bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2420: The association between severe treatment-related lymphopenia and progression free survival in patients with newly diagnosed squamous cell head and neck cancer.
Authors: Campian Jian L. , Sarai Guneet , Ye Xiaobu , Marur Shanthi , Grossman Stuart A.
Published: August 2014

Background:

Severe treatment-related lymphopenia (TRL) occurs in 50% of glioblastoma and pancreatic cancer patients and is associated with early death from tumor progression. We sought to determine if similar findings were seen in head and neck squamous cell carcinoma (HNSCC).

Patients and Methods:

Eligible patients for this retrospective study had: 1) HNSCC cancer diagnosed in 2007-2009, 2) good performance status, 3) platinum-based chemoradiation, and 4) follow-up at Johns Hopkins. Serial total lymphocyte counts (TLC), overall survival (OS) and progression-free survival (PFS) were analyzed accounting for known prognostic factors.

Results:

Fifty-six adults met eligibility criteria: median age: 57 years, female: 21%, HPV+: 61%, surgery prior to chemoradiation: 16%, stage IVA-IVB: 77%, T stage 3-4: 40% and N stage 2b-3: 56%. Changes in TLC are shown below:

Median TLC:Baseline Median TLC: 2 months post-Rx % with TLC <500: 2 months post-Rx Overall 1660 448 61% HPV- (n = 22) 1770 550 36% HPV+ (n = 34) 1610 320 76%

14/56 patients (25%) had tumor recurrence and 9 (16%) died of their tumor. HPV+ patients had longer PFS (p=0.01) and OS (p=0.006) than HPV- patients. 10/22 HPV- patients had disease progression and 7 died. HPV- patients who developed grade III-IV TRL two months after beginning chemoradiation had a strikingly higher hazard rate for disease progression than those whose TLC remained higher (multivariate analysis HR 6.2, 95%CI: 1.1-35.2; p=0.039). Too few events occurred in the HPV+ cohort for analysis.

Conclusion:

TLCs were normal before chemoradiation. However, two months after chemoradiation ∼60% of patients had severe TLC regardless of HPV status. HPV- patients with severe TRL were much more likely to have disease progression than those without TRL. Prospective studies are needed to confirm these findings which are similar to those reported in other cancers. Preservation of the immune system during chemoradiation may be important to improving PFS and OS.

Citation Format: Jian L. Campian, Guneet Sarai, Xiaobu Ye, Shanthi Marur, Stuart A. Grossman. The association between severe treatment-related lymphopenia and progression free survival in patients with newly diagnosed squamous cell head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2420. doi:10.1158/1538-7445.AM2013-2420

Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
Abstract 2422: Circulating miRNAs as potential biomarkers in prostate cancer pathogenesis.
Authors: Srivastava Anvesha , Ramalinga Malathi , Kumar Deepak , Dimtchev Alexander , Chijioke Juliet , Soldin Offie , Li Xin , Marian Catalin , Suy Simeng , Collins Sean P. , Kumar Deepak
Published: August 2014

Introduction: According to the American Cancer Society, prostate cancer (PCa) is a major health concern for U.S. men and is expected to result in approximately 241,740 new cases and about 28,170 deaths in 2012. African American (AA) men have a 60 percent higher incidence rate and twice the death rate of Caucasian men (CA). Whereas metastasis is the most common cause of death, no specific markers have been assigned to the severity and ethnic biasness of the disease. Deregulation of miRNAs has been observed in various human tumors which emphasize their role in cancer pathogenesis, progression and metastasis. This study was aimed to investigate potential miRNAs involved in PCa. The study also aims to identify potential biomarkers and/or their therapeutic targets which can provide insight into the severity and ethnic biasness of disease.

Methods: Study was divided into three phases: (I) Candidate serum miRNAs were detected by using PCR microarray (learning set of 6 AA and 6 CA PCa patients). (II) Discriminating performance of candidate miRNAs were validated by real-time RT-PCR in serum [AA (20 PCa patients and 16 controls) and CA (16 PCa patients and 16 controls). (III) Functional studies on candidate miRNA(s) were subsequently performed using gain-and loss-of-function approach in cellular assays.

Results: In the discovery phase we obtained 5 miRNAs which were differentially expressed in the AA prostate cancer patients. After careful examination miR-212 and few others were selected for future validation. In the validation set there was an overall high expression of miR-212 in serum of PCa patients as compared to normal individuals (p=0.0110). Subdivision on the basis of ethnicity showed serum expression levels of miR-212 in AA were significantly modulated (p=0.034) as compared to CA PCa patients. Functional validation of miR-212 was done in PC3 and LnCap cell lines.

Conclusion: Our results showed a potential role for miR-212 in the regulation of gene expression which in turn can define the aggressiveness associated in African American PCa patients.

