Genome-Wide DNA Methylation Profiling Reveals Low Methylation Variability in Moyamoya Disease.

Moyamoya disease (MMD) is a chronic cerebrovascular disorder that can lead to stroke and neurological dysfunctions. Given the largely sporadic nature and the role of gene-environment interactions in various diseases, we examined epigenetic modifications in MMD. We performed genome-wide DNA methylation using Illumina 850 K Methylation EPIC BeadChip, in two racially distinct adult female cohorts: a non-Asian cohort (13 MMD patients and 7 healthy controls) and an Asian cohort (14 MMD patients and 3 healthy controls). An additional external cohort with both sexes (females: 5 MMD patients and 5 healthy controls, males: 5 MMD patients and 5 healthy controls) was included for validation. Our findings revealed strikingly low DNA methylation variability between MMD patients and healthy controls, in both MMD female cohorts. In the non-Asian cohort, only 6 probes showed increased variability versus 647 probes that showed decreased variability. Similarly, in the Asian cohort, the MMD group also displayed a reduced methylation variability across all 2845 probes. Subsequent analysis showed that these differentially variable probes are located on genes involved in key biological processes such as methylation and transcription, DNA repair, cytoskeletal remodeling, natural killer cell signaling, cellular growth, and migration. These findings mark the first observation of low methylation variability in any disease, contrasting with the high variability observed in other disorders. This reduced methylation variability in MMD may hinder patients' adaptability to environmental shifts, such as hemodynamic stress, thereby influencing vascular homeostasis and contributing to MMD pathology. These findings offer new insights into the mechanisms of MMD and potential treatment strategies.

Pharmacologic Inhibition of EIF4A Blocks NRF2 Synthesis to Prevent Osteosarcoma Metastasis.

PURPOSE: Effective therapies for metastatic osteosarcoma (OS) remain a critical unmet need. Targeting mRNA translation in metastatic OS offers a promising option, as selective translation drives the synthesis of cytoprotective proteins under harsh microenvironmental conditions to facilitate metastatic competence. EXPERIMENTAL DESIGN: We assessed the expression levels of eukaryotic translation factors in OS, revealing the high expression of the eukaryotic initiation factor 4A1 (EIF4A1). Using a panel of metastatic OS cell lines and patient-derived xenograft (PDX) models, EIF4A1 inhibitors were evaluated for their ability to block proliferation and reduce survival under oxidative stress, mimicking harsh conditions of the lung microenvironment. Inhibitors were also evaluated for their antimetastatic activity using the ex vivo pulmonary metastasis assay and in vivo metastasis models. Proteomics was performed to catalog which cytoprotective proteins or pathways were affected by EIF4A1 inhibition. RESULTS: CR-1-31B, a rocaglate-based EIF4A1 inhibitor, exhibited nanomolar cytotoxicity against all metastatic OS models tested. CR-1-31B exacerbated oxidative stress and apoptosis when OS cells were co-treated with tert-butylhydroquinone, a chemical oxidative stress inducer. CR-1-31B potently inhibited OS growth in the pulmonary metastasis assay model and in experimental and spontaneous models of OS lung metastasis. Proteomic analysis revealed that tert-butylhydroquinone-mediated upregulation of the NRF2 antioxidant factor was blocked by co-treatment with CR-1-31B. Genetic inactivation of NRF2 phenocopied the antimetastatic activity of CR-1-31B. Finally, the clinical-grade EIF4A1 phase-1-to-2 inhibitor, zotatifin, similarly blocked NRF2 synthesis and the OS metastatic phenotype. CONCLUSIONS: Collectively, our data reveal that pharmacologic targeting of EIF4A1 is highly effective in blocking OS metastasis by blunting the NRF2 antioxidant response.

Efficacy of the Allosteric MEK Inhibitor Trametinib in Relapsed and Refractory Juvenile Myelomonocytic Leukemia: a Report from the Children's Oncology Group.

