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1.
Clin Cancer Res ; 29(21): 4479-4491, 2023 11 01.
Article in English | MEDLINE | ID: mdl-37616468

ABSTRACT

PURPOSE: Deregulated metabolism in cancer cells represents a vulnerability that may be therapeutically exploited to benefit patients. One such target is nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage pathway. NAMPT is necessary for efficient NAD+ production and may be exploited in cells with increased metabolic demands. We have identified NAMPT as a dependency in rhabdomyosarcoma (RMS), a malignancy for which novel therapies are critically needed. Here we describe the effect of NAMPT inhibition on RMS proliferation and metabolism in vitro and in vivo. EXPERIMENTAL DESIGN: Assays of proliferation and cell death were used to determine the effects of pharmacologic NAMPT inhibition in a panel of ten molecularly diverse RMS cell lines. Mechanism of the clinical NAMPTi OT-82 was determined using measures of NAD+ and downstream NAD+-dependent functions, including energy metabolism. We used orthotopic xenograft models to examine tolerability, efficacy, and drug mechanism in vivo. RESULTS: Across all ten RMS cell lines, OT-82 depleted NAD+ and inhibited cell growth at concentrations ≤1 nmol/L. Significant impairment of glycolysis was a universal finding, with some cell lines also exhibiting diminished oxidative phosphorylation. Most cell lines experienced profound depletion of ATP with subsequent irreversible necrotic cell death. Importantly, loss of NAD and glycolytic activity were confirmed in orthotopic in vivo models, which exhibited complete tumor regressions with OT-82 treatment delivered on the clinical schedule. CONCLUSIONS: RMS is highly vulnerable to NAMPT inhibition. These findings underscore the need for further clinical study of this class of agents for this malignancy.


Subject(s)
NAD , Rhabdomyosarcoma , Humans , NAD/metabolism , Cytokines/metabolism , Nicotinamide Phosphoribosyltransferase/metabolism , Pyrazoles , Necrosis , Rhabdomyosarcoma/drug therapy , Cell Line, Tumor
2.
JAMA Dermatol ; 159(10): 1112-1118, 2023 10 01.
Article in English | MEDLINE | ID: mdl-37585199

ABSTRACT

Importance: Knowledge about the prevalence and tumor types of CDKN2A-related melanoma-astrocytoma syndrome (MAS) is limited and could improve disease recognition. Objective: To estimate the prevalence and describe the tumor types of MAS. Design, Setting, and Participants: This retrospective cohort study analyzed all available MAS cases from medical centers in the US (2 sites) and Europe (2 sites) and from biomedical population genomic databases (UK Biobank [United Kingdom], Geisinger MyCode [US]) between January 1, 1976, and December 31, 2020. Patients with MAS with CDKN2A germline pathogenic variants and 1 or more neural tumors were included. Data were analyzed from June 1, 2022, to January 31, 2023. Main Outcomes and Measures: Disease prevalence and tumor frequency. Results: Prevalence of MAS ranged from 1 in 170 503 (n = 1 case; 95% CI, 1:30 098-1:965 887) in Geisinger MyCode (n = 170 503; mean [SD] age, 58.9 [19.1] years; 60.6% women; 96.2% White) to 1 in 39 149 (n = 12 cases; 95% CI, 1:22 396-1:68 434) in UK Biobank (n = 469 789; mean [SD] age, 70.0 [8.0] years; 54.2% women; 94.8% White). Among UK Biobank patients with MAS (n = 12) identified using an unbiased genomic ascertainment approach, brain neoplasms (4 of 12, 33%; 1 glioblastoma, 1 gliosarcoma, 1 astrocytoma, 1 unspecified type) and schwannomas (3 of 12, 25%) were the most common malignant and benign neural tumors, while cutaneous melanoma (2 of 12, 17%) and head and neck squamous cell carcinoma (2 of 12, 17%) were the most common nonneural malignant neoplasms. In a separate case series of 14 patients with MAS from the US and Europe, brain neoplasms (4 of 14, 29%; 2 glioblastomas, 2 unspecified type) and malignant peripheral nerve sheath tumor (2 of 14, 14%) were the most common neural cancers, while cutaneous melanoma (4 of 14, 29%) and sarcomas (2 of 14, 14%; 1 liposarcoma, 1 unspecified type) were the most common nonneural cancers. Cutaneous neurofibromas (7 of 14, 50%) and schwannomas (2 of 14, 14%) were also common. In 1 US family, a father and son with MAS had clinical diagnoses of neurofibromatosis type 1 (NF1). Genetic testing of the son detected a pathogenic CDKN2A splicing variant (c.151-1G>C) and was negative for NF1 genetic alterations. In UK Biobank, 2 in 150 (1.3%) individuals with clinical NF1 diagnoses had likely pathogenic variants in CDKN2A, including 1 individual with no detected variants in the NF1 gene. Conclusions and Relevance: This cohort study estimates the prevalence and describes the tumors of MAS. Additional studies are needed in genetically diverse populations to further define population prevalence and disease phenotypes.


