bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1733: EGF816, a novel covalent inhibitor of mutant-selective epidermal growth factor receptor, overcomes T790M-mediated resistance in NSCLC
Authors: Kasibhatla Shailaja , Li Jie , Tompkins Celin , Vaillancourt Mei-Ting , Anderson Jennifer , Pferdekamper AnneMarie Culazzo , Li Chun , Long Oliver , McNeill Mathew , Epple Robert , Liao Debbie , Murphy Eric , Bender Steve , Jia Yong , Lelais Gerald
Published: May 2015

Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond to EGFR tyrosine kinase inhibitors (TKI) but ultimately develop resistance to these therapies. The most common mechanism of resistance is a second site gate-keeper mutation within exon 20 of EGFR (T790M), followed by MET and other receptor tyrosine kinase amplification/activation. We developed a covalent mutant-selective EGFR inhibitor, EGF816 that potently inhibits both activating EGFR mutations as well as the T790M resistance mutation while sparing wild-type EGFR. EGF816 demonstrated strong tumor regressions in several EGFR activating and resistant tumor models in vivo. These include H1975 (L858R; T790M), HCC827 (exon 19 del) and H3255 (L858R) that are representative of the relevant clinical settings. In all of the models EGF816 inhibited tumor growth in a dose dependent manner and achieved regressions of established tumors at well tolerated doses. In single dose studies, EGF816 showed sustained inhibition of pEGFR, consistent with the irreversible binding mechanism of EGF816. EGF816 also performs exceptionally well in long term dosing studies providing durable responses in the preclinical models. Together, this data indicates that EGF816 exhibits excellent anti-tumor activity in the relevant patient derived tumor cell lines at well tolerated doses and is expected to provide long term duration of responses compared to current EGFR TKI therapy in the clinic.

Citation Format: Shailaja Kasibhatla, Jie Li, Celin Tompkins, Mei-Ting Vaillancourt, Jennifer Anderson, AnneMarie Culazzo Pferdekamper, Chun Li, Oliver Long, Mathew McNeill, Robert Epple, Debbie Liao, Eric Murphy, Steve Bender, Yong Jia, Gerald Lelais. EGF816, a novel covalent inhibitor of mutant-selective epidermal growth factor receptor, overcomes T790M-mediated resistance in NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2014-1733
Abstract 1735: Discovery of novel and selective reversible inhibitors of EGFR containing the T790M drug resistance mutation with activity in vitro and in vivo
Authors: Schaefer Gabriele , Hanan Emily J. , Chan Emily , Shao Lily , Chen Yuan , Knight Jamie , Yauch Robert L. , Schmidt Stephen , Sideris Steven , Malek Shiva , Heffron Timothy P.
Published: May 2015

Activating mutations in the epidermal growth factor receptor (EGFR) characterize a subset of non-small cell lung cancers (NSCLC) with exquisite sensitivity to tyrosine kinase inhibitors like erlotinib or gefitinib. However, after an initial dramatic response, patients harboring these mutations progress within 8-14 months on therapy. In approximately 60% of patients, the resistance to TKIs is associated with the acquisition of a secondary mutation within the EGFR kinase domain. The mutation in the kinase domain leads to the substitution of a bulky methionine for threonine at position 790 (T790M) also referred to as the gatekeeper residue.

Using structure based-design we identified and optimized a series of non-covalent, and highly selective pyridinyl-aminopyrimidine inhibitors of EGFR containing the T790M drug resistant mutation that spared the wild-type EGFR enzyme. These pyridinyl-aminopyrimidine compounds potently inhibited phosphorylation of T790M containing EGFR in enzymatic assays and cell based assays. Strong inhibition of enzyme activity resulted in anti-proliferative activity in H1975 and PC9 erlotinib resistant cells but treatment with these molecules did not affect cell lines containing wild-type EGFR. Moreover, oral dosing of mice bearing either H1975 or PC9_erlotinib resistant xenograft tumors resulted in robust inhibition of tumor growth that was accompanied by profound and dose-dependent inhibition of EGFR activity as well as downstream signaling molecules such as ERK1/2 and AKT.

These results suggest that these pyridinyl-aminopyrimidine inhibitors have the potential to benefit NSCLC patients who have developed resistance to first-generation EGFR inhibitors due to the acquisition of the T790M mutation, without the skin rash and gastrointestinal toxicity associated with wild-type EGFR inhibition.

*EJH and GS contributed equally to this work.

Citation Format: Gabriele Schaefer, Emily J. Hanan, Emily Chan, Lily Shao, Yuan Chen, Jamie Knight, Robert L. Yauch, Stephen Schmidt, Steven Sideris, Shiva Malek, Timothy P. Heffron. Discovery of novel and selective reversible inhibitors of EGFR containing the T790M drug resistance mutation with activity in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1735. doi:10.1158/1538-7445.AM2014-1735
Abstract 1737: Allosteric FGFR2 inhibitor RPT835 impacts on tumor growth and neovascularization
Authors: Tsimafeyeu Ilya , Stepanova Evgenia , Solomko Eliso , Kochenkov Dmitry , Peretolchina Nina , Ryabaya Oxana , Byakhov Mikhail , Tjulandin Sergey
Published: May 2015

Encouraged by the clinical validation of fibroblast growth factor receptor 2 (FGFR2) as the target for the treatment of several tumors, we sought to identify pharmacological agents that could target this receptor by an allosteric mechanism. We report the efficacy of RPT835, novel highly potent inhibitor of the FGFR2 extracellular domain (RusPharmTech, LLC), in vitro and in vivo.

