Publications by authors named "Jurgen Moll"

48 Publications

BI-3406, a Potent and Selective SOS1-KRAS Interaction Inhibitor, Is Effective in KRAS-Driven Cancers through Combined MEK Inhibition.

Cancer Discov 2020 Aug 19. Epub 2020 Aug 19.

Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.

is the most frequently mutated driver of pancreatic, colorectal, and non-small cell lung cancers. Direct KRAS blockade has proved challenging, and inhibition of a key downstream effector pathway, the RAF-MEK-ERK cascade, has shown limited success because of activation of feedback networks that keep the pathway in check. We hypothesized that inhibiting SOS1, a KRAS activator and important feedback node, represents an effective approach to treat KRAS-driven cancers. We report the discovery of a highly potent, selective, and orally bioavailable small-molecule SOS1 inhibitor, BI-3406, that binds to the catalytic domain of SOS1, thereby preventing the interaction with KRAS. BI-3406 reduces formation of GTP-loaded RAS and limits cellular proliferation of a broad range of KRAS-driven cancers. Importantly, BI-3406 attenuates feedback reactivation induced by MEK inhibitors and thereby enhances sensitivity of KRAS-dependent cancers to MEK inhibition. Combined SOS1 and MEK inhibition represents a novel and effective therapeutic concept to address KRAS-driven tumors. SIGNIFICANCE: To date, there are no effective targeted pan-KRAS therapies. In-depth characterization of BI-3406 activity and identification of MEK inhibitors as effective combination partners provide an attractive therapeutic concept for the majority of KRAS-mutant cancers, including those fueled by the most prevalent mutant KRAS oncoproteins, G12D, G12V, G12C, and G13D.
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http://dx.doi.org/10.1158/2159-8290.CD-20-0142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892644PMC
August 2020

Selective and Potent CDK8/19 Inhibitors Enhance NK-Cell Activity and Promote Tumor Surveillance.

Mol Cancer Ther 2020 04 5;19(4):1018-1030. Epub 2020 Feb 5.

Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.

Natural killer (NK) cells play a pivotal role in controlling cancer. Multiple extracellular receptors and internal signaling nodes tightly regulate NK activation. Cyclin-dependent kinases of the mediator complex (CDK8 and CDK19) were described as a signaling intermediates in NK cells. Here, we report for the first time the development and use of CDK8/19 inhibitors to suppress phosphorylation of STAT1 in NK cells and to augment the production of the cytolytic molecules perforin and granzyme B (GZMB). Functionally, this resulted in enhanced NK-cell-mediated lysis of primary leukemia cells. Treatment with the CDK8/19 inhibitor BI-1347 increased the response rate and survival of mice bearing melanoma and breast cancer xenografts. In addition, CDK8/19 inhibition augmented the antitumoral activity of anti-PD-1 antibody and SMAC mimetic therapy, both agents that promote T-cell-mediated antitumor immunity. Treatment with the SMAC mimetic compound BI-8382 resulted in an increased number of NK cells infiltrating EMT6 tumors. Combination of the CDK8/19 inhibitor BI-1347, which augments the amount of degranulation enzymes, with the SMAC mimetic BI-8382 resulted in increased survival of mice carrying the EMT6 breast cancer model. The observed survival benefit was dependent on an intermittent treatment schedule of BI-1347, suggesting the importance of circumventing a hyporesponsive state of NK cells. These results suggest that CDK8/19 inhibitors can be combined with modulators of the adaptive immune system to inhibit the growth of solid tumors, independent of their activity on cancer cells, but rather through promoting NK-cell function.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661742PMC
April 2020

A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.

Cell 2018 01 7;172(1-2):373-386.e10. Epub 2017 Dec 7.

Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands; Cancer Genomics Netherlands, Oncode Institute, 3584 CG Utrecht, the Netherlands. Electronic address:

Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.
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http://dx.doi.org/10.1016/j.cell.2017.11.010DOI Listing
January 2018

Spindle Misorientation of Cerebral and Cerebellar Progenitors Is a Mechanistic Cause of Megalencephaly.

Stem Cell Reports 2017 10 21;9(4):1071-1080. Epub 2017 Sep 21.

Leibniz Institute on Aging, Fritz Lipmann Institute, 07745 Jena, Germany. Electronic address:

Misoriented division of neuroprogenitors, by loss-of-function studies of centrosome or spindle components, has been linked to the developmental brain defects microcephaly and lissencephaly. As these approaches also affect centrosome biogenesis, spindle assembly, or cell-cycle progression, the resulting pathologies cannot be attributed solely to spindle misorientation. To address this issue, we employed a truncation of the spindle-orienting protein RHAMM. This truncation of the RHAMM centrosome-targeting domain does not have an impact on centrosome biogenesis or on spindle assembly in vivo. The RHAMM mutants exhibit misorientation of the division plane of neuroprogenitors, without affecting the division rate of these cells, resulting against expectation in megalencephaly associated with cerebral cortex thickening, cerebellum enlargement, and premature cerebellum differentiation. We conclude that RHAMM associates with the spindle of neuroprogenitor cells via its centrosome-targeting domain, where it regulates differentiation in the developing brain by orienting the spindle.
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http://dx.doi.org/10.1016/j.stemcr.2017.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639290PMC
October 2017

The IGF1R/INSR Inhibitor BI 885578 Selectively Inhibits Growth of IGF2-Overexpressing Colorectal Cancer Tumors and Potentiates the Efficacy of Anti-VEGF Therapy.

