Kenneth S. Chen

Somatic mutations in DROSHA and DICER1 impair microRNA biogenesis through distinct mechanisms in Wilms tumours

Wilms tumour is the most common childhood kidney cancer. Here we report the whole-exome sequencing of 44 Wilms tumours, identifying missense mutations in the microRNA (miRNA)-processing enzymes DROSHA and DICER1, and novel mutations in MYCN, SMARCA4 and ARID1A. Examination of tumour miRNA expression, in vitro processing assays and genomic editing in human cells demonstrates that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. DICER1 RNase IIIB mutations preferentially impair processing of miRNAs deriving from the 5′-arm of pre-miRNA hairpins, while DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Both DROSHA and DICER1 mutations impair expression of tumour-suppressing miRNAs, including the let-7 family, important regulators of MYCN, LIN28 and other Wilms tumour oncogenes. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogramme miRNA expression in human cancer and suggest that these defects define a distinct subclass of Wilms tumours.

A Large T Cell Invagination with CD2 Enrichment Resets Receptor Engagement in the Immunological Synapse

T cell activation is driven by the TCR and complemented by costimulation. We have studied the dynamics of ligand-engagement of the costimulatory receptor CD2 in T cell/APC couples. Thousands of ligand-engaged CD2 molecules were included in a large T cell invagination at the center of the cellular interface within 1 min of cell couple formation. The structure and regulation of this invagination shared numerous features with phagocytosis and macropinocytosis. Three observations further characterize the invagination and the inclusion of CD2: 1) numerous ligand-engaged receptors were enriched in and internalized through the T cell invagination, none as prominently as CD2; 2) dissolution of the T cell invagination and CD2 engagement were required for effective proximal T cell signaling; and 3) the T cell invagination was uniquely sensitive to the affinity of the TCR for peptide-MHC. Based on this characterization, we speculate that the T cell invagination, aided by CD2 enrichment, internalizes parts of the TCR signaling machinery to reset T cell signaling upon agonist-mediated, stable APC contact.

DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

BackgroundAberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development.MethodsWe used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing.ResultsMethylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location.ConclusionUnderstanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy.

Hearing loss and vestibular dysfunction among children with cancer after receiving aminoglycosides

Children undergoing cancer therapy often receive aminoglycosides to treat febrile neutropenia or gram‐negative infections. The magnitude of the risk of developing aminoglycoside‐induced ototoxicity and the dose threshold at which that risk significantly increases are unknown.

A novel TP53-KPNA3 translocation defines a de novo treatment-resistant clone in osteosarcoma

Osteosarcoma is the most common primary bone cancer. It can be cured by aggressive surgery and chemotherapy, but outcomes for metastatic or chemoresistant disease remain dismal. Cancer sequencing studies have shown that the p53 pathway is dysregulated in nearly every case, often by translocation; however, no studies of osteosarcoma evolution or intratumor heterogeneity have been done to date. We studied a patient with chemoresistant, metastatic disease over the course of 3 years. We performed exome sequencing on germline DNA and DNA collected from tumor at three separate time points. We compared variant calls and variant allele frequencies between different samples. We identified subclonal mutations in several different genes in the primary tumor sample and found that one particular subclone dominated subsequent tumor samples at relapse. This clone was marked by a novel TP53-KPNA3 translocation and loss of the opposite-strand wild-type TP53 allele. Future research must focus on the functional significance of such clones and strategies to eliminate them.