Citation Format: Anvesha Srivastava, Malathi Ramalinga, Deepak Kumar, Alexander Dimtchev, Juliet Chijioke, Offie Soldin, Xin Li, Catalin Marian, Simeng Suy, Sean P. Collins, Deepak Kumar. Circulating miRNAs as potential biomarkers in prostate cancer pathogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2422. doi:10.1158/1538-7445.AM2013-2422
Abstract 2424: CD40 expression in human solid tumors and NCI60 tumor cell lines.
Authors: Nguyen Dat , Tomaszewski Joseph E. , Hewitt Stephen M. , Doroshow James H. , Yang Sherry X.
Published: August 2014

Cell-surface CD40 is a member of the tumor necrosis factor receptor superfamily (TNFRSF) expressed and functionally well characterized in immune and hematopoietic cells. However, its expression and role in human solid tumors remain to be evaluated. As a molecular target currently with the antibody-based cancer therapy, it is important to characterize expression of CD40 across a range of human solid tumors and cancer cell lines. This could help improve our understanding of biology of CD40 molecule in human cancers especially epithelial tumors, and in turn increase the odds of success of targeting CD40 pathway. Expression of CD40 in the NCI60 human tumor cell lines and common types of human solid tumors were examined by immunohistochemistry in formalin-fixed and paraffin-embedded tumor specimens. CD40, predominantly membranous signal, was constitutively expressed in 15 lines, respectively derived from carcinomas of the breast, colorectum, non-small cell lung, ovary, prostate, and renal cell, of the NCI60 tumor cell line panel, and confirmed by Western blotting and gene expression microarray data available at http://dtp.nci.nih.gov/mtweb/search.jsp. CD40 expression was largely in accordance with the mRNA expression data measured by gene expression profiling. The status of CD40 was verified in MDA-MB-231, T-47D and HCT-116 cells (CD40-positive), and MCF-7 and HT-29 cells (CD40-negative) by Western blot. Moreover, CD40 was detected in tumor cells in 17% (6/34) of breast cancer, 24% (8/34) colorectal cancer, 54% (23/43) non-small cell lung cancer, and 47% (17/36) ovarian cancer. CD40 was also spotted in infiltrating lymphocytes in the tumor stroma. The data indicate that CD40 is expressed in human solid tumors with varying expression frequencies and one quarter of cancer cell lines examined. In addition, we have established a ready-for-use immunohistochemistry assay with an antibody that specifically detects CD40 in formalin-fixed and paraffin-embedded human tumor samples. Its application holds promise to evaluate the prognostic role of CD40 in human cancers.

Citation Format: Dat Nguyen, Joseph E. Tomaszewski, Stephen M. Hewitt, James H. Doroshow, Sherry X. Yang. CD40 expression in human solid tumors and NCI60 tumor cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2424. doi:10.1158/1538-7445.AM2013-2424
Abstract 2427: Molecular and pharmacokinetic studies of dicycloplatin, a new platinum analogue, suggest it is a promising chemotherapeutic agent.
Authors: Yang Xuqing , Song Qinhua , Mueller Michael D. , Yu Jing Jie
Published: August 2014

Dicycloplatin (DCP), a new platinum compound recently approved by the Chinese FDA, is a platinum-based supramolecule. The host-part (carboplatin) and a guest-part (cyclobutane-1,1-dicarboxylic acid or CBDC) are linked by hydrogen bonds. DCP has a stable chemical structure and good water solubility. Pharmacokinetic studies show that the prototype DCP concentration 2-h after administration is still high (17.1 μg/ml), after 26.9 μg/ml at 0.5 hour. In comparison, 5.01 μg/ml of carboplatin prototype is obtained 2-h after administration. Preclinical in vitro and in vivo studies and a phase I clinical trial demonstrated that DCP possesses significant antitumor activity, with fewer adverse effects than carboplatin. Molecular investigation of DCP-treated ovarian cancer A2780 cells showed activation of several kinases, including Chk2, p53, BRCA1 and other downstream factors. The increase in activated p-Chk2, p-p53 and p-BRCA1 after 1-h DCP exposure at IC50 dose occurred in a time-dependent manner. Quantitative analysis of phosphorylated Chk2 and BRCA1 expression revealed that the amount of dicycloplatin-induced phosphorylation of the two factors doubled at 48-h and tripled for p53 at 24-h, compared to controls. We observed that the DCP-induced activation profile and cisplatin-activated mechanisms are similar. We conclude that dicycloplatin is a promising platinum chemotherapeutic compound.

Citation Format: Xuqing Yang, Qinhua Song, Michael D. Mueller, Jing Jie Yu. Molecular and pharmacokinetic studies of dicycloplatin, a new platinum analogue, suggest it is a promising chemotherapeutic agent. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2427. doi:10.1158/1538-7445.AM2013-2427
Abstract 2428: Molecular determinants of dasatinib response in uterine carcinoma.
Authors: Huang Jie , Hu Wei , Bottsford-Miller Justin , Liu Tao , Zand Behrouz , Coleman Robert L. , Sood Anil K.
Published: August 2014

Despite the many attractive features of EphA2 as a therapeutic target in cancer, approaches to block it have lagged behind. Dasatinib is a multi-kinase inhibitor and may be useful for targeting EphA2. However, reliable predictive markers of response are needed. Additionally, the underlying mechanism deciding whether responsive or resistant to dasatinib and biological impact of EphA2 function in the context of EphA2 targeted therapy in cancer are not known.

We undertook a large number of studies to fully characterize the effects of dasatinib using in vitro (cell viability, RPPAs, gene expression, and confocal imaging) and in vivo uterine cancer models. Molecular markers for response prediction were identified by using RPPA and immunohistochemistry.

In vitro, the effects of dasatinib treatment varied among the different endometrial cancer cell lines tested, (IC50 values ranged from 30 nM to 17,792 nM), which was associated with varying levels of EphA2 and tyrosine phosphorylation and serine897 phosphorylation of EphA2, and with varying status of caveolin-1 (CAV-1), PTEN and RAS/MAPK . A set of markers that are essential for dasatinib response was identified, including EphA2, CAV-1, pAKTs473, BRAF/CRAF (MAPK pathway) and ppS6s240/244 (mTOR pathway). A novel mechanism for response was identified by which dasatinib can drive the BRAF and EphA2 to CAV-1 at the plasma membrane, resulting in breaking off the BRAF/CRAF heterodimer induced by dasatinib, and blocking compensatory activation of the MAPK pathway. In vivo data using orthotopic mouse models of uterine carcinoma were entirely concordant with our in vitro findings.