Mol Cancer Ther 2017 10 20;16(10):2223-2233. Epub 2017 Jul 20.

Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria.

Clinical studies of pharmacologic agents targeting the insulin-like growth factor (IGF) pathway in unselected cancer patients have so far demonstrated modest efficacy outcomes, with objective responses being rare. As such, the identification of selection biomarkers for enrichment of potential responders represents a high priority for future trials of these agents. Several reports have described high IGF2 expression in a subset of colorectal cancers, with focal amplification being responsible for some of these cases. We defined a novel cut-off value for IGF2 overexpression based on differential expression between colorectal tumors and normal tissue samples. Analysis of two independent colorectal cancer datasets revealed IGF2 to be overexpressed at a frequency of 13% to 22%. An screen of 34 colorectal cancer cell lines revealed IGF2 expression to significantly correlate with sensitivity to the IGF1R/INSR inhibitor BI 885578. Furthermore, autocrine IGF2 constitutively activated IGF1R and Akt phosphorylation, which was inhibited by BI 885578 treatment. BI 885578 significantly delayed the growth of IGF2-high colorectal cancer xenograft tumors in mice, while combination with a VEGF-A antibody increased efficacy and induced tumor regression. Besides colorectal cancer, IGF2 overexpression was detected in more than 10% of bladder carcinoma, hepatocellular carcinoma and non-small cell lung cancer patient samples. Meanwhile, IGF2-high non-colorectal cancer cells lines displayed constitutive IGF1R phosphorylation and were sensitive to BI 885578. Our findings suggest that IGF2 may represent an attractive patient selection biomarker for IGF pathway inhibitors and that combination with VEGF-targeting agents may further improve clinical outcomes. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0336DOI Listing
October 2017

Impaired Planar Germ Cell Division in the Testis, Caused by Dissociation of RHAMM from the Spindle, Results in Hypofertility and Seminoma.

Cancer Res 2016 11 19;76(21):6382-6395. Epub 2016 Aug 19.

Leibniz Institute on Aging - Fritz Lipmann Institute, Jena, Germany.

Hypofertility is a risk factor for the development of testicular germ cell tumors (TGCT), but the initiating event linking these pathologies is unknown. We hypothesized that excessive planar division of undifferentiated germ cells promotes their self-renewal and TGCT development. However, our results obtained from mouse models and seminoma patients demonstrated the opposite. Defective planar divisions of undifferentiated germ cells caused their premature exit from the seminiferous tubule niche, resulting in germ cell depletion, hypofertility, intratubular germ cell neoplasias, and seminoma development. Oriented divisions of germ cells, which determine their fate, were regulated by spindle-associated RHAMM-a function we found to be abolished in 96% of human seminomas. Mechanistically, RHAMM expression is regulated by the testis-specific polyadenylation protein CFIm25, which is downregulated in the human seminomas. These results suggested that spindle misorientation is oncogenic, not by promoting self-renewing germ cell divisions within the niche, but by prematurely displacing proliferating cells from their normal epithelial milieu. Furthermore, they suggested RHAMM loss-of-function and spindle misorientation as an initiating event underlying both hypofertility and TGCT initiation. These findings identify spindle-associated RHAMM as an intrinsic regulator of male germ cell fate and as a gatekeeper preventing initiation of TGCTs. Cancer Res; 76(21); 6382-95. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0179DOI Listing
November 2016

Cell Proliferation Method: Click Chemistry Based on BrdU Coupling for Multiplex Antibody Staining.

Curr Protoc Cytom 2015 Apr 1;72:7.34.1-7.34.17. Epub 2015 Apr 1.

Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.

Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry.
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http://dx.doi.org/10.1002/0471142956.cy0734s72DOI Listing
April 2015

RHAMM deficiency disrupts folliculogenesis resulting in female hypofertility.

Biol Open 2015 Mar 6;4(4):562-71. Epub 2015 Mar 6.

Leibniz Institute for Age Research - Fritz Lipmann Institute, Beutenbergstrasse 11, D-07745 Jena, Germany

The postnatal mammalian ovary contains the primary follicles, each comprising an immature oocyte surrounded by a layer of somatic granulosa cells. Oocytes reach meiotic and developmental competence via folliculogenesis. During this process, the granulosa cells proliferate massively around the oocyte, form an extensive extracellular matrix (ECM) and differentiate into cumulus cells. As the ECM component hyaluronic acid (HA) is thought to form the backbone of the oocyte-granulosa cell complex, we deleted the relevant domain of the Receptor for HA Mediated Motility (RHAMM) gene in the mouse. This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected. We report that wild-type RHAMM localises at the mitotic spindle of granulosa cells, surrounding the oocyte. Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility. These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility.
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http://dx.doi.org/10.1242/bio.201410892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4400598PMC
March 2015

Efficacy and mechanism of action of volasertib, a potent and selective inhibitor of Polo-like kinases, in preclinical models of acute myeloid leukemia.