Subject(s)
Astrocytoma , Brain Neoplasms , Melanoma , Neurilemmoma , Neurofibromatosis 1 , Skin Neoplasms , Humans , Female , Middle Aged , Aged , Male , Melanoma/epidemiology , Melanoma/genetics , Neurofibromatosis 1/diagnosis , Retrospective Studies , Cohort Studies , Prevalence , Skin Neoplasms/epidemiology , Skin Neoplasms/genetics , Astrocytoma/epidemiology , Astrocytoma/genetics , Phenotype , Brain Neoplasms/epidemiology , Brain Neoplasms/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Melanoma, Cutaneous Malignant
3.
Clin Cancer Res ; 29(17): 3329-3339, 2023 09 01.
Article in English | MEDLINE | ID: mdl-37398992

ABSTRACT

PURPOSE: Antibodies against insulin-like growth factor (IGF) type 1 receptor have shown meaningful but transient tumor responses in patients with rhabdomyosarcoma (RMS). The SRC family member YES has been shown to mediate IGF type 1 receptor (IGF-1R) antibody acquired resistance, and cotargeting IGF-1R and YES resulted in sustained responses in murine RMS models. We conducted a phase I trial of the anti-IGF-1R antibody ganitumab combined with dasatinib, a multi-kinase inhibitor targeting YES, in patients with RMS (NCT03041701). PATIENTS AND METHODS: Patients with relapsed/refractory alveolar or embryonal RMS and measurable disease were eligible. All patients received ganitumab 18 mg/kg intravenously every 2 weeks. Dasatinib dose was 60 mg/m2/dose (max 100 mg) oral once daily [dose level (DL)1] or 60 mg/m2/dose (max 70 mg) twice daily (DL2). A 3+3 dose escalation design was used, and maximum tolerated dose (MTD) was determined on the basis of cycle 1 dose-limiting toxicities (DLT). RESULTS: Thirteen eligible patients, median age 18 years (range 8-29) enrolled. Median number of prior systemic therapies was 3; all had received prior radiation. Of 11 toxicity-evaluable patients, 1/6 had a DLT at DL1 (diarrhea) and 2/5 had a DLT at DL2 (pneumonitis, hematuria) confirming DL1 as MTD. Of nine response-evaluable patients, one had a confirmed partial response for four cycles, and one had stable disease for six cycles. Genomic studies from cell-free DNA correlated with disease response. CONCLUSIONS: The combination of dasatinib 60 mg/m2/dose daily and ganitumab 18 mg/kg every 2 weeks was safe and tolerable. This combination had a disease control rate of 22% at 5 months.


Subject(s)
Rhabdomyosarcoma , src-Family Kinases , Humans , Animals , Mice , Child , Adolescent , Young Adult , Adult , Dasatinib/adverse effects , Insulin-Like Growth Factor I , Receptor, IGF Type 1 , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Maximum Tolerated Dose
4.
J Biol Chem ; 299(3): 102992, 2023 03.
Article in English | MEDLINE | ID: mdl-36758799

