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Frequency of pathological changes

Whole exome sequencing data with SNVs.

PDF file - 57K, SuperArray Analysis of Oncogenes, Tumor Suppressors, and Stem Cell-Related Genes in MPM Lines Following Selective PRC2 Knockdown or 5 �M DZNep.

PDF file - 661K, TaqMan Primers, shRNA, and Antibodies.

PDF file - 102K, Effects of DZNep treatment on cultured mesothelioma cell lines relative to normal mesothelia cells. Pyrosequencing analysis of NBL2 DNA repeat in normal mesothelia (LP9) and MPM cell lines cultured in the presence or absence of DZNep.

PDf file - 63K, qRT-PCR and immunoblot analysis of EZH2 and EED levels in MPM lines following transduction with shRNA targeting EZH2, EED, or sham sequences. Effects of knock-down of EZH2 and EED on proliferation of MPM lines.

PDF file - 63K, Genes Simultaneously Modulated by DZNep in MES1 and H28.

PDF file - 661K, Photomicrographs of NMES1 and NMES2 normal pleura cultures, and MES1 through MES4 mesothelioma cell lines established at the NCI.

Purpose: Patients with inflammatory bowel diseases, that is, ulcerative colitis and Crohn's disease (CD), face an increased risk of developing colorectal cancer (CRC). Evidence, mainly from ulcerative colitis, suggests that TP53 mutations represent an initial step in the progression from inflamed colonic epithelium to CRC. However, the pathways involved in the evolution of CRC in patients with CD are poorly characterized.Experimental Design: Here, we analyzed 73 tissue samples from 28 patients with CD-CRC, including precursor lesions, by targeted next-generation sequencing of 563 cancer-related genes and array-based comparative genomic hybridization. The results were compared with 24 sporadic CRCs with similar histomorphology (i.e., mucinous adenocarcinomas), and to The Cancer Genome Atlas data (TCGA).Results: CD-CRCs showed somatic copy-number alterations (SCNAs) similar to sporadic CRCs with one notable exception: the gain of 5p was significantly more prevalent in CD-CRCs. CD-CRCs had a distinct mutation signature: TP53 (76% in CD-CRCs vs. 33% in sporadic mucinous CRCs), KRAS (24% vs. 50%), APC (17% vs. 75%), and SMAD3 (3% vs. 29%). TP53 mutations and SCNAs were early and frequent events in CD progression, while APC, KRAS, and SMAD2/4 mutations occurred later. In four patients with CD-CRC, at least one mutation and/or SCNAs were already present in non-dysplastic colonic mucosa, indicating occult tumor evolution.Conclusions: Molecular profiling of CD-CRCs and precursor lesions revealed an inflammation-associated landscape of genome alterations: 5p gains and TP53 mutations occurred early in tumor development. Detection of these aberrations in precursor lesions may help predicting disease progression and distinguishes CD-associated from sporadic colorectal neoplasia. Clin Cancer Res; 24(20); 4997–5011. ©2018 AACR.

Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequently observed in a wide variety of hematologic malignancies, including myeloid and T-cell leukemias. In this study, we generated Idh2R140Q transgenic mice to examine the role of the Idh2R140Q mutation in leukemia. No leukemia developed in Idh2R140Q transgenic mice, suggesting a need for additional genetic events for leukemia development. Because myeloid cells from NUP98-HOXD13 fusion (NHD13) transgenic mice frequently acquire somatic Idh mutations when they transform to acute myeloid leukemia, we generated Idh2R140Q/NHD13 double transgenic mice. Idh2R140Q/NHD13 transgenic mice developed an immature T-cell leukemia with an immunophenotype similar to double-negative 1 (DN1) or DN2 thymocytes. Idh2R140Q/NHD13 leukemic cells were enriched for an early thymic precursor transcriptional signature, and the gene expression profile for Idh2R140Q/NHD13 DN1/DN2 T-ALL closely matched that of human early/immature T-cell precursor (EITP) acute lymphoblastic leukemia (ALL). Moreover, recurrent mutations found in patients with EITP ALL, including KRAS, PTPN11, JAK3, SH2B3, and EZH2 were also found in Idh2R140Q/NHD13 DN1/DN2 T-ALL. In vitro treatment of Idh2R140Q/NHD13 thymocytes with enasidenib, a selective inhibitor of mutant IDH2, led to a marked decrease in leukemic cell proliferation. These findings demonstrate that Idh2R140Q/NHD13 mice can serve as a useful in vivo model for the study of early/immature thymocyte precursor acute lymphoblastic leukemia development and therapy.Significance:T-cell leukemia induced in Idh2R140Q/NUP98-HOXD13 mice is immunophenotypically, transcriptionally, and genetically similar to human EITP ALL, providing a model for studying disease development and treatment.