Concordant with this activity, in human umbilical vein endothelial cells (HUVEC) and mouse endothelial cells (SVEC-4-10), RPT835 inhibited FGF-stimulated proliferation with IC50 values of 11 and 10 nmol/L, respectively. Inhibition of HUVEC cells proliferation by brivanib as positive control was significantly lower (IC50=289 nmol/L, P<0.001). Once-daily oral administration of RPT835 significantly ablated experimental FGF-induced angiogenesis in vivo and reduced endothelial cell migration, capillary-like tubular structure and mature vessel formation in comparison with bevacizumab (P<0.001). The growth of established human tumor xenograft of breast cancer (HS578T) in athymic mice was inhibited by RPT835 with chronic administration of 30 mg per kg per day. Differences with control group were significant on 34 day of study. There was no significant impact of RPT835 on colon (HCT-116, 10 mg/kg) and lung (NCI-H226, 30 mg/kg) cancer growth.

Collectively, the data obtained with RPT835 are consistent with potent inhibition of FGF signaling, angiogenesis, and breast cancer growth.

Citation Format: Ilya Tsimafeyeu, Evgenia Stepanova, Eliso Solomko, Dmitry Kochenkov, Nina Peretolchina, Oxana Ryabaya, Mikhail Byakhov, Sergey Tjulandin. Allosteric FGFR2 inhibitor RPT835 impacts on tumor growth and neovascularization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1737. doi:10.1158/1538-7445.AM2014-1737
Abstract 1748: Novel Ig1 inhibitors of the Axl tyrosine kinase that block Gas6-inducible receptor activation
Authors: Welsh William J. , Kholodovych Vladyslav , Birge Raymond , Comollo Tom , Kimani Stanley , Singh Kamal
Published: May 2015

Tyro-3, Axl, and Mer (abbreviated TAMs) are a family of three homologous receptor tyrosine kinases (RTKs) expressed predominantly on myeloid-derived hematopoietic cells and epithelial cells that function as inhibitory receptors that dampen inflammatory responses and maintain tissue tolerance. Both TAMs and their ligands (vitamin K activated proteins that consist of Gas6 and Protein S) are overexpressed in many human malignancies and expression is associated with more aggressive cancer staging, drug resistance, and poorer patient survival. Based on their clinical importance, there has been considerable effort to generate anti-TAM cancer therapeutics in the form of small molecule tyrosine kinase inhibitors, monoclonal antibodies, and soluble receptor traps that capture TAM ligands and prevent receptor activation. However, whereas several tyrosine kinase inhibitors have been reported, the high degree of sequence conservation in the TAM kinase domain is associated with lack of specificity and off target effects. To generate more selective inhibitors of TAMs, we have targeted the Ig1 domain of Axl, a ligand-binding region in the extracellular domain that gives TAMs their unique structural features. From the X-ray crystallographic structure of Gas6 in complex with the human Axl ectodomain (PDB 2c5d), two unique sites were targeted that include (i) the major binding pocket around Glu59 in Axl that interacts with Arg310 and Lys312 in Gas6, and (ii) the main hydrophobic interface between the Ig1 and the Lg1 that is required for Gas6-inducible dimerization. Virtual screening of the ZINC molecular data base was performed, and several drug-like compounds were selected based on ligand-Gas6 docking. We report on the characterization of two novel types of Axl inhibitors that include a triazolo pyridazine compound that targets the Ig1 binding pocket, and a 3-cyanocoumarin compound that targets the Ig1-Lg1 interface. Both compounds show activity on human Axl reporter lines, and therefore may represent a new class of Axl inhibitors with potential applications for anticancer therapy.

Citation Format: William J. Welsh, Vladyslav Kholodovych, Raymond Birge, Tom Comollo, Stanley Kimani, Kamal Singh. Novel Ig1 inhibitors of the Axl tyrosine kinase that block Gas6-inducible receptor activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1748. doi:10.1158/1538-7445.AM2014-1748
Abstract 1751: Preclinical development of a novel multi-targeted kinase inhibitor, MG516, for the treatment of sarcoma
Authors: Ivy Kathryn S. , Patwardhan Parag P. , Schwartz Gary K.
Published: May 2015

Receptor tyrosine kinases (RTKs) are key regulators of tumor development as well as metastasis, aiding in the epithelial-mesenchymal transition, migration and angiogenesis. RTK signaling pathways such as VEGFR, PDGFR, and c-Met have been shown to be critical for cell survival, proliferation and metastasis in sarcomas. RTK inhibitors have been used with moderate success to block tumor cell survival and proliferation, however, internal compensatory signaling pathways give rise to drug resistance in a majority of cases. MG516, is a novel multi-targeted tyrosine kinase inhibitor that blocks RTK signaling pathways including c-Met, Ephrin receptors (Eph), and VEGFR with low nanomolar IC50 values. We evaluated the RTK inhibition profile and anti-proliferative effect of MG516 in a diverse panel of sarcoma cell lines including malignant peripheral nerve sheath tumor (MPNST), Ewing's sarcoma (A673) and leiomyosarcoma (SKLMS). MG516 inhibits tumor cell proliferation in vitro in a dose dependent manner with IC50s ranging from 500 to 2500 nM. We used a human phopho-RTK array kit, which allows the screening of 49 different phosphorylated human RTKs, in a panel of sarcoma cell lines. Our data indicates that MG516 provides robust blockade of a wide panel of RTKs including c-Met, EphB3, EphA10, PDGFR-β, Axl and IGF1-R at nanomolar to low micromolar concentrations. Western blot analysis showed a significant blockade of downstream effectors including p-AKT, p-FAK and p-STAT3. These results demonstrate the efficacy of MG516 in blocking RTKs and their downstream signaling pathways. Since PDGFR-β, c-Met and IGF1-R have been shown to be involved in tumor initiation, development and metastasis in a variety of sarcoma subtypes, our data would strongly support further development of MG516 as a novel agent in the treatment of sarcomas.

