Publications by authors named "Giordano Caponigro"

28 Publications

  • Page 1 of 1

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors.

Clin Cancer Res 2021 Apr 22;27(7):2061-2073. Epub 2020 Dec 22.

Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

Purpose: Targeting RAF for antitumor therapy in RAS-mutant tumors holds promise. Herein, we describe in detail novel properties of the type II RAF inhibitor, LXH254.

Experimental Design: LXH254 was profiled in biochemical, , and assays, including examining the activities of the drug in a large panel of cancer-derived cell lines and a comprehensive set of models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF.

Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations coexpressed with mutant K/NRAS, and mutants, but had only modest activity in mutants. In RAS-mutant lines, loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Finally, , LXH254 drove complete regressions of isogenic variants of RAS-mutant cells lacking ARAF expression, while parental lines were only modestly sensitive.

Conclusions: LXH254 is a novel RAF inhibitor, which is able to inhibit dimerized BRAF and CRAF, as well as monomeric BRAF, while largely sparing ARAF.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2563DOI Listing
April 2021

Combinations with Allosteric SHP2 Inhibitor TNO155 to Block Receptor Tyrosine Kinase Signaling.

Clin Cancer Res 2021 01 12;27(1):342-354. Epub 2020 Oct 12.

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

Purpose: SHP2 inhibitors offer an appealing and novel approach to inhibit receptor tyrosine kinase (RTK) signaling, which is the oncogenic driver in many tumors or is frequently feedback activated in response to targeted therapies including RTK inhibitors and MAPK inhibitors. We seek to evaluate the efficacy and synergistic mechanisms of combinations with a novel SHP2 inhibitor, TNO155, to inform their clinical development.

Experimental Design: The combinations of TNO155 with EGFR inhibitors (EGFRi), BRAFi, KRASi, CDK4/6i, and anti-programmed cell death-1 (PD-1) antibody were tested in appropriate cancer models and , and their effects on downstream signaling were examined.

Results: In EGFR-mutant lung cancer models, combination benefit of TNO155 and the EGFRi nazartinib was observed, coincident with sustained ERK inhibition. In BRAF colorectal cancer models, TNO155 synergized with BRAF plus MEK inhibitors by blocking ERK feedback activation by different RTKs. In KRAS cancer cells, TNO155 effectively blocked the feedback activation of wild-type KRAS or other RAS isoforms induced by KRASi and greatly enhanced efficacy. In addition, TNO155 and the CDK4/6 inhibitor ribociclib showed combination benefit in a large panel of lung and colorectal cancer patient-derived xenografts, including those with KRAS mutations. Finally, TNO155 effectively inhibited RAS activation by colony-stimulating factor 1 receptor, which is critical for the maturation of immunosuppressive tumor-associated macrophages, and showed combination activity with anti-PD-1 antibody.

Conclusions: Our findings suggest TNO155 is an effective agent for blocking both tumor-promoting and immune-suppressive RTK signaling in RTK- and MAPK-driven cancers and their tumor microenvironment. Our data provide the rationale for evaluating these combinations clinically.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2718DOI Listing
January 2021

Resistance to allosteric SHP2 inhibition in FGFR-driven cancers through rapid feedback activation of FGFR.

Oncotarget 2020 Jan 21;11(3):265-281. Epub 2020 Jan 21.

Novartis Institutes for Biomedical Research, Oncology Disease Area, Cambridge, Massachusetts, USA.

SHP2 mediates RAS activation downstream of multiple receptor tyrosine kinases (RTKs) and cancer cell lines dependent on RTKs are in general dependent on SHP2. Profiling of the allosteric SHP2 inhibitor SHP099 across cancer cell lines harboring various RTK dependencies reveals that FGFR-dependent cells are often insensitive to SHP099 when compared to EGFR-dependent cells. We find that FGFR-driven cells depend on SHP2 but exhibit resistance to SHP2 inhibitors and . Treatment of such models with SHP2 inhibitors results in an initial decrease in phosphorylated ERK1/2 (p-ERK) levels, however p-ERK levels rapidly rebound within two hours. This p-ERK rebound is blocked by FGFR inhibitors or high doses of SHP2 inhibitors. Mechanistically, compared with EGFR-driven cells, FGFR-driven cells tend to express high levels of RTK negative regulators such as the SPRY family proteins, which are rapidly downregulated upon ERK inhibition. Moreover, over-expression of SPRY4 in FGFR-driven cells prevents MAPK pathway reactivation and sensitizes them to SHP2 inhibitors. We also identified two novel combination approaches to enhance the efficacy of SHP2 inhibitors, either with a distinct site 2 allosteric SHP2 inhibitor or with a RAS-SOS1 interaction inhibitor. Our findings suggest the rapid FGFR feedback activation following initial pathway inhibition by SHP2 inhibitors may promote the open conformation of SHP2 and lead to resistance to SHP2 inhibitors. These findings may assist to refine patient selection and predict resistance mechanisms in the clinical development of SHP2 inhibitors and to suggest strategies for discovering SHP2 inhibitors that are more effective against upstream feedback activation.
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http://dx.doi.org/10.18632/oncotarget.27435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980623PMC
January 2020

