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PDF file 57K, Table S2, related to Figure 5. Expression values/read counts from list of genes of RNA-SEQ results shown in Figure 5 and S2

PDF file 115K, Figure legends of Supplemental Figures: S1, S2, S3, S4, S5, S6, S7

PDF file 723K, Figure S1, related to Figure 1. DHX9 is a binding partner of KIF1Bβ apoptotic domain

PDF file 5985K, Figure S6, related to Figure 7. DHX9 is localized to the nucleus in mouse sympathetic neurons but not in human neuroblastoma tumors deficient in KIF1Bbeta

PDF file 60K, Supplemental Method of Kaplan Meier overall survival curves related to Supplementary Figure S6D

PDF file 1540K, Figure S7, related to Figure 7G and Discussion. Model proposing mechanism of KIF1Bβ mediated apoptosis in a developing neuroblast competing for limiting trophic factor NGF

PDF file 408K, Figure S4, related to Figure 4. Quantification of Exportin-2 protein level

PDF file 1031K, Figure S5, related to Figure 6. Induction of KIF1Bβ during NGF withdrawal increases nuclear DHX9 and XAF1 expression

PDF file 4071K, Figure S3, related to Figure 3. KIF1Bbeta promotes DHX9 nuclear localization

PDF file 3837K, Figure S2, related to Figure 2. DHX9 is required for KIF1Bbeta-induced apoptosis

Objective: Vascular endothelial growth factor (VEGF), apart from its predominant roles in angiogenesis, can enhance cancer cellproliferation, but its mechanisms remain elusive. The purpose of the present study was therefore to identify how VEGF regulatescancer cell proliferation. Methods: VEGF effects on cancer cell proliferation were investigated with the VEGF receptor 2 inhibitor, Ki8751, and the breastcancer cell lines, MCF-7 and MDA-MB-231, using flow cytometry, mass spectrometry, immunoblotting, and confocal microscopy.Data were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test. Results: VEGF blockade by Ki8751 significantly reduced cancer cell proliferation, and enhanced breast cancer cell apoptosis.Mass spectrometric analyses revealed that Ki8751 treatment significantly upregulated the expression of mitochondrial proteins,suggesting the involvement of mitochondrial biogenesis. Confocal microscopy and flow cytometric analyses showed that Ki8751treatment robustly increased the mitochondrial masses of both cancer cells, induced endomitosis, and arrested cancer cells in the highaneuploid phase. VEGFR2 knockdown by shRNAs showed similar effects to those of Ki8751, confirming the specificity of Ki8751treatment. Enhanced mitochondrial biogenesis increased mitochondrial oxidative phosphorylation and stimulated reactive oxygenspecies (ROS) production, which induced cancer cell apoptosis. Furthermore, Ki8751 treatment downregulated the phosphorylationof Akt and PGC1α, and translocated PGC1α into the nucleus. The PGC1α alterations increased mitochondrial transcription factorA (TFAM) expression and subsequently increased mitochondrial biogenesis. Conclusions: VEGF enhances cancer cell proliferation by decreasing Akt-PGC1α-TFAM signaling-mediated mitochondrialbiogenesis, ROS production, and cell apoptosis. These findings suggested the anticancer potential of Ki8751 via increasedmitochondrial biogenesis and ROS production.