ABSTRACT

The ADP-ribosylation factor (Arf) GTPases and their regulatory proteins are implicated in cancer progression. NAV-2729 was previously identified as a specific inhibitor of Arf6 that reduced progression of uveal melanoma in an orthotopic xenograft. Here, our goal was to assess the inhibitory effects of NAV-2729 on the proliferation of additional cell types. We found NAV-2729 inhibited proliferation of multiple cell lines, but Arf6 expression did not correlate with NAV-2729 sensitivity, and knockdown of Arf6 affected neither cell viability nor sensitivity to NAV-2729. Furthermore, binding to native Arf6 was not detected; however, we determined that NAV-2729 inhibited both Arf exchange factors and Arf GTPase-activating proteins. ASAP1, a GTPase-activating protein linked to cancer progression, was further investigated. We demonstrated that NAV-2729 bound to the PH domain of ASAP1 and changed ASAP1 cellular distribution. However, ASAP1 knockdown did not fully recapitulate the cytoskeletal effects of NAV-2729 nor affect cell proliferation. Finally, our screens identified 48 other possible targets of NAV-2729. These results illustrate the complexities of defining targets of small molecules and identify NAV-2729 as a model PH domain-binding inhibitor.


Subject(s)
ADP-Ribosylation Factors , Neoplasms , Humans , ADP-Ribosylation Factors/metabolism , Chlorobenzenes , Pyrazoles , GTPase-Activating Proteins/metabolism , ADP-Ribosylation Factor 1/metabolism
5.
Clin Cancer Res ; 29(2): 472-487, 2023 01 17.
Article in English | MEDLINE | ID: mdl-36322002

ABSTRACT

PURPOSE: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. EXPERIMENTAL DESIGN: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. RESULTS: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. CONCLUSIONS: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.


Subject(s)
Rhabdomyosarcoma , Humans , Animals , Mice , Child , Cell Line, Tumor , Mice, SCID , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/pathology , Protein Kinase Inhibitors/pharmacology , Mitogen-Activated Protein Kinase Kinases
6.
Am J Med Genet C Semin Med Genet ; 190(4): 541-560, 2022 12.
Article in English | MEDLINE | ID: mdl-36533679

ABSTRACT

The RASopathies are a group of clinically defined developmental syndromes caused by germline variants of the RAS/mitogen-activated protein (MAPK) cascade. The prototypic RASopathy is Noonan syndrome, which has phenotypic overlap with related disorders such as cardiofaciocutaneous syndrome, Costello syndrome, Noonan syndrome with multiple lentigines, and others. In this state-of-the-art review, we summarize current knowledge on unmet therapeutic needs in these diseases and novel treatment approaches informed by insights from RAS/MAPK-associated cancer therapies, in particular through inhibition of MEK1/2 and mTOR in patients with severe disease manifestations. We explore the possibilities of integrating a larger arsenal of molecules currently under development into future care plans. Lastly, we describe both medical and ethical challenges and opportunities for future clinical trials in the field.


Subject(s)
Costello Syndrome , Heart Defects, Congenital , Noonan Syndrome , Humans , Prospective Studies , MAP Kinase Signaling System , Noonan Syndrome/drug therapy , Noonan Syndrome/genetics , Costello Syndrome/genetics , Costello Syndrome/therapy , ras Proteins/genetics
7.
Cell Rep ; 40(12): 111363, 2022 09 20.
Article in English | MEDLINE | ID: mdl-36130486

ABSTRACT

Loss-of-function mutations in the polycomb repressive complex 2 (PRC2) occur frequently in malignant peripheral nerve sheath tumor, an aggressive sarcoma that arises from NF1-deficient Schwann cells. To define the oncogenic mechanisms underlying PRC2 loss, we use engineered cells that dynamically reassemble a competent PRC2 coupled with single-cell sequencing from clinical samples. We discover a two-pronged oncogenic process: first, PRC2 loss leads to remodeling of the bivalent chromatin and enhancer landscape, causing the upregulation of developmentally regulated transcription factors that enforce a transcriptional circuit serving as the cell's core vulnerability. Second, PRC2 loss reduces type I interferon signaling and antigen presentation as downstream consequences of hyperactivated Ras and its cross talk with STAT/IRF transcription factors. Mapping of the transcriptional program of these PRC2-deficient tumor cells onto a constructed developmental trajectory of normal Schwann cells reveals that changes induced by PRC2 loss enforce a cellular profile characteristic of a primitive mesenchymal neural crest stem cell.


