MYC up-regulation confers vulnerability to dual inhibition of CDK12 and CDK13 in high-risk Group 3 medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is the most common cerebellar malignancy during childhood. Among MB, MYC-amplified Group 3 tumors display the worst prognosis. MYC is an oncogenic transcription factor currently thought to be undruggable. Nevertheless, targeting MYC-dependent processes (i.e. transcription and RNA processing regulation) represents a promising approach. METHODS: We have tested the sensitivity of MYC-driven Group 3 MB cells to a pool of transcription and splicing inhibitors that display a wide spectrum of targets. Among them, we focus on THZ531, an inhibitor of the transcriptional cyclin-dependent kinases (CDK) 12 and 13. High-throughput RNA-sequencing analyses followed by bioinformatics and functional analyses were carried out to elucidate the molecular mechanism(s) underlying the susceptibility of Group 3 MB to CDK12/13 chemical inhibition. Data from International Cancer Genome Consortium (ICGC) and other public databases were mined to evaluate the functional relevance of the cellular pathway/s affected by the treatment with THZ531 in Group 3 MB patients. RESULTS: We found that pharmacological inhibition of CDK12/13 is highly selective for MYC-high Group 3 MB cells with respect to MYC-low MB cells. We identified a subset of genes enriched in functional terms related to the DNA damage response (DDR) that are up-regulated in Group 3 MB and repressed by CDK12/13 inhibition. Accordingly, MYC- and CDK12/13-dependent higher expression of DDR genes in Group 3 MB cells limits the toxic effects of endogenous DNA lesions in these cells. More importantly, chemical inhibition of CDK12/13 impaired the DDR and induced irreparable DNA damage exclusively in MYC-high Group 3 MB cells. The augmented sensitivity of MYC-high MB cells to CDK12/13 inhibition relies on the higher elongation rate of the RNA polymerase II in DDR genes. Lastly, combined treatments with THZ531 and DNA damage-inducing agents synergically suppressed viability of MYC-high Group 3 MB cells. CONCLUSIONS: Our study demonstrates that CDK12/13 activity represents an exploitable vulnerability in MYC-high Group 3 MB and may pave the ground for new therapeutic approaches for this high-risk brain tumor.

Non-Canonical Splicing and Its Implications in Brain Physiology and Cancer.

The advance of experimental and computational techniques has allowed us to highlight the existence of numerous different mechanisms of RNA maturation, which have been so far unknown. Besides canonical splicing, consisting of the removal of introns from pre-mRNA molecules, non-canonical splicing events may occur to further increase the regulatory and coding potential of the human genome. Among these, splicing of microexons, recursive splicing and biogenesis of circular and chimeric RNAs through back-splicing and trans-splicing processes, respectively, all contribute to expanding the repertoire of RNA transcripts with newly acquired regulatory functions. Interestingly, these non-canonical splicing events seem to occur more frequently in the central nervous system, affecting neuronal development and differentiation programs with important implications on brain physiology. Coherently, dysregulation of non-canonical RNA processing events is associated with brain disorders, including brain tumours. Herein, we summarize the current knowledge on molecular and regulatory mechanisms underlying canonical and non-canonical splicing events with particular emphasis on cis-acting elements and trans-acting factors that all together orchestrate splicing catalysis reactions and decisions. Lastly, we review the impact of non-canonical splicing on brain physiology and pathology and how unconventional splicing mechanisms may be targeted or exploited for novel therapeutic strategies in cancer.

The pathogenesis of mesothelioma is driven by a dysregulated translatome.

Malignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no 'druggable' driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease.

Long non-coding RNA uc.291 controls epithelial differentiation by interfering with the ACTL6A/BAF complex.

The mechanisms that regulate the switch between epidermal progenitor state and differentiation are not fully understood. Recent findings indicate that the chromatin remodelling BAF complex (Brg1-associated factor complex or SWI/SNF complex) and the transcription factor p63 mutually recruit one another to open chromatin during epidermal differentiation. Here, we identify a long non-coding transcript that includes an ultraconserved element, uc.291, which physically interacts with ACTL6A and modulates chromatin remodelling to allow differentiation. Loss of uc.291 expression, both in primary keratinocytes and in three-dimensional skin equivalents, inhibits differentiation as indicated by epidermal differentiation complex genes down-regulation. ChIP experiments reveal that upon uc.291 depletion, ACTL6A is bound to the differentiation gene promoters and inhibits BAF complex targeting to induce terminal differentiation genes. In the presence of uc.291, the ACTL6A inhibitory effect is released, allowing chromatin changes to promote the expression of differentiation genes. Thus, uc.291 interacts with ACTL6A to modulate chromatin remodelling activity, allowing the transcription of late differentiation genes.

ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining.

Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients' stratification and for the development of personalised therapeutic strategies.

p53 mutants cooperate with HIF-1 in transcriptional regulation of extracellular matrix components to promote tumor progression.

Mutations in the TP53 gene and microenvironmentally driven activation of hypoxia-inducible factor-1 (HIF-1) typically occur in later stages of tumorigenesis. An ongoing challenge is the identification of molecular determinants of advanced cancer pathogenesis to design alternative last-line therapeutic options. Here, we report that p53 mutants influence the tumor microenvironment by cooperating with HIF-1 to promote cancer progression. We demonstrate that in non-small cell lung cancer (NSCLC), p53 mutants exert a gain-of-function (GOF) effect on HIF-1, thus regulating a selective gene expression signature involved in protumorigenic functions. Hypoxia-mediated activation of HIF-1 leads to the formation of a p53 mutant/HIF-1 complex that physically binds the SWI/SNF chromatin remodeling complex, promoting expression of a selective subset of hypoxia-responsive genes. Depletion of p53 mutants impairs the HIF-mediated up-regulation of extracellular matrix (ECM) components, including type VIIa1 collagen and laminin-γ2, thus affecting tumorigenic potential of NSCLC cells in vitro and in mouse models in vivo. Analysis of surgically resected human NSCLC revealed that expression of this ECM gene signature was highly correlated with hypoxic tumors exclusively in patients carrying p53 mutations and was associated with poor prognosis. Our data reveal a GOF effect of p53 mutants in hypoxic tumors and suggest synergistic activities of p53 and HIF-1. These findings have important implications for cancer progression and might provide innovative last-line treatment options for advanced NSCLC.

TAp73 contributes to the oxidative stress response by regulating protein synthesis.

TAp73 is a transcription factor that plays key roles in brain development, aging, and cancer. At the cellular level, TAp73 is a critical homeostasis-maintaining factor, particularly following oxidative stress. Although major studies focused on TAp73 transcriptional activities have indicated a contribution of TAp73 to cellular metabolism, the mechanisms underlying its role in redox homeostasis have not been completely elucidated. Here we show that TAp73 contributes to the oxidative stress response by participating in the control of protein synthesis. Regulation of mRNA translation occupies a central position in cellular homeostasis during the stress response, often by reducing global rates of protein synthesis and promoting translation of specific mRNAs. TAp73 depletion results in aberrant ribosomal RNA (rRNA) processing and impaired protein synthesis. In particular, polysomal profiles show that TAp73 promotes the integration of mRNAs that encode rRNA-processing factors in polysomes, supporting their translation. Concurrently, TAp73 depletion causes increased sensitivity to oxidative stress that correlates with reduced ATP levels, hyperactivation of AMPK, and translational defects. TAp73 is important for maintaining active translation of mitochondrial transcripts in response to oxidative stress, thus promoting mitochondrial activity. Our results indicate that TAp73 contributes to redox homeostasis by affecting the translational machinery, facilitating the translation of specific mitochondrial transcripts. This study identifies a mechanism by which TAp73 contributes to the oxidative stress response and describes a completely unexpected role for TAp73 in regulating protein synthesis.

Ultraconserved long non-coding RNA uc.63 in breast cancer.

Transcribed-ultraconserved regions (T-UCRs) are long non-coding RNAs (lncRNA) encoded by a subset of long ultraconserved stretches in the human genome. Recent studies revealed that the expression of several T-UCRs is altered in cancer and growing evidences underline the importance of T-UCRs in oncogenesis, offering also potential new strategies for diagnosis and prognosis. We found that overexpression of one specific T-UCRs named uc.63 is associated with bad outcome in luminal A subtype of breast cancer patients. uc.63 is localized in the third intron of exportin-1 gene (XPO1) and is transcribed in the same orientation of its host gene. Interestingly, silencing of uc.63 induces apoptosis in vitro. However, silencing of host gene XPO1 does not cause the same effect suggesting that the transcription of uc.63 is independent of XPO1. Our results reveal an important role of uc.63 in promoting breast cancer cells survival and offer the prospect to identify a signature associated with poor prognosis.

Metabolic shift toward oxidative phosphorylation in docetaxel resistant prostate cancer cells.

