The CD73 immune checkpoint promotes tumor cell metabolic fitness.

CD73 is an ectonucleotidase overexpressed on tumor cells that suppresses anti-tumor immunity. Accordingly, several CD73 inhibitors are currently being evaluated in the clinic, including in large randomized clinical trials. Yet, the tumor cell-intrinsic impact of CD73 remain largely uncharacterized. Using metabolomics, we discovered that CD73 significantly enhances tumor cell mitochondrial respiration and aspartate biosynthesis. Importantly, rescuing aspartate biosynthesis was sufficient to restore proliferation of CD73-deficient tumors in immune deficient mice. Seahorse analysis of a large panel of mouse and human tumor cells demonstrated that CD73 enhanced oxidative phosphorylation (OXPHOS) and glycolytic reserve. Targeting CD73 decreased tumor cell metabolic fitness, increased genomic instability and suppressed poly ADP ribose polymerase (PARP) activity. Our study thus uncovered an important immune-independent function for CD73 in promoting tumor cell metabolism, and provides the rationale for previously unforeseen combination therapies incorporating CD73 inhibition.

The APE2 nuclease is essential for DNA double-strand break repair by microhomology-mediated end joining.

Microhomology-mediated end joining (MMEJ) is an intrinsically mutagenic pathway of DNA double-strand break (DSB) repair essential for proliferation of homologous recombination (HR)-deficient tumors. Although targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as an MMEJ effector. We show that loss of APE2 inhibits MMEJ at deprotected telomeres and at intra-chromosomal DSBs and is epistatic with Pol Theta for MMEJ activity. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease activity, and further identify an uncharacterized domain required for its recruitment to DSBs. We conclude that this previously unappreciated role of APE2 in MMEJ contributes to the addiction of HRD cells to APE2, which could be exploited in the treatment of cancer.

Pre-activation of autophagy impacts response to olaparib in prostate cancer cells.

Poly (ADP-ribose) polymerase 1 (PARP1) plays an essential role in DNA repair and is targeted by anticancer therapies using PARP inhibitors (PARPi) such as olaparib. PARPi treatment in prostate cancer (PC) is currently used as a monotherapy or in combination with standard therapies (hormonotherapy) in clinical trials for patients with DNA damage response mutation. Unfortunately, 20% of these patients did not respond to this new treatment. This resistance mechanism in PC is still not well understood. Here, we report that autophagy affects differently the response of PC cell lines to olaparib depending on its activation status. Pre-activation of autophagy before olaparib resulted in an increase of DNA repair activity by homologous recombination (HR) to repair double-strand breaks induced by olaparib and enhanced cell proliferation. When autophagy was activated after olaparib treatment, or completely inhibited, PC cells demonstrated an increased sensitivity to this PARPi. This autophagy-mediated resistance is, in part, regulated by the nuclear localization of sequestrosome 1 (SQSTM1/p62). Decrease of SQSTM1/p62 nuclear localization due to autophagy pre-activation leads to an increase of filamin A (FLNA) protein expression and BRCA1/Rad51 recruitment involved in the HR pathway. Our results reveal that autophagy basal levels may in part determine amenability to PARPi treatment.

Targeting IKKε in Androgen-Independent Prostate Cancer Causes Phenotypic Senescence and Genomic Instability.

Advanced prostate cancer will often progress to a lethal, castration-resistant state. We previously demonstrated that IKKε expression correlated with the aggressiveness of prostate cancer disease. Here, we address the potential of IKKε as a therapeutic target in prostate cancer. We examined cell fate decisions (proliferation, cell death, and senescence) in IKKε-depleted PC-3 cells, which exhibited delayed cell proliferation and a senescent phenotype, but did not undergo cell death. Using IKKε/TBK1 inhibitors, BX795 and Amlexanox, we measured their effects on cell fate decisions in androgen-sensitive prostate cancer and androgen-independent prostate cancer cell lines. Cell-cycle analyses revealed a G2-M cell-cycle arrest and a higher proportion of cells with 8N DNA content in androgen-independent prostate cancer cells only. Androgen-independent prostate cancer cells also displayed increased senescence-associated (SA)-ß-galactosidase activity; increased γH2AX foci; genomic instability; and altered p15, p16, and p21 expression. In our mouse model, IKKε inhibitors also decreased tumor growth of androgen-independent prostate cancer xenografts but not 22Rv1 androgen-sensitive prostate cancer xenografts. Our study suggests that targeting IKKε with BX795 or Amlexanox in androgen-independent prostate cancer cells induces a senescence phenotype and demonstrates in vivo antitumor activity. These results strengthen the potential of exploiting IKKε as a therapeutic target.

NR1D1 regulation by Ran GTPase via miR4472 identifies an essential vulnerability linked to aneuploidy in ovarian cancer.

While aneuploidy is a main enabling characteristic of cancers, it also creates specific vulnerabilities. Here we demonstrate that Ran inhibition targets epithelial ovarian cancer (EOC) survival through its characteristic aneuploidy. We show that induction of aneuploidy in rare diploid EOC cell lines or normal cells renders them highly dependent on Ran. We also establish an inverse correlation between Ran and the tumor suppressor NR1D1 and reveal the critical role of Ran/NR1D1 axis in aneuploidy-associated endogenous DNA damage repair. Mechanistically, we show that Ran, through the maturation of miR4472, destabilizes the mRNA of NR1D1 impacting several DNA repair pathways. We showed that NR1D1 interacts with both PARP1 and BRCA1 leading to the inhibition of DNA repair. Concordantly, loss of Ran was associated with NR1D1 induction, accumulation of DNA damages, and lethality of aneuploid EOC cells. Our findings suggest a synthetic lethal strategy targeting aneuploid cells based on their dependency to Ran.

A functionally impaired missense variant identified in French Canadian families implicates FANCI as a candidate ovarian cancer-predisposing gene.

BACKGROUND: Familial ovarian cancer (OC) cases not harbouring pathogenic variants in either of the BRCA1 and BRCA2 OC-predisposing genes, which function in homologous recombination (HR) of DNA, could involve pathogenic variants in other DNA repair pathway genes. METHODS: Whole exome sequencing was used to identify rare variants in HR genes in a BRCA1 and BRCA2 pathogenic variant negative OC family of French Canadian (FC) ancestry, a population exhibiting genetic drift. OC cases and cancer-free individuals from FC and non-FC populations were investigated for carrier frequency of FANCI c.1813C>T; p.L605F, the top-ranking candidate. Gene and protein expression were investigated in cancer cell lines and tissue microarrays, respectively. RESULTS: In FC subjects, c.1813C>T was more common in familial (7.1%, 3/42) than sporadic (1.6%, 7/439) OC cases (P = 0.048). Carriers were detected in 2.5% (74/2950) of cancer-free females though female/male carriers were more likely to have a first-degree relative with OC (121/5249, 2.3%; Spearman correlation = 0.037; P = 0.011), suggesting a role in risk. Many of the cancer-free females had host factors known to reduce risk to OC which could influence cancer risk in this population. There was an increased carrier frequency of FANCI c.1813C>T in BRCA1 and BRCA2 pathogenic variant negative OC families, when including the discovery family, compared to cancer-free females (3/23, 13%; OR = 5.8; 95%CI = 1.7-19; P = 0.005). In non-FC subjects, 10 candidate FANCI variants were identified in 4.1% (21/516) of Australian OC cases negative for pathogenic variants in BRCA1 and BRCA2, including 10 carriers of FANCI c.1813C>T. Candidate variants were significantly more common in familial OC than in sporadic OC (P = 0.04). Localization of FANCD2, part of the FANCI-FANCD2 (ID2) binding complex in the Fanconi anaemia (FA) pathway, to sites of induced DNA damage was severely impeded in cells expressing the p.L605F isoform. This isoform was expressed at a reduced level, destabilized by DNA damaging agent treatment in both HeLa and OC cell lines, and exhibited sensitivity to cisplatin but not to a poly (ADP-ribose) polymerase inhibitor. By tissue microarray analyses, FANCI protein was consistently expressed in fallopian tube epithelial cells and only expressed at low-to-moderate levels in 88% (83/94) of OC samples. CONCLUSIONS: This is the first study to describe candidate OC variants in FANCI, a member of the ID2 complex of the FA DNA repair pathway. Our data suggest that pathogenic FANCI variants may modify OC risk in cancer families.