Juvenile myelomonocytic leukemia (JMML) is a hematologic malignancy of young children caused by mutations that increase Ras signaling output. Hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment, but patients with relapsed or refractory (advanced) disease have dismal outcomes. This phase II trial evaluated the safety and efficacy of trametinib, an oral MEK1/2 inhibitor, in patients with advanced JMML. Ten infants and children were enrolled, and the objective response rate was 50%. Four patients with refractory disease proceeded to HSCT after receiving trametinib. Three additional patients completed all 12 cycles permitted on study and continue to receive off-protocol trametinib without HSCT. The remaining three patients had progressive disease with two demonstrating molecular evolution by the end of cycle 2. Transcriptomic and proteomic analyses provided novel insights into the mechanisms of response and resistance to trametinib in JMML. ClinicalTrials.gov Identifier: NCT03190915. Significance: Trametinib was safe and effective in young children with relapsed or refractory JMML, a lethal disease with poor survival rates. Seven of 10 patients completed the maximum 12 cycles of therapy or used trametinib as a bridge to HSCT and are alive with a median follow-up of 24 months. See related commentary by Ben-Crentsil and Padron, p. 1574.

Comprehensive Profiling of Secreted Factors in the Cerebrospinal Fluid of Moyamoya Disease Patients.

Moyamoya disease (MMD) is characterized by progressive occlusion of the intracranial internal carotid arteries, leading to ischemic and hemorrhagic events. Significant clinical differences exist between ischemic and hemorrhagic MMD. To understand the molecular profiles in the cerebrospinal fluid (CSF) of MMD patients, we investigated 62 secreted factors in both MMD subtypes (ischemic and hemorrhagic) and examined their relationship with preoperative perfusion status, the extent of postoperative angiographic revascularization, and functional outcomes. Intraoperative CSF was collected from 32 control and 71 MMD patients (37 ischemic and 34 hemorrhagic). Multiplex Luminex assay analysis showed that 41 molecules were significantly elevated in both MMD subtypes when compared to controls, including platelet-derived growth factor-BB (PDGF-BB), plasminogen activator inhibitor 1 (PAI-1), and intercellular adhesion molecule 1 (ICAM1) (p < 0.001). Many of these secreted proteins have not been previously reported in MMD, including interleukins (IL-2, IL-4, IL-5, IL-7, IL-8, IL-9, IL-17, IL-18, IL-22, and IL-23) and C-X-C motif chemokines (CXCL1 and CXCL9). Pathway analysis indicated that both MMD subtypes exhibited similar cellular/molecular functions and pathways, including cellular activation, migration, and inflammatory response. While neuroinflammation and dendritic cell pathways were activated in MMD patients, lipid signaling pathways involving nuclear receptors, peroxisome proliferator-activated receptor (PPAR), and liver X receptors (LXR)/retinoid X receptors (RXR) signaling were inhibited. IL-13 and IL-2 were negatively correlated with preoperative cerebral perfusion status, while 7 factors were positively correlated with the extent of postoperative revascularization. These elevated cytokines, chemokines, and growth factors in CSF may contribute to the pathogenesis of MMD and represent potential future therapeutic targets.

Osteosarcoma PDX-Derived Cell Line Models for Preclinical Drug Evaluation Demonstrate Metastasis Inhibition by Dinaciclib through a Genome-Targeted Approach.

PURPOSE: Models to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, multiple models are needed to fully elucidate key aspects of disease biology and to recapitulate clinically relevant phenotypes. EXPERIMENTAL DESIGN: Matched patient samples, patient-derived xenografts (PDX), and PDX-derived cell lines were comprehensively evaluated using whole-genome sequencing and RNA sequencing. The in vivo metastatic phenotype of the PDX-derived cell lines was characterized in both an intravenous and an orthotopic murine model. As a proof-of-concept study, we tested the preclinical effectiveness of a cyclin-dependent kinase inhibitor on the growth of metastatic tumors in an orthotopic amputation model. RESULTS: PDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication in a subset of cell lines. The cell lines were heterogeneous in their metastatic capacity, and heterogeneous tissue tropism was observed in both intravenous and orthotopic models. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden. CONCLUSIONS: The variation in metastasis predilection sites between osteosarcoma PDX-derived cell lines demonstrates their ability to recapitulate the spectrum of the disease observed in patients. We describe here a panel of new osteosarcoma PDX-derived cell lines that we believe will be of wide use to the osteosarcoma research community.

Pre-and post-HSCT use of TKI therapy for fusion-driven B-ALL: A case series of five pediatric, adolescent and young adult patients.

