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Lysophosphatidic acid (LPA) is a bioactive lipid enriched in the tumor microenvironment of immunosuppressive malignancies such as ovarian cancer. Although LPA enhances the tumorigenic attributes of cancer cells, the immunomodulatory activity of this phospholipid messenger remains largely unexplored. Here, we report that LPA operates as a negative regulator of type I interferon (IFN) responses in ovarian cancer. Ablation of the LPA-generating enzyme autotaxin (ATX) in ovarian cancer cells reprogrammed the tumor immune microenvironment, extended host survival, and improved the effects of therapies that elicit protective responses driven by type I IFN. Mechanistically, LPA sensing by dendritic cells triggered PGE2 biosynthesis that suppressed type I IFN signaling via autocrine EP4 engagement. Moreover, we identified an LPA-controlled, immune-derived gene signature associated with poor responses to combined PARP inhibition and PD-1 blockade in patients with ovarian cancer. Controlling LPA production or sensing in tumors may therefore be useful to improve cancer immunotherapies that rely on robust induction of type I IFN.Significance:This study uncovers that ATX–LPA is a central immunosuppressive pathway in the ovarian tumor microenvironment. Ablating this axis sensitizes ovarian cancer hosts to various immunotherapies by unleashing protective type I IFN responses. Understanding the immunoregulatory programs induced by LPA could lead to new biomarkers predicting resistance to immunotherapy in patients with cancer.See related commentary by Conejo-Garcia and Curiel, p. 1841.This article is highlighted in the In This Issue feature, p. 1825

Supplementary Figure from Tumor-Derived Lysophosphatidic Acid Blunts Protective Type I Interferon Responses in Ovarian Cancer

The incidence of anal squamous cell carcinoma (ASCC) has been increasing, particularly in populations with HIV. Human papillomavirus (HPV) is the causal factor in 85% to 90% of ASCCs, but few studies evaluated HPV genotypes and integrations in relation to genomic alterations in ASCC. Using whole-exome sequence data for primary (n = 56) and recurrent (n = 31) ASCC from 72 patients, we detected HPV DNA in 87.5% of ASCC, of which HPV-16, HPV-18, and HPV-6 were detected in 56%, 22%, and 33% of HIV-positive (n = 9) compared with 83%, 3.2%, and 1.6% of HIV-negative cases (n = 63), respectively. Recurrent copy-number variations (CNV) involving genes with documented roles in cancer included amplification of PI3KCA and deletion of APC in primary and recurrent tumors; amplifications of CCND1, MYC, and NOTCH1 and deletions of BRCA2 and RB1 in primary tumors; and deletions of ATR, FANCD2, and FHIT in recurrent tumors. DNA damage response genes were enriched among recurrently deleted genes in recurrent ASCCs (P = 0.001). HPV integrations were detected in 29 of 76 (38%) ASCCs and were more frequent in stage III–IV versus stage I–II tumors. HPV integrations were detected near MYC and CCND1 amplifications and recurrent targets included NFI and MUC genes. These results suggest HPV genotypes in ASCC differ by HIV status, HPV integration is associated with ASCC progression, and DNA damage response genes are commonly disrupted in recurrent ASCCs.Implications:These data provide the largest whole-exome sequencing study of the ASCC genomic landscape to date and identify HPV genotypes, integrations, and recurrent CNVs in primary or recurrent ASCCs.

PDF - 90KB, Legends for Supplemental Figures 1 through 6.

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PDF - 502KB, Supplementary figure 1. USP1 inhibitor SJB2-043 inhibits activity of native USP1 in a dose-dependent manner and causes a decrease in ID1, ID2 and ID3 levels in K562 cells. Supplementary figure 2. Knockdown of USP1 results in growth inhibition, increased apoptosis and cell cycle arrest in K562 cells. Supplementary figure 3. Analogs of USP1 inhibitor lead to ID1 destabilization in leukemic cells. Supplementary figure 4. Caspase inhibitor does not block the degradation of ID1 by USP1 inhibitors. Supplementary figure 5. Effects of USP1 inhibitors on growth of primary human cord blood CD34+ cells. Supplementary figure 6. USP1 inhibitors cause increase in Ub-FANCD2, Ub-PCNA, and p21, and decrease the HR activity.

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All supplemental text and 9 supplementary figures

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Supplemental Table S1. Clinical characteristics and outcomes by HIV status. Supplemental Table S2. HPV integration sites detected in patient WXS data. Supplemental Table S3. Curated list of 981 cancer-related genes considered in this study.