Carboplatin response in preclinical models for ovarian cancer: comparison of 2D monolayers, spheroids, ex vivo tumors and in vivo models.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer. Among the key challenges in developing effective therapeutics is the poor translation of preclinical models used in the drug discovery pipeline. This leaves drug attrition rates and costs at an unacceptably high level. Previous work has highlighted the discrepancies in therapeutic response between current in vitro and in vivo models. To address this, we conducted a comparison study to differentiate the carboplatin chemotherapy response across four different model systems including 2D monolayers, 3D spheroids, 3D ex vivo tumors and mouse xenograft models. We used six previously characterized EOC cell lines of varying chemosensitivity and performed viability assays for each model. In vivo results from the mouse model correlated with 2D response in 3/6 cell lines while they correlated with 3D spheroids and the ex vivo model in 4/6 and 5/5 cell lines, respectively. Our results emphasize the variability in therapeutic response across models and demonstrate that the carboplatin response in EOC cell lines cultured in a 3D ex vivo model correlates best with the in vivo response. These results highlight a more feasible, reliable, and cost-effective preclinical model with the highest translational potential for drug screening and prediction studies in EOC.

Carboplatin sensitivity in epithelial ovarian cancer cell lines: The impact of model systems.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy in North America, underscoring the need for the development of new therapeutic strategies for the management of this disease. Although many drugs are pre-clinically tested every year, only a few are selected to be evaluated in clinical trials, and only a small number of these are successfully incorporated into standard care. Inaccuracies with the initial in vitro drug testing may be responsible for some of these failures. Drug testing is often performed using 2D monolayer cultures or 3D spheroid models. Here, we investigate the impact that these different in vitro models have on the carboplatin response of four EOC cell lines, and in particular how different 3D models (polydimethylsiloxane-based microfluidic chips and ultra low attachment plates) influence drug sensitivity within the same cell line. Our results show that carboplatin responses were observed in both the 3D spheroid models tested using apoptosis/cell death markers by flow cytometry. Contrary to previously reported observations, these were not associated with a significant decrease in spheroid size. For the majority of the EOC cell lines (3 out of 4) a similar carboplatin response was observed when comparing both spheroid methods. Interestingly, two cell lines classified as resistant to carboplatin in 2D cultures became sensitive in the 3D models, and one sensitive cell line in 2D culture showed resistance in 3D spheroids. Our results highlight the challenges of choosing the appropriate pre-clinical models for drug testing.

Long-term fluorescence hyperspectral imaging of on-chip treated co-culture tumour spheroids to follow clonal evolution.

Multicellular tumour spheroids are an ideal in vitro tumour model to study clonal heterogeneity and drug resistance in cancer research because different cell types can be mixed at will. However, measuring the individual response of each cell population over time is challenging: current methods are either destructive, such as flow cytometry, or cannot image throughout a spheroid, such as confocal microscopy. Our group previously developed a wide-field fluorescence hyperspectral imaging system to study spheroids formed and cultured in microfluidic chips. In the present study, two subclones of a single parental ovarian cancer cell line transfected to express different fluorophores were produced and co-culture spheroids were formed on-chip using ratios forming highly asymmetric subpopulations. We performed a 3D proliferation assay on each cell population forming the spheroids that matched the 2D growth behaviour. Response assays to PARP inhibitors and platinum-based drugs were also performed to follow the clonal evolution of mixed populations. Our experiments show that hyperspectral imaging can detect spheroid response before observing a decrease in spheroid diameter. Hyperspectral imaging and microfluidic-based spheroid assays provide a versatile solution to study clonal heterogeneity, able to measure response in subpopulations presenting as little as 10% of the initial spheroid.

Exploiting interconnected synthetic lethal interactions between PARP inhibition and cancer cell reversible senescence.