Tumor Intrinsic Efficacy by SHP2 and RTK Inhibitors in KRAS-Mutant Cancers.

Mol Cancer Ther 2019 12 22;18(12):2368-2380. Epub 2019 Aug 22.

Disease Area Oncology, Novartis Institute of Biomedical Research, Cambridge, Massachusetts.

, an oncogene mutated in nearly one third of human cancers, remains a pharmacologic challenge for direct inhibition except for recent advances in selective inhibitors targeting the G12C variant. Here, we report that selective inhibition of the protein tyrosine phosphatase, SHP2, can impair the proliferation of KRAS-mutant cancer cells and using cell line xenografts and primary human tumors. , sensitivity of KRAS-mutant cells toward the allosteric SHP2 inhibitor, SHP099, is not apparent when cells are grown on plastic in 2D monolayer, but is revealed when cells are grown as 3D multicellular spheroids. This antitumor activity is also observed in mouse models. Interrogation of the MAPK pathway in SHP099-treated KRAS-mutant cancer models demonstrated similar modulation of p-ERK and DUSP6 transcripts in 2D, 3D, and , suggesting a MAPK pathway-dependent mechanism and possible non-MAPK pathway-dependent mechanisms in tumor cells or tumor microenvironment for the efficacy. For the KRAS MIAPaCa-2 model, we demonstrate that the efficacy is cancer cell intrinsic as there is minimal antiangiogenic activity by SHP099, and the effects of SHP099 is recapitulated by genetic depletion of SHP2 in cancer cells. Furthermore, we demonstrate that SHP099 efficacy in KRAS-mutant models can be recapitulated with RTK inhibitors, suggesting RTK activity is responsible for the SHP2 activation. Taken together, these data reveal that many KRAS-mutant cancers depend on upstream signaling from RTK and SHP2, and provide a new therapeutic framework for treating KRAS-mutant cancers with SHP2 inhibitors.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0170DOI Listing
December 2019

Next-generation characterization of the Cancer Cell Line Encyclopedia.

Nature 2019 05 8;569(7757):503-508. Epub 2019 May 8.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Large panels of comprehensively characterized human cancer models, including the Cancer Cell Line Encyclopedia (CCLE), have provided a rigorous framework with which to study genetic variants, candidate targets, and small-molecule and biological therapeutics and to identify new marker-driven cancer dependencies. To improve our understanding of the molecular features that contribute to cancer phenotypes, including drug responses, here we have expanded the characterizations of cancer cell lines to include genetic, RNA splicing, DNA methylation, histone H3 modification, microRNA expression and reverse-phase protein array data for 1,072 cell lines from individuals of various lineages and ethnicities. Integration of these data with functional characterizations such as drug-sensitivity, short hairpin RNA knockdown and CRISPR-Cas9 knockout data reveals potential targets for cancer drugs and associated biomarkers. Together, this dataset and an accompanying public data portal provide a resource for the acceleration of cancer research using model cancer cell lines.
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http://dx.doi.org/10.1038/s41586-019-1186-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697103PMC
May 2019

SHP2 Inhibition Overcomes RTK-Mediated Pathway Reactivation in KRAS-Mutant Tumors Treated with MEK Inhibitors.

Mol Cancer Ther 2019 07 8;18(7):1323-1334. Epub 2019 May 8.

