Lethal Mechanism Research Articles

STAT3 contributes to cisplatin resistance, modulating EMT markers, and the mTOR signaling in lung adenocarcinoma.

Lung cancer is the second leading cause of cancer death worldwide and is strongly associated with cisplatin resistance. The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells and coordinates critical cellular processes as survival, self-renewal, and inflammation. In several types of cancer, STAT3 controls the development, immunogenicity, and malignant behavior of tumor cells while it dictates the responsiveness to radio- and chemotherapy. It is known that STAT3 phosphorylation at Ser727 by mechanistic target of rapamycin (mTOR) is necessary for its maximal activation, but the crosstalk between STAT3 and mTOR signaling in cisplatin resistance remains elusive. In this study, using a proteomic approach, we revealed important targets and signaling pathways altered in cisplatin-resistant A549 lung adenocarcinoma cells. STAT3 had increased expression in a resistance context, which can be associated with a poor prognosis. STAT3 knockout (SKO) resulted in a decreased mesenchymal phenotype in A549 cells, observed by clonogenic potential and by the expression of epithelial-mesenchymal transition markers. Importantly, SKO cells did not acquire the mTOR pathway overactivation induced by cisplatin resistance. Consistently, SKO cells were more responsive to mTOR inhibition by rapamycin and presented impairment of the feedback activation loop in Akt. Therefore, rapamycin was even more potent in inhibiting the clonogenic potential in SKO cells and sensitized to cisplatin treatment. Mechanistically, STAT3 partially coordinated the cisplatin resistance phenotype via the mTOR pathway in non-small cell lung cancer. Thus, our findings reveal important targets and highlight the significance of the crosstalk between STAT3 and mTOR signaling in cisplatin resistance. The synergic inhibition of STAT3 and mTOR potentially unveil a potential mechanism of synthetic lethality to be explored for human lung cancer treatment.

Paralogous synthetic lethality underlies genetic dependencies of the cancer-mutated gene STAG2.

STAG2, a component of the mitotically essential cohesin complex, is highly mutated in several different tumour types, including glioblastoma and bladder cancer. Whereas cohesin has roles in many cancer-related pathways, such as chromosome instability, DNA repair and gene expression, the complex nature of cohesin function has made it difficult to determine how STAG2 loss might either promote tumorigenesis or be leveraged therapeutically across divergent cancer types. Here, we have performed whole-genome CRISPR-Cas9 screens for STAG2-dependent genetic interactions in three distinct cellular backgrounds. Surprisingly, STAG1, the paralog of STAG2, was the only negative genetic interaction that was shared across all three backgrounds. We also uncovered a paralogous synthetic lethal mechanism behind a genetic interaction between STAG2 and the iron regulatory gene IREB2 Finally, investigation of an unusually strong context-dependent genetic interaction in HAP1 cells revealed factors that could be important for alleviating cohesin loading stress. Together, our results reveal new facets of STAG2 and cohesin function across a variety of genetic contexts.

Overview of Ferroptosis and Synthetic Lethality Strategies.

Ferroptosis, a term first proposed in 2012, is iron-dependent, non-apoptotic regulatory cell death induced by erastin. Ferroptosis was originally discovered during synthetic lethal screening for drugs sensitive to RAS mutant cells, and is closely related to synthetic lethality. Ferroptosis sensitizes cancer stem cells and tumors that undergo epithelial−mesenchymal transition and are resistant to anticancer drugs or targeted therapy. Therefore, ferroptosis-inducing molecules are attractive new research targets. In contrast, synthetic lethal strategies approach mechanisms and genetic abnormalities that cannot be directly targeted by conventional therapeutic strategies, such as RAS mutations, hypoxia, and abnormalities in the metabolic environment. They also target the environment and conditions specific to malignant cells, have a low toxicity to normal cells, and can be used in combination with known drugs to produce new ones. However, the concept of synthetic lethality has not been widely adopted with ferroptosis. In this review, we surveyed the literature on ferroptosis-related factors and synthetic lethality to examine the potential therapeutic targets in ferroptosis-related molecules, focusing on factors related to synthetic lethality, discovery methods, clinical application stages, and issues in drug discovery.