Background Cell metabolism drives T cell functions, while platelets regulate overall CD4+ T cell immune responses. Objective To investigate if platelets influence cell metabolism and thus regulate CD4+ T effector memory cell (Tem) responses. Methods Human CD4+ Tem cells were activated with αCD3/αCD28 and cultured without or with platelets or platelet-derived mediators. Results Polyclonal stimulation induced rapid and marked Th1 and Treg cell activation of CD4+ Tem cells. Platelet co-culture enhanced Th1 response transiently, while it persistently enhanced Treg cell activation of Tem cells, with an enhancement that plateaued by day 3. Platelet factor 4 (PF4) was the key platelet-derived mediator regulating CD4+ Tem cell responses, which involved cellular metabolisms as indicated by mass spectrometric analyses. PF4 exerted its effects via its receptor CXCR3, attenuated Akt activity, and reduced PGC1α phosphorylation, and resulted in elevations of PGC1α function and mitochondrial transcription factor A (TFAM) synthesis. The latter increased mitochondrial biogenesis, and subsequently enhanced Th1 and Treg responses. Consistent with these observations, inhibition of mitochondrial function by rotenone counteracted the enhancements by recombinant PF4, and TFAM overexpression by TFAM-adenovirus infection mimicked PF4 effects. Furthermore, increased mitochondrial mass elevated oxygen consumption, and enhanced adenosine triphosphate and reactive oxygen species production, which, in turn, stimulated Th1 (T-bet) and Treg (FoxP3) transcription factor expression and corresponding CD4+ T effector cell responses. Conclusions Platelets enhance CD4+ T cell responses of Tem cells through PF4-dependent and Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis. Hence, PF4 may be a promising intervention target of platelet-regulated immune responses.

Childhood neuroblastoma has a remarkable variability in outcome. Age at diagnosis is one of the most important prognostic factors, with children less than 1 year old having favorable outcomes. Here we study single-cell and single-nuclei transcriptomes of neuroblastoma with different clinical risk groups and stages, including healthy adrenal gland. We compare tumor cell populations with embryonic mouse sympatho-adrenal derivatives, and post-natal human adrenal gland. We provide evidence that low and high-risk neuroblastoma have different cell identities, representing two disease entities. Low-risk neuroblastoma presents a transcriptome that resembles sympatho- and chromaffin cells, whereas malignant cells enriched in high-risk neuroblastoma resembles a subtype of TRKB+ cholinergic progenitor population identified in human post-natal gland. Analyses of these populations reveal different gene expression programs for worst and better survival in correlation with age at diagnosis. Our findings reveal two cellular identities and a composition of human neuroblastoma tumors reflecting clinical heterogeneity and outcome., Childhood neuroblastoma can be separated into high and low risk groups, with prognosis depending on age at diagnosis. Here, the authors show that low and high risk neuroblastoma tumours are composed of different cell types with different malignancy potential.

Mitochondria are the main consumers of oxygen within the cell. How mitochondria sense oxygen levels remains unknown. Here we show an oxygen-sensitive regulation of TFAM, an activator of mitochondrial transcription and replication, whose alteration is linked to tumours arising in the von Hippel–Lindau syndrome. TFAM is hydroxylated by EGLN3 and subsequently bound by the von Hippel–Lindau tumour-suppressor protein, which stabilizes TFAM by preventing mitochondrial proteolysis. Cells lacking wild-type VHL or in which EGLN3 is inactivated have reduced mitochondrial mass. Tumorigenic VHL variants leading to different clinical manifestations fail to bind hydroxylated TFAM. In contrast, cells harbouring the Chuvash polycythaemia VHLR200W mutation, involved in hypoxia-sensing disorders without tumour development, are capable of binding hydroxylated TFAM. Accordingly, VHL-related tumours, such as pheochromocytoma and renal cell carcinoma cells, display low mitochondrial content, suggesting that impaired mitochondrial biogenesis is linked to VHL tumorigenesis. Finally, inhibiting proteolysis by targeting LONP1 increases mitochondrial content in VHL-deficient cells and sensitizes therapy-resistant tumours to sorafenib treatment. Our results offer pharmacological avenues to sensitize therapy-resistant VHL tumours by focusing on the mitochondria.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Here we investigate the presence and possible roles of aberrant splicing and splicing-dependent RNA-fusion transcripts in neuroblastoma. In addition, we attend to establish whether the spliceosome can be targeted to treat neuroblastoma. Through analysis of RNA-sequenced neuroblastoma we show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, producing a truncated BAG2-protein which inhibited retinoic acid induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism generating altered gene products in neuroblastoma and show that the spliceosome is a potential target for clinical intervention.

