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1.
Nat Commun ; 15(1): 48, 2024 01 02.
Article in English | MEDLINE | ID: mdl-38167722

ABSTRACT

CD3 bispecific antibody (CD3 bsAb) therapy is clinically approved for refractory hematological malignancies, but responses in solid tumors have been limited so far. One of the main hurdles in solid tumors is the lack of sufficient T-cell infiltrate. Here, we show that pre-treatment vaccination, even when composed of tumor-unrelated antigens, induces CXCR3-mediated T-cell influx in immunologically 'cold' tumor models in male mice. In the absence of CD3 bsAb, the infiltrate is confined to the tumor invasive margin, whereas subsequent CD3 bsAb administration induces infiltration of activated effector CD8 T cells into the tumor cell nests. This combination therapy installs a broadly inflamed Th1-type tumor microenvironment, resulting in effective tumor eradication. Multiple vaccination formulations, including synthetic long peptides and viruses, empower CD3 bsAb therapy. Our results imply that eliciting tumor infiltration with vaccine-induced tumor-(un)related T cells can greatly improve the efficacy of CD3 bsAbs in solid tumors.


Subject(s)
Antibodies, Bispecific , Neoplasms , Vaccines , Male , Animals , Mice , T-Lymphocytes , CD3 Complex , Neoplasms/drug therapy , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Antigens, Neoplasm , Tumor Microenvironment
2.
Life Sci Alliance ; 5(11)2022 Nov.
Article in English | MEDLINE | ID: mdl-36271507

ABSTRACT

CD3 bispecific antibodies (bsAbs) show great promise as anticancer therapeutics. Here, we show in-depth mechanistic studies of a CD3 bsAb in solid cancer, using DuoBody-CD3x5T4. Cross-linking T cells with tumor cells expressing the oncofetal antigen 5T4 was required to induce cytotoxicity. Naive and memory CD4+ and CD8+ T cells were equally effective at mediating cytotoxicity, and DuoBody-CD3x5T4 induced partial differentiation of naive T-cell subsets into memory-like cells. Tumor cell kill was associated with T-cell activation, proliferation, and production of cytokines, granzyme B, and perforin. Genetic knockout of FAS or IFNGR1 in 5T4+ tumor cells abrogated tumor cell kill. In the presence of 5T4+ tumor cells, bystander kill of 5T4- but not of 5T4-IFNGR1- tumor cells was observed. In humanized xenograft models, DuoBody-CD3x5T4 antitumor activity was associated with intratumoral and peripheral blood T-cell activation. Lastly, in dissociated patient-derived tumor samples, DuoBody-CD3x5T4 activated tumor-infiltrating lymphocytes and induced tumor-cell cytotoxicity, even when most tumor-infiltrating lymphocytes expressed PD-1. These data provide an in-depth view on the mechanism of action of a CD3 bsAb in preclinical models of solid cancer.


Subject(s)
Antibodies, Bispecific , Neoplasms , Humans , Antibodies, Bispecific/pharmacology , CD8-Positive T-Lymphocytes , Granzymes/pharmacology , CD3 Complex/pharmacology , Cytotoxicity, Immunologic , Perforin/pharmacology , Programmed Cell Death 1 Receptor , Neoplasms/drug therapy , Cytokines
3.
Clin Cancer Res ; 27(19): 5389-5400, 2021 10 01.
Article in English | MEDLINE | ID: mdl-34230026