BACKGROUND: The development of tyrosine kinase inhibitors (TKIs) has significantly improved survival rates among patients with Philadelphia chromosome (Ph+) B cell acute lymphoblastic leukemia (B-ALL). Ph-like B-ALL patients lack the BCR::ABL1 translocation but share gene expression profiles with Ph+ B-ALL. The role of TKIs for Ph-like patients pre- and post-hematopoietic stem cell transplantation (HSCT) is not yet clear. CASE: Here we present five cases of pediatric, adolescent, and young adult patients who presented with Ph-like B-ALL or CML in B-ALL blast phase who were treated with personalized TKI regimens pre- and post-HSCT. CONCLUSION: This report describes several novel Ph-like fusions as well as combinations of TKIs with chemotherapy or immunotherapy not yet reported in the pediatric population. This case series provides real-world experience highlighting the potential application of pre- and post-HSCT use of TKIs in a subset of patients with targetable fusions.

UHRF1 is a mediator of KRAS driven oncogenesis in lung adenocarcinoma.

KRAS is a frequent driver in lung cancer. To identify KRAS-specific vulnerabilities in lung cancer, we performed RNAi screens in primary spheroids derived from a Kras mutant mouse lung cancer model and discovered an epigenetic regulator Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). In human lung cancer models UHRF1 knock-out selectively impaired growth and induced apoptosis only in KRAS mutant cells. Genome-wide methylation and gene expression analysis of UHRF1-depleted KRAS mutant cells revealed global DNA hypomethylation leading to upregulation of tumor suppressor genes (TSGs). A focused CRISPR/Cas9 screen validated several of these TSGs as mediators of UHRF1-driven tumorigenesis. In vivo, UHRF1 knock-out inhibited tumor growth of KRAS-driven mouse lung cancer models. Finally, in lung cancer patients high UHRF1 expression is anti-correlated with TSG expression and predicts worse outcomes for patients with KRAS mutant tumors. These results nominate UHRF1 as a KRAS-specific vulnerability and potential target for therapeutic intervention.

Decoding the molecular crosstalk between grafted stem cells and the stroke-injured brain.

Stem cell therapy shows promise for multiple disorders; however, the molecular crosstalk between grafted cells and host tissue is largely unknown. Here, we take a step toward addressing this question. Using translating ribosome affinity purification (TRAP) with sequencing tools, we simultaneously decode the transcriptomes of graft and host for human neural stem cells (hNSCs) transplanted into the stroke-injured rat brain. Employing pathway analysis tools, we investigate the interactions between the two transcriptomes to predict molecular pathways linking host and graft genes; as proof of concept, we predict host-secreted factors that signal to the graft and the downstream molecular cascades they trigger in the graft. We identify a potential host-graft crosstalk pathway where BMP6 from the stroke-injured brain induces graft secretion of noggin, a known brain repair factor. Decoding the molecular interplay between graft and host is a critical step toward deciphering the molecular mechanisms of stem cell action.

Development and characterization of new patient-derived xenograft (PDX) models of osteosarcoma with distinct metastatic capacities.

Models to study metastatic disease in rare cancers are needed to advance preclinical therapeutics and to gain insight into disease biology, especially for highly aggressive cancers with a propensity for metastatic spread. Osteosarcoma is a rare cancer with a complex genomic landscape in which outcomes for patients with metastatic disease are poor. As osteosarcoma genomes are highly heterogeneous, a large panel of models is needed to fully elucidate key aspects of disease biology and to recapitulate clinically-relevant phenotypes. We describe the development and characterization of osteosarcoma patient-derived xenografts (PDXs) and a panel of PDX-derived cell lines. Matched patient samples, PDXs, and PDX-derived cell lines were comprehensively evaluated using whole genome sequencing and RNA sequencing. PDXs and PDX-derived cell lines largely maintained the expression profiles of the patient from which they were derived despite the emergence of whole-genome duplication (WGD) in a subset of cell lines. These cell line models were heterogeneous in their metastatic capacity and their tissue tropism as observed in both intravenous and orthotopic models. As proof-of-concept study, we used one of these models to test the preclinical effectiveness of a CDK inhibitor on the growth of metastatic tumors in an orthotopic amputation model. Single-agent dinaciclib was effective at dramatically reducing the metastatic burden in this model.

A 3D Osteosarcoma Model with Bone-Mimicking Cues Reveals a Critical Role of Bone Mineral and Informs Drug Discovery.