Citation Format: Kathryn S. Ivy, Parag P. Patwardhan, Gary K. Schwartz. Preclinical development of a novel multi-targeted kinase inhibitor, MG516, for the treatment of sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1751. doi:10.1158/1538-7445.AM2014-1751
Abstract 1754: Cycloartane anticancer activity
Authors: Lowe Henry Isaac Cloore , Toyang Ngeh J. , Watson Charah , Bryant Joseph
Published: May 2015

New cancer diagnoses remain on the rise despite significant improvements in early detection and treatment. The role of Kinases in cancer onset and progression has made kinases a target for the control of some cancers. The MRCKα/β kinases have recently been implicated in cancer onset and progression and as such could serve as a potential drug target. The discovery that kinases are most effectively inhibited by small molecules has also resulted in an increased search for small molecule kinase inhibitors. Cycloartanes are small molecules found in many medicinal plants including the Jamaican Ball Moss (Tillandsia recurvata). Recent studies on T. recurvata have demonstrated that it possesses significant anticancer activity. Cycloartane-3,24,25-triol, an analog of a cycloartane identified in Ball moss was also shown to have inhibitory activity against MRCKα kinase. This study was as such set up to determine the MRCKα/β kinase inhibition activity of other cycloartanes in Ball Moss and their analogs. The kinase inhibitory activity of 6 cycloartanes was investigated using the ligand-kinase binding assay while the WST-1 reagent assay was used to determine the antiproliferative activity of the cycloartanes against some prostate and breast cancer cell lines. Cycloart-23-ene-3,25-diol (1), Cycloartane-3,24,25-triol (2), Cycloart-25-ene-3,24-diol (3), 3,23-Dioxo-9,19-cyclolanost-24-en-26-oic acid (4), 24,25-Dihydroxycycloartan-3-one (5) inhibited the MRCKα kinase with Kd of 0.21 μM, 0.25µM, 0.36 µM, 3.0 µM, and 2.1 µM respectively. Hydroxycycloart-23-en-3-one,25, (6) showed no inhibition against the MRCKα kinase. Compounds 1, 3, 4, 5 inhibited the MRCKβ kinase with Kd of 4.7 μM, 1.10 µM, 3.2 µM, and 9.8 µM, respectively. Three of the six cycloartanes exhibited antiproliferation activity against two prostate and breast cancer cell lines each.

In conclusion, cycloart-23-ene-3,25-diol (1) showed the most promising activity against the MRCKα/β kinase out of the 6 cycloartanes screened demonstrating an interesting structure activity relationship profile when compared with the other molecules. Cycloart-23-ene-3,25-diol (1) deserves further studies to determine its in vivo efficacy and safety.

Citation Format: Henry Isaac Cloore Lowe, Ngeh J. Toyang, Charah Watson, Joseph Bryant. Cycloartane anticancer activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1754. doi:10.1158/1538-7445.AM2014-1754
Abstract 1761: PDE10, a novel therapeutic target for lung cancer
Authors: Zhu Bing , Li Nan , Ramirez-Alcantara Veronica , Canzoneri Joshua , Gurpinar Evrim , Fajardo Alexandra , Lee Kevin , Sigler Sara , Gary Bernard , Thomas Meagan , Keeton Adam , Chen Xi , Grizzle William , Piazza Gary
Published: May 2015

Phosphodiesterase 10A (PDE10) is a relatively new PDE isozyme that has been mostly studied in the brain as a therapeutic target for certain psychiatric and neurological conditions, but has not been associated with tumorigenesis. While other investigators have reported low levels of PDE10 in most adult peripheral tissues, we found a large percentage of human lung tumors express high levels of PDE10 compared with normal lung tissue as determined by real-time PCR analysis of cDNA arrays. Statistical analysis revealed that 68% of lung tumors (n=19) displayed more than two-fold increase compared with normal lung tissue, while 26% showed greater than a 10-fold difference. High levels of PDE10 mRNA and protein were also found in human non-small cell lung cancer (NSCLC) cell lines compared with normal airway epithelial cells. PDE10 knockdown by small interference RNA significantly suppressed lung tumor cell growth, as did PDE10 selective inhibitors, papaverine, PQ-10 and Pf-2545920. A novel PDE10 inhibitor, MCI-020 also inhibited the growth of NSCLC cells with a high degree of selectivity for tumor cells. Consistent with a mechanism involving PDE10 inhibition, PQ-10 and MCI-020 increased cGMP levels in a concentration-dependent manner as determined using a cGMP biosensor assay. MCI-020 also activated cGMP-dependent protein kinase (PKG) in NSCLCs as evident by increased phosphorylation of VASP-Serine239. With attractive pharmacokinetic properties, MCI-020 was studied further and found to display an unusual characteristic of achieving high concentrations in lung tissue compared with other tissues and plasma following oral administration. MCI-020 was well tolerated in mice at a dose of 250 mg/kg bid and strongly inhibited tumor formation in an orthotopic model of lung cancer at a dose of 150 mg/kg bid. These observations suggest that PDE10 is essential for lung tumor cell growth and represents a novel therapeutic target for lung cancer.