ABSTRACT

PURPOSE: Combining anti-PD-1 + anti-CTLA-4 immune-checkpoint blockade (ICB) shows improved patient benefit, but it is associated with severe immune-related adverse events and exceedingly high cost. Therefore, there is a dire need to predict which patients respond to monotherapy and which require combination ICB treatment. EXPERIMENTAL DESIGN: In patient-derived melanoma xenografts (PDX), human tumor microenvironment (TME) cells were swiftly replaced by murine cells upon transplantation. Using our XenofilteR deconvolution algorithm we curated human tumor cell RNA reads, which were subsequently subtracted in silico from bulk (tumor cell + TME) patients' melanoma RNA. This produced a purely tumor cell-intrinsic signature ("InTumor") and a signature comprising tumor cell-extrinsic RNA reads ("ExTumor"). RESULTS: We show that whereas the InTumor signature predicts response to anti-PD-1, the ExTumor predicts anti-CTLA-4 benefit. In PDX, InTumorLO, but not InTumorHI, tumors are effectively eliminated by cytotoxic T cells. When used in conjunction, the InTumor and ExTumor signatures identify not only patients who have a substantially higher chance of responding to combination treatment than to either monotherapy, but also those who are likely to benefit little from anti-CTLA-4 on top of anti-PD-1. CONCLUSIONS: These signatures may be exploited to distinguish melanoma patients who need combination ICB blockade from those who likely benefit from either monotherapy.


Subject(s)
Melanoma , Programmed Cell Death 1 Receptor , Animals , CTLA-4 Antigen , Humans , Immune Checkpoint Inhibitors , Melanoma/drug therapy , Melanoma/genetics , Mice , Programmed Cell Death 1 Receptor/therapeutic use , RNA , Tumor Microenvironment
4.
Cancers (Basel) ; 13(2)2021 Jan 14.
Article in English | MEDLINE | ID: mdl-33466732

ABSTRACT

Immunotherapy of cancer with CD3-bispecific antibodies is an approved therapeutic option for some hematological malignancies and is under clinical investigation for solid cancers. However, the treatment of solid tumors faces more pronounced hurdles, such as increased on-target off-tumor toxicities, sparse T-cell infiltration and impaired T-cell quality due to the presence of an immunosuppressive tumor microenvironment, which affect the safety and limit efficacy of CD3-bispecific antibody therapy. In this review, we provide a brief status update of the CD3-bispecific antibody therapy field and identify intrinsic hurdles in solid cancers. Furthermore, we describe potential combinatorial approaches to overcome these challenges in order to generate selective and more effective responses.

5.
Cell Syst ; 9(4): 366-374.e5, 2019 10 23.
Article in English | MEDLINE | ID: mdl-31521607

ABSTRACT

Aberrant kinase activity has been linked to a variety of disorders; however, methods to probe kinase activation states in cells have been lacking. Until now, kinase activity has mainly been deduced from either protein expression or substrate phosphorylation levels. Here, we describe a strategy to directly infer kinase activation through targeted quantification of T-loop phosphorylation, which serves as a critical activation switch in a majority of protein kinases. Combining selective phosphopeptide enrichment with robust targeted mass spectrometry, we provide highly specific assays for 248 peptides, covering 221 phosphosites in the T-loop region of 178 human kinases. Using these assays, we monitored the activation of 63 kinases through 73 T-loop phosphosites across different cell types, primary cells, and patient-derived tissue material. The sensitivity of our assays is highlighted by the reproducible detection of TNF-α-induced RIPK1 activation and the detection of 46 T-loop phosphorylation sites from a breast tumor needle biopsy.


Subject(s)
Breast Neoplasms/diagnosis , High-Throughput Screening Assays/methods , Peptides/metabolism , Proteomics/methods , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Biopsy, Needle , Enzyme Activation , Female , Humans , Jurkat Cells , Mass Spectrometry , Phosphorylation , Reproducibility of Results , Sensitivity and Specificity , Tumor Necrosis Factor-alpha/metabolism
6.
Mol Cancer Ther ; 18(9): 1637-1648, 2019 09.
Article in English | MEDLINE | ID: mdl-31270153

