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Purpose: Despite promising preclinical studies, toxicities have precluded combinations of chemotherapy and DNA damage response (DDR) inhibitors. We hypothesized that tumor-targeted chemotherapy delivery might enable clinical translation of such combinations. Patients and Methods: In a phase I trial, we combined sacituzumab govitecan, antibody–drug conjugate (ADC) that delivers topoisomerase-1 inhibitor SN-38 to tumors expressing Trop-2, with ataxia telangiectasia and Rad3-related (ATR) inhibitor berzosertib. Twelve patients were enrolled across three dose levels. Results: Treatment was well tolerated, with improved safety over conventional chemotherapy-based combinations, allowing escalation to the highest dose. No dose-limiting toxicities or clinically relevant ≥grade 4 adverse events occurred. Tumor regressions were observed in 2 patients with neuroendocrine prostate cancer, and a patient with small cell lung cancer transformed from EGFR-mutant non–small cell lung cancer. Conclusions: ADC-based delivery of cytotoxic payloads represents a new paradigm to increase efficacy of DDR inhibitors.

SCLC is a highly aggressive tumor with a 5-year survival rate of less than 6%. A heterogeneous disease, SCLC is classified into four subtypes that include tumors with neuroendocrine and non-neuroendocrine features. Immune checkpoint blockade has been recently added for the frontline treatment of SCLC; however, this therapy has only led to modest clinical improvements. The lack of clinical benefit in a cancer type known to have a high tumor mutational burden has been attributed to poor T-cell infiltration and low expression of MHC-class I in most SCLC tumors. In an attempt to devise a more effective immunotherapeutic regimen, this study investigated an alternate approach on the basis of the use of the clinical-stage interleukin-15 superagonist, N-803.Preclinical models of SCLC spanning all molecular subtypes were used to evaluate the susceptibility of SCLC to natural killer (NK)-mediated lysis in vitro, including NK cells activated by N-803. Antitumor activity of N-803 was evaluated in vivo with a xenograft model of SCLC.In vitro and in vivo data revealed differences in susceptibility of SCLC subtypes to lysis by NK cells and that NK cells activated by N-803 effectively lyse SCLC tumor cells across all variant subtypes, regardless of their expression of MHC-class I.These findings highlight the potential of a novel immune-based intervention using a cytokine-based therapeutic option for the treatment of SCLC. We hypothesize that N-803 may provide benefit to most patients with SCLC, including those with immunologically cold tumors lacking MHC expression.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. Oncogenic MYC amplifications drive SCLC heterogeneity, but the genetic mechanisms of MYC amplification and phenotypic plasticity, characterized by neuroendocrine and nonneuroendocrine cell states, are not known. Here, we integrate whole-genome sequencing, long-range optical mapping, single-cell DNA sequencing, and fluorescence in situ hybridization to find extrachromosomal DNA (ecDNA) as a primary source of SCLC oncogene amplifications and driver fusions. ecDNAs bring to proximity enhancer elements and oncogenes, creating SCLC transcription-amplifying units, driving exceptionally high MYC gene dosage. We demonstrate that cell-free nucleosome profiling can noninvasively detect ecDNA amplifications in plasma, facilitating its genome-wide interrogation in SCLC and other cancers. Altogether, our work provides the first comprehensive map of SCLC ecDNA and describes a new mechanism that governs MYC-driven SCLC heterogeneity. ecDNA-enabled transcriptional flexibility may explain the significantly worse survival outcomes of SCLC harboring complex ecDNA amplifications. Significance: MYC drives SCLC progression, but the genetic basis of MYC-driven SCLC evolution is unknown. Using SCLC as a paradigm, we report how ecDNA amplifications function as MYC-amplifying units, fostering tumor plasticity and a high degree of tumor heterogeneity. This article is highlighted in the In This Issue feature, p. 799