Citation Format: Bing Zhu, Nan Li, Veronica Ramirez-Alcantara, Joshua Canzoneri, Evrim Gurpinar, Alexandra Fajardo, Kevin Lee, Sara Sigler, Bernard Gary, Meagan Thomas, Adam Keeton, Xi Chen, William Grizzle, Gary Piazza. PDE10, a novel therapeutic target for lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1761. doi:10.1158/1538-7445.AM2014-1761
Abstract 1765: Ras and EF-hand domain containing as a novel tissue biomarker and a therapeutic target for lung cancer
Authors: Daigo Yataro , Takano Atsushi , Nakamura Yusuke
Published: May 2015

Lung cancer is the most common cause of cancer-related death in the world and its incidence has been increasing. The number of patients responding well to current standard therapies is still small, therefore further development of new agents targeting cancer-specific molecules with no or minimum risk of adverse effect is urgently awaited. To screen molecules, particularly oncoantigens which could be useful for the development of diagnostic biomarkers and new drugs, we have established an effective screening strategy as follows; i) To identify up-regulated genes in 120 lung cancers by genome-wide screening using the cDNA microarray representing 27,648 genes and pure populations of tumor cells taken from cancer tissues by laser microdissection, ii) To verify the candidate genes for their very low or absent expression in normal tissues by northern-blot analyses, iii) To validate the clinicopathological significance of their expression with tissue microarray containing hundreds of archived lung cancers, iv) To verify whether the target genes are essential for the growth or survival of cancer cells by RNAi and cell growth assays. As a result of this process, we identified 40 druggable oncoproteins. We further identified the Ras and EF-hand domain containing (RASEF), which contains a Rab GTPase domain and is considered as a member of Rab GTPase protein family. Immunohistochemical staining using tumor tissue microarrays consisting of 341 archived non-small cell lung cancers revealed the association of strong RASEF positivity with poor prognosis (P = 0.0034 by multivariate analysis). Reduction in RASEF expression by siRNA suppressed lung cancer cell proliferation, while induction of RASEF conferred growth-promoting activity of mammalian cells. RASEF could bind to extracellular signal-regulated kinase (ERK) 1/2 and appeared to enhance the ERK1/2 signaling. In addition, inhibition of interaction between RASEF and ERK1/2 using cell-permeable peptide that corresponded to the ERK1/2-interacting site of RASEF protein, suppressed growth of lung cancer cells. RASEF may play important roles in lung carcinogenesis, and could be useful as a prognostic biomarker and a target for the development of new molecular therapies.

Citation Format: Yataro Daigo, Atsushi Takano, Yusuke Nakamura. Ras and EF-hand domain containing as a novel tissue biomarker and a therapeutic target for lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1765. doi:10.1158/1538-7445.AM2014-1765
Abstract 1773: Phosphodiesterase 10A: A novel target for selective inhibition of colon tumor cell growth and Wnt/β-catenin signaling
Authors: Li Nan , Lee Kevin , Xi Yaguang , Zhu Bing , Gary Bernary D. , Ramírez-Alcántara Verónica , Gurpinar Evrim , Canzoneri Joshua C. , Fajardo Alexandra , Sigler Sara , Piazza John T. , Chen Xi , Andrews Joel , Lu Wenyan , Li Yonghe , Russo Suzanne , Yet Larry , Keeton Adam B. , Grizzle William E. , Piazza Gary A.
Published: May 2015

The cyclic nucleotide phosphodiesterase10A (PDE10) has been studied as a therapeutic target for certain psychiatric and neurological conditions, although a potential role in tumorigenesis has not been reported. Here we report that PDE10 is elevated in human colon tumor cell lines compared with normal colonocytes. Similarly, PDE10 is elevated in colon tumors from human clinical specimens as well as intestinal tumors from the ApcMin/+ mouse model compared with non-involved tissue or normal intestinal mucosa, respectively. An isozyme and tumor-selective role of PDE10 was evident by the ability of specific inhibitors and siRNA knockdown to suppress colon tumor cell growth without significantly affecting the growth of normal colonocytes. Stable knockdown of PDE10 by shRNA also inhibits colony formation and increases doubling time of colon tumor cells. Inhibition of PDE10 results in the selective activation of cGMP/PKG signaling in colon tumor cells to suppress β-catenin levels and T-cell factor (TCF) transcriptional activity. Conversely, ectopic expression of PDE10 in normal and precancerous colonocytes increases proliferation and activates TCF transcriptional activity. These observations demonstrate that PDE10 is essential for colon tumor cell growth and suggest that inhibitors may have therapeutic potential for the treatment or prevention of colorectal cancer.

Citation Format: Nan Li, Kevin Lee, Yaguang Xi, Bing Zhu, Bernary D. Gary, Verónica Ramírez-Alcántara, Evrim Gurpinar, Joshua C. Canzoneri, Alexandra Fajardo, Sara Sigler, John T. Piazza, Xi Chen, Joel Andrews, Wenyan Lu, Yonghe Li, Suzanne Russo, Larry Yet, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Phosphodiesterase 10A: A novel target for selective inhibition of colon tumor cell growth and Wnt/β-catenin signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1773. doi:10.1158/1538-7445.AM2014-1773
Abstract 1775: Identification of small molecule inhibitors of HSF1 stress pathway activation in cancer cells
Authors: Billy Emmanuel de , Chessum Nicola , Poele Robert Te , Smith Jennifer , Zani Lorenzo , Sharp Swee , Stubbs Mark , Aherne Wynne , Jones Keith , Workman Paul
Published: May 2015