Objective: Vascular endothelial growth factor (VEGF), apart from its predominant roles in angiogenesis, can enhance cancer cell proliferation, but its mechanisms remain elusive. The purpose of the present study was therefore to identify how VEGF regulates cancer cell proliferation. Methods: VEGF effects on cancer cell proliferation were investigated with the VEGF receptor 2 inhibitor, Ki8751, and the breast cancer cell lines, MCF-7 and MDA-MB-231, using flow cytometry, mass spectrometry, immunoblotting, and confocal microscopy. Data were analyzed using one-way analysis of variance followed by Tukey’s multiple comparison test. Results: VEGF blockade by Ki8751 significantly reduced cancer cell proliferation, and enhanced breast cancer cell apoptosis. Mass spectrometric analyses revealed that Ki8751 treatment significantly upregulated the expression of mitochondrial proteins, suggesting the involvement of mitochondrial biogenesis. Confocal microscopy and flow cytometric analyses showed that Ki8751 treatment robustly increased the mitochondrial masses of both cancer cells, induced endomitosis, and arrested cancer cells in the high aneuploid phase. VEGFR2 knockdown by shRNAs showed similar effects to those of Ki8751, confirming the specificity of Ki8751 treatment. Enhanced mitochondrial biogenesis increased mitochondrial oxidative phosphorylation and stimulated reactive oxygen species (ROS) production, which induced cancer cell apoptosis. Furthermore, Ki8751 treatment downregulated the phosphorylation of Akt and PGC1α, and translocated PGC1α into the nucleus. The PGC1α alterations increased mitochondrial transcription factor A (TFAM) expression and subsequently increased mitochondrial biogenesis. Conclusions: VEGF enhances cancer cell proliferation by decreasing Akt-PGC1α-TFAM signaling-mediated mitochondrial biogenesis, ROS production, and cell apoptosis. These findings suggested the anticancer potential of Ki8751 via increased mitochondrial biogenesis and ROS production.

Cell metabolism drives T cell functions, while platelets regulate overall CD4To investigate if platelets influence cell metabolism and thus regulate CD4Human CD4Polyclonal stimulation induced rapid and marked Th1 and Treg cell activation of CD4Platelets enhance CD4

We recently identified pathogenic KIF1Bβ mutations in sympathetic nervous system malignancies that are defective in developmental apoptosis. Here we deleted KIF1Bβ in the mouse sympathetic nervous system and observed impaired sympathetic nervous function and misexpression of genes required for sympathoadrenal lineage differentiation. We discovered that KIF1Bβ is required for nerve growth factor (NGF)-dependent neuronal differentiation through anterograde transport of the NGF receptor TRKA. Moreover, pathogenic KIF1Bβ mutations identified in neuroblastoma impair TRKA transport. Expression of neuronal differentiation markers is ablated in both KIF1Bβ-deficient mouse neuroblasts and human neuroblastomas that lack KIF1Bβ. Transcriptomic analyses show that unfavorable neuroblastomas resemble mouse sympathetic neuroblasts lacking KIF1Bβ independent of MYCN amplification and the loss of genes neighboring KIF1B on chromosome 1p36. Thus, defective precursor cell differentiation, a common trait of aggressive childhood malignancies, is a pathogenic effect of KIF1Bβ loss in neuroblastomas. Furthermore, neuropathy-associated KIF1Bβ mutations impede cargo transport, providing a direct link between neuroblastomas and neurodegeneration.

