Oncotarget

Effective treatment for Pancreatic Cancer remains a major challenge due to its resistance to radiation/chemotherapy and poor drug permeability. Moreover, treatment induced normal tissue toxicity, mainly to the duodenum and gastrointestinal epithelium, is common and is a dose limiting event, while toxicity to the pancreas is relatively rare1-3. Gastrointestinal toxicity, however, often results in interruption, reduction or premature withdrawal of anti-cancer therapy which is a very significant factor impacting the overall survival of patients being treated. Therefore, development of a therapeutic strategy to selectively sensitize tumor tissue without inducing normal tissue toxicity is important. In this manuscript, we show that the novel small molecule BCN057 can modulate chemo-sensitivity of oncogenic RAS pancreatic cancer cells while conversely protecting normal intestinal epithelium from off target toxicity. In particular, BCN 057 protects Lgr5 positive intestinal stem cells, thereby preserving barrier function. Further, it is demonstrated that BCN057 inhibits GSK3{beta} and thereby induces a pro-apoptotic phosphorylation pattern on c-Jun in KRAS G12D mutant pancreatic cancer cells (Panc-1) leading to the restoration of PTEN expression and consequent apoptosis. This appears to be a new mechanistic observation for the oncogenic RAS phenotype. Lastly, concurrent with its GSK3{beta} inhibition, BCN057 is a small molecule inhibitor of PD-1 expression on human T-lymphocytes co-cultured with human pancreatic cancer cells. In summary, BCN057 can promote synthetic lethality specifically to malignant cells and therefore should be considered to improve the therapeutic ratio in pancreatic and epithelial cancer treatment in conjunction with chemotherapy and radiation.

In the era of personalized medicine as well as precision medicine, targeted therapy has become an integral part of cancer treatment in conjunction with conventional chemo- and radiotherapy. We designed aptamer-siRNA chimeras that can specifically target cancers expressing EpCAM, a stem cell marker and deliver the specific siRNA required for therapy response. The siRNAs were chosen against PLK1, BCL2 and STAT3 as these oncogenes play prominent role in tumour progression of several cancers. Targeted delivery of EpCAM-siRNA chimeras resulted in cell death in several cancer cell lines such as cancers of the breast, lung, head and neck, liver and retinoblastoma. In vivo analysis of EpCAM-siRNA chimera mediated silencing on RB xenografts tumour model showed increased tumor reduction in all the three EpCAM-siRNA treated conditions. However, regulation of PLK1 exhibited higher efficacy in tumour reduction. Therefore. We studied signaling mechanism using global phosphoproteomics analysis. An increased P53 mediated downstream signalling pathway might have enabled increased apoptosis in the cancer cells. In conclusion, this study demonstrated the efficacy of EpCAM aptamer chimeras coupled to siRNA gene silencing for targeted anti-cancer therapy.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC=\"FIGDIR/small/656199v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (48K):\norg.highwire.dtl.DTLVardef@1ce89daorg.highwire.dtl.DTLVardef@bc5daeorg.highwire.dtl.DTLVardef@aa4ceforg.highwire.dtl.DTLVardef@a1159e_HPS_FORMAT_FIGEXP M_FIG C_FIG Illustration showing how EpCAM aptamer-mediated silencing of PLK1 could control the cell cycle progression at multiple number of check points and induce apoptosis involving hyper and hypophosphorylation of variety of signalling molecules

BackgroundSLIT2 has been shown to serve as a tumor suppressor in breast, lung, colon, and liver cancers. Additionally, expression of SLIT2 has been shown to be epigenetically regulated in prostate cancer. Therefore, we sought to determine transcriptional regulation of SLIT2 in pancreatic ductal adenocarcinoma. MethodsRNA expression of SLIT2, SLIT3, and ROBO1 was examined in a panel of pancreatic ductal adenocarcinoma cell lines while protein expression of ROBO1 and SLIT2 was examined in tumor tissue. Methylation of the SLIT2 promoter was determined using Sequenom while histone modifications were queried by chromatin immunoprecipitation. Reexpression of SLIT2 was tested by treatment with 5-aza-2deoxycytidine and Trichostatin A. ResultsPancreatic cancer cell lines fall into three distinct groups based on SLIT2 and ROBO1 expression. The SLIT2 promoter is methylated in pancreatic ductal adenocarcinoma and SLIT2 expression is dependent on the level of methylation at specific CpG sites. Treatment with 5-aza-2deoxycytidine (but not Trichostatin A) led to SLIT2 reexpression. The SLIT2 promoter is bivalent in pancreatic ductal adenocarcinoma and histone marks around the transcriptional start site are responsible for transcription. ConclusionsLoss of SLIT2 expression modulated by epigenetic silencing may play a role in pancreatic ductal adenocarcinoma progression.

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with poor prognosis for metastasis and recurrent disease. Large scale sequencing endeavors demonstrate that RMS tumors have limited mutations and a dearth of driver mutations that are precisely targetable. However, IGF2 signaling is known to be grossly altered in RMS. The IGF2 signalling molecule binds both its innate IGF1 receptor as well as the insulin-receptor-variant-A (IR-A) with high affinity. Mitogenic and proliferative signalling via the canonical IGF2 pathway is therefore augmented by IR-A. The insulin receptor (IR) which is a transmembrane tyrosine-kinase receptor exists in two alternatively spliced isoforms, IR-A and IR-B. In this study, we show that RMS patients express increased IR-A compared to control tissues that express predominantly the IR-B isoform. We also found that Hif1a is significantly increased in RMS tumors, portraying their hypoxic phenotype. Furthermore, the alternative-splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a hypoxia-responsive-element, which is also the binding site for the RNA-binding protein CUG-BP1. We designed Splice-Switching-Oligonucleotides (SSO) against this binding site to decrease the levels of IR-A in RMS cell-lines and consequently rescue the IR-B expression levels. SSO treatment resulted in significant reductions in proliferation, migration and angiogenesis. Our data show promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. Our data reveal that RMS tumors use IR alternative-splicing as yet another survival strategy which can be exploited as therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.

In this work I present evidence that frameshift mutations represent substantial oncogenic potential across multiple tumor types and may change our understanding of the function of some genes with well established tumor suppressor. I analyzed data deposited in Cbio portal and show that frameshifts, even when they result in the removal of a substantial part of a protein have the potential to create recurring large domains with unknown function. Based on this analysis I propose a novel categorization of genes according to their association with cancer that is more reflective of a complex nature that goes beyond the simple division to tumor suppressors and oncogenes.

Multiple Myeloma is a plasma cell malignancy for which there is currently no cure, despite many novel therapies. TRAIL (Tumour necrosis factor-related apoptosis inducing ligand) is a promising anti-tumour agent although TRAIL-insensitive cells readily emerge when used as a single agent and its effects are limited due to many TRAIL-resistant cells which emerge soon after treatment. EZH2 is a H3K27 histone methyltransferase found to be overexpressed in many cancers including Multiple Myeloma. We tested the hypothesis that epigenetic reprogramming using the EZH2 inhibitor GSK343 would enhance TRAIL sensitivity, overcome TRAIL resistance, and target TRAIL-resistant quiescent cell populations. We show that GSK343 is a potent TRAIL sensitiser in TRAIL-sensitive RPMI 8226, NCI-H 929 and U266, and in TRAIL-resistant OPM-2 and JJN3, the latter showing very potent synergistic induction of apoptosis, primarily via caspase-8 activation but also via caspase-9. GSK343-enhancement of TRAIL responses was further enhanced in a 3D cell culture model of Multiple Myeloma in NCI-H 929 and U266. We show that in TRAIL-resistant sub-populations of Multiple Myeloma cells, GSK343 responses were completely attenuated in RPMI 8226 although synergistic enhancement of apoptosis was observed in NCI-H 929. Furthermore, following isolation of PKH26Hi quiescent cell populations, TRAIL responses and enhancement of TRAIL responses by GSK343 were completely attenuated. These studies show that EZH2 inhibition enhances TRAIL responses both in TRAIL-sensitive and TRAIL-resistant MM cell lines suspension culture and also in 3D cell culture to model the semi-solid Multiple Myeloma lesions in bone. Pre-existing TRAIL resistance was also enhanced by EZH2, and although synergistic enhancement of TRAIL responses by GSK343 was seen in NCI-H 929, responses were completely lost in TRAIL-resistant RPMI 8226 and also in quiescent cells. These studies highlight that although EZH2 inhibitors enhance TRAIL responses, acquired TRAIL resistance, and the presence of quiescent cells may mediate TRAIL-insensitivity in response to GSK343.

Fanconi anaemia (FA) is a rare, recessive, genetic disorder characterised by a predisposition to cancer. Patients with FA are 700 times more likely to develop head and neck squamouscell carcinomas including oral epithelial dysplasia (OED), a potentially malignant disorder of the oral mucosa. This increased likelihood suggests that the molecular mechanism responsible for dysplastic transformation may involve defects in the FA pathway. In this study, the significance of ataxia telangiectasia and Rad3-related protein (ATR), which is responsible for homologous recombination repair (HRR), was investigated. ATR protects against mutations in both normal squamous cells and cancer cells by inducing HRR, and thus is associated with radiotherapy resistance. This investigation was designed to study the effects of heat on DNA repair specific to the FA pathway. Western blotting was carried out to determine whether heat affects the ATR pathway, followed by survival assays to determine the viability of cells after heat treatment and then compared to cells treated with a specific ATR inhibitor. This project aims to discover whether heat could be used as a non-invasive treatment to increase the sensitivity of tumour cells towards radiotherapy leading to an improved treatment plan for patients suffering from head and neck cancers.

P53 suppresses tumorigenesis through multiple cellular functions/mechanisms. Recently, Janic A, et al. reported that DNA repair pathways are critical mediators of p53-dependent tumor suppression. We showed, by mining cBioPortal data of a range of human cancers, that the tendency of mutual exclusivity of mutations in p53 and DNA repair genes only exist in very limited human cancer types. In the majority of human cancers, p53 mutations are equally distributed between DNA repair gene mutation positive and negative cases and in a number of human cancers, p53 and DNA repair gene mutations have a tendency of co-occurrence. These different correlation patterns of p53 and DNA repair gene mutations in human malignancies may reflect different critical molecular/cellular pathways activated by p53 in different organs or cell types to suppress tumorigenesis.

Genomic instability fuels genomic alterations that befit cancer cells with necessary adaptations to keep proliferating and overcome the impact of host anti-tumor immunity and cytotoxic therapy. Since DNA breaks are required for genomic rearrangements to take place, we hypothesized that dysregulated nuclease activity mediates genomic instability in cancer. Using an integrated genomics protocol, we identified a four gene deoxyribonuclease signature correlating with genomic instability in six human cancers which included adenocarcinomas of esophagus (EAC), lung, prostate, stomach, pancreas and triple negative breast cancer. Functional screens confirmed the role of these nucleases in genomic instability and growth of cancer cells. Apurinic/apyrimidinic nuclease 1 (APE1), identified as top nuclease in functional screen, was further investigated in five cell lines representing four solid tumors (EAC, lung, prostate and breast cancer). We demonstrate that chemical as well as transgenic suppression of APE1 impaired growth/colony formation and increased cytotoxicity of chemotherapeutic agent, whereas inhibited spontaneous as well as chemotherapy-induced DNA breaks, homologous recombination (HR) activity and genomic instability in all cancer cell types tested. Treatment with APE1 inhibitor also impaired tumor growth and significantly increased efficacy of a chemotherapeutic agent in a subcutaneous mouse model of EAC. Overexpression of APE1 in normal esophageal epithelial cells increased DNA breaks and HR activity, leading to massive mutational, copy number as well as karyotypic instability. Evaluation of by whole genome sequencing identified HR as the top mutational process activated by APE1. Normal cells overexpressing APE1 grew as tumors in mice and tumors removed from mice displayed additional karyotypic changes, providing evidence of genomic instability in vivo. Overall, our data demonstrate that elevated APE1 dysregulates HR activity, G2/M checkpoint and genome stability thus contributing to tumorigenesis and chemoresistance in cancer. Therefore, inhibitors of APE1 have potential to inhibit growth and increase cytotoxicity of chemotherapeutic agents while minimizing spontaneous as well as chemotherapy-induced genomic damage and instability in EAC and other solid tumors.

Glioblastoma (GBM) is the most aggressive primary adult brain tumor, with a median survival of approximately 15 months. Despite novel therapeutic approaches, median survival has remained largely unchanged since the standard of care therapy for GBM was established nearly 15 years ago. Phosphatase and tensin homolog (PTEN) is a prognostic biomarker of GBM. PTEN mutation is associated with defects in homologous recombination (HR), making it a candidate for targeted therapy by synthetic lethality (SL). The SL concept has been clinically validated in HR-deficient breast and ovarian cancers upon treatment with poly(ADP-ribose) polymerase (PARP 1) inhibitors (PARPi). This inhibitor, as well as poly(ADP-ribose) glycohydrolase (PARG) inhibitors (PARGi), dysregulate PARylation post-translational modification, which plays a major role in DNA repair and genomic stability. To determine whether PARPi/PARGi promotes SL in GBM, this study investigated the effects of PARPi (veliparib and olaparib) and PARGi in GBM cells with wildtype versus mutant PTEN. Sensitivity to these drugs was analyzed in function of PTEN status. Specifically, PTEN-wildtype cells displayed higher levels of DNA damage after PARPi treatment compared to PTEN-mutant cells. However, focusing on DNA double-strand break (DSB) repair, there was no indication of efficient activation of non-homologous end joining (NHEJ) or homologous recombination (HR). These findings highlight the complex relationship between PARylation and PTEN. Thus, our results do not support the SL between PARP/PARG inhibition and PTEN mutations in GBM cells in absence of other DNA damaging agents.Competing Interest StatementThe authors have declared no competing interest.View Full Text

The fundamental transcription factor p53 regulates cellular processes and integrates signals of cellular stress, triggering a coordinated response to ensure survival of cells restored to healthy function and programmed death of those that couldnt be repaired. Unsurprisingly, this is one of the most mutated genes in human cancers, with most changes occurring in the DNA-binding domain of the protein. In this work, we take a genome-wide approach and use available resources to identify high confidence p53-target genes, that we examine in three breast cancer cell lines with different p53 status, wild type (MCF-7) and different mutations in the DNA-binding domain (MDA-MB231, T47D). Comparison of p53-targets expression in response to DNA damage by RNAseq and cellular assays reveals that MDA-MB231 have a severely impaired p53-dependent pathway functionality while T47D are much less affected. MDA-MB231 are more resistant to DNA damage yet unable to repair and able to override cell cycle arrest leading to survival while T47D are sensitive only to high dose and exposure to genotoxic agents. This data shows the variability of effects of different p53 mutations and highlight the importance of understanding the mechanisms of p53 in the context of genotoxicity-based treatment.

About 80% of patients with HPV negative head and neck squamous cell carcinoma are diagnosed at stage III or IV. In these cases, Surgical resection followed by (chemo-)radiotherapy is the standard treatment for most cases. However, many patients present with unresectable and/or resistant disease and even metastases, limiting therapeutic options. A common molecular defect in these tumours is the deactivation of the tumour suppressor gene TP53, frequently through inactivating mutations. Restoring TP53 function, combined with the standard chemoradiotherapy may enhance the therapeutic outcomes. In this study, the efficacy of two TP53 reactivating compound, APR-246 and COTI-2, was evaluated in combination with chemoradiotherapy on two different human HNSCC cell lines. Results highlight a synergistic effect of the combination treatment, significantly reducing clonogenic survival, spheroid growth and subcutaneous tumour growth in a preclinical murine model. Mechanistic investigation suggests that this effect is linked to redox imbalance caused by the generation of reactive oxygen species. This appears to play a key role in the Fenton reaction, further facilitated by an increase in DMT1 or decrease in FTH1 expression, leading to elevated cytosolic iron and lipid peroxide levels. Additionally, the reactive oxygen species may contribute towards the increase in both single and double strands breaks observed in several western blots. Overall, these results suggest that combining TP53 reactivation with chemoradiotherapy could trigger ferroptosis, improving tumour control in HNSCC.

The immune checkpoint molecule B7-H3 is regarded as one of the most promising therapeutic targets for the treatment of human cancers. B7-H3 is highly expressed in many cancers and its expression has been associated to impaired antitumor immunity and poor patient prognosis. In immunocompetent mouse tumor models, genetic deletion of B7-H3 in tumor cells enhances antitumor immune response leading to tumor shrinkage. The underlying mechanisms of B7-H3 inhibitory function remain largely uncharacterized and the identity of potential cognate(s) receptor(s) of B7-H3 is still to be defined. To better understand B7-H3 function in vivo, several studies have employed MJ18, a monoclonal antibody reported to bind murine B7-H3 and blocks its immune-inhibitory function. In this brief research report, we show that 1) MJ18 does not bind B7-H3, 2) MJ18 binds the Fc receptor Fc{gamma}RIIB on surface of murine splenocytes, and 3) MJ18 does not induce tumor regression in a mouse model responsive to B7-H3 knockout. Given the high profile of B7-H3 as therapeutic target for human cancers, our work emphasizes that murine B7-H3 studies using the MJ18 antibody should be interpreted with caution. Finally, we hope that our study will motivate the scientific community to establish much-needed validated research tools to study B7-H3 biology in mouse models.

Genome instability is a hallmark of cancer and are driven by mutations in oncogenes and tumor suppressor genes. Despite successes seen with select targeted therapeutics, this type of personalized medicine is only beneficial for a small subpopulation of cancer patients who have one of a few actionable genetic changes. Most tumors also contain hundreds of passenger mutations that offered no fitness advantage or disadvantage during tumor evolution. Mutations in known pharmacogenetic (PGx) loci for which germline variants encode variability in drug response can cause somatically acquired drug sensitivity. The NUDT15 gene is a known PGx locus that participates in the rate-limiting metabolism of thiopurines. People with two defective germline alleles of NUDT15 are hypersensitive to the toxic effects of thiopurines. NUDT15 is located adjacent to the Retinoblastoma (RB1) tumor suppressor gene, which often undergoes homozygous deletion in retinoblastomas and other epithelial cancers. We observed that RB1 undergoes homozygous deletions in 9.4% of prostate adenocarcinomas and 2.5% of ovarian cancers, and in nearly all of these cases NUDT15 is also lost. Moreover, 44% of prostate adenocarcinomas and over 60% of ovarian cancers have lost one allele of NUDT15, which predicts that a majority of all prostate and ovarian cancers have somatically acquired hypersensitivity to thiopurine treatment. We performed a retrospective analysis of >16,000 patients in the US Veterans Administration health care system and found concurrent xanthine oxidase inhibition (XOi) and thiopurine usage for non-cancer indications is significantly associated with reduced incidence of prostate cancer. The hazard ratio for the development of prostate cancer in patients treated with thiopurines and XOi was 0.562 (0.301-1.051) for the unmatched cohort and 0.389 (0.185-0.819) for the propensity score matched cohort. We experimentally depleted NUDT15 from ovarian and prostate cancer cell lines and observed a dramatic sensitization to thiopurine-induced and DNA damage-dependent toxicity. These results indicate that somatic loss of NUDT15 predicts therapeutic sensitivity to a low cost and well tolerated drug with a broad therapeutic window.

Radioresistant cancers often exhibit upregulated DNA damage response (DDR) proteins including ataxia telangiectasia mutated (ATM), recovering more readily from radiation-induced DNA double-strand breaks (DSBs). ATM inhibitors (ATMi) are therefore being explored as adjuvants in radiotherapy to both enhance radiosensitivity and minimise normal tissue complications. The molecule AZD1390 has been developed as an inhibitor of ATM and is undergoing assessment in clinical trials. However, traditional markers of DNA DSB repair, such as {gamma}H2AX, represent stages of DDR downstream from the action of ATM. The ATMi AZD1390, developed by AstraZeneca, ultimately prevents phosphorylation of the H2AX histone variant. In order to quantify ATMi AZD1390 action, the novel and highly complementary assay SensiTive Recognition of Individual DNA Ends (STRIDE) was employed to directly quantify DSBs in comparison to {gamma}H2AX after X-ray irradiation of 4T1 cells. Findings revealed that ATM inhibition via AZD1390 delays DSB repair initiation and appears to play a greater role in suppressing DDR beyond ATM inhibition alone. In X-ray irradiated conditions, obfuscation of DSBs commenced between 30- to 45-minutes post-irradiation without AZD1390 versus 45 to 60-minutes for cells pretreated with AZD1390 and entirely prevented {gamma}H2AX in the majority of cells. STRIDE and {gamma}H2AX exhibited almost no co-localization indicating that they provide distinct and complementary information. DSB formation was also assessed in cells fixed pre-insult to minimise any biological response, providing unprecedented assessment of DSB formation. These results highlight the potential of STRIDE to accurately measure DNA damage response kinetics, paving the way for more precise mechanistic studies into the role of ATM in radiotherapy.

Replication protein A (RPA) plays essential roles in DNA replication, repair, recombination and the DNA-damage response (DDR). We have developed second generation RPA inhibitors (RPAis) that block the RPA-DNA interaction. These DNA-binding inhibitors (DBis) can elicit a state of cellular RPA exhaustion resulting in single agent in vitro anticancer activity across a broad spectrum of cancers and in vivo activity in two non-small cell lung cancer models. The cellular response to RPAi treatment suggests a threshold exists before RPA inhibition induces cell death. Chemical RPA exhaustion potentiates the anticancer activity of other DDR inhibitors as well as traditional DNA damaging cancer therapeutics. Consistent with the chemical RPA exhaustion model, we demonstrate that the effects of RPAi on replication fork dynamics and DNA damage signaling are similar to other known DDR inhibitors. In accordance with the RPA threshold model, retrospective analysis of lung cancer patient data demonstrates high RPA expression as a negative prognostic biomarker for overall survival in smoking-related lung cancers. Similarly, relative expression of RPA is a predictive marker for response to chemotherapy. These observations are consistent with the increase in RPA expression serving as an adaptive mechanism that allows tolerance of the genotoxic stress resulting from carcinogen exposure. These data demonstrate a unique mechanism of action of RPAis eliciting a state of RPA exhaustion that impacts the DDR and may provide an effective therapeutic option for difficult to treat lung cancers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/404640v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@d662e4org.highwire.dtl.DTLVardef@f68b11org.highwire.dtl.DTLVardef@241c0corg.highwire.dtl.DTLVardef@ad7398_HPS_FORMAT_FIGEXP M_FIG C_FIG

The synthesized molybdenum complex, [cis-MoO2(BHAN)2] (BHAN= {beta}-hydroxy--naphthaldehyde), exhibits remarkable efficacy in safeguarding DNA against radiation-induced damage. Comparative studies reveal that the complex offers superior protection to radiolysed DNA compared to the ligand (BHAN). Notably, at a concentration of 2 mM, the complex demonstrates the capability to shield 90% of damaged plasmid DNA from a 20 Gy radiation exposure. Additionally, it also affords significant protection against radiation-induced damage to cellular DNA (CTDNA) from gamma rays. These findings underscore the significant potential of cis-MoO2(BHAN)2 as an effective radioprotector for normal tissues in the context of radiotherapy. The results of this study contribute valuable insights into the advancement of radioprotective strategies, presenting a noteworthy breakthrough with implications for future medical advancements.

