Alterations in DNA damage repair (DDR) pathways can impair cisplatin efficacy. MicroRNAs (miRNAs) are actively involved in DDR regulation and in parallel have been suggested as potential biomarkers for the prediction of response to platinum- based chemotherapy (CT) in non- small cell lung cancer (NSCLC). In the present study we used a bioinformatics approach to identify differentially expressed miRNAs associated with the efficacy of platinum-based chemotherapy in NSCLC. We assembled four miRNA microarray expression datasets from NSCLC patients treated with CT included in the Gene Expression Omnibus (GEO) repository. Analysis in Limma package in R revealed 3.883 differentially expressed (DE) miRNAs between responders (n=71) and non-responders (n=99). After meta-analysis by random-effects, we identified a total of 697 miRNAs that were consistently up or down-regulated in at least two studies. By using more stringent effect size thresholds, we identified 43 DE miRNAs with log fold change ≥1.3 in at least two datasets. The prognostic significance of the selected miRNAs was further validated through survival analysis by KM plotter in stage III NSCLC. Twenty-two of the DE miRNAs were revealed to have prognostic significance in NSCLC. Integrated function and target pathway enrichment analysis revealed significant associations to a number of pathways and functions related to DDR, such as p53, HIPPO and FOXO3. Finally, we extracted a miRNA signature consisting of 8-miRNAs (miR-181a, let-7g, miR-26a, miR-145, miR-30d, miR-99a, miR-214, miR-107) that were down-regulated in non-responders and are involved in at least three DDR pathways. Comparative expression analysis on tumor and matched normal tissues from operable NSCLC patients treated with platinum-based chemotherapy will be performed to evaluate the clinical relevance of the signature.

(1) Background: Poly (ADP-ribose) polymerases (PARPs) have pleiotropic roles including canonical DNA-damage response (DDR) pathways. PARP inhibition is initially proposed as a synthetic lethal interactor with cancer harboring homologous recombination deficiency (HRD), thus becoming a key therapeutic option for genetically-defined subsets of patients. Recently, there has been increasing evidence supporting the expansion of PARP-targeted therapy beyond HRD. Besides, synthetic lethality pathways for PARP-targeted therapy are being studied extensively due to the rapidly developed resistance to PARP inhibitors (PARPi).

(2) Methods: We perform integrative pharmaco-transcriptomic analyses by correlating the drug response profiles of clinically-approved PARPi olaparib with the transcriptomes of solid cancer cell lines (n=659) to establish PARPis responsive gene signatures, which are then evaluated for their reliability using independent drug response datasets, and applied to identify tumor subsets primed for PARPi and potential targets synergistically interacting with PARPi.

(3) Results: Based on the pharmaco-transcriptomic correlation analysis, we delineate gene signatures to predict the sensitivity and resistance to olaparib in pan-solid cancer cells, which is confirmed by independent drug response datasets. In further exploring the PARPi sensitivity signature, we identify IDH1/2 (isocitrate dehydrogenase 1/2)-mutated low-grade glioma (LGG) and NEUROD1-driven small cell lung cancer (SCLC) as potential subsets for prioritized PARPi, highlighting relaunching PARPi as a promising and innovative strategy to target these malignancy subtypes. Interestingly, the PARPi responsive signatures display a high degree of heterogeneity in the correlation with the curated HRD signatures across TCGA pan-solid cancer cohort, suggesting that these signatures predictive of PARPi responsiveness are HRD-independent. With the PARPi resistance signature, we identified several potentially synthetic lethal interactors with PARPi, e.g. dasatinib, EGFR, or MEK inhibitors.

(4) Conclusions: The established PARPi responsive (sensitive/resistant) signatures in solid tumors exhibit robustness in identifying cancer subtypes that are highly primed for PARP-targeted therapy, and combined targets that synergistically augment the efficacy of PARPi.