J Pharmacol Exp Ther 2015 Mar 9;352(3):579-89. Epub 2015 Jan 9.

Boehringer Ingelheim RCV GmbH & Co KG, Vienna, Austria (D.R., M.A.I., U.T.-G., A.W., F.S., M.H.H., C.A., L.G., M.R., P.G.-C., J.M., G.R.A., N.K.); Institute of Molecular Biotechnology, Vienna, Austria (C.B., C.S., D.W.G.); and Research Institute of Molecular Pathology, Vienna, Austria (M.R., J.Z.).

Polo-like kinase 1 (Plk1), a member of the Polo-like kinase family of serine/threonine kinases, is a key regulator of multiple steps in mitosis. Here we report on the pharmacological profile of volasertib, a potent and selective Plk inhibitor, in multiple preclinical models of acute myeloid leukemia (AML) including established cell lines, bone marrow samples from AML patients in short-term culture, and subcutaneous as well as disseminated in vivo models in immune-deficient mice. Our results indicate that volasertib is highly efficacious as a single agent and in combination with established and emerging AML drugs, including the antimetabolite cytarabine, hypomethylating agents (decitabine, azacitidine), and quizartinib, a signal transduction inhibitor targeting FLT3. Collectively, these preclinical data support the use of volasertib as a new therapeutic approach for the treatment of AML patients, and provide a foundation for combination approaches that may further improve and prolong clinical responses.
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http://dx.doi.org/10.1124/jpet.114.221150DOI Listing
March 2015

Drug resistance.

Drug Discov Today Technol 2014 Mar;11:1-3

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http://dx.doi.org/10.1016/j.ddtec.2014.03.013DOI Listing
March 2014

Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models.

Cancers (Basel) 2013 Jul 24;5(3):901-18. Epub 2013 Jul 24.

Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway, Ireland.

Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.
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http://dx.doi.org/10.3390/cancers5030901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3795371PMC
July 2013

Targeting aurora kinases with danusertib (PHA-739358) inhibits growth of liver metastases from gastroenteropancreatic neuroendocrine tumors in an orthotopic xenograft model.

Clin Cancer Res 2012 Sep 2;18(17):4621-32. Epub 2012 Jul 2.

I. Medizinische Klinik, Diagnostische und Interventionelle Radiologie, II. Medizinische Klinik, Onkologisches Zentrum, Universitätsklinikum Hamburg-Eppendorf; Labor Lademannbogen, Hamburg, Germany.

Purpose: Aurora kinases play a crucial role in cell-cycle control. Uncontrolled expression of aurora kinases causes aneuploidy and tumor growth. As conservative treatment options for advanced gastroenteropancreatic neuroendocrine tumors (GEP-NET) are disappointing, aurora kinases may be an interesting target for novel therapeutic strategies.

Experimental Design: Human GEP-NETs were tested for aurora kinase expression. The efficacy of the new aurora kinase inhibitor danusertib was evaluated in two human GEP-NET cell lines (BON1 and QGP) in vitro and in vivo.

Results: The majority of ten insulinomas and all 33 nonfunctional pancreatic or midgut GEP-NETs expressed aurora A despite a mostly high degree of cell differentiation. Both human GEP-NET cell lines expressed aurora kinase A and B, and high Ser10 phosphorylation of histone H3 revealed increased aurora B activity. Remarkably, danusertib led to cell-cycle arrest and completely inhibited cell proliferation of the GEP-NET cells in vitro. Decreased phosphorylation of histone H3 indicated effective aurora B inhibition. In a subcutaneous murine xenograft model, danusertib significantly reduced tumor growth in vivo compared with controls or mice treated with streptozotocine/5-fluorouracil. As a consequence, decreased levels of tumor marker chromogranin A were found in mouse serum samples. In a newly developed orthotopic model for GEP-NET liver metastases by intrasplenic tumor cell transplantation, dynamic MRI proved significant growth inhibition of BON1- and QGP-derived liver metastases.

Conclusions: These results show that danusertib may impose a new therapeutic strategy for aurora kinase expressing metastasized GEP-NETs.
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http://dx.doi.org/10.1158/1078-0432.CCR-11-2968DOI Listing
September 2012

Treatment of human pre-B acute lymphoblastic leukemia with the Aurora kinase inhibitor PHA-739358 (Danusertib).

Mol Cancer 2012 Jun 21;11:42. Epub 2012 Jun 21.

Section of Molecular Carcinogenesis, Division of Hematology/Oncology and The Saban Research Institute of Children's Hospital Los Angeles, Los Angeles, CA 90027, USA.

Background: Treatment of Philadelphia chromosome-positive acute lymphoblastic leukemias (Ph-positive ALL) with clinically approved inhibitors of the Bcr/Abl tyrosine kinase frequently results in the emergence of a leukemic clone carrying the T315I mutation in Bcr/Abl, which confers resistance to these drugs. PHA-739358, an Aurora kinase inhibitor, was reported to inhibit the Bcr/Abl T315I mutant in CML cells but no preclinical studies have examined this in detail in human ALL.

Results: We compared the sensitivity of human Bcr/Abl T315I, Bcr/Abl wild type and non-Bcr/Abl ALL cells to this drug. PHA-739358 inhibited proliferation and induced apoptosis independently of Bcr/Abl, the T315I mutation, or presence of the tumor suppressor p53, but the degree of effectiveness varied between different ALL samples. Since short-term treatment with a single dose of drug only transiently inhibited proliferation, we tested combination treatments of PHA-739358 with the farnesyltransferase inhibitor Lonafarnib, with vincristine and with dasatinib. All combinations reduced viability and cell numbers compared to treatment with a single drug. Clonogenic assays showed that 25 nM PHA-739358 significantly reduced the colony growth potential of Ph-positive ALL cells, and combined treatment with a second drug abrogated colony growth in this assay. PHA-739358 further effectively blocked Bcr/Abl tyrosine kinase activity and Aurora kinase B in vivo, and mice transplanted with human Bcr/Abl T315I ALL cells treated with a 3x 7-day cycle of PHA-739358 as mono-treatment had significantly longer survival.

Conclusions: PHA-739358 represents an alternative drug for the treatment of both Ph-positive and negative ALL, although combined treatment with a second drug may be needed to eradicate the leukemic cells.
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http://dx.doi.org/10.1186/1476-4598-11-42DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3489684PMC
June 2012

NMS-P937, an orally available, specific small-molecule polo-like kinase 1 inhibitor with antitumor activity in solid and hematologic malignancies.

Mol Cancer Ther 2012 Apr 7;11(4):1006-16. Epub 2012 Feb 7.

Nerviano Medical Sciences Srl, Nerviano, Milan, Italy.

Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase considered to be the master player of cell-cycle regulation during mitosis. It is indeed involved in centrosome maturation, bipolar spindle formation, chromosome separation, and cytokinesis. PLK1 is overexpressed in a variety of human tumors and its overexpression often correlates with poor prognosis. Although five different PLKs are described in humans, depletion or inhibition of kinase activity of PLK1 is sufficient to induce cell-cycle arrest and apoptosis in cancer cell lines and in xenograft tumor models. NMS-P937 is a novel, orally available PLK1-specific inhibitor. The compound shows high potency in proliferation assays having low nanomolar activity on a large number of cell lines, both from solid and hematologic tumors. NMS-P937 potently causes a mitotic cell-cycle arrest followed by apoptosis in cancer cell lines and inhibits xenograft tumor growth with clear PLK1-related mechanism of action at well-tolerated doses in mice after oral administration. In addition, NMS-P937 shows potential for combination in clinical settings with approved cytotoxic drugs, causing tumor regression in HT29 human colon adenocarcinoma xenografts upon combination with irinotecan and prolonged survival of animals in a disseminated model of acute myelogenous leukemia in combination with cytarabine. NMS-P937, with its favorable pharmacologic parameters, good oral bioavailability in rodent and nonrodent species, and proven antitumor activity in different preclinical models using a variety of dosing regimens, potentially provides a high degree of flexibility in dosing schedules and warrants investigation in clinical settings.
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http://dx.doi.org/10.1158/1535-7163.MCT-11-0765DOI Listing
April 2012

Transcriptional analysis of the Aurora inhibitor Danusertib leading to biomarker identification in TP53 wild type cells.

Gene 2012 Feb 2;494(2):202-8. Epub 2011 Sep 2.

Business Unit Oncology, Nerviano Medical Sciences srl, Nerviano (MI), Italy.

Aurora kinases represent an appealing target for anticancer therapies and several Aurora inhibitors are in clinical development, including the potent pan-Aurora inhibitor Danusertib. Treatment with Aurora inhibitors has been shown to induce diverse biological responses in different tumor cells, in part depending on TP53 status. To characterize the effects of Danusertib at the transcriptional level we carried out gene expression profiling of wt and TP53 mutant tumor cells showing differential cell cycle response upon drug treatment. We found that treatment with Danusertib induces a strong transcriptional response only in TP53 wt cells, with an overlapping pattern of expression of TP53-dependent genes among the three cell lines tested, while a prevalent signature could not be identified in the two TP53 mutant cells, suggesting that TP53 status is a key determinant for the observed transcriptional effects. This work led to the identification of a number of genes consistently modulated by Aurora treatment in TP53 cells. One of these is GDF15, a secreted protein belonging to the TGF-β superfamily, for which we found a potential role in resistance to Danusertib, and which could represent a potential biomarker for Danusertib treatment in TP53 WT tumors and in surrogate tissues such as blood or skin.
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http://dx.doi.org/10.1016/j.gene.2011.08.014DOI Listing
February 2012

Discovery of a novel tumour metastasis-promoting gene, NVM-1.

J Pathol 2011 Sep 8;225(1):96-105. Epub 2011 Jul 8.