The efficacy of dasatinib therapy in uterine carcinoma depends on EphA2 phosphorylation at its S897 site and high CAV-1 levels are predictive of response to dasatinib. These findings may have clinical implications for ongoing dasatinib-based clinical trials.

Citation Format: Jie Huang, Wei Hu, Justin Bottsford-Miller, Tao Liu, Behrouz Zand, Robert L. Coleman, Anil K. Sood. Molecular determinants of dasatinib response in uterine carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2428. doi:10.1158/1538-7445.AM2013-2428
Abstract 243: ABCB5 is functionally required for melanoma growth.
Authors: Wilson Brian J. , Saab Karim R. , Schatton Tobias , Ma Jie , Murphy George F. , Gasser Martin , Waaga-Gasser Ana Maria , Frank Natasha Y. , Frank Markus H.
Published: August 2014

Melanoma remains a disease with relatively few beneficial treatments currently available, for late-stage patients. The ATP-binding cassette (ABC) transporter ABCB5 has recently been identified as a cell-surface marker for malignant melanoma initiating cells (MMICs; Schatton et al, Nature 2008), opening a new paradigm for investigation of this aggressive cancer. We have previously demonstrated that these ABCB5+ MMICs can evade the host immune system, and also express VEGFR-1, required for efficient tumor formation. However, the possibility that ABCB5 itself plays a functional role in melanoma tumorigenic growth and progression has not been investigated, until now.

To test whether ABCB5 plays a functional role in melanoma growth, or is solely a marker of the cancer stem-cell subset, we generated ABCB5-shRNA knockdown (ABCB5-KD) cell populations in melanoma model cell-lines. To distinguish a bona fide role in intrinsic tumorigenesis from immuno-modulatory effects we subcutaneously injected these cells, versus control cells, into highly immunocompromised NOD/SCID Il2rγ-/- mice and followed tumor progression. We observed a marked and significant downregulation in the ability of ABCB5-KD cells to maintain efficient tumor growth, implying a direct role for ABCB5 in melanoma progression.

To obtain mechanistic data to explain this phenotype we generated RNA from ABCB5-KD cells versus controls, and performed microarray analysis of global genomic changes in transcript levels of these cells. We then validated potential “ABCB5-pathway” genes, which also change in the reciprocal manner in an ABCB5 overexpression system that we have developed.

Our study links ABCB5 to a direct role in melanoma carcinogenesis, identifies genes and pathways of interest, and uncovers ABCB5 as a putative master-regulator of melanoma.

Citation Format: Brian J. Wilson, Karim R. Saab, Tobias Schatton, Jie Ma, George F. Murphy, Martin Gasser, Ana Maria Waaga-Gasser, Natasha Y. Frank, Markus H. Frank. ABCB5 is functionally required for melanoma growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 243. doi:10.1158/1538-7445.AM2013-243
Abstract 2431: IGF1R/IR axis is a novel treatment target in ER-negative breast cancer.
Authors: Kai Kazuharu , Kondo Kimie , Ueno Naoto
Published: August 2014

Background/Hypothesis: Insulin growth factor-1 receptor (IGF1R) and insulin receptor (IR) are overexpressed in a subpopulation of breast cancers (BCs). However, the clinical significance of IGF1R/IR is not fully understood, especially in ER-negative BC, which is aggressive and lacks molecularly targeted therapies. The objective of this study was to rationally develop treatment strategies for ER-negative BC with IGF1R/IR inhibitor. Method: We treated 6 ER-negative BC cell lines that express IGF1R/IR and have p53 mutations, a genetic hallmark of ER-negative BC, with KW2450, a dual tyrosine-kinase inhibitor against IGFR/IR. Western blot analysis was performed to confirm IGF1R, IR, or phosphor-IGFR/IR expression. To evaluate growth-inhibition effect, Cell-titer-blue assay and xenograft model were performed in vitro and in vivo. For apoptosis assay, Annexin V staining and western blotting of cleaved-caspase 8 and -PARP were performed. The amounts of intracellular reactive oxygen species (ROS) were measured as a candidate marker in predicting the efficacy of KW2450. Result: The values of IC50 are shown in the table. In cell-cycle analysis, all 6 cell lines treated with KW2450 accumulated in the G2/M phase and underwent cell death (sub-G1) in a dose-dependent manner. MCF7 and ZR15.1 cells, which have wild-type p53, had the same response to KW2450, which suggests that this phenotype is independent of p53 function. We attributed this cell death to apoptosis following apoptosis assay using one of the most sensitive cell lines, MDA-MB-468. We found that ROS accumulation correlated with a susceptibility to cell death at the dose of 0.5 μm, as shown below. In the xenograft model, we used SUM149 cells, a widely-used model, and saw a significant tumor growth-inhibitory effect at 20 or 40 mg/kg/day compared with vehicle control. Conclusion: ER-negative BCs that express IGF1R or IR are sensitive to KW2450. ROS accumulation with KW2450 treatment may be a good predictive marker of patient benefit from KW2450 treatment.