Heat Shock Factor 1 (HSF1) is a key transcription factor involved in proteostasis and response to stress, as well as being implicated in many diseases including cancer. Up-regulation of its activity by environmental stress or oncogenesis leads to transcriptional induction of genes involved in diverse cellular processes supporting the cancer state, including proteostasis, proliferation, survival and metastasis. Inhibition of HSF1 is therefore potentially beneficial for cancer treatment, but HSF1 is not technically druggable. We developed a high-throughput cell-based reporter gene assay to screen a library of small-molecule kinase inhibitors and identified 3 compound series as validated inhibitors of HSF1 activation by the HSP90 inhibitor 17-AAG. Further characterization of imidazo[1,2-b]pyridazine compounds showed inhibition of HSF1 target gene expression and the ability to mimic other features of the HSF1 knockdown phenotype. Using these tool compounds and by siRNA knockdown of HSF1 expression we demonstrate that HSF1 inhibition leads to cell cycle arrest and enhances the antiproliferative effect of 17-AAG. In addition, we show that HSP90 inhibition induces HSF1 phosphorylation at two serine residues, Ser326 and Ser320, highlighting the importance of Ser320 phosphorylation in HSP72 up-regulation by 17-AAG-induced HSF1 activation, and show that this activation is inhibited by our tool compounds. Our findings support the optimization and development of small-molecule inhibitors of the HSF1 pathway for cancer treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Emmanuel de Billy, Nicola Chessum, Robert Te Poele, Jennifer Smith, Lorenzo Zani, Swee Sharp, Mark Stubbs, Wynne Aherne, Keith Jones, Paul Workman. Identification of small molecule inhibitors of HSF1 stress pathway activation in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1775. doi:10.1158/1538-7445.AM2014-1775
Abstract 1788: 19-Substituted benzoquinone ansamycins. Hsp90 inhibitors with decreased off-target toxicity
Authors: Chang Chuan-Hsin , Drechsel Derek A. , Kitson Russell R.A. , Siegel David , You Qiang , Backos Donald S. , Ju Cynthia , Moody Christopher J. , Ross David
Published: May 2015

Benzoquinone ansamycin (BQA) Hsp90 inhibitors such as 17-DMAG and 17-AAG have off-target toxicities in clinical trials including hepatotoxicity. Mechanisms underlying the toxicity of quinones are a function of their ability to redox cycle and/or arylate cellular nucleophiles at the unsubstituted 19-position of the molecule. Therefore, we designed 19-substituted BQAs to prevent glutathione conjugation and non-specific interactions with protein thiols as an approach to reduce the hepatotoxicity and minimize off-target effects of the BQA class of Hsp90 inhibitors. In this study, the results showed that 19-substituted BQAs did not react with glutathione at the 19-position, while marked reactivity was observed using parent BQAs. Importantly, while parent 17-DMAG induced cell death in primary and cultured mouse hepatocytes, 19-phenyl and 19-methyl 17-DMAG showed reduced toxicity, validating the overall approach. There was no significant difference between the redox cycling ability of either 19-phenyl or 19-methyl 17-DMAG with their parental BQAs in both mouse and human liver microsomes. Accordingly, this suggests that arylation reactions at the unsubstituted 19-position are predominantly responsible for hepatotoxicity. 19-substituted17-DMAG inhibited purified Hsp90 ATPase activity in an NQO1-dependent manner that demonstrated increased inhibitory efficacy of the hydroquinone ansamycin relative to its parent quinone. In human breast cancer cells, 19-phenyl BQAs induced growth inhibition in an NQO1-dependent manner with molecular signatures of Hsp90 inhibition, including decreases in client proteins and compensatory induction of Hsp70. These data indicate that 19-substituted BQAs may be useful Hsp90 inhibitors with decreased off target toxicity (Supported by NCI grant CA51210)

Citation Format: Chuan-Hsin Chang, Derek A. Drechsel, Russell R.A. Kitson, David Siegel, Qiang You, Donald S. Backos, Cynthia Ju, Christopher J. Moody, David Ross. 19-Substituted benzoquinone ansamycins. Hsp90 inhibitors with decreased off-target toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2014-1788
Abstract 1789: Profiling HSP90 inhibitors in cellular extracts on a mass spectrometry chemoproteomics platform
Authors: Nomanbhoy Tyzoon K. , Nordin Brian E. , Rosenblum Jonathan , Liu Yongsheng
Published: May 2015

HSP90 is a ubiquitous molecular chaperone that couples ATP hydrolysis to the folding and maturation of numerous client proteins. Because many of these client proteins are essential to cancer cell growth and survival, HSP90 inhibitors have undergone clinical investigation in numerous oncology indications. Here, using an LC-MS/MS platform previously developed to characterize ATP-competitive inhibitors of protein kinases, several HSP90 inhibitors were profiled in native cell lysates. This method utilizes an ATP derivative that covalently labels lysine residues in the ATP-binding site of any amenable enzyme. The specificity of inhibitors for all four paralogs of HSP90 (HSP90α, HSP90β, Grp94 and Trap-1) found in human cells was simultaneously monitored at their endogenous relative abundances. Interestingly, the concentration of HSP90 paralogs in human cell lysates was significantly higher than reported inhibitor binding constants, therefore complete inhibition was only observed at much higher concentrations than would have been predicted. In addition to the four HSP90 paralogs, inhibition data was collected on over 400 protein kinases and other nucleotide-binding proteins. This led to the discovery that NVP-AUY922 has off-target activity toward PMS2, an enzyme, like HSP90 itself, belonging to the GHKL ATPase superfamily. In addition to directly profiling compound targets in lysates, downstream effects of HSP90 inhibition were monitored by treating live cells and analyzing the resulting cell lysates at defined time points. Many proteins were found to be much less abundant in the compound-treated cells, including known HSP90 client proteins and novel, putative client proteins.