University of Heidelberg, Medical Faculty Mannheim, Germany; KIT Campus Nord, Institut für Toxikologie und Genetik, Karlsruhe, Germany.

We have previously reported that over-expression of a panel of 119 genes correlates with the metastatic potential of pancreatic carcinoma cells. We sought to identify and functionally characterize candidate tumour metastasis promoting genes among this library using a secondary phenotype-assisted screen. Here we report the discovery of the metastasis-promoting function of a hitherto not characterized gene located on chromosome 14 (ORF138), which we have named 'novel metastasis-promoting gene 1' (NVM-1). The NVM-1 transcript is extensively alternatively spliced, is expressed endogenously in a number of different tissues, and is strongly over-expressed at the protein level in a variety of human tumour types. Importantly, NVM-1 expression stimulates the migratory and invasive behaviour of tumour cells and promotes metastasis formation in experimental animals in vivo. Up-regulation of FMNL2 and MT1E and down-regulation of TIMP4 and MHC-I is observed as a consequence of NVM-1 expression. Together these data identify NVM-1 as a gene that is functionally involved in tumour metastasis, and suggest that NVM-1 may constitute a promising therapeutic target for inhibition of tumour metastasis.
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http://dx.doi.org/10.1002/path.2924DOI Listing
September 2011

Abcg2 overexpression represents a novel mechanism for acquired resistance to the multi-kinase inhibitor Danusertib in BCR-ABL-positive cells in vitro.

PLoS One 2011 Apr 26;6(4):e19164. Epub 2011 Apr 26.

Klinik für Onkologie, Hämatologie und Knochenmarktransplantation mit Sektion Pneumologie, Universitäres Cancer Center Hamburg, Universitäts-Klinikum Hamburg-Eppendorf, Hamburg, Germany.

The success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance in vitro. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance in vitro, raising hope that this drug combination may also achieve more durable disease control in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019164PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082549PMC
April 2011

Targeting aneuploid cancer cells.

Expert Opin Ther Targets 2011 May 12;15(5):595-608. Epub 2011 Feb 12.

Nerviano Medical Sciences, Nerviano (Mi), Italy.

Introduction: Most cancers are characterized by some degree of aneuploidy, although its relevance for tumor initiation or progression and the nature of the initial trigger are still not well understood. It was Theodor Boveri who first suggested a link between aneuploidy and cancer at the beginning of the last century, but it is only recently that the molecular mechanisms involved have started to be uncovered.

Areas Covered: The molecular mechanisms that are at the origin of aneuploidy and their cellular consequences. Based on these new findings molecular targets have emerged which could lead to a specific treatment of at least some types of aneuploid tumors.

Expert Opinion: Therapeutic intervention specifically for aneuploid cells is a very promising approach, however, although new promising targets have been spotted they still need to be tested for proof of concept. Targeting the spindle checkpoint could be an interesting approach for cancer therapy, however, as for other mitotic targets, the open question of the therapeutic window and sensitivity of normal hemopoietic cells has to be considered carefully. Future challenges will not only include identifying and validating druggable targets related to the relevant pathways, but also finding predictive biomarkers to define the responding patient population(s).
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http://dx.doi.org/10.1517/14728222.2011.558007DOI Listing
May 2011

Miniaturizing bromodeoxyuridine incorporation enables the usage of flow cytometry for cell cycle analysis of adherent tissue culture cells for high throughput screening.

Cytometry A 2010 Oct 8;77(10):953-61. Epub 2010 Sep 8.

Cell Biology Department, Nerviano Medical Sciences Srl, Nerviano, Italy.

Analysis of cell cycle progression by 5-bromo-2'-deoxyuridine (BrdU) incorporation is commonly used for evaluating the mode of action of anticancer drugs, but usually requires a high number of cells and large amounts of monoclonal antibodies. In addition, manual sample handling is not suitable for high throughput. To circumvent these limitations, we have developed a miniaturized method to measure BrdU incorporation into DNA directly in 96-wells plates. Adherent cells were grown in 96-well plates in the absence or presence of compounds of interest. After BrdU pulse labeling or pulse chase, cells were harvested, transferred to polymerase chain reaction (PCR) V-bottom plates, and fixed by adding methanol. DNA denaturation was performed directly in the plates by heat using a PCR thermocycler. BrdU incorporation was detected by indirect immunocytochemical staining, and cellular DNA was counterstained using propidium iodide. Samples were acquired by a BD FACSCalibur with BD Multiwells Auto sampler or BD HTS. We defined a dynamic range of the optimal cell number, for which cells maintained exponential growth up to 72 h. The assay was robust up to 30,000 cells per well. BrdU dot plots of cell cycle phases showed an excellent separation of cell populations, and DNA histograms showed a low coefficient of variation. Thermal denaturation was suitable for 96-well plates to detect BrdU incorporation with a good signal-to-noise ratio, and cluster analysis allowed fingerprint readouts for drug sensitivity and mechanism of action as exemplified for paclitaxel and doxorubicin. This method provided rapid high-throughput BrdU/DNA content analysis with high accuracy and reproducibility, accompanied by a reduction in reagent consumption. A critical step was identified as the standardization of DNA denaturation using a PCR thermocycler. Here,we show some applications of this method for cell cycle studies in drug discovery.
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http://dx.doi.org/10.1002/cyto.a.20962DOI Listing
October 2010

Targeting the mitotic checkpoint for cancer therapy with NMS-P715, an inhibitor of MPS1 kinase.

Cancer Res 2010 Dec;70(24):10255-64

Department of Cell Biology-Oncology, Nerviano Medical Sciences, Viale Pasteur 10, Nerviano 20014, Italy.

MPS1 kinase is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. It has been found aberrantly overexpressed in a wide range of human tumors and is necessary for tumoral cell proliferation. Here we report the identification and characterization of NMS-P715, a selective and orally bioavailable MPS1 small-molecule inhibitor, which selectively reduces cancer cell proliferation, leaving normal cells almost unaffected. NMS-P715 accelerates mitosis and affects kinetochore components localization causing massive aneuploidy and cell death in a variety of tumoral cell lines and inhibits tumor growth in preclinical cancer models. Inhibiting the SAC could represent a promising new approach to selectively target cancer cells.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-2101DOI Listing
December 2010

4,5-Dihydro-1H-pyrazolo[4,3-h]quinazolines as potent and selective Polo-like kinase 1 (PLK1) inhibitors.

Bioorg Med Chem Lett 2010 Nov 17;20(22):6489-94. Epub 2010 Sep 17.

Nerviano Medical Sciences srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano, Milan, Italy.

A series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives was optimized as Polo-like kinase 1 inhibitors. Extensive SAR afforded a highly potent and selective PLK1 compound. The compound showed good antiproliferative activity when tested in a panel of tumor cell lines with PLK1 related mechanism of action and with good in vivo antitumor efficacy in two xenograft models after i.v. administration.
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http://dx.doi.org/10.1016/j.bmcl.2010.09.060DOI Listing
November 2010

Cdc7 kinase inhibitors: 5-heteroaryl-3-carboxamido-2-aryl pyrroles as potential antitumor agents. 1. Lead finding.

J Med Chem 2010 Oct;53(20):7296-315

Nerviano Medical Sciences Srl, Business Unit Oncology, Viale Pasteur 10, 20014 Nerviano, MI, Italy.

Cdc7 serine/threonine kinase is a key regulator of DNA synthesis in eukaryotic organisms. Cdc7 inhibition through siRNA or prototype small molecules causes p53 independent apoptosis in tumor cells while reversibly arresting cell cycle progression in primary fibroblasts. This implies that Cdc7 kinase could be considered a potential target for anticancer therapy. We previously reported that pyrrolopyridinones (e.g., 1) are potent and selective inhibitors of Cdc7 kinase, with good cellular potency and in vitro ADME properties but with suboptimal pharmacokinetic profiles. Here we report on a new chemical class of 5-heteroaryl-3-carboxamido-2-substituted pyrroles (1A) that offers advantages of chemistry diversification and synthetic simplification. This work led to the identification of compound 18, with biochemical data and ADME profile similar to those of compound 1 but characterized by superior efficacy in an in vivo model. Derivative 18 represents a new lead compound worthy of further investigation toward the ultimate goal of identifying a clinical candidate.
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http://dx.doi.org/10.1021/jm100504dDOI Listing
October 2010

Destabilizing aneuploidy by targeting cell cycle and mitotic checkpoint proteins in cancer cells.

Curr Drug Targets 2010 Oct;11(10):1325-35

Nerviano Medical Sciences S.r.l., Viale Pasteur 10, I-20014 Nerviano (Mi), Italy.

Aneuploidy is one of the major hallmarks of cancer cells and several paths towards aneuploidy have been described. However, the relevance for tumor initiation or progression and how tumors deal with the initial aneuploidy related stress response is still unclear and recent results suggest that aneuploidy can even have tumor suppressive effects under certain conditions. The molecular mechanisms leading to and sustaining growth of aneuploid cells are just at the beginning to be understood and might provide new targets for cancer drug development. We will discuss some of the ideas to specifically kill aneuploid cells by targeting key regulators of mitosis.
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http://dx.doi.org/10.2174/1389450111007011325DOI Listing
October 2010

Dual targeting of CDK and tropomyosin receptor kinase families by the oral inhibitor PHA-848125, an agent with broad-spectrum antitumor efficacy.

Mol Cancer Ther 2010 Aug 3;9(8):2243-54. Epub 2010 Aug 3.

Cell Biology Department, BU Oncology, Nerviano Medical Sciences, v.le Pasteur 10, Nerviano, Milan 20014, Italy.

Altered expression and activity of cyclin-dependent kinase (CDK) and tropomyosin receptor kinase (TRK) families are observed in a wide variety of tumors. In those malignancies with aberrant CDK activation, the retinoblastoma protein (pRb) pathway is deregulated, leading to uncontrolled cell proliferation. Constitutive activation of TRKs is instead linked to cancer cell survival and dissemination. Here, we show that the novel small-molecule PHA-848125, a potent dual inhibitor of CDKs and TRKs, possesses significant antitumor activity. The compound inhibits cell proliferation of a wide panel of tumoral cell lines with submicromolar IC(50). PHA-848125-treated cells show cell cycle arrest in G(1) and reduced DNA synthesis, accompanied by inhibition of pRb phosphorylation and modulation of other CDK-dependent markers. The compound additionally inhibits phosphorylation of TRKA and its substrates in cells, which functionally express this receptor. Following oral administration, PHA-848125 has significant antitumor activity in various human xenografts and carcinogen-induced tumors as well as in disseminated primary leukemia models, with plasma concentrations in rodents in the same range as those found active in inhibiting cancer cell proliferation. Mechanism of action was also confirmed in vivo as assessed in tumor biopsies from treated mice. These results show that the dual CDK-TRK inhibitor PHA-848125 has the potential for being a novel and efficacious targeted drug for cancer treatment.
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http://dx.doi.org/10.1158/1535-7163.MCT-10-0190DOI Listing
August 2010

Targeting cell division cycle 7 kinase: a new approach for cancer therapy.

Clin Cancer Res 2010 Sep 20;16(18):4503-8. Epub 2010 Jul 20.

Nerviano Medical Sciences Oncology, Nerviano, Milan, Italy.

The cell division cycle 7 (Cdc7) is a serine-threonine kinase, originally discovered in budding yeast, required to initiate DNA replication. Human Cdc7 phosphorylates the minichromosome maintenance protein 2 (Mcm2), a component of the DNA replicative helicase needed for genome duplication. Inhibition of Cdc7 in cancer cells impairs progression through S phase, inducing a p53-independent apoptotic cell death, whereas in normal cells, it does not affect cell viability. Small molecule compounds able to interfere with Cdc7 activity have been identified and shown to be effective in controlling tumor growth in animal models. Two Cdc7 inhibitors are currently in phase I clinical development. Inhibition of Cdc7 kinase activity in cancer cells restricts DNA replication and induces apoptotic cell death by an unprecedented molecular mechanism of action.
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http://dx.doi.org/10.1158/1078-0432.CCR-10-0185DOI Listing
September 2010

Identification of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as a new class of orally and selective Polo-like kinase 1 inhibitors.

J Med Chem 2010 May;53(9):3532-51

Nerviano Medical Sciences Srl, Oncology, Viale Pasteur 10, 20014 Nerviano, (Mi), Italy.

Polo-like kinase 1 (Plk1) is a fundamental regulator of mitotic progression whose overexpression is often associated with oncogenesis and therefore is recognized as an attractive therapeutic target in the treatment of proliferative diseases. Here we discuss the structure-activity relationship of the 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline class of compounds that emerged from a high throughput screening (HTS) campaign as potent inhibitors of Plk1 kinase. Furthermore, we describe the discovery of 49, 8-{[2-methoxy-5-(4-methylpiperazin-1-yl)phenyl]amino}-1-methyl-4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline-3-carboxamide, as a highly potent and specific ATP mimetic inhibitor of Plk1 (IC(50) = 0.007 microM) as well as its crystal structure in complex with the methylated Plk1(36-345) construct. Compound 49 was active in cell proliferation against different tumor cell lines with IC(50) values in the submicromolar range and active in vivo in the HCT116 xenograft model where it showed 82% tumor growth inhibition after repeated oral administration.
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http://dx.doi.org/10.1021/jm901713nDOI Listing
May 2010

Identification of Myb-binding protein 1A (MYBBP1A) as a novel substrate for aurora B kinase.

J Biol Chem 2010 Apr 22;285(16):11775-85. Epub 2010 Feb 22.

Nerviano Medical Sciences, Nerviano 20014 MI, Italy.

Aurora kinases are mitotic enzymes involved in centrosome maturation and separation, spindle assembly and stability, and chromosome condensation, segregation, and cytokinesis and represent well known targets for cancer therapy because their deregulation has been linked to tumorigenesis. The availability of suitable markers is of crucial importance to investigate the functions of Auroras and monitor kinase inhibition in in vivo models and in clinical trials. Extending the knowledge on Aurora substrates could help to better understand their biology and could be a source for clinical biomarkers. Using biochemical, mass spectrometric, and cellular approaches, we identified MYBBP1A as a novel Aurora B substrate and serine 1303 as the major phosphorylation site. MYBBP1A is phosphorylated in nocodazole-arrested cells and is dephosphorylated upon Aurora B silencing or by treatment with Danusertib, a small molecule inhibitor of Aurora kinases. Furthermore, we show that MYBBP1A depletion by RNA interference causes mitotic progression delay and spindle assembly defects. MYBBP1A has until now been described as a nucleolar protein, mainly involved in transcriptional regulation. The results presented herein show MYBBP1A as a novel Aurora B kinase substrate and reveal a not yet recognized link of this nucleolar protein to mitosis.
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http://dx.doi.org/10.1074/jbc.M109.068312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852913PMC
April 2010

A phase I dose-escalation study of danusertib (PHA-739358) administered as a 24-hour infusion with and without granulocyte colony-stimulating factor in a 14-day cycle in patients with advanced solid tumors.