Molecular/genetic profiles and IC50s to KW-2450 in breast cancer cell lines, TN; triple-negative Cell line SUM149 MDA-MB-231 MDA-MB-468 BT-549 HCC70 SUM190 Subtype TN TN TN TN TN HER2+ p53 genotype M237I R280K R273H R249S R248Q Q317stop IC50 (μM) 0.23 0.35 0.27 0.28 0.26 1.72 % of dead cells 8.1 ± 0.2 7.3 ± 0.5 27.3 ± 0.9 NA NA NA % of ROS+ cells 3.6 ± 0.8 5.9 ± 0.4 62.5 ± 4.6 NA NA NA

Citation Format: Kazuharu Kai, Kimie Kondo, Naoto Ueno. IGF1R/IR axis is a novel treatment target in ER-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2431. doi:10.1158/1538-7445.AM2013-2431
Abstract 2445: Cucurbitacin B suppresses breast cancer growth by inhibiting TGFβ-mediated integrin-HER2 signaling.
Authors: Gupta Parul , Srivastava Sanjay K.
Published: August 2014

Integrins have recently been shown to play an important role in promotion of cell motility and survival in various cancers through cooperative signaling with EGFRs. Integrins transmit cell survival signals through ILK-Paxillin complex and further play role in activating AKT signaling. Curcubitacin B (CuB) is a steroidal class of chemical present in the plants belonging to cucurbitaceae family. CuB has shown cytotoxic potential in several cancers by causing G2/M cell cycle arrest and inhibition of JAK/STAT3 pathway. However, the exact mechanism of CuB and its role in metastasis and cell survival is not known. Our results show that CuB significantly suppressed the growth of MDA-MB-231, SKBR3, MCF-7, MDA-MB-231 (HER2) and MCF-7 (HER2), five different breast cancer cells in a concentration and time-dependent manner. CuB also induced apoptosis and down-regulated Integrin linked kinase 1(ILK1) expression. In addition, CuB down-regulated Integrin β4 (ITGB4) and Integrin α 6 (ITGA6) protein expression and suppressed the phosphorylation of AKT at Ser473, paxillin at Tyr118. Previous studies have shown that Transforming growth factor β (TGFβ) mediates crosstalk between ITGB4 and HER2, through Src kinase to enhance cell survival and motility. Interestingly we observed significant downregulation of Src, EGFR and HER2 in various breast cancer cells, suggesting that CuB suppresses cooperative signaling of Integrins and HER2. We also observed that TGFβ pretreatment imparted protection against CuB induced cytotoxic effects, indicating that CuB modulates TGFβ mediated Integrin signaling. Taken together our results indicate the anti-cancer effects of CuB in breast cancer cells by targeting ITGB4/ITGA6, Src, ILK and TGFβ. Further detailed mechanistic work is in progress. [Supported in part by R01 grants CA129038 (to S.K.S) awarded by the National Cancer Institute].

Citation Format: Parul Gupta, Sanjay K. Srivastava. Cucurbitacin B suppresses breast cancer growth by inhibiting TGFβ-mediated integrin-HER2 signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2445. doi:10.1158/1538-7445.AM2013-2445
Abstract 2447: Activation of Chk1 critical for Piperine induced cell cycle arrest and apoptosis in melanoma cells.
Authors: Fofaria Neel M. , Srivastava Sanjay K.
Published: August 2014

The National Cancer Institute estimates that about 76,250 new cases of melanomas will be diagnosed in 2012 in the United States leading to at least 9810 related deaths. Therefore, novel strategies to control and treat melanoma are of prime importance. In this study, we determined the cytotoxic effects of Piperine, a major constituent of black (Piper nigrum Linn) and long pepper (Piper longum Linn), in melanoma cell lines. Piperine treatment inhibited the growth of SKMEL-28 and B16-F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine was correlated with the down-regulation of cyclin D1 and induction of p21. The growth arrest was associated with DNA damage as indicated by phosphorylation of H2A.X at Ser139, activation of ataxia telangiectasia and Rad3-related protein (ATR) and checkpoint kinase 1 (Chk1). The G1 arrest by piperine was also associated with the down-regulation of E2F1 and decreased phosphorylation of retinoblastoma protein (Rb), hence indicating the inhibition of the dynamic transcription complex. In addition, apoptosis induced by piperine was associated with down-regulation of XIAP and Bid (uncleaved) and cleavage of Caspase-3 and PARP. Pretreatment of SK-MEL-28 with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Silencing of Chk1 in SK-MEL28 cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine treatment showing the involvement of Chk1. Through immunofluorescence of Phospho Chk1 (Ser 256), we found significant activation of Chk1 in the cells that were treated with piperine hence, establishing the role of Chk1 activation. Furthermore, our results showed that piperine treatment caused ROS generation in melanoma cells. Blocking ROS generation by Tiron pre-treatment prevented the cells from piperine mediated cell cycle arrest. Moreover, western-blotting results showed that Tiron pre treatment prevented the activation of Chk1 hence, confirming the role played by Chk1 in piperine mediated cell cycle arrest. Taken together, our results demonstrate that piperine induced G1 phase cell cycle arrest by generating ROS and inducing DNA damage with activation of Chk1 being a critical event. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute].

Citation Format: Neel M. Fofaria, Sanjay K. Srivastava. Activation of Chk1 critical for Piperine induced cell cycle arrest and apoptosis in melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2447. doi:10.1158/1538-7445.AM2013-2447
Abstract 245: Phenotypically diverse cancer cell subpopulations in a luminal-like breast cancer xenograft model are associated with different signaling pathways.
Authors: Skrbo Nirma , Andersen Kristin , Kristian Alexandr , Antberg Linn , Hjortland Geir Olav , Egenbråten Olav , James Peter , Mælandsmo Gunhild Mari , Sørlie Therese
Published: August 2014

High degree of intratumor cellular diversity represent a major challenge when attempting to cure cancer. The presence of tumor cell subpopulations with enhanced in vivo tumorigenic capacity (tumor initiating cells, TICs), and high resistance to conventional cancer therapy, compared with the “bulk” tumor cell populations, has for the last decade been the focus of many breast cancer research groups. The cellular heterogeneity of an orthotopic luminal-like breast cancer xenograft model was investigated using IHC, flow cytometry, whole genome expression profiling and mass spectrometry-based proteomics combined with in vivo tumorigenicity and targeted therapy assays. Epithelial cell adhesion molecule, EpCAM, highly specific for the human tumor epithelial cells, was used to separate human tumor cells from the mouse stromal compartment.

Further flow analysis of the EpCAM positive tumor cell population revealed diverse expression of several cell surface markers, including CD24 and SSEA-4 (stage specific embryonic antigen 4).

SSEA-4-/CD24-, SSEA-4+/CD24- and SSEA-4-/CD24+ populations were capable of initiating tumors in NOD SCID mice while SSEA-4+/CD24+ cells were non-tumorigenic. Tumors resulting from the SSEA-4+/CD24- subpopulation did not express CD24, while tumors arising from the SSEA-4-/CD24- and SSEA-4-/CD24+ populations, contained all four subpopulations.

As measured by whole genome expression analysis and mass spectrometry-based proteomics, the molecular differences were most pronounced between tumorigenic subpopulations and the non-tumorigenic subpopulation. While the mRNA expression data revealed a high degree of similarity among the four subpopulations, the proteomics data suggested that several signaling pathways might have distinct and different activity across the populations. The effects of targeted therapy against ER and WNT signaling on heterogeneity and tumorigenicity have been evaluated by in vivo experiments.

Citation Format: Nirma Skrbo, Kristin Andersen, Alexandr Kristian, Linn Antberg, Geir Olav Hjortland, Olav Egenbråten, Peter James, Gunhild Mari Mælandsmo, Therese Sørlie. Phenotypically diverse cancer cell subpopulations in a luminal-like breast cancer xenograft model are associated with different signaling pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 245. doi:10.1158/1538-7445.AM2013-245
Abstract 2450: Either pan class I PI3K inhibitor or PI3K alpha selective inhibitor sensitizes head and neck squamous cell carcinoma cells to Erlotinib, an EGFR TKI.
Authors: Gao Ming , Lu Yen-Shen , Hsiao Li-Ping , Chen Suz Wen , Cheng Ann-Lii
Published: August 2014

The epidermal growth factor receptor (EGFR) is overexpressed in 90% of head and neck squamous cell carcinoma (HNSCC), and is correlated with poor prognosis. EGFR is therefore a potential therapeutic target for HNSCC. However, most HNSCC patients do not respond well to small molecular EGFR inhibitors. One of the possible mechanisms of EGFR TKI resistance is compensatory collateral activation of PI3K/Akt pathway. This study sought to explore whether NVP-BKM120, a selective pan class I PI3 kinase inhibitor, and NVP-BYL719, a novel PI3 kinase alpha selective inhibitor, may reverse the resistance of HNSCC cells toward erlotinib, an EGFR TKI. Cells were treated with the PI3K inhibitors, either BKM120 or BYL719, alone, erlotinib alone, or in combination, and viability was measured at 72 hours with MTT assay. Combination effect was evaluated by median effect analysis. Synergism, indicated by a combination index (CI) value <1, were seen in all five cell lines (SAS, HSC3, UMCC1, CA922 and FaDu) studied. By immunoblotting analysis, combination of Erlotinib and either PI3K inhibitor resulted in dowregulation of p-EGFR and p-AKT expression in SAS cells. When SAS and HSC3 cells were treated with erlotinib in combination with either BKM120 or BYL719, an additional increase in the subG1 population by flow cytometry analysis were observed, indicating an increase of apoptotic cell death. Our data suggest that additional PI3K blockade might be an effective strategy to overcome resistance of HNSCC cells toward EGFR TKIs, and the beneficial effect is similar between pan Class I PI3K inhibitor and PI3K alpha selective inhibitor. (The study was supported by the grant of DOH101-TD-B-111-001).

Citation Format: Ming Gao, Yen-Shen Lu, Li-Ping Hsiao, Suz Wen Chen, Ann-Lii Cheng. Either pan class I PI3K inhibitor or PI3K alpha selective inhibitor sensitizes head and neck squamous cell carcinoma cells to Erlotinib, an EGFR TKI. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2450. doi:10.1158/1538-7445.AM2013-2450
Abstract 2453: Optimizing a modular synthetic approach for producing SH7139, a new drug candidate for treating B-cell lymphomas.
Authors: Balhorn Monique , Hok Saphon , Balhorn Rod
Published: August 2014