Subject(s)
Interferon Type I , Neurofibrosarcoma , Carcinogenesis , Chromatin , Humans , Interferon Regulatory Factors/genetics , Interferon Type I/genetics , Neurofibrosarcoma/genetics , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism
8.
Neuro Oncol ; 24(11): 1845-1856, 2022 11 02.
Article in English | MEDLINE | ID: mdl-35788692

ABSTRACT

The wide variety of clinical manifestations of the genetic syndrome neurofibromatosis type 1 (NF1) are driven by overactivation of the RAS pathway. Mitogen-activated protein kinase kinase inhibitors (MEKi) block downstream targets of RAS. The recent regulatory approvals of the MEKi selumetinib for inoperable symptomatic plexiform neurofibromas in children with NF1 have made it the first medical therapy approved for this indication in the United States, the European Union, and elsewhere. Several recently published and ongoing clinical trials have demonstrated that MEKi may have potential benefits for a variety of other NF1 manifestations, and there is broad interest in the field regarding the appropriate clinical use of these agents. In this review, we present the current evidence regarding the use of existing MEKi for a variety of NF1-related manifestations, including tumor (neurofibromas, malignant peripheral nerve sheath tumors, low-grade glioma, and juvenile myelomonocytic leukemia) and non-tumor (bone, pain, and neurocognitive) manifestations. We discuss the potential utility of MEKi in related genetic conditions characterized by overactivation of the RAS pathway (RASopathies). In addition, we review practical treatment considerations for the use of MEKi as well as provide consensus recommendations regarding their clinical use from a panel of experts.


Subject(s)
Mitogen-Activated Protein Kinase Kinases , Neurofibroma, Plexiform , Neurofibromatosis 1 , Protein Kinase Inhibitors , Child , Humans , Consensus , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Neurofibroma, Plexiform/drug therapy , Neurofibromatosis 1/drug therapy , Neurofibromatosis 1/pathology , Protein Kinase Inhibitors/pharmacology
10.
Oncogene ; 41(21): 2973-2983, 2022 05.
Article in English | MEDLINE | ID: mdl-35459782

ABSTRACT

Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with RMS.


Subject(s)
Rhabdomyosarcoma, Embryonal , Rhabdomyosarcoma , Farnesyltranstransferase/genetics , Genes, ras , Humans , Prenylation , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Rhabdomyosarcoma/drug therapy , Rhabdomyosarcoma/genetics
11.
Biochim Biophys Acta Mol Cell Res ; 1869(8): 119264, 2022 08.
Article in English | MEDLINE | ID: mdl-35381293

ABSTRACT

Osteosarcoma (OS) and Pax-Foxo1 fusion negative rhabdomyosarcoma (FN-RMS) are pediatric sarcomas with poor prognoses in patients with advanced disease. In both malignancies, an actin binding protein has been linked to poor prognosis. Integrin adhesion complexes (IACs) are closely coupled to actin networks and IAC-mediated signaling has been implicated in the progression of carcinomas. However, the relationship of IACs and actin cytoskeleton remodeling with cell signaling is understudied in pediatric sarcomas. Here, we tested the hypothesis that IAC dynamics affect ERK activation in OS and FN-RMS cell lines. Adhesion dependence of ERK activation differed among the OS and FN-RMS cells examined. In the OS cell lines, adhesion did not have a consistent effect on phospho-ERK (pERK). ERK phosphorylation in response to fetal calf serum or 1 ng/ml EGF was nearly as efficient in OS cell lines and one FN-RMS cell line in suspension as cells adherent to poly-l-lysine (PL) or fibronectin (FN). By contrast, adhesion to plastic, PL or FN increased ERK phosphorylation and was greater than additive with a 15 min exposure to 1 ng/ml EGF in three FN-RMS cell lines. Increases in pERK were partly dependent on FAK and PAK1/2 but independent of IAC maturation. As far as we are aware, this examination of adhesion-dependent signaling is the first in pediatric sarcomas and has led to the discovery of differences from the prevailing paradigms and differences in the degree of coupling between components in the signaling pathways among the cell lines.


Subject(s)
Epidermal Growth Factor , Sarcoma , Cell Adhesion , Cell Line , Child , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Phosphorylation , Sarcoma/genetics
12.
Dis Model Mech ; 15(2)2022 02 01.
Article in English | MEDLINE | ID: mdl-35178568

ABSTRACT

The RASopathies are a group of disorders caused by a germline mutation in one of the genes encoding a component of the RAS/MAPK pathway. These disorders, including neurofibromatosis type 1, Noonan syndrome, cardiofaciocutaneous syndrome, Costello syndrome and Legius syndrome, among others, have overlapping clinical features due to RAS/MAPK dysfunction. Although several of the RASopathies are very rare, collectively, these disorders are relatively common. In this Review, we discuss the pathogenesis of the RASopathy-associated genetic variants and the knowledge gained about RAS/MAPK signaling that resulted from studying RASopathies. We also describe the cell and animal models of the RASopathies and explore emerging RASopathy genes. Preclinical and clinical experiences with targeted agents as therapeutics for RASopathies are also discussed. Finally, we review how the recently developed drugs targeting RAS/MAPK-driven malignancies, such as inhibitors of RAS activation, direct RAS inhibitors and RAS/MAPK pathway inhibitors, might be leveraged for patients with RASopathies.


Subject(s)
Costello Syndrome , Neurofibromatosis 1 , Noonan Syndrome , Animals , Costello Syndrome/genetics , Failure to Thrive/genetics , Humans , Neurofibromatosis 1/genetics , Noonan Syndrome/genetics , ras Proteins/genetics
13.
Mol Cancer Ther ; 21(1): 170-183, 2022 01.
Article in English | MEDLINE | ID: mdl-34737198

ABSTRACT

Oncogenic RAS signaling is an attractive target for fusion-negative rhabdomyosarcoma (FN-RMS). Our study validates the role of the ERK MAPK effector pathway in mediating RAS dependency in a panel of H/NRASQ61X mutant RMS cells and correlates in vivo efficacy of the MEK inhibitor trametinib with pharmacodynamics of ERK activity. A screen is used to identify trametinib-sensitizing targets, and combinations are evaluated in cells and tumor xenografts. We find that the ERK MAPK pathway is central to H/NRASQ61X dependency in RMS cells; however, there is poor in vivo response to clinically relevant exposures with trametinib, which correlates with inefficient suppression of ERK activity. CRISPR screening points to vertical inhibition of the RAF-MEK-ERK cascade by cosuppression of MEK and either CRAF or ERK. CRAF is central to rebound pathway activation following MEK or ERK inhibition. Concurrent CRAF suppression and MEK or ERK inhibition, or concurrent pan-RAF and MEK/ERK inhibition (pan-RAFi + MEKi/ERKi), or concurrent MEK and ERK inhibition (MEKi + ERKi) all synergistically block ERK activity and induce myogenic differentiation and apoptosis. In vivo assessment of pan-RAFi + ERKi or MEKi + ERKi potently suppress growth of H/NRASQ61X RMS tumor xenografts, with pan-RAFi + ERKi being more effective and better tolerated. We conclude that CRAF reactivation limits the activity of single-agent MEK/ERK inhibitors in FN-RMS. Vertical targeting of the RAF-MEK-ERK cascade and particularly cotargeting of CRAF and MEK or ERK, or the combination of pan-RAF inhibitors with MEK or ERK inhibitors, have synergistic activity and potently suppress H/NRASQ61X mutant RMS tumor growth.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Signaling System/genetics , Proto-Oncogene Proteins B-raf/metabolism , Rhabdomyosarcoma/genetics , Animals , Apoptosis , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Female , Genes, ras , Humans , Mice , Rhabdomyosarcoma/pathology , Transfection
14.
Cell Rep ; 37(8): 110047, 2021 11 23.
Article in English | MEDLINE | ID: mdl-34818552

ABSTRACT

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.


Subject(s)
Neoplasms/genetics , Neoplasms/immunology , Transcriptome/genetics , Adolescent , Antigens, Neoplasm , Cell Line, Tumor , Child , Child, Preschool , Female , Gene Expression/genetics , Gene Expression Profiling/methods , Humans , Immune Checkpoint Proteins/genetics , Immune Checkpoint Proteins/immunology , Immunogenetics/methods , Immunotherapy, Adoptive , Infant , Lymphocytes, Tumor-Infiltrating/immunology , Male , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Transcriptome/immunology , Tumor Microenvironment , Exome Sequencing/methods
15.
J Clin Oncol ; 39(26): 2859-2871, 2021 09 10.
Article in English | MEDLINE | ID: mdl-34166060

ABSTRACT

PURPOSE: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome. PATIENTS AND METHODS: Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed. RESULTS: DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In FOXO1 fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. BCOR (15%), NF1 (15%), and TP53 (13%) mutations were found at a higher incidence than previously reported and TP53 mutations were associated with worse outcomes in both fusion-negative and FOXO1 fusion-positive cases. Interestingly, mutations in RAS isoforms predominated in infants < 1 year (64% of cases). Mutation of MYOD1 was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data. CONCLUSION: This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.