Radiation therapy is a mainstay of cancer treatment but does not always lead to complete tumor regression. Here we combine radiotherapy with blockade of the ‘don’t-eat-me’ cell-surface molecule CD47 in small cell lung cancer (SCLC), a highly metastatic form of lung cancer. CD47 blockade potently enhances the local antitumor effects of radiotherapy in preclinical models of SCLC. Notably, CD47 blockade also stimulates off-target ‘abscopal’ effects inhibiting non-irradiated SCLC tumors in mice receiving radiation. These abscopal effects are independent of T cells but require macrophages that migrate into non-irradiated tumor sites in response to inflammatory signals produced by radiation and are locally activated by CD47 blockade to phagocytose cancer cells. Similar abscopal antitumor effects were observed in other cancer models treated with radiation and CD47 blockade. The systemic activation of antitumor macrophages following radiotherapy and CD47 blockade may be particularly important in patients with cancer who suffer from metastatic disease.

SummaryAntibody–drug conjugates (ADC) allow the delivery of cytotoxic chemotherapeutic agents to tumors. Two ADC delivering topoisomerase I (TOP1) poisons (Enhertu and Trodelvy) have recently been FDA-approved for Her2- and Trop2-expressing solid tumors. In a recent study, a TOP1-anti B7-H4 ADC was described and shown to be synergistic with a novel PARP1-selective inhibitor.See related article by Kinneer et al., p. 1086

Small cell lung cancers (SCLC) are highly aggressive, and currently there are no available targeted therapies. To identify clinically actionable drug combinations, we analyzed our previously reported chemogenomics screens and identified a synergistically cytotoxic combination of the topoisomerase I (TOP1) inhibitor topotecan and cycle-dependent kinase 7 (CDK7) inhibitor THZ1. Topotecan causes cell death by generating TOP1-induced DNA breaks and DNA-protein cross-links (TOP1-DPC) that require proteolysis by the ubiquitin-proteasome pathway for their repair. We find that inhibition of the transcriptional kinase CDK7 by THZ1 induces ubiquitin-mediated proteasomal degradation of RNA polymerase II and prevents the proteasomal degradation of TOP1-DPCs. We provide a mechanistic basis for combinatorial targeting of transcription using selective inhibitors of CDK7 and TOP1 in clinical trials to advance SCLC therapeutics.

Background: The “central vein sign” (CVS), a linear hypointensity on T2*-weighted imaging corresponding to a central vein/venule, is associated with multiple sclerosis (MS) lesions. The effect of lesion-size exclusion criteria on MS diagnostic accuracy has not been extensively studied. Objective: Investigate the optimal lesion-size exclusion criteria for CVS use in MS diagnosis. Methods: Cross-sectional study of 163 MS and 51 non-MS, and radiological/histopathological correlation of 5 MS and 1 control autopsy cases. The effects of lesion-size exclusion on MS diagnosis using the CVS, and intralesional vein detection on histopathology were evaluated. Results: CVS+ lesions were larger compared to CVS− lesions, with effect modification by MS diagnosis (mean difference +7.7 mm3, p = 0.004). CVS percentage-based criteria with no lesion-size exclusion showed the highest diagnostic accuracy in differentiating MS cases. However, a simple count of three or more CVS+ lesions greater than 3.5 mm is highly accurate and can be rapidly implemented (sensitivity 93%; specificity 88%). On magnetic resonance imaging (MRI)-histopathological correlation, the CVS had high specificity for identifying intralesional veins (0/7 false positives). Conclusion: Lesion-size measures add important information when using CVS+ lesion counts for MS diagnosis. The CVS is a specific biomarker corresponding to intralesional veins on histopathology.

Trial protocol dose limiting toxicity criteria

Study Representativeness

Pharmacokinetic assessment

Purpose:Despite promising preclinical studies, toxicities have precluded combinations of chemotherapy and DNA damage response (DDR) inhibitors. We hypothesized that tumor-targeted chemotherapy delivery might enable clinical translation of such combinations.Patients and Methods:In a phase I trial, we combined sacituzumab govitecan, antibody–drug conjugate (ADC) that delivers topoisomerase-1 inhibitor SN-38 to tumors expressing Trop-2, with ataxia telangiectasia and Rad3-related (ATR) inhibitor berzosertib. Twelve patients were enrolled across three dose levels.Results:Treatment was well tolerated, with improved safety over conventional chemotherapy-based combinations, allowing escalation to the highest dose. No dose-limiting toxicities or clinically relevant ≥grade 4 adverse events occurred. Tumor regressions were observed in 2 patients with neuroendocrine prostate cancer, and a patient with small cell lung cancer transformed from EGFR-mutant non–small cell lung cancer.Conclusions:ADC-based delivery of cytotoxic payloads represents a new paradigm to increase efficacy of DDR inhibitors.

