κB-Ras/RalGAP complexes limit the activity of Ral GTPases, which function in EGFR/Ras signaling. RalGAP expression is down-regulated in pancreatic cancer; however, the role of RalGAP and Ral GTPases in tumor development in vivo remained unclear. Here, we show that pancreatic RalGAPβ deficiency alone is sufficient to induce inflammation and neoplasia in vivo. We identify that this phenotype is triggered by disturbance of the secretory pathway and polarized exocytosis in acinar cells, demonstrating that RalGAP complexes uphold spatial control of Ral activity. We furthermore show that RALGAPβ deficiency results in defective primary cilium assembly, a process required for efficient acinar regeneration upon inflammation. Only primary cilium formation depends on κB-Ras proteins, suggesting that κB-Ras proteins are not essential for all RalGAP complex-controlled processes. In combination with an oncogenic KRAS G12D mutation, RalGAPβ deficiency leads to a dramatic shortening of tumor latency and median survival. Our results highlight an important role of RalGAP/Ral signaling in upholding acinar cell identity and preventing pancreatic cancer development.

Inhibition of the dual function cell cycle and transcription kinase CDK7 is known to affect the viability of cancer cells, but the mechanisms underlying cell line-specific growth control remain poorly understood. Here, we employed a previously developed, highly specific small molecule inhibitor that non-covalently blocks ATP binding to CDK7 (LDC4297) to study the mechanisms underlying cell line-specific growth using a panel of genetically heterogeneous human pancreatic tumor lines as model system. Although LDC4297 diminished both transcription rates and CDK T-loop phosphorylation in a comparable manner, some PDAC lines displayed significantly higher sensitivity than others. We focused our analyses on two well-responsive lines (Mia-Paca2 and Panc89) that, however, showed significant differences in their viability upon extended exposure to limiting LDC4297 concentrations. Biochemical and RNAseq analysis revealed striking differences in gene expression and cell cycle control. Especially the downregulation of a group of cell cycle control genes, among them CDK1/2 and CDC25A/C, correlated well to the observed viability differences in Panc89 versus Mia-Paca2 cells. A parallel downregulation of regulatory pathways supported the hypothesis of a feedforward programmatic effect of CDK7 inhibitors, eventually causing hypersensitivity of PDAC lines.

Rhabdoid tumors (RT) are embryonal neoplasms occurring most frequently in the central nervous system where they are termed atypical teratoid rhabdoid tumor (ATRT). A common hallmark of RT is homozygous loss of the BAF complex subunit SMARCB1. RT patients have a poor prognosis with an overall survival time of 17 months and >60% of patients suffer from relapses. The lack of an optimal treatment strategy could be attributed to the heterogeneity within and between different subgroups of ATRT. Despite the recent advancements in characterizing RT at a molecular level, the cellular origin of RT remains elusive. Thus, this study focused on the identification of the cellular origin of MYC-RT and underlying epigenetic deregulations which account for the cellular heterogeneity in these tumors. We showed that Smarcb1 abrogation in Sox2-positive progenitor cells at E6.5 give rise to RT of the MYC and SHH subgroup in genetically engineered mouse models (GEMM). To uncover distinct cells of origin (COO) for the SHH and MYC subgroups, unbiased computational approaches were used to compare single-cell transcriptomes of GEMMs with single-cell reference maps of murine early embryogenesis. While SHH tumors arise from mid/hindbrain progenitor cells, primordial germ cells (PGCs) emerge as COO of both intracranial and extracranial MYC tumors. PGCs as COO of MYC-RT were validated in vivo by using PGC-specific Smarcb1 knockout mouse model. We further characterized a deregulated transcriptome in MYC-RT compared to PGCs, which is sustained by a subset of epigenetically driven tumor cells. Deregulated expression of genes driving methylation/demethylation processes in MYC tumors and regression of these tumors upon treatment with decitabine in vitro and in vivo, indicates that DNA methylation plays a key role in cellular transformation and development of MYC-RT.

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRasG12D-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling.

