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Drug screening and whole genome sequencing of primary cells and cell lines from ovarian cancer patients to associate genomic aberrations with in vitro drug sensitivities

ABSTRACT: Identifying drugs for cancers with poor prognosis like ovarian cancer (OC) is hindered by inappropriate cell models and the limitations of current viability assays. We developed an automated confocal microscopy-based live cell assay (DeathPro) that robustly de-convolves cell death and proliferation over time by employing a specific image processing strategy. Using DeathPro, we screened nine OC patient-derived cell lines and one normal cell line (HOSEpiC) with 22 drugs or combinations. Whole genome was performed to analyze if the cell lines harbor OC-typical genomic aberrations and to relate genomic features to drug sensitivities. Commonly observed alterations in OC patients like TP53 mutations and MYC or PI3KCA amplifications were absent in the normal cell line but frequently detected in the OC cell lines. Moreover, genome-derived homologous recombination deficiency (HRD) scores of the OC cell lines correlated with their responses to carboplatin, olaparib and DNA demethylation drugs. Overall, we propose an imaging-based platform to guide drug development and genome-drug sensitivity analysis.

PROVIDER: EGAS00001002239 | EGA |

REPOSITORIES: EGA

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Project description:Silencing of genes that suppress the malignant phenotype by DNA methylation spurred an interest in the clinical use of epigenetic reprogramming agents. Single therapy is unlikely to be curative in the context of a heterogeneous disease such as Diffuse Large B cell Lymphomas (DLBCL). The combination of DNA demethylating drugs could increase the chance to respond to classical and new treatments. We found that DLBCL cell lines respond heterogeneously to DNA demethylating agents. In sensitive cell lines, 5-aza-2’-deoxycytidine induced a genomic signature similar to that of doxorubicin, the most important drug of the combinatorial chemotherapy regimen for DLBCL treatment. Accordingly, the combination of 5-aza-2’-deoxycytidine and doxorubicin proved to be synergistic in cell killing in vitro and in vivo for DLBCL cell lines individually responsive to these drugs. In doxorubicin resistant cell lines, long-term exposure to low-dose of 5-aza-2’-deoxycytidine induces DNA demethylation and subsequent doxorubicin sensitization in vitro and in vivo. This later effect correlates with SMAD1 demethylation. SMAD1 is epigenetically silenced in doxorubicin-resistant DLBCL cells and DLBCL patients with poor prognostic. In addition, we found that DNA demethylating agents can sensitize primary DLBCL cells to doxorubicin. Primary cells obtained from a DLBCL patient treated with 5-azacytidine shows SMAD1 demethylation and ex vivo sensitization to multiple drugs. Therefore, DNA demethylating drugs can reprogram otherwise resistant DLBCL cells to respond to chemotherapy agents without increasing the toxicity to normal tissues. Our data also indicate that DNA methylation and consequent suppression of SMAD1 expression represent a previously undescribed molecular mechanism of chemoresistance in DLBCL that can be further exploit for therapy. A microarray study using genomic DNA from different DLBCL cell lines before any treatment. Two to four biological replicates by cell line. The HELP data wil be used to find genes hypermethylated in resistant cell lines compared to sensitive cell lines to doxorubicin and other drugs.

Project description:Silencing of genes that suppress the malignant phenotype by DNA methylation spurred an interest in the clinical use of epigenetic reprogramming agents. Single therapy is unlikely to be curative in the context of a heterogeneous disease such as Diffuse Large B cell Lymphomas (DLBCL). The combination of DNA demethylating drugs could increase the chance to respond to classical and new treatments. We found that DLBCL cell lines respond heterogeneously to DNA demethylating agents. In sensitive cell lines, 5-aza-2M-bM-^@M-^Y-deoxycytidine induced a genomic signature similar to that of doxorubicin, the most important drug of the combinatorial chemotherapy regimen for DLBCL treatment. Accordingly, the combination of 5-aza-2M-bM-^@M-^Y-deoxycytidine and doxorubicin proved to be synergistic in cell killing in vitro and in vivo for DLBCL cell lines individually responsive to these drugs. In doxorubicin resistant cell lines, long-term exposure to low-dose of 5-aza-2M-bM-^@M-^Y-deoxycytidine induces DNA demethylation and subsequent doxorubicin sensitization in vitro and in vivo. This later effect correlates with SMAD1 demethylation. SMAD1 is epigenetically silenced in doxorubicin-resistant DLBCL cells and DLBCL patients with poor prognostic. In addition, we found that DNA demethylating agents can sensitize primary DLBCL cells to doxorubicin. Primary cells obtained from a DLBCL patient treated with 5-azacytidine shows SMAD1 demethylation and ex vivo sensitization to multiple drugs. Therefore, DNA demethylating drugs can reprogram otherwise resistant DLBCL cells to respond to chemotherapy agents without increasing the toxicity to normal tissues. Our data also indicate that DNA methylation and consequent suppression of SMAD1 expression represent a previously undescribed molecular mechanism of chemoresistance in DLBCL that can be further exploit for therapy. A microarray study using double-stranded cDNA from different DLBCL cell lines before any treatment. Two biological replicates by cell line. The gene expression will be used to find gene expression signatures between sensitive and resistant cell lines to chemotherapeutics.

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Drug screening and whole genome sequencing of primary cells and cell lines from ovarian cancer patients to associate genomic aberrations with in vitro drug sensitivities

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