Citation Format: Tyzoon K. Nomanbhoy, Brian E. Nordin, Jonathan Rosenblum, Yongsheng Liu. Profiling HSP90 inhibitors in cellular extracts on a mass spectrometry chemoproteomics platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1789. doi:10.1158/1538-7445.AM2014-1789
Abstract 1790: Small molecule inhibitors of QSOX1 suppress tumor cell growth and invasion
Authors: Hanavan Paul D. , Faigel Douglas O. , Ma Chen-Ting , Sergienko Eduard , Meurice Nathalie , Petit Joachim L. , Ruiz Yvette W. , Katchman Benjamin A. , Lake Douglas F.
Published: May 2015

Introduction:

QSOX1 is a flavin-dependent sulfhydryl oxidase and the only known human enzyme with both disulfide generating and transferring activities. Using tissue microarrays, we previously demonstrated that QSOX1 is over-expressed in multiple tumor types including breast, pancreas, prostate and myeloma. Loss of QSOX1 leads to a significant decrease in tumor cell proliferation and invasion. The over-expression of QSOX1 in tumor cells compared to non-malignant cells combined with its role in tumor cell proliferation and invasion make it an attractive target for potential anti-neoplastic agents.

Methods:

Using a high throughput screening assay to detect sulfhydryl oxidase activity, we screened a library of compounds for inhibition of the QSOX1 enzyme. We used ROS-GloTM, a luciferase-based assay that detects H2O2, to determine if any compounds in the library inhibited QSOX1-mediated disulfide bond formation using reduced RNAse A as a substrate. QSOX1 generates H2O2 as a by-product of disulfide bond formation. To determine if compounds that showed activity in the enzymatic assay were also active against tumor cells, we treated pancreatic, breast, and myeloma cells with the compounds in a dose-dependent fashion, using non-malignant cells as controls. We assayed growth/viability by MTT and CellTiter-Glo assays. Invasive activity of tumor cells was assessed using matrigel-coated inserts in a modified Boyden chamber assay.

Results:

High throughput library screening identified 4 compounds that specifically inhibited QSOX1 activity (designated A-D) compared to a glucose oxidase counter-screen. Growth/viability assays showed that compound D suppressed the growth of tumor cells with IC50s between 300nM-750nM, depending on tumor cell line. In non-malignant cells the IC50s for compound D were more than 10-fold higher than in tumor cells. Compounds A and D also inhibited invasion of breast and pancreatic tumor cells through Matrigel.

Conclusion:

This is the first report of chemical compounds that inhibit QSOX1 enzymatic activity. The discovery of QSOX1 inhibitors with cellular efficacy establishes the chemical tractability of the target. Our data demonstrate that these First-In-Class compounds have potential as anti-neoplastic therapeutics via inhibition of QSOX1.

Citation Format: Paul D. Hanavan, Douglas O. Faigel, Chen-Ting Ma, Eduard Sergienko, Nathalie Meurice, Joachim L. Petit, Yvette W. Ruiz, Benjamin A. Katchman, Douglas F. Lake. Small molecule inhibitors of QSOX1 suppress tumor cell growth and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1790. doi:10.1158/1538-7445.AM2014-1790
Abstract 1791: 19-Substituted benzoquinone ansamycin Hsp90 inhibitors: Effects on Hsp90 co-chaperones and Hsp90-Hsf1 complexes in cellular systems
Authors: Drechsel Derek A. , Chang Chuan-Hsin , Kitson Russell , Siegel David , Moody Christopher J. , Ross David
Published: May 2015

The 19-substituted benzoquinone ansamycin (BQA) class of Hsp90 inhibitors were developed to decrease the off-target toxicity of their parent unsubstituted BQAs, geldanamcyin, 17-AAG, and 17-DMAG. We have shown that 19-BQAs do not react with thiols and show decreased toxicity to liver cell systems relative to parent BQAs. As a class, Hsp90 inhibitors exhibit anti-cancer activity by decreasing the levels of Hsp90 client proteins critical to cell growth and survival. Some Hsp90 inhibitors also disrupt interaction with Hsp90 co-chaperones, including cdc37, contributing to inhibited growth. Upon inhibition, the transcription factor heat shock factor 1 (Hsf1) dissociates from Hsp90 leading to a compensatory induction of other heat shock proteins, including Hsp70 which is commonly used as a molecular biomarker of cellular Hsp90 inhibition. We undertook a detailed study of the mechanism of action of 19-BQAs in HER2-positive breast cancer cells (BT474) using 19-phenyl- and 19-methyl-DMAG as model compounds. Their ability to disrupt association of Hsp90 with the co-chaperones p23 and cdc37, the kinetics of decreased Hsp90-Hsf1 levels, nuclear Hsf1 accumulation and increases in Hsp70 levels were defined. Both 19-phenyl- and 19-methyl -DMAG disrupted Hsp90-p23 association, confirming binding of the compounds at the N-terminal ATPase site of Hsp90. 19-Phenyl-DMAG and 19-methyl-DMAG also disrupted association of Hsp90 with cdc37, although 19-phenyl-DMAG was more potent. A detailed time course of decreased levels of Hsp90 client proteins and Hsp induction showed that increased Hsp70 was a more sensitive cellular marker as it could be detected at lower drug concentrations. Increases in Hsp70 did not appear to be a generalized stress response, since 19-substituted-BQAs caused dissociation of the Hsp90-Hsf1 dimer, nuclear translocation of Hsf-1, and subsequent increases in Hsp70 levels in a temporal manner. These data indicate that 19-substituted BQAs may exhibit their growth inhibitory effects in breast cancer cells through disruption of Hsp90 and its co-chaperones, particularly cdc37. The N-terminal ATPase site binding and Hsp90 inhibitory activity of these compounds was confirmed through disruption of Hsp90-p23 and Hsp90-Hsf1 associations, respectively (Supported by NCI grant CA51210).