The scale-up of synthetic methods for producing small molecule therapeutics can often prove to be problematic and expensive. Synthetic mechanisms can be inefficient and purification methods used to select specific isomers or remove reactants or other contaminants can often be difficult to scale. The modular approach we developed for creating small molecule targeting agents called SHALs (Selective High Affinity Ligands) as therapeutics and diagnostics for B-cell lymphoma circumvents many of these problems by using off the shelf components as “recognition elements” that collectively contribute to the molecules high affinity and selectivity and miniPEG and common amino acids to build up the scaffold using solid phase chemistry. Here we present the results of our efforts to scale up the synthesis of the first of these promising new drug candidates, SH7139, to provide the compounds needed for pre-IND animal testing and a pet dog lymphoma clinical trial. Working with AmbioPharm Inc,, a CRO with extensive expertise in the GMP synthesis of peptide pharmaceuticals, we recently completed small pilot production scale syntheses of SH7139 and several closely related analogs. The simple solid phase peptide-based chemistry used to synthesize the compounds coupled with preparative HPLC adapted well to scale-up and resulted in the production of highly purified compounds identical in structure (as determined by mass spectrometry and nuclear magnetic resonance spectroscopy) to the bench scale syntheses we performed in house. However, one unexpected and notable difference between the scaled-up synthetic products and their bench-scale synthesis counterparts was their solubility. SH7139 and two of its analogs, which are essentially insoluble in water and PBS when synthesized in house, were observed to be highly soluble (>100mg/ml) when synthesized using the scale-up protocol. While the reason for this is still under investigation, microscopic analyses of the dried product suggest the solubility difference may relate to the lyophilization step used to produce the dried product. The majority of the insoluble SH7139 we produce in bench-scale syntheses is amorphous in form, while the soluble SH7139 produced by AmbioPharm appears to be in a semi-crystalline or glass-like state. This research was supported, in part, by NIH/NCI SBIR grant 1R43CA159843-01 to Rod Balhorn.

Citation Format: Monique Balhorn, Saphon Hok, Rod Balhorn. Optimizing a modular synthetic approach for producing SH7139, a new drug candidate for treating B-cell lymphomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2453. doi:10.1158/1538-7445.AM2013-2453
Abstract 2459: Discovery of small molecule TAK1 inhibitors with antiproliferative activity in cell lines derived from hematological malignancies.
Authors: Toader Dorin , Saeh Jamal C. , Guan Nin , Powell Francoise , Chen Raymond , Reimer Corinne , Byth Kate
Published: August 2014

Transforming growth factor β-activated kinase 1 (TAK1), a member of the MAPKKK family, is a key mediator of proinflammatory and stress signaling. It was shown previously that inhibition of TAK1 via the use of siRNA or small-molecule kinase inhibitors can inactivate NF-κB, down-regulate p38, and activate the intrinsic caspase pathway, resulting in profound induction of apoptosis. Herein we report the discovery of 5-fluoro-4-(imidazo[1,2-b]pyridazin-3-yl)-N-phenylpyrimidin-2-amines as TAK1 ATP competitive small molecule inhibitors. These compounds inhibit the kinase activity of TAK1 in vitro with low nanomolar potency. Evidence is presented that supports a mechanism of action consistent with inhibition of TAK1 kinase activity within the NF-κB pathway. Optimization of potency and selectivity for this series from hit to lead will be discussed, including the structure-based design and crystallographic determination of the binding mode. The outcome of efforts aimed at improving the in vivo pharmacokinetics of compounds is described. Results of kinase selectivity profiling for both an in vitro probe compound - AZ TAK1 - and an in vivo probe compound will be presented. Furthermore, we report low nanomolar antiproliferative activities in cell lines derived from Haematological Malignancies for these compounds. This cellular activity profile is suggesting that TAK1 inhibition presents therapeutic potential in Haematological Cancers.

Citation Format: Dorin Toader, Jamal C. Saeh, Nin Guan, Francoise Powell, Raymond Chen, Corinne Reimer, Kate Byth. Discovery of small molecule TAK1 inhibitors with antiproliferative activity in cell lines derived from hematological malignancies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2459. doi:10.1158/1538-7445.AM2013-2459
Abstract 2463: Discovery of imidazo[1,5-a]pyrazine derived potent, selective and orally bioavailable ACK1 inhibitors.
Authors: Jin Meizhong , Wang Jing , Kleinberg Andrew , Kadalbajoo Mridula , Siu Kam W. , Cooke Andrew , Bittner Mark , Yao Yan , Thelemann April , Ji Qunsheng , Bhagwat Shripad , Mulvihill Kristen M. , Rechka Josef A. , Pachter Jonathan A. , Crew Andrew P. , Epstein David , Mulvihill Mark J.
Published: August 2014

Activated Cdc42-associated kinase (ACK1) is a non-receptor tyrosine kinase originally identified by virtue of its binding to GTP-bound small GTPase Cdc42. Considerable attention has been paid to ACK1’s involvement in cancer in recent years. For example, gene amplification and over-expression of ACK1 were found in multiple cancers including lung, ovarian and prostate cancers and were associated with poor prognosis and metastatic phenotypes. Activated ACK1 has been shown to phosphorylate and activate androgen receptor function and to promote the progression of prostate cancer. More recently, activated ACK1 was found to phosphorylate and promote the activation of Akt, a protein kinase that plays a central role in growth, proliferation and cell survival in various cancers. Taken together, these literature data suggest that ACK1 is a potential target for cancer treatment. Several series of ACK1 inhibitors have been previously disclosed in literature. Unfortunately, compounds from these series suffer from poor oral pharmacokinetic (PK) properties which have prevented them from being utilized further for in vivo studies. Therefore, there is a clear need for potent, selective and orally bioavailable small molecule ACK1 inhibitors to further probe its role in cancer, both in the in vitro and in vivo setting. This report describes the medicinal chemistry effort towards a series of novel imidazo[1,5-a]pyrazine derived inhibitors of ACK1. Virtual screening led to the discovery of the initial hit, and subsequent exploration of structure-activity relationships and optimization of drug metabolism and pharmacokinetic properties led to the identification of potent, selective and orally bioavailable ACK1 inhibitors.

