Immunogenicity of Non-Mutated Ovarian Cancer-Specific Antigens.

Epithelial ovarian cancer (EOC) has not significantly benefited from advances in immunotherapy, mainly because of the lack of well-defined actionable antigen targets. Using proteogenomic analyses of primary EOC tumors, we previously identified 91 aberrantly expressed tumor-specific antigens (TSAs) originating from unmutated genomic sequences. Most of these TSAs derive from non-exonic regions, and their expression results from cancer-specific epigenetic changes. The present study aimed to evaluate the immunogenicity of 48 TSAs selected according to two criteria: presentation by highly prevalent HLA allotypes and expression in a significant fraction of EOC tumors. Using targeted mass spectrometry analyses, we found that pulsing with synthetic TSA peptides leads to a high-level presentation on dendritic cells. TSA abundance correlated with the predicted binding affinity to the HLA allotype. We stimulated naïve CD8 T cells from healthy blood donors with TSA-pulsed dendritic cells and assessed their expansion with two assays: MHC-peptide tetramer staining and TCR Vß CDR3 sequencing. We report that these TSAs can expand sizeable populations of CD8 T cells and, therefore, represent attractive targets for EOC immunotherapy.

Autophagy degrades immunogenic endogenous retroelements induced by 5-azacytidine in acute myeloid leukemia.

The hypomethylating agent 5-azacytidine (AZA) is the first-line treatment for AML patients unfit for intensive chemotherapy. The effect of AZA results in part from T-cell cytotoxic responses against MHC-I-associated peptides (MAPs) deriving from hypermethylated genomic regions such as cancer-testis antigens (CTAs), or endogenous retroelements (EREs). However, evidence supporting higher ERE MAPs presentation after AZA treatment is lacking. Therefore, using proteogenomics, we examined the impact of AZA on the repertoire of MAPs and their source transcripts. AZA-treated AML upregulated both CTA and ERE transcripts, but only CTA MAPs were presented at greater levels. Upregulated ERE transcripts triggered innate immune responses against double-stranded RNAs but were degraded by autophagy, and not processed into MAPs. Autophagy resulted from the formation of protein aggregates caused by AZA-dependent inhibition of DNMT2. Autophagy inhibition had an additive effect with AZA on AML cell proliferation and survival, increased ERE levels, increased pro-inflammatory responses, and generated immunogenic tumor-specific ERE-derived MAPs. Finally, autophagy was associated with a lower abundance of CD8+ T-cell markers in AML patients expressing high levels of EREs. This work demonstrates that AZA-induced EREs are degraded by autophagy and shows that inhibiting autophagy can improve the immune recognition of AML blasts in treated patients.

Development of Olaparib-Resistance Prostate Cancer Cell Lines to Identify Mechanisms Associated with Acquired Resistance.

BACKGROUND: Poly (ADP-ribose) polymerase inhibitors (PARPi) were initially deployed to target breast and ovarian tumors with mutations in DNA damage response genes. Recently, PARPi have been shown to be beneficial in the treatment of prostate cancer (PC) patients having exhausted conventional therapeutics. Despite demonstrating promising response rates, all patients treated with PARPi eventually develop resistance. However, PARPi resistance in PC is not well understood, and further studies are required to understand PARPi resistance in PC to propose strategies to circumvent resistance. METHODS: Starting from well-established olaparib-sensitive PC cell lines (LNCaP, C4-2B and DU145), we derived olaparib-resistant (OR) PC cell lines and performed a microarray analysis. RESULTS: The olaparib IC50 values of OR cell lines increased significantly as compared to the parental cell lines. Gene expression analyses revealed that different pathways, including DNA repair, cell cycle regulation and autophagy, were affected by acquired resistance. A total of 195 and 87 genes were significantly upregulated and downregulated, respectively, in all three OR cell lines compared to their parental counterparts. Among these genes, we selected BRCC3, ROCK2 and ATG2B for validation. We showed that ROCK2 expression, basal autophagy and homologous recombination (HR) efficiency were increased in all OR cell lines. CONCLUSIONS: Our study provides a new in vitro model to study PARPi resistance in PC and suggests new possible targets to reverse resistance and prolong the benefits of PARPi treatment.