Subject(s)
Biomarkers, Tumor/genetics , Gene Amplification , Gene Deletion , Genomics , INDEL Mutation , Rhabdomyosarcoma, Alveolar/genetics , Rhabdomyosarcoma, Alveolar/therapy , Rhabdomyosarcoma, Embryonal/genetics , Rhabdomyosarcoma, Embryonal/therapy , Adolescent , Adult , Child , Child, Preschool , DNA Mutational Analysis , Databases, Genetic , Disease Progression , Female , Gene Expression Profiling , Genetic Predisposition to Disease , Humans , Infant , Infant, Newborn , Male , Phenotype , Predictive Value of Tests , Progression-Free Survival , Rhabdomyosarcoma, Alveolar/mortality , Rhabdomyosarcoma, Alveolar/pathology , Rhabdomyosarcoma, Embryonal/mortality , Rhabdomyosarcoma, Embryonal/pathology , Risk Assessment , Risk Factors , Time Factors , Transcriptome , United Kingdom , United States , Young Adult
16.
Nat Commun ; 12(1): 192, 2021 01 08.
Article in English | MEDLINE | ID: mdl-33420019

ABSTRACT

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.


Subject(s)
Carcinogenesis/metabolism , Cell Differentiation , MyoD Protein/metabolism , Oncogene Proteins, Fusion/metabolism , Rhabdomyosarcoma/genetics , Rhabdomyosarcoma/metabolism , Snail Family Transcription Factors/metabolism , Animals , Carcinogenesis/genetics , Cell Differentiation/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Female , Gene Expression Regulation, Neoplastic , Gene Knockdown Techniques , Heterografts , Humans , MEF2 Transcription Factors/metabolism , Male , Mice , Mice, SCID , Muscle Development/genetics , MyoD Protein/genetics , Myogenin/metabolism , Oncogene Proteins, Fusion/genetics , Oncogenes , Rhabdomyosarcoma/pathology , Rhabdomyosarcoma, Alveolar/genetics , Rhabdomyosarcoma, Embryonal/genetics , Snail Family Transcription Factors/genetics , Transcriptome
17.
Mol Cancer Ther ; 20(2): 307-319, 2021 02.
Article in English | MEDLINE | ID: mdl-33158997

ABSTRACT

Relapsed pediatric rhabdomyosarcomas (RMS) and neuroblastomas (NBs) have a poor prognosis despite multimodality therapy. In addition, the current standard of care for these cancers includes vinca alkaloids that have severe toxicity profiles, further underscoring the need for novel therapies for these malignancies. Here, we show that the small-molecule rigosertib inhibits the growth of RMS and NB cell lines by arresting cells in mitosis, which leads to cell death. Our data indicate that rigosertib, like the vinca alkaloids, exerts its effects mainly by interfering with mitotic spindle assembly. Although rigosertib has the ability to inhibit oncogenic RAS signaling, we provide evidence that rigosertib does not induce cell death through inhibition of the RAS pathway in RAS-mutated RMS and NB cells. However, the combination of rigosertib and the MEK inhibitor trametinib, which has efficacy in RAS-mutated tumors, synergistically inhibits the growth of an RMS cell line, suggesting a new avenue for combination therapy. Importantly, rigosertib treatment delays tumor growth and prolongs survival in a xenograft model of RMS. In conclusion, rigosertib, through its impact on the mitotic spindle, represents a potential therapeutic for RMS.