Supplementary Figures and tables.

PDF file - 97K, Supplementary Materials and Methods.

PDF file - 62K, Demographic Data Pertaining to the Patients from whom NCI-SB-MES 1-4 lines Were Established.

PDF file - 245K, EZH2 expression in tumors correlates with cell line expression.

Purpose: Polycomb group (PcG) proteins are critical epigenetic mediators of stem cell pluripotency, which have been implicated in the pathogenesis of human cancers. This study was undertaken to examine the frequency and clinical relevance of PcG protein expression in malignant pleural mesotheliomas (MPM).Experimental Design: Microarray, quantitative reverse transcriptase PCR (qRT-PCR), immunoblot, and immunohistochemistry techniques were used to examine PcG protein expression in cultured MPM, mesothelioma specimens, and normal mesothelial cells. Lentiviral short hairpin RNA techniques were used to inhibit EZH2 and EED expression in MPM cells. Proliferation, migration, clonogenicity, and tumorigenicity of MPM cells either exhibiting knockdown of EZH2 or EED, or exposed to 3-deazaneplanocin A (DZNep), and respective controls were assessed by cell count, scratch and soft agar assays, and murine xenograft experiments. Microarray and qRT-PCR techniques were used to examine gene expression profiles mediated by knockdown of EZH2 or EED, or DZNep.Results:EZH2 and EED, which encode components of polycomb repressor complex-2 (PRC-2), were overexpressed in MPM lines relative to normal mesothelial cells. EZH2 was overexpressed in approximately 85% of MPMs compared with normal pleura, correlating with diminished patient survival. Overexpression of EZH2 coincided with decreased levels of miR-101 and miR-26a. Knockdown of EZH2 orEED, or DZNep treatment, decreased global H3K27Me3 levels, and significantly inhibited proliferation, migration, clonogenicity, and tumorigenicity of MPM cells. Common as well as differential gene expression profiles were observed following knockdown of PRC-2 members or DZNep treatment.Conclusions: Pharmacologic inhibition of PRC-2 expression/activity is a novel strategy for mesothelioma therapy. Clin Cancer Res; 18(1); 77–90. ©2011 AACR.

Supplementary Figure S1. Overview of tissue samples analyzed for each patient. Supplementary Figure S2. Topology and histomorphology of Crohn''s disease associated colorectal carcinomas (CD-CRCs; n = 32) from our cohort. Supplementary Figure S3. Representative histology images of Crohn''s disease associated colorectal carcinomas. Supplementary Figure S4. Analysis of microsatellite instability. Supplementary Figure S5. Association of microsatellite instability with carcinoma site and histomorphology in Crohn''s disease associated colorectal carcinomas (CD-CRCs; n = 32) from our cohort. Supplementary Figure S6. Frequency plots of copy number profiles reconstructed from the TCGA CRC dataset (Affymetrix SNP 6 data). Supplementary Figure S7. Frequency plot of copy number profiles reconstructed from targeted sequencing data. Supplementary Figure S8. Comparison of variant calling by MuTect and Strelka algorithms. Supplementary Figure S9. Number of genome alterations and base substitution patterns in Crohn''s disease associated CRCs compared to sporadic mucinous CRCs. Supplementary Figure S10. Graphical summary of TP53 mutations from Crohn''s disease associated CRCs (n = 29) and sporadic mucinous CRCs (n = 24). Supplementary Figure S11. Graphical summary of KRAS mutations from Crohn''s disease associated CRCs (n = 29) and sporadic mucinous CRCs (n = 24). Supplementary Figure S12. Graphical summary of APC mutations from Crohn''s disease associated CRCs (n = 29) and sporadic mucinous CRCs (n = 24). Supplementary Figure S13. Genomic alteration patterns in selected pathways in Crohn''s disease associated CRCs (n = 29). Supplementary Figure S14. Genomic alteration patterns in selected pathways in sporadic mucinous CRCs (n = 24). Supplementary Figure S15. Genome alterations during tumor progression in Crohn''s disease. Supplementary Figure S16. Overview of all detected mutations in patients with multiple synchronous lesions. Supplementary Figure S17. Patient C12 Supplementary Figure S18. Patient C26. Supplementary Figure S19. Patient C18. Supplementary Figure S20. Patient C05. Supplementary Figure S21. Patient C02. Supplementary Figure S22. Patient C11. Supplementary Figure S23. Patient C07 Supplementary Figure S24. Patient C06. Supplementary Figure S25. Mutant allele fractions by gene. Supplementary Figure S26-S29. Representative images of histology (H&E, left) and TP53 immunohistochemistry (right) in TP53-mutated and TP53 wild-type tissues comprising non-dysplastic (normal, inflamed) colonic mucosa and dysplasia.

Supplementary Table S1. Genes included in OncoVar assay. Supplementary Table S2. Individual clinico-pathologic characteristics of patients with Crohn's disease associated colorectal carcinoma (CD-CRC). Supplementary Table S3. Individual clinico-pathologic characteristics of patients with sporadic mucinous colorectal cancer (MUC-CRC). Supplementary Table S4. Summary of fluorescence in situ hybridization (FISH) results. Supplementary Table S5. Tumor cell content and expected mutant allele fraction (MAF) of founding clones in Crohn's disease associated CRC and sporadic mucinous CRC. Supplementary Table S6. Epithelial cell content and expected mutant allele fraction (MAF) of founding clones in normal colonic mucosa, inflamed mucosa and dysplastic lesions from patients with Crohn's disease associated CRC. Supplementary Table S7. Analysis of microsatellite instability in non-dysplastic and dysplastic colonic mucosa. Supplementary Table S8. Summary of genome alterations in precursor lesions comprising non-dysplastic and dysplastic colonic mucosa.

PDF file - 65K, Top 20 Gene Sets Enriched in MES1 and H28 Cells following DZNep Treatment.

PDF file - 54K, Top 20 Gene Ontologies Modulated by DZNep in MES1 and H28 Cells.

PDF file - 66K, Summary of percentages of mesothelioma or normal mesothelia specimens expressing EZH2 in figure 2D.

PDF file - 880K, Volcano plots for 28 FDA approved cancer drugs of interest.

XLSX file - 713K, Excel sheet containing additional data for main Figure 4.

XLSX file - 271K, Excel sheet containing additional data for the main text.

XLSX file - 337K, Excel sheet containing the list of compounds from Supplemental Figure 9.

PDF file - 3388K, Supplementary Figure 1: Variant filtering decision tree. Supplementary Figure 2: Distribution of variants by annotation. Supplementary Figure 3: The NCI-60 cell lines. Supplementary Figure 4: Distribution of variants by type. Supplementary Figure 5: Phylogenic conservation of POL epsilon-P286. Supplementary Figure 6: A snapshot of the WES dataset in Variant Analysis by Ingenuity Systems (http://www.ingenuity.com/NCI60_WES). Supplementary Figure 7: Pattern comparison for cell with wild-type TP53 status. Supplementary Figure 8: Receiver operating curves (ROC) for cross-validated drug predictors.

PDF file - 4628K, Volcano plots for 46 cancer genes of interest.

PDF file - 125K

Minichromosome maintenance proteins (Mcm2-7) form a hexameric complex that unwinds DNA ahead of a replicative fork. The deficiency of Mcm proteins leads to replicative stress and consequent genomic instability. Mice with a germline insertion of a Cre cassette into the 3'UTR of the Mcm2 gene (designated Mcm2

SUMMARY We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extra-cranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches., Graphical Abstract, In brief Brohl et al. perform immunogenomics analysis of a cohort of 788 pediatric extracranial solid tumors and cell lines, representing 14 diagnoses, utilizing RNA sequencing. They broadly describe prognostically relevant immunophenotypes and provide proof of concept of a transcriptomics-informed adoptive cellular therapy approach, validating PRAME as a multi-pediatric cancer immunotherapy target.

Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are frequently observed in a wide variety of hematologic malignancies, including myeloid and T-cell leukemias. In this study, we generated Idh2R140Q transgenic mice to examine the role of the Idh2R140Q mutation in leukemia. No leukemia developed in Idh2R140Q transgenic mice, suggesting a need for additional genetic events for leukemia development. Because myeloid cells from NUP98-HOXD13 fusion (NHD13) transgenic mice frequently acquire somatic Idh mutations when they transform to acute myeloid leukemia, we generated Idh2R140Q/NHD13 double transgenic mice. Idh2R140Q/NHD13 transgenic mice developed an immature T-cell leukemia with an immunophenotype similar to double-negative 1 (DN1) or DN2 thymocytes. Idh2R140Q/NHD13 leukemic cells were enriched for an early thymic precursor transcriptional signature, and the gene expression profile for Idh2R140Q/NHD13 DN1/DN2 T-ALL closely matched that of human early/immature T-cell precursor (EITP) acute lymphoblastic leukemia (ALL). Moreover, recurrent mutations found in patients with EITP ALL, including KRAS, PTPN11, JAK3, SH2B3, and EZH2 were also found in Idh2R140Q/NHD13 DN1/DN2 T-ALL. In vitro treatment of Idh2R140Q/NHD13 thymocytes with enasidenib, a selective inhibitor of mutant IDH2, led to a marked decrease in leukemic cell proliferation. These findings demonstrate that Idh2R140Q/NHD13 mice can serve as a useful in vivo model for the study of early/immature thymocyte precursor acute lymphoblastic leukemia development and therapy. Significance: T-cell leukemia induced in Idh2R140Q/NUP98-HOXD13 mice is immunophenotypically, transcriptionally, and genetically similar to human EITP ALL, providing a model for studying disease development and treatment.

Transgenic mice that express either a NUP98–PHF23 (NP23) or NUP98-HOXD13 (NHD13) fusion in the hematopoietic compartment develop a wide spectrum of leukemias, including myeloid, erythroid, megakaryocytic and lymphoid, at age 9–14 months. NP23-NHD13 double transgenic mice were generated by interbreeding NP23 and NHD13 mice. Remarkably, 100% of the NP23-NHD13 double transgenic mice developed acute myeloid leukemia (AML) within three months, characterized by replacement of the thymus with leukemic myeloblasts. The marked infiltration of thymus led to the intriguing hypothesis that AML generated in NP23-NHD13 mice arose in the thymus, as opposed to the bone marrow (BM). Transplantation of CD4-CD8- double negative (DN) thymocytes (which were also negative for Mac1 and Gr1) from leukemic NHD13/NP23 mice demonstrated that DN thymocytes could transmit AML, and limiting dilution studies showed that leukemia initiating cells were increased 14-fold in the thymus compared to BM. Further thymocyte fractionation demonstrated that DN1 and DN2, but not DN3 or DN4 fractions transmitted AML, and a marked expansion (100-fold) of Lineage-Sca1 + Kit + (LSK) cells in the thymus of the NP23-NHD13 mice. Taken together, these results show that the thymus of NP23-NHD13 mice acts as a reservoir for AML initiating cells and that thymic progenitors can transmit AML.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Purpose: Patients with inflammatory bowel diseases, that is, ulcerative colitis and Crohn's disease (CD), face an increased risk of developing colorectal cancer (CRC). Evidence, mainly from ulcerative colitis, suggests that TP53 mutations represent an initial step in the progression from inflamed colonic epithelium to CRC. However, the pathways involved in the evolution of CRC in patients with CD are poorly characterized. Experimental Design: Here, we analyzed 73 tissue samples from 28 patients with CD-CRC, including precursor lesions, by targeted next-generation sequencing of 563 cancer-related genes and array-based comparative genomic hybridization. The results were compared with 24 sporadic CRCs with similar histomorphology (i.e., mucinous adenocarcinomas), and to The Cancer Genome Atlas data (TCGA). Results: CD-CRCs showed somatic copy-number alterations (SCNAs) similar to sporadic CRCs with one notable exception: the gain of 5p was significantly more prevalent in CD-CRCs. CD-CRCs had a distinct mutation signature: TP53 (76% in CD-CRCs vs. 33% in sporadic mucinous CRCs), KRAS (24% vs. 50%), APC (17% vs. 75%), and SMAD3 (3% vs. 29%). TP53 mutations and SCNAs were early and frequent events in CD progression, while APC, KRAS, and SMAD2/4 mutations occurred later. In four patients with CD-CRC, at least one mutation and/or SCNAs were already present in non-dysplastic colonic mucosa, indicating occult tumor evolution. Conclusions: Molecular profiling of CD-CRCs and precursor lesions revealed an inflammation-associated landscape of genome alterations: 5p gains and TP53 mutations occurred early in tumor development. Detection of these aberrations in precursor lesions may help predicting disease progression and distinguishes CD-associated from sporadic colorectal neoplasia. Clin Cancer Res; 24(20); 4997–5011. ©2018 AACR.