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

FGFR1 was recently shown to be activated as part of a compensatory response to prolonged treatment with the MEK inhibitor trametinib in several KRAS-mutant lung and pancreatic cancer cell lines. We hypothesize that other receptor tyrosine kinases (RTK) are also feedback-activated in this context. Herein, we profile a large panel of KRAS-mutant cancer cell lines for the contribution of RTKs to the feedback activation of phospho-MEK following MEK inhibition, using an SHP2 inhibitor (SHP099) that blocks RAS activation mediated by multiple RTKs. We find that RTK-driven feedback activation widely exists in KRAS-mutant cancer cells, to a less extent in those harboring the G13D variant, and involves several RTKs, including EGFR, FGFR, and MET. We further demonstrate that this pathway feedback activation is mediated through mutant KRAS, at least for the G12C, G12D, and G12V variants, and wild-type KRAS can also contribute significantly to the feedback activation. Finally, SHP099 and MEK inhibitors exhibit combination benefits inhibiting KRAS-mutant cancer cell proliferation and These findings provide a rationale for exploration of combining SHP2 and MAPK pathway inhibitors for treating KRAS-mutant cancers in the clinic.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0852DOI Listing
July 2019

The potent and selective cyclin-dependent kinases 4 and 6 inhibitor ribociclib (LEE011) is a versatile combination partner in preclinical cancer models.

Oncotarget 2018 Oct 16;9(81):35226-35240. Epub 2018 Oct 16.

Novartis Institutes for BioMedical Research, Oncology Disease Area, Cambridge, MA, USA.

Inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) is associated with robust antitumor activity. Ribociclib (LEE011) is an orally bioavailable CDK4/6 inhibitor that is approved for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer, in combination with an aromatase inhibitor, and is currently being evaluated in several additional trials. Here, we report the preclinical profile of ribociclib. When tested across a large panel of kinase active site binding assays, ribociclib and palbociclib were highly selective for CDK4, while abemaciclib showed affinity to several other kinases. Both ribociclib and abemaciclib showed slightly higher potency in -dependent cells than in -dependent cells, while palbociclib did not show such a difference. Profiling CDK4/6 inhibitors in large-scale cancer cell line screens confirmed that loss of function is a negative predictor of sensitivity. We also found that routinely used cellular viability assays measuring adenosine triphosphate levels as a proxy for cell numbers underestimated the effects of CDK4/6 inhibition, which contrasts with assays that assess cell number more directly. Robust antitumor efficacy and combination benefit was detected when ribociclib was added to encorafenib, nazartinib, or endocrine therapies in patient-derived xenografts.
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http://dx.doi.org/10.18632/oncotarget.26215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219668PMC
October 2018

SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors.

Nat Med 2018 05 5;24(4):512-517. Epub 2018 Mar 5.

Cancer Center, Massachusetts General Hospital, Charlestown, Massachusetts, USA.

Most anaplastic lymphoma kinase (ALK)-rearranged non-small-cell lung tumors initially respond to small-molecule ALK inhibitors, but drug resistance often develops. Of tumors that develop resistance to highly potent second-generation ALK inhibitors, approximately half harbor resistance mutations in ALK, while the other half have other mechanisms underlying resistance. Members of the latter group often have activation of at least one of several different tyrosine kinases driving resistance. Such tumors are not expected to respond to lorlatinib-a third-generation inhibitor targeting ALK that is able to overcome all clinically identified resistant mutations in ALK-and further therapeutic options are limited. Herein, we deployed a shRNA screen of 1,000 genes in multiple ALK-inhibitor-resistant patient-derived cells (PDCs) to discover those that confer sensitivity to ALK inhibition. This approach identified SHP2, a nonreceptor protein tyrosine phosphatase, as a common targetable resistance node in multiple PDCs. SHP2 provides a parallel survival input downstream of multiple tyrosine kinases that promote resistance to ALK inhibitors. Treatment with SHP099, the recently discovered small-molecule inhibitor of SHP2, in combination with the ALK tyrosine kinase inhibitor (TKI) ceritinib halted the growth of resistant PDCs through preventing compensatory RAS and ERK1 and ERK2 (ERK1/2) reactivation. These findings suggest that combined ALK and SHP2 inhibition may be a promising therapeutic strategy for resistant cancers driven by several different ALK-independent mechanisms underlying resistance.
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http://dx.doi.org/10.1038/nm.4497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6343825PMC
May 2018

Allele-Specific Mechanisms of Activation of MEK1 Mutants Determine Their Properties.