Subject(s)
Glycine/analogs & derivatives , Neuroblastoma/drug therapy , Rhabdomyosarcoma/drug therapy , Spindle Apparatus/metabolism , Sulfones/therapeutic use , Apoptosis , Glycine/pharmacology , Glycine/therapeutic use , Humans , Sulfones/pharmacology
18.
Sci Adv ; 6(40)2020 09.
Article in English | MEDLINE | ID: mdl-32998886

ABSTRACT

Adenosine diphosphate-ribosylation factor (Arf) guanosine triphosphatase-activating proteins (GAPs) are enzymes that need to bind to membranes to catalyze the hydrolysis of guanosine triphosphate (GTP) bound to the small GTP-binding protein Arf. Binding of the pleckstrin homology (PH) domain of the ArfGAP With SH3 domain, ankyrin repeat and PH domain 1 (ASAP1) to membranes containing phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is key for maximum GTP hydrolysis but not fully understood. By combining nuclear magnetic resonance, neutron reflectometry, and molecular dynamics simulation, we show that binding of multiple PI(4,5)P2 molecules to the ASAP1 PH domain (i) triggers a functionally relevant allosteric conformational switch and (ii) maintains the PH domain in a well-defined orientation, allowing critical contacts with an Arf1 mimic to occur. Our model provides a framework to understand how binding of the ASAP1 PH domain to PI(4,5)P2 at the membrane may play a role in the regulation of ASAP1.

19.
Nat Commun ; 11(1): 911, 2020 02 14.
Article in English | MEDLINE | ID: mdl-32060262

ABSTRACT

Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis.


Subject(s)
DNA-Binding Proteins/metabolism , Muscle Development , Muscle, Skeletal/metabolism , MyoD Protein/metabolism , Myogenin/metabolism , Rhabdomyosarcoma, Embryonal/metabolism , Transcription Factors/metabolism , Animals , Carcinogenesis , DNA-Binding Proteins/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Mice, SCID , MyoD Protein/genetics , Myoblasts/cytology , Myoblasts/metabolism , Myogenin/genetics , Rhabdomyosarcoma, Embryonal/genetics , Rhabdomyosarcoma, Embryonal/physiopathology , Transcription Factors/genetics
20.
Am J Med Genet A ; 182(4): 866-876, 2020 04.
Article in English | MEDLINE | ID: mdl-31913576

ABSTRACT

RASopathies caused by germline pathogenic variants in genes that encode RAS pathway proteins. These disorders include neurofibromatosis type 1 (NF1), Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFC), and Costello syndrome (CS), and others. RASopathies are characterized by heterogenous manifestations, including congenital heart disease, failure to thrive, and increased risk of cancers. Previous work led by the NCI Pediatric Oncology Branch has altered the natural course of one of the key manifestations of the RASopathy NF1. Through the conduct of a longitudinal cohort study and early phase clinical trials, the MEK inhibitor selumetinib was identified as the first active therapy for the NF1-related peripheral nerve sheath tumors called plexiform neurofibromas (PNs). As a result, selumetinib was granted breakthrough therapy designation by the FDA for the treatment of PN. Other RASopathy manifestations may also benefit from RAS targeted therapies. The overall goal of Advancing RAS/RASopathy Therapies (ART), a new NCI initiative, is to develop effective therapies and prevention strategies for the clinical manifestations of the non-NF1 RASopathies and for tumors characterized by somatic RAS mutations. This report reflects discussions from a February 2019 initiation meeting for this project, which had broad international collaboration from basic and clinical researchers and patient advocates.


Subject(s)
Costello Syndrome/therapy , Ectodermal Dysplasia/therapy , Failure to Thrive/therapy , Heart Defects, Congenital/therapy , Molecular Targeted Therapy , Mutation , Neurofibromatosis 1/therapy , Noonan Syndrome/therapy , ras Proteins/antagonists & inhibitors , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/genetics , Costello Syndrome/genetics , Costello Syndrome/pathology , Ectodermal Dysplasia/genetics , Ectodermal Dysplasia/pathology , Facies , Failure to Thrive/genetics , Failure to Thrive/pathology , Heart Defects, Congenital/genetics , Heart Defects, Congenital/pathology , Humans , Intersectoral Collaboration , National Cancer Institute (U.S.) , Neurofibromatosis 1/genetics , Neurofibromatosis 1/pathology , Noonan Syndrome/genetics , Noonan Syndrome/pathology , Research Report , Signal Transduction , United States , ras Proteins/genetics
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