Function of four mitochondrial genes in fumigation lethal mechanisms of Allyl Isothiocyanate against Sitophilus zeamais adults

Allyl isothiocyanate (AITC) is a promising alternative to chemical fumigants, and mitochondrial dysfunction has been proposed to play a crucial role in its lethal mechanisms; however, the specific lethal mechanisms of AITC remain unknown. Four mitochondrial electron transport chain genes, nd5, nd6, cox1, and cox5, were selected from adult Sitophilus zeamais and processed with RNA interference experiments. Then, the biochemical and biophysical effects were compared between double-stranded RNA (dsRNA)-mediated insects and wild-type insects after AITC fumigation at the concentration of LC50 values. The bioactivity of AITC against dsnd6-mediated insects increased, while the bioactivity against dcox1-mediated insects decreased. Compared with the wild-type insects, the increase of reactive oxygen species (ROS) levels by AITC in mitochondria from dsnd6-mediated insects increased by 18.95%, while that of dscox1-mediated insects decreased by 27.45%. The effects of AITC on mRNA expression levels of detoxifying enzymes including CAT (down-regulation effect) and CuZnSOD (overexpression effect) partly recovered in the dsnd5-mediated insects, while a greater effect was observed for dscox1-mediated insects. Molecular docking results indicated that ASN511 at the cox1 subunit was the binding site of AITC by one hydrogen bond, with a bond distance of 2.1 Å. These findings provide insight for further applications of AITC and could provide a novel strategy to investigate lethal mechanisms of insecticides.

Analyzing the Opportunities to Target DNA Double-Strand Breaks Repair and Replicative Stress Responses to Improve Therapeutic Index of Colorectal Cancer.

Simple SummaryColorectal cancer (CRC) is among the most common cancers and the third leading cause of cancer deaths worldwide. Despite the identification of alterations in DNA repair genes and the resulting genomic instability in sub-populations of CRC, therapies that exploit defects in DNA repair pathways or high level of replicative stress have been explored only in breast, ovarian, and other tumor types, but not yet systematically in CRC. Here, we discuss how targeting genes involved in the responses to replication stress and the repair of DNA double-strand breaks (DSBs) could provide new therapeutic opportunities to treat CRCs and have the potential to confer increased sensitivity to current chemotherapy regimens, thus, expanding the spectrum of therapy options, and potentially improving clinical outcomes for CRC patients.Despite the ample improvements of CRC molecular landscape, the therapeutic options still rely on conventional chemotherapy-based regimens for early disease, and few targeted agents are recommended for clinical use in the metastatic setting. Moreover, the impact of cytotoxic, targeted agents, and immunotherapy combinations in the metastatic scenario is not fully satisfactory, especially the outcomes for patients who develop resistance to these treatments need to be improved. Here, we examine the opportunity to consider therapeutic agents targeting DNA repair and DNA replication stress response as strategies to exploit genetic or functional defects in the DNA damage response (DDR) pathways through synthetic lethal mechanisms, still not explored in CRC. These include the multiple actors involved in the repair of DNA double-strand breaks (DSBs) through homologous recombination (HR), classical non-homologous end joining (NHEJ), and microhomology-mediated end-joining (MMEJ), inhibitors of the base excision repair (BER) protein poly (ADP-ribose) polymerase (PARP), as well as inhibitors of the DNA damage kinases ataxia-telangiectasia and Rad3 related (ATR), CHK1, WEE1, and ataxia-telangiectasia mutated (ATM). We also review the biomarkers that guide the use of these agents, and current clinical trials with targeted DDR therapies.

Selective Modulation of a Pan-Essential Protein as a Therapeutic Strategy in Cancer.

Cancer dependency maps, which use CRISPR/Cas9 depletion screens to profile the landscape of genetic dependencies in hundreds of cancer cell lines, have identified context-specific dependencies that could be therapeutically exploited. An ideal therapy is both lethal and precise, but these depletion screens cannot readily distinguish between gene effects that are cytostatic or cytotoxic. Here, we use a diverse panel of functional genomic screening assays to identify NXT1 as a selective and rapidly lethal in vivo relevant genetic dependency in MYCN-amplified neuroblastoma. NXT1 heterodimerizes with NXF1, and together they form the principal mRNA nuclear export machinery. We describe a previously unrecognized mechanism of synthetic lethality between NXT1 and its paralog NXT2: their common essential binding partner NXF1 is lost only in the absence of both. We propose a potential therapeutic strategy for tumor-selective elimination of a protein that, if targeted directly, is expected to cause widespread toxicity. SIGNIFICANCE: We provide a framework for identifying new therapeutic targets from functional genomic screens. We nominate NXT1 as a selective lethal target in neuroblastoma and propose a therapeutic approach where the essential protein NXF1 can be selectively eliminated in tumor cells by exploiting the NXT1-NXT2 paralog relationship.See related commentary by Wang and Abdel-Wahab, p. 2129.This article is highlighted in the In This Issue feature, p. 2113.

Hereditary Prostate Cancer: Genes Related, Target Therapy and Prevention.