Drug resistance of cancer cells is recognized as the primary cause of failure of chemotherapeutic treatment in most human cancers. Growing evidences support the idea that deregulated cellular metabolism is linked to such resistance. Indeed, both components of the glycolytic and mitochondrial pathways are involved in altered metabolism linked to chemoresistance of several cancers. Here we investigated the drug-induced metabolic adaptations able to confer advantages to docetaxel resistant prostate cancer (PCa) cells. We found that docetaxel-resistant PC3 cells (PC3-DR) acquire a pro-invasive behavior undergoing epithelial-to-mesenchymal-transition (EMT) and a decrease of both intracellular ROS and cell growth. Metabolic analyses revealed that PC3-DR cells have a more efficient respiratory phenotype than sensitive cells, involving utilization of glucose, glutamine and lactate by the mitochondrial oxidative phosphorylation (OXPHOS). Consequently, targeting mitochondrial complex I by metformin administration, impairs proliferation and invasiveness of PC3-DR cells without effects on parental cells. Furthermore, stromal fibroblasts, which cause a "reverse Warburg" phenotype in PCa cells, reduce docetaxel toxicity in both sensitive and resistant PCa cells. However, re-expression of miR-205, a microRNA strongly down-regulated in EMT and associated to docetaxel resistance, is able to shift OXPHOS to a Warburg metabolism, thereby resulting in an elevated docetaxel toxicity in PCa cells. Taken together, these findings suggest that resistance to docetaxel induces a shift from Warburg to OXPHOS, mandatory for conferring a survival advantage to resistant cells, suggesting that impairing such metabolic reprogramming could be a successful therapeutic approach.

Senescent stroma promotes prostate cancer progression: the role of miR-210.

We focused our interest on senescent human-derived fibroblasts in the progression of prostate cancer. Hypoxic senescent fibroblasts promote prostate cancer aggressiveness by inducing epithelial to mesenchymal transition (EMT) and by secreting energy-rich compounds to support cancer cell growth. Hypoxic senescent fibroblasts additionally increase: i) the recruitment of monocytes and their M2-macrophage polarization, ii) the recruitment of bone marrow-derived endothelial precursor cells, facilitating their vasculogenic ability and iii) capillary morphogenesis, proliferation and invasion of human mature endothelial cells. In addition, we highlight that overexpression of the hypoxia-induced miR-210 in young fibroblasts increases their senescence-associated features and converts them into cancer associated fibroblast (CAF)-like cells, able to promote cancer cells EMT, to support angiogenesis and to recruit endothelial precursor cells and monocytes/macrophages.

From the molecular structure to spectroscopic and material properties: computational investigation of a bent-core nematic liquid crystal.

We present a computational investigation of the nematic phase of the bent-core liquid crystal A131. We use an integrated approach that bridges density functional theory calculations of molecular geometry and torsional potentials to elastic properties through the molecular conformational and orientational distribution function. This unique capability to simultaneously access different length scales enables us to consistently describe molecular and material properties. We can reassign (13)C NMR chemical shifts and analyze the dependence of phase properties on molecular shape. Focusing on the elastic constants we can draw some general conclusions on the unconventional behavior of bent-core nematics and highlight the crucial role of a properly-bent shape.

Carbonic anhydrase IX from cancer-associated fibroblasts drives epithelial-mesenchymal transition in prostate carcinoma cells.

Extracellular acidification, a mandatory feature of several malignancies, has been mainly correlated with metabolic reprogramming of tumor cells toward Warburg metabolism, as well as to the expression of carbonic anydrases or proton pumps by malignant tumor cells. We report herein that for aggressive prostate carcinoma, acknowledged to be reprogrammed toward an anabolic phenotype and to upload lactate to drive proliferation, extracellular acidification is mainly mediated by stromal cells engaged in a molecular cross-talk circuitry with cancer cells. Indeed, cancer-associated fibroblasts, upon their activation by cancer delivered soluble factors, rapidly express carbonic anhydrase IX (CA IX). While expression of CAIX in cancer cells has already been correlated with poor prognosis in various human tumors, the novelty of our findings is the upregulation of CAIX in stromal cells upon activation. The de novo expression of CA IX, which is not addicted to hypoxic conditions, is driven by redox-based stabilization of hypoxia-inducible factor-1. Extracellular acidification due to carbonic anhydrase IX is mandatory to elicit activation of stromal fibroblasts delivered metalloprotease-2 and -9, driving in cancer cells the epithelial-mesenchymal transition epigenetic program, a key event associated with increased motility, survival and stemness. Both genetic silencing and pharmacological inhibition of CA IX (with sulfonamide/sulfamides potent inhibitors) or metalloprotease-9 are sufficient to impede epithelial-mesenchymal transition and invasiveness of prostate cancer cells induced by contact with cancer-associated fibroblasts. We also confirmed in vivo the upstream hierarchical role of stromal CA IX to drive successful metastatic spread of prostate carcinoma cells. These data include stromal cells, as cancer-associated fibroblasts as ideal targets for carbonic anhydrase IX-directed anticancer therapies.