Cancer Discov 2018 05 26;8(5):648-661. Epub 2018 Feb 26.

Program in Molecular Pharmacology, Memorial Sloan Kettering Cancer Center, New York, New York.

Mutations at multiple sites in occur in cancer, suggesting that their mechanisms of activation might be different. We analyzed 17 tumor-associated MEK1 mutants and found that they drove ERK signaling autonomously or in a RAS/RAF-dependent manner. The latter are sensitive to feedback inhibition of RAF, which limits their functional output, and often cooccur with or mutations. They act as amplifiers of RAF signaling. In contrast, another class of mutants deletes a hitherto unrecognized negative regulatory segment of MEK1, is RAF- and phosphorylation-independent, is unaffected by feedback inhibition of upstream signaling, and drives high ERK output and transformation in the absence of RAF activity. Moreover, these RAF-independent mutants are insensitive to allosteric MEK inhibitors, which preferentially bind to the inactivated form of MEK1. All the mutants are sensitive to an ATP-competitive MEK inhibitor. Thus, our study comprises a novel therapeutic strategy for tumors driven by RAF-independent MEK1 mutants. Mutants with which MEK1 mutants coexist and their sensitivity to inhibitors are determined by allele-specific properties. This study shows the importance of functional characterization of mutant alleles in single oncogenes and identifies a new class of MEK1 mutants, insensitive to current MEK1 inhibitors but treatable with a new ATP-competitive inhibitor. .
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http://dx.doi.org/10.1158/2159-8290.CD-17-1452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112572PMC
May 2018

Antitumor Properties of RAF709, a Highly Selective and Potent Inhibitor of RAF Kinase Dimers, in Tumors Driven by Mutant RAS or BRAF.

Cancer Res 2018 03 17;78(6):1537-1548. Epub 2018 Jan 17.

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

Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAF alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors. In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling. .
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http://dx.doi.org/10.1158/0008-5472.CAN-17-2033DOI Listing
March 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

BRAF-inhibitor Associated MEK Mutations Increase RAF-Dependent and -Independent Enzymatic Activity.

Mol Cancer Res 2017 10 27;15(10):1431-1444. Epub 2017 Jun 27.

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

Alterations in occur in cancers, both in the treatment-naïve state and following targeted therapies, most notably BRAF and MEK inhibitors in -V600E-mutant melanoma and colorectal cancer. Efforts were undertaken to understand the effects of these mutations, based upon protein structural location, and MEK1/2 activity. Two categories of MEK1/2 alterations were evaluated, those associated with either the allosteric pocket or helix-A. Clinically, MEK1/2 alterations of the allosteric pocket are rare and we demonstrate that they confer resistance to MEK inhibitors, while retaining sensitivity to BRAF inhibition. Most mutations described in patients fall within, or are associated with, helix-A. Mutations in this region reduce sensitivity to both BRAF and MEK inhibition and display elevated phospho-ERK1/2 levels, independent from increases in phospho-MEK1/2. Biochemical experiments with a representative helix-A variant, -Q56P, reveal both increased catalytic efficiency of the activated enzyme, and phosphorylation-independent activity relative to wild-type MEK1. Consistent with these findings, MEK1/2 alterations in helix A retain sensitivity to downstream antagonism via pharmacologic inhibition of ERK1/2. This work highlights the importance of classifying mutations based on structural and phenotypic consequences, both in terms of pathway signaling output and response to pharmacologic inhibition. This study suggests that alternate modes of target inhibition, such as ERK inhibition, will be required to effectively treat tumors harboring these MEK1/2-resistant alleles. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0211DOI Listing
October 2017

Phase I Dose-Escalation and -Expansion Study of the BRAF Inhibitor Encorafenib (LGX818) in Metastatic -Mutant Melanoma.

Clin Cancer Res 2017 Sep 13;23(18):5339-5348. Epub 2017 Jun 13.

University Hospital Zurich, Zurich, Switzerland.

Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with BRAFi treatment-naïve and pretreated -mutant melanoma. The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50-700 mg) or twice-daily (75-150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic -mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib. Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of V600E-mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar-plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months [95% confidence interval (CI), 7.4-not reached (NR)]. In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9-3.7). Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-2923DOI Listing
September 2017

Dual and Inhibition Demonstrates Synergy against Neuroblastoma.

Clin Cancer Res 2017 Jun 16;23(11):2856-2868. Epub 2016 Dec 16.

Division of Oncology and Center for Childhood Cancer Research, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Anaplastic lymphoma kinase () is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an screen for synergistic drug combinations that target neuroblastomas with mutations in to determine whether drug combinations could enhance antitumor efficacy. We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of ceritinib and ribociclib on proliferation, cell cycle, viability, caspase activation, and the cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing ceritinib alone, ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug-drug interactions. The combination of ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor ceritinib demonstrated higher cytotoxicity ( = 0.008) and synergy scores ( = 0.006) in cell lines with mutations as compared with cell lines lacking mutations or alterations in Compared with either drug alone, combination therapy enhanced growth inhibition, cell-cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with -F1174L and F1245C resistance mutations and prevented the emergence of resistance. Murine ribociclib and ceritinib plasma concentrations were unaltered by combination therapy. This preclinical combination drug screen with validation has provided the rationale for a first-in-children trial of combination ceritinib and ribociclib in a molecularly selected pediatric population. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457336PMC
June 2017

Preclinical Therapeutic Synergy of MEK1/2 and CDK4/6 Inhibition in Neuroblastoma.

Clin Cancer Res 2017 Apr 11;23(7):1785-1796. Epub 2016 Oct 11.

Division of Oncology and Center for Childhood Cancer Research, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition. We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies. Sensitivity to binimetinib and ribociclib was inversely related ( = -0.58, = 0.009). amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs. Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1131DOI Listing
April 2017

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

Personalized Preclinical Trials in BRAF Inhibitor-Resistant Patient-Derived Xenograft Models Identify Second-Line Combination Therapies.

Clin Cancer Res 2016 Apr 16;22(7):1592-602. Epub 2015 Dec 16.

Molecular and Cellular Oncogenesis Program, Tumor Microenvironment and Metastasis Program, and Melanoma Research Center, The Wistar Institute, Philadelphia, Pennsylvania.

Purpose: To test second-line personalized medicine combination therapies, based on genomic and proteomic data, in patient-derived xenograft (PDX) models.

Experimental Design: We established 12 PDXs from BRAF inhibitor-progressed melanoma patients. Following expansion, PDXs were analyzed using targeted sequencing and reverse-phase protein arrays. By using multi-arm preclinical trial designs, we identified efficacious precision medicine approaches.

Results: We identified alterations previously described as drivers of resistance: NRAS mutations in 3 PDXs, MAP2K1 (MEK1) mutations in 2, BRAF amplification in 4, and aberrant PTEN in 7. At the protein level, re-activation of phospho-MAPK predominated, with parallel activation of PI3K in a subset. Second-line efficacy of the pan-PI3K inhibitor BKM120 with either BRAF (encorafenib)/MEK (binimetinib) inhibitor combination or the ERK inhibitor VX-11e was confirmed in vivo Amplification of MET was observed in 3 PDX models, a higher frequency than expected and a possible novel mechanism of resistance. Importantly, MET amplification alone did not predict sensitivity to the MET inhibitor capmatinib. In contrast, capmatinib as single agent resulted in significant but transient tumor regression in a PDX with resistance to BRAF/MEK combination therapy and high pMET. The triple combination capmatinib/encorafenib/binimetinib resulted in complete and sustained tumor regression in all animals.

Conclusions: Genomic and proteomic data integration identifies dual-core pathway inhibition as well as MET as combinatorial targets. These studies provide evidence for biomarker development to appropriately select personalized therapies of patients and avoid treatment failures. See related commentary by Hartsough and Aplin, p. 1550.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-1762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4818716PMC
April 2016

Global chromatin profiling reveals NSD2 mutations in pediatric acute lymphoblastic leukemia.

Nat Genet 2013 Nov 29;45(11):1386-91. Epub 2013 Sep 29.

1] The Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA. [2].