Prostate cancer (PCa) is globally the second most diagnosed cancer type and the most common cause of cancer-related deaths in men. Family history of PCa, hereditary breast and ovarian cancer (HBOC) and Lynch syndromes (LS), are among the most important risk factors compared to age, race, ethnicity and environmental factors for PCa development. Hereditary prostate cancer (HPCa) has the highest heritability of any major cancer in men. The proportion of PCa attributable to hereditary factors has been estimated in the range of 5–15%. To date, the genes more consistently associated to HPCa susceptibility include mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) and homologous recombination genes (BRCA1/2, ATM, PALB2, CHEK2). Additional genes are also recommended to be integrated into specific research, including HOXB13, BRP1 and NSB1. Importantly, BRCA1/BRCA2 and ATM mutated patients potentially benefit from Poly (ADP-ribose) polymerase PARP inhibitors, through a mechanism of synthetic lethality, causing selective tumor cell cytotoxicity in cell lines. Moreover, the detection of germline alterations in MMR genes has therapeutic implications, as it may help to predict immunotherapy benefits. Here, we discuss the current knowledge of the genetic basis for inherited predisposition to PCa, the potential target therapy, and the role of active surveillance as a management strategy for patients with low-risk PCa. Finally, the current PCa guideline recommendations are reviewed.

Pathophysiology, Diagnosis, and Management of Coronary No-Reflow Phenomenon.

Coronary no-reflow phenomenon is a lethal mechanism of ongoing myocardial injury, following successful revascularization of an infarct-related coronary artery. Incidence of this phenomenon is high following percutaneous intervention, and is associated with adverse in-hospital and long-term outcomes. Several mechanisms such as ischemia-reperfusion injury and distal microthromboembolism in genetically susceptible patients and those with preexisting endothelial dysfunction have been implicated. However, the exact mechanism in humans is still poorly understood. Several investigative and treatment strategies within and outside the cardiac catheterization laboratory have been proposed, but have not uniformly shown success in reducing mortality or in preventing adverse left ventricular remodeling resulting from this condition. The aim of this article is to provide a brief and concise review of the current understanding of the pathophysiology, clinical predictors, and investigations and management of coronary no-reflow phenomenon.

Overcoming Radiation Resistance by Iron-Platinum Metal Alloy Nanoparticles in Human Copper Transport 1-Overexpressing Cancer Cells via Mitochondrial Disturbance.

BackgroundRadiation therapy remains an important treatment modality in cancer therapy, however, resistance is a major problem for treatment failure. Elevated expression of glutathione is known to associate with radiation resistance. We used glutathione overexpressing small cell lung cancer cell lines, SR3A-13 and SR3A-14, established by transfection with γ-glutamylcysteine synthetase (γ-GCS) cDNA, as a model for investigating strategies of overcoming radiation resistance. These radiation-resistant cells exhibit upregulated human copper transporter 1 (hCtr1), which also transports cisplatin. This study was initiated to investigate the effect and the underlying mechanism of iron-platinum nanoparticles (FePt NPs) on radiation sensitization in cancer cells.Materials and MethodsUptakes of FePt NPs in these cells were studied by plasma optical emission spectrometry and transmission electron microscopy. Effects of the combination of FePt NPs and ionizing radiation were investigated by colony formation assay and animal experiment. Intracellular reactive oxygen species (ROS) were assessed by using fluorescent probes and imaged by a fluorescence-activated-cell-sorting caliber flow cytometer. Oxygen consumption rate (OCR) in mitochondria after FePt NP and IR treatment was investigated by a Seahorse XF24 cell energy metabolism analyzer.ResultsThese hCtr1-overexpressing cells exhibited elevated resistance to IR and the resistance could be overcome by FePt NPs via enhanced uptake of FePt NPs. Overexpression of hCtr1 was responsible for the increased uptake/transport of FePt NPs as demonstrated by using hCtr1-transfected parental SR3A (SR3A-hCtr1-WT) cells. Increased ROS and drastic mitochondrial damages with substantial reduction of oxygen consumption rate were observed in FePt NPs and IR-treated cells, indicating that structural and functional insults of mitochondria are the lethal mechanism of FePt NPs. Furthermore, FePt NPs also increased the efficacy of radiotherapy in mice bearing SR3A-hCtr1-WT-xenograft tumors.ConclusionThese results suggest that FePt NPs can potentially be a novel strategy to improve radiotherapeutic efficacy in hCtr1-overexpressing cancer cells via enhanced uptake and mitochondria targeting.