A photosensitive liquid crystal studied by 14N NMR, 2H NMR, and DFT calculations.

An azobenzene derivative, namely diheptylazobenzene, showing the nematic and smectic A liquid crystalline phases, was investigated by means of a combined approach based on NMR and DFT calculations. (14)N NMR quadrupole- and chemical-shift-perturbed spectra were acquired in the whole mesophasic range, providing both experimental quadrupolar splittings and chemical shift anisotropy values. On the same mesogen, deuterium labelled at the α-position of the hydrocarbon chain, (2)H NMR quadrupole-perturbed spectra were recorded. The analysis of these NMR data was performed with the help of ab initio calculations, in vacuo and by taking into account the effect of the anisotropic environment typical of liquid crystals, by using the IEF-PCM model. The geometry optimizations of the azomesogen in the trans and cis configurations were performed by DFT calculations employing the combination of B3LYP functional with the 6-311G(d) basis set. The analysis of experimental NMR data was performed by considering the trans configuration as the most populated one and the corresponding quadrupolar tensors and chemical shielding tensors were determined at the DFT level of theory. The main result of this work is the determination of a relatively high and temperature-dependent molecular biaxiality of the trans state of this azomesogen.

Reciprocal metabolic reprogramming through lactate shuttle coordinately influences tumor-stroma interplay.

Cancer-associated fibroblasts (CAF) engage in tumor progression by promoting the ability of cancer cells to undergo epithelial-mesenchymal transition (EMT), and also by enhancing stem cells traits and metastatic dissemination. Here we show that the reciprocal interplay between CAFs and prostate cancer cells goes beyond the engagement of EMT to include mutual metabolic reprogramming. Gene expression analysis of CAFs cultured ex vivo or human prostate fibroblasts obtained from benign prostate hyperplasia revealed that CAFs undergo Warburg metabolism and mitochondrial oxidative stress. This metabolic reprogramming toward a Warburg phenotype occurred as a result of contact with prostate cancer cells. Intercellular contact activated the stromal fibroblasts, triggering increased expression of glucose transporter GLUT1, lactate production, and extrusion of lactate by de novo expressed monocarboxylate transporter-4 (MCT4). Conversely, prostate cancer cells, upon contact with CAFs, were reprogrammed toward aerobic metabolism, with a decrease in GLUT1 expression and an increase in lactate upload via the lactate transporter MCT1. Metabolic reprogramming of both stromal and cancer cells was under strict control of the hypoxia-inducible factor 1 (HIF1), which drove redox- and SIRT3-dependent stabilization of HIF1 in normoxic conditions. Prostate cancer cells gradually became independent of glucose consumption, while developing a dependence on lactate upload to drive anabolic pathways and thereby cell growth. In agreement, pharmacologic inhibition of MCT1-mediated lactate upload dramatically affected prostate cancer cell survival and tumor outgrowth. Hence, cancer cells allocate Warburg metabolism to their corrupted CAFs, exploiting their byproducts to grow in a low glucose environment, symbiotically adapting with stromal cells to glucose availability.

Integrated NMR and computational study of push-pull NLO probes: interplay of solvent and structural effects.

In this study we combined QM calculations and NMR measurements to understand at a detailed level the complex interplay of structural/electronic properties with the effects of the solvent in the NLO activity of push-pull systems, quantified in terms of variations of the static hyperpolarizability. Different parameters (bond lengths and bond length alternation, vibrational frequencies, electronic charge distribution) are introduced and tested to rationalize both the solvent sensitivity of three molecular systems (namely, p-nitroaniline, ethyl 4-ammino benzoate, and 5-nitro-1H-indole) and the differences among them. This analysis has finally allowed us to establish a clear correlation between the charge transfer behavior of the systems, their NLO properties, and NMR parameters also validating simplified but effective chemical analyses based on resonance limit forms.

Polarity-sensitive fluorescent probes in lipid bilayers: bridging spectroscopic behavior and microenvironment properties.

We have studied the emission features of the fluorescent polarity-sensitive probes known as Prodan and Laurdan in a liquid-crystalline DPPC bilayer. To this purpose, we have combined high-level quantum mechanical electronic structure calculations with a molecular field theory for the positional-orientational-conformational distribution of the probes, in their ground and excited states, inside of the lipid bilayer, taking into account at both levels the nonuniformity and anisotropy of the environment. Thus, we can interpret the features of the fluorescence spectra of Prodan and Laurdan in relation to the position and orientation of their chromophore in the bilayer. We have found that the environment polarity is not sufficient to explain the large red shifts experimentally observed and that specific effects due to hydrogen bonding must be considered. We show that the orientation of the probe is important in determining the accessibility to water of the H-bond-acceptor group; in the case of Laurdan interesting conformational effects are highlighted.