The paucity of recurrent mutations has hampered efforts to understand and treat neuroblastoma. Alternative splicing and splicing-dependent RNA-fusions represent mechanisms able to increase the gene product repertoire but their role in neuroblastoma remains largely unexplored. Through analysis of RNA-sequenced neuroblastoma we here show that elevated expression of splicing factors is a strong predictor of poor clinical outcome. Furthermore, we identified >900 primarily intrachromosomal fusions containing canonical splicing sites. Fusions included transcripts from well-known oncogenes, were enriched for proximal genes and in chromosomal regions commonly gained or lost in neuroblastoma. As a proof-of-principle that these fusions can generate altered gene products, we characterized a ZNF451-BAG2 fusion, generating a truncated BAG2-protein which inhibited retinoic acid-induced differentiation. Spliceosome inhibition impeded neuroblastoma fusion expression, induced apoptosis and inhibited xenograft tumor growth. Our findings elucidate a splicing-dependent mechanism producing altered gene products in neuroblastoma and suggest that the spliceosome is a tractable therapeutical target.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system. Current treatment modalities do not fully exploit the genetic basis between the different molecular subtypes and little is known about the targets discovered in recent mutational and genetic studies. Neuroblastomas with poor prognosis are often characterized by 1p36 deletion, containing the kinesin gene KIF1B. Its beta isoform, KIF1Bβ, is required for NGF withdrawal-dependent apoptosis, mediated by the induction of XIAP-associated Factor 1 (XAF1). Here, we showed that XAF1 low expression correlates with poor survival and disease status. KIF1Bβ deletion results in loss of XAF1 expression, suggesting that XAF1 is indeed a downstream target of KIF1Bβ. XAF1 silencing protects from NGF withdrawal and from KIF1Bβ-mediated apoptosis. Overexpression of XAF1 impairs tumor progression whereas knockdown of XAF1 promotes tumor growth, suggesting that XAF1 may be a candidate tumor suppressor in neuroblastoma and its associated pathway may be important for developing future interventions.

KIF1Bβ is a candidate 1p36 tumor suppressor that regulates apoptosis in the developing sympathetic nervous system. We found that KIF1Bβ activates the Ca(2+)-dependent phosphatase calcineurin (CN) by stabilizing the CN-calmodulin complex, relieving enzymatic autoinhibition and enabling CN substrate recognition. CN is the key mediator of cellular responses to Ca(2+) signals and its deregulation is implicated in cancer, cardiac, neurodegenerative, and immune disease. We show that KIF1Bβ affects mitochondrial dynamics through CN-dependent dephosphorylation of Dynamin-related protein 1 (DRP1), causing mitochondrial fission and apoptosis. Furthermore, KIF1Bβ actuates recognition of all known CN substrates, implying a general mechanism for KIF1Bβ in Ca(2+) signaling and how Ca(2+)-dependent signaling is executed by CN. Pathogenic KIF1Bβ mutations previously identified in neuroblastomas and pheochromocytomas all fail to activate CN or stimulate DRP1 dephosphorylation. Importantly, KIF1Bβ and DRP1 are silenced in 1p36 hemizygous-deleted neuroblastomas, indicating that deregulation of calcineurin and mitochondrial dynamics contributes to high-risk and poor-prognosis neuroblastoma.

Our principal aim was not to explore the role of EPAS1 /HIF2α in neuroblastoma (NB). Neither do we claim to present conclusive data showing that HIF2α is a bona fide NB tumor suppressor. We simply followed our analysis of the transcriptional response induced by 5-aza-deoxycytidine (AZA)+retinoic acid (RA) treatment to the analysis of large ( n = 498 + 649) sequenced/microarrayed NB cohorts. We show that EPAS1 /HIF2α expression strongly correlates with better outcome and low-risk NB, while being inversely correlated with key features of high-risk NB. Thus, our analysis indicates a possible tumor suppressor role and question whether HIF2α is a NB oncogene (1). The claim that we have not measured HIF2α protein levels in NB (2) is simply … [↵][1]1To whom correspondence should be addressed. Email: johan.holmberg{at}ki.se. [1]: #xref-corresp-1-1

Neuroblastoma is a pediatric cancer characterized by variable outcomes ranging from spontaneous regression to life-threatening progression. High-risk neuroblastoma patients receive myeloablative chemotherapy with hematopoietic stem-cell transplant followed by adjuvant retinoid differentiation treatment. However, the overall survival remains low; hence, there is an urgent need for alternative therapeutic approaches. One feature of high-risk neuroblastoma is the high level of DNA methylation of putative tumor suppressors. Combining the reversibility of DNA methylation with the differentiation-promoting activity of retinoic acid (RA) could provide an alternative strategy to treat high-risk neuroblastoma. Here we show that treatment with the DNA-demethylating drug 5-Aza-deoxycytidine (AZA) restores high-risk neuroblastoma sensitivity to RA. Combined systemic distribution of AZA and RA impedes tumor growth and prolongs survival. Genome-wide analysis of treated tumors reveals that this combined treatment rapidly induces a HIF2α-associated hypoxia-like transcriptional response followed by an increase in neuronal gene expression and a decrease in cell-cycle gene expression. A small-molecule inhibitor of HIF2α activity diminishes the tumor response to AZA+RA treatment, indicating that the increase in HIF2α levels is a key component in tumor response to AZA+RA. The link between increased HIF2α levels and inhibited tumor growth is reflected in large neuroblastoma patient datasets. Therein, high levels of HIF2α, but not HIF1α, significantly correlate with expression of neuronal differentiation genes and better prognosis but negatively correlate with key features of high-risk tumors, such as MYCN amplification. Thus, contrary to previous studies, our findings indicate an unanticipated tumor-suppressive role for HIF2α in neuroblastoma.