Supplemental Table S4. Functional ontology enrichment of genes within recurrent CNVs in all ASCC tumors, primary ASCC tumors, and recurrent or metastatic ASCC tumors

Inhibitor of DNA binding 1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types, including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin-specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a ubiquitin-rhodamine–based high-throughput screening, we identified small-molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose-dependent manner. A known USP1 inhibitor, pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary acute myelogenous leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small-molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy. Mol Cancer Ther; 12(12); 2651–62. ©2013 AACR.

Supplementary Table 1

Supplemental Figure S1. HPV-18 genome coverage in ASCC. Supplemental Figure S2. Copy number variation in 81 ASCCs.

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Supplementary Table 3

Supplementary Table 1 showing reagents and antibodies used in the study.

Synergism of prexasertib and olaparib in ovarian cancer cell lines

Supplementary Table 2

Purpose: To evaluate the prognostic utility of immunohistochemical assessment of key proteins in multiple DNA repair pathways in triple-negative breast cancer (TNBC; estrogen receptor negative, progesterone receptor negative, and HER2/neu negative by immunohistochemistry).Experimental Design: Archived clinically annotated tumor specimens from 112 women with TNBC were immunostained with antibodies against DNA repair proteins and scored using digital image analysis. The cohort was divided into training and test sets for development of a multiantibody model. Scores were combined with clinical data to assess association with outcome.Results: Low XPF (P = 0.005), pMK2 (P = 0.01), MLH; P = 0.002), and FANCD2 (P = 0.001) were each associated with shorter time to recurrence (TTR) in univariate analysis. A 4-antibody model could segregate high-risk and low-risk groups on the basis of TTR in both the training (relative risk [RR] = 3.52; P = 9.05E-07) and test (RR 2.67; P = 0.019) cohorts.Conclusions: DNA repair proteins may be useful as prognostic markers in TNBC. Further study in larger, uniformly treated cohorts with additional clinical parameters is warranted. Clin Cancer Res; 16(23); 5796–804. ©2010 AACR.

Genetic analyses of BRCA1 mutant cell lines and PDX models.

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Overall Clinical Response in All Evaluable Patients

Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo. Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778–90. ©2016 AACR.

Homologous recombination (HR)-deficient cancers are sensitive to poly-ADP ribose polymerase inhibitors (PARPi), which have shown clinical efficacy in the treatment of high-grade serous cancers (HGSC). However, the majority of patients will relapse, and acquired PARPi resistance is emerging as a pressing clinical problem. Here we generated seven single-cell clones with acquired PARPi resistance derived from a PARPi-sensitive TP53−/− and BRCA1−/− epithelial cell line generated using CRISPR/Cas9. These clones showed diverse resistance mechanisms, and some clones presented with multiple mechanisms of resistance at the same time. Genomic analysis of the clones revealed unique transcriptional and mutational profiles and increased genomic instability in comparison with a PARPi-sensitive cell line. Clonal evolutionary analyses suggested that acquired PARPi resistance arose via clonal selection from an intrinsically unstable and heterogenous cell population in the sensitive cell line, which contained preexisting drug-tolerant cells. Similarly, clonal and spatial heterogeneity in tumor biopsies from a clinical patient with BRCA1-mutant HGSC with acquired PARPi resistance was observed. In an imaging-based drug screening, the clones showed heterogenous responses to targeted therapeutic agents, indicating that not all PARPi-resistant clones can be targeted with just one therapy. Furthermore, PARPi-resistant clones showed mechanism-dependent vulnerabilities to the selected agents, demonstrating that a deeper understanding on the mechanisms of resistance could lead to improved targeting and biomarkers for HGSC with acquired PARPi resistance.Significance:This study shows that BRCA1-deficient cells can give rise to multiple genomically and functionally heterogenous PARPi-resistant clones, which are associated with various vulnerabilities that can be targeted in a mechanism-specific manner.

Supplementary Figure Legends

Legend for Supplementary Figures S1-S6.