ABSTRACT

Combined BRAF and MEK inhibition is a standard of care in patients with advanced BRAF-mutant melanoma, but acquired resistance remains a challenge that limits response durability. Here, we quantitated in vivo ERK1/2 activity and tumor response associated with resistance to combined BRAF and MEK inhibition in mutant BRAF xenografts. We found that ERK1/2 pathway reactivation preceded the growth of resistant tumors. Moreover, we detected a subset of cells that not only persisted throughout long-term treatment but restored ERK1/2 signaling and grew upon drug removal. Cell lines derived from combination-resistant tumors (CRT) exhibited elevated ERK1/2 phosphorylation, which were sensitive to ERK1/2 inhibition. In some CRTs, we detected a tandem duplication of the BRAF kinase domain. Monitoring ERK1/2 activity in vivo was efficacious in predicting tumor response during intermittent treatment. We observed maintained expression of the mitotic regulator, polo-like kinase 1 (Plk1), in melanoma resistant to BRAF and MEK inhibitors. Plk1 inhibition induced apoptosis in CRTs, leading to slowed growth of BRAF and MEK inhibitor-resistant tumors in vivo These data demonstrate the utility of in vivo ERK1/2 pathway reporting as a tool to optimize clinical dosing schemes and establish suppression of Plk1 as potential salvage therapy for BRAF inhibitor and MEK inhibitor-resistant melanoma.


Subject(s)
Drug Resistance, Neoplasm/drug effects , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Melanoma/drug therapy , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic/drug effects , Humans , Melanoma/genetics , Melanoma/metabolism , Mice, Nude , Mitogen-Activated Protein Kinase Kinases/metabolism , Mutation , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins B-raf/metabolism , Xenograft Model Antitumor Assays/methods , Polo-Like Kinase 1
7.
Nat Cell Biol ; 21(6): 778-790, 2019 06.
Article in English | MEDLINE | ID: mdl-31160710

ABSTRACT

Phosphorylation networks intimately regulate mechanisms of response to therapies. Mapping the phospho-catalytic profile of kinases in cells or tissues remains a challenge. Here, we introduce a practical high-throughput system to measure the enzymatic activity of kinases using biological peptide targets as phospho-sensors to reveal kinase dependencies in tumour biopsies and cell lines. A 228-peptide screen was developed to detect the activity of >60 kinases, including ABLs, AKTs, CDKs and MAPKs. Focusing on BRAFV600E tumours, we found mechanisms of intrinsic resistance to BRAFV600E-targeted therapy in colorectal cancer, including targetable parallel activation of PDPK1 and PRKCA. Furthermore, mapping the phospho-catalytic signatures of melanoma specimens identifies RPS6KB1 and PIM1 as emerging druggable vulnerabilities predictive of poor outcome in BRAFV600E patients. The results show that therapeutic resistance can be caused by the concerted upregulation of interdependent pathways. Our kinase activity-mapping system is a versatile strategy that innovates the exploration of actionable kinases for precision medicine.


Subject(s)
3-Phosphoinositide-Dependent Protein Kinases/genetics , Colorectal Neoplasms/drug therapy , Melanoma/drug therapy , Protein Kinase C-alpha/genetics , Proto-Oncogene Proteins c-pim-1/genetics , Adult , Aged , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Drug Resistance, Neoplasm/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Indoles/chemistry , Kaplan-Meier Estimate , MAP Kinase Signaling System/genetics , Male , Melanoma/genetics , Melanoma/pathology , Middle Aged , Peptides/chemistry , Peptides/therapeutic use , Phosphorylation/drug effects , Protein Kinase Inhibitors/therapeutic use , Sulfonamides/therapeutic use
8.
BMC Bioinformatics ; 19(1): 366, 2018 Oct 04.
Article in English | MEDLINE | ID: mdl-30286710

ABSTRACT

BACKGROUND: Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary. RESULTS: We developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations. CONCLUSIONS: XenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR .


Subject(s)
High-Throughput Nucleotide Sequencing/methods , Animals , Computers , Databases, Genetic , Humans , Mice
10.
Mol Syst Biol ; 14(3): e7858, 2018 03 05.
Article in English | MEDLINE | ID: mdl-29507054

