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Sauveur-Michel Maira,
Sabina Pecchi,
Alan Huang,
Matthew Burger,
Mark Knapp,
Dario Sterker,
Christian Schnell,
Daniel Guthy,
Tobi Nagel,
Marion Wiesmann, [......],
Alain De Pover,
Daniel Menezes,
Georg Martiny-Baron,
Doriano Fabbro,
Christopher J Wilson, Robert Schlegel,
Francesco Hofmann,
Carlos García-Echeverría,
William R Sellers,
Charles F Voliva
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ABSTRACT: Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional phosphoinositide 3-kinase (PI3K) inhibitors from different chemical classes with a different selectivity profile. The key to achieve these objectives was to couple a structure-based design approach with intensive pharmacologic evaluation of selected compounds during the medicinal chemistry optimization process. Here, we report on the biologic characterization of the 2-morpholino pyrimidine derivative pan-PI3K inhibitor NVP-BKM120. This compound inhibits all four class I PI3K isoforms in biochemical assays with at least 50-fold selectivity against other protein kinases. The compound is also active against the most common somatic PI3Kα mutations but does not significantly inhibit the related class III (Vps34) and class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic models and relevant tumor cell lines, as well as downstream effectors in a concentration-dependent and pathway-specific manner. Tested in a panel of 353 cell lines, NVP-BKM120 exhibited preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant models. NVP-BKM120 shows dose-dependent in vivo pharmacodynamic activity as measured by significant inhibition of p-Akt and tumor growth inhibition in mechanistic xenograft models. NVP-BKM120 behaves synergistically when combined with either targeted agents such as MEK or HER2 inhibitors or with cytotoxic agents such as docetaxel or temozolomide. The pharmacological, biologic, and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is undergoing phase II clinical trials in patients with cancer.
Molecular Cancer Therapeutics 12/2011; 11(2):317-28. · 5.23 Impact Factor
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Alix Scholer-Dahirel,
Michael R Schlabach,
Alice Loo,
Linda Bagdasarian,
Ronald Meyer,
Ribo Guo,
Steve Woolfenden,
Kristine K Yu,
Judit Markovits,
Karen Killary,
Dmitry Sonkin,
Yung-Mae Yao,
Markus Warmuth,
William R Sellers, Robert Schlegel,
Frank Stegmeier,
Rebecca E Mosher,
Margaret E McLaughlin
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ABSTRACT: Persistent expression of certain oncogenes is required for tumor maintenance. This phenotype is referred to as oncogene addiction and has been clinically validated by anticancer therapies that specifically inhibit oncoproteins such as BCR-ABL, c-Kit, HER2, PDGFR, and EGFR. Identifying additional genes that are required for tumor maintenance may lead to new targets for anticancer drugs. Although the role of aberrant Wnt pathway activation in the initiation of colorectal cancer has been clearly established, it remains unclear whether sustained Wnt pathway activation is required for colorectal tumor maintenance. To address this question, we used inducible β-catenin shRNAs to temporally control Wnt pathway activation in vivo. Here, we show that active Wnt/β-catenin signaling is required for maintenance of colorectal tumor xenografts harboring APC mutations. Reduced tumor growth upon β-catenin inhibition was due to cell cycle arrest and differentiation. Upon reactivation of the Wnt/β-catenin pathway colorectal cancer cells resumed proliferation and reacquired a crypt progenitor phenotype. In human colonic adenocarcinomas, high levels of nuclear β-catenin correlated with crypt progenitor but not differentiation markers, suggesting that the Wnt/β-catenin pathway may also control colorectal tumor cell fate during the maintenance phase of tumors in patients. These results support efforts to treat human colorectal cancer by pharmacological inhibition of the Wnt/β-catenin pathway.
Proceedings of the National Academy of Sciences 09/2011; 108(41):17135-40. · 9.68 Impact Factor
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Pixu Liu,
Hailing Cheng,
Stephanie Santiago,
Maria Raeder,
Fan Zhang,
Adam Isabella,
Janet Yang,
Derek J Semaan,
Changzhong Chen,
Edward A Fox,
Nathanael S Gray,
John Monahan, Robert Schlegel,
Rameen Beroukhim,
Gordon B Mills,
Jean J Zhao
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ABSTRACT: PIK3CA gain-of-function mutations are a common oncogenic event in human malignancy, making phosphatidylinositol 3-kinase (PI3K) a target for cancer therapy. Despite the promise of targeted therapy, resistance often develops, leading to treatment failure. To elucidate mechanisms of resistance to PI3K-targeted therapy, we constructed a mouse model of breast cancer conditionally expressing human PIK3CA(H1047R). Notably, most PIK3CA(H1047R)-driven mammary tumors recurred after PIK3CA(H1047R) inactivation. Genomic analyses of recurrent tumors revealed multiple lesions, including focal amplification of Met or Myc (also known as c-Met and c-Myc, respectively). Whereas Met amplification led to tumor survival dependent on activation of endogenous PI3K, tumors with Myc amplification became independent of the PI3K pathway. Functional analyses showed that Myc contributed to oncogene independence and resistance to PI3K inhibition. Notably, PIK3CA mutations and c-MYC elevation co-occur in a substantial fraction of human breast tumors. Together, these data suggest that c-MYC elevation represents a potential mechanism by which tumors develop resistance to current PI3K-targeted therapies.