A Big Catch for Germ Cell Tumour Research

Testicular germ cell tumour (TGCT) is the most common cancer in young men, and the incidence of TGCT is rising worldwide for unknown reasons [1], [2]. Treatments for TGCT are overall quite effective, but at the cost of significant toxicity [3], creating a powerful incentive for the development of more specific, molecularly guided therapies. TGCTs are generally thought to arise from a pluripotent fetal or embryonic germ cell [4]. Reflecting this pluripotency, these tumours can present in a wide range of histologic forms. Seminomas are TGCTs that retain features of pluripotent, primitive germ cells. In contrast, non-seminoma TGCTs exhibit differentiation into forms resembling somatic tissues (teratomas) or extraembryonic structures such as yolk sac (yolk sac tumour) or placenta (choriocarcinoma) (Figure 1). Family members of TGCT patients have a markedly increased risk of developing TGCT, strongly implicating an underlying genetic basis. Recent genome-wide association studies of TGCT have identified SNPs near ATF7IP, BAK1, DMRT1, KITLG, SPRY4, and TERT-CLPTM1L that increase TGCT risk [5]–[7]; however, the mechanisms associating most of these loci with tumourigenesis remain unclear. Figure 1 Animal models of TGCT and correlation to human histologic subtypes. Researchers seeking to identify such mechanisms have been hindered by the limited TGCT animal models available thus far. Two heritable TGCT models have been previously described: one in mouse and one in zebrafish. The mouse model arose through the observation by Leroy Stevens in the late 1950s that testicular teratomas arise spontaneously at low frequency during embryonic development in mice of the 129/Sv strain [8]. This discovery, which ultimately led to the experimental derivation of embryonic stem cells [9], [10], has also proved to be a useful model of teratoma formation. A number of genes have been identified as modifier loci that increase teratoma incidence in the 129/Sv background, including Tp53, Dmrt1, and Dnd1, an RNA-binding protein that is central to germ cell maintenance [11]–[13]. Forward genetic screening led to the discovery of a second in vivo TGCT model. Zebrafish carrying nonsense mutations in alk6b/bmpr1bb, an ortholog of the human bone morphogenetic protein (BMP) receptor BMPR1B, develop TGCTs resembling human seminomas [14], [15]. This finding illuminated the importance of BMP signaling in germ cell development and implicated disruption of BMP signaling in human germ cell tumourigenesis [16]. These two models have provided insight into the roles of pluripotency and differentiation pathways in TGCT development; however, their direct correlation to human tumourigenesis has been limited, as genes such as DND1 and BMPR1B have not been found to be mutated in human TGCTs [14], [17]. In this issue of PLOS Genetics, Basten and coworkers describe a new zebrafish TGCT model with a direct connection to human TGCT mutations [18]. Zebrafish with homozygous mutations in the ciliary protein lrrc50 were previously described to have kidney cysts homologous to human polycystic kidney disease [19]. In this paper, the authors report that male lrrc50 heterozygotes develop testicular tumours late in life with near complete penetrance. Morphologically, these tumours, similar to those arising in bmpr1bb-deficient zebrafish, contain sheets of uniform, undifferentiated germ cells, resembling human seminoma. Loss of heterozygosity at the lrrc50 locus was found in some tumours, consistent with a role of lrrc50 as a tumour suppressor. The authors then conducted a mutational analysis of LRRC50 in a collection of human seminoma samples and identified different mutations in two pedigrees with family history of seminomas, as well as heterozygosity for a different germline LRRC50 mutation in five of 38 patients with sporadic seminomas. LRRC50 is thus the first gene specifically linked to seminoma predisposition in humans. The mutations were found to be functional nulls through their inability to complement lrrc50 knockdown in zebrafish embryos, an elegant example of the utility of the fish system for both gene discovery (forward genetics) and functional genomics (reverse genetics). lrrc50 has heretofore been characterized solely as a ciliary motor protein, and its connection to GCT suppression is intriguing. Cilia have not previously been thought to be present in spermatogonia, but the authors show that normal spermatogonia do indeed have a cilium and that LRRC50 colocalized with the axoneme in spermatogonial stem cells. In addition, the authors provide evidence that its role may not be solely structural by showing that its expression is cell cycle–regulated and that it localizes with condensed chromosomes. The development of both renal cysts in homozygotes and seminomas in heterozygotes may implicate an underlying role for lrrc50 in early genitourinary development. Furthermore, the primary cilium has emerged as a signaling center for Hedgehog, Wnt, and other developmental pathways [20], and this, along with the TGCT phenotype of bmpr1bb mutants, raises the interesting possibility that interplay of these developmental signaling pathways is central to TGCT tumour suppression. Follow-up mechanistic studies will be critical to testing these hypotheses. This paper provides another example of the power of zebrafish forward genetic screens for discovery of genes with novel roles in cancer and other diseases, and is a welcome addition to the list of animal models of TGCT. Some caveats apply when comparing analogous tumours arising in animals separated by a large evolutionary distance; for example, the fish seminomas are benign compared to human seminomas, and may well arise at a different stage of germ cell development. More significantly, neither the fish nor the mouse models currently reflect the biology of human non-seminomatous GCTs, such as embryonal carcinoma, choriocarcinoma, or yolk sac tumour. Clinically the non-seminomas are more likely to be metastatic and resistant to standard treatments, meaning that new models of these GCT subtypes are urgently needed to provide insight into tumour biology, as well as platforms for testing new therapeutic strategies for these cancers.