Citation Format: Derek A. Drechsel, Chuan-Hsin Chang, Russell Kitson, David Siegel, Christopher J. Moody, David Ross. 19-Substituted benzoquinone ansamycin Hsp90 inhibitors: Effects on Hsp90 co-chaperones and Hsp90-Hsf1 complexes in cellular systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2014-1791
Abstract 1794: The HSP90 inhibitor, AT13387, combined with erlotinib improves response in EGFR-driven xenograft models of NSCLC
Authors: Smyth Tomoko , Lewis Jon , Hearn Keisha , Thompson Neil , Lyons John , Wallis Nicola G.
Published: May 2015

Background: Although Epidermal Growth Factor Receptor (EGFR)-driven non-small cell lung cancer (NSCLC) can be successfully treated with EGFR inhibitors such as erlotinib, relapse is common due to the development of resistance. Methods to improve response and delay resistance are therefore of value. Inhibition of the chaperone, HSP90, leads to the depletion of many client proteins, including EGFR, and has the capacity to simultaneously affect many signalling pathways, offering an alternative strategy for targeting EGFR-driven disease. AT13387 is a potent, second generation HSP90 inhibitor currently being tested in Phase II clinical trials. Here we investigated the effects of combining AT13387 and erlotinib in models of EGFR-driven NSCLC.

Results: AT13387 single-agent treatment was effective in EGFR-driven NSCLC xenograft models, significantly inhibiting tumor growth of both HCC827 and NCI-H1650 xenograft tumors when dosed once weekly (see table). As expected, treatment with erlotinib caused significant tumor regression in all HCC827 tumors, but was less effective in the NCI-H1650 model; its PTEN status conferring a reduction in sensitivity. However, combination of AT13387 with erlotinib led to a further enhancement of tumor growth inhibition over either of the monotherapies in both of these models. All HCC827 tumors treated with the combination regressed further (by at least 80%), and regression was also now observed in all NCI-H1650 tumors. These data demonstrate that combination with AT13387 has the potential to increase response, despite the differing initial sensitivities to erlotinib of these models. The combination was well tolerated.

Effects of AT13387 and erlotinib on EGFR-driven xenograftsCell lineGenetic BackgroundT/C(Median RTV, treated / control)AT1338755 mg/kg ip 1qwErlotinib12.5 mg/kg po qdCombinationHCC827(Day 32)EGFR Del E746_A75026%17%(47% regression)6%(84% regression)NCI-H1650(Day 31)EGFR Del E746_A750/PTEN del35%36%6%(50% regression)

Conclusions: AT13387 was shown to improve response when combined with erlotinib in EGFR-driven xenograft models. These data suggest that there is therapeutic potential in an upfront combination of an HSP90 inhibitor, such as AT13387, with erlotinib and support clinical investigation of such a combination.

Citation Format: Tomoko Smyth, Jon Lewis, Keisha Hearn, Neil Thompson, John Lyons, Nicola G. Wallis. The HSP90 inhibitor, AT13387, combined with erlotinib improves response in EGFR-driven xenograft models of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1794. doi:10.1158/1538-7445.AM2014-1794
Abstract 18: MicroRNA-103 induces DNA damage and radiosensitizes the tumor vasculature
Authors: Anand Sudarshan , Advani Sunil J. , Cheresh David A.
Published: May 2015

While it is well known that tumor vasculature is a key determinant of resistance to radiotherapy, there remains a paucity of agents that sensitize this compartment to clinical doses of radiation. Using an expression screen for ‘apoptomiRs’ induced by high dose radiation, we have identified microRNA-103 as a potent, genotoxic stress responsive apoptomiR in endothelial cells. Functionally, ectopic expression of miR-103 mimics genotoxic stress causing DNA double strand breaks leading to cell death and decreased retinal angiogenesis in mice . Mechanistically, we show that miR-103 targets DNA repair pathway components - the exonuclease TREX1 and the adaptor FANCF. Finally, miR-103 injection in mice decreased tumor burden in a human colorectal cancer xenograft model where it enhanced the effects of low dose radiation. Our data uncovers a microRNA mediated pathway of DNA damage unique to proliferating endothelium that can be exploited for radiosensitization of tumor vasculature.

Citation Format: Sudarshan Anand, Sunil J. Advani, David A. Cheresh. MicroRNA-103 induces DNA damage and radiosensitizes the tumor vasculature. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 18. doi:10.1158/1538-7445.AM2014-18
Abstract 1810: PI-1840, a novel non-covalent and rapidly reversible proteasome inhibitor with anti-tumor activity
Authors: Sebti Said M. , Kazi Aslamuzzaman , Ozcan Sevil , Tecleab Awet G. , Sun Ying , Lawrence Harshani
Published: May 2015

Non-covalent proteasome inhibitors that are less toxic and more effective against solid tumors than currently used proteasome inhibitors are desirable. Through HTS and hit-to-lead optimization efforts we have identified PI-1840, a potent and selective inhibitor for chymotrypsin-like (CT-L) (IC50 value = 27 ± 0.14 nM) over T-L and PGPH (IC50 values >100 µM) activities of the proteasome. Furthermore, PI-1840 is over 100-fold more selective for the constitutive proteasome over the immunoproteasome. Dialysis and mass spectrometry studies demonstrate that PI-1840 is a non-covalent and rapidly reversible CT-L inhibitor. PI-1840 inhibits CT-L activity, induces the accumulation of proteasome substrates p27, Bax and IκB-α, inhibits survival pathways and proliferation, and induces apoptosis in human cancer cells. The mdm2/p53 disruptor, nutlin, sensitizes cancer cells to PI-1840 in a p53-dependent manner. Furthermore, the pan Bcl-2 antagonist BH3-M6 also sensitizes cancer cells to PI-1840. Finally, in vivo, PI-1840 but not Bortezomib (clinically used proteasome inhibitor) suppresses the growth in nude mice of human breast xenografts. These results warrant further evaluation of non-covalent and rapidly reversible proteasome inhibitor as potential anticancer agents against solid tumors.