Nature medicine 08/2011; 17(9):1116-20. · 27.14 Impact Factor
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Cory M Johannessen,
Jesse S Boehm,
So Young Kim,
Sapana R Thomas,
Leslie Wardwell,
Laura A Johnson,
Caroline M Emery,
Nicolas Stransky,
Alexandria P Cogdill,
Jordi Barretina, [......],
Thomas M Roberts,
Todd Golub,
Keith T Flaherty,
Reinhard Dummer,
Barbara L Weber,
William R Sellers, Robert Schlegel,
Jennifer A Wargo,
William C Hahn,
Levi A Garraway
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ABSTRACT: Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
Nature 12/2010; 468(7326):968-72. · 36.28 Impact Factor
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Alex Gaither,
Dale Porter,
Yao Yao,
Jason Borawski,
Guang Yang,
Jerry Donovan,
David Sage,
Joanna Slisz,
Mary Tran,
Christopher Straub,
Tim Ramsey,
Vadim Iourgenko,
Alan Huang,
Yan Chen, Robert Schlegel,
Mark Labow,
Stephen Fawell,
William R Sellers,
Leigh Zawel
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ABSTRACT: Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor alpha (TNFalpha) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself. Here, we show that in a subset of highly sensitive tumor cell lines, activity of LBW242 is dependent on TNFalpha signaling. Mechanistic studies indicate that in this context, XIAP is a positive modulator of TNFalpha induction whereas cellular inhibitor of apoptosis protein 1 negatively regulates TNFalpha-mediated apoptosis.
Cancer Research 01/2008; 67(24):11493-8. · 7.86 Impact Factor
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Yan Chen,
Christine Miller,
Rebecca Mosher,
Xumei Zhao,
Jim Deeds,
Mike Morrissey,
Barb Bryant,
David Yang,
Ron Meyer,
Frank Cronin,
Bobbie S Gostout,
Karen Smith-McCune, Robert Schlegel
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ABSTRACT: The Pap test has effectively reduced the incidence and mortality of cervical cancer. However, because of the morphological basis of this test, sensitivity and specificity are less than ideal, a situation that complicates the clinical management of women diagnosed with low-grade cervical abnormalities. In an attempt to understand the molecular basis of cervical tumorigenesis and to discover molecular markers for accurate cervical cancer screening, we used cDNA microarrays containing >30,000 Unigene clones to examine the gene expression patterns of 34 cervical tissues from different clinically defined stages. It was found that global gene expression patterns separated normal cervical tissues and low-grade squamous intraepithelial lesions from cervical cancers and most of the high-grade squamous intraepithelial lesions (HSILs). Among the top 62 genes/(expressed sequence tags) that were overexpressed in tumors and HSIL tissues, 35 were confirmed using in situ hybridization on cervical tissue micorarrays. Many of these genes were overexpressed in high-grade dysplastic and malignant cervical epithelium or in stroma adjacent to the diseased tissues, with cellular proliferation and extracellular matrix-associated genes being the most common. In general, the extent of gene overexpression increased as the lesions progressed from low-grade squamous intraepithelial lesions to HSILs and finally to cancer. It is hoped that with additional development, some of these markers will improve the interpretation of cervical screening tests and provide useful information for patient management decisions.
Cancer Research 04/2003; 63(8):1927-35. · 7.86 Impact Factor
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ABSTRACT: Human papillomaviruses (HPVs) infect cervical epithelial cells and induce both benign and precancerous lesions. High-risk HPVs promote the development of cervical cancer in vivo and can immortalize cervical epithelial cells in vitro, whereas low-risk HPVs cannot. We used cDNA microarrays and quantitative polymerase chain reaction to compare cellular gene expression in primary cervical epithelial cells during a time course after retroviral transduction with either low-risk or high-risk E6/E7 genes. At early passages, cervical cells transduced with high-risk E6/E7 genes demonstrated increased expression of the cell cycle-regulated genes CDC2 and ubiquitin carrier E2-C. At later passages, these same cells exhibited dramatic increases in insulin-like growth factor-binding protein-3 (IGFBP-3) mRNA and both secreted an intracellular protein, with mRNA levels increasing approximately 85-fold. Corroborating these in vitro studies, in situ hybridization of cervical biopsies with an IGFBP-3 riboprobe revealed high levels of expression in high-grade squamous intraepithelial neoplasia but not in normal cervical epithelium. Our in vitro results indicate that overexpression of IGFBP-3 is a late event after E6/E7 expression, and analysis of cervical lesions indicates that overexpression of this gene is also seen in vivo.
American Journal Of Pathology 09/2002; 161(2):603-10. · 4.89 Impact Factor
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ABSTRACT: Cervical carcinoma cells display high telomerase activity and usually contain and express integrated copies of the human papillomavirus (HPV) genome. Recent studies have demonstrated that the E6 oncogene of malignancy-associated HPVs increases cellular telomerase activity, predominantly via transcriptional activation of the catalytic subunit of telomerase, hTERT. To examine the relationship between E6 oncoprotein expression and telomerase expression during cellular immortalization, we transduced primary human cervical epithelial cells with the HPV E6/E7 genes and monitored temporal changes in viral oncoprotein expression, cellular hTERT RNA expression, and cellular telomerase activity. Quantitation of the individual E6 and E7 proteins, using a newly developed immunoprecipitation/immunoblotting technique, demonstrated that both oncoproteins were expressed at stable levels during successive passages of cervical cells. In contrast, the levels of hTERT mRNA and telomerase activity increased progressively and dramatically during passaging. Late-passage immortalized cells (passage 30) showed a 25-fold increase in hTERT mRNA and a 300-fold increase in telomerase activity compared to early-passage (passage 4) cells. Thus, neither hTERT mRNA expression nor telomerase activity are directly proportional to the level of E6 oncoprotein, indicating that E6 is not the sole determinant of the high levels of telomerase in cervical cells during immortalization.
American Journal Of Pathology 05/2002; 160(4):1251-7. · 4.89 Impact Factor
