Supplementary Table 2 from Small Ubiquitin-Related Modifier Pathway Is a Major Determinant of Doxorubicin Cytotoxicity in <i>Saccharomyces cerevisiae</i>
Ovarian cancer (OC) is the fifth leading cause of cancer death in the US, presenting a low mutational burden and a diverse degree of infiltrating T cells. Neoantigens derived from somatic mutations represent an attractive immunotherapeutic target, however, mouse models for the development of personalized immunotherapies are still poor and do not fully recapitulate the individualized nature of OC in patients. To address this hurdle, our study established a patient-derived xenograft (PDX) of an OC patient as segue to studying neoantigen-driven autologous T cell response. We first evaluated tumor mutations in the primary tumor (Pr) and two successive passages (P0, P1) via WES. As tumors from P0 and P1 have a higher mutational count than Pr, we hypothesize that this difference is caused by the loss of immune-surveillance in immunocompromised mice, which allows for the outgrowth of previously repressed clones. By using the variant allele frequency (VAF) to cluster 123 shared mutations, we identified three clusters with unique VAF dynamics (Fig 1A-D). Tumors from Pr, P0 and P1 had a conserved functional with a strong conservation of antigen presenting pathways (Fig 2A-E). In PDX P0 we identified 184 non-synonymous mutations, leading to 30 potential neoantigens with high affinity for the patient's HLAs (Fig 3A-B). Neoantigens were ranked based on differential predicted HLA affinity of the WT versus mutated peptide (Fig 3C). T cell activation by neoantigens was tested in vitro via ELISA and flow cytometry. Interferon-γ production and upregulation of CD137 identified a core set of six neoantigens selectively recognized by patient's autologous CD8+ T cells (Fig 4A-B). Of those, 3/6 neoantigens were common between PDX and primary tumor, corroborating the role of the patient's own immune system in repressing the expansion of selected tumor clones (Fig 5). In vivo ACT studies showed that mice injected with neoantigen-stimulated autologous PBMCs (ACT_MUT) have reduced tumor growth when compared to mice injected with unstimulated cells (ACT_NP) (Fig 6A). ACT_MUT mice have higher levels of circulating T cells 15 days post-ACT and higher intratumoral T cells at end point than ACT_NP (Fig 6 B-C). We then sought to identify the TCR moieties that determine T cell response. Single cell TCRSeq analyses on the two strongest neoantigens identified multiple TCR activated by a single cancer neoantigen (Fig 7A-B), suggesting oligoclonal T cell activation. We tested this hypothesis by generating a motif with the most expanded clones and comparing it with the motif from clones that did not expend. Results indicate a significant difference in the frequency of amino acid in multiple CDR3 locations, suggesting the presence of an oligoclonal response to neoantigenic T cell stimulation. In conclusion, we have successfully established PDX models of OC that can be used to study and predict autologous T cell response to neoantigens.Citation Format: Muzamil Y. Want, Anna Konstorum, Ruea-Yea Huang, Richard Koya, Sebastiano Battaglia. Neoantigens retention by patient derived xenograft model mediate autologous T cells activation in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1076.
Chromosomal aberrations induced by ethyl methanesulfonate (EMS) in Chinese hamster ovary cells were potentiated by subsequent exposure to sodium arsenite (AS), 3-aminobenzamide (3AB), or caffeine (CAF). The coclastogenicity of AS was most evident when this drug was applied for 3 or 6 h immediately after EMS was removed, whereas caffeine acted primarily after 12–18 h. The coclastogenicity of 3AB was not stage dependent. AS and 3AB increased chromatid exchanges more than chromatid breaks, whereas caffeine mainly increased chromatid breaks. Thus the coclastogenicities of AS, 3AB, and CAF differ in their time of action and the types of aberrations they potentiate.
ABSTRACT We report a meta-analysis of breast, prostate, ovarian, and endometrial cancer genome-wide association data (effective sample size: 237,483 cases/317,006 controls). This identified 465 independent lead variants ( P <5×10 −8 ) across 192 genomic regions. Four lead variants were >1Mb from previously identified risk loci for the four cancers and an additional 23 lead variant-cancer associations were novel for one of the cancers. Bayesian models supported pleiotropic effects involving at least two cancers at 222/465 lead variants in 118/192 regions. Gene-level association analysis identified 13 shared susceptibility genes ( P <2.6×10 −6 ) in 13 regions not previously implicated in any of the four cancers and not uncovered by our variant-level meta-analysis. Several lead variants had opposite effects across cancers, including a cluster of such variants in the TP53 pathway. Fifty-four lead variants were associated with blood cell traits and suggested genetic overlaps with clonal hematopoiesis. Our study highlights the remarkable pervasiveness of pleiotropy across hormone-related cancers, further illuminating their shared genetic and mechanistic origins at variant- and gene-level resolution.