Clin Cancer Res 2009 Nov 13;15(21):6694-701. Epub 2009 Oct 13.

Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.

Purpose: This study was conducted to assess the safety, tolerability, pharmacokinetics, and pharmacodynamics of the i.v. pan-aurora kinase inhibitor PHA-739358, danusertib, in patients with advanced solid tumors.

Experimental Design: In part 1, patients received escalating doses of danusertib (24-hour infusion every 14 days) without filgrastim (granulocyte colony-stimulating factor, G-CSF). Febrile neutropenia was the dose-limiting toxicity without G-CSF. Further dose escalation was done in part 2 with G-CSF. Blood samples were collected for danusertib pharmacokinetics and pharmacodynamics. Skin biopsies were collected to assess histone H3 phosphorylation (pH3).

Results: Fifty-six patients were treated, 40 in part 1 and 16 in part 2. Febrile neutropenia was the dose-limiting toxicity in part 1 without G-CSF. Most other adverse events were grade 1 to 2, occurring at doses >or=360 mg/m(2) with similar incidence in parts 1 and 2. The maximum tolerated dose without G-CSF is 500 mg/m(2). The recommended phase 2 dose in part 2 with G-CSF is 750 mg/m(2). Danusertib showed dose-proportional pharmacokinetics in parts 1 and 2 with a median half-life of 18 to 26 hours. pH3 modulation in skin biopsies was observed at >or=500 mg/m(2). One patient with refractory small cell lung cancer (1,000 mg/m(2) with G-CSF) had an objective response lasting 23 weeks. One patient with refractory ovarian cancer had 27% tumor regression and 30% CA125 decline.

Conclusions: Danusertib was well tolerated with target inhibition in skin at >or=500 mg/m(2). Preliminary evidence of antitumor activity, including a partial response and several occurrences of prolonged stable disease, was seen across a variety of advanced refractory cancers. Phase II studies are ongoing.
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http://dx.doi.org/10.1158/1078-0432.CCR-09-1445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2826106PMC
November 2009

Aurora kinase inhibitor PHA-739358 suppresses growth of hepatocellular carcinoma in vitro and in a xenograft mouse model.

Neoplasia 2009 Sep;11(9):934-44

I. Medizinische Klinik, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany.

Patients with advanced stages of hepatocellular carcinoma (HCC) face a poor prognosis. Although encouraging clinical results have been obtained with multikinase inhibitor sorafenib, the development of improved therapeutic strategies for HCC remains an urgent goal. Aurora kinases are key regulators of the cell cycle, and their uncontrolled expression promotes aneuploidy and tumor development. In tissue microarray analyses, we detected aurora-A kinase expression in all of the examined 93 human HCC samples, whereas aurora-B kinase expression levels significantly correlated with the proliferation index of HCCs. In addition, two human HCC cell lines (Huh-7 and HepG2) were tested positive for aurora-A and -B and revealed Ser10 phosphorylation of histone H3, indicating an increased aurora-B kinase activity. The antiproliferative features of a novel aurora kinase inhibitor, PHA-739358, currently under investigation in phase 2 clinical trials for other solid tumors, were examined in vitro and in vivo. At concentrations exceeding 50 nM, PHA-739358 completely suppressed tumor cell proliferation in cell culture experiments and strongly decreased histone H3 phosphorylation. Cell cycle inhibition and endoreduplication were observed at 50 nM, whereas higher concentrations led to a complete G(2)/M-phase arrest. In vivo, administration of PHA-739358 resulted in significant tumor growth inhibition at a well-tolerated dose. In combination with sorafenib, additive effects were observed. Remarkably, when tumors restarted to grow under sorafenib monotherapy, subsequent treatment with PHA-739358 induced tumor shrinkage by up to 81%. Thus, targeting aurora kinases with PHA-739358 is a promising therapeutic strategy administered alone or in combination with sorafenib for patients with advanced stages of HCC.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2735802PMC
http://dx.doi.org/10.1593/neo.09664DOI Listing
September 2009

Is there a future for Aurora kinase inhibitors for anticancer therapy?

Curr Opin Drug Discov Devel 2009 Jul;12(4):533-42

Nerviano Medical Sciences Srl, Nerviano, Milano, Italy.

The development of Aurora kinase inhibitors is a competitive research field, with many inhibitors currently being evaluated in preclinical and clinical studies. Progress during the past few years, both preclinically and clinically, has increased the evidence supporting Aurora kinases as promising molecular targets for the treatment of cancer. Aurora kinase inhibitors differ based on their selectivity within the Aurora kinase family and their cross-reactivities with other kinases. Additional factors that will contribute to the success or failure of the Aurora kinase inhibitors include: routes of administration, drug-like properties, workable combinations with approved drugs, adequate clinical development paths, and the identification of the appropriate patient population. The clinical trial results that are emerging for the most advanced inhibitors are promising, and it is probable that clinical proof of concept will be achieved, and that Aurora kinase inhibitors will be part of treatment for cancer in the future.
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July 2009