Publications by authors named "Ensar Halilovic"

27 Publications

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Inhibition of MDM2 promotes anti-tumor responses in p53 wild-type cancer cells through their interaction with the immune and stromal microenvironment.

Cancer Res 2021 Jan 27. Epub 2021 Jan 27.

Oncology, Novartis Institutes for BioMedical Research, Inc.

p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell cycle arrest or induction of apoptosis. However, the effects of p53 in anti-tumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell crosstalk, we studied murine syngeneic models treated with HDM201, a potent and selective second generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells (DC) increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, anti-tumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering anti-tumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0189DOI Listing
January 2021

Reduced Mitochondrial Apoptotic Priming Drives Resistance to BH3 Mimetics in Acute Myeloid Leukemia.

Cancer Cell 2020 Dec 19;38(6):872-890.e6. Epub 2020 Nov 19.

Department of Medical Oncology, Dana-Farber Cancer Institute, 440 Brookline Avenue, M430, Boston, MA 02215, USA; Harvard Medical School, Boston, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA. Electronic address:

Acquired resistance to BH3 mimetic antagonists of BCL-2 and MCL-1 is an important clinical problem. Using acute myelogenous leukemia (AML) patient-derived xenograft (PDX) models of acquired resistance to BCL-2 (venetoclax) and MCL-1 (S63845) antagonists, we identify common principles of resistance and persistent vulnerabilities to overcome resistance. BH3 mimetic resistance is characterized by decreased mitochondrial apoptotic priming as measured by BH3 profiling, both in PDX models and human clinical samples, due to alterations in BCL-2 family proteins that vary among cases, but not to acquired mutations in leukemia genes. BCL-2 inhibition drives sequestered pro-apoptotic proteins to MCL-1 and vice versa, explaining why in vivo combinations of BCL-2 and MCL-1 antagonists are more effective when concurrent rather than sequential. Finally, drug-induced mitochondrial priming measured by dynamic BH3 profiling (DBP) identifies drugs that are persistently active in BH3 mimetic-resistant myeloblasts, including FLT-3 inhibitors and SMAC mimetics.
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http://dx.doi.org/10.1016/j.ccell.2020.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988687PMC
December 2020

MDM2 inhibition in combination with endocrine therapy and CDK4/6 inhibition for the treatment of ER-positive breast cancer.

Breast Cancer Res 2020 08 12;22(1):87. Epub 2020 Aug 12.

Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, 2010, Australia.

Background: Resistance to endocrine therapy is a major clinical challenge in the management of oestrogen receptor (ER)-positive breast cancer. In this setting, p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment-resistant ER-positive breast cancer.

Methods: We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate anti-tumour effects in p53 wildtype and p53 mutant ER-positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and endocrine-resistant ER-positive breast cancer.

Results: We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant-resistant patient-derived xenograft model.

Conclusions: We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine-resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programmes.
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http://dx.doi.org/10.1186/s13058-020-01318-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425060PMC
August 2020

Preclinical evaluation of drug combinations identifies co-inhibition of Bcl-2/XL/W and MDM2 as a potential therapy in uveal melanoma.

Eur J Cancer 2020 02 9;126:93-103. Epub 2020 Jan 9.

Uveal Melanoma Translational Group, Department of Translational Research, Institut Curie, PSL Research University, Paris, France. Electronic address:

Introduction: Uveal melanoma (UM) is a rare and malignant intraocular tumour with a dismal prognosis. Despite a good control of the primary tumour by radiation or surgery, up to 50% of patients subsequently develop metastasis for which no efficient treatment is yet available.

Methodology: To identify therapeutic opportunities, we performed an in vitro screen of 30 combinations of different inhibitors of pathways that are dysregulated in UM. Effects of drug combinations on viability, cell cycle and apoptosis were assessed in eight UM cell lines. The best synergistic combinations were further evaluated in six UM patient-derived xenografts (PDXs).

Results: We demonstrated that the Bcl-2/X/W inhibitor (ABT263) sensitised the UM cell lines to other inhibitors, mainly to mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinase (MEK) and murine double minute 2 (MDM2) inhibitors. mTOR (RAD001) and MEK1/2 (trametinib) inhibitors were efficient as single agents, but their combinations with ABT263 displayed no synergism in UM PDXs. In contrast, the combination of ABT263 with MDM2 inhibitor (HDM201) showed a trend for a synergistic effect.

