Giselle Y. Lopez

Transformation by the ( R )-enantiomer of 2-hydroxyglutarate linked to EGLN activation

The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation. Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). Here we show that (R)-2HG, but not (S)-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the (R)-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy.

Isocitrate dehydrogenase mutations in gliomas: mechanisms, biomarkers and therapeutic target

PURPOSE OF REVIEW Isocitrate dehydrogenases, IDH1 and IDH2, decarboxylate isocitrate to α-ketoglutarate (α-KG) and reduce NADP to NADPH. Point mutations of IDH1 and IDH2 have been discovered in gliomas. IDH mutations cause loss of native enzymatic activities and confer novel activity of converting α-KG to 2-hydroxyglutarate (2-HG). The mechanisms of IDH mutations in gliomagenesis, and their value as diagnostic, prognostic marker and therapeutic target have been extensively studied. This review is to summarize the findings of these studies. RECENT FINDINGS Crystal structural studies revealed conformation changes in mutant IDHs, which may explain the gain of function by mutant IDHs. The product of mutant IDHs, 2-HG, is an inhibitor of α-KG-dependent dioxygenases, which may cause genome-wide epigenetic changes in human gliomas. IDH mutations are a favorable prognostic factor for human glioma and can be used as biomarker for differential diagnosis and subclassification rather than predictor of response to treatment. Preliminary data suggested that inhibiting production of the substrate of mutant IDH enzymes caused slow-down of glioma cell growth. SUMMARY As valuable diagnostic and prognostic markers of human gliomas, there is still a lack of knowledge on biological functions of mutant IDHs, making targeting IDHs in glioma both difficult and unsecured.

IDH1R132 mutation identified in one human melanoma metastasis, but not correlated with metastases to the brain

Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are enzymes which convert isocitrate to alpha-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP+to NADPH). IDH1/2 were recently identified as mutated in a large percentage of progressive gliomas. These mutations occur at IDH1(R132) or the homologous IDH2(R172). Melanomas share some genetic features with IDH1/2-mutated gliomas, such as frequent TP53 mutation. We sought to test whether melanoma is associated with IDH1/2 mutations. Seventy-eight human melanoma samples were analyzed for IDH1(R132) and IDH2(R172) mutation status. A somatic, heterozygous IDH1 c.C394T (p.R132C) mutation was identified in one human melanoma metastasis to the lung. Having identified this mutation in one metastasis, we sought to test the hypothesis that certain selective pressures in the brain environment may specifically favor the cell growth or survival of tumor cells with mutations in IDH1/2, regardless of primary tumor site. To address this, we analyzed IDH1(R132) and IDH2(R172) mutation status 53 metastatic brain tumors, including nine melanoma metastases. Results revealed no mutations in any samples. This lack of mutations would suggest that mutations in IDH1(R132) or IDH2(R172) may be necessary for the formation of tumors in a cell-lineage dependent manner, with a particularly strong selective pressure for mutations in progressive gliomas; this also suggests the lack of a particular selective pressure for growth in brain tissue in general. Studies on the cell-lineages of tumors with IDH1/2 mutations may help clarify the role of these mutations in the development of brain tumors.

EGFR phosphorylation of DCBLD2 recruits TRAF6 and stimulates AKT-promoted tumorigenesis

Aberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation.

Gastrin-Releasing Peptide, Immune Responses, and Lung Disease

Gastrin‐releasing peptide (GRP) is produced by pulmonary neuroendocrine cells (PNECs), with highest numbers of GRP‐positive cells present in fetal lung. Normally GRP‐positive PNECs are relatively infrequent after birth, but PNEC hyperplasia is frequently associated with chronic lung diseases. To address the hypothesis that GRP mediates chronic lung injury, we present the cumulative evidence implicating GRP in bronchopulmonary dysplasia (BPD), the chronic lung disease of premature infants who survive acute respiratory distress syndrome. The availability of well‐characterized animal models of BPD was a critical tool for demonstrating that GRP plays a direct role in the early pathogenesis of this disease. Potential mechanisms by which GRP contributes to injury are analyzed, with the main focus on innate immunity. Autoreactive T cells may contribute to lung injury late in the course of disease. A working model is proposed with GRP triggering multiple cell types in both the innate and adaptive immune systems, promoting cascades culminating in chronic lung disease. These observations represent a paradigm shift in the understanding of the early pathogenesis of BPD, and suggest that GRP blockade could be a novel treatment to prevent this lung disease in premature infants.