Senescence is a tumor suppression mechanism defined by stable proliferation arrest. Here we demonstrate that the known synthetic lethal interaction between poly(ADP-ribose) polymerase 1 inhibitors (PARPi) and DNA repair triggers p53-independent ovarian cancer cell senescence defined by senescence-associated phenotypic hallmarks including DNA-SCARS, inflammatory secretome, Bcl-XL-mediated apoptosis resistance, and proliferation restriction via Chk2 and p21 (CDKN1A). The concept of senescence as irreversible remains controversial and here we show that PARPi-senescent cells re-initiate proliferation upon drug withdrawal, potentially explaining the requirement for sustained PARPi therapy in the clinic. Importantly, PARPi-induced senescence renders ovarian and breast cancer cells transiently susceptible to second-phase synthetic lethal approaches targeting the senescence state using senolytic drugs. The combination of PARPi and a senolytic is effective in preclinical models of ovarian and breast cancer suggesting that coupling these synthetic lethalities provides a rational approach to their clinical use and may together be more effective in limiting resistance.

Genomic consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes.

We studied the whole-genome point mutation and structural variation patterns of 133 tumors (59 high-grade serous (HGSC), 35 clear cell (CCOC), 29 endometrioid (ENOC), and 10 adult granulosa cell (GCT)) as a substrate for class discovery in ovarian cancer. Ab initio clustering of integrated point mutation and structural variation signatures identified seven subgroups both between and within histotypes. Prevalence of foldback inversions identified a prognostically significant HGSC group associated with inferior survival. This finding was recapitulated in two independent cohorts (n = 576 cases), transcending BRCA1 and BRCA2 mutation and gene expression features of HGSC. CCOC cancers grouped according to APOBEC deamination (26%) and age-related mutational signatures (40%). ENOCs were divided by cases with microsatellite instability (28%), with a distinct mismatch-repair mutation signature. Taken together, our work establishes the potency of the somatic genome, reflective of diverse DNA repair deficiencies, to stratify ovarian cancers into distinct biological strata within the major histotypes.

Cumulative defects in DNA repair pathways drive the PARP inhibitor response in high-grade serous epithelial ovarian cancer cell lines.

PARP inhibitors (PARPi), such as Olaparib, have shown promising results in high-grade serous (HGS) epithelial ovarian cancer (EOC) treatment. PARPi sensitivity has been mainly associated with homologous recombination (HR) deficiency, but clinical trials have shown that predicting actual patient response is complex. Here, we investigated gene expression microarray, HR functionality and Olaparib sensitivity of 18 different HGS EOC cell lines and demonstrate that PARPi sensitivity is not only associated with HR defects. Gene target validation show that down regulation of genes in the nucleotide excision repair (NER) and mismatch repair (MMR) pathways (ERCC8 and MLH1, respectively) increases PARPi response. The highest sensitivity was observed when genes in both the HR and either NER or MMR pathways were concomitantly down regulated. Using clinical samples, patients with these concurrent down regulations could be identified. Based on these results, a novel model to predict PARPi sensitivity is herein proposed. This model implies that the extreme responders identified in clinical trials have deficiencies in HR and either NER or MMR.

Roles for APRIN (PDS5B) in homologous recombination and in ovarian cancer prediction.

APRIN (PDS5 cohesin associated factor B) interacts with both the cohesin complex and the BRCA2 tumor suppressor. How APRIN influences cohesion and DNA repair processes is not well understood. Here, we show that APRIN is recruited to DNA damage sites. We find that APRIN interacts directly with RAD51, PALB2 and BRCA2. APRIN stimulates RAD51-mediated DNA strand invasion. APRIN also binds DNA with an affinity for D-loop structures and single-strand (ss) DNA. APRIN is a new homologous recombination (HR) mediator as it counteracts the RPA inhibitory effect on RAD51 loading to ssDNA. We show that APRIN strongly improves the annealing of complementary-strand DNA and that it can stimulate this process in synergy with BRCA2. Unlike cohesin constituents, its depletion has no impact on class switch recombination, supporting a specific role for this protein in HR. Furthermore, we show that low APRIN expression levels correlate with a better survival in ovarian cancer patients and that APRIN depletion sensitizes cells to the PARP inhibitor Olaparib in xenografted zebrafish. Our findings establish APRIN as an important and specific actor of HR, with cohesin-independent functions.

Proximal and distal regulation of the HYAL1 gene cluster by the estrogen receptor α in breast cancer cells.

