ABSTRACT
Glycerol kinase (GK) is a multifunctional enzyme located at the interface of carbohydrate and fat metabolism. It contributes to both central carbon metabolism and adipogenesis; specifically, through its role as the ATP-stimulated translocation promoter (ASTP). GK overexpression leads to increased ASTP activity and increased fat storage in H4IIE cells. We performed metabolic flux analysis in human GK-overexpressing H4IIE cells and found that overexpressing cells had significantly altered fluxes through central carbon and lipid metabolism including increased flux through the pentose phosphate pathway and increased production of lipids. We also observed an equal contribution of glycerol to carbohydrate metabolism in all cell lines, suggesting that GK's alternate functions rather than its enzymatic function are important for these processes. To further elucidate the contributions of the enzymatic (phosphorylation) and alternative (ASTP) functions of GK in adipogenesis, we performed experiments on mammalian GK and E. coli GK. We determined that the ASTP function of GK (which is absent in E. coli GK) plays a greater role than the enzymatic activity in these processes. These studies further emphasize GK's diverse functionality and provides fundamental insights into the multiple protein functions of glycerol kinase.
Subject(s)
Adipogenesis/genetics , Carrier Proteins/genetics , Glycerol Kinase/genetics , Lipid Metabolism/genetics , Animals , Carbohydrate Metabolism/genetics , Carrier Proteins/chemistry , Escherichia coli/enzymology , Gene Expression Regulation, Enzymologic , Glycerol/metabolism , Glycerol Kinase/chemistry , Humans , Promoter Regions, Genetic , RatsABSTRACT
The BRAFV600E-specific inhibitor vemurafenib blocks mitogen-activated protein kinase pathway and induces cell cycle arrest at G0/G1 phase leading to apoptosis of melanomas. To gain an understanding of the dynamics of cell cycle regulation during vemurafenib therapy, we analyzed several vemurafenib-resistant human melanoma sublines derived from BRAFV600E harboring vemurafenib-sensitive parental lines. Vemurafenib provoked G0/G1 phase arrest in parental but not in vemurafenib-resistant sublines. We hypothesized that refractoriness of vemurafenib-resistant sublines to vemurafenib-mediated cell cycle inhibition can be partially rescued by the chromatin modifier suberoylanilide hydroxamic acid. Suberoylanilide hydroxamic acid promoted G2/M arrest at expense of S phase irrespective of vemurafenib sensitivity. In parental lines, combination of suberoylanilide hydroxamic acid and vemurafenib induced both G0/G1 arrest and apoptosis, whereas in vemurafenib-resistant sublines combination induced G0/G1 as well as G2/M arrest resulting in dramatic cytostasis. Vemurafenib-resistant sublines exhibited extracellular signal-regulated protein kinases 1 and 2 but not AKT and hyperphosphorylation. Gene expression profiling revealed mitogen-activated protein kinase hyperactivation and deregulations of cyclins and cyclin-dependent kinases in vemurafenib-resistant sublines, all of which were reversed by suberoylanilide hydroxamic acid; changes that may explain the cytostatic effects of suberoylanilide hydroxamic acid. These results suggest that unresponsiveness of vemurafenib-resistant sublines to the biological effects of vemurafenib may be amenable by suberoylanilide hydroxamic acid. These in vitro results, while require further investigation, may provide rational biological basis for combination therapy in the management of vemurafenib-resistant melanoma.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Cycle/drug effects , Drug Resistance, Neoplasm/drug effects , Hydroxamic Acids/pharmacology , Indoles/pharmacology , Melanoma/pathology , Sulfonamides/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Immunoblotting , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Real-Time Polymerase Chain Reaction , Transcriptome/drug effects , Vemurafenib , VorinostatABSTRACT
Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in â¼7% of human malignancies and â¼60% of melanomas. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRß (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRß RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRß-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRß or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRß or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.