Conclusion: We showed that inhibition of Bcl-2/X/W sensitised the UM cell lines to other treatments encouraging investigation of the underlying mechanisms. Furthermore, our findings highlighted Bcl-2/X/W and MDM2 co-inhibition as a promising strategy in UM.
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http://dx.doi.org/10.1016/j.ejca.2019.12.012DOI Listing
February 2020

MDM2 antagonists overcome intrinsic resistance to CDK4/6 inhibition by inducing p21.

Sci Transl Med 2019 08;11(505)

Department of Veterans Affairs, Tennessee Valley Healthcare System, Nashville, TN 37212, USA.

Intrinsic resistance of unknown mechanism impedes the clinical utility of inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) in malignancies other than breast cancer. Here, we used melanoma patient-derived xenografts (PDXs) to study the mechanisms for CDK4/6i resistance in preclinical settings. We observed that melanoma PDXs resistant to CDK4/6i frequently displayed activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, and inhibition of this pathway improved CDK4/6i response in a p21-dependent manner. We showed that a target of p21, CDK2, was necessary for proliferation in CDK4/6i-treated cells. Upon treatment with CDK4/6i, melanoma cells up-regulated cyclin D1, which sequestered p21 and another CDK inhibitor, p27, leaving a shortage of p21 and p27 available to bind and inhibit CDK2. Therefore, we tested whether induction of p21 in resistant melanoma cells would render them responsive to CDK4/6i. Because p21 is transcriptionally driven by p53, we coadministered CDK4/6i with a murine double minute (MDM2) antagonist to stabilize p53, allowing p21 accumulation. This resulted in improved antitumor activity in PDXs and in murine melanoma. Furthermore, coadministration of CDK4/6 and MDM2 antagonists with standard of care therapy caused tumor regression. Notably, the molecular features associated with response to CDK4/6 and MDM2 inhibitors in PDXs were recapitulated by an ex vivo organotypic slice culture assay, which could potentially be adopted in the clinic for patient stratification. Our findings provide a rationale for cotargeting CDK4/6 and MDM2 in melanoma.
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http://dx.doi.org/10.1126/scitranslmed.aav7171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584132PMC
August 2019

Combining BH3-mimetics to target both BCL-2 and MCL1 has potent activity in pre-clinical models of acute myeloid leukemia.

Leukemia 2019 04 10;33(4):905-917. Epub 2018 Sep 10.

Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.

Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy.
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http://dx.doi.org/10.1038/s41375-018-0261-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484700PMC
April 2019

Dose and Schedule Determine Distinct Molecular Mechanisms Underlying the Efficacy of the p53-MDM2 Inhibitor HDM201.

Cancer Res 2018 11 22;78(21):6257-6267. Epub 2018 Aug 22.

Disease Area Oncology, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts.

Activation of p53 by inhibitors of the p53-MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53-MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 , and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2-p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure-response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53-MDM2 inhibitors. Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. .
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http://dx.doi.org/10.1158/0008-5472.CAN-18-0338DOI Listing
November 2018

A Comprehensive Patient-Derived Xenograft Collection Representing the Heterogeneity of Melanoma.

Cell Rep 2017 Nov;21(7):1953-1967

Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA.

Therapy of advanced melanoma is changing dramatically. Following mutational and biological subclassification of this heterogeneous cancer, several targeted and immune therapies were approved and increased survival significantly. To facilitate further advancements through pre-clinical in vivo modeling, we have established 459 patient-derived xenografts (PDX) and live tissue samples from 384 patients representing the full spectrum of clinical, therapeutic, mutational, and biological heterogeneity of melanoma. PDX have been characterized using targeted sequencing and protein arrays and are clinically annotated. This exhaustive live tissue resource includes PDX from 57 samples resistant to targeted therapy, 61 samples from responders and non-responders to immune checkpoint blockade, and 31 samples from brain metastasis. Uveal, mucosal, and acral subtypes are represented as well. We show examples of pre-clinical trials that highlight how the PDX collection can be used to develop and optimize precision therapies, biomarkers of response, and the targeting of rare genetic subgroups.
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http://dx.doi.org/10.1016/j.celrep.2017.10.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5726788PMC
November 2017

Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human mutant neuroblastoma cell lines and xenograft models.