Thiazole orange (TO) as a light-switch probe: a combined quantum-mechanical and spectroscopic study.

A Density Functional Theory (DFT) study of the absorbance and fluorescence emission characteristics of the cyanine thiazole orange (TO) in solution and when intercalated in DNA was carried out in combination with spectrophotometric and spectrofluorometric experiments under different conditions (temperature, concentration, solvent viscosity). T-jump relaxation kinetics of the TO monomer-dimer conversion enabled the thermodynamic parameters of this process to be evaluated. The overall data collected provided information on the features of the "light-switch" by the fluorescent TO and the comparison between experimental and calculated photo-physical properties allowed us to explain and rationalize both shifts and quenching/enhancing effects on fluorescence due to solvation, dimerisation and intercalation in the DNA.

Density-functional-theory investigation of conformations, 13C shielding, and magnetic field interactions in a V-shaped phenylene bis carboxylate homologous series.

Density functional theory (DFT) has been employed in a conformational study of a bent-core PBBC homologous series. IR spectra, GIAO-DFT chemical shielding tensors (CSTs), and molecular susceptibility tensors (MSTs) are theoretically calculated for various optimized conformations established by searching the potential energy surface of each PBBC V-shaped molecule. The IR results could aid the understanding of vibration normal modes, while the MST results can illuminate the alignment properties of the V-shaped molecules in an external magnetic field. CSTs of the aromatic carbons, and those previously measured by the 2D-NMR SUPER technique (with some (13)C NMR peaks reassigned for correctness based on new DFT calculations), were found to be in good agreement. These verified experimental CSTs are then used to revisit the (13)C NMR data to yield structural and local orientational order parameters for two members of the PBBC series. The PBBC series studied using the combined DFT-(13)C NMR approach strongly supports the notion that lesser populated conformational states found by DFT could be reached in the studied mesogens 10DClPBBC and 11ClPBBC upon decreasing temperature, as revealed by the change in the bend angle determined by NMR and identified with those of the DFT molecular structures optimized for various conformers.

Carbon-13 NMR chemical shifts in columnar liquid crystals.

In this article, we present quantum chemical density functional theory (DFT) calculations of the NMR (13)C chemical shift (CS) tensors in 2,3,6,7,10,11-hexahexylthiotriphenylene (HHTT). The DFT calculations are performed on a smaller model molecule where the hexyl chains were reduced to methyl groups (HMTT). These tensors are compared with our previously reported experimental results carried out under magic-angle spinning (MAS) conditions. The phase diagram of HHTT is K ↔ H ↔ D(hd) ↔ I, where H is a helical phase and D(hd) is a columnar liquid crystal. The motivation for the present study was to explain experimentally observed and puzzling thermal history effects, which resulted in different behavior in the helical phase upon cooling and heating. In particular, the CS tensors for the aromatic carbons measured in the helical phase upon heating from the solid phase were essentially unaffected, while the cooling from the columnar liquid crystal resulted in a significant averaging. We investigate the effect on the CS tensors of (i) conformational transitions, and (ii) relative molecular orientations within the columns for dimer and trimer configurations. Finally a motional wobbling (PIZZA) model for the dynamic averaging of the CS tensor in the helical phase is suggested.

What is solvatochromism?

Solvatochromism is commonly used in many fields of chemical and biological research to study bulk and local polarity in macrosystems (membranes, etc.), or even the conformation and binding of proteins. Despite its wide use, solvatochromism still remains a largely unknown phenomenon due to the extremely complex coupling of many different interactions and dynamical processes which characterize it. In this study we analyze the influence of different solvents on the photophysical properties of selected charge-transfer probes (4-AP, PRODAN, and FR0). The purpose is to achieve a microscopic understanding of the intermolecular effects which govern the absorption and fluorescence properties of solvated molecular probes, such as solvent-induced structural modifications, polarization effects, solubility, solute-solvent hydrogen-bonding interactions, and solute aggregation. To this aim we have exploited a time dependent density functional theory (TDDFT) approach coupled to complementary solvation approaches (continuum, discrete and mixed discrete and continuum). Such an integration has allowed us to clearly disentangle the complex interplay between specific and nonspecific interactions of the solvent with the probes and show that strong H-bonding effects not only can lead to large solvatochromic shifts but also can affect the nature of the emitting species with resulting reduction of the quantum yield.