Mutations in microRNA processing genes in Wilms tumors derepress the IGF2 regulator PLAG1

Many childhood Wilms tumors are driven by mutations in the microRNA biogenesis machinery, but the mechanism by which these mutations drive tumorigenesis is unknown. Here we show that the transcription factor pleomorphic adenoma gene 1 (PLAG1) is a microRNA target gene that is overexpressed in Wilms tumors with mutations in microRNA processing genes. Wilms tumors can also overexpress PLAG1 through copy number alterations, and PLAG1 expression correlates with prognosis in Wilms tumors. PLAG1 overexpression accelerates growth of Wilms tumor cells in vitro and induces neoplastic growth in the developing mouse kidney in vivo. In both settings, PLAG1 transactivates insulin-like growth factor 2 (IGF2), a key Wilms tumor oncogene, and drives mammalian target of rapamycin complex 1 (mTORC1) signaling. These data link microRNA impairment to the PLAG1-IGF2 pathway, providing new insight into the manner in which common Wilms tumor mutations drive disease pathogenesis.

Invited reply to Amikacin ototoxicity in children with cancer: The problem is the administration schedule

To the Editor: We appreciate the comments of Drs. Castagnola, Dallorso, and Haupt [1], concerning the effect of administration schedule on aminoglycoside-associated toxicity. At the time when the patients in our study were exposed to amikacin, thrice-daily dosing was the standard starting regimen in our medical center. In fact, since that time we have moved to once-daily dosing as a standard, based on meta-analyses demonstrating improvement in efficacy and nephrotoxicity, as stated in their letter [2,3]. These meta-analyses also demonstrated a non-significant trend toward improvement in ototoxicity. We concur that changing to once-daily aminoglycoside dosing is important for minimizing toxicity, and that future studies of aminoglycoside ototoxicity should focus on this new standard of care. Nevertheless, toxicity remains a concern for children with prolonged aminoglycoside exposure regardless of dosing regimen, and pediatric oncologists should be aware of the long-term toxicities of supportive care measures such as antibiotics.

Invited reply to Amikacin ototoxicity in children with cancer

To the Editor: We appreciate the comments of Drs. Castagnola, Dallorso, and Haupt [1], concerning the effect of administration schedule on aminoglycoside-associated toxicity. At the time when the patients in our study were exposed to amikacin, thrice-daily dosing was the standard starting regimen in our medical center. In fact, since that time we have moved to once-daily dosing as a standard, based on meta-analyses demonstrating improvement in efficacy and nephrotoxicity, as stated in their letter [2,3]. These meta-analyses also demonstrated a non-significant trend toward improvement in ototoxicity. We concur that changing to once-daily aminoglycoside dosing is important for minimizing toxicity, and that future studies of aminoglycoside ototoxicity should focus on this new standard of care. Nevertheless, toxicity remains a concern for children with prolonged aminoglycoside exposure regardless of dosing regimen, and pediatric oncologists should be aware of the long-term toxicities of supportive care measures such as antibiotics.

EGF receptor and mTORC1 are novel therapeutic targets in nonseminomatous germ cell tumors

Germ cell tumors (GCT) are malignant tumors that arise from pluripotent embryonic germ cells and occur in children and young adults. GCTs are treated with cisplatin-based regimens which, while overall effective, fail to cure all patients and cause significant adverse late effects. The seminoma and nonseminoma forms of GCT exhibit distinct differentiation states, clinical behavior, and response to treatment; however, the molecular mechanisms of GCT differentiation are not fully understood. We tested whether the activity of the mTORC1 and MAPK pathways were differentially active in the two classes of GCT. Here we show that nonseminomatous germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma) from both children and adults display activation of the mTORC1 pathway, while seminomas do not. In seminomas, high levels of REDD1 may negatively regulate mTORC1 activity. In NSGCTs, on the other hand, EGF and FGF2 ligands can stimulate mTORC1 and MAPK signaling, and members of the EGF and FGF receptor families are more highly expressed. Finally, proliferation of NSGCT cells in vitro and in vivo is significantly inhibited by combined treatment with the clinically available agents erlotinib and rapamycin, which target EGFR and mTORC1 signaling, respectively. These results provide an understanding of the signaling network that drives GCT growth and a rationale for therapeutic targeting of GCTs with agents that antagonize the EGFR and mTORC1 pathways. Mol Cancer Ther; 17(5); 1079–89. ©2018 AACR.