Citation Format: Meizhong Jin, Jing Wang, Andrew Kleinberg, Mridula Kadalbajoo, Kam W. Siu, Andrew Cooke, Mark Bittner, Yan Yao, April Thelemann, Qunsheng Ji, Shripad Bhagwat, Kristen M. Mulvihill, Josef A. Rechka, Jonathan A. Pachter, Andrew P. Crew, David Epstein, Mark J. Mulvihill. Discovery of imidazo[1,5-a]pyrazine derived potent, selective and orally bioavailable ACK1 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2463. doi:10.1158/1538-7445.AM2013-2463
Abstract 2464: Flavone derivatives as small-molecule probes of cytochrome P450 enzymes: Inhibitory activity and selectivity.
Authors: Liu Jiawang , Taylor Shannon , Dupart Patrick , Arnold Corey , Foroozesh Maryam
Published: August 2014

Cytochrome P450 enzymes are the most important enzymes involved in the conversion of environmental procarcinogens such as polycyclic aromatic hydrocarbons (PAHs) from fuels, cigarette smoke, and charcoal-grilled foods into carcinogenic species. These carcinogenic species have the potential to bond to cellular macromolecules such as DNA and can lead to cancer formation. Therefore, selectively inhibiting certain P450 enzymes involved in carcinogenesis is a potential approach to cancer prevention. To investigate the 3D-structural features of the active site cavities of our target P450s, our approach in this project is to use the natural P450 substrate, flavone, as a lead compound. Here we report the synthesis of six novel flavonoids (5,6-pyranflavone, 6,5-pyranflavone, 7,8-pyranflavone, 2’,3’-pyranflavone, 3’,2’-pyranflaovne, and 4’,3’-pyranflavone) and the evaluation of their inhibitory activities, mechanisms of action, and selectivity towards P450 enzymes 1A1, 1A2, 2A6, and 2B1. All of these target compounds were found to be competitive inhibitors of these P450 enzymes. A high inhibitory activity represents a high affinity between the compound and the enzyme. Our data show that the α-naphthoflavone-like flavone derivative, 7,8-pyranflavone, has a high affinity for P450 1A2 (IC50: 0.05 μM), and a relatively low affinity for P450 1A1 (IC50: 0.25 μM). On the contrary, β-naphthoflavone-like flavone derivatives, 5,6-pyranflavone and 6,5-pyranflavone, show higher affinities for P450 1A1 compared to P450 1A2. However, none of these three compounds shows any inhibitory activity towards P450s 2A6 and 2B1. Interestingly, the C-ring modified derivatives (2’,3’-pyranflavone, 3’,2’-pyranflaovne, and 4’,3’-pyranflavone) have relatively low inhibitory activities toward the P450s 1A1 and 1A2, while showing a weak inhibition of P450 2B1 (IC50s: ca. 7.0 μM). These structural data suggest that the length of the P450 1A1 active site cavity is probably longer than that of P450 1A2, while the width of the P450 1A2 active site cavity may be larger than that of P450 1A1. Further studies of 7,8-pyranflavone, 5,6-pyranflavone, and 6,5-pyranflavone will provide us with more detailed structural information about the target P450 enzymes.

Citation Format: Jiawang Liu, Shannon Taylor, Patrick Dupart, Corey Arnold, Maryam Foroozesh. Flavone derivatives as small-molecule probes of cytochrome P450 enzymes: Inhibitory activity and selectivity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2464. doi:10.1158/1538-7445.AM2013-2464
Abstract 2467: Development of new N-Arylbenzamides as STAT3 dimerization inhibitors.
Authors: Lawrence Nicholas J. , Pireddu Roberta , Ge Yiyu , Urlam Murali K. , Zhang Xiaolei , Sun Ying , Lawrence Harshani R. , Guida Wayne C. , Sebti Said
Published: August 2014

The association of aberrant STAT3 activation with many types of human malignancies and solid tumors makes STAT3 an attractive molecular target for the development of novel cancer therapeutics. STAT3 dimerization via two reciprocal phosphotyrosine (pTyr)-Src-homology 2 (SH2) interaction is required for its binding to specific DNA-response elements in the promoters of target genes. This dimerization process offers a potentially selective way to directly target STAT3. Nevertheless the design of small molecules capable of disrupting protein-protein interactions remains a significant challenge to medicinal chemistry. We describe the development of a new class of small molecule capable of disrupting the STAT3:STAT3 protein-protein interaction. We identified the STAT3 inhibitor S3I-201 (NSC-74859) from the NCI chemical collection by using structure-based virtual screening with a model based on the X-ray crystal structure of the STAT3β homodimer. We developed a series of phosphonic and salicylic acids based on the structure of S3I-201 incorporating an N-arylbenzamide scaffold. The series of phosphonic and salicylic acids with a shorter amide linker lacking the O-tosyl group has improved STAT3 inhibitory activity [as measured by a competitive, fluorescence-polarization (FP)-based assay]. The equivalent in vitro potencies observed by the replacement of phosphonic acid moiety of with 5-amino-2-hydroxybenzoic acid group validates 5-amino-2-hydroxybenzoic acid as a phosphotyrosine mimic in this series. The binding mode of selected compounds, as predicted by molecular modeling, will be discussed. Our docking studies indicated that the phosphonic and salicylic acid groups interact in the pTyr-705 binding site in a broadly similar manner, while the other substituents occupy the STAT3 pY+1 and pY-X hydrophobic pockets. One of the most potent compounds in vitro (in the FP assay) inhibits STAT3 dimerization in intact cells and suppresses malignant transformation in human cancer cells that depend on STAT3. The in vitro and cell based potency of the N-arylbenzamides warrants further development of this scaffold as STAT3 inhibitors.