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung cancer. Oncogenic MYC amplifications drive SCLC heterogeneity, but the genetic mechanisms of MYC amplification and phenotypic plasticity, characterized by neuroendocrine and nonneuroendocrine cell states, are not known. Here, we integrate whole-genome sequencing, long-range optical mapping, single-cell DNA sequencing, and fluorescence in situ hybridization to find extrachromosomal DNA (ecDNA) as a primary source of SCLC oncogene amplifications and driver fusions. ecDNAs bring to proximity enhancer elements and oncogenes, creating SCLC transcription-amplifying units, driving exceptionally high MYC gene dosage. We demonstrate that cell-free nucleosome profiling can noninvasively detect ecDNA amplifications in plasma, facilitating its genome-wide interrogation in SCLC and other cancers. Altogether, our work provides the first comprehensive map of SCLC ecDNA and describes a new mechanism that governs MYC-driven SCLC heterogeneity. ecDNA-enabled transcriptional flexibility may explain the significantly worse survival outcomes of SCLC harboring complex ecDNA amplifications.Significance:MYC drives SCLC progression, but the genetic basis of MYC-driven SCLC evolution is unknown. Using SCLC as a paradigm, we report how ecDNA amplifications function as MYC-amplifying units, fostering tumor plasticity and a high degree of tumor heterogeneity.This article is highlighted in the In This Issue feature, p. 799

Fusion analysis results calculated with STARfusion and summary of RLF exon1 and exon2 expression for the CCLE cohort. Table summarizes the RLF-MYCL expression qPCR results.

ecDNA in SCLC, related to Figure 1

Endogenous replication stress is a major driver of genomic instability. Current assessments of replication stress are low throughput precluding its comprehensive assessment across tumors. Here we develop and validate a transcriptional profile of replication stress by leveraging established cellular characteristics that portend replication stress. The repstress gene signature defines a subset of tumors across lineages characterized by activated oncogenes, aneuploidy, extrachromosomal DNA amplification, immune evasion, high genomic instability, and poor survival, and importantly predicts response to agents targeting replication stress more robustly than previously reported transcriptomic measures of replication stress. Repstress score profiles the dual roles of replication stress during tumorigenesis and in established cancers and defines distinct molecular subtypes within cancers that may be more vulnerable to drugs targeting this dependency. Altogether, our study provides a molecular profile of replication stress, providing novel biological insights of the replication stress phenotype, with clinical implications.Significance:We develop a transcriptional profile of replication stress which characterizes replication stress and its cellular response, revealing phenotypes of replication stress across cancer types. We envision the repstress score to serve as an effective discovery platform to predict efficacy of agents targeting replication stress and clinical outcomes.

Supplementary Data

Cell line AmpliconArchitect assemblies, related to figure 1 and Figure 2

Supplementary Figure from CDK7 Inhibition Synergizes with Topoisomerase I Inhibition in Small Cell Lung Cancer Cells by Inducing Ubiquitin-Mediated Proteolysis of RNA Polymerase II

Supplementary Data from PLX038: A Long-Acting Topoisomerase I Inhibitor With Robust Antitumor Activity in ATM-Deficient Tumors and Potent Synergy With PARP Inhibitors

Supplementary Figure/Table Legends from A Phase I/II Trial of Belinostat in Combination with Cisplatin, Doxorubicin, and Cyclophosphamide in Thymic Epithelial Tumors: A Clinical and Translational Study

PDF file - 105K, Methodological details of 18F-FDG PET/CT image acquisition and analysis