Mice homozygous for a hypomorphic allele of DNA replication factor minichromosome maintenance protein 2 (designated Mcm2(cre/cre)) develop precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) with 4–32 small interstitial deletions per tumor. Mice that express a NUP98-HOXD13 (NHD13) transgene develop multiple types of leukemia, including myeloid and T and B lymphocyte. All Mcm2(cre/cre) NHD13(+) mice develop pre-T LBL, and 26% develop an unrelated, concurrent B cell precursor acute lymphoblastic leukemia (BCP-ALL). Copy number alteration (CNA) analysis demonstrated that pre-T LBLs were characterized by homozygous deletions of Pten and Tcf3 and partial deletions of Notch1 leading to Notch1 activation. In contrast, BCP-ALLs were characterized by recurrent deletions involving Pax5 and Ptpn1 and copy number gain of Abl1 and Nup214 resulting in a Nup214-Abl1 fusion. We present a model in which Mcm2 deficiency leads to replicative stress, DNA double strand breaks (DSBs), and resultant CNAs due to errors in DNA DSB repair. CNAs that involve critical oncogenic pathways are then selected in vivo as malignant lymphoblasts because of a fitness advantage. Some CNAs, such as those involving Abl1 and Notch1, represent attractive targets for therapy.

B-1 and B-2 lymphocytes are derived from distinct developmental pathways and represent layered arms of the innate and adaptive immune systems, respectively. In contrast to a majority of murine B-cell malignancies, which stain positive with the B220 antibody, we discovered a novel form of B-cell leukemia in NUP98-PHF23 (NP23) transgenic mice. The immunophenotype (Lin- B220- CD19+ AA4.1+) was identical to that of progenitor (pro) B-1 cells, and VH gene usage was skewed toward 3' V regions, similar to murine fetal liver B cells. Moreover, the gene expression profile of these leukemias was most similar to that of fetal liver pro-B fraction BC, a known source of B-1 B cells, further supporting a pro-B-1 origin of these leukemias. The NP23 pro-B-1 acute lymphoblastic leukemias (ALLs) acquired spontaneous mutations in both Bcor and Janus kinase (Jak) pathway (Jak1/2/3 and Stat5a) genes, supporting a hypothesis that mutations in 3 critical pathways (stem-cell self-renewal, B-cell differentiation, and cytokine signaling) collaborate to induce B-cell precursor (BCP) ALL. Finally, the thymic stromal lymphopoietin (Tslp) cytokine is required for murine B-1 development, and chromosomal rearrangements resulting in overexpression of the TSLP receptor (CRLF2) are present in some patients with high-risk BCP-ALL (referred to as CRLF2r ALL). Gene expression profiles of NP23 pro-B-1 ALL were more similar to that of CRLF2r ALL than non-CRLF2r ALL, and analysis of VH gene usage from patients with CRLF2r ALL demonstrated preferential usage of VH regions used by human B-1 B cells, leading to the suggestion that this subset of patients with BCP-ALL has a malignancy of B-1, rather than B-2, B-cell origin.

