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Mutant p53(s) are widely considered as oncogenes and promote several gain-of-function oncogenic activities. p53 mutations correlate with higher rates of metastasis and poor survival, therefore, it is paramount to inhibit mutant p53 protein either directly or indirectly. Although some compounds have been developed, none of them have achieved a desirable level of specificity. Some of these compounds only targeted specific mutations. In search of less-toxic compounds, we tested plant-derived compounds on mutant p53 triple-negative breast cancer cell lines. Here, we show that the compounds tested reduced the protein levels of one of the more frequent oncogenic p53 mutants (R249S, hot spot mutation), and its important targets that promote invasion and metastasis, including GMPS and IMPDH1. All compounds tested perturbed the invasion potential of the breast cancer cell line. These compounds downregulated several nucleotide metabolism genes (NMGs) which are essential for cell cycle progression. We observed S-phase arrest correlating to reduced cell proliferation and increased replication stress. Moreover, we also show a reduction of key ETS transcription family members including ETS2, ETS1, ETV1, and ETV4, which are involved in invasion and metastasis. We propose that these compounds may inhibit invasion by interfering with multiple pathways. Our findings exemplify that these tested compounds could inhibit invasion and cell growth in TNBC in a nucleotide-dependent manner.

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.

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.

© 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.

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.