<div>Abstract<p>To identify novel genes that mediate cellular resistance to cisplatin, we have screened the collection of <i>Saccharomyces cerevisiae</i> deletion strains. We have found reproducibly 22 genes/open reading frames (ORF), which when deleted, confer resistance to cisplatin at a concentration that is lethal to wild-type cells. Complementation of individual deletion strains with the corresponding wild-type gene abolished cisplatin resistance, confirming that specific gene deletions caused the resistance. Twenty of the genes/ORFs identified have not been previously linked to cisplatin resistance and belong to several distinct functional groups. Major functional groups encode proteins involved in nucleotide metabolism, mRNA catabolism, RNA-polymerase-II–dependent gene regulation and vacuolar transport systems. In addition, proteins that function in ubiquitination, sphingolipid biogenesis, cyclic AMP–dependent signaling, DNA repair, and genome stability are also associated with cisplatin resistance. More than half of the identified genes are known to have sequences or functional homology to mammalian counterparts. Some deletion strains are cross-resistant to selected cytotoxic agents whereas hypersensitive to others. The sensitivity of certain resistant strains to other cytotoxic agents suggests that our findings may point to particular drug combinations that can overcome resistance caused by inactivation of specific genes.</p></div>
Supplementary Materials and Methods from Small Ubiquitin-Related Modifier Pathway Is a Major Determinant of Doxorubicin Cytotoxicity in <i>Saccharomyces cerevisiae</i>
Despite the identification of several ovarian cancer (OC) predisposition genes, a large proportion of familial OC risk remains unexplained. We adopted a two-stage design to identify new OC predisposition genes. We first carried out a large germline whole-exome sequencing study on 158 patients from 140 families with significant OC history, but without evidence of genetic predisposition due to BRCA1/2. We then evaluated the potential candidate genes in a large case-control association study involving 381 OC cases in the Cancer Genome Atlas project and 27,173 population controls from the Exome Aggregation Consortium. Two new putative OC risk genes were identified, namely, ANKRD11, a putative tumor suppressor, and POLE, an enzyme involved in DNA repair and replication. These two genes likely confer moderate OC risk. We performed in vitro experiments and showed an ANKRD11 mutation identified in our patients markedly lowered the protein expression by compromising protein stability. Upon future validation and functional characterization, these genes may shed light on cancer etiology along with improving ascertainment power and preventive care of individuals at high risk of OC.
Background Immune checkpoint blockers (ICBs) have been approved by the Food and Drug Administration to be used alone in front-line therapies or in combination with other regimens for certain advanced cancers. Since ICB only works in a subset of patients and has limited efficacy in treating ovarian cancer (OVC), developing preclinical models that help to understand which patients may derive benefit from ICB would be of tremendous benefit in OVC. Methods Here, we generated preclinical human OVC models from freshly resected tumors, which include six patient-derived xenografts (PDXs) from six different patient tumors, three transplantable OVC PD spheroid lines (PD-sphs), and 3 cell lines (PD-CLs). We tested the therapeutic combination of anti-PD1/CTLA4 antibodies with (1) autologous tumor-associated leukocytes (TALs) on the growth of PD-sphs in a coculture system in vitro, (2) with adoptively transferred autologous peripheral blood mononuclear cells or TALs in patient-derived OVC models using partially humanized mice, NSG-HHDxSGM3 (N-HSGM3). Results We show that PD-1 and CTLA-4 dual blockade when combined with autologous TALs effectively reduced PD-sph number in a co-culture system and led to regression of established PD-CLs and PDXs in the N-HSGM3 mice. Combinatorial PD-1 and CTLA-4 blockade increased the frequency and function of tumor-specific CD8 T cells. These CD8 T cells persisted in the tumor microenvironment, exhibited memory phenotype and protected animals from tumor growth on tumor rechallenge. Gene expression analysis of tumors resistant to dual PD1/CTLA4 blockade treatment identified upregulation of antigen processing and presentation pathways and downregulation of extracellular matrix organization genes. Conclusions These findings describe a novel platform for developing patient-derived preclinical tumor models suitable for rationally testing combinatorial ICB in the context of autologous tumor-reactive T cells. This platform can be further developed for testing additional targeted therapies relevant to OVC.
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