Insecticidal action of the botanical insecticide wilforine on Mythimna separata (Walker) related with the changes of ryanodine receptor expression

The detailed molecular mechanism of wilforine, a novel botanical insecticidal component, remains unclear, except for the knowledge that it affects the calcium signaling pathway. The aim of the current study was to examine the underlying molecular mechanism of wilforine in Mythimna separata (Walker) by transcriptome and RNA interference (RNAi), with chlorantraniliprole as control. RNA sequencing showed that the relative expression of genes related to the calcium signaling pathway and muscle contraction in M. separata treated with wilforine significantly changed and was further validated by qRT-PCR. Interestingly, the expression level of the ryanodine receptor (MsRyR) gene was downregulated by wilforine at relatively high concentrations and long treatment time, contrary to that observed using chlorantraniliprole. Furthermore, a putative MsRyR was cloned using a 16,258-bp contiguous sequence containing a 308-bp 5′-untranslated region and 578-bp 3′-untranslated region by RT-PCR and RACE. The results of the RNAi experiment showed that injection of dsMsRyR significantly reduced MsRyR mRNA levels, and growth and development were inhibited. Importantly, silencing of the MsRyR gene resulted in decreased susceptibility to both wilforine and chlorantraniliprole. Together with the results of our previous studies on toxic symptoms and muscle tissue lesions between wilforine and chlorantraniliprole, we propose that RyR Ca2+ release channel dysfunction is closely related with significant lethal mechanisms of wilforine.

Comparative transcriptome analysis of lethality in response to RNA interference of the oriental river prawn (Macrobrachium nipponense)

A previous study identified slow-tonic S2 tropomyosin and slow tropomyosin isoform as sex-related genes in Macrobrachium nipponense. Their functions were analyzed using RNA interference. However, more than half of the specimens died approximately 8–12 h after injection of the respective double-stranded RNAs (dsRNAs), and HE staining indicated that the heart and gills were the most likely tissues responsible for the resultant deaths. In the current study, we conducted a comparative transcriptomic study of the gills and hearts of M. nipponense to identify potential target genes associated with acute death after dsRNA injection. A total of 68,772 annotated unigenes were generated. In the heart, differentially expressed genes (DEGs) were mainly enriched in glycolysis/gluconeogenesis and oxidative phosphorylation, while the most relevant pathways in the gills were lysosome, phagosome, and peroxisome. Ten DEGs were screened out and analyzed under lethal hypoxic stress. Among these, fructose 1, 6-biphosphate-aldolase (FBA), glyceraldehyde 3-phosphate dehydrogenase (GDPDH), alcohol dehydrogenase class-3 (ADC3), ATP-synthase subunit 9 (ATPS9), and acid ceramidase-like (ACL) were all differentially expressed under hypoxic conditions. This study shed light on the lethal mechanism caused by interference with tropomyosin genes in M. nipponense, and identifies the related pathways and key genes that could help to improve stress resistance and tolerance in M. nipponense.

In search of novel synthetic lethality anti-cancer drug targets in intrahepatic cholangiocarcinoma: MTAP genomic loss.

337 Background: The association of BRCA tumor suppressor gene (TSG) inactivation and PARP inhibitor efficacy through a mechanism of synthetic lethality (SL) is now well-established. Recently, the PRMT5 arginine methyltransferase dependence in cells with MTAP (S-methyl-5'-thioadenosine phosphorylase) genomic alterations (GA) has been proposed as a new SL based anti-tumor strategy and under consideration for development for intrahepatic cholangiocarcinoma (IC). Methods: 2,170 cases of clinically advanced IC were submitted for comprehensive genomic profiling (CGP) to detect genomic alterations (GA), tumor mutational burden (TMB) and microsatellite instability (MSI) using a hybrid capture based assay (F1CDx). PD-L1 expression in tumor cells (Dako 22C3) was measured by IHC and scored using the tumor proportion score (TPS) method. Results: 328 (15%) ICs featured MTAP inactivation (MTAP-IC). 48% of MTAP-IC cases were female and 52% were male with a median age of 66 years (range 26-93 years). MTAP-IC featured 6.3 GA per sample. The median TMB was 2.5 mut/Mb with only 2% of cases with TMB ≥10 mut/Mb. No (0%) MTAP-IC cases were MSI-high. Of 112 MTAP-IC IHC stained for PD-L1, 4% were PD-L1 high (> 50%), 14% were PD-L1 low (1-49%) and 82% were PD-L1 negative. 99% of MTAP inactivation was by copy number (CN) loss and 1% was due to non-CN mutation. In MTAP-IC, 99% featured CDKN2A loss and 95% featured CDKN2B loss (9p21 co-deletion). TP53 was mutated in 31% and KRAS in 24% (3% G12C). Other currently non-targetable GA included ARID1A (19%), BAP1 (16%) and SMAD4 (13%). Among potentially targetable GA, the MTAP-IC featured 12% FGFR2 GA (8% rearrangements), 10% BRAF (8% V600E), 7% IDH1, 7% PIK3CA, 4% BRCA2, 4% ERBB2 (2% amplification) and 3% MET. Conclusions: Comprehensive genomic profiling elucidated that MTAP GA are frequent in IC and features relatively high GA/tumor, including 9p21 co-deletion, but relatively low TMB and PD-L1 expression. Isolated MTAP deletion without CDKN2A/B co-deletion was extremely rare. In addition to the potential targeting of PRMT5 in MTAP-IC tumors, co-altered genes known to be targets in IC such as FGFR2, IDH1 and BRAF are still prevalent in MTAP-IC, indicating potential for combining targeted therapies in some MTAP-IC patients.

TBHQ-Overview of Multiple Mechanisms against Oxidative Stress for Attenuating Methamphetamine-Induced Neurotoxicity.

Methamphetamine is a derivative of amphetamines, a highly addictive central stimulant with multiple systemic toxicity including the brain, heart, liver, lung, and spleen. It has adverse effects such as apoptosis and breakdown of the blood-brain barrier. Methamphetamine is a fatal and toxic chemical substance, and its lethal mechanism has been widely studied in recent years. The possible mechanism is that methamphetamine can cause cardiotoxicity and neurotoxicity mainly by inducing oxidative stress so as to generate heat, eliminate people's hunger and thirst, and maintain a state of excitement so that people can continue to exercise. According to many research, there is no doubt that methamphetamine triggers neurotoxicity by inducing reactive oxygen species (ROS) production and redox imbalance. This review summarized the mechanisms of methamphetamine-induced neurotoxicity including apoptosis and blood-brain barrier breakdown through oxidative stress and analyzed several possible antioxidative mechanisms of tert-butylhydroquinone (TBHQ) which is a kind of food additive with antioxidative effects. As a nuclear factor E2-related factor 2 (Nrf2) agonist, TBHQ may inhibit neurotoxicity caused by oxidative stress through the following three mechanisms: the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, the astrocytes activation, and the glutathione pathway. The mechanism about methamphetamine's toxic effects and its antioxidative therapeutic drugs would become a research hotspot in this field and has very important research significance.

Mitochondrial F1FO-ATPase and permeability transition pore response to sulfide in the midgut gland of Mytilus galloprovincialis

The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca2+ increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial F1FO-ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg2+ by Ca2+ in the F1FO-ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg2+- or Ca2+-dependent F1FO-ATPase activities, namely the same affinity for ATP and bi-site activation kinetics by the ATP substrate, in spite of the higher enzyme activity and coupling efficiency of the Mg2+-dependent F1FO-ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery. Other than being a mitochondrial poison and environmental contaminant, sulfide at low concentrations acts as gaseous mediator and can induce post-translational modifications of proteins. The sulfide donor NaHS, at micromolar concentrations, does not alter the two F1FO-ATPase activities, but desensitizes the mPTP to Ca2+ input. Unexpectedly, NaHS, under the conditions tested, points out a chemical refractoriness of both F1FO-ATPase activities and a failed relationship between the Ca2+-dependent F1FO-ATPase and the mPTP in mussels. The findings suggest that mPTP role and regulation may be different in different taxa and that the F1FO-ATPase insensitivity to NaHS may allow mussels to cope with environmental sulfide.

Clear cell renal cell carcinoma ontogeny and mechanisms of lethality.

The molecular features that define clear cell renal cell carcinoma (ccRCC) initiation and progression are being increasingly defined. The TRACERx Renal studies and others that have described the interaction between tumour genomics and remodelling of the tumour microenvironment provide important new insights into the molecular drivers underlying ccRCC ontogeny and progression. Our understanding of common genomic and chromosomal copy number abnormalities in ccRCC, including chromosome 3p loss, provides a mechanistic framework with which to organize these abnormalities into those that drive tumour initiation events, those that drive tumour progression and those that confer lethality. Truncal mutations in ccRCC, including those in VHL, SET2, PBRM1 and BAP1, may engender genomic instability and promote defects in DNA repair pathways. The molecular features that arise from these defects enable categorization of ccRCC into clinically and therapeutically relevant subtypes. Consideration of the interaction of these subtypes with the tumour microenvironment reveals that specific mutations seem to modulate immune cell populations in ccRCC tumours. These findings present opportunities for disease prevention, early detection, prognostication and treatment.

Cadmium exposure alters expression of protective enzymes and protein processing genes in venom glands of the wolf spider Pardosa pseudoannulata.

