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This file contains Supplementary Figure S1 (Correlation between downregulation of EGFR transcriptional target genes, and sensitivity to erlotinib at the high concentration after 6 hours); Supplementary Figure S2 (EGFR inhibitor and DNA damaging agents induce directionally opposing changes in EGR1 expression that are differentially associated with drug sensitivity); Supplementary Figure S3 (Representative 3D response surface plots for the gemcitabine-erlotinib combination in the indicated cell lines); Supplementary Figure S4 (Representative 3D response surface plots for the vorinostat-talazoparib combination in the indicated cell lines); Supplementary Table S1 (Validation of array-based measurements by qRT-PCR); Supplementary Table S2 (Genes with concerted changes in the same direction [upregulation or downregulation] 24 hours after treatment with each of the 15 drugs); and Supplementary Table S4 (Selected genes potentially mediating drug insensitivity to agents in the NCI TPW dataset).

The intracellular effects and overall efficacies of anticancer therapies can vary significantly by tumor type. To identify patterns of drug-induced gene modulation that occur in different cancer cell types, we measured gene-expression changes across the NCI-60 cell line panel after exposure to 15 anticancer agents. The results were integrated into a combined database and set of interactive analysis tools, designated the NCI Transcriptional Pharmacodynamics Workbench (NCI TPW), that allows exploration of gene-expression modulation by molecular pathway, drug target, and association with drug sensitivity. We identified common transcriptional responses across agents and cell types and uncovered gene-expression changes associated with drug sensitivity. We also demonstrated the value of this tool for investigating clinically relevant molecular hypotheses and identifying candidate biomarkers of drug activity. The NCI TPW, publicly available at https://tpwb.nci.nih.gov, provides a comprehensive resource to facilitate understanding of tumor cell characteristics that define sensitivity to commonly used anticancer drugs.Significance:The NCI Transcriptional Pharmacodynamics Workbench represents the most extensive compilation to date of directly measured longitudinal transcriptional responses to anticancer agents across a thoroughly characterized ensemble of cancer cell lines.

Aims The histone deacetylase inhibitor belinostat has activity in various cancers. Because belinostat is metabolized by the liver, reduced hepatic clearance could lead to excessive drug accumulation and increased toxicity. Safety data in patients with liver dysfunction are needed for this drug to reach its full potential in the clinic. Methods We performed a phase 1 trial to determine the safety, maximum tolerated dose (MTD) and pharmacokinetics of belinostat in patients with advanced cancer and varying degrees of liver dysfunction. Results Seventy-two patients were enrolled and divided into cohorts based on liver function. In patients with mild dysfunction, the MTD was the same as the recommended phase 2 dose (1000 mg/m2 /day). Belinostat was well tolerated in patients with moderate and severe liver dysfunction, although the trial was closed before the MTD in these cohorts could be determined. The mean clearance of belinostat was 661 mL/min/m2 in patients with normal liver function, compared to 542, 505 and 444 mL/min/m2 in patients with mild, moderate and severe hepatic dysfunction. Although this trial was not designed to assess clinical activity, of the 47 patients evaluable for response, 13 patients (28%) experienced stable disease. Conclusion While a statistically significant difference in clearance indicates increased belinostat exposure with worsening liver function, no relationship was observed between belinostat exposure and toxicity. An assessment of belinostat metabolites revealed significant differences in metabolic pathway capability in patients with differing levels of liver dysfunction. Further studies are needed to establish formal dosing guidelines in this patient population.

11519 Background: ASPS constitutes < 1% of soft tissue sarcomas and frequently presents in adolescents and young adults. There are no approved therapies for ASPS. We are currently evaluating the clinical activity of atezolizumab (atezo), an anti-PD-L1 antibody, in patients (pts) with advanced ASPS. Methods: This is a multicenter, open-label, single-arm phase II study where atezo is administered at a fixed dose of 1200 mg in adults or 15 mg/kg (1200 mg max) in pediatric pts age ≥2 once Q21 days. The primary objective is to determine the objective response rate (ORR) of atezo using RECIST 1.1. Secondary objectives include duration of response and correlating response with the immune effects of atezo in blood and paired tumor biopsies (pre- and post-treatment). Tumor specimens were analyzed with multiplex immunofluorescence immuno-oncology panels to quantify CD8+, PD-1+, and PD-L1+ cells/mm2 in the tumor microenvironment. CD8+ density was calculated as the total number of CD8+ cells divided by the entire area (mm2) of the tumor and invasive margins of the biopsy. Results: As of February 4, 2021, 44 pts have been enrolled. The median age in the study was 31 years (range, 12–70) with equal male: female distribution. 54.5% of pts were Caucasian. Baseline ECOG ≤1 was present in 97.7%. The median time on study was 11.5 months (range, 0.8–40.3 months). At data cutoff, response evaluation was available for 43 pts with an ORR of 37.2% (16/43). One pt experienced a complete response and 15 pts experienced a partial response (PR), of which 14 were confirmed. The median time to confirmed response was 3.5 months (range, 2.1–14.9 months). The median duration of confirmed response was 16.5 months (range, 4.9–38.1 months). Stable disease (SD) was present in 58.1% (25/43). One or more grade 3 adverse events potentially related to atezo were identified in 16.3% (7/43) pts. These include diarrhea, hypothyroidism, transaminitis, anemia, vertigo, extremity pain, myalgia, pneumonitis, rash, and stroke (n = 1 each). No grade 4 or 5 events have been reported. Among 8 cases with evaluable biopsy pairs, both baseline and C3D1 specimens in all cases demonstrated CD8+ T cell infiltration and PD-L1 expression. PD-1 expression was detected at baseline in 5 cases and at C3D1 in 7 cases. In 6 cases (3 SDs and 3 PRs), treatment did not change CD8+ cell density. In the other 2 cases (both PRs), CD8+ density increased > 3x above baseline by C3D1. Analysis of T cell activation using pharmacodynamic response biomarkers, along with whole exome and RNA-seq to evaluate the genomic and transcriptomic landscape of ASPS, are ongoing. Conclusions: Atezo is well tolerated and demonstrates promising single agent activity with durable responses in advanced ASPS. Preliminary tumor biomarker analysis confirms the presence of multiple PD-1/PD-L1 immune checkpoint (IC) components, indicating that advanced ASPS is an ideal candidate for therapeutic IC inhibition. Funded by NCI Contract No HHSN261200800001E. Clinical trial information: NCT03141684.

Objective To introduce a novel preclinical animal model of psoriatic arthritis (PsA) in R26Stat3Cstopfl/fl CD4Cre mice, and to investigate the role of Th17 cytokines in the disease pathogenesis. Methods We characterized a novel murine model of Th17-driven cutaneous and synovio-entheseal disease directed by T cell-specific expression of a hyperactive Stat3 allele. By crossing R26Stat3Cstopfl/fl CD4Cre mice onto an interleukin-22 (IL-22)-knockout background or treating the mice with a neutralizing antibody against IL-17, we interrogated how these Th17 cytokines could contribute to the pathogenesis of PsA. Results R26Stat3Cstopfl/fl CD4Cre mice developed acanthosis, hyperkeratosis, and parakeratosis of the skin, as well as enthesitis/tendinitis and periarticular bone erosion in different joints, accompanied by osteopenia. T cell-specific expression of a hyperactive Stat3C allele was found to drive the augmented Th17 response in these animals. Careful characterization of the mouse bone marrow revealed an increase in osteoclast progenitor (OCP) and RANKL-producing cells, which contributed to the osteopenia phenotype observed in the mutant animals. Abrogation of the Th17 cytokines IL-17 or IL-22 improved both the skin and bone phenotype in R26Stat3Cstopfl/fl CD4Cre mice, revealing a central role of Th17 cells in the regulation of OCP and RANKL expression on stromal cells. Conclusion Perturbation of the IL-23/Th17 axis instigates Th17-mediated inflammation in R26Stat3Cstopfl/fl CD4Cre mice, leading to cutaneous and synovio-entheseal inflammation and bone pathologic features highly reminiscent of human PsA. Both IL-17A and IL-22 produced by Th17 cells appear to play critical roles in promoting the cutaneous and musculoskeletal inflammation that characterizes PsA.

The intracellular effects and overall efficacies of anticancer therapies can vary significantly by tumor type. To identify patterns of drug-induced gene modulation that occur in different cancer cell types, we measured gene-expression changes across the NCI-60 cell line panel after exposure to 15 anticancer agents. The results were integrated into a combined database and set of interactive analysis tools, designated the NCI Transcriptional Pharmacodynamics Workbench (NCI TPW), that allows exploration of gene-expression modulation by molecular pathway, drug target, and association with drug sensitivity. We identified common transcriptional responses across agents and cell types and uncovered gene-expression changes associated with drug sensitivity. We also demonstrated the value of this tool for investigating clinically relevant molecular hypotheses and identifying candidate biomarkers of drug activity. The NCI TPW, publicly available at https://tpwb.nci.nih.gov, provides a comprehensive resource to facilitate understanding of tumor cell characteristics that define sensitivity to commonly used anticancer drugs. Significance: The NCI Transcriptional Pharmacodynamics Workbench represents the most extensive compilation to date of directly measured longitudinal transcriptional responses to anticancer agents across a thoroughly characterized ensemble of cancer cell lines.

Summary T-cell lymphomas (TCL) are aggressive lymphomas usually treated with CHOP (cyclophsophamide, doxorubicin, vincristine, prednisolone)-like regimens upfront. Recent data suggest that TCL are driven by epigenetic defects, potentially rendering them sensitive to epigenetic therapies. We explored the therapeutic merits of a combined epigenetic platform using histone deacetylase inhibitors (HDACIs) and DNA methyltransferase inhibitors (DNMT) in in vitro and in vivo models of TCL. The 50% inhibitory concentration (IC50) values revealed romidepsin was the most potent HDACI, with an IC50 in the low nanomolar range. The combination with a hypomethylating agent produced synergy across all cell lines, which was confirmed in cytotoxicity and apoptosis assays. An in vivo xenograft study demonstrated inhibition of tumour growth in the combination cohort compared to the single agent. Gene expression array and global methylation profiling revealed differentially expressed genes and modulated pathways for each of the single treatment conditions and the combination. Most of the effects induced by the single agent treatment were maintained in the combination group. In total, 944 unique genes were modulated by the combination treatment, supporting the hypothesis of molecular synergism. These data suggest combinations of hypomethylating agents and HDACIs are synergistic in models of TCL, which is supported at the molecular level.

Cutaneous T-cell lymphoma is a heterogeneous group of lymphomas characterized by the accumulation of malignant T cells in the skin. The molecular and cellular etiology of this malignancy remains enigmatic, and what role antigenic stimulation plays in the initiation and/or progression of the disease remains to be elucidated. Deep sequencing of the tumor genome showed a highly heterogeneous landscape of genetic perturbations, and transcriptome analysis of transformed T cells further highlighted the heterogeneity of this disease. Nonetheless, using data harvested from high-throughput transcriptional profiling allowed us to develop a reliable signature of this malignancy. Focusing on a key cytokine signaling pathway previously implicated in cutaneous T-cell lymphoma pathogenesis, JAK/STAT signaling, we used conditional gene targeting to develop a fully penetrant small animal model of this disease that recapitulates many key features of mycosis fungoides, a common variant of cutaneous T-cell lymphoma. Using this mouse model, we show that T-cell receptor engagement is critical for malignant transformation of the T lymphocytes and that progression of the disease is dependent on microbiota.

Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.