Differential Agonist Properties That Promotes Antitumor Immunity
Authors: Kotanides Helen , Sattler Rose Marie , Lebron Maria B. , Carpenito Carmine , Shen Juqun , Li Jingxing , Surguladze David , Haidar Jaafar N. , Burns Colleen , Shen Leyi , Inigo Ivan , Pennello Anthony L. , Forest Amelie , Chen Xinlei , Chin Darin , Sonyi Andreas , Topper Michael , Boucher Lauren , Sharma Prachi , Zhang Yiwei , Burtrum Douglas , Novosiadly Ruslan D. , Ludwig Dale L. , Plowman Gregory D. , Kalos Michael
Published: March 2024

Abstract
The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy.