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Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm characterized by activating mutations in the related receptor tyrosine kinases KIT and PDGFRA. GIST relies on expression of these unamplified receptor tyrosine kinase (RTK) genes through a large enhancer domain, resulting in high expression levels of the oncogene required for tumor growth. Although kinase inhibition is an effective therapy for many patients with GIST, disease progression from kinase-resistant mutations is common and no other effective classes of systemic therapy exist. In this study, we identify regulatory regions of the KIT enhancer essential for KIT gene expression and GIST cell viability. Given the dependence of GIST upon enhancer-driven expression of RTKs, we hypothesized that the enhancer domains could be therapeutically targeted by a BET bromodomain inhibitor (BBI). Treatment of GIST cells with BBIs led to cell-cycle arrest, apoptosis, and cell death, with unique sensitivity in GIST cells arising from attenuation of the KIT enhancer domain and reduced KIT gene expression. BBI treatment in KIT-dependent GIST cells produced genome-wide changes in the H3K27ac enhancer landscape and gene expression program, which was also seen with direct KIT inhibition using a tyrosine kinase inhibitor (TKI). Combination treatment with BBI and TKI led to superior cytotoxic effects in vitro and in vivo, with BBI preventing tumor growth in TKI-resistant xenografts. Resistance to select BBI in GIST was attributable to drug efflux pumps. These results define a therapeutic vulnerability and clinical strategy for targeting oncogenic kinase dependency in GIST.Significance:Expression and activity of mutant KIT is essential for driving the majority of GIST neoplasms, which can be therapeutically targeted using BET bromodomain inhibitors.

Figure S1. Evaluation of the KIT locus in GIST cell lines. Figure S2. Effect of imatinib and JQ1 treatment on cell cycle and apoptosis in GIST cell lines. Figure S3. Effect of JQ1 and imatinib on KIT mRNA, protein and signaling. Figure S4. KIT rescue with enhancer independence in GIST430 and effects of HAND1 overexpression on KIT levels and drug sensitivity. Figure S5. Differences in GIST cell line enhancers and effects of drug treatment at the KIT locus in GIST430. Figure S6. H3K27ac ChIP-seq signal and RNA expression changes of exemplary genes following imatinib and/or JQ1 treatment in GIST. Figure S7. Expression and dependency of genes at the KIT locus in GIST cells. Figure S8. Combinatorial effects of bromodomain and KIT inhibition. Figure S9. Consequences of bromodomain inhibition in GIST xenograft models. Table S1. Oligonucleotide Sequences. Table S2. IC50 values in GIST cell lines.

Objective A ventricle-to-brain index (VBI) >0.35 is associated with low scores on the Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III) in preterm infants with birth weight Study Design The present study is a single-center retrospective cohort study. Results The study included 270 preterm infants born at 230/7 to 286/7 weeks of gestational age. The ICC of VBI between independent measurements by two study radiologists on the first 50 patients was 0.934. Factors associated with the value of VBI included severe intraventricular hemorrhage, bronchopulmonary dysplasia, and systemic steroid administration for BPD but not postmenstrual age. In multivariate analysis, VBI was negatively and independently associated with cognitive (p = 0.002), language (p = 0.004), and motor (p Conclusion In this very preterm cohort the measurement of VBI had excellent reliability. Moreover, VBI measurements were negatively associated with motor, language, and cognitive BSID-III scores. Key Points

Gastrointestinal stromal tumor (GIST) is a mesenchymal neoplasm characterized by activating mutations in the related receptor tyrosine kinases KIT and PDGFRA. GIST relies on expression of these unamplified receptor tyrosine kinase (RTK) genes through a large enhancer domain, resulting in high expression levels of the oncogene required for tumor growth. Although kinase inhibition is an effective therapy for many patients with GIST, disease progression from kinase-resistant mutations is common and no other effective classes of systemic therapy exist. In this study, we identify regulatory regions of the KIT enhancer essential for KIT gene expression and GIST cell viability. Given the dependence of GIST upon enhancer-driven expression of RTKs, we hypothesized that the enhancer domains could be therapeutically targeted by a BET bromodomain inhibitor (BBI). Treatment of GIST cells with BBIs led to cell-cycle arrest, apoptosis, and cell death, with unique sensitivity in GIST cells arising from attenuation of the KIT enhancer domain and reduced KIT gene expression. BBI treatment in KIT-dependent GIST cells produced genome-wide changes in the H3K27ac enhancer landscape and gene expression program, which was also seen with direct KIT inhibition using a tyrosine kinase inhibitor (TKI). Combination treatment with BBI and TKI led to superior cytotoxic effects in vitro and in vivo, with BBI preventing tumor growth in TKI-resistant xenografts. Resistance to select BBI in GIST was attributable to drug efflux pumps. These results define a therapeutic vulnerability and clinical strategy for targeting oncogenic kinase dependency in GIST. Significance: Expression and activity of mutant KIT is essential for driving the majority of GIST neoplasms, which can be therapeutically targeted using BET bromodomain inhibitors.

Despite decades of effort, the sensitivity of patient tumors to individual drugs is often not predictable on the basis of molecular markers alone. Therefore, unbiased, high-throughput approaches to match patient tumors to effective drugs, without requiring a priori molecular hypotheses, are critically needed. Here, we improved upon a method that we previously reported and developed called high-throughput dynamic BH3 profiling (HT-DBP). HT-DBP is a microscopy-based, single-cell resolution assay that enables chemical screens of hundreds to thousands of candidate drugs on freshly isolated tumor cells. The method identifies chemical inducers of mitochondrial apoptotic signaling, a mechanism of cell death. HT-DBP requires only 24 hours of ex vivo culture, which enables a more immediate study of fresh primary tumor cells and minimizes adaptive changes that occur with prolonged ex vivo culture. Effective compounds identified by HT-DBP induced tumor regression in genetically engineered and patient-derived xenograft (PDX) models of breast cancer. We additionally found that chemical vulnerabilities changed as cancer cells expanded ex vivo. Furthermore, using PDX models of colon cancer and resected tumors from colon cancer patients, our data demonstrated that HT-DBP could be used to generate personalized pharmacotypes. Thus, HT-DBP appears to be an ex vivo functional method with sufficient scale to simultaneously function as a companion diagnostic, therapeutic personalization, and discovery tool.

Mesenchymal tumor subpopulations secrete pro-tumorigenic cytokines and promote treatment resistance1-4. This phenomenon has been implicated in chemorefractory small cell lung cancer and resistance to targeted therapies5-8, but remains incompletely defined. Here, we identify a subclass of endogenous retroviruses (ERVs) that engages innate immune signaling in these cells. Stimulated 3 prime antisense retroviral coding sequences (SPARCS) are oriented inversely in 3' untranslated regions of specific genes enriched for regulation by STAT1 and EZH2. Derepression of these loci results in double-stranded RNA generation following IFN-γ exposure due to bi-directional transcription from the STAT1-activated gene promoter and the 5' long terminal repeat of the antisense ERV. Engagement of MAVS and STING activates downstream TBK1, IRF3, and STAT1 signaling, sustaining a positive feedback loop. SPARCS induction in human tumors is tightly associated with major histocompatibility complex class 1 expression, mesenchymal markers, and downregulation of chromatin modifying enzymes, including EZH2. Analysis of cell lines with high inducible SPARCS expression reveals strong association with an AXL/MET-positive mesenchymal cell state. While SPARCS-high tumors are immune infiltrated, they also exhibit multiple features of an immune-suppressed microenviroment. Together, these data unveil a subclass of ERVs whose derepression triggers pathologic innate immune signaling in cancer, with important implications for cancer immunotherapy.

Phenotypic differences between primary patient tumors and models of tumors that are amenable to chemical screening represents a key barrier to drug discovery and drug repurposing. High-throughput technologies that can accurately identify active drugs using primary patient tumors are therefore valuable additions to the drug development toolbox and may have direct clinical utility as predictive biomarkers. Here we report a method called high-throughput dynamic BH3 profiling (HT-DBP), a robust microscopy-based assay with single-cell resolution that enables chemical screens of hundreds of candidate drugs on freshy isolated tumor cells to identify chemical inducers of mitochondrial apoptotic signaling. HT-DBP requires only 24 hours of ex vivo culture which enables the direct study of fresh primary tumor cells and minimizes adaptive changes that occur with prolonged ex vivo culture. Using 1650 compounds, we performed HT-DBP on freshly isolated cells from the MMTV-PyMT genetically engineered mouse model of breast cancer to identify drugs that sensitize these tumors for apoptosis. Selected compounds identified by HT-DBP induced regressions in the MMTV-PyMT genetically engineered mouse model of breast cancer in vivo, and in a patient derived xenograft model of breast cancer. To evaluate how chemical vulnerabilities evolve in cell culture, we performed HT-DBP on a cancer cell line from MMTV-PyMT tumors. We observe different patterns of chemical vulnerabilities between freshly isolated tumor cells and in the derived cell lines, which is consistent with the mixed track record of cancer cell line chemical screens. We demonstrate that HT-DBP can be used to generate personalized apoptotic chemical vulnerabilities (which we refer to as pharmacotypes) for a set of colon cancer PDX models. We find that a PDX model derived from a primary site tumor and a metastatic lesion from the same patient have different pharmacotypes. Using annotations of small molecule targets, we can identify proteins and signaling pathways that represent apoptotic vulnerabilities in colon PDX models. Finally, we apply HT-DBP to primary human thyroid tumors and sarcomas to identify potential active therapies. In sum, HT-DBP can efficiently predict therapeutic sensitivity upon short-term ex vivo drug exposure and may empower functional precision medicine approaches in the clinic. Citation Format: Patrick D. Bhola, Eman Ahmed, Jennifer Guerriero, Ewa Sicinska, Emily Su, Jing Ni, Elizaveta Lavrova, Otari Chipashvili, Timothy Hagan, Kimmie Ng, Andrew Aguirre, Jochen Lorch, Suzanne George, George Demetri, Jean Zhao, Anthony Letai. High-throughput dynamic BH3 profiling identifies active cancer therapies in solid tumors [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 4478.

Given the rapid development of new small molecule cancer therapeutics, there is a growing need for predictive diagnostics to match cancer patients with optimal therapies. We previously developed a precision medicine technology with a functional phenotypic readout called dynamic BH3 profiling (DBP). DBP exposes cancer cells to drugs and measures induction of apoptotic cell death signaling after 24 hours ex vivo. Nonetheless, the application of DBP to core biopsies from metastatic tumors or other limited samples remains a technical challenge. Here, we adapt the DBP protocol for use on samples with small numbers of cells such as core biopsies. We maximize information returned per cell by imaging mitochondrial integrity in response to BH3 peptide exposure over time. We first show that the adapted protocol works in limited numbers of cancer cell lines, and in limited cells from the MMTV-PyMT genetically engineered mouse model of breast cancer. Specifically, we show that our ex vivo DBP predictions of the MMTV-PyMT mouse tumor matches known in vivo response. Finally, we apply our modified protocol to patient derived xenografts of colon cancer and primary patient colon tumors. We expect that our adapted protocol will find utility as a clinical biomarker, and as a method to optimize pre-clinical drug testing. Citation Format: Rebecca German, Elizaveta Lavrova, Timothy Hagan, Otari Chipashvili, Ewa Sicinska, James Cleary, Kimmie Ng, Anthony Letai, Patrick Bhola. Identifying cancer drug sensitivity using live cell imaging dynamic BH3 profiling of solid tumor core biopsies [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 260.

e22547 Background: TRK inhibitor drugs such as the highly selective larotrectinib (Laro), have proven highly effective in malignancies harboring fusions of NTRK1, 2, or 3. Resistance in patients (pts) with progressive disease (PD) after response to initial TRK inhibitor therapy has been attributed to secondary mutations in the solvent front or gatekeeper domains of the NTRK fusion gene. LOXO-195 (L195) is a 2nd generation TRK inhibitor that overcomes these mutations. Mechanisms of resistance to L195 have not yet been well characterized. Methods: We analyzed molecular mechanisms of resistance in one adult pt with undifferentiated pleomorphic sarcoma (UPS) who had serial responses and PD on Laro and L195. Targeted DNA sequencing, RNA sequencing and multicolor cyclic immunofluorescence (CyCIF) were performed on pre- and post-PD specimens on both drugs. Results: The patient was enrolled on the phase 2 clinical trial of Laro (NCT02576431) with TPM3-NTRK1 fusion UPS. Multifocal PD and resistance to Laro developed after major objective response RECIST -74.9%, of 10 months (mo); the resistant tumor harbored both the initial TPM3-NTRK1 fusion but also evolved a new solvent front mutation in NTRK1 [c.1783G > A (p.G595R)]. A single pt protocol (NCT03206931) was designed to treat with L195. After an initial response to L195, PD limited to 2 sites developed; both sites were resected at 5 and 10 mo. The pt continues on L195 with systemic disease control 20 mo after initiation (in total 30 mo since Laro initiation). Analysis of tumor samples pre- and post-PD on L195 identified the emergence of a KRAS G12V mutation, with associated activation of the KRAS signaling pathway and a significant infiltration by inflammatory cells. A cell line and pt-derived xenografts (PDX), all harboring the initial TPM3-NTRK1 fusion, were generated from this pt. Conclusions: Resection of oligoclonal PD and continuation of L195 post-PD can be an effective treatment strategy. Oncogenic activation of the KRAS pathway is a possible mechanism of resistance to L195. Our studies indicate that the tumor microenvironment of TRK-fusion sarcomas resistant to TRK inhibitors may increase inflammatory cell infiltrates, which may provide clues for future combination therapy.

Bacteria go the distance in cancer The bacterial species Fusobacterium nucleatum is associated with a subset of human colorectal cancers, but its role in tumorigenesis is unclear. Studying patient samples, Bullman et al. found that F. nucleatum and certain co-occurring bacteria were present not only in primary tumors but also in distant metastases. Preliminary evidence suggests that the bacterium is localized primarily within the metastatic cancer cells rather than in the stroma. Antibiotic treatment of mice carrying xenografts of F. nucleatum –positive human colorectal cancer slowed tumor growth, consistent with a causal role for the bacterium in tumorigenesis. Science , this issue p. 1443

Previous studies indicate that specific extracts and the pure triterpene glycoside actein obtained from black cohosh inhibit growth of human breast cancer cells. Our aim is to identify alterations in gene expression induced by treatment with a methanolic extract (MeOH) of black cohosh.We treated MDA-MB-453 human breast cancer cells with the MeOH extract at 40 microg/ml and collected RNA at 6 and 24 h; we confirmed the microarray results with real-time RT-PCR for 18 genes.At 6 h after treatment there was significant increase in expression of ER stress (GRP78), apoptotic (GDF15), lipid biosynthetic (INSIG1 and HSD17B7) and Phase 1 (CYP1A1) genes and, at 24 h, decrease in expression of cell cycle (HELLS and PLK4) genes.Since the MeOH extract activated genes that enhance apoptosis and repressed cell cycle genes, it may be useful in the prevention and therapy of breast cancer.