Subject(s)
Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Genes, ras/genetics , Melanoma/drug therapy , Melanoma/enzymology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/metabolism , Base Sequence , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Enzyme Activation/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Humans , Indoles/pharmacology , Indoles/therapeutic use , MAP Kinase Signaling System/drug effects , Melanoma/genetics , Melanoma/pathology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Mutation/genetics , Oligonucleotide Array Sequence Analysis , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Receptor, Platelet-Derived Growth Factor beta/biosynthesis , Receptor, Platelet-Derived Growth Factor beta/genetics , Sulfonamides/pharmacology , Sulfonamides/therapeutic use , Up-Regulation/drug effects , VemurafenibABSTRACT
BACKGROUND: Hundreds of ongoing clinical trials combine radiation therapy, mostly delivered as stereotactic body radiotherapy (SBRT), with immune checkpoint blockade. However, our understanding of the effect of radiotherapy on the intratumoral immune balance is inadequate, hindering the optimal design of trials that combine radiation therapy with immunotherapy. Our objective was to characterize the intratumoral immune balance of the malignant prostate after SBRT in patients. METHODS: Sixteen patients with high-risk, non-metastatic prostate cancer at comparable Gleason Grade disease underwent radical prostatectomy with (n = 9) or without (n = 7) neoadjuvant SBRT delivered in three fractions of 8 Gy over 5 days completed 2 weeks before surgery. Freshly resected prostate specimens were processed to obtain single-cell suspensions, and immune-phenotyped for major lymphoid and myeloid cell subsets by staining with two separate 14-antibody panels and multicolor flow cytometry analysis. RESULTS: Malignant prostates 2 weeks after SBRT had an immune infiltrate dominated by myeloid cells, whereas malignant prostates without preoperative treatment were more lymphoid-biased (myeloid CD45+ cells 48.4 ± 19.7% vs. 25.4 ± 7.0%; adjusted p-value = 0.11; and CD45+ lymphocytes 51.6 ± 19.7% vs. 74.5 ± 7.0%; p = 0.11; CD3+ T cells 35.2 ± 23.8% vs. 60.9 ± 9.7%; p = 0.12; mean ± SD). CONCLUSION: SBRT drives a significant lymphoid to myeloid shift in the prostate-tumor immune infiltrate. This may be of interest when combining SBRT with immunotherapies, particularly in prostate cancer.
Subject(s)
Immunotherapy/methods , Myeloid Cells/pathology , Prostatectomy/methods , Prostatic Neoplasms/therapy , Radiosurgery/methods , Humans , Injections, Intralymphatic , Male , Middle Aged , Neoadjuvant Therapy , Neoplasm Grading , Prostate , Prostatic Neoplasms/pathology , Quality of LifeABSTRACT
Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 muM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 muM, and three were moderately sensitive with IC50 values between 1 and 10 muM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity.
Subject(s)
Indoles/pharmacology , Melanoma/genetics , Mutation/genetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Sulfonamides/pharmacology , Xenograft Model Antitumor Assays , Amino Acid Substitution/genetics , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Diagnostic Imaging , Drug Resistance, Neoplasm/drug effects , Genome, Human/genetics , Humans , MAP Kinase Signaling System/drug effects , Melanoma/enzymology , Melanoma/pathology , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , VemurafenibABSTRACT
PURPOSE: This study aimed to evaluate the feasibility and safety of prostate stereotactic body radiation therapy (SBRT) neoadjuvant to radical prostatectomy (RP) in a phase 1 trial. The primary endpoint was treatment completion rate without severe acute surgical complications. Secondary endpoints included patient-reported quality of life and physician-reported toxicities. METHODS AND MATERIALS: Patients with nonmetastatic high-risk or locally advanced prostate cancer received 24 Gy in 3 fractions to the prostate and seminal vesicles over 5 days, completed 2 weeks before RP. Patients with pN1 disease were treated after multidisciplinary discussion and shared decision making. Patient-reported quality of life (International Prostate Symptom Score and Expanded Prostate Cancer Index Composite 26-item version questionnaires) and physician-reported toxicity (Common Terminology Criteria for Adverse Events, version 4.03) were assessed before SBRT, immediately before surgery, and at 3-month intervals for 1 year. RESULTS: Twelve patients were enrolled, and 11 completed treatment (1 patient had advanced disease on prostate-specific membrane antigen positron emission tomography after enrollment but before treatment). There were no significant surgical complications. After RP, 2 patients underwent additional radiation therapy to nodes with androgen suppression for pN1 disease. Median follow-up after completion of treatment was 20.1 months, with 9 of 11 patients having a follow-up period of >12 months. Two patients had biochemical recurrence (prostate-specific antigen ≥0.05) within the first 12 months, with an additional 2 patients found to have biochemical recurrence after the 12-month period. The highest Common Terminology Criteria for Adverse Events genitourinary grades were 0, 1, 2, and 3 (n = 1, 4, 4, and 2, respectively), and the highest gastrointestinal grades were 0, 1, and 2 (n = 9, 1, and 1, respectively). At 12 months, incontinence was the only grade ≥2 toxicity. One and 2 of 9 patients had grade 2 and 3 incontinence, respectively. On the Expanded Prostate Cancer Index Composite (26-item version), the mean/median changes in scores from baseline to 12 months were -32.8/-31.1 for urinary incontinence, -1.6/-6.2 for urinary irritative/obstructive, -2.1/0 for bowel, -34.4/-37.5 for sexual function, and -10.6/-2.5 for hormonal. The mean/median change in International Prostate Symptom Score from baseline to 12 months was 0.5/0.5. CONCLUSIONS: RP after neoadjuvant SBRT appears to be feasible and safe at the dose tested. The severity of urinary incontinence may be higher than RP alone.
Subject(s)
Neoadjuvant Therapy/methods , Prostatectomy , Prostatic Neoplasms/radiotherapy , Prostatic Neoplasms/surgery , Radiosurgery , Feasibility Studies , Follow-Up Studies , Humans , Male , Prostate/radiation effects , Prostatic Neoplasms/pathology , Quality of Life , Seminal Vesicles/radiation effects , Urinary Incontinence/etiologyABSTRACT
BACKGROUND: Metastatic castration resistant prostate cancer (mCRPC) is incurable and progression after drugs that target the androgen receptor-signaling axis is inevitable. Thus, there is an urgent need to develop more effective treatments beyond hormonal manipulation. We sought to identify activated kinases in mCRPC as therapeutic targets for existing, approved agents, with the goal of identifying candidate drugs for rapid translation into proof of concept Phase II trials in mCRPC. METHODS: To identify evidence of activation of druggable kinases in these patients, we compared mRNA expression from metastatic biopsies of patients with mCRPC (n = 101) to mRNA expression in localized prostate from TCGA and used this analysis to infer differential kinase activity. In addition, we assessed the differential phosphorylation levels for key MAPK pathway kinases between mCRPC and localized prostate cancers. RESULTS: Transcriptomic profiling of 101 patients with mCRPC as compared to patients with localized prostate cancer identified evidence of hyperactive ERK1, and whole genome sequencing revealed frequent amplifications of members of the MAPK pathway in 32% of this cohort. Next, we confirmed elevated levels of phosphorylated ERK1/2 in castration resistant prostate cancer as compared to untreated primary prostate cancer. We observed that the presence of detectable phosphorylated ERK1/2 in the primary tumor is associated with biochemical failure after radical prostatectomy independent of clinicopathologic features. ERK1 is the immediate downstream target of MEK1/2, which is druggable with trametinib, an approved therapeutic for melanoma. Trametinib elicited a profound biochemical and clinical response in a patient who had failed multiple prior treatments for mCRPC. CONCLUSIONS: We conclude that pharmacologic targeting of the MEK/ERK pathway may be a viable treatment strategy for patients with refractory metastatic prostate cancer. An ongoing Phase II trial tests this hypothesis.