Purpose: Schlafen 11 (SLFN11), a putative DNA/RNA helicase is a dominant genomic determinant of response to DNA-damaging agents and is frequently not expressed in cancer cells. Whether histone deacetylase (HDAC) inhibitors can be used to release SLFN11 and sensitize SLFN11-inactivated cancers to DNA-targeted agents is tested here.Experimental Design: SLFN11 expression was examined in The Cancer Genome Atlas (TCGA), in cancer cell line databases and in patients treated with romidepsin. Isogenic cells overexpressing or genetically inactivated for SLFN11 were used to investigate the effect of HDAC inhibitors on SLFN11 expression and sensitivity to DNA-damaging agents.Results: SLFN11 expression is suppressed in a broad fraction of common cancers and cancer cell lines. In cancer cells not expressing SLFN11, transfection of SLFN11 sensitized the cells to camptothecin, topotecan, hydroxyurea, and cisplatin but not to paclitaxel. SLFN11 mRNA and protein levels were strongly induced by class I (romidepsin, entinostat), but not class II (roclinostat) HDAC inhibitors in a broad panel of cancer cells. SLFN11 expression was also enhanced in peripheral blood mononuclear cells of patients with circulating cutaneous T-cell lymphoma treated with romidepsin. Consistent with the epigenetic regulation of SLFN11, camptothecin and class I HDAC inhibitors were synergistic in many of the cell lines tested.Conclusions: This study reports the prevalent epigenetic regulation of SLFN11 and the dominant stimulatory effect of HDAC inhibitors on SLFN11 expression. Our results provide a rationale for combining class I HDAC inhibitors and DNA-damaging agents to overcome epigenetic inactivation of SLFN11-mediated resistance to DNA-targeted agents. Clin Cancer Res; 24(8); 1944–53. ©2018 AACR.

Intratumoral leukocyte composition of mesothelioma representing pre- and post-therapy immunotherapy, evaluated by multiplex IHC.

Purpose:LMB-100 is a recombinant immunotoxin (iTox) consisting of a mesothelin-binding Fab for targeting and a modified Pseudomonas exotoxin A payload. Preclinical studies showed that combining taxanes with iTox results in synergistic antitumor activity. The objectives of this phase I/II study were to determine the MTD of LMB-100 when administered with nanoalbumin bound (nab)-paclitaxel to patients with previously treated advanced pancreatic adenocarcinoma and to assess the objective response rate.Patients and Methods:Patients (n = 20) received fixed-dose nab-paclitaxel (125 mg/m2 on days 1 and 8) with LMB-100 (65 or 100 μg/kg on days 1, 3, and 5) in 21-day cycles for 1–3 cycles.Results:Fourteen patients were treated on the dose escalation and an additional six in the phase II expansion. MTD of 65 μg/kg was established for the combination. Dose-limiting toxicity resulting from capillary leak syndrome (CLS) was seen in two of five patients treated at 100 μg/kg and one of six evaluable phase I patients receiving the MTD. Severity of CLS was associated with increases in apoptotic circulating endothelial cells. LMB-100 exposure was unaffected by anti-LMB-100 antibody formation in five of 13 patients during cycle 2. Seven of 17 evaluable patients experienced >50% decrease in CA 19-9, including three with previous exposure to nab-paclitaxel. One patient developed an objective partial response. Patients with biomarker responses had higher tumor mesothelin expression.Conclusions:Although clinical activity was observed, the combination was not well tolerated and alternative drug combinations with LMB-100 will be pursued.

Table S1: Dose escalation schema; Table S2: Disease and treatment characteristics and outcomes of patients who underwent multimodality therapy; Table S3: Cycle 1 pharmacokinetic parameters of belinostat.

Mean plasma concentration vs. time curve at belinostat 250 mg/m2.

Supplementary Fig. S1. Romidepsin derepresses SLFN11 expression in cancer cell lines from the NCI-60. Supplementary Fig. S2. Forcing SLFN11 expression sensitizes human colon cancer HCT116 cells to camptothecin. Supplementary Fig. S3. Induction of SLFN11 independently of FLI1, rapid reversal of the romidepsin effect and short half-life of SLN11 transcripts. Supplementary Fig. 4. Derepression of SLFN11 by entinostat sensitizes H82 and HH cells to camptothecin and exhibit additive effect in H446 cells.