ABSTRACT

Novel therapies are undergoing clinical trials, for example, the Hsp90 inhibitor, XL888, in combination with BRAF inhibitors for the treatment of therapy-resistant melanomas. Unfortunately, our data show that this combination elicits a heterogeneous response in a panel of melanoma cell lines including PDX-derived models. We sought to understand the mechanisms underlying the differential responses and suggest a patient stratification strategy. Thermal proteome profiling (TPP) identified the protein targets of XL888 in a pair of sensitive and unresponsive cell lines. Unbiased proteomics and phosphoproteomics analyses identified CDK2 as a driver of resistance to both BRAF and Hsp90 inhibitors and its expression is regulated by the transcription factor MITF upon XL888 treatment. The CDK2 inhibitor, dinaciclib, attenuated resistance to both classes of inhibitors and combinations thereof. Notably, we found that MITF expression correlates with CDK2 upregulation in patients; thus, dinaciclib would warrant consideration for treatment of patients unresponsive to BRAF-MEK and/or Hsp90 inhibitors and/or harboring MITF amplification/overexpression.


Subject(s)
Azabicyclo Compounds/pharmacology , Cyclin-Dependent Kinase 2/metabolism , Drug Resistance, Neoplasm , Imidazoles/pharmacology , Melanoma/metabolism , Microphthalmia-Associated Transcription Factor/metabolism , Oximes/pharmacology , Phthalic Acids/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Cyclic N-Oxides , Cyclin-Dependent Kinase 2/genetics , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic/drug effects , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Humans , Indolizines , Melanoma/drug therapy , Melanoma/genetics , Microphthalmia-Associated Transcription Factor/genetics , Phosphoproteins/metabolism , Proteomics , Pyridinium Compounds/pharmacology , Up-Regulation
11.
Cancer Cell ; 33(2): 322-336.e8, 2018 02 12.
Article in English | MEDLINE | ID: mdl-29438700

ABSTRACT

Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive trimethylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases-the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)-disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.


Subject(s)
Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Melanoma/genetics , Animals , Histones/metabolism , Humans , Lysine/genetics , Lysine/metabolism , Methylation , Mice, Nude , Promoter Regions, Genetic/genetics
12.
Nat Med ; 24(2): 203-212, 2018 02.
Article in English | MEDLINE | ID: mdl-29334371

ABSTRACT

Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.


Subject(s)
Immunoconjugates/pharmacology , Melanoma/drug therapy , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins/immunology , Receptor Protein-Tyrosine Kinases/immunology , Animals , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/immunology , Genetic Heterogeneity/drug effects , Humans , Immunoconjugates/immunology , Melanoma/genetics , Melanoma/immunology , Melanoma/pathology , Mice , Oligopeptides/chemistry , Oligopeptides/immunology , Oligopeptides/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/immunology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/immunology , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/pharmacology , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine Kinase
13.
Cancer Res ; 77(21): e62-e66, 2017 11 01.
Article in English | MEDLINE | ID: mdl-29092942

ABSTRACT

Patient-derived tumor xenograft (PDX) mouse models have emerged as an important oncology research platform to study tumor evolution, mechanisms of drug response and resistance, and tailoring chemotherapeutic approaches for individual patients. The lack of robust standards for reporting on PDX models has hampered the ability of researchers to find relevant PDX models and associated data. Here we present the PDX models minimal information standard (PDX-MI) for reporting on the generation, quality assurance, and use of PDX models. PDX-MI defines the minimal information for describing the clinical attributes of a patient's tumor, the processes of implantation and passaging of tumors in a host mouse strain, quality assurance methods, and the use of PDX models in cancer research. Adherence to PDX-MI standards will facilitate accurate search results for oncology models and their associated data across distributed repository databases and promote reproducibility in research studies using these models. Cancer Res; 77(21); e62-66. ©2017 AACR.


Subject(s)
Neoplasms , Xenograft Model Antitumor Assays/statistics & numerical data , Animals , Databases as Topic , Disease Models, Animal , Humans , Mice , Neoplasms/drug therapy , Neoplasms/genetics , Patients
14.
Nature ; 550(7675): 270-274, 2017 10 12.
Article in English | MEDLINE | ID: mdl-28976960

ABSTRACT

Observations from cultured cells, animal models and patients raise the possibility that the dependency of tumours on the therapeutic drugs to which they have acquired resistance represents a vulnerability with potential applications in cancer treatment. However, for this drug addiction trait to become of clinical interest, we must first define the mechanism that underlies it. We performed an unbiased CRISPR-Cas9 knockout screen on melanoma cells that were both resistant and addicted to inhibition of the serine/threonine-protein kinase BRAF, in order to functionally mine their genome for 'addiction genes'. Here we describe a signalling pathway comprising ERK2 kinase and JUNB and FRA1 transcription factors, disruption of which allowed addicted tumour cells to survive on treatment discontinuation. This occurred in both cultured cells and mice and was irrespective of the acquired drug resistance mechanism. In melanoma and lung cancer cells, death induced by drug withdrawal was preceded by a specific ERK2-dependent phenotype switch, alongside transcriptional reprogramming reminiscent of the epithelial-mesenchymal transition. In melanoma cells, this reprogramming caused the shutdown of microphthalmia-associated transcription factor (MITF), a lineage survival oncoprotein; restoring this protein reversed phenotype switching and prevented the lethality associated with drug addiction. In patients with melanoma that had progressed during treatment with a BRAF inhibitor, treatment cessation was followed by increased expression of the receptor tyrosine kinase AXL, which is associated with the phenotype switch. Drug discontinuation synergized with the melanoma chemotherapeutic agent dacarbazine by further suppressing MITF and its prosurvival target, B-cell lymphoma 2 (BCL-2), and by inducing DNA damage in cancer cells. Our results uncover a pathway that underpins drug addiction in cancer cells, which may help to guide the use of alternating therapeutic strategies for enhanced clinical responses in drug-resistant cancers.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Death/drug effects , Drug Resistance, Neoplasm/drug effects , Lung Neoplasms/pathology , Melanoma/pathology , Mitogen-Activated Protein Kinase 1/metabolism , Phenotype , Animals , Antineoplastic Agents/administration & dosage , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm/physiology , Epithelial-Mesenchymal Transition , Female , Gene Editing , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/enzymology , MAP Kinase Signaling System/drug effects , Melanoma/drug therapy , Melanoma/enzymology , Mice , Mice, Knockout , Microphthalmia-Associated Transcription Factor/metabolism , Molecular Targeted Therapy , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Transcription Factors/metabolism
16.
Nat Rev Cancer ; 17(4): 254-268, 2017 04.
Article in English | MEDLINE | ID: mdl-28104906

ABSTRACT

Patient-derived xenografts (PDXs) have emerged as an important platform to elucidate new treatments and biomarkers in oncology. PDX models are used to address clinically relevant questions, including the contribution of tumour heterogeneity to therapeutic responsiveness, the patterns of cancer evolutionary dynamics during tumour progression and under drug pressure, and the mechanisms of resistance to treatment. The ability of PDX models to predict clinical outcomes is being improved through mouse humanization strategies and the implementation of co-clinical trials, within which patients and PDXs reciprocally inform therapeutic decisions. This Opinion article discusses aspects of PDX modelling that are relevant to these questions and highlights the merits of shared PDX resources to advance cancer medicine from the perspective of EurOPDX, an international initiative devoted to PDX-based research.


Subject(s)
Neoplasms/therapy , Precision Medicine , Xenograft Model Antitumor Assays , Animals , Biomarkers, Tumor/analysis , Clinical Trials as Topic , Disease Models, Animal , Drug Resistance, Neoplasm , Humans , Immunotherapy , Mice , Neoplasm Metastasis , Neoplasms/pathology , Neoplastic Stem Cells/physiology
17.
Cell Rep ; 16(1): 263-277, 2016 06 28.
Article in English | MEDLINE | ID: mdl-27320919

ABSTRACT

The therapeutic landscape of melanoma is improving rapidly. Targeted inhibitors show promising results, but drug resistance often limits durable clinical responses. There is a need for in vivo systems that allow for mechanistic drug resistance studies and (combinatorial) treatment optimization. Therefore, we established a large collection of patient-derived xenografts (PDXs), derived from BRAF(V600E), NRAS(Q61), or BRAF(WT)/NRAS(WT) melanoma metastases prior to treatment with BRAF inhibitor and after resistance had occurred. Taking advantage of PDXs as a limitless source, we screened tumor lysates for resistance mechanisms. We identified a BRAF(V600E) protein harboring a kinase domain duplication (BRAF(V600E/DK)) in ∼10% of the cases, both in PDXs and in an independent patient cohort. While BRAF(V600E/DK) depletion restored sensitivity to BRAF inhibition, a pan-RAF dimerization inhibitor effectively eliminated BRAF(V600E/DK)-expressing cells. These results illustrate the utility of this PDX platform and warrant clinical validation of BRAF dimerization inhibitors for this group of melanoma patients.


Subject(s)
Gene Duplication , Melanoma/drug therapy , Melanoma/genetics , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Xenograft Model Antitumor Assays , Animals , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Chromosome Aberrations , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation, Neoplastic , Humans , Indoles/pharmacology , Indoles/therapeutic use , MAP Kinase Signaling System/drug effects , Melanoma/pathology , Mice , Mutation/genetics , Neoplasm Metastasis , Protein Domains , Protein Multimerization , Reproducibility of Results , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Vemurafenib
19.
EMBO Mol Med ; 7(9): 1104-18, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26105199

ABSTRACT

The development of targeted inhibitors, like vemurafenib, has greatly improved the clinical outcome of BRAF(V600E) metastatic melanoma. However, resistance to such compounds represents a formidable problem. Using whole-exome sequencing and functional analyses, we have investigated the nature and pleiotropy of vemurafenib resistance in a melanoma patient carrying multiple drug-resistant metastases. Resistance was caused by a plethora of mechanisms, all of which reactivated the MAPK pathway. In addition to three independent amplifications and an aberrant form of BRAF(V600E), we identified a new activating insertion in MEK1. This MEK1(T55delins) (RT) mutation could be traced back to a fraction of the pre-treatment lesion and not only provided protection against vemurafenib but also promoted local invasion of transplanted melanomas. Analysis of patient-derived xenografts (PDX) from therapy-refractory metastases revealed that multiple resistance mechanisms were present within one metastasis. This heterogeneity, both inter- and intra-tumorally, caused an incomplete capture in the PDX of the resistance mechanisms observed in the patient. In conclusion, vemurafenib resistance in a single patient can be established through distinct events, which may be preexisting. Furthermore, our results indicate that PDX may not harbor the full genetic heterogeneity seen in the patient's melanoma.


Subject(s)
Antineoplastic Agents/therapeutic use , Drug Resistance, Neoplasm/genetics , Indoles/therapeutic use , Melanoma/drug therapy , Mutation , Neoplasm Metastasis/drug therapy , Skin Neoplasms/drug therapy , Sulfonamides/therapeutic use , Antineoplastic Agents/pharmacology , Genetic Variation , Heterografts , Humans , Indoles/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Sequence Analysis, DNA , Skin Neoplasms/complications , Sulfonamides/pharmacology , Vemurafenib
20.
Genome Biol ; 16: 49, 2015 Feb 27.
Article in English | MEDLINE | ID: mdl-25887352

ABSTRACT

Current methods for detection of copy number variants (CNV) and aberrations (CNA) from targeted sequencing data are based on the depth of coverage of captured exons. Accurate CNA determination is complicated by uneven genomic distribution and non-uniform capture efficiency of targeted exons. Here we present CopywriteR, which eludes these problems by exploiting 'off-target' sequence reads. CopywriteR allows for extracting uniformly distributed copy number information, can be used without reference, and can be applied to sequencing data obtained from various techniques including chromatin immunoprecipitation and target enrichment on small gene panels. CopywriteR outperforms existing methods and constitutes a widely applicable alternative to available tools.


Subject(s)
DNA Copy Number Variations/genetics , Exome/genetics , Genome, Human , Algorithms , Exons/genetics , High-Throughput Nucleotide Sequencing , Humans , Sequence Analysis, DNA
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