PD-1/PD-L1 checkpoint inhibitors in combination with olaparib display antitumor activity in ovarian cancer patient-derived three-dimensional spheroid cultures.

Immune checkpoint inhibitors (ICIs) that target programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown modest activity as monotherapies for the treatment of ovarian cancer (OC). The rationale for using these therapies in combination with poly (ADP-ribose) polymerase inhibitors (PARP-Is) has been described, and their in vivo application will benefit from ex vivo platforms that aid in the prediction of patient response or resistance to therapy. This study examined the effectiveness of detecting patient-specific immune-related activity in OC using three-dimensional (3D) spheroids. Immune-related cell composition and PD-1/PD-L1 expression status were evaluated using cells dissociated from fresh OC tissue from two patients prior to and following 3D culture. The patient sample with the greatest increase in the proportion of PD-L1 + cells also possessed more activated cytotoxic T cells and mature DCs compared to the other patient sample. Upon cytokine stimulation, patient samples demonstrated increases in cytotoxic T cell activation and DC major histocompatibility complex (MHC) class-II expression. Pembrolizumab increased cytokine secretion, enhanced olaparib cytotoxicity, and reduced spheroid viability in a T cell-dependent manner. Furthermore, durvalumab and olaparib combination treatment increased cell death in a synergistic manner. This work demonstrates that immune cell activity and functional modulation can be accurately detected using our ex vivo 3D spheroid platform, and it presents evidence for their utility to demonstrate sensitivity to ICIs alone or in combination with PARP-Is in a preclinical setting.

Single-molecule analysis of subtelomeres and telomeres in Alternative Lengthening of Telomeres (ALT) cells.

BACKGROUND: Telomeric DNA is typically comprised of G-rich tandem repeat motifs and maintained by telomerase (Greider CW, Blackburn EH; Cell 51:887-898; 1987). In eukaryotes lacking telomerase, a variety of DNA repair and DNA recombination based pathways for telomere maintenance have evolved in organisms normally dependent upon telomerase for telomere elongation (Webb CJ, Wu Y, Zakian VA; Cold Spring Harb Perspect Biol 5:a012666; 2013); collectively called Alternative Lengthening of Telomeres (ALT) pathways. By measuring (TTAGGG) n tract lengths from the same large DNA molecules that were optically mapped, we simultaneously analyzed telomere length dynamics and subtelomere-linked structural changes at a large number of specific subtelomeric loci in the ALT-positive cell lines U2OS, SK-MEL-2 and Saos-2. RESULTS: Our results revealed loci-specific ALT telomere features. For example, while each subtelomere included examples of single molecules with terminal (TTAGGG) n tracts as well as examples of recombinant telomeric single molecules, the ratio of these molecules was subtelomere-specific, ranging from 33:1 (19p) to 1:25 (19q) in U2OS. The Saos-2 cell line shows a similar percentage of recombinant telomeres. The frequency of recombinant subtelomeres of SK-MEL-2 (11%) is about half that of U2OS and Saos-2 (24 and 19% respectively). Terminal (TTAGGG) n tract lengths and heterogeneity levels, the frequencies of telomere signal-free ends, and the frequency and size of retained internal telomere-like sequences (ITSs) at recombinant telomere fusion junctions all varied according to the specific subtelomere involved in a particular cell line. Very large linear extrachromosomal telomere repeat (ECTR) DNA molecules were found in all three cell lines; these are in principle capable of templating synthesis of new long telomere tracts via break-induced repair (BIR) long-tract DNA synthesis mechanisms and contributing to the very long telomere tract length and heterogeneity characteristic of ALT cells. Many of longest telomere tracts (both end-telomeres and linear ECTRs) displayed punctate CRISPR/Cas9-dependent (TTAGGG) n labeling patterns indicative of interspersion of stretches of non-canonical telomere repeats. CONCLUSION: Identifying individual subtelomeres and characterizing linked telomere (TTAGGG) n tract lengths and structural changes using our new single-molecule methodologies reveals the structural consequences of telomere damage, repair and recombination mechanisms in human ALT cells in unprecedented molecular detail and significant differences in different ALT-positive cell lines.

High-throughput single-molecule telomere characterization.

We have developed a novel method that enables global subtelomere and haplotype-resolved analysis of telomere lengths at the single-molecule level. An in vitro CRISPR/Cas9 RNA-directed nickase system directs the specific labeling of human (TTAGGG)n DNA tracts in genomes that have also been barcoded using a separate nickase enzyme that recognizes a 7-bp motif genome-wide. High-throughput imaging and analysis of large DNA single molecules from genomes labeled in this fashion using a nanochannel array system permits mapping through subtelomere repeat element (SRE) regions to unique chromosomal DNA while simultaneously measuring the (TTAGGG)n tract length at the end of each large telomere-terminal DNA segment. The methodology also permits subtelomere and haplotype-resolved analyses of SRE organization and variation, providing a window into the population dynamics and potential functions of these complex and structurally variant telomere-adjacent DNA regions. At its current stage of development, the assay can be used to identify and characterize telomere length distributions of 30-35 discrete telomeres simultaneously and accurately. The assay's utility is demonstrated using early versus late passage and senescent human diploid fibroblasts, documenting the anticipated telomere attrition on a global telomere-by-telomere basis as well as identifying subtelomere-specific biases for critically short telomeres. Similarly, we present the first global single-telomere-resolved analyses of two cancer cell lines.