Clinico‐pathological description of three paediatric medulloblastoma cases with MLL2/3 gene mutations

Medulloblastoma is the most common paediatric malignant tumour. To identify altered genetic events in a comprehensive manner, we recently performed exome sequencing of a series of medulloblastomas [1]. This study identified mutations in genes involved in chromatin modification in 20% of patients examined, including the myeloid/lymphoid or mixed lineage leukemia (MLL) family genes MLL2 and MLL3, which were not previously known to be associated with medulloblastoma [1]. The majority of those alterations were nonsense or frameshift mutations, indicating that MLL2 and MLL3 are new medulloblastoma tumour suppressor genes [1]. Subsequent exome sequencing studies further validated MLL2 pathway mutations as medulloblastoma driver events [2-4]. In this report, we present detailed histopathological characteristics of three cases with MLL2/3 gene mutations. The male patient discussed in case #1 initially presented as a 5-year-old with a profound frontal headache associated with nausea and vomiting, following receipt of an immunization booster. Five days later the headache returned, and he was noted to have a gait imbalance; a magnetic resonance imaging scan showed a fourth ventricular mass (Figure 1A). Histopathological analysis revealed a medulloblastoma. Therapy consisted of craniospinal irradiation with a posterior fossa boost and chemotherapy consisting of a bone marrow transplant protocol of vincristine, amifostine, cisplatin, and cyclophosphamide. He is now 5 years post therapy without evidence of disease. Figure A) CT demonstrates a hyperdense 3x4 centimeter midline posterior fossa mass that appears to fill the fourth ventricle, containing punctate calcifications and cystic components. B) Haematoxylin and eosin staining of histological sections from Case #1, ... Case #2 is of a male patient who presented as an 11-year-old who began to experience decreased appetite and headaches that awoke him, associated with nausea and vomiting. A computed tomography scan showed marked hydrocephalus with a 4 cm mass in the posterior fossa. Histopathological analysis identified a medulloblastoma. Post-operatively, he underwent cranio-spinal radiation therapy and chemotherapy with vincristine, cisplatin, and cyclophosphamide supplemented with hyperalimentation via gastric tube placement. Now at six years post-diagnosis, he is doing well at recent follow-up. Case #3 is a female patient who presented as a 7-year-old with a three-week history of headache associated with morning nausea and vomiting, dizziness and recent onset of double vision. Radiographic studies revealed an enhancing mass lesion in the fourth ventricle. Axial and sagittal gadolinium-enhanced images demonstrated diffuse leptomeningeal spread of disease. Histopathological analysis disclosed a medulloblastoma. Cytological examination of her post-operative cerebrospinal fluid revealed malignant cytology. The patient began craniospinal X-ray therapy. Three months following initial diagnosis, she died of disease. Postmortem examination of the brain and spinal cord revealed extensive spread along the subarachnoid space of the cerebellum, forebrain, brain stem, and spinal cord. The term medulloblastoma describes a series of heterogeneous brain tumours originating in the cerebellum. This heterogeneity is reflected at two levels: (1) tumours are histopathologically and molecularly distinct; (2) there is a lack of tight correlation between histopathological and molecular subtypes, as tumours within each histopathological subtype are also molecularly heterogeneous. Accordingly, additional genetic alterations, and analysis of the histopathological characteristics associated with them, may provide information for improving tumour subclassification. As a first step toward that purpose, we present three medulloblastoma cases with MLL2/3 mutations. Intriguingly, all three cases demonstrate features of a moderate to severe large-cell/anaplastic subtype (Figure 1B). However, despite these similarities, clinical outcomes varied. Patient #3 had both MLL2 and MLL3 mutations and, unlike the first two patients, had a poor clinical outcome. However, Patient #3 also had MYC amplification (frequently associated with a poor prognosis [5]). The role of MLL2/MLL3 complexes in medulloblastoma are unknown, yet genetic and biological evidence supports a tumour suppressor role [1-4, 6], and studies have identified MLL2/3 gene mutations in a variety of other cancers. MLL family genes are essential for histone modification and play roles in regulating other developmentally critical pathways [7, 8]. One of these pathways impacted by MLL2, retinoic acid signaling [9], may in turn impact orthodenticle homeobox 2 (OTX2) expression [10]. Because increased OTX2 expression was noted (Table 1, Figure 1C), it is tempting to postulate that MLL2/3 inactivation, and the subsequence changes in histone methylation, may present a mechanism for OTX2 overexpression, and thus dysregulation of OTX2-associated pathways. Additionally, it is possible that loss of MLL2/MLL3 function impairs cell differentiation and renders cells susceptible to transformation. All cases presented here demonstrated anaplastic features, geographic necrosis and characteristics of the same histopathological subclass. Molecular subclassification, completed for Cases #1 and #2, revealed Group 3 classification for both cases (classification based on Northcott et al. 2011[11]). Because of the presence of MYC amplification and the extremely poor prognosis, it is likely that the tumour in Case #3 is also a Group 3 tumor. It is expected that improved subclassification will provide guidance for therapy and risk assessment in the clinical setting. MLL2/3 mutations add one more genetic variable for subclassification of medulloblastomas. MLL2/3 pathway mutations were found to be distributed among various histological groups in previous studies [2, 4]. Additionally, studies have found MLL2/3 mutations to be distributed among various molecular subgroups [2-4]. To clarify the subclassification issue, more detailed histopathological analysis of a large number of patients with MLL2/3 mutations will be necessary. We favour the possibility that dysregulation of the MLL2/3 pathway affects the histopathological and clinical characteristics of medulloblastoma, and we suggest an analysis of more cases is warranted.