Purpose: The cross-talk between tumor cells, myeloid cells, and T cells can play a critical role in tumor pathogenesis and response to immunotherapies. Although the etiology of mesothelioma is well understood, the impact of mesothelioma tumor cells on the surrounding immune microenvironment is less well studied. In this study, the effect of the mesothelioma tumor microenvironment on circulating and infiltrating granulocytes and T cells is investigated.Experimental Design: Tumor tissues and peripheral blood from mesothelioma patients were evaluated for presence of granulocytes, which were then tested for their T-cell suppression potential. Different cocultures of granulocytes and/or mesothelioma tumor cells and/or T cells were set up to identify the mechanism of T-cell inhibition.Results: Analysis of human tumors showed that the mesothelioma microenvironment is enriched in infiltrating granulocytes, which inhibit T-cell proliferation and activation. Characterization of the whole blood at diagnosis identified similar, circulating, immunosuppressive CD11b+CD15+HLADR− granulocytes at increased frequency compared with healthy controls. Culture of healthy-donor granulocytes with human mesothelioma cells showed that GM-CSF upregulates NOX2 expression and the release of reactive oxygen species (ROS) from granulocytes, resulting in T-cell suppression. Immunohistochemistry and transcriptomic analysis revealed that a majority of mesothelioma tumors express GM-CSF and that higher GM-CSF expression correlated with clinical progression. Blockade of GM-CSF with neutralizing antibody, or ROS inhibition, restored T-cell proliferation, suggesting that targeting of GM-CSF could be of therapeutic benefit in these patients.Conclusions: Our study presents the mechanism behind the cross-talk between mesothelioma tumors and the immune microenvironment and indicates that targeting GM-CSF could be a novel treatment strategy to augment immunotherapy in patients with mesothelioma. Clin Cancer Res; 24(12); 2859–72. ©2018 AACR.

Dose adjustments.

Purpose:Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with poor prognosis. CRS-207 is a live-attenuated Listeria monocytogenes engineered to express mesothelin, a tumor-associated antigen highly expressed in MPM. CRS-207 induces antitumor immune responses and increases susceptibility of neoplastic cells to immune-mediated killing.Patients and Methods:Patients with unresectable MPM, ECOG 0 or 1, and adequate organ and pulmonary function were enrolled in this multicenter, open-label phase Ib study. They received two priming infusions of 1 × 109 CFU CRS-207, followed by pemetrexed/cisplatin chemotherapy, and CRS-207 booster infusions. Primary objectives were safety and induction of immune response. Secondary/exploratory objectives included tumor response, progression-free survival (PFS), overall survival (OS), immune subset analysis, and gene-expression profiling of tumor.Results:Of 35 evaluable patients, 89% (31/35) had disease control with one complete response (3%), 19 partial responses (54%), and 10 stable disease (29%). The estimated median duration of response was 5.0 months (95% CI, 3.9–11.5). The median PFS and OS were 7.5 (95% CI, 7.0–9.9) and 14.7 (95% CI, 11.2–21.9) months, respectively. Tumor size reduction was observed post–CRS-207 infusion prior to chemotherapy in 11 of 35 (31%) patients. No unexpected treatment-related serious adverse events or deaths were observed. IHC analysis of pre- and post–CRS-207 treatment tumor biopsies revealed possible reinvigoration and proliferation of T cells, increased infiltration of dendritic and natural killer cells, increased CD8:Treg ratio, and a shift from immunosuppressive M2-like to proinflammatory M1-like macrophages following CRS-207 administration.Conclusions:Combination of CRS-207 and chemotherapy induced significant changes in the local tumor microenvironment and objective tumor responses in a majority of treated patients.

Supplemental Figure 4

Mesothelioma granulocytes suppress T cell activation

Supplementary Figs and Tables

Limitations of the camptothecins and advantages of the indenoisoquinoline TOP1 inhibitors