Chromosomal and genome abnormalities at the 3p21.3 locus are frequent events linked to epithelial cancers, including ovarian and breast cancers. Genes encoded in the 3p21.3 cluster include HYAL1, HYAL2 and HYAL3 members of hyaluronidases involved in the breakdown of hyaluronan, an abundant component of the vertebrate extracellular matrix. However, the transcriptional regulation of HYAL genes is poorly defined. Here, we identified the estrogen receptor ERα as a negative regulator of HYAL1 expression in breast cancer cells. Integrative data mining using METABRIC dataset revealed a significant inverse correlation between ERα and HYAL1 gene expression in human breast tumors. ChIP-Seq analysis identified several ERα binding sites within the 3p21.3 locus, supporting the role of estrogen as an upstream signal that diversely regulates the expression of 3p21.3 genes at both proximal and distal locations. Of these, HYAL1 was repressed by estrogen through ERα binding to a consensus estrogen response element (ERE) located in the proximal promoter of HYAL1 and flanked by an Sp1 binding site, required to achieve optimal estrogen repression. The repressive chromatin mark H3K27me3 was increased at the proximal HYAL1 ERE but not at other EREs contained in the cluster, providing a mechanism to selectively downregulate HYAL1. The HYAL1 repression was also specific to ERα and not to ERß, whose expression did not correlate with HYAL1 in human breast tumors. This study identifies HYAL1 as an ERα target gene and provides a functional framework for the direct effect of estrogen on 3p21.3 genes in breast cancer cells.

Novel high-grade serous epithelial ovarian cancer cell lines that reflect the molecular diversity of both the sporadic and hereditary disease.

Few cell line models of epithelial ovarian cancer (EOC) have been developed for the high-grade serous (HGS) subtype, which is the most common and lethal form of gynaecological cancer. Here we describe the establishment of six new EOC cell lines spontaneously derived from HGS tumors (TOV2978G, TOV3041G and TOV3291G) or ascites (OV866(2), OV4453 and OV4485). Exome sequencing revealed somatic TP53 mutations in five of the cell lines. One cell line has a novel BRCA1 splice-site mutation, and another, a recurrent BRCA2 nonsense mutation, both of germline origin. The novel BRCA1 mutation induced abnormal splicing, mRNA instability, resulting in the absence of BRCA1 protein. None of the cell lines harbor mutations in KRAS or BRAF, which are characteristic of other EOC subtypes. SNP arrays showed that all of the cell lines exhibited structural chromosomal abnormalities, copy number alterations and regions of loss of heterozygosity, consistent with those described for HGS. Four cell lines were able to produce 3D-spheroids, two exhibited anchorage-independent growth, and three (including the BRCA1 and BRCA2 mutated cell lines) formed tumors in SCID mice. These novel HGS EOC cell lines and their detailed characterization provide new research tools for investigating the most common and lethal form of EOC.

Chemotherapy reduces PARP1 in cancers of the ovary: implications for future clinical trials involving PARP inhibitors.

BACKGROUND: PARP inhibitors have shown promising clinical results in cancer patients carrying BRCA1/2 mutations. Their clinical efficacy could logically be influenced by PARP1 protein levels in patient tumors. METHODS: We screened three cohorts of patients with ovarian cancer, totaling 313 samples, and evaluated PARP1 protein expression by immunohistochemistry with further validation by western blotting. RESULTS: We observed that up to 60 % of tumors showed little PARP1 protein expression. In serous ovarian tumors, comparing intratumoral PARP1 expression between chemo-naïve and post-chemotherapy patients revealed a decrease in intratumoral PARP1 following chemotherapy in all three cohorts (immunohistochemistry: p < 0.001, n = 239; western blot: p = 0.012, n = 74). The findings were further confirmed in a selection of matched samples from the same patients before and after chemotherapy. CONCLUSION: Our data suggest that patients should be screened for PARP1 expression prior to therapy with PARP inhibitors. Further, the observed reduction of intratumoral PARP1 post-chemotherapy suggests that treating chemo-naïve patients with PARP inhibitors prior to the administration of chemotherapy, or concurrently, might increase the responsiveness to PARP1 inhibition. Thus, a change in the timing of PARP inhibitor administration may be warranted for future clinical trials.