Epigenetic dysregulation is an emerging hallmark of cancers. We developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancers. When applied to 115 lines from the Cancer Cell Line Encyclopedia, this approach identified distinct molecular chromatin signatures. One signature was characterized by increased histone 3 lysine 36 (H3K36) dimethylation, exhibited by several lines harboring translocations in NSD2, which encodes a methyltransferase. A previously unknown NSD2 p.Glu1099Lys (p.E1099K) variant was identified in nontranslocated acute lymphoblastic leukemia (ALL) cell lines sharing this signature. Ectopic expression of the variant induced a chromatin signature characteristic of NSD2 hyperactivation and promoted transformation. NSD2 knockdown selectively inhibited the proliferation of NSD2-mutant lines and impaired the in vivo growth of an NSD2-mutant ALL xenograft. Sequencing analysis of >1,000 pediatric cancer genomes identified the NSD2 p.E1099K alteration in 14% of t(12;21) ETV6-RUNX1-containing ALLs. These findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes.
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http://dx.doi.org/10.1038/ng.2777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262138PMC
November 2013

Dual CDK4/CDK6 inhibition induces cell-cycle arrest and senescence in neuroblastoma.

Clin Cancer Res 2013 Nov 17;19(22):6173-82. Epub 2013 Sep 17.

Authors' Affiliations: Division of Oncology and Center for Childhood Cancer Research; Division of Pathology, Children's Hospital of Philadelphia; Department of Pediatrics; Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; and Novartis Institutes for Biomedical Research, Cambridge, Massachusetts.

Purpose: Neuroblastoma is a pediatric cancer that continues to exact significant morbidity and mortality. Recently, a number of cell-cycle proteins, particularly those within the Cyclin D/CDK4/CDK6/RB network, have been shown to exert oncogenic roles in neuroblastoma, suggesting that their therapeutic exploitation might improve patient outcomes.

Experimental Procedures: We evaluated the effect of dual CDK4/CDK6 inhibition on neuroblastoma viability using LEE011 (Novartis Oncology), a highly specific CDK4/6 inhibitor.

Results: Treatment with LEE011 significantly reduced proliferation in 12 of 17 human neuroblastoma-derived cell lines by inducing cytostasis at nanomolar concentrations (mean IC50 = 307 ± 68 nmol/L in sensitive lines). LEE011 caused cell-cycle arrest and cellular senescence that was attributed to dose-dependent decreases in phosphorylated RB and FOXM1, respectively. In addition, responsiveness of neuroblastoma xenografts to LEE011 translated to the in vivo setting in that there was a direct correlation of in vitro IC50 values with degree of subcutaneous xenograft growth delay. Although our data indicate that neuroblastomas sensitive to LEE011 were more likely to contain genomic amplification of MYCN (P = 0.01), the identification of additional clinically accessible biomarkers is of high importance.

Conclusions: Taken together, our data show that LEE011 is active in a large subset of neuroblastoma cell line and xenograft models, and supports the clinical development of this CDK4/6 inhibitor as a therapy for patients with this disease. Clin Cancer Res; 19(22); 6173-82. ©2013 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-1675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3844928PMC
November 2013

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

Nature 2012 Mar 28;483(7391):603-7. Epub 2012 Mar 28.

The Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA.

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of 'personalized' therapeutic regimens.
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http://dx.doi.org/10.1038/nature11003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3320027PMC
March 2012

Advances in the preclinical testing of cancer therapeutic hypotheses.

Nat Rev Drug Discov 2011 03;10(3):179-87

Novartis Institutes For BioMedical Research, Oncology Research and Oncology Translational Medicine, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

The genetic and epigenetic underpinnings of cancer are becoming increasingly clear owing to impressive and well-coordinated ventures occurring worldwide. As our understanding of the molecular alterations driving human cancer increases, there is an opportunity to direct the clinical application of cancer therapeutics with improved accuracy. The often empirical treatment of cancer--which was initially based on inhibiting DNA synthesis and cellular division--while having led to a number of remarkable successes, remains prone to a high rate of clinical failure that results partly from a lack of understanding of how best to implement drugs in the clinic. Consequently, it is vital that robust translational strategies be developed preclinically to both reduce failure rates in the clinic and shorten the time required to identify patient populations most likely to benefit from a given therapeutic. Here, we review both historical and current uses of preclinical model systems, being mindful that a combination of approaches will be needed to address all meritorious therapeutic hypotheses.
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http://dx.doi.org/10.1038/nrd3385DOI Listing
March 2011

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

Nature 2010 Dec 24;468(7326):968-72. Epub 2010 Nov 24.

Broad Institute of Harvard and Massachusetts Institute of Technology, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA.

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.
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http://dx.doi.org/10.1038/nature09627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3058384PMC
December 2010

Single-vector inducible lentiviral RNAi system for oncology target validation.

