Multiple Myeloma (MM) is an incurable haematological malignancy characterised by the clonal proliferation of plasma cells. Deletion of chromosome 1p (del1p) is a common genetic event associated with high-risk MM. Located within this region at 1p13.2 is TRIM33, a chromatin-associated E3 ligase which can function as a transcriptional co-repressor. Recent studies have shown that TRIM33 functions in the PARP-dependent DNA Damage Response (DDR). However, its molecular function during the DDR remains unclear. Here, we investigated the impact of TRIM33 loss on genome stability and the DDR in MM.

Using the publicly available CoMMpass (Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile) dataset, we identified 69 (9.5%) out of 730 newly diagnosed MM patients that had a copy number loss of TRIM33. Kaplan-Meier analysis revealed that these patients have a poorer overall survival compared to those without TRIM33 loss (median 52.3 months vs 72.6 months; p<0.0001). In addition, these patients have a significantly higher number of structural variants (median 38 vs 26; p<0.0001) indicative of increased chromosomal instability. We show that TRIM33 is rapidly recruited to chromatin following 2Gy irradiation, where it transiently interacts with the chromatin remodelling enzyme ALC1. Western blotting and immunofluorescence staining revealed an increase in double-strand break markers γH2AX and 53BP1 (p<0.001) in TRIM33 knockdown cells, along with increased expression of key HR protein RAD51 (p<0.01). MM patients with loss of TRIM33 similarly exhibited significantly higher gene expression of 53BP1 and RAD51 (p<0.0001) indicating a potential HR dysregulation.

We have demonstrated that TRIM33 loss results in chromosomal instability and increased endogenous DNA damage in MM, typical of a DDR defect. Further understanding the role of TRIM33 in the DDR may lead to opportunities to therapeutically exploit DDR defects in patients with TRIM33 loss.

Background

Glioblastoma (GBM) is a treatment refractory cancer of extreme unmet need which exhibits treatment resistance due to a subpopulation of GBM cancer stem cells which have constitutive DNA damage response activation driven by elevated replication stress (RS). RS response inhibition is potently cytotoxic to GSC, however mechanistic understanding will be key to biomarker discovery and successful clinical translation.

Aims/Objectives

We investigated response to combined ATR and PARP inhibition (CAiPi) to gain mechanistic insight and inform biomarker development. Feasibility of this approach was explored in an in vivo orthotopic GBM model.

Methods/Results

Responses to CAiPi in a panel of primary paired GBM GSCs vs differentiated progeny were heterogenous. CAiPi is selectively GSC cytotoxic in a subpopulation of tumours. A population of treatment-sensitive GSCs with increased numbers of 53BP1 nuclear bodies in G1 phase with CAiPi were identified, indicative of under-replication of DNA in S phase. DNA fibre analysis identified increased new origin firing with PARPi, which was correlated with increased PARP trapping. Inhibition of origin firing by exposure to roscovitine rescued the CAiPi cytotoxic phenotype, suggesting origin firing has an important role in selective GSC cytotoxicity. In vivo PK studies have demonstrated effective tumour penetration by both drugs.

Conclusions

Selective GSC cytotoxicity is induced by CAiPi via dysregulation of replication, by both DNA under-replication resulting in DNA lesions, and the novel finding of increased new origin firing in GSC due to PARPi. In vivo survival studies of CAiPi are planned.

The last several years have witnessed a tremendous advance in the knowledge of DNA repair and cell cycle mechanisms for the purpose of increasing the treatment efficacy of radiotherapy and DNA damaging agents. Thereby, targeting DNA damage and repair pathways and cell cycle checkpoints become an attractive rationale to optimize treatment strategies through identifying new targets. However, the improved knowledge has increased the complexity of DNA damage response (DDR) and checkpoint pathways which extremely proved challenges in the development of cell cycle and DNA repair targeting drugs. To this end, a novel approach of synthesizing new compounds has been recently introduced which involved accommodating two chemical entities that target several molecules into a single structure. Here we combined 5-aminosalicylic acid and 4-thiazolinone, which both reported to affect DDR and cell cycle progression, in a single structural framework to generate two derivatives named HH32 and HH33. The transcriptomic, in silico, and in vitro analysis has been used to uncover the anti-cancer potential of these two compounds. Both compounds exhibited a high cytotoxic effect against a panel of eight cancer cell lines from different tissue origins and showed a low toxicity profile on normal cells compared to Doxorubicin. The in-silico molecular docking predicts a strong binding of the HH32 and HH33 to cell cycle regulators like CDC2-cyclin B, CDK2-cyclin A complexes, and retinoblastoma. Interestingly, the transcriptomic analysis revealed that DNA double-strand repair and cell cycle are the most affected pathways by HH33 compound. These findings were validated using in vitro models and demonstrated the induction of DNA double-strand breaks and the stimulation of ATM/ATR signaling pathway by HH32 and HH33. In addition to the potent effect of HH compounds on cell cycle progression mediated through upregulation of cyclin-dependent kinase inhibitors and downregulation of G2/M phase cell cycle markers which ultimately arrest the cells at G2/M phase and promote apoptosis. In conclusion, the pleiotropic biological effect of HH32 and HH33 compounds on cancer cells suggests the requirement for assessing their anti-cancer activities in preclinical models which may lead to a new area in the development of potentially therapeutic drugs.

The chromatin-remodeling complex SWI/SNF is the most mutated remodeler that is currently described in many tumor types. Traditionally, it has been associated with a tumor suppressive role, leading the cellular machinery towards differentiation pathways and DNA repair processes. ARID1A is the most mutated SWI/SNF subunit across all human malignancies. It is also considered as one of the top mutated genes in lung adenocarcinoma (LUAD) and an important driver gene. However, there is a lack of phenotypical studies that confirm the tumor suppressive role of ARID1A in LUAD.

We have observed that ARID1A depletion in LUAD cell lines significantly impaired cell viability and promoted apoptosis. At first glance, these results contradicted its initially defined tumor suppressor status and could not be explained by synthetic lethal events involving other SWI/SNF subunits or driver genes. In addition, when we down-regulated ARID1A in a normal lung cell line, we did not see a significant reduction of cell viability, suggesting a tumor context dependency of ARID1A. Moreover, after performing RNA-seq in A549 after ARID1A-knockdown, we observed some up-regulated pathways related with apoptosis and genotoxic stress responses. We found that the depletion of ARID1A enhanced DNA damage in cells and triggered a severe ER stress response that promoted apoptosis. In addition, the protein levels of other subunits of the SWI/SNF complex decreased upon ARID1A, which could explain a decrease of the DNA repair processes.

Overall, we conclude that some LUAD cell lines are dependent on ARID1A expression in a tumor-dependent manner. In those contexts, ARID1A loss triggers a DNA damage-induced apoptosis, which could open new therapeutic opportunities.

Glioblastomas (GBMs) are one of the most malignant brain tumors in adults. This is partly due to the potential presence of so-called glioma stem-like cells (GSCs), which are characterized by the expression of stemness markers and a resistance to radio- and chemotherapy. Previous work from us showed that after combination treatment of GSCs with Arsenic Trioxide and Gossypol, the protein BRCA1-associated ATM-activator 1 (BRAT1) was one of the most downregulated proteins. This protein is largely undescribed, but it has been shown to regulate DNA damage signaling through interaction with ATM, BRCA1 and DNA-PKcs in initial stages of DNA damage response. An unpublished analysis of the The Cancer Genome Atlas and The Human Protein Atlas databases showed an increased expression of BRAT1 in GBMs compared to healthy tissues and that an increased expression is negatively correlated with patient survival. Because of these findings, our goal is to analyze the radio-sensitizing effect of BRAT1 on FCS-grown (i.e. differentiated) highly radio-resistant GBM cells and GSCs. Here, using stable knockdowns of BRAT1, we show that it is needed for effective DNA repair after irradiation using a γH2AX-foci assay, whereas it is dispensable for cellular proliferation. A cell death analysis using Annexin V/propidium iodide staining revealed a first hint that BRAT1 downregulation sensitizes GBM cells to irradiation. Moreover, through immunofluorescent staining we showed that BRAT1 is needed for BRCA1 recruitment to DNA damage sites. Future experiments will aim at systematically analyzing the downstream effects of BRAT1 depletion and to determine further interactors. Thus, we hope to gain a deeper understanding of the mechanism of radio-resistance in GSCs, also in order to individually determine the effectiveness of radiotherapy.

Radium-223 dichloride (Ra-223, Xofigo^®) is the first approved α-particle-emitting radionuclide for the treatment of symptomatic bone metastases in patients with castration-resistant prostate cancer (CRPC) with no known evidence of visceral metastases. Ra-223 is a calcium-mimetic that preferentially binds with the bone mineral hydroxyapatite at areas of high bone turnover, such as bone metastases. This highly localized radiotherapy causes a high frequency of unrepairable double-stranded DNA breaks (DSBs), resulting in a potent antitumor effect on bone metastases. Recent evidence has suggested that patients with mutations in the DNA damage response pathway (DDR), may have differential outcomes to Ra-223 treatment (1).

DDR comprises a dynamic network of signalling pathways for the maintenance of genomic integrity. Ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) are critical proteins which orchestrate the DDR and their activation is dependent on the type of DNA lesion. ATM is the primary responder to DSBs whilst ATR is activated by a range of lesions including single strand DNA structures at resected ends of DBSs and after replication fork stalling.

In this study, we evaluated the impact of combining DDR kinase inhibitors with Ra-223 to investigate whether a greater radiosensitisation response occurs in comparison to standard X-rays in PC3 and DU145 human prostate cell lines and normal prostate epithelial RWPE-1 cells.

Cell assays including clonogenic survival, DNA damage assays and flow cytometry were used to assess the effect of DDR kinase inhibitors in combination with ionising radiation. Cells were pre-treated with DDR inhibitors one-hour before exposure to 2Gy X-rays or an equivalent dose of 0.25Gy Ra-223.

Our data show that, in all prostate models, DDR kinase inhibitors in combination with Ra-223 significantly enhanced radiosensitivity (p<0.005) response in comparison to combined treatment with X-rays. Furthermore, a greater quantity of residual DSBs at 24 hours post combination treatment was observed after Ra-223 exposure in comparison to X-ray exposure (p<0.001). Promisingly, this combined treatment had minimal effect on RWPE-1 normal cells.

Our findings strongly support the combination of DNA damage induction by Ra-223 with DDR kinase inhibitors as a novel potential treatment option for mCRPC patients in order to improve clinical outcome.

References:

1. Velho PI, Qazi F, Hassan S, et al. Efficacy of Radium-223 in Bone-metastatic Castration-resistant Prostate Cancer with and Without Homologous Repair Gene Defects. European Urology. 2019; 76: 170-176.

Nucleotide excision repair (NER) is an essential DNA damage repair pathway that removes bulky DNA lesions formed by exposure to ultraviolet light, environmental toxins, and platinum (Pt)-based chemotherapeutic drugs that are a standard of care for many cancer types. Mutation or decreased NER gene expression in cancer correlates with improved patient survival after Pt-based chemotherapy. However, the impact of most missense mutations in NER genes is unknown, and few approaches exist to reliably identify nonrecurrent passenger mutations with functional consequences. In this study, a multidisciplinary strategy will be developed to predict, validate, and characterize NER-defective mutations in the essential NER scaffold protein Xeroderma Pigmentosum Complementation Group A (XPA). Computational analyses were used to score NER-deficient versus NER-proficient mutations for further study. Predicted NER-deficient XPA mutants are being expressed in human XPA-deficient cells and screened for both NER activity and cisplatin sensitivity. In-depth biophysical and structural studies are being implemented to elucidate mechanisms of dysfunction. Identifying NER-deficient mutations that may sensitize tumors to Pt-based chemotherapies represents a promising strategy to stratify patients for optimal treatment strategies.

Molecular network activation states alter dynamically in biology and diseases. In cancer stem cells (CSCs), epithelial-mesenchymal transition (EMT) networks play an important role to acquisition of the drug resistance and cancer malignant feature. To reveal the network pathways in EMT and CSCs, gene expression in diffuse- and intestinal-type gastric cancer (GC) have been analyzed. The several canonical pathways have been found to be altered in diffuse- and intestinal-type GC. Canonical pathway on Cell Cycle: G1/S Checkpoint Regulation was activated in diffuse-type GC, and Cyclins and Cell Cycle Regulation was activated in intestinal-type GC. In Cell Cycle: G1/S Checkpoint Regulation, DNA damage induces p53, which was predicted to be activated in diffuse-type GC. Canonical pathway related to Role of BRCA1 in DNA Damage Response was activated in intestinal-type GC, where BRCA1 which is related to G1/S phase transition was up-regulated. Cell cycle regulation may be altered in EMT condition in diffuse-type GC.

Background:

PARP inhibitors (PARPi) exploit defects in homologous recombination repair (HRR) to selectively kill tumour cells. Continuous, PARP inhibition is required for cytotoxicity. PARPis rucaparib, olaparib and niraparib have been approved for use in ovarian cancer on continuous schedules. Previous studies demonstrate prolonged PARP inhibition by rucaparb¹.

Aim:

To determine if persistent PARP inhibition is a class effect.

Methods:

IGROV-1 (human ovarian cancer) cells were treated with 1µM of rucaparib, olaparib, niraparib, talazoparib or pamiparib for 1h before drug was washed off and replaced with fresh media for 0, 1, 24, 48 or 72h prior to harvesting. Cellular PARP activity was measured using a GCLP-validated assay² in comparison with untreated controls and where 1µM inhibitor was added to the reaction.

Results:

Rucaparib, olaparib, niraparib, talazoparib and pamiparib each inhibited PARP activity in permeabilised cells >99% when 1µM was present during the reaction. After 2 h in drug-free medium rucaparib-induced PARP inhibition was maintained at 92.3 ± 4.3% but was much less with talazoparib (58.6 ±5.0%), pamiparib (56.0 ± 4.5%) olaparib (48.3 ± 19.8%) and niraparib (37.3 ± 11.6%). PARP inhibition in rucaparib-treated cells was maintained for 72h in drug-free medium (77.7 ± 12.3%). This sustained PARP inhibition was not observed with the other PARPis. PARP inhibition was only 12.3 ± 5.2% and 12.5 ± 4.9% 72h after talazoparib and pamiparib, respectively, and undetectable with olaparib and niraparib.

Conclusion:

Rucaparib is unique in its ability to cause persistent PARP inhibition and it is not a class effect. These data have clinical implications for the different uses of PARPi, as a single agent use to exploit HRR defects versus chemo- and radiosensitisation.

¹ Murray, J.; et al. BJC, 110, 1977-1984 (2014)

² Plummer ER, et al Clin Cancer Res. 11 3402-3409 (2005)

Despite showing great clinical promise, response rates to immune checkpoint blockade (ICB) vary greatly and biomarkers of response are lacking. A recent Phase II clinical trial in patients with deficiency in the DNA mismatch repair (MMR) pathway indicated that MMR status predicted clinical benefit with the PD-1 inhibitor, pembrolizumab. These findings have led to the first tissue-agnostic approval for anti PD-1 therapy for unresectable or metastatic solid tumours with MMR deficiency. However, it is becoming increasingly clear that many MMR-deficient tumours fail to respond to ICBs with ~50% refractory to treatment. Furthermore, there is a wide diversity of clinical benefit among responders. However why this is the case and how this can be clinically translated remains largely unknown.

Our exciting preliminary data suggest that loss of specific MMR genes results in a differential increased expression of the immune checkpoint molecule, PD-L1. Significantly, we observed an upregulation of PD-L1 expression in cells silenced for the MMR genes, MLH1, MSH2, PMS2 and MSH3, as expected. However, we did not observe an increased expression of PD-L1 upon MSH6 loss. This differential expression amongst MMR gene loss was further validated at both RNA and cell surface level.

Upon investigation of the molecular mechanism regulating PD-L1 expression after MMR loss, we observed that that phosphorylation of STAT1 positively correlates with PD-L1 expression whilst STAT3 phosphorylation was negatively correlated, such that increased STAT1 phosphorylation was observed upon MLH1 and PMS2 loss and not in MSH6-deficient cells whilst STAT3 phosphorylation was only observed upon MSH6 loss. Significantly, inhibition of STAT3, both pharmacologically and genetically, reinstated PD-L1 expression in MSH6-deficient cells.

Therefore, we have evidence that loss of specific MMR genes can trigger differential expression of PD-L1 through a STAT1/STAT3 mediated pathway and we hypothesize that it is this differential expression that may in part determine sensitivity to treatment with ICB.