Inherited KIF1B loss-of-function mutations in neuroblastomas and pheochromocytomas implicate the kinesin KIF1B as a 1p36.2 tumor suppressor. However, the mechanism of tumor suppression is unknown. We found that KIF1B isoform β (KIF1Bβ) interacts with RNA helicase A (DHX9), causing nuclear accumulation of DHX9, followed by subsequent induction of the proapoptotic XIAP-associated factor 1 (XAF1) and, consequently, apoptosis. Pheochromocytoma and neuroblastoma arise from neural crest progenitors that compete for growth factors such as nerve growth factor (NGF) during development. KIF1Bβ is required for developmental apoptosis induced by competition for NGF. We show that DHX9 is induced by and required for apoptosis stimulated by NGF deprivation. Moreover, neuroblastomas with chromosomal deletion of 1p36 exhibit loss of KIF1Bβ expression and impaired DHX9 nuclear localization, implicating the loss of DHX9 nuclear activity in neuroblastoma pathogenesis. Significance: KIF1Bβ has neuroblastoma tumor-suppressor properties and promotes and requires nuclear-localized DHX9 for its apoptotic function by activating XAF1 expression. Loss of KIF1Bβ alters subcellular localization of DHX9 and diminishes NGF dependence of sympathetic neurons, leading to reduced culling of neural progenitors, and, therefore, might predispose to tumor formation. Cancer Discov; 4(4); 434–51. ©2014 AACR. See related commentary by Bernards, p. 392 This article is highlighted in the In This Issue feature, p. 377

Peppi Koivunen,1 Stuart M Fell,2,3 Wenyun Lu,4 Joshua D Rabinowitz,4 Andrew L Kung,5,6 Susanne Schlisio,2,71Biocenter Oulu, Faculty of Biochemistry and Molecular Medicine, Oulu Center for Cell-Matrix Research, University of Oulu, Oulu, Finland; 2Ludwig Institute for Cancer Research Ltd, Stockholm, Sweden; 3Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden; 4Department of Chemistry and Integrative Genomics, Princeton University, Princeton, NJ, 5Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, 6Department of Pediatrics, Columbia University Medical Center, New York, NY, USA; 7Department of Microbiology and Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Abstract: The cellular response to hypoxia is primarily regulated by the hypoxia-inducible factors (HIFs). HIF-1α is also a major mediator of tumor physiology, and its abundance is correlated with therapeutic resistance in a broad range of cancers. Accumulation of HIF-1α under hypoxia is mainly controlled by the oxygen-sensing HIF prolyl 4-hydroxylases (EGLNs, also known as PHDs). Here, we identified a high level of normoxicHIF-1αprotein in various cancer cell lines. EGLNs require oxygen and 2-oxoglutarate for enzymatic activity. We tested the ability of several cell-permeable 2-oxoglutarate analogs to regulate the abundance ofHIF-1αprotein. We identified 3-oxoglutarate as a potent regulator ofHIF-1αin normoxic conditions. In contrast to 2-oxoglutarate, 3-oxoglutarate decreased the abundance ofHIF-1αprotein in several cancer cell lines in normoxia and diminishedHIF-1αlevels independent of EGLN enzymatic activity. Furthermore, we observed that 3-oxoglutarate was detrimental to cancer cell survival. We show that esterified 3-oxoglutarate, in combination with the cancer chemotherapeutic drug vincristine, induces apoptosis and inhibits tumor growth in vitro and in vivo. Our data imply that a novel treatment strategy targetingHIF-1αin combination with the use of existing cytotoxic agents could serve as potent, future antitumor chemotherapies. Keywords: cancer, EGLN, HIF, hypoxia, 3-oxoglutarate, prolyl hydroxylase