Subject(s)
MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 3/antagonists & inhibitors , Prostatic Neoplasms, Castration-Resistant/drug therapy , Protein Kinase Inhibitors/therapeutic use , Aged , Antineoplastic Agents , Biopsy , Disease-Free Survival , Gene Amplification , Gene Expression Regulation, Neoplastic , Humans , MAP Kinase Signaling System/genetics , Male , Middle Aged , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Molecular Targeted Therapy/methods , Phosphorylation/drug effects , Prospective Studies , Prostate/pathology , Prostatic Neoplasms, Castration-Resistant/mortality , Prostatic Neoplasms, Castration-Resistant/pathology , Protein Kinase Inhibitors/pharmacology , Pyridones/pharmacology , Pyridones/therapeutic use , Pyrimidinones/pharmacology , Pyrimidinones/therapeutic use , RNA-SeqABSTRACT
Hermansky-Pudlak syndrome (HPS) comprises a constellation of human autosomal recessive disorders characterized by albinism and platelet storage pool deficiency. At least eight types of HPS have been defined based on the identity of the mutated gene. These genes encode components of four ubiquitously expressed protein complexes, named Adaptor Protein (AP)-3 and Biogenesis of Lysosome-related Organelles Complex (BLOC)-1 through -3. In patients of Puerto Rican origin, the molecular diagnosis can be based on analysis of two founder mutations. On the other hand, identification of the HPS type in other patients relies on the sequencing of all candidate genes. In this work, we have developed a biochemical assay to minimize the number of candidate genes to be sequenced per patient. The assay consists of immunoblotting analysis of extracts prepared from skin fibroblasts, using antibodies to one subunit per protein complex. The assay allowed us to determine which complex was defective in each of a group of HPS patients with unknown genetic lesions, thus subsequent sequencing was limited to genes encoding the corresponding subunits. Because no mutations within the two genes encoding BLOC-3 subunits could be found in two patients displaying reduced BLOC-3 levels, the possible existence of additional subunits was considered. Through size-exclusion chromatography and sedimentation velocity analysis, the native molecular mass of BLOC-3 was estimated to be 140+/-30 kDa, a value most consistent with the idea that BLOC-3 is a HPS1HPS4 heterodimer (approximately 156 kDa) albeit not inconsistent with the putative existence of a relatively small third subunit.
Subject(s)
Blotting, Western/methods , Hermanski-Pudlak Syndrome/diagnosis , Hermanski-Pudlak Syndrome/genetics , Adaptor Protein Complex 3/genetics , Adaptor Protein Complex 3/immunology , Amino Acid Sequence , Animals , Antibody Specificity , Base Sequence , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/immunology , DNA/genetics , Hermanski-Pudlak Syndrome/classification , Humans , Mice , Molecular Sequence Data , Molecular Weight , Mutation , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/immunology , Proteins/genetics , Proteins/immunology , Sequence Homology, Amino AcidABSTRACT
Dysbindin was identified as a dystrobrevin-binding protein potentially involved in the pathogenesis of muscular dystrophy. Subsequently, genetic studies have implicated variants of the human dysbindin-encoding gene, DTNBP1, in the pathogeneses of Hermansky-Pudlak syndrome and schizophrenia. The protein is a stable component of a multisubunit complex termed BLOC-1 (biogenesis of lysosome-related organelles complex-1). In the present study, the significance of the dystrobrevin-dysbindin interaction for BLOC-1 function was examined. Yeast two-hybrid analyses, and binding assays using recombinant proteins, demonstrated direct interaction involving coiled-coil-forming regions in both dysbindin and the dystrobrevins. However, recombinant proteins bearing the coiled-coil-forming regions of the dystrobrevins failed to bind endogenous BLOC-1 from HeLa cells or mouse brain or muscle, under conditions in which they bound the Dp71 isoform of dystrophin. Immunoprecipitation of endogenous dysbindin from brain or muscle resulted in robust co-immunoprecipitation of the pallidin subunit of BLOC-1 but no specific co-immunoprecipitation of dystrobrevin isoforms. Within BLOC-1, dysbindin is engaged in interactions with three other subunits, named pallidin, snapin and muted. We herein provide evidence that the same 69-residue region of dysbindin that is sufficient for dystrobrevin binding in vitro also contains the binding sites for pallidin and snapin, and at least part of the muted-binding interface. Functional, histological and immunohistochemical analyses failed to detect any sign of muscle pathology in BLOC-1-deficient, homozygous pallid mice. Taken together, these results suggest that dysbindin assembled into BLOC-1 is not a physiological binding partner of the dystrobrevins, likely due to engagement of its dystrobrevin-binding region in interactions with other subunits.
Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Dystrophin-Associated Proteins/metabolism , Animals , Carrier Proteins/genetics , Dystrophin/deficiency , Dystrophin/genetics , Dystrophin/metabolism , HeLa Cells , Humans , Immunohistochemistry , Lectins/metabolism , Mice , Mice, Knockout , Molecular Sequence Data , Muscles/cytology , Muscles/metabolism , Nerve Tissue Proteins , Protein Binding , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Two-Hybrid System TechniquesABSTRACT
Tumor necrosis factor-alpha (TNF-alpha) is a major mediator of inflammation and it is involved in many neurological disorders such as multiple sclerosis. Levels of TNF-alpha and lymphotoxin-alpha have been found elevated in plaques, bloods, and cerebral spinal fluids from multiple sclerosis patients. The expression of myelin basic protein (MBP), a major protein of the myelin sheath, is affected by cytokines secreted by activated immune cells. To determine the signal transduction pathway involving tumor necrosis factor's action in myelination and demyelination, we have cloned and analyzed cis-elements on promoters of the human and mouse MBP genes. There are two putative nuclear factors kappa-B (NF-kappaB) cis-elements on the human and one on the mouse gene promoter. In an electrophoretic mobility shift assay, all three NF-kappaB cis-elements showed binding to a protein, which was recognized by an antibody against NF-kappaB P65 component. The specificity of the binding was demonstrated in a competitive assay using NF-kappaB consensus oligonucleotides. A two base pair site-directed mutation on the mouse NF-kappaB cis-element abolished its binding activity. We created a DNA construct by linking the mouse MBP gene promoter containing the NF-kappaB cis-element to luciferase gene. Transfection of this construct into a human oligodendroglioma cell line showed TNF-alpha increased the transgene expression. Furthermore the mutation of NF-kappaB site abolished TNF-alpha -induction of the transgene. The data demonstrate that NF-kappaB is the mediator between tumor necrosis factor's action and MBP gene expression. Elucidating the molecular mechanisms underlying TNF-alpha regulation of MBP gene expression provides new scientific bases for the development of therapy against oligodendrocyte-specific and myelin-related disorders such as multiple sclerosis.
Subject(s)
Multiple Sclerosis/genetics , Myelin Basic Protein/genetics , Myelin Sheath/genetics , NF-kappa B/genetics , Nervous System/metabolism , Transcription, Genetic/genetics , Tumor Necrosis Factor-alpha/genetics , Animals , DNA-Binding Proteins/genetics , Gene Expression Regulation/genetics , Humans , Mice , Multiple Sclerosis/metabolism , Multiple Sclerosis/physiopathology , Mutagenesis, Site-Directed/genetics , Myelin Basic Protein/metabolism , Myelin Proteolipid Protein/genetics , Myelin Proteolipid Protein/metabolism , Myelin Sheath/metabolism , Myelin Sheath/pathology , NF-kappa B/metabolism , Nervous System/physiopathology , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligodendroglioma , Promoter Regions, Genetic/genetics , Signal Transduction/genetics , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/metabolismABSTRACT
BRAF(V600E)-inhibitors (BRAFi; e.g., vemurafenib) and modern immune-based therapies such as PD-1/PD-L1 and CTLA-4 checkpoints blockade and adoptive cell transfer (ACT) have significantly improved the care of melanoma patients. Having these two effective (BRAFi and immunotherapy) therapies raises the question whether there is a rational biological basis for using them in combination. We developed an in vitro model to determine whether tumor resistance mechanisms to a small molecule inhibitor of a driver oncogene, and to cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-delivered apoptotic death signals were exclusive or intersecting. We generated melanoma sublines resistant to BRAFi vemurafenib and to CTL recognizing the MART-1 melanoma antigen. Vemurafenib-resistant (VemR) sublines were cross-resistant to MART CTL and NK cells indicating that a common apoptotic pathway governing tumor response to both modalities was disrupted. Pretreatment of VemR melanomas with a histone deacetylase inhibitor (HDACi) restored sensitivity to MART CTL and NK apoptosis by skewing the apoptotic gene programs towards a proapoptotic phenotype. Our in vitro findings suggest that during the course of acquisition of BRAFi resistance, melanomas develop cross-resistance to CTL- and NK-killing. Further, aberrant apoptotic pathways, amenable by an FDA-approved chromatin remodeling drug, regulate tumor resistance mechanisms to immune effector cells. These results may provide rational molecular basis for further investigations to combine these therapies clinically.