Purpose:DNA-damaging agents comprise the backbone of systemic treatment for many tumor types; however, few reliable predictive biomarkers are available to guide use of these agents. In muscle-invasive bladder cancer (MIBC), cisplatin-based chemotherapy improves survival, yet response varies widely among patients. Here, we sought to define the role of the nucleotide excision repair (NER) gene ERCC2 as a biomarker predictive of response to cisplatin in MIBC.Experimental Design:Somatic missense mutations in ERCC2 are associated with improved response to cisplatin-based chemotherapy; however, clinically identified ERCC2 mutations are distributed throughout the gene, and the impact of individual ERCC2 variants on NER capacity and cisplatin sensitivity is unknown. We developed a microscopy-based NER assay to profile ERCC2 mutations observed retrospectively in prior studies and prospectively within the context of an institution-wide tumor profiling initiative. In addition, we created the first ERCC2-deficient bladder cancer preclinical model for studying the impact of ERCC2 loss of function.Results:We used our functional assay to test the NER capacity of clinically observed ERCC2 mutations and found that most ERCC2 helicase domain mutations cannot support NER. Furthermore, we show that introducing an ERCC2 mutation into a bladder cancer cell line abrogates NER activity and is sufficient to drive cisplatin sensitivity in an orthotopic xenograft model.Conclusions:Our data support a direct role for ERCC2 mutations in driving cisplatin response, define the functional landscape of ERCC2 mutations in bladder cancer, and provide an opportunity to apply combined genomic and functional approaches to prospectively guide therapy decisions in bladder cancer.See related commentary by Grivas, p. 907

Supplementary Table 3

RNA-seq analyses of global exon splicing events (S1); Cell line therapy sensitivities (S2); Hazard ratios for survival of patients with serous ovarian carcinomas associated with BRCA1 mutation, age at diagnosis and stage (S3); Characteristics of studies included in survival analysis from Fig. 5A (S4).

Table 1

Characterization of high-grade serous ovarian cancer models

Supplementary Data from Targeting DNA Repair with Combined Inhibition of NHEJ and MMEJ Induces Synthetic Lethality in TP53-Mutant Cancers

Supplementary Table 2 showing the positive and negative GSEA results for each cell line.

DNA repair pathway inhibitors are a new class of anticancer drugs that are advancing in clinical trials. Peposertib is an inhibitor of DNA-dependent protein kinase (DNA-PK), which is a key driver of nonhomologous end-joining (NHEJ). To identify regulators of response to peposertib, we performed a genome-wide CRISPR knockout screen and found that loss of POLQ (polymerase theta, POLθ) and other genes in the microhomology-mediated end-joining (MMEJ) pathway are key predictors of sensitivity to DNA-PK inhibition. Simultaneous disruption of two DNA repair pathways via combined treatment with peposertib plus a POLθ inhibitor novobiocin exhibited synergistic synthetic lethality resulting from accumulation of toxic levels of DNA double-strand break end resection. TP53-mutant tumor cells were resistant to peposertib but maintained elevated expression of POLQ and increased sensitivity to novobiocin. Consequently, the combination of peposertib plus novobiocin resulted in synthetic lethality in TP53-deficient tumor cell lines, organoid cultures, and patient-derived xenograft models. Thus, the combination of a targeted DNA-PK/NHEJ inhibitor with a targeted POLθ/MMEJ inhibitor may provide a rational treatment strategy for TP53-mutant solid tumors.Significance:Combined inhibition of NHEJ and MMEJ using two nontoxic, targeted DNA repair inhibitors can effectively induce toxic DNA damage to treat TP53-deficient cancers.

Supplementary Figures and Methods // Supplementary Figure 1: The DDB2 proteo-probe assay measures cellular nucleotide excision repair (NER) capacity. Low magnification (20X) images of wild-type (WT) or mutant ERCC2-complemented XP cells before, 5 minutes after, and 150 minutes after UV exposure (see Figure 1 for high magnification [63X] images). DDB2 proteoprobe binding to UV-induced (6,4)-photoproducts is visualized using an anti-FLAG antibody (red). Nuclei are stained with DAPI (blue). ERCC2 expression is confirmed with an anti-Myc antibody (green). R683W is a common germline pathogenic allele, D312N is a common non-pathogenic germline allele, and G607A is a mutation identified in a cisplatin responsive tumor. Supplementary Figure 2: Sensitivity profiles for WT and mutant ERCC2-complemented cells for non-cisplatin agents. A. Carboplatin. B. Doxorubicin (Adriamycin) is a DNA intercalating agents that results in creation of double-strand DNA breaks. C. Rucaparib is a poly(ADP-ribose) polymerase inhibitor that demonstrates enhanced toxicity in homologous recombination (HR)-deficient cells. D. Ionizing radiation (IR). R683W is a common pathogenic germline allele in patients with the xeroderma pigmentosum complementation group D (XPD). D312N is a common, non-pathogenic germline ERCC2 allele in the general population. Y14C and G607A are helicase domain mutations identified in cisplatin responsive patients and are associated with NER loss and cisplatin sensitivity (Figure 2). Supplementary Figure 3: ERCC2 mutations identified in cisplatin non-responders are functionally deleterious. A. Cisplatin sensitivity curves for WT or mutant ERCC2-complemented cells. Whereas WT ERCC2 rescues cisplatin sensitivity, the sensitivity profiles for S44L and S246F, identified in two cisplatin non-responders from the FCCC cohort, are similar to mock-infected cells. B. The S44L and S246F mutants also fail to rescue sensitivity to UV sensitivity. C. Immunoblot showing expression of FLAG-tagged WT or mutant ERCC2. Supplementary Figure 4: Identification of an ERCC2 E86Q mutation in matched pre- and post-treatment tumor samples. The E86Q mutation was not identified in the pre-treatment sample by the standard mutation-calling algorithm due to the low number of reads, but was later identified by "force-calling" after the E86Q mutation was identified in the post-treatment sample. Correcting for tumor purity, the E86Q mutation was inferred to be clonal in both the pre- and post-treatment samples. Supplementary Figure 5: Phylogenetic tree showing relationship between pre- and post-chemotherapy tumors from a patient with an ERCC2 E86Q mutation. The pre- and post-chemotherapy tumors share several mutations, indicating a common ancestor. Shared mutations in other select cancer genes are noted. Supplementary Figure 6: CRISPR/Cas9 technology was used to create a KU19-19 cell line harboring an in-frame deletion at the endogenous ERCC2 allele. A. An sgRNA guide was designed to target the region near T484, a common site of ERCC2 mutations in MIBC tumors. B. Integrated Genomics Viewer (IGV) screenshot showing sequence of WT ERCC2 allele in KU19-19 parental cell line. C. The CRISPR-edited cell line KE1 harbors an in-frame deletion at T484. Supplementary Figure 7: An ERCC2 mutation confers an NER defect in a bladder cancer cell line. Low magnification (20X) images from a DDB2 proteoprobe assay comparing KU19-19 (WT ERCC2) and KE1 (mutant ERCC2) cell lines (see Figure 4 for high magnification [63X] images). Whereas the KU19-19 cell line efficiently resolves DDB2 foci, the foci persist in NER-deficient KE1 cells. Supplementary Figure 8: KU19-19 (WT ERCC2) and KE1 (mutant ERCC2) cell lines have similar sensitivity to the PARP inhibitor olaparib. Supplementary Figure 9: Overall survival for patients with ERCC2 wild-type (blue) versus mutant (red) tumors in the updated TCGA bladder tumor cohort (Robertson et al. Cell 2017; 171:1-17). There was no significant difference in survival between the groups (P=0.095). This figure was created using cBioPortal (Gao et al. Sci Signaling 2013; 6(269):pl1 and Cerami et al. Cancer Discov 2012; 2(5):401).

Purpose:PARP inhibitors are approved for the treatment of high-grade serous ovarian cancers (HGSOC). Therapeutic resistance, resulting from restoration of homologous recombination (HR) repair or replication fork stabilization, is a pressing clinical problem. We assessed the activity of prexasertib, a checkpoint kinase 1 (CHK1) inhibitor known to cause replication catastrophe, as monotherapy and in combination with the PARP inhibitor olaparib in preclinical models of HGSOC, including those with acquired PARP inhibitor resistance.Experimental Design:Prexasertib was tested as a single agent or in combination with olaparib in 14 clinically annotated and molecularly characterized luciferized HGSOC patient-derived xenograft (PDX) models and in a panel of ovarian cancer cell lines. The ability of prexasertib to impair HR repair and replication fork stability was also assessed.Results:Prexasertib monotherapy demonstrated antitumor activity across the 14 PDX models. Thirteen models were resistant to olaparib monotherapy, including 4 carrying BRCA1 mutation. The combination of olaparib with prexasertib was synergistic and produced significant tumor growth inhibition in an olaparib-resistant model and further augmented the degree and durability of response in the olaparib-sensitive model. HGSOC cell lines, including those with acquired PARP inhibitor resistance, were also sensitive to prexasertib, associated with induction of DNA damage and replication stress. Prexasertib also sensitized these cell lines to PARP inhibition and compromised both HR repair and replication fork stability.Conclusions:Prexasertib exhibits monotherapy activity in PARP inhibitor–resistant HGSOC PDX and cell line models, reverses restored HR and replication fork stability, and synergizes with PARP inhibition.