The TATA-box Binding Protein (TBP) plays a central role in regulating gene expression and is the first step in the process of pre-initiation complex (PIC) formation on promoter DNA. The lifetime of TBP at the promoter site is controlled by several cofactors including the Modifier of transcription 1 (Mot1), an essential TBP-associated ATPase. Based on ensemble measurements, Mot1 can use adenosine triphosphate (ATP) hydrolysis to displace TBP from DNA and various models for how this activity is coupled to transcriptional regulation have been proposed. However, the underlying molecular mechanism of Mot1 action is not well understood. In this work, the interaction of Mot1 with the DNA/TBP complex was investigated by single-pair Förster resonance energy transfer (spFRET). Upon Mot1 binding to the DNA/TBP complex, a transition in the DNA/TBP conformation was observed. Hydrolysis of ATP by Mot1 led to a conformational change but was not sufficient to efficiently disrupt the complex. SpFRET measurements of dual-labeled DNA suggest that Mot1's ATPase activity primes incorrectly oriented TBP for dissociation from DNA and additional Mot1 in solution is necessary for TBP unbinding. These findings provide a framework for understanding how the efficiency of Mot1's catalytic activity is tuned to establish a dynamic pool of TBP without interfering with stable and functional TBP-containing complexes.

DNA damage can induce autophagy; however, the underlying mechanism remains largely unknown.Here we report that DNA damage leads to autophagy through ATR/Chk1/RhoBmediated lysosomal recruitment of TSC complex and subsequent mTORC1 inhibition.DNA damage caused by ultraviolet light (UV) or alkylating agent methyl methanesulphonate (MMS) results in phosphorylation of small GTPase RhoB by Chk1.Phosphorylation of RhoB enhances its interaction with the TSC2, and promotes its sumoylation by PIAS1, which is required for RhoB/TSC complex to translocate to lysosomes.As a result, mTORC1 is inhibited, and autophagy is activated.Knockout of RhoB severely attenuates lysosomal translocation of TSC complex and the DNA damage-induced autophagy.Reintroducing wildtype but not sumoylation-resistant RhoB into RhoB -/-cells restores the onset of autophagy.Hence, our study identifies a molecular mechanism for translocation of TSC complex to lysosomes in response to DNA damage, which depends on ATR/Chk1-mediated RhoB phosphorylation and sumoylation.

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. PMID: 29930094 Funding information This work was supported by: NIGMS NIH HHS, United States Grant ID: T32 GM087237 NCI NIH HHS, United States Grant ID: DP2 CA195769

Charcot-Marie-Tooth disease (CMT) represents a heterogeneous group of hereditary peripheral neuropathies. We previously reported a CMT locus on chromosome 19q13.3 segregating with the disease in a large Costa Rican family with axonal neuropathy and autosomal recessive pattern of inheritance (CMT2B2). We proposed a homozygous missense variant in the Mediator complex 25 (MED25) gene as causative of the disease. Nevertheless, the fact that no other CMT individuals with MED25 variants were reported to date led us to reevaluate the original family. Using exome sequencing, we now identified a homozygous nonsense variant (p.Gln517ter) in the last exon of an adjacent gene, the polynucleotide kinase 3′-phosphatase (PNKP) gene. It encodes a DNA repair protein recently associated with recessive ataxia with oculomotor apraxia type 4 (AOA4) and microcephaly, seizures, and developmental delay (MCSZ). Subsequently, five unrelated Costa Rican CMT2 subjects initially identified as being heterozygous for the same MED25 variant were found to be also compound heterozygote for PNKP. All were heterozygous for the same variant found homozygous in the large family and a second one previously associated with ataxia (p.Thr408del). Detailed clinical reassessment of the initial family and the new individuals revealed in all an adult-onset slowly progressive CMT2 associated with signs of cerebellar dysfunction such as slurred speech and oculomotor involvement, but neither microcephaly, seizures, nor developmental delay. We propose that PKNP variants are the major causative variant for the CMT2 phenotype in these individuals and that the milder clinical manifestation is due to an allelic effect.