ABSTRACT
UNLABELLED: BRAF inhibitors (BRAFi) induce antitumor responses in nearly 60% of patients with advanced V600E/KBRAF melanomas. Somatic activating MEK1 mutations are thought to be rare in melanomas, but their potential concurrence with V600E/KBRAF may be selected for by BRAFi. We sequenced MEK1/2 exon 3 in melanomas at baseline and upon disease progression. Of 31 baseline V600E/KBRAF melanomas, 5 (16%) carried concurrent somatic BRAF/MEK1 activating mutations. Three of 5 patients with BRAF/MEK1 double-mutant baseline melanomas showed objective tumor responses, consistent with the overall 60% frequency. No MEK1 mutation was found in disease progression melanomas, except when it was already identified at baseline. MEK1-mutant expression in V600E/KBRAF melanoma cell lines resulted in no significant alterations in p-ERK1/2 levels or growth-inhibitory sensitivities to BRAFi, MEK1/2 inhibitor (MEKi), or their combination. Thus, activating MEK1 exon 3 mutations identified herein and concurrent with V600E/KBRAF do not cause BRAFi resistance in melanoma. SIGNIFICANCE: As BRAF inhibitors gain widespread use for treatment of advanced melanoma, biomarkers for drug sensitivity or resistance are urgently needed. We identify here concurrent activating mutations in BRAF and MEK1 in melanomas and show that the presence of a downstream mutation in MEK1 does not necessarily make BRAFmutant melanomas resistant to BRAF inhibitors.
Subject(s)
Drug Resistance, Neoplasm/genetics , MAP Kinase Kinase 1/genetics , Melanoma/genetics , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/genetics , Adult , Aged , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Exons , Female , HEK293 Cells , Humans , Imidazoles/therapeutic use , Indoles/therapeutic use , Male , Melanoma/drug therapy , Middle Aged , Mutation , Oximes/therapeutic use , Sulfonamides/therapeutic use , VemurafenibABSTRACT
BACKGROUND: The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF(V600) mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway. METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance. CONCLUSIONS/SIGNIFICANCE: Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.
Subject(s)
Drug Resistance, Neoplasm/drug effects , Melanoma/enzymology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , TOR Serine-Threonine Kinases/antagonists & inhibitors , Benzimidazoles/pharmacology , Carrier Proteins/genetics , Cell Line, Tumor , Gene Silencing/drug effects , Humans , Indoles/pharmacology , Inhibitory Concentration 50 , MAP Kinase Signaling System/drug effects , Melanoma/drug therapy , Melanoma/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Mutation/genetics , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/metabolism , Rapamycin-Insensitive Companion of mTOR Protein , Ribosomal Protein S6 Kinases/genetics , Sirolimus/pharmacology , Sulfonamides/pharmacology , TOR Serine-Threonine Kinases/metabolism , VemurafenibSubject(s)
Cecum , Colonic Diseases/diagnosis , Colonoscopy/methods , Intubation/standards , Postoperative Complications/epidemiology , Female , Humans , MaleABSTRACT
Late endosomes and lysosomes of mammalian cells in interphase tend to concentrate in the perinuclear region that harbors the microtubule-organizing center. We have previously reported abnormal distribution of these organelles - as judged by reduced percentages of cells displaying pronounced perinuclear accumulation - in mutant fibroblasts lacking BLOC-3 (for ;biogenesis of lysosome-related organelles complex 3'). BLOC-3 is a protein complex that contains the products of the genes mutated in Hermansky-Pudlak syndrome types 1 and 4. Here, we developed a method based on image analysis to estimate the extent of organelle clustering in the perinuclear region of cultured cells. Using this method, we corroborated that the perinuclear clustering of late endocytic organelles containing Lamp1 (for ;lysosome-associated membrane protein 1') is reduced in BLOC-3-deficient murine fibroblasts, and found that it is apparently normal in fibroblasts deficient in BLOC-1 or BLOC-2, which are another two protein complexes associated with Hermansky-Pudlak syndrome. Wild-type and mutant fibroblasts were transfected to express human LAMP1 fused at its cytoplasmic tail to green fluorescence protein (GFP). At low expression levels, LAMP1-GFP was targeted correctly to late endocytic organelles in both wild-type and mutant cells. High levels of LAMP1-GFP overexpression elicited aberrant aggregation of late endocytic organelles, a phenomenon that probably involved formation of anti-parallel dimers of LAMP1-GFP as it was not observed in cells expressing comparable levels of a non-dimerizing mutant variant, LAMP1-mGFP. To test whether BLOC-3 plays a role in the movement of late endocytic organelles, time-lapse fluorescence microscopy experiments were performed using live cells expressing low levels of LAMP1-GFP or LAMP1-mGFP. Although active movement of late endocytic organelles was observed in both wild-type and mutant fibroblasts, quantitative analyses revealed a relatively lower frequency of microtubule-dependent movement events, either towards or away from the perinuclear region, within BLOC-3-deficient cells. By contrast, neither the duration nor the speed of these microtubule-dependent events seemed to be affected by the lack of BLOC-3 function. These results suggest that BLOC-3 function is required, directly or indirectly, for optimal attachment of late endocytic organelles to microtubule-dependent motors.
Subject(s)
Carrier Proteins/metabolism , Endocytosis , Endosomes/metabolism , Fibroblasts/metabolism , Lysosomal-Associated Membrane Protein 1/metabolism , Membrane Proteins/metabolism , Vesicular Transport Proteins/metabolism , Animals , Cell Nucleus/metabolism , Fibroblasts/cytology , Genes, Reporter , Guanine Nucleotide Exchange Factors , Mice , Mice, Inbred C57BL , Microscopy, Fluorescence , Mutation/genetics , Recombinant Fusion Proteins , TransfectionABSTRACT
Hermansky-Pudlak syndrome (HPS) defines a group of autosomal recessive disorders characterized by defects in lysosome-related organelles such as melanosomes and platelet dense granules. The genes that are defective in each of the different forms of HPS in humans, or in HPS-like disorders in mice, are thought to encode components of a putative molecular machinery required for the formation of specialized organelles of the lysosomal system. This review discusses the biochemical and functional properties of the products of identified HPS genes, which include subunits of the AP-3 complex and the novel proteins HPS1p, HPS3p, HPS4p, pallidin and muted.
Subject(s)
Hermanski-Pudlak Syndrome/metabolism , Hermanski-Pudlak Syndrome/pathology , Lysosomes/metabolism , Lysosomes/pathology , Humans , Melanosomes/metabolism , Melanosomes/pathology , Protein Transport/physiologyABSTRACT
Hermansky-Pudlak syndrome (HPS) defines a group of autosomal recessive disorders characterized by deficiencies in lysosome-related organelles such as melanosomes and platelet-dense granules. Several HPS genes encode proteins of unknown function including HPS1, HPS3, and HPS4. Here we have identified and characterized endogenous HPS3 and HPS4 proteins from HeLa cells. Both proteins were found in soluble and membrane-associated forms. Sedimentation-velocity and coimmunoprecipitation experiments revealed that HPS4 but not HPS3 associates with HPS1 in a complex, which we term biogenesis of lysosome-related organelles complex 3 (BLOC-3). Mutant fibroblasts deficient in either HPS1 or HPS4 displayed abnormal localization of lysosomes and late endosomes, which were less concentrated at the juxtanuclear region in mutant cells than in control fibroblasts. The coat-color phenotype of young homozygous double-mutant mice deficient in subunits of BLOC-3 (HPS1) and BLOC-1 (pallidin) was indistinguishable from that of BLOC-1 single mutants. Taken together, these observations suggest that HPS1 and HPS4 are components of a protein complex that regulates the intracellular localization of lysosomes and late endosomes and may function in a BLOC-1-dependent pathway for melanosome biogenesis.