Germline mutations in the von Hippel‐Lindau disease (VHL) and succinate dehydrogenase subunit B (SDHB) genes can cause inherited phaeochromocytoma and/or renal cell carcinoma (RCC). Dysregulation of the hypoxia-inducible factor (HIF) transcription factors has been linked to VHL and SDHB-related RCC; both HIF dysregulation and disordered function of a prolyl hydroxylase domain isoform 3 (PHD3/EGLN3)-related pathway of neuronal apoptosis have been linked to the development of phaeochromocytoma. The 2-oxoglutarate-dependent prolyl hydroxylase enzymes PHD1 (EGLN2), PHD2 (EGLN1) and PHD3 (EGLN3) have a key role in regulating the stability of HIF-a subunits (and hence expression of the HIF-a transcription factors). A germline PHD2 mutation has been reported in association with congenital erythrocytosis and recurrent extra-adrenal phaeochromocytoma. We undertook mutation analysis of PHD1, PHD2 and PHD3 in two cohorts of patients with features of inherited phaeochromocytoma (nZ82) and inherited RCC (nZ64) and no evidence of germline mutations in known susceptibility genes. No confirmed pathogenic mutations were detected suggesting that mutations in these genes are not a frequent cause of inherited phaeochromocytoma or RCC. Endocrine-Related Cancer (2011) 18 73‐83

• Introduction • Lessons from a rare disease • Specificity of function within the EglN prolyl hydroxylases • Failure of EglN3-mediated apoptosis in the genesis of phaeochromocytoma • Understanding the mechanistic basis of EglN3 killing • Connecting EglN activity to the Warburg conundrum • Future directions Oxygen-sensing mechanisms are often dysfunctional in tumours. Oxygen sensing is mediated partly via prolyl hydroxylation. The EglN prolyl hydroxylases are well characterized in regulating the hypoxia inducible factor α (HIF-α) hypoxic response, but also are implicated in HIF-independent processes. EglN3 executes apoptosis in neural precursors during development and failure of EglN3 developmental apoptosis can lead to certain forms of sympathetic nervous system tumours. Mutations in metabolic/mitochondrial enzymes (SDH, FH, IDH) impair EglN activity and predisposes to certain cancers. This is because the EglNs not only require molecular oxygen to execute hydroxylation, but also equally require the electron donor α-ketoglutarate, a metabolite from the Krebs cycle. Therefore EglN enzymes are considered oxygen, and also, metabolic sensors. α-Ketoglutarate is crucial for EglN hydroxylation activity, whereas the metabolites succinate and fumarate are inhibitors of the EglN enzymes. Since EglN activity is dependent upon metabolites that take part in the Krebs cycle, these enzymes are directly tied into the cellular metabolic network. Cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis), an observation that was first made by Otto Warburg in 1924. Despite the striking difference in ATP production, cancer cells might favour aerobic glycolysis to escape from EglN hydroxylation, resulting in the accumulation of oncogenic HIFα and/or resistance to EglN3-mediated apoptosis.

VHL, NF-1, c-Ret, and Succinate Dehydrogenase Subunits B and D act on a developmental apoptotic pathway that is activated when nerve growth factor (NGF) becomes limiting for neuronal progenitor cells and requires the EglN3 prolyl hydroxylase as a downstream effector. Germline mutations of these genes cause familial pheochromocytoma and other neural crest-derived tumors. Using an unbiased shRNA screen we found that the kinesin KIF1Bβ acts downstream from EglN3 and is both necessary and sufficient for neuronal apoptosis when NGF becomes limiting. KIF1Bβ maps to chromosome 1p36.2, which is frequently deleted in neural crest-derived tumors including neuroblastomas. We identified inherited loss-of-function KIF1Bβ missense mutations in neuroblastomas and pheochromocytomas and an acquired loss-of-function mutation in a medulloblastoma, arguing that KIF1Bβ is a pathogenic target of these deletions.