Single-pair Förster resonance energy transfer (spFRET) has become an important tool for investigating conformational dynamics in biological systems. To extract dynamic information from the spFRET traces measured with total internal reflection fluorescence microscopy, we extended the hidden Markov model (HMM) approach. In our extended HMM analysis, we incorporated the photon-shot noise from camera-based systems into the HMM. Thus, the variance in Förster resonance energy transfer (FRET) efficiency of the various states, which is typically a fitted parameter, is explicitly included in the analysis estimated from the number of detected photons. It is also possible to include an additional broadening of the FRET state, which would then only reflect the inherent flexibility of the dynamic biological systems. This approach is useful when comparing the dynamics of individual molecules for which the total intensities vary significantly. We used spFRET with the extended HMM analysis to investigate the dynamics of TATA-box-binding protein (TBP) on promoter DNA in the presence of negative cofactor 2 (NC2). We compared the dynamics of two promoters as well as DNAs of different length and labeling location. For the adenovirus major late promoter, four FRET states were observed; three states correspond to different conformations of the DNA in the TBP-DNA-NC2 complex and a four-state model in which the complex has shifted along the DNA. The HMM analysis revealed that the states are connected via a linear, four-well model. For the H2B promoter, more complex dynamics were observed. By clustering the FRET states detected with the HMM analysis, we could compare the general dynamics observed for the two promoter sequences. We observed that the dynamics from a stretched DNA conformation to a bent conformation for the two promoters were similar, whereas the bent conformation of the TBP-DNA-NC2 complex for the H2B promoter is approximately three times more stable than for the adenovirus major late promoter.

Rhabdoid tumors are caused by the deletion of SMARCB1, whose protein encodes the SMARCB1 subunit of the chromatin remodeling complex SWI/SNF that is involved in global chromatin organization and gene expression control. Simultaneously inhibiting the main players involved in the deregulated transcription machinery is a promising option for preventing exaggerated tumor cell proliferation and survival as it may bypass compensatory mechanisms. In support of this hypothesis, we report efficient impairment of cellular proliferation and strong induction of cell death elicited by inhibition of bromodomain protein BRD4 and transcription kinase CDK9 using small molecular compounds. Combination of both compounds efficiently represses antiapoptotic genes and the oncogene MYC. Our results provide a novel approach for the treatment of RT.

CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb) that controls the transition of RNA polymerase II (RNAPII) into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21) and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed-with significant delay-by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription.

SMARCA4 (BRG1) and SMARCB1 (INI1) are tumor suppressor genes that are crucially involved in the formation of malignant rhabdoid tumors, such as atypical teratoid/rhabdoid tumor (AT/RT). AT/RTs typically affect infants and occur at various sites of the CNS with a particular frequency in the cerebellum. Here, granule neurons and their progenitors represent the most abundant cell type and are known to give rise to a subset of medulloblastoma, a histologically similar embryonal brain tumor. To test how Smarc proteins influence the development of granule neurons and whether this population may serve as cellular origin for AT/RTs, we specifically deleted Smarca4 and Smarcb1 in cerebellar granule cell precursors. Respective mutant mice displayed severe ataxia and motor coordination deficits, but did not develop any tumors. In fact, they suffered from a severely hypoplastic cerebellum due to a significant inhibition of granule neuron precursor proliferation. Molecularly, this was accompanied by an enhanced activity of Wnt/β-catenin signaling that, by itself, is known to cause a nearly identical phenotype. We further used an hGFAP-cre allele, which deleted Smarcb1 much earlier and in a wider neural precursor population, but we still did not detect any tumor formation in the CNS. In summary, our results emphasize cell-type-dependent roles of Smarc proteins and argue against cerebellar granule cells and other progeny of hGFAP-positive neural precursors as the cellular origin for AT/RTs.

Rhabdoid tumors are highly aggressive tumors occurring in infants and very young children. Despite multimodal and intensive therapy prognosis remains poor. Molecular analyses have uncovered several deregulated pathways, among them the CDK4/6‐Rb‐, the WNT‐ and the Sonic hedgehog (SHH) pathways. The SHH pathway is activated in rhabdoid tumors by GLI1 overexpression. Here, we demonstrate that arsenic trioxide (ATO) inhibits tumor cell growth of malignant rhabdoid tumors in vitro and in a mouse xenograft model by suppressing Gli1. Our data uncover ATO as a promising therapeutic approach to improve prognosis for rhabdoid tumor patients.

Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low- and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G1/S and G2/M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis.