Search

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brain.

Colorectal cancer (CRC) is a multi-stage process initiated through the formation of a benign adenoma, progressing to an invasive carcinoma and finally metastatic spread. Tumour cells must adapt their metabolism to support the energetic and biosynthetic demands associated with disease progression. As such, targeting cancer cell metabolism is a promising therapeutic avenue in CRC. However, to identify tractable nodes of metabolic vulnerability specific to CRC stage, we must understand how metabolism changes during CRC development. Here, we use a unique model system – comprising human early adenoma to late adenocarcinoma. We show that adenoma cells transition to elevated glycolysis at the early stages of tumour progression but maintain oxidative metabolism. Progressed adenocarcinoma cells rely more on glutamine-derived carbon to fuel the TCA cycle, whereas glycolysis and TCA cycle activity remain tightly coupled in early adenoma cells. Adenocarcinoma cells are more flexible with respect to fuel source, enabling them to proliferate in nutrient-poor environments. Despite this plasticity, we identify asparagine (ASN) synthesis as a node of metabolic vulnerability in late-stage adenocarcinoma cells. We show that loss of asparagine synthetase (ASNS) blocks their proliferation, whereas early adenoma cells are largely resistant to ASN deprivation. Mechanistically, we show that late-stage adenocarcinoma cells are dependent on ASNS to support mTORC1 signalling and maximal glycolytic and oxidative capacity. Resistance to ASNS loss in early adenoma cells is likely due to a feedback loop, absent in late-stage cells, allowing them to sense and regulate ASN levels and supplement ASN by autophagy. Together, our study defines metabolic changes during CRC development and highlights ASN synthesis as a targetable metabolic vulnerability in later stage disease.

CYC116 is a selective Aurora kinase inhibitor that has been tested in a Phase I study in patients with advanced solid tumors. Although CYC116 has shown desirable preclinical efficacy, the potential for emergence of resistance has not been explored. We established several CYC116 resistant clones from isogenic HCT116 p53+/+ and HCT116 p53−/− cell line pairs. We also generated resistant clones towards ZM447439 (quinazoline derivative), a model Aurora inhibitor. The selected clones were 10-80 fold resistant to CYC116 and cross-resistant to other synthetic Aurora inhibitors including AZD1152, VX-680, and MLN8054. Resistant clones displayed multidrug resistant phenotypes, tested by using 13 major cytostatics. All clones were highly resistant to etoposide followed by other drugs. Interestingly, all CYC116 clones but not ZM447439 became polyploid. ZM447439, but not CYC116 induced three novel mutations in Aurora B. Leu152Ser significantly affected ZM447439 binding, but not CYC116. Gene expression studies revealed differential expression of more than 200 genes. Some of these genes expression profiles were also observed in CYC116 resistant primary tumors. Bcl-xL (BCL2L1) was found to be overexpressed in CYC116 clones and its knockdown resensitized the p53+/+ resistant clone to CYC116. Finally Bcl-xL overexpressing p53+/+ CYC116 clones were highly sensitive to navitoclax (ABT-263) compared to parent cells. The data shed light on the genetic basis for resistance to Aurora kinase inhibitors which could be used to predict clinical response, to select patients who might benefit from therapy and to suggest suitable drug combinations for a particular patient population.

Targeted inhibition of proteins modulating epigenetic changes is an increasingly important priority in cancer therapeutics, and many small molecule inhibitors are currently being developed. In the case of neuroblastoma (NB), a paediatric solid tumour with a paucity of intragenic mutations, epigenetic deregulation may be especially important. In this study we validate the histone methyltransferase G9a/EHMT2 as being associated with indicators of poor prognosis in NB. Immunological analysis of G9a protein shows it to be more highly expressed in NB cell-lines with MYCN amplification, which is a primary determinant of dismal outcome in NB patients. Furthermore, G9a protein in primary tumours is expressed at higher levels in poorly differentiated/undifferentiated NB, and correlates with high EZH2 expression, a known co-operative oncoprotein in NB. Our functional analyses demonstrate that siRNA-mediated G9a depletion inhibits cell growth in all NB cell lines, but, strikingly, only triggers apoptosis in NB cells with MYCN amplification, suggesting a synthetic lethal relationship between G9a and MYCN. This pattern of sensitivity is also evident when using small molecule inhibitors of G9a, UNC0638 and UNC0642. The increased efficacy of G9a inhibition in the presence of MYCN-overexpression is also demonstrated in the SHEP-21N isogenic model with tet-regulatable MYCN. Finally, using RNA sequencing, we identify several potential tumour suppressor genes that are reactivated by G9a inhibition in NB, including the CLU, FLCN, AMHR2 and AKR1C1-3. Together, our study underlines the under-appreciated role of G9a in NB, especially in MYCN-amplified tumours.

Mutations in the tumor suppressor gene TP53 contribute to the development of approximately half of all human cancers. One mechanism by which mutant p53 (mtp53) acts is through interaction with other transcription factors, which can either enhance or repress the transcription of their target genes. Mtp53 preferentially interacts with the erythroblastosis virus E26 oncogene homologue 2 (ETS2), an ETS transcription factor, and increases its protein stability. To study the mechanism underlying ETS2 degradation, we knocked down ubiquitin ligases known to interact with ETS2. We observed that knockdown of the constitutive photomorphogenesis protein 1 (COP1) and its binding partner De-etiolated 1 (DET1) significantly increased ETS2 stability, and conversely, their ectopic expression led to increased ETS2 ubiquitination and degradation. Surprisingly, we observed that DET1 binds to ETS2 independently of COP1, and we demonstrated that mutation of multiple sites required for ETS2 degradation abrogated the interaction between DET1 and ETS2. Furthermore, we demonstrate that mtp53 prevents the COP1/DET1 complex from ubiquitinating ETS2 and thereby marking it for destruction. Mechanistically, we show that mtp53 destabilizes DET1 and also disrupts the DET1/ETS2 complex thereby preventing ETS2 degradation. Our study reveals a hitherto unknown function in which DET1 mediates the interaction with the substrates of its cognate ubiquitin ligase complex and provides an explanation for the ability of mtp53 to protect ETS2.

Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra-and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues. However, our previous work demonstrated that Wnt3a/Rspo2 treatment of some neuroblastoma cell lines can, paradoxically, decrease c-MYC and MYCN proteins. This prompted us to define the neuroblastoma-specific Wnt3a/Rspo2-driven transcriptome using RNA sequencing, and characterise the accompanying changes in cell biology.Here we report the identification of ninety Wnt target genes, and show that Wnt signalling is upstream of numerous transcription factors and signalling pathways in neuroblastoma. Using live-cell imaging, we show that Wnt signalling can drive differentiation of SK-N-BE(2)-C and SH-SY5Y cell-lines, but, conversely, proliferation of SK-N-AS cells. We show that cell-lines that differentiate show induction of pro-differentiation BMP4 and EPAS1 proteins, which is not apparent in the SK-N-AS cells. In contrast, SK-N-AS cells show increased CCND1, phosphorylated RB and E2F1 in response to Wnt3a/Rspo2, consistent with their proliferative response, and these proteins are not increased in differentiating lines. By meta-analysis of the expression of our 90 genes in primary tumour gene expression databases, we demonstrate discrete expression patterns of our Wnt genes in patient cohorts with different prognosis. Furthermore our analysis reveals interconnectivity within subsets of our Wnt genes, with one subset comprised of novel putative drivers of neuronal differentiation repressed by MYCN. Assessment of β-catenin immunohistochemistry shows high levels of β-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.

Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers., Highlights • The small molecule inhibitor YK-4-279 can trigger p53-independent apoptosis in neuroblastoma cell lines. • YK-4-279 induces mitotic arrest with multipolar, fragmented and unseparated spindles. • YK-4-279 acts differently from paclitaxel and vincristine. • YK-4-279 can overcome resistance to vincristine and synergize with other inhibitors of mitosis.

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value

Drug resistance is the major obstacle to successful cancer therapy. Our study focuses on resistance to Aurora kinase inhibitors tested as anti-cancer drugs in clinical trials. We have used 2D electrophoresis in the pH ranges of 4-7 and 6-11 followed by protein identification using MALDI-TOF/TOF to compare the protein composition of HCT116 colon cancer cells either sensitive to CYC116 and ZM447439 inhibitors or resistant toward these drugs. The analysis also included p53(+/+) and p53(-/-) phenotypes of HCT116 cells. Our findings demonstrate that platelet-activating factor acetylhydrolase and GTP-binding nuclear protein Ran contribute to the development of resistance to ZM447439 only where resistance is related to p53. On the other hand, serine hydroxymethyltransferase was found to promote the tumor growth in cells resistant to CYC116 without the influence of p53. Computer modeling of interaction networks highlighted a direct link of the p53-independent mechanism of resistance to CYC116 with autophagy. Importantly, serine hydroxymethyltransferase, serpin B5, and calretinin represent the target proteins that may help overcome resistance in combination therapies. In addition, serpin B5 and calretinin appear to be candidate biomarkers that may be accessible in patients for monitoring of cancer therapy with ease.

A series of uridine analogues modified at the 5-position with the 5-[alkoxy-(4-nitrophenyl)-methyl] moiety was synthesized. Nucleosides were formed as a mixture of two diastereoisomers, which were separated and tested for their cytotoxic activity in vitro against different cancer cell lines and for antimicrobial activity. Relationships between structure and the above mentioned activities were studied. The cytotoxic activity was slightly increased in some cases by transformation of bases to nucleosides. Depending on the length of the alkyl chain increased cytotoxic and antimicrobial activity were noted. The cytotoxic activity of the nucleosides was not due to cell cycle alterations, DNA and/or RNA synthesis.

© 2015 Macmillan Publishers Limited. All rights reserved. Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. Here we report that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduces NMG expression and substantially depletes nucleotide pools, which attenuates GTP-dependent protein activity and cell invasion. Addition of exogenous guanosine or GTP restores the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. In addition, mtp53 creates a dependency on the nucleoside salvage pathway enzyme deoxycytidine kinase for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53-harbouring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleoside salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development.

Aurora kinases are a recently discovered family of kinases (A, BC) consisting of highly conserved serine\threonine protein kinases found to be involved in multiple mitotic events: regulation of spindle assembly checkpoint pathway, function of centrosomes and cytoskeleton, and cytokinesis. Aberrant expression of Aurora kinases may lead to cancer. For this reason the Aurora kinases are potential targets in the treatment of cancer. In this review we discuss the biology of these kinases: structure, function, regulation and association with cancer.A literature search.Many of the multiple functions of mitosis are mediated by the Aurora kinases. Their aberrant expression can lead to the deregulation of cell division and cancer. For this reason, the Aurora kinases are currently one of the most interesting targets for cancer therapy. Some Aurora kinase inhibitors in the clinic have proven effectively on a wide range of tumor types. The clinical data are very encouraging and promising for development of novel class of structurally different Aurora kinase inhibitors. Hopefully the Aurora kinases will be potentially useful in drug targeted cancer treatment.

MDM2 is an E3 ubiquitin ligase that binds the N-terminus of p53 and promotes its ubiquitin-dependent degradation. Elevated levels of MDM2 due to overexpression or gene amplification can contribute to tumor development by suppressing p53 activity. Since MDM2 is an oncogene, we explored the possibility that other genetic lesions, namely missense mutations, might alter its activities. We selected mutations in MDM2 that reside in one of the 4 key regions of the protein: p53 binding domain, acidic domain, zinc finger domain, and the RING domain. Unexpectedly, we observed that individual mutations in several of these domains compromised the ability of MDM2 to degrade p53. Mutations in the N-terminal p53 binding domain prevented the formation of a p53-MDM2 complex, thereby protecting p53 from degradation. Additionally, as would be predicted, several cancer-associated mutations in the RING finger domain disrupted the ubiquitin ligase activity of MDM2 and prevented p53 degradation. Interestingly, we observed that amino acid substitutions at the same codon differentially affected MDM2 activity. Our data reveal that mutations in this oncogene can have the paradoxical effect of suppressing its activity. Further understanding of how these mutations perturb MDM2 function may yield novel approaches to inhibiting its activity.

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. We found that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduced NMG expression and substantially depleted nucleotide pools. Decreased GTP levels correlated with reduced activation of GTP dependent proteins (GTPases) and reduced invasiveness. Addition of exogenous GTP restored the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. Additionally, mtp53 creates a dependency on the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53 harboring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleotide salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development. Citation Format: Luis A. Martinez, Madhusudhan Kollareddy. Regulation of nucleotide metabolism by mutant p53 contributes to its gain-of-function activities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2108. doi:10.1158/1538-7445.AM2015-2108