Germline von Hippel-Lindau tumour suppressor gene (VHL) mutations cause renal cell carcinomas, haemangioblastomas and phaeochromocytomas in humans. Mutations in VHL also occur in sporadic renal cell carcinomas. The protein encoded by VHL, VHL, is part of the ubiquitin ligase that downregulates the heterodimeric transcription factor Hif under well-oxygenated conditions. Here we show that acute VHL inactivation causes a senescent-like phenotype in vitro and in vivo. This phenotype was independent of p53 and Hif but dependent on the retinoblastoma protein (Rb) and the SWI2/SNF2 chromatin remodeller p400. Rb activation occurred through a decrease in Skp2 messenger RNA, which resulted in the upregulation of p27 in a Hif-independent fashion. Our results suggest that senescence induced by VHL inactivation is a tumour-suppressive mechanism that must be overcome to develop VHL-associated neoplasias.

SummaryGermline NF1, c-RET, SDH, and VHL mutations cause familial pheochromocytoma. Pheochromocytomas derive from sympathetic neuronal precursor cells. Many of these cells undergo c-Jun-dependent apoptosis during normal development as NGF becomes limiting. NF1 encodes a GAP for the NGF receptor TrkA, and NF1 mutations promote survival after NGF withdrawal. We found that pheochromocytoma-associated c-RET and VHL mutations lead to increased JunB, which blunts neuronal apoptosis after NGF withdrawal. We also found that the prolyl hydroxylase EglN3 acts downstream of c-Jun and is specifically required among the three EglN family members for apoptosis in this setting. Moreover, EglN3 proapoptotic activity requires SDH activity because EglN3 is feedback inhibited by succinate. These studies suggest that failure of developmental apoptosis plays a role in pheochromocytoma pathogenesis.

E2F transcription factors play a critical role in the control of cell cycle progression, regulating the expression of genes involved in DNA replication, DNA repair, mitosis, and cell fate. This involves both positive-acting and negative-acting E2F proteins, the latter group including the E2F6 protein, which has been shown to function as an Rb-independent repressor of E2F-target gene transcription. In an effort to better delineate the context of E2F6 function, including the mechanisms of E2F6 functional specificity, we used chromatin immunoprecipitation assays to assess when and with what genes E2F6 associates during a cell cycle. We find that E2F6 associates specifically with the E2F target genes that are activated at G1/S; this interaction occurs during S phase of the cell cycle. In sharp contrast, E2F6 does not bind to E2F-regulated genes activated at G2/M. In the absence of E2F6, E2F4 can bind to the G1/S-regulated promoters and compensate for loss of E2F6 function. Indeed, inhibition of both E2F4 and E2F6 activity results in specific derepression of these genes during S phase. We conclude that E2F6 functions as a repressor of E2F-dependent transcription during S phase and given the specificity for the G1/S-regulated genes, we propose that E2F6 functions to distinguish G1/S and G2/M transcription during the cell cycle.

Dephosphorylation of SpoIIAA-P by SpoIIE is strictly dependent on the presence of the bivalent metal ions Mn2+ or Mg2+. Replacement by Ala of one of the four Asp residues, invariant in all representatives of protein phosphatase 2C, completely abolished the SpoIIE phosphatase activity in vitro, whilst replacement of the Asp residues by another acidic amino acid, Glu, had varying effects on the activities of the resulting mutated proteins. D610E and D795E exhibited some residual activity while D628E and D745E were without enzymatic activity. The results suggest that the functional model in which metal-associated water molecules are involved in the dephosphorylation reaction catalyzed by human protein phosphatase 2C alpha can also be applied to the bacterial protein phosphatase 2C-like protein.