Elife 2017 04 20;6. Epub 2017 Apr 20.

Disease Area Oncology, Novartis Institutes for BioMedical Research, Cambridge, United States.

The efficacy of ALK inhibitors in patients with -mutant neuroblastoma is limited, highlighting the need to improve their effectiveness in these patients. To this end, we sought to develop a combination strategy to enhance the antitumor activity of ALK inhibitor monotherapy in human neuroblastoma cell lines and xenograft models expressing activated ALK. Herein, we report that combined inhibition of ALK and MDM2 induced a complementary set of anti-proliferative and pro-apoptotic proteins. Consequently, this combination treatment synergistically inhibited proliferation of wild-type neuroblastoma cells harboring amplification or mutations in vitro, and resulted in complete and durable responses in neuroblastoma xenografts derived from these cells. We further demonstrate that concurrent inhibition of MDM2 and ALK was able to overcome ceritinib resistance conferred by MYCN upregulation in vitro and in vivo. Together, combined inhibition of ALK and MDM2 may provide an effective treatment for wild-type neuroblastoma with aberrations.
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http://dx.doi.org/10.7554/eLife.17137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435462PMC
April 2017

The HDM2 (MDM2) Inhibitor NVP-CGM097 Inhibits Tumor Cell Proliferation and Shows Additive Effects with 5-Fluorouracil on the p53-p21-Rb-E2F1 Cascade in the p53wild type Neuroendocrine Tumor Cell Line GOT1.

Neuroendocrinology 2018 21;106(1):1-19. Epub 2016 Nov 21.

Department of Internal Medicine II, Campus Grosshadern, University Hospital, Ludwig Maximilian University of Munich, Munich, Germany.

Background/aims: The tumor suppressor p53 is depleted in many tumor cells by the E3 ubiquitin ligase mouse double minute 2 homolog (MDM2) through MDM2/p53 interaction. A novel target for inhibiting p53 degradation and for causing reexpression of p53wild type is inhibition of MDM2. The small molecule NVP-CGM097 is a novel MDM2 inhibitor. We investigated MDM2 inhibition as a target in neuroendocrine tumor cells in vitro.

Methods: Human neuroendocrine tumor cell lines from the pancreas (BON1), lung (NCI-H727), and midgut (GOT1) were incubated with the MDM2 inhibitor NVP-CGM097 (Novartis) at concentrations from 4 to 2,500 nM.

Results: While p53wild type GOT1 cells were sensitive to NVP-CGM097, p53mutated BON1 and p53mutated NCI-H727 cells were resistant to NVP-CGM097. Incubation of GOT1 cells with NVP-CGM097 at 100, 500, and 2,500 nM for 96 h caused a significant decline in cell viability to 84.9 ± 9.2% (p < 0.05), 77.4 ± 6.6% (p < 0.01), and 47.7 ± 9.2% (p < 0.01). In a Western blot analysis of GOT1 cells, NVP-CGM097 caused a dose-dependent increase in the expression of p53 and p21 tumor suppressor proteins and a decrease in phospho-Rb and E2F1. Experiments of co-incubation of NVP-CGM097 with 5-fluorouracil, temozolomide, or everolimus each showed additive antiproliferative effects in GOT1 cells. NVP-CGM097 and 5-fluorouracil increased p53 and p21 expression in an additive manner.

Conclusions: MDM2 inhibition seems a promising novel therapeutic target in neuroendocrine tumors harboring p53wild type. Further investigations should examine the potential role of MDM2 inhibitors in neuroendocrine tumor treatment.
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http://dx.doi.org/10.1159/000453369DOI Listing
January 2019

High-Order Drug Combinations Are Required to Effectively Kill Colorectal Cancer Cells.

Cancer Res 2016 12 22;76(23):6950-6963. Epub 2016 Sep 22.

Translational Clinical Oncology, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

Like classical chemotherapy regimens used to treat cancer, targeted therapies will also rely upon polypharmacology, but tools are still lacking to predict which combinations of molecularly targeted drugs may be most efficacious. In this study, we used image-based proliferation and apoptosis assays in colorectal cancer cell lines to systematically investigate the efficacy of combinations of two to six drugs that target critical oncogenic pathways. Drug pairs targeting key signaling pathways resulted in synergies across a broad spectrum of genetic backgrounds but often yielded only cytostatic responses. Enhanced cytotoxicity was observed when additional processes including apoptosis and cell cycle were targeted as part of the combination. In some cases, where cell lines were resistant to paired and tripled drugs, increased expression of antiapoptotic proteins was observed, requiring a fourth-order combination to induce cytotoxicity. Our results illustrate how high-order drug combinations are needed to kill drug-resistant cancer cells, and they also show how systematic drug combination screening together with a molecular understanding of drug responses may help define optimal cocktails to overcome aggressive cancers. Cancer Res; 76(23); 6950-63. ©2016 AACR.
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http://dx.doi.org/10.1158/0008-5472.CAN-15-3425DOI Listing
December 2016

Dual inhibition of protein kinase C and p53-MDM2 or PKC and mTORC1 are novel efficient therapeutic approaches for uveal melanoma.

Oncotarget 2016 Jun;7(23):33542-56

Laboratory of Preclinical Investigation, Department of Translational Research, PSL University, Institut Curie, Paris, France.

Uveal melanoma (UM) is the most common cancer of the eye in adults. Many UM patients develop metastases for which no curative treatment has been identified. Novel therapeutic approaches are therefore urgently needed. UM is characterized by mutations in the genes GNAQ and GNA11 which activate the PKC pathway, leading to the use of PKC inhibitors as a rational strategy to treat UM tumors. Encouraging clinical activity has been noted in UM patients treated with PKC inhibitors. However, it is likely that curative treatment regimens will require a combination of targeted therapeutic agents. Employing a large panel of UM patient-derived xenograft models (PDXs), several PKC inhibitor-based combinations were tested in vivo using the PKC inhibitor AEB071. The most promising approaches were further investigated in vitro using our unique panel of UM cell lines. When combined with AEB071, the two agents CGM097 (p53-MDM2 inhibitor) and RAD001 (mTORC1 inhibitor) demonstrated greater activity than single agents, with tumor regression observed in several UM PDXs. Follow-up studies in UM cell lines on these two drug associations confirmed their combination activity and ability to induce cell death. While no effective treatment currently exists for metastatic uveal melanoma, we have discovered using our unique panel of preclinical models that combinations between PKC/mTOR inhibitors and PKC/p53-MDM2 inhibitors are two novel and very effective therapeutic approaches for this disease. Together, our study reveals that combining PKC and p53-MDM2 or mTORC1 inhibitors may provide significant clinical benefit for UM patients.
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http://dx.doi.org/10.18632/oncotarget.9552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085101PMC
June 2016

The Public Repository of Xenografts Enables Discovery and Randomized Phase II-like Trials in Mice.

Cancer Cell 2016 04;29(4):574-586

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, 450 Brookline Avenue, Dana 510B, MA 02215, USA.

More than 90% of drugs with preclinical activity fail in human trials, largely due to insufficient efficacy. We hypothesized that adequately powered trials of patient-derived xenografts (PDX) in mice could efficiently define therapeutic activity across heterogeneous tumors. To address this hypothesis, we established a large, publicly available repository of well-characterized leukemia and lymphoma PDXs that undergo orthotopic engraftment, called the Public Repository of Xenografts (PRoXe). PRoXe includes all de-identified information relevant to the primary specimens and the PDXs derived from them. Using this repository, we demonstrate that large studies of acute leukemia PDXs that mimic human randomized clinical trials can characterize drug efficacy and generate transcriptional, functional, and proteomic biomarkers in both treatment-naive and relapsed/refractory disease.
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http://dx.doi.org/10.1016/j.ccell.2016.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5177991PMC
April 2016

Inhibition of Wild-Type p53-Expressing AML by the Novel Small Molecule HDM2 Inhibitor CGM097.

Mol Cancer Ther 2015 Oct 23;14(10):2249-59. Epub 2015 Jul 23.

Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts.