Cell Cycle 2009 Feb 25;8(3):498-504. Epub 2009 Feb 25.

Oncology Research, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA.

The use of RNA interference (RNAi) has enabled loss-of-function studies in mammalian cancer cells and has hence become critical for identifying and validating cancer drug targets. Current transient siRNA and stable shRNA systems, however, have limited utility in accurately assessing the cancer dependency due to their short-lived effects and limited in vivo utility, respectively. In this study, a single-vector lentiviral, Tet-inducible shRNA system (pLKO-Tet-On) was generated to allow for the rapid generation of multiple stable cell lines with regulatable shRNA expression. We demonstrate the advantages and versatility of this system by targeting two polycomb group proteins, Bmi-1 and Mel-18, in a number of cancer cell lines. Our data show that pLKO-Tet-On-mediated knockdown is tightly regulated by the inducer tetracycline and its derivative, doxycycline, in a concentration- and time-dependent manner. Furthermore, target gene expression is fully restored upon withdrawal of the inducing agent. An additional, 17 distinct gene products have been targeted by inducible shRNAs with robust regulation in all cases. Importantly, we functionally validate the ability of the pLKO-Tet-On vector to reversibly silence targeted transcripts in vivo. The versatile and robust inducible lentiviral RNAi system reported herein can therefore serve as a powerful tool to rapidly reveal tumor cell dependence.
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http://dx.doi.org/10.4161/cc.8.3.7701DOI Listing
February 2009

Down-regulation of class II phosphoinositide 3-kinase alpha expression below a critical threshold induces apoptotic cell death.

Mol Cancer Res 2008 Apr;6(4):614-23

Van Andel Research Institute, Grand Rapids, MI 49503, USA.

Members of the phosphoinositide 3-kinase (PI3K) family collectively control multiple cellular responses, including proliferation, growth, chemotaxis, and survival. These diverse effects can partly be attributed to the broad range of downstream effectors being regulated by the products of these lipid kinases, the 3'-phosphoinositides. However, an additional layer of complexity is introduced by the existence of multiple PI3K enzyme isoforms. Much has been learned over the last years on the roles of the classes I and III PI3K members in cellular signaling, but little is known about the isoform-specific tasks done by the class II PI3Ks (C2alpha, beta, and gamma). In this study, we used quantitative reverse transcription-PCR and RNA interference in mammalian cells to gain further insight into the function of these lesser studied PI3K enzymes. We find that PI3K-C2alpha, but not PI3K-C2beta, has an important role in controlling cell survival and by using a panel of RNA interference reagents, we were able to determine a critical threshold of PI3K-C2alpha mRNA levels, below which the apoptotic program is switched on, via the intrinsic cell death pathway. In addition, knockdown of PI3K-C2alpha to levels that by themselves do not induce apoptosis sensitize cells to the anticancer agent Taxol (paclitaxel). Lastly, we report that lowering the levels of PI3K-C2alpha in a number of cancer cell lines reduces their proliferation and cell viability, arguing that PI3K inhibitors targeting not only the class Ialpha isoform but also class IIalpha may contribute to an effective anticancer strategy.
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http://dx.doi.org/10.1158/1541-7786.MCR-07-0262DOI Listing
April 2008

Functional analysis of expressed peptides that bind yeast STE proteins.

J Biotechnol 2003 Aug;103(3):213-25

677 7th Ave., Salt Lake City, UT 84103, USA.

Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes.
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http://dx.doi.org/10.1016/s0168-1656(03)00127-5DOI Listing
August 2003

Expression levels of transdominant peptides and proteins in Saccharomyces cerevisiae.

Yeast 2002 Jan;19(1):1-7

Deltagen Proteomics, Inc., 615 Arapeen Drive, Suite 300, Salt Lake City, UT 84108, USA.

From libraries of peptides and protein fragments, several inhibitors that block pheromone response in Saccharomyces cerevisiae have been isolated previously. In many cases, the inhibitors are displayed as part of a scaffold, such as green fluorescent protein. Each of the inhibitors has a characteristic physiological strength or genetic penetrance. In this report, the roles of expression level and display scaffold on the activities of a subset of pheromone-response pathway inhibitors were examined. Special consideration was given to the relationship between expression levels of specific inhibitors, which may exceed 50 microM in some instances, and penetrance.
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http://dx.doi.org/10.1002/yea.795DOI Listing
January 2002
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