Cadmium (Cd) pollution is currently the most serious type of heavy metal pollution throughout the world. Previous studies have shown that Cd elevates the mortality of paddy field spiders, but the lethal mechanism remains to be explored profoundly. In the present study, we measured the activities of protective enzymes (acetylcholinesterase, glutathione peroxidase, phenol oxidase) and a heavy metal chelating protein (metallothionein) in the pond wolf spider Pardosa pseudoannulata after Cd exposure. The results indicated that Cd initially increased the enzyme activities and protein concentration of the spider after 10- and 20-day exposure before inhibiting them at 30-day exposure. Further analysis showed that the enzyme activities in the cephalothorax were inhibited to some extent. Since the cephalothorax region contains important venom glands, we performed transcriptome sequencing (RNA-seq) analysis of the venom glands collected from the spiders after long-term Cd exposure. RNA-seq yielded a total of 2826 differentially expressed genes (DEGs), and most of the DEGs were annotated into the process of protein synthesis, processing and degradation. Furthermore, a mass of genes involved in protein recognition and endoplasmic reticulum (ER) -associated protein degradation were down-regulated. The reduction of protease activities supports the view that protein synthesis and degradation in organelles and cytoplasm were dramatically inhibited. Collectively, our outcomes illustrate that Cd poses adverse effects on the expression of protective enzymes and protein, which potentially down-regulates the immune function in the venom glands of the spiders via the alteration of protein processing and degradation in the ER.

Abstract 2379: Elaborating the mechanism of PARP synthetic lethality in ATM loss

Abstract PARP inhibitors have transformed the treatment for ovarian cancer, which is known to have high incidence of homologous recombination (HR) deficiency through the loss of BRCA1 or BRCA2 genes. 30-35% of ovarian cancer patients have defects in BRCA1/2 genes caused by germline, somatic or epigenetic alterations. PARP inhibitors induce cell death in HR-deficient cells through the mechanism of synthetic lethality where cancer cells cannot tolerate the loss of both SSB and DSB repair machinery. Although genetic or epigenetic alterations in other pathways such as Fanconi anemia, or in non-authentic homologous recombination repair (HRR) genes such as PTEN, IDH, and CDK12 have been reported to contribute to HR deficiency, mechanistic understanding of HR loss remains to be determined. Another DNA damage repair (DDR) protein, ATM, a kinase that phosphorylates and activates major DNA damage checkpoints, i.e. CHK2 & BRCA1, has also being increasingly recognized recently as a synthetic lethal partner with PARP inhibitors in multiple tumor indications such as prostate, lung and colorectal cancers. ATM functions in both cell cycle arrest and DNA repair in response to DSBs, however how cells respond to inhibition of PARP in the absence of ATM is not yet fully understood. In this study, we demonstrated that loss of ATM function, i.e. via generating an ATM KO by CRISPR/Cas9 in two genetic backgrounds, i.e. in DLD-1 and HeLa cell lines, induced HR-deficiency indicated by the impairment of Rad51-foci formation and increased phosphorylation of histone H2AX in ATM KO (-/-) cell lines. Furthermore DLD-1 ATM KO (-/-) cell line demonstrated increased sensitivity to niraparib (i.e. 45-fold difference in sensitivity compared to the DLD-1 parental, HR-proficient cell line) in the 14day colony formation assay. In comparison, BRCA2 (-/-) KO in DLD-1 gave a 125-fold increase compared to parental cell line. In DLD-1 ATM KO (-/-) cell line, we have also detected that loss of ATM was associated with low BRCA1 and BRCA2 protein expression suggestive of downstream effect on impairment of HR-mediated DNA checkpoint signaling in ATM loss. Thus, PARPi synthetic lethality phenotype observed in tumors with ATM loss could be regulated through the combination effect coming from the loss of both of ATM & BRCA1/2 and the subsequent effect on both HR gene functions. Our findings highlight that ATM has a critical role in DNA repair for the synergism with PARP inhibitors. Citation Format: Asli Muvaffak, Kevin G. Coleman. Elaborating the mechanism of PARP synthetic lethality in ATM loss [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2379.

Abstract 1422: Clinical association of miR-155-5p with breast cancer and its relevance for treatment with PARP inhibitors

Abstract INTRODUCTION. miR-155-5p is a well-known oncogenic microRNA frequently overexpressed in human cancers, including breast cancer (BC), but its putative role as biomarker remains controversial. Thus, we aimed at evaluating miR-155-5p levels in a large BC cohort, and its association with clinical parameters. Then, since miR-155-5p was found to be epigenetically controlled by BRCA1, and to increase the tumor mutation load, we analysed miR-155-5p correlation with defects in Homologous Recombination (HR) genes (i.e. HRD), and its capability to alter the response to the first-in-class PARP (Poly-[ADP-ribose]-polymerase-1 (PARP-1)) inhibitor Olaparib (AZD2281). METHODS. We measured miR-155-5p expression in a retrospective cohort of breast cancer patients (CSS cohort, n=283; median follow-up=81 months) by RT-qPCR. Then, we used the TCGA Breast Cancer (TCGA-BC) dataset (n=1095) to validate our results, and evaluate the association between miR-155-5p and mutations in HR genes. Last, we ectopically modulated miR-155-5p in three wtBRCA1/2 cell lines (i.e. MDA-MB-231, MDA-MB-468, MDA-MB-453) and one mutBRCA1 cell line (MDA-MB-436) by using mirVana miR-155-5p mimic or inhibitor, and we measured cell viability before and after treatment with Olaparib or vehicle by PrestoBlue viability reagent. RESULTS. High miR-155-5p levels were associated with unfavourable prognostic indicators including high tumor grade (overall P=0.0007; G2 vs G3 P=0.0053), reduced expression of ER (r= -0.243; P=0.0002) and PgR (r= -0.240; P<0.0001), and high Ki-67 expression (r=0.215; P=0.0005). Consistently, miR-155-5p was differentially expressed across the BC subgroups identified by the surrogate molecular classification in the CSS-cohort (P=0.0008) and by the PAM50 panel in the TCGA-BC dataset (P=0.0001), with the Triple Negative and HER2-amplified tumors showing the highest levels. The analysis of the TCGA BRCA dataset also found higher miR-155 levels in the patient subgroup carrying somatic mutations in HR genes compared to the subgroup without somatic mutations in HR genes (P<0.0001). Last, the in vitro drug testing confirmed that mutBRCA1 MDA-MB-436 cells, exhibiting miR-155-5p endogenous levels 1000-fold higher than the other TNBC cell lines, had a higher sensitivity to a short-term (72hrs) treatment with Olaparib alone (IC50 25.80, 95%CI 24.52-27.15) compared to the other cell lines. Importantly, ectopic overexpression of miR-155-5p in wtBRCA1/2 cell lines, followed by Olaparib exposure, resulted in a significant reduction in viability (p<0.001) compared to Olaparib alone in all cell lines. However, miR-155-5p inhibition in BRCA1mut MDA-MB-436 cell line did not affect the higher sensitivity to PARP inhibition, suggesting that sensitivity to PARPi is not limited to HRD. CONCLUSIONS. Overall, our data point to a role of miR-155-5p in Homologous Recombination Deficiency (HRD), and suggest a putative novel mechanism of synthetic lethality mediated by PARP inhibition and miR-155-5p to promote cancer cell death. Citation Format: Barbara Pasculli, Raffaela Barbano, Andrea Fontana, Tommaso Biagini, Maria Pia Di Viesti, Michelina Rendina, Vanna Maria Valori, Maria Morritti, Sara Bravaccini, Sara Ravaioli, Evaristo Maiello, Paolo Graziano, Roberto Murgo, Massimiliano Copetti, Tommaso Mazza, Vito Michele Fazio, Manel Esteller, Paola Parrella. Clinical association of miR-155-5p with breast cancer and its relevance for treatment with PARP inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1422.

Abstract 2601: Expression and clinicopathological association of RABs in pancreatic cancer

Abstract Rab proteins form the largest subfamily of small GTPases and play an indispensable role in the regulation of vesicle trafficking. They control a wide variety of endocytic, transcytic and exocytic transport pathways and thus play important roles in cellular growth and behavior. Altered expression and functions of Rab proteins have been reported in various malignancies and implicated in tumorigenic processes. Pancreatic cancer (PC) is a highly lethal malignancy and molecular mechanisms associated with its pathobiology are relatively less understood. Here we mined The Cancer Genome Atlas (TCGA) Database for differential expression and clinicopathological association of RAB genes in pancreatic cancer. Out of the 62 RAB genes analyzed, 5 (RAB3A, RAB26, RAB25, RAB21, and RAB22A) were found to have significant upregulation in cancer, while 5 (RAB6B, RAB8B, RABL2A, RABL2B and RAB32) exhibited significant downregulation. Racially disparate expression was also reported for some RABs. RAB3A and RAB26 had significantly higher and lower expression, respectively, in African American (AA) and Asian PC as compared to Caucasian Americans (CA) PC, whereas RAB25 was significantly higher in Asian PC than CA PC. No clear trend of altered expression was observed with increasing stage and grade, although stage- and grade-specific differences were recorded for some RABs. RAB21, RAB22A, and RAB8B showed significantly higher expression in PC from middle-aged patients (41-60 years) compared to older age groups, but no gender-associated differential expression was observed. PC from occasional drinkers had significantly higher expression of RAB21 compared to daily or weekly drinker, whereas RAB25 expression was significantly higher in social drinkers, compared to occasional ones. Expression of RABL2A was significantly reduced in PC of diabetic patients whereas RAB26 was significantly down in pancreatitis patients. Most importantly, a significant association of high expression of RAB21, RAB22A and RAB25, and low expression of RAB6B, RABL2A, and RABL2B was observed with poorer survival of PC patients. Together, our study is suggestive of potential diagnostic and prognostic significance of RABs in PC and warrants further investigations to define their functional and mechanistic significance. Citation Format: Shashi Anand, Haseeb Zubair, Mohammad Aslam Khan, Seema Singh, Ajay P. Singh. Expression and clinicopathological association of RABs in pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2601.

Abstract 1058: Targeting chromatin remodeling-associated genetic vulnerabilities in cancer: PBRM1 defects are synthetic lethal with PARP and ATR inhibitors

Abstract Aim: Polybromo-1 (PBRM1), a specific subunit of the pBAF chromatin remodeling complex, is frequently inactivated in cancer. For example, 40% of clear cell Renal Cell Carcinoma (ccRCC) and 15% of cholangiocarcinoma present deleterious PBRM1 mutations. There is currently no precision medicine-based therapeutic approach that targets PBRM1 defects. To identify novel, targeted therapeutic strategies for PBRM1-defective cancers, we carried out high-throughput functional genomics and drug screenings followed by in vitro and in vivo validation studies. Methods: High-throughput siRNA-drug sensitization and drug sensitivity screens evaluating > 150 cancer-relevant small molecules in dose-response were performed in Pbrm1 siRNA-transfected mouse embryonic stem cells (mES) and isogenic PBRM1-KO or -WT HAP1 cells, respectively. After identification of PBRM1-selective small molecules, revalidation was carried out in a series of in-house-generated isogenic models of PBRM1 deficiency - including 786-O (ccRCC), A498 (ccRCC), U2OS (osteosarcoma) and H1299 (non-small cell lung cancer) human cancer cell lines - and non-isogenic ccRCC models, using multiple clinical compounds. Mechanistic dissection was performed using immunofluorescence, RT-qPCR, western blotting, DNA fiber assay, transcriptomics, proteomics and DRIP-sequencing to evaluate markers of DNA damage response (DDR), replication stress and cell-autonomous innate immune signaling. Preclinical data were integrated with TCGA tumor data. Results: Parallel high-throughput drug screens independently identified PARP inhibitors (PARPi) as being synthetic lethal with PBRM1 defects - a cell type-independent effect which was exacerbated by ATR inhibitors (ATRi) and which we revalidated in vitro in isogenic and non-isogenic systems and in vivo in a xenograft model. PBRM1 defects were associated with increased replication fork stress (higher γH2AX and RPA foci levels, decreased replication fork speed and increased ATM checkpoint activation), R-loop accumulation and enhanced genomic instability in vitro; these effects were exacerbated upon PARPi exposure. In patient tumor samples, we also found that PBRM1-mutant cancers possessed a higher mutational load. Finally, we found that ATRi selectively activated the cGAS/STING cytosolic DNA sensing pathway in PBRM1-deficient cells, resulting in increased expression of type I interferon genes. Conclusion: PBRM1-defective cancer cells present increased replication fork stress, R-loop formation, genome instability and are selectively sensitive to PARPi and ATRi through a synthetic lethal mechanism that is cell type-independent. Our data provide the pre-clinical rationale for assessing PARPi as a monotherapy or in combination with ATRi or immune-modulating agents in molecularly-selected patients with PBRM1-defective cancers. Citation Format: Roman Merial Chabanon, Daphné Morel, Léo Colmet-Daage, Thomas Eychenne, Nicolas Dorvault, Ilirjana Bajrami, Marlène Garrido, Suzanna Hopkins, Cornelia Meisenberg, Andrew Lamb, Theo Roumeliotis, Samuel Jouny, Clémence Astier, Asha Konde, Geneviève Almouzni, Jyoti Choudhary, Jean-Charles Soria, Jessica Downs, Christopher J. Lord, Sophie Postel-Vinay. Targeting chromatin remodeling-associated genetic vulnerabilities in cancer: PBRM1 defects are synthetic lethal with PARP and ATR inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1058.