The tumor suppressor p53 is a key regulator of apoptosis and functions upstream in the apoptotic cascade by both indirectly and directly regulating Bcl-2 family proteins. In cells expressing wild-type (WT) p53, the HDM2 protein binds to p53 and blocks its activity. Inhibition of HDM2:p53 interaction activates p53 and causes apoptosis or cell-cycle arrest. Here, we investigated the ability of the novel HDM2 inhibitor CGM097 to potently and selectively kill WT p53-expressing AML cells. The antileukemic effects of CGM097 were studied using cell-based proliferation assays (human AML cell lines, primary AML patient cells, and normal bone marrow samples), apoptosis, and cell-cycle assays, ELISA, immunoblotting, and an AML patient-derived in vivo mouse model. CGM097 potently and selectively inhibited the proliferation of human AML cell lines and the majority of primary AML cells expressing WT p53, but not mutant p53, in a target-specific manner. Several patient samples that harbored mutant p53 were comparatively unresponsive to CGM097. Synergy was observed when CGM097 was combined with FLT3 inhibition against oncogenic FLT3-expressing cells cultured both in the absence as well as the presence of cytoprotective stromal-secreted cytokines, as well as when combined with MEK inhibition in cells with activated MAPK signaling. Finally, CGM097 was effective in reducing leukemia burden in vivo. These data suggest that CGM097 is a promising treatment for AML characterized as harboring WT p53 as a single agent, as well as in combination with other therapies targeting oncogene-activated pathways that drive AML.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4596780PMC
October 2015

A distinct p53 target gene set predicts for response to the selective p53-HDM2 inhibitor NVP-CGM097.

Elife 2015 May 12;4. Epub 2015 May 12.

Disease Area Oncology, Novartis Institutes for BioMedical Research, Basel, Switzerland.

Biomarkers for patient selection are essential for the successful and rapid development of emerging targeted anti-cancer therapeutics. In this study, we report the discovery of a novel patient selection strategy for the p53-HDM2 inhibitor NVP-CGM097, currently under evaluation in clinical trials. By intersecting high-throughput cell line sensitivity data with genomic data, we have identified a gene expression signature consisting of 13 up-regulated genes that predicts for sensitivity to NVP-CGM097 in both cell lines and in patient-derived tumor xenograft models. Interestingly, these 13 genes are known p53 downstream target genes, suggesting that the identified gene signature reflects the presence of at least a partially activated p53 pathway in NVP-CGM097-sensitive tumors. Together, our findings provide evidence for the use of this newly identified predictive gene signature to refine the selection of patients with wild-type p53 tumors and increase the likelihood of response to treatment with p53-HDM2 inhibitors, such as NVP-CGM097.
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http://dx.doi.org/10.7554/eLife.06498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4468608PMC
May 2015

Relief of profound feedback inhibition of mitogenic signaling by RAF inhibitors attenuates their activity in BRAFV600E melanomas.

Cancer Cell 2012 Nov;22(5):668-82

Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

BRAF(V600E) drives tumors by dysregulating ERK signaling. In these tumors, we show that high levels of ERK-dependent negative feedback potently suppress ligand-dependent mitogenic signaling and Ras function. BRAF(V600E) activation is Ras independent and it signals as a RAF-inhibitor-sensitive monomer. RAF inhibitors potently inhibit RAF monomers and ERK signaling, causing relief of ERK-dependent feedback, reactivation of ligand-dependent signal transduction, increased Ras-GTP, and generation of RAF-inhibitor-resistant RAF dimers. This results in a rebound in ERK activity and culminates in a new steady state, wherein ERK signaling is elevated compared to its initial nadir after RAF inhibition. In this state, ERK signaling is RAF inhibitor resistant, and MEK inhibitor sensitive, and combined inhibition results in enhancement of ERK pathway inhibition and antitumor activity.
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http://dx.doi.org/10.1016/j.ccr.2012.10.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3713778PMC
November 2012

PIK3CA mutation uncouples tumor growth and cyclin D1 regulation from MEK/ERK and mutant KRAS signaling.

Cancer Res 2010 Sep 10;70(17):6804-14. Epub 2010 Aug 10.

Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Mutational activation of KRAS is a common event in human tumors. Identification of the key signaling pathways downstream of mutant KRAS is essential for our understanding of how to pharmacologically target these cancers in patients. We show that PD0325901, a small-molecule MEK inhibitor, decreases MEK/ERK pathway signaling and destabilizes cyclin D1, resulting in significant anticancer activity in a subset of KRAS mutant tumors in vitro and in vivo. Mutational activation of PIK3CA, which commonly co-occurs with KRAS mutation, provides resistance to MEK inhibition through reactivation of AKT signaling. Genetic ablation of the mutant PIK3CA allele in MEK inhibitor-resistant cells restores MEK pathway sensitivity, and re-expression of mutant PIK3CA reinstates the resistance, highlighting the importance of this mutation in resistance to therapy in human cancers. In KRAS mutant tumors, PIK3CA mutation restores cyclin D1 expression and G(1)-S cell cycle progression so that they are no longer dependent on KRAS and MEK/ERK signaling. Furthermore, the growth of KRAS mutant tumors with coexistent PIK3CA mutations in vivo is profoundly inhibited with combined pharmacologic inhibition of MEK and AKT. These data suggest that tumors with both KRAS and phosphoinositide 3-kinase mutations are unlikely to respond to the inhibition of the MEK pathway alone but will require effective inhibition of both MEK and phosphoinositide 3-kinase/AKT pathway signaling.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-0409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3178450PMC
September 2010

The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner.

Proc Natl Acad Sci U S A 2010 Aug 28;107(33):14903-8. Epub 2010 Jul 28.

Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA.

Tumors with mutant BRAF and some with mutant RAS are dependent upon ERK signaling for proliferation, and their growth is suppressed by MAPK/ERK kinase (MEK) inhibitors. In contrast, tumor cells with human EGF receptor (HER) kinase activation proliferate in a MEK-independent manner. These findings have led to the development of RAF and MEK inhibitors as anticancer agents. Like MEK inhibitors, the RAF inhibitor PLX4032 inhibits the proliferation of BRAF(V600E) tumor cells but not that of HER kinase-dependent tumors. However, tumors with RAS mutation that are sensitive to MEK inhibition are insensitive to PLX4032. MEK inhibitors inhibit ERK phosphorylation in all normal and tumor cells, whereas PLX4032 inhibits ERK signaling only in tumor cells expressing BRAF(V600E). In contrast, the drug activates MEK and ERK phosphorylation in cells with wild-type BRAF. In BRAF(V600E) tumor cells, MEK and RAF inhibitors affect the expression of a common set of genes. PLX4032 inhibits ERK signaling output in mutant BRAF cells, whereas it transiently activates the expression of these genes in tumor cells with wild-type RAF. Thus, PLX4032 inhibits ERK signaling output in a mutant BRAF-selective manner. These data explain why the drug selectively inhibits the growth of mutant BRAF tumors and suggest that it will not cause toxicity resulting from the inhibition of ERK signaling in normal cells. This selectivity may lead to a broader therapeutic index and help explain the greater antitumor activity observed with this drug than with MEK inhibitors.
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http://dx.doi.org/10.1073/pnas.1008990107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930420PMC
August 2010

4E-BP1 is a key effector of the oncogenic activation of the AKT and ERK signaling pathways that integrates their function in tumors.

Cancer Cell 2010 Jul;18(1):39-51

Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

PIK3CA and PTEN alterations are common in human cancer, but only a fraction of such tumors are dependent upon AKT signaling. AKT independence is associated with redundant activation of cap-dependent translation mediated by convergent regulation of the translational repressor 4E-BP1 by the AKT and ERK pathways. This provides mechanistic bases for the limited activity of AKT and MEK inhibitors in tumors with comutation of both pathways and the profound synergy observed with combined inhibition. Whereas such tumors are sensitive to a dominant active 4E-BP1 mutant, knockdown of 4E-BP1 expression reduces their dependence on AKT/ERK signaling for translation or survival. Thus, 4E-BP1 plays a prominent role in mediating the effects of these pathways in tumors in which they are activated by mutation.
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http://dx.doi.org/10.1016/j.ccr.2010.05.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3286650PMC
July 2010

Genomic and biological characterization of exon 4 KRAS mutations in human cancer.

Cancer Res 2010 Jul 22;70(14):5901-11. Epub 2010 Jun 22.

Departments of Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-0192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943514PMC
July 2010

Genetic predictors of MEK dependence in non-small cell lung cancer.