Osteosarcoma (OS) is an aggressive bone cancer for which survival has not improved over three decades. While biomaterials have been widely used to engineer 3D soft-tissue tumor models, the potential of engineering 3D biomaterials-based OS models for comprehensive interrogation of OS pathology and drug discovery remains untapped. Bone is characterized by high mineral content, yet the role of bone mineral in OS progression and drug response remains unknown. Here, a microribbon-based OS model with bone-mimicking compositions is developed to elucidate the role of 3D culture and hydroxyapatite in OS signaling and drug response. The results reveal that hydroxyapatite in 3D is critical to support retention of OS signaling and drug resistance similar to patient tissues and mouse orthotopic tumors. The physiological relevance of this 3D model is validated using four established OS cell lines, seven patient-derived xenograft (PDX) cell lines and two animal models. Integrating 3D OS PDX models with RNA-sequencing identified 3D-specific druggable target, which predicts drug response in mouse orthotopic model. These results establish microribbon-based 3D OS models as a novel experimental tool to enable discovery of novel therapeutics that would be otherwise missed with 2D model and may serve as platforms to study patient-specific OS heterogeneity and drug resistance mechanisms.

Electrical modulation of transplanted stem cells improves functional recovery in a rodent model of stroke.

Stroke is a leading cause of long-term disability worldwide, intensifying the need for effective recovery therapies. Stem cells are a promising stroke therapeutic, but creating ideal conditions for treatment is essential. Here we developed a conductive polymer system for stem cell delivery and electrical modulation in animals. Using this system, electrical modulation of human stem cell transplants improve functional stroke recovery in rodents. Increased endogenous stem cell production corresponds with improved function. Transcriptome analysis identified stanniocalcin 2 (STC2) as one of the genes most significantly upregulated by electrical stimulation. Lentiviral upregulation and downregulation of STC2 in the transplanted stem cells demonstrate that this glycoprotein is an essential mediator in the functional improvements seen with electrical modulation. Moreover, intraventricular administration of recombinant STC2 post-stroke confers functional benefits. In summation, our conductive polymer system enables electrical modulation of stem cells as a potential method to improve recovery and identify important therapeutic targets.

Unique Subtype of Microglia in Degenerative Thalamus After Cortical Stroke.

BACKGROUND AND PURPOSE: Stroke disrupts neuronal functions in both local and remotely connected regions, leading to network-wide deficits that can hinder recovery. The thalamus is particularly affected, with progressive development of neurodegeneration accompanied by inflammatory responses. However, the complexity of the involved inflammatory responses is poorly understood. Herein we investigated the spatiotemporal changes in the secondary degenerative thalamus after cortical stroke, using targeted transcriptome approach in conjunction with histology and flow cytometry. METHODS: Cortical ischemic stroke was generated by permanent occlusion of the left middle cerebral artery in male C57BL6J mice. Neurodegeneration, neuroinflammatory responses, and microglial activation were examined in naive and stroke mice at from poststroke days (PD) 1 to 84, in both ipsilesional somatosensory cortex and ipsilesional thalamus. NanoString neuropathology panel (780 genes) was used to examine transcriptome changes at PD7 and PD28. Fluorescence activated cell sorting was used to collect CD11c+ microglia from ipsilesional thalamus, and gene expressions were validated by quantitative real-time polymerase chain reaction. RESULTS: Neurodegeneration in the thalamus was detected at PD7 and progressively worsened by PD28. This was accompanied by rapid microglial activation detected as early as PD1, which preceded the neurodegenerative changes. Transcriptome analysis showed higher number of differentially expressed genes in ipsilesional thalamus at PD28. Notably, neuroinflammation was the top activated pathway, and microglia was the most enriched cell type. Itgax (CD11c) was the most significantly increased gene, and its expression was highly detected in microglia. Flow-sorted CD11c+ microglia from degenerative thalamus indicated molecular signatures similar to neurodegenerative disease-associated microglia; these included downregulated Tmem119 and CX3CR1 and upregulated ApoE, Axl, LpL, CSF1, and Cst7. CONCLUSIONS: Our findings demonstrate the dynamic changes of microglia after stroke and highlight the importance of investigating stroke network-wide deficits. Importantly, we report the existence of a unique subtype of microglia (CD11c+) with neurodegenerative disease-associated microglia features in the degenerative thalamus after stroke.

NUP98-NSD1 Driven MDS/MPN in Childhood Masquerading as JMML.

Overlapping myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are clonal hematopoietic disorders with features of myelodysplasia and myeloproliferation. The only well-characterized MDS/MPN in children is juvenile myelomonocytic leukemia, an aggressive disorder of infants and toddlers. The biochemical hallmark of this disease is hyperactivation of the Ras/MAPK signaling pathway caused by mutations in Ras pathway genes in more than 90% of patients. Translocations involving receptor tyrosine kinases have been identified in rare cases. Here, we report a 2-year-old patient who presented with MDS/MPN driven by a cytogenetically cryptic NUP98-NSD1 fusion, a translocation thought to exclusively occur in patients with acute myeloid leukemia.