Developmental apoptosis of neural crest precursors is crucial in determining the final number of terminally differentiated cells such as sympathetic neurons. During neural development, cells undergo apoptosis as NGF becomes limiting. Aberrant developmental apoptosis is implicated in pediatric sympathetic nervous system tumors. When NGF becomes limiting, a developmental apoptotic pathway is activated which requires KIF1Bbeta. KIF1Bbeta is necessary and sufficient for apoptosis during NGF withdrawal. KIF1Bbeta maps to 1p36.2, a frequently deleted region in neuroblastomas, and a neural crest-derived cancer. We identified that KIF1Bbeta-induced apoptosis requires RNA/DNA helicase DHX9. KIF1Bbeta interacts with DHX9 to enhance translocation and accumulation of cytoplasmic DHX9 in the nucleus, resulting in transcription of apoptotic XAF1. Transcription-incompetent DHX9 is unable to potentiate KIF1Bbeta-induced cell death. Knockdown of DHX9 also protects from KIF1Bbeta-induced cell death whereas KIF1Bbeta loss-of-function domains or patient-associated point mutants are unable to translocate and accumulate cytoplasmic DHX9 in the nucleus, impairing XAF1 expression. Furthermore, XAF1 silencing protects from KIF1Bbeta-induced and NGF withdrawal-mediated apoptosis in vitro as well as promotes tumor growth in vivo whereas XAF1 overexpression is necessary and sufficient to induce apoptosis in vitro and delays tumor growth in vivo. Conditional knockout of KIF1Bbeta in the superior cervical ganglia neurons of mouse pups also specifically ablates XAF1 expression in vivo and ex vivo, suggesting that KIF1Bbeta and XAF1 are tightly regulated and act along the same pathway. Clinically, tissue microarray analysis of pre-treatment or post-treatment neuroblastoma patients who are 1p-intact or 1p-deleted, revealed strong prognostic significance of XAF1 expression in disease stratification. Our findings provide a mechanistic understanding on the development of neuroblastoma through unexpected candidates of tumorigenesis. Citation Format: Zhang'e Choo, Rachel Yu Lin Koh, Karin Wallis, Timothy Jia Wei Koh, Chik Hong Kuick, Veronica Sobrado, Amos Hong Pheng Loh, Shui Yen Soh, Susanne Schlisio, Kenneth Tou En Chang, Zhi Xiong Chen. Unusual suspects identified in neuroblastoma: An unexpected tumor suppression pathway. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A47.

The VHL tumor suppressor protein (pVHL) is part of an E3 ubiquitin ligase that targets HIF for destruction. pVHL-defective renal carcinoma cells exhibit increased NF-kappaB activity but the mechanism is unclear. NF-kappaB affects tumorigenesis and therapeutic resistance in some settings. We found that pVHL associates with the NF-kappaB agonist Card9 but does not target Card9 for destruction. Instead, pVHL serves as an adaptor that promotes the phosphorylation of the Card9 C terminus by CK2. Elimination of these sites markedly enhanced Card9's ability to activate NF-kappaB in VHL(+/+) cells, and Card9 siRNA normalized NF-kappaB activity in VHL(-/-) cells and restored their sensitivity to cytokine-induced apoptosis. Furthermore, downregulation of Card9 in VHL(-/-) cancer cells reduced their tumorigenic potential. Therefore pVHL can serve as an adaptor for both a ubiquitin conjugating enzyme and a kinase. The latter activity, which promotes Card9 phosphorylation, links pVHL to control of NF-kappaB activity and tumorigenesis.

To explore mechanisms for specificity of function within the family of E2F transcription factors, we have identified proteins that interact with individual E2F proteins. A two-hybrid screen identified RYBP (Ring1- and YY1-binding protein) as a protein that interacts specifically with the E2F2 and E2F3 family members, dependent on the marked box domain in these proteins. The Cdc6 promoter contains adjacent E2F- and YY1-binding sites, and both are required for promoter activity. In addition, YY1 and RYBP, in combination with either E2F2 or E2F3, can stimulate Cdc6 promoter activity synergistically, dependent on the marked box domain of E2F3. Using chromatin immunoprecipitation assays, we show that both E2F2 and E2F3, as well as YY1 and RYBP, associate with the Cdc6 promoter at G(1)/S of the cell cycle. In contrast, we detect no interaction of E2F1 with the Cdc6 promoter. We suggest that the ability of RYBP to mediate an interaction between E2F2 or E2F3 and YY1 is an important component of Cdc6 activation and provides a basis for specificity of E2F function.