Cancer Res 2008 Nov;68(22):9375-83

Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non-small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a "driver" mutation in the cells in which it is expressed, MEK inhibition in (V600E)BRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-2223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2649746PMC
November 2008

Therapeutic strategies for inhibiting oncogenic BRAF signaling.

Curr Opin Pharmacol 2008 Aug 3;8(4):419-26. Epub 2008 Aug 3.

Program in Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

Mitogen-activated protein kinase (MAPK) activation is a common property of human cancers and is often due to activating mutations in the BRAF and RAS genes. BRAF kinase domain mutations, the vast majority of which are V600E, occur in approximately 8% of human tumors. These mutations are non-overlapping in distribution with RAS mutations and are observed most frequently in melanoma but also in tumors arising in the colon, thyroid, lung and other sites. V600E BRAF mutation stimulates extracellular signal-regulated kinase (ERK) signaling, induces proliferation and is capable of promoting transformation. Given the frequent occurrence of BRAF mutations in human cancer and the continued requirement for BRAF activity in tumors in which it is mutated, efforts are underway to develop targeted inhibitors of BRAF and its downstream effectors. These agents offer the possibility of greater therapeutic efficacy than the currently available systemic therapies for tumors driven by activating mutations in the MAPK pathway.
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http://dx.doi.org/10.1016/j.coph.2008.06.014DOI Listing
August 2008

Centrosomal anchoring of protein kinase C betaII by pericentrin controls microtubule organization, spindle function, and cytokinesis.

J Biol Chem 2004 Feb 1;279(6):4829-39. Epub 2003 Nov 1.

Department of Pharmacology, School of Medicine, University of California at San Diego, La Jolla, California 92093-0640, USA.

Location is a critical determinant in dictating the cellular function of protein kinase C (PKC). Scaffold proteins contribute to localization by poising PKC at specific intracellular sites. Using a yeast two-hybrid screen, we identified the centrosomal protein pericentrin as a scaffold that tethers PKC betaII to centrosomes. Co-immunoprecipitation studies reveal that the native proteins interact in cells. Co-overexpression studies show that the interaction is mediated by the C1A domain of PKC and a segment of pericentrin within residues 494-593. Immunofluorescence analysis reveals that endogenous PKC betaII colocalizes with pericentrin at centrosomes. Disruption of this interaction by expression of the interacting region of pericentrin results in release of PKC from the centrosome, microtubule disorganization, and cytokinesis failure. Overexpression of this disrupting fragment has no effect in cells lacking PKC betaII, indicating a specific regulatory role of this isozyme in centrosome function. These results reveal a novel role for PKC betaII in cytokinesis and indicate that this function is mediated by an interaction with pericentrin at centrosomes.
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http://dx.doi.org/10.1074/jbc.M311196200DOI Listing
February 2004

A novel human protein of the maternal centriole is required for the final stages of cytokinesis and entry into S phase.

J Cell Biol 2003 May 5;161(3):535-45. Epub 2003 May 5.

University of Massachusetts Medical School, Department of Molecular Medicine, Worcester, MA 01605, USA.

Centrosomes nucleate microtubules and contribute to mitotic spindle organization and function. They also participate in cytokinesis and cell cycle progression in ways that are poorly understood. Here we describe a novel human protein called centriolin that localizes to the maternal centriole and functions in both cytokinesis and cell cycle progression. Centriolin silencing induces cytokinesis failure by a novel mechanism whereby cells remain interconnected by long intercellular bridges. Most cells continue to cycle, reenter mitosis, and form multicellular syncytia. Some ultimately divide or undergo apoptosis specifically during the protracted period of cytokinesis. At later times, viable cells arrest in G1/G0. The cytokinesis activity is localized to a centriolin domain that shares homology with Nud1p and Cdc11p, budding and fission yeast proteins that anchor regulatory pathways involved in progression through the late stages of mitosis. The Nud1p-like domain of centriolin binds Bub2p, another component of the budding yeast pathway. We conclude that centriolin is required for a late stage of vertebrate cytokinesis, perhaps the final cell cleavage event, and plays a role in progression into S phase.
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http://dx.doi.org/10.1083/jcb.200301105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2172935PMC
May 2003