The Mir181ab1 cluster promotes KRAS-driven oncogenesis and progression in lung and pancreas.

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.

Antitumor activity of an engineered decoy receptor targeting CLCF1-CNTFR signaling in lung adenocarcinoma.

Proinflammatory cytokines in the tumor microenvironment can promote tumor growth, yet their value as therapeutic targets remains underexploited. We validated the functional significance of the cardiotrophin-like cytokine factor 1 (CLCF1)-ciliary neurotrophic factor receptor (CNTFR) signaling axis in lung adenocarcinoma (LUAD) and generated a high-affinity soluble receptor (eCNTFR-Fc) that sequesters CLCF1, thereby inhibiting its oncogenic effects. eCNTFR-Fc inhibits tumor growth in multiple xenograft models and in an autochthonous, highly aggressive genetically engineered mouse model of LUAD, driven by activation of oncogenic Kras and loss of Trp53. Abrogation of CLCF1 through eCNTFR-Fc appears most effective in tumors driven by oncogenic KRAS. We observed a correlation between the effectiveness of eCNTFR-Fc and the presence of KRAS mutations that retain the intrinsic capacity to hydrolyze guanosine triphosphate, suggesting that the mechanism of action may be related to altered guanosine triphosphate loading. Overall, we nominate blockade of CLCF1-CNTFR signaling as a novel therapeutic opportunity for LUAD and potentially for other tumor types in which CLCF1 is present in the tumor microenvironment.

Comparative Tumor RNA Sequencing Analysis for Difficult-to-Treat Pediatric and Young Adult Patients With Cancer.

Importance: Pediatric cancers are epigenetic diseases; therefore, considering tumor gene expression information is necessary for a complete understanding of the tumorigenic processes. Objective: To evaluate the feasibility and utility of incorporating comparative gene expression information into the precision medicine framework for difficult-to-treat pediatric and young adult patients with cancer. Design, Setting, and Participants: This cohort study was conducted as a consortium between the University of California, Santa Cruz (UCSC) Treehouse Childhood Cancer Initiative and clinical genomic trials. RNA sequencing (RNA-Seq) data were obtained from the following 4 clinical sites and analyzed at UCSC: British Columbia Children's Hospital (n = 31), Lucile Packard Children's Hospital at Stanford University (n = 80), CHOC Children's Hospital and Hyundai Cancer Institute (n = 46), and the Pacific Pediatric Neuro-Oncology Consortium (n = 24). The study dates were January 1, 2016, to March 22, 2017. Exposures: Participants underwent tumor RNA-Seq profiling as part of 4 separate clinical trials at partner hospitals. The UCSC either downloaded RNA-Seq data from a partner institution for analysis in the cloud or provided a Docker pipeline that performed the same analysis at a partner institution. The UCSC then compared each participant's tumor RNA-Seq profile with more than 11 000 uniformly analyzed tumor profiles from pediatric and young adult patients with cancer, downloaded from public data repositories. These comparisons were used to identify genes and pathways that are significantly overexpressed in each patient's tumor. Results of the UCSC analysis were presented to clinical partners. Main Outcomes and Measures: Feasibility of a third-party institution (UCSC Treehouse Childhood Cancer Initiative) to obtain tumor RNA-Seq data from patients, conduct comparative analysis, and present analysis results to clinicians; and proportion of patients for whom comparative tumor gene expression analysis provided useful clinical and biological information. Results: Among 144 samples from children and young adults (median age at diagnosis, 9 years; range, 0-26 years; 72 of 118 [61.0%] male [26 patients sex unknown]) with a relapsed, refractory, or rare cancer treated on precision medicine protocols, RNA-Seq-derived gene expression was potentially useful for 99 of 144 samples (68.8%) compared with DNA mutation information that was potentially useful for only 34 of 74 samples (45.9%). Conclusions and Relevance: This study's findings suggest that tumor RNA-Seq comparisons may be feasible and highlight the potential clinical utility of incorporating such comparisons into the clinical genomic interpretation framework for difficult-to-treat pediatric and young adult patients with cancer. The study also highlights for the first time to date the potential clinical utility of harmonized publicly available genomic data sets.