Citation Format: Nicholas J. Lawrence, Roberta Pireddu, Yiyu Ge, Murali K. Urlam, Xiaolei Zhang, Ying Sun, Harshani R. Lawrence, Wayne C. Guida, Said Sebti. Development of new N-Arylbenzamides as STAT3 dimerization inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2467. doi:10.1158/1538-7445.AM2013-2467
Abstract 2472: Small-molecule-induced reactivation of mutant p53 in cancer cells.
Authors: Liu Xiangrui , Wilcken Rainer , Joerger Andreas , Chuckowree Irina , Spencer John , Fersht Alan
Published: August 2014

The tumour suppressor p53 inhibits tumour growth by induction of cell cycle arrest, apoptosis or senescence. However, p53 mutations occur frequently in human tumours. About one-third of the mutations lower the melting temperature of the protein and cause rapidly unfolds at body temperature. The p53 cancer mutant Y220C is an excellent paradigm for rescuing the function of conformationally unstable p53 mutants because it has a unique surface crevice that can be targeted by small-molecule stabilizers. Through biophysical and cell-based screening, we identified a compound, PK7088, which binds to the Y220C mutant with a dissociation constant of 140 μM. The binding mode of this compound class was determined by X-ray crystallography. In cancer cells carrying the Y220C mutant, PK7088 increased the amount of folded mutant protein with wild-type like conformation, as monitored by immunofluorescence, and restored its transcriptional functions. It induced p53-Y220C-dependent growth inhibition, cell-cycle arrest and apoptosis. This was confirmed by p53 silencing. Most notably, PK7088 increased the expression levels of p21 and the proapoptotic NOXA protein. PK7088 worked synergistically with Nutlin-3 on up-regulating p21 and NOXA expression, whereas Nutlin-3 on its own had no effect, consistent with its mechanism of action. PK7088 also restored non-transcriptional apoptotic functions of p53 by triggering nuclear export of BAX to the mitochondria. These findings provide novel insights into p53 mutant reactivation and may facilitate the development of more potent and selective small-molecule drugs for specific mutant p53 rescue.

Citation Format: Xiangrui Liu, Rainer Wilcken, Andreas Joerger, Irina Chuckowree, John Spencer, Alan Fersht. Small-molecule-induced reactivation of mutant p53 in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2472. doi:10.1158/1538-7445.AM2013-2472
Abstract 2473: Fragment-based discovery of inhibitors of replication protein A protein-protein interactions.
Authors: Waterson Alex G. , Patrone James D. , Kennedy J. Phillip , Pelz Nicholas F. , Frank Andreas O. , Vandgamudi Bhavatarini , Feldkamp Michael D. , Souza-Fagundes Elaine M. , Rossanese Olivia W. , Chazin Walter J. , Fesik Stephen W.
Published: August 2014

Replication Protein A (RPA) is a heterotrimeric protein that binds to and protects ssDNA and plays an integral role in initiating the cellular response to DNA damage. This response is mediated via protein-protein interactions between a basic cleft on the RPA70N subunit and a number of protein partners, including ATRIP, Rad9, Mre11, and p53. RNAi against RPA has shown an expected toxicity against cancer cell lines, possibly due to abrogation of the ssDNA binding function of RPA. Specific disruption of the protein-protein interactions between the RPA07N subunit and its binding partners has the potential to produce a more selective cytotoxic response in cancer cells. To more accurately dissect the therapeutic relevance of disrupting only the protein-protein interaction functions of RPA, we sought to discover potent small molecule probes that bind to the basic cleft of RPA70N.

Inhibition of protein-protein interactions is considered a difficult task. An NMR-based fragment screen has identified more than 130 fragment molecules that bind to the RPA70N protein-protein interaction cleft with affinities that range from 500 μM to 2 mM and corresponding ligand efficiencies from 0.18 to 0.30. Selected fragments, representing several distinct chemotypes, have been optimized for binding to the protein. Using X-ray crystallography, the binding modes of these fragments have been defined. Fragments were found to bind primarily to two main sites within the basic cleft of RPA70N. Additional structure-guided optimizations have been carried out and ternary co-crystal structures have been generated to guide fragment linking strategies. Together, these activities have led to the creation of multiple lead series of inhibitors of the RPA:ATRIP interaction, with binding affinities improved by several fold over the initial fragments. The SAR and biological activities of the fragments and lead compounds will be discussed.

Citation Format: Alex G. Waterson, James D. Patrone, J. Phillip Kennedy, Nicholas F. Pelz, Andreas O. Frank, Bhavatarini Vandgamudi, Michael D. Feldkamp, Elaine M. Souza-Fagundes, Olivia W. Rossanese, Walter J. Chazin, Stephen W. Fesik. Fragment-based discovery of inhibitors of replication protein A protein-protein interactions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2473. doi:10.1158/1538-7445.AM2013-2473