Citation Format: Said M. Sebti, Aslamuzzaman Kazi, Sevil Ozcan, Awet G. Tecleab, Ying Sun, Harshani Lawrence. PI-1840, a novel non-covalent and rapidly reversible proteasome inhibitor with anti-tumor activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1810. doi:10.1158/1538-7445.AM2014-1810
Abstract 1812: XCE853: A novel PDI inhibitor that inhibits proliferation of human tumor cells in vitro, ex-vivo and in vivo
Authors: Prevost Gregoire P. , Xu Shili , Garrido Marine , Serova Maria , Vewer Olivier D. , Gespach Christian , Briand Jean-François , Tijeras-Raballand Annemilaï , Gutmann Mathieu , Hendrix An , Sabbah Michele , Chachereau Anne , Gramont Armand de , Neamati Nouri , Carniato Denis , Pitty Marc-Henry , Foster Paul
Published: May 2015

XCE853 was originally identified within a family of synthetic compounds displaying a preferential antiproliferative activity on drug resistant human cancer cells (Gutmann et al, AACR annual meeting, 2013). XCE853 induced an irreversible cytolysis of human tumor cells after a short in vitro exposure independently of efflux pumps leading to a tumor cell death by autophagy and particles release (vesicles or protein aggregates).

The interest for XCE853 has been reinforced by three in vitro observations: 1) XCE853 inhibits the recombinant human enzyme PDI recognized as one novel target linked to drug resistance 2) XCE853 has a unique antiproliferative profile compared to the NCI database compounds when using the NCI Compare assay and 3) XCE853 has no activity on human normal prostate cells even at high concentrations whereas XCE shows an IC50 in the nanomolar range on human tumoral cells.

In addition, the ex-vivo approach using fresh human tumor explants cultivated in 3 dimensions with low concentrations of XCE853 has shown a strong decrease of the proliferation (KI-67 labeling) with an apoptosis induction (Caspase-3 labeling) of Head & Neck cancers following 24h of exposure.

Finally, XCE853 which displayed an excellent oral bioavailability in mouse is able to block growth of human ovarian cancer using in vivo xenograft model leading to a complete tumor growth arrest even after the stop of the treatment.

Altogether, these data support further efforts on this drug candidate to initiate the preclinical studies and to define the most relevant human tumor types.

Citation Format: Gregoire P. Prevost, Shili Xu, Marine Garrido, Maria Serova, Olivier DE Vewer, Christian Gespach, Jean-François Briand, Annemilaï Tijeras-Raballand, Mathieu Gutmann, An Hendrix, Michele Sabbah, Anne Chachereau, Armand de Gramont, Nouri Neamati, Denis Carniato, Marc-Henry Pitty, Paul Foster. XCE853: A novel PDI inhibitor that inhibits proliferation of human tumor cells in vitro, ex-vivo and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2014-1812
Abstract 1813: A novel designed small peptide enhances chemotherapy sensitivity of leukemia cells in stromal microenvironment
Authors: Li Xiaojin , Guo Hua , Yang Yanlian , Meng Jie , Liu Jian , Wang Chen , Xu Haiyan
Published: May 2015

Many types of leukemia cells highly express chemokine receptor (CXCR4) that actively responses to CXCL12, a chemokine constitutively secreted by stromal cells in bone marrow. Activation of CXCR4 induces leukemia cells trafficking and homing to the marrow microenvironment, where CXCL12 retains these cells in close contact with marrow stromal cells that provide growth, self-renewal, and drug resistance signals, which lead to poor prognosis and relapse. We report here a novel designed peptide targeting to CXCR4, named as XW1108-3. The patented sequence is GGQGCRFRNTVDD WISITRAL. We use flow cytometry, cell migration and adhesion assay to study the affinity of XW1108-3 to leukemia cells and the inhibitory effect on CXCL12-induced cell behaviors. The XW1108-3 shows high affinity to different types of leukemia cell highly expressing CXCR4 including HL-60, NB4, THP-1 and U937, the binding was maintained for up to 4 hours. The XW1108-3 treatment significantly inhibited CXCL12- or murine stromal cell MS-5-induced migration of leukemia cells in a concentration dependent manner in the transwell assay and significantly reduced the leukemia cells adhesion to MS-5 cell layer. Mechanistic study with western blot and confocal microscopy assay demonstrates that

XW1108-3 treatment down regulates CXCL12-induced phosphorylation of Erk, Akt and p38 and affects F-actin organization, resulting in the suppression of the cell response to CXCL12 activation. It is notable that XW1108-3 treatment did not induce apoptosis of leukemia cells or human fibroblasts and endothelial cells even at 10 µM, instead enhanced the level of 10-hydroxy camptothecin-induced apoptosis of different leukemia cells co-incubated with the stromal cell MS-5 layer, thereby exhibiting great clinical implications in leukemia chemotherapies and immune therapies.

Citation Format: Xiaojin Li, Hua Guo, Yanlian Yang, Jie Meng, Jian Liu, Chen Wang, Haiyan Xu. A novel designed small peptide enhances chemotherapy sensitivity of leukemia cells in stromal microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1813. doi:10.1158/1538-7445.AM2014-1813