MDM2 is an oncogenic E3 ligase found to be overexpressed in a number of human cancers, leading to poor prognosis. MDM2 overexpression inhibits the function of the tumour suppressor p53, which plays a critical role in safeguarding the integrity of the genome. MDM2 ubiquitinates p53 and tags it for proteasomal degradation. MDM2 also exhibits p53-independent oncogenic activities through targeting other tumour suppressor proteins, such as Foxo3a and Rb. Thus, aberrant regulation of Mdm2 is a key factor in promotion of tumor formation and progression and represents an important cancer therapeutic target. Here, we describe an integrated structure- based approach to develop potential lead compounds for inhibition of MDM2 oncogenic activity. We established a structure-based virtual screening strategy and used the crystal

structure of the MDM2:MDMX RING domain heterodimer to predict the potential “druggable”

pocket on Mdm2. An in silico screening of a small molecule compound library was employed to identify the candidate compounds that could interact with the MDM2:MDMX heterodimer RING domain. Additionally, using biochemical and cellular assays, the candidate compounds were examined for their ability to inhibit MDM2 E3 ligase activity, to induce apoptosis and to inhibit cell proliferation in cancer cell lines. This study reveals that inhibition of the MDM2:MDMX RING heterodimer could be a plausible approach for the development of MDM2 inhibitors as potential anti-cancer therapeutic agents.

Introduction

Myeloproliferative neoplasms (MPNs) are a group of haematological malignancies arising from haematopoietic stem cells with acquired driver mutations in JAK2, MPL and CALR. Increased replication stress is seen in the presence of JAK2V617F. Genes involved in the DNA double-strand break (DSB) repair pathways – BRCA-dependent homologous recombination repair (HRR) and DNA-dependent protein kinase-mediated non-homologous end-joining (D-NHEJ) are upregulated in MPN cells expressing mutated JAK2.

Aims

Using JAK2V617F and CALR (del 52) mutant cell lines:

• Determine the effect of single-agent DNA damage repair (DDR) inhibitors on cell viability and apoptosis.
• Evaluate the efficacy of DDR inhibitors in combination with hydroxyurea or ruxolitinib.

Materials and Methods

Cell lines expressing JAK2 (V617F)- HEL and CALR (del52)- MARIMO were treated with a drug panel comprising hydroxyurea, ruxolitinib, methotrexate, AZD6738 (ATRi), NU7441 (DNA-PKi), Olaparib (PARPi) and VE-821 (ATRi). AlamarBlue assay for cell proliferation and annexin V/ propidium iodide staining for flow cytometry were used to evaluate the toxicity.

Results

In JAK2 and CALR mutated cell lines, ATR inhibition by AZD6738 or VE-821, DNA-PKs inhibition by NU7441 and hydroxyurea each reduced viability, whereas PARP inhibition by olaparib had a minimal effect. The combination of ATR inhibition and hydroxyurea demonstrated high synergism in both apoptosis induction and proliferation arrest. Ruxolitinib alone had a modest effect in the presence of JAK2V617F and a minimal effect in CALR (del 52) mutated cells. Synergistic toxicity was observed for ruxolitinib and AZD6738/ VE-821 combination in JAK2 mutated cell line.

Conclusions

DDR inhibition reduces viability in cells expressing the driver mutations seen in MPNs. Most notably, ATR kinase inhibitors have a synergistic effect with the current standard-of-care treatment hydroxyurea. This study provides preliminary evidence that ATR inhibitors combined with standard therapies may be exploited in MPNs harbouring JAK2 and CALR mutations.

Glioblastoma multiforme (GBM) is a highly aggressive brain cancer. The standard course of treatment is a combination of radiation and chemotherapy. Even with the dual treatment, the 5-year survival rate of patients with GBM is between 4-7%. Therefore, there is an urgent need to develop novel therapies to increase the survivorship. A possible cause of the low survival rate for GBM patients is the presence of neoplastic cells with efficient DNA repair abilities. These cells have been shown to be resilient against chemotherapy and radiation. They often continue to grow unchecked leading to lethal secondary tumor disease.

XRN2 is upregulated in GBMs as compared to normal and other brain cancer types. XRN2 is a 5’-3’ exonuclease that resolve DNA:RNA hybrids (R loops) that arise during transcription, especially at the 3’ end of genes. R-loop biology can affect gene expression by modulating the access of genes to transcription factors, miRNA transcription, and methylation status of genes. Our preliminary data have shown that loss of XRN2 sensitizes to a variety of DNA damaging agents but in particular ionizing radiation. In addition, XRN2 is required for DNA repair in specifically DNA double stranded break repair.

To understand how XRN2 modulates DNA repair, we conducted RNA-Seq analyses of two GBM cell lines with and without XRN2 expression and found that XRN2 can regulate genes involved in DNA repair pathway. We have conducted a mini-cherry picked screen of the XRN2 targets and found at least 6 genes to be required for DNA double stranded break repair. A subset of the 6 genes were also found to be sensitive to DNA damage agents such as ionizing radiation, etoposide, and Parp-1 inhibition. One XRN2-target is POLA2. We have found that POLA2 is required for homologous recombination and non-homologous end-joining repair, the two major repair pathways for DNA double stranded breaks. Another XRN2- target is CENPI. We found that CENPI is required for homologous recombination repair and not non-homologous end-joining; showing that XRN2 targets have differential impacts in DNA repair.

Our goal is to develop a patient signature that can better predict patient outcome and if possible a new synergetic treatment plan to increase the efficacy of radio-therapies.

Ionizing radiation (IR) is widely used in cancer treatment as it induces vast DNA damage leading ultimately to tumour cell death. The mechanisms involved in x-ray-induced cell death have been deeply studied, while little is known about the impact of IR of higher linear energy transfer (LET) on cell biology and the critical enzymes and mechanisms that are responsive to this. We have recently performed a focused small interfering RNA (siRNA) screen to identify proteins involved in cell survival in response to high-LET α-particles and protons, versus low-LET x-rays and protons. From this screening, we have validated that depletion of the ubiquitin-specific protease 9X (USP9X) in HeLa and oropharyngeal squamous cell carcinoma (UMSCC74A) cells using siRNA leads to significantly decreased survival of cells after exposure to high-LET radiation, whilst no effect was observed after low-LET radiation (protons and x-rays) treatment. We consequently investigated the mechanism through which this occurs and found that USP9X inhibition does not interfere with DNA damage (double strand breaks and complex DNA damage) repair post-irradiation, nor does it induce apoptosis, autophagy or senescence. Instead, we observed that USP9X depletion destabilizes key centrosome proteins (CEP55 and CEP131) causing centrosome amplification and ultimately cell death in response to high-LET protons.

Triple-negative breast cancer (TNBC), representing 15% of breast carcinomas, is an aggressive breast cancer subtype with a high probability of metastasis and limited treatment options. Noticeably, BRCA-deficiency occurs in 25% of the TNBCs and results in deficient homologous recombination (HR) repair. Interestingly, PARP inhibitors (PARPi) have shown synthetic lethality in a BRCA-deficient context, however, their efficacy is frequently hampered by intrinsic or acquired resistance mechanisms involving restoration of the HR. In that regard, the role of some CDKs proven to regulate key HR actors was of interest to us.
We aim at understanding the rewiring pathways determining resistance to PARPi in BRCA-deficient cancers and assessing the role of transcriptional regulating CDKs such as CDK7, CDK9 or CDK12 in the transcriptional regulation of key HR genes. Our ultimate goal is to determine whether and which CDK inhibitors could be effective approaches to repress HR gene expression and induce pharmacological HR-deficiency. As such, these CDK-inhibitors could be molecules of choice allowing sensitization of tumors that would otherwise respond poorly to DNA damaging treatment. With this purpose, we use in vitro and in vivo (PDX) models of TNBC and study the attenuation of HR response in tumor cells and PDX models treated with CDK-inhibitors. Our final aim is to determine the most efficient combination CDK-I + PARP-I. Our HR read outs are RAD51 and BRCA1 foci formation upon PARP-I treatment. We also measure the modification of RNA and protein expression levels induced by CDK-I treatment on a series of diagnostic HR genes (BRCA2, PALB2, ATR, FAND2), as a measure of HR repression.
We will present data comparing the relative efficiency of 3 CDK-I, dinaciclib, NVP-2 and SR-4835, which have different specificities and inhibit different CDKs with variable efficacy.

Due to oncogene expression and altered metabolism, reactive oxygen species (ROS) production is augmented in cancer cells resulting in oxidative DNA damage. 8‑oxoguanine (8-oxoG) is one of the most abundant oxidative DNA lesions. This premutagenic lesion is eliminated from duplex DNA by 8‑Oxoguanine DNA Glycosylase (OGG1), a key player in the base excision repair (BER) pathway. Here, we validate OGG1 as a potential anti-cancer target. OGG1 depletion impairs the growth of A3 T-cell lymphoblastic acute leukemia both in vitro and in vivo, but is well tolerated in non-transformed immortalized cells¹. To further validate our findings, we developed TH5487, a potent small-molecule inhibitor that targets OGG1's active site [1,2]. We show that TH5487 suppresses the growth of a wide range of tumor cells, with a favorable therapeutic index compared to non‑transformed cells [1]. Mechanistically, TH5487 treatment inhibits the repair of potassium bromate-induced 8-oxo(d)G lesions, affects OGG1-chromatin dynamics, and hinders OGG1 recruitment to DNA damage regions [3]. Importantly, TH5487 induces replication stress and proliferation arrest¹. This study presents a novel mechanistic strategy to exploit ROS elevation in cancer by inhibiting OGG1.

References:

1. Visnes, T.; Benítez-Buelga, C.; Cázares-Körner, A.; Sanjiv, K.; Hanna, B.M.; Mortusewicz, O.; Rajagopal, V.; Albers, J.J.; Hagey, D.W.; Bekkhus, T. et al. Targeting OGG1 arrests cancer cell proliferation by inducing replication stress. Nucleic acids research 2020; 48, 12234–12251.
2. Visnes, T.; Cázares-Körner, A.; Hao, W.; Wallner, O.; Masuyer, G.; Loseva, O.; Mortusewicz, O.; Wiita, E.; Sarno, A.; Manoilov, A. et al. Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation. Science 2018, 362, 834–839.
3. Hanna, B.M.F.; Helleday, T.; Mortusewicz, O. OGG1 inhibitor TH5487 alters OGG1 chromatin dynamics and prevents incisions. Biomolecules 2020, 10, 1–10.

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by approved by the regional experimental animal Ethical Committee in Stockholm 2010/63 (N8914)

Introduction: Mitotic slippage (MS) - mitosis failure and reversal to interphase with a doubled genome , is a typical response of TP53-mutant tumors resistant to genotoxic therapy. MS and associated micronucleation may play a role in escaping cell death via sorting of the intrinsically damaged DNA. The mechanisms of this MS-aided cancer resistance are poorly understood.

Methods: For experimental studies the metastatic triple-negative breast adenocarcinoma MDA-MB-231 cell line, was treated with 100 nM DOX (doxorubicin) for 24 h. After drug removal, cells were sampled over a 3-week period post-treatment until the appearance of escape clones.

Results: After DOX treatment these cells polyploidize, display premature senescence, sort the damaged DNA into the cytoplasm and acquire an amoeboid phenotype and finally bud the depolyploidized progeny, restarting the mitotic cycling. We found selective release into the cytoplasm of telomere fragments enriched in telomerase reverse transcriptase (hTERT), telomere capping protein TRF2, and DNA double-strand breaks marked by γH2AX. This occurs along with the alternative lengthening of telomeres (ALT) and DNA repair by homologous recombination (HR) in the nuclear promyelocytic leukemia (PML) bodies, where we found colocalization of PML bodies with TRF2, RAD51 and meiotic nuclease SPO11. The cells in repeated MS cycles activate meiotic genes (meiotic recombinase DMC1, meiotic cohesin REC8, meiotic kinase MOS) and display holocentric chromosomes characteristic for inverted meiosis (IM).

Conclusion: Present results investigating the link between DNA damage response, cellular senescence, MS, and the processes of ALT and IM in chemoresistant cancer cells, all converging on telomeres, open a new avenue for further research and possible targeting.

References: Salmina K., et al. “‘Mitotic Slippage’ and Extranuclear DNA in Cancer Chemoresistance: A Focus on Telomeres.” Int. J. Mol. Sci. 2020, 21, 2779.

Acknowledgements: This work was supported by a ERDF project No. 1.1.1.2/VIAA/3/19/463

Contact information: kristine@biomed.lu.lv

Methotrexate (MTX) is a widely used anticancer agent whose DNA binding properties are well known. Despite its consolidated usage in the therapeutics of cancer, the physicochemical features of MTX binding to healthy and neoplastic DNA are still not fully understood. Therefore, this work showcases the electrochemical study of MTX binding to distinct DNA sequences through voltametric approaches.