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.

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 Keratin 7 and E-Cadherin Signature Is Highly Predictive of Tubo-Ovarian High-Grade Serous Carcinoma Prognosis.

During tubo-ovarian high-grade serous carcinoma (HGSC) progression, tumoral cells undergo phenotypic changes in their epithelial marker profiles, which are essential for dissemination processes. Here, we set out to determine whether standard epithelial markers can predict HGSC patient prognosis. Levels of E-CADH, KRT7, KRT18, KRT19 were quantified in 18 HGSC cell lines by Western blot and in a Discovery cohort tissue microarray (TMA) (n = 101 patients) using immunofluorescence. E-CADH and KRT7 levels were subsequently analyzed in the TMA of the Canadian Ovarian Experimental Unified Resource cohort (COEUR, n = 1158 patients) and in public datasets. Epithelial marker expression was highly variable in HGSC cell lines and tissues. In the Discovery cohort, high levels of KRT7 and KRT19 were associated with an unfavorable prognosis, whereas high E-CADH expression indicated a better outcome. Expression of KRT7 and E-CADH gave a robust combination to predict overall survival (OS, p = 0.004) and progression free survival (PFS, p = 5.5 × 10-4) by Kaplan-Meier analysis. In the COEUR cohort, the E-CADH-KRT7 signature was a strong independent prognostic biomarker (OS, HR = 1.6, p = 2.9 × 10-4; PFS, HR = 1.3, p = 0.008) and predicted a poor patient response to chemotherapy (p = 1.3 × 10-4). Our results identify a combination of two epithelial markers as highly significant indicators of HGSC patient prognosis and treatment response.

Dual Inhibition of Autophagy and PI3K/AKT/MTOR Pathway as a Therapeutic Strategy in Head and Neck Squamous Cell Carcinoma.

Genomic analyses of head and neck squamous cell carcinoma (HNSCC) have highlighted alterations in the phosphatidylinositol 3-kinase (PI3K) signaling pathway, presenting a therapeutic target for multiple ongoing clinical trials with PI3K or PI3K/MTOR inhibitors. However, these inhibitors can potentially increase autophagy in HNSCC and indirectly support cancer cell survival. Here, we sought to understand the relationship between the PI3K signaling pathway and autophagy during their dual inhibition in a panel of HNSCC cell lines. We used acridine orange staining, immunoblotting, and tandem sensor Red Fluorescent Protein- Green Fluorescent Protein-, microtubule-associated protein 1 light chain 3 beta (RFP-GFP-LC3B) expression analysis to show that PI3K inhibitors increase autophagosomes in HNSCC cells, but that chloroquine treatment effectively inhibits the autophagy that is induced by PI3K inhibitors. Using the Bliss independence model, we determined that the combination of chloroquine with PI3K inhibitors works in synergy to decrease cancer cell proliferation, independent of the PIK3CA status of the cell line. Our results indicate that a strategy focusing on autophagy inhibition enhances the efficacy of therapeutics already in clinical trials. Our results suggest a broader application for this combination therapy that can be promptly translated to in vivo studies.

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.

Fluorescence hyperspectral imaging for live monitoring of multiple spheroids in microfluidic chips.

Tumor spheroids represent a realistic 3D in vitro cancer model because they provide a missing link between monolayer cell culture and live tissues. While microfluidic chips can easily form and assay thousands of spheroids simultaneously, few commercial instruments are available to analyze this massive amount of data. Available techniques to measure spheroid response to external stimuli, such as confocal imaging and flow cytometry, are either not appropriate for 3D cultures, or destructive. We designed a wide-field hyperspectral imaging system to analyze multiple spheroids trapped in a microfluidic chip in a single acquisition. The system and its fluorescence quantification algorithm were assessed using liquid phantoms mimicking spheroid optical properties. Spectral unmixing was tested on three overlapping spectral entities. Hyperspectral images of co-culture spheroids expressing two fluorophores were compared with confocal microscopy and spheroid growth was measured over time. The system can spectrally analyze multiple fluorescent markers simultaneously and allows multiple time-points assays, providing a fast and versatile solution for analyzing lab on a chip devices.