Carboplatin, cisplatin and oxaliplatin are structurally similar anticancer drugs and their mechanism of action is permanent coordination to DNA and interfering with DNA repair. However, these drugs have different anticancer activities in human tumors, adverse effects and dose limiting toxicities. Therefore, we performed complex proteomic profiling in order to characterize cellular effects of all three platinum based anticancer drugs. To identify the molecular basis of different pharmacological properties, expressional characteristics of cells exposed to vincristine and daunorubicin were used in parallel as prototypes of drugs with different mechanism of action. T-lymphoblastic leukemia CCRF-CEM cells were treated with 5x IC50 concentrations of individual drugs for time equivalent to half-time to induction apoptosis. Untreated (control) cells were cultured with heavy amino acid labeled media in order to enable relative quantification of peptide expression using SILAC technology, LC-MS/MS and LC-MALDI-TOF/TOF. Results were processed by Proteinscape software equipped by Mascot and Phenyx analytical algorithms. We identified 646 ± 133 proteins by ESI and 312 ± 85 proteins by MALDI in average of all fifteen replicates. Identified results were further processed by in-house written script to remove outlier peptides and to normalize individual runs. All replicates were summed, and statistic analysis was done. To see up- and down- regulated proteins, the hierarchical clustering and PCA analysis was performed. Hierarchical clustering revealed that cisplatin and carboplatin shares the same cluster what is conclusive with literature. Surprisingly, the cluster of daunorubicin and vincristine was close to cisplatin and carboplatin and the oxaliplatin was in the separated cluster from rest of four drugs. PCA analysis revealed 30 proteins with contribution higher than 1 %. The highest contribution (7.5 %) to drug clustering have proteins involved in DNA remodeling. These proteins could be used as specific biomarkers of therapy by platinum based compounds. Citation Format: Petr Dzubak, Tomas Ozdian, Dusan Holub, Gabriela Rylova, Jiri Rehulka, Madhusudhan kollareddy, Daniela Mlcuchova, Marian Hajduch. Proteomic profiling of platinum based anticancer drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4652. doi:10.1158/1538-7445.AM2013-4652

Aurora kinase inhibitors are being developed as anti-cancer drugs and more than 10 small molecules are currently undergoing clinical trials. CYC116, an Aurora kinase inhibitor, discovered and developed by Cyclacel Ltd. is currently in phase I clinical trial in patients with advanced solid tumors. CYC116 showed significant anti-proliferative activity in preclinical studies. However tumor cell resistance mechanisms towards this Aurora kinase inhibitor have not been yet established. We developed several HCT116 p53 wild-type and HCT116 p53 null resistant clones towards CYC116 by exposing the cells to high drug concentrations. In order to compare resistance mechanisms, another Aurora kinase inhibitor, ZM447439, was used as a reference compound. Here we present the preliminary data characterizing CYC116-resistant clones. In MTT cytotoxicity assays all the clones showed CYC116 resistance and cross resistance to other Aurora kinase inhibitors, and to some anticancer drugs, which suggest a multidrug resistance phenotype. However the degree of resistance is variable between the clones. All CYC116-resistant clones irrespective of their p53 background became tetraploid. This was not observed for the ZM447439-resistant clones. PH3 (ser10), an Aurora kinase B phosphorylation site, was not inhibited in all resistant clones, suggesting that this kinase is still active. DNA and RNA synthesis were also not inhibited in these clones. Flow cytometry based studies revealed no up regulation of PgP drug transporter in all CYC116-resistant clones, whereas two HCT116 p53 null ZM447439-resistant clones displayed an increase in PgP levels. Up regulation of MRP was only detected in one HCT116 p53 null CYC116-resistant clone, which expressed high levels of this drug transporter. From the initial data it is obvious that there were no common resistance mechanisms towards the studied Aurora kinase inhibitors. We are continuing the characterization of these resistant clones, which include microarray gene expression studies, 2-D electrophoresis and mass spectrometry studies, Aurora A and B sequencing (target mutations), and other drug transporter (e.g. BCRP & LRP) expression studies. These will further help the understanding of the molecular basis of acquired tumor resistance to CYC116 and will aid us in predicting the clinical response as well as in designing combination treatment regimens for overcoming resistance hence furthering clinical development. This work was kindly supported by the Czech ministry of education. Grant number: MSM6198959216 and LC07017. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 633.

Summary The Aurora kinases (serine/threonine kinases) were discovered in 1995 during studies of mutant alleles associated with abnormal spindle pole formation in Drosophila melanogaster. They soon became the focus of much attention because of their importance in human biology and association with cancer. Aurora kinases are essential for cell division and are primarily active during mitosis. Following their identification as potential targets for cancer chemotherapy, many Aurora kinase inhibitors have been discovered, and are currently under development. The binding modes of Aurora kinase inhibitors to Aurora kinases share specific hydrogen bonds between the inhibitor core and the back bone of the kinase hinge region, while others parts of the molecules may point to different parts of the active site via noncovalent interactions. Currently there are about 30 Aurora kinase inhibitors in different stages of pre-clinical and clinical development. This review summarizes the characteristics and status of Aurora kinase inhibitors in preclinical, Phase I, and Phase II clinical studies, with particular emphasis on the mechanisms of action and resistance to these promising anticancer agents. We also discuss the validity of Aurora kinases as oncology targets, on/off-target toxicities, and other important aspects of overall clinical performance and future of Aurora kinase inhibitors.