Radiation-induced bystander effects (RIBE) in human hematopoietic stem and progenitor cells may initiate myeloid neoplasms (MN). Here, the occurrence of RIBE caused by genotoxic signaling from irradiated human mesenchymal stromal cells (MSC) on human bone marrow CD34+ cells was investigated. For this purpose, healthy MSC were irradiated in order to generate conditioned medium containing potential genotoxic signaling factors. Afterwards, healthy CD34+ cells from the same donors were grown in conditioned medium and RIBE were analyzed. Increased DNA damage and chromosomal instability were detected in CD34+ cells grown in MSC conditioned medium when compared to CD34+ cells grown in control medium. Furthermore, reactive oxygen species and distinct proteome alterations, e.g., heat-shock protein GRP78, that might be secreted into the extracellular medium, were identified as potential RIBE mediators. In summary, our data provide evidence that irradiated MSC induce genetic instability in human CD34+ cells potentially resulting in the initiation of MN. Furthermore, the identification of key bystander signals, such as GRP78, may lay the framework for the development of next-generation anti-leukemic drugs.

BackgroundHomozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene is a frequent genetic alteration in cancer. MTAP, which creates adenine from 5-methylthioadenosine (MTA), is constitutively expressed in all tissues throughout the body. Previously, we described a novel strategy to specifically target MTAP-deleted cancer cells by combining the antipurine prodrug 2-fluoroadenine (2FA) with MTA. In vitro, this combination efficiently killed MTAP- cancer cells, but in vivo the combination was much less effective in vivo. Here, we explored the role of xanthine oxidase (XO) in this process. Materials and MethodsVarious combinations of 2FA, MTA, and the xanthine oxidase inhibitor febuxostat (FX) were tested in various cancer cell lines grown in vitro and in mice. LC-MS/MS was used to examine the levels and ratio of intracellular 2-FA-containing nucleotides compared to adenine-containing nucleotides. Results and conclusionsThe treatment of cells with 2FA+MTA in vitro resulted in much higher 2FANP/ANP ratios than the same treatment in vivo. The addition of XO to culture media in vitro effectively abolished the killing by 2FA, and this effect was fully reversed by the addition of febuxostat (FX), a xanthine oxidase inhibitor. In vivo, the addition of FX to 2FA results in increased cell killing and toxicity and a 1000% increase in the amount of 2FA converted to 2-FA-monophosphate (2FAMP). Xenograft studies using MTAP- HT1080 and MiaPaCa-2 cell lines have shown that a 2FA/MTA/FX cocktail can cause tumor regression in vivo. These studies suggest that the combination of 2FA/MTA/FX should be explored as a treatment for MTAP- cancer.

Ewing sarcoma is the second most common bone malignancy in children and adolescents. Patients with upfront metastatic or recurrent disease have poor outcomes with 5-year survival rates of <30%. CDC7, also known as DDK (DBF4-dependent kinase), is a serine-threonine kinase that, in coordination with its activation subunit ASK (or DBF4), is involved in a diverse array of cellular functions including the regulation of DNA replication initiation and activation of the replication stress response. Due to DDKs diverse roles during replication, coupled with an increased level of genomic instability and R-loop-mediated replication stress within Ewing sarcoma cells, we hypothesized that Ewing sarcoma cells would be particularly vulnerable to DDK inhibitors. Here, we show that treatment with two selective DDK inhibitors, TAK-931 and XL413, results in apoptosis and a significant reduction in cell viability in EWS-FLI1-harboring Ewing sarcoma cell lines. We show that low dose DDK inhibition in Ewing sarcoma cells causes an accumulation of cells in late-S phase with a reduced replication capacity. There is also evidence of premature mitotic entry indicating an inability to properly complete DNA replication in a timely manner upon DDK inhibition. Also, there is a significant increase in the formation of micronuclei and other aberrant mitotic structures upon DDK inhibition in Ewing sarcoma cells indicating a failure to properly progress through S-phase followed by improper mitotic entry/progression, resulting in mitotic catastrophe. Interestingly, we observed minimal signs of mitotic accumulation, despite clear evidence of replication and mitotic stress, suggesting a failure to properly enforce the mitotic checkpoint. Together, these results suggest that Ewing sarcoma cells rely on the activity of DDK to maintain cell viability and suggest that DDK inhibition may prove to be a viable therapeutic strategy for patients with Ewing sarcoma.