Supratentorial Tanycytic Ependymoma in an Adult Male: Case Report and Review of Literature

Ependymomas, tumors of the ependymal cells, are very rare and usually present in the pediatric population. Furthermore, there are even rarer variants of ependymomas that can include cellular, papillary, clear cell, and tanycytic subtypes. We present a case of a supratentorial tanycytic ependymoma in an adult male and review the literature in regard to this rare primary central nervous system neoplasm.

EGFR PHOSPHORYLATION OF DCBLD2 RECRUITS TRAF6 AND STIMULATES AKT-PROMOTED TUMORIGENESIS

Aberrant activation of EGFR in human cancers promotes tumorigenesis through stimulation of AKT signaling. Here, we determined that the discoidina neuropilin-like membrane protein DCBLD2 is upregulated in clinical specimens of glioblastomas and head and neck cancers (HNCs) and is required for EGFR-stimulated tumorigenesis. In multiple cancer cell lines, EGFR activated phosphorylation of tyrosine 750 (Y750) of DCBLD2, which is located within a recently identified binding motif for TNF receptor-associated factor 6 (TRAF6). Consequently, phosphorylation of DCBLD2 Y750 recruited TRAF6, leading to increased TRAF6 E3 ubiquitin ligase activity and subsequent activation of AKT, thereby enhancing EGFR-driven tumorigenesis. Moreover, evaluation of patient samples of gliomas and HNCs revealed an association among EGFR activation, DCBLD2 phosphorylation, and poor prognoses. Together, our findings uncover a pathway in which DCBLD2 functions as a signal relay for oncogenic EGFR signaling to promote tumorigenesis and suggest DCBLD2 and TRAF6 as potential therapeutic targets for human cancers that are associated with EGFR activation. Research Article Oncology