Breast cancer (BC) is a highly heterogeneous disease, both at the pathological and molecular level, and several chromatin-associated proteins play crucial roles in BC initiation and progression. Here, we demonstrate the role of PSIP1 (PC4 and SF2 interacting protein)/p75 (LEDGF) in BC progression. PSIP1/p75, previously identified as a chromatin-adaptor protein, is found to be upregulated in basal-like/triple negative breast cancer (TNBC) patient samples and cell lines. Immunohistochemistry in tissue arrays showed elevated levels of PSIP1 in metastatic invasive ductal carcinoma. Survival data analyses revealed that the levels of PSIP1 showed a negative association with TNBC patient survival. Depletion of PSIP1/p75 significantly reduced the tumorigenicity and metastatic properties of TNBC cell lines while its over-expression promoted tumorigenicity. Further, gene expression studies revealed that PSIP1 regulates the expression of genes controlling cell-cycle progression, cell migration and invasion. Finally, by interacting with RNA polymerase II, PSIP1/p75 facilitates the association of RNA pol II to the promoter of cell cycle genes and thereby regulates their transcription. Our findings demonstrate an important role of PSIP1/p75 in TNBC tumorigenicity by promoting the expression of genes that control the cell cycle and tumor metastasis.

Malignant transformation is a multistep process that is dictated by the acquisition of multiple genomic aberrations that provide growth and survival advantage. During the post genomic era, high throughput genomic sequencing has advanced exponentially, leading to identification of countless cancer associated mutations with potential for targeted therapy. Mouse models of cancer serve as excellent tools to examine the functionality of gene mutations and their contribution to the malignant process. However, it remains unclear whether the genetic events that occur during transformation are similar in mice and humans. To address that, we chose several transgenic mouse models of hematopoietic malignancies and identified acquired mutations in these mice by means of targeted re-sequencing of known cancer-associated genes as well as whole exome sequencing. We found that mutations that are typically found in acute myeloid leukemia or T cell acute lymphoblastic leukemia patients are also common in mouse models of the respective disease. Moreover, we found that the most frequent mutations found in a mouse model of lymphoma occur in a set of epigenetic modifier genes, implicating this pathway in the generation of lymphoma. These results demonstrate that genetically engineered mouse models (GEMM) mimic the genetic evolution of human cancer and serve as excellent platforms for target discovery and validation.

Purpose: New therapeutic approaches are needed for patients with thyroid cancer refractory to radioiodine treatment. An inhibitor of bromodomain and extraterminal domain (BET) proteins, JQ1, shows potent antitumor effects in hematological cancers and solid tumors. To evaluate whether JQ1 is effective against thyroid cancer, we examined antitumor efficacy of JQ1 using the ThrbPV/PVKrasG12D mouse, a model of anaplastic thyroid cancer. Experimental Design: We treated ThrbPV/PVKrasG12D mice with vehicle or JQ1 at a dose of 50 mg/kg body weight/day starting at the age of 8 weeks for a 10-week period and monitored thyroid tumor progression. Results: JQ1 markedly inhibited thyroid tumor growth and prolonged survival of these mice. Global differential gene expression analysis showed that JQ1 suppressed the cMyc (hereafter referred to as Myc) transcription program by inhibiting mRNA expression of Myc, ccnd1, and other related genes. JQ1-suppressed Myc expression was accompanied by chromatin remodeling as evidenced by increased expression of histones and hexamethylene bis-acetamide inducible 1, a suppressor of RNA polymerase II transcription elongation. Analyses showed that JQ1 decreased MYC abundance in thyroid tumors and attenuated the cyclin D1–CDK4–Rb–E2F3 signaling to decrease tumor growth. Further analysis indicated that JQ1 inhibited the recruitment of BDR4 to the promoter complex of the Myc and Ccnd1 genes in rat thyroid follicular PCCL3 cells, resulting in decreased MYC expression at the mRNA and protein levels to inhibit tumor cell proliferation. Conclusions: These preclinical findings suggest that BET inhibitors may be an effective agent to reduce thyroid tumor burden for the treatment of refractory thyroid cancer. Clin Cancer Res; 23(2); 430–40. ©2016 AACR.

Approximately 10% of NUP98-PHF23 (NP23) mice develop an aggressive acute lymphoblastic leukemia of B-1 lymphocyte progenitor origin (pro-B1 ALL), accompanied by somatic frameshift mutations of the BCL6 interacting corepressor (Bcor) gene, most commonly within a 9-bp “hotspot” in Bcor exon 8. To determine whether experimentally engineered Bcor mutations would lead to pro-B1 ALL, we used clustered, regularly interspaced, short palindromic repeats–associated protein 9 to introduce a Bcor frameshift mutation into NP23 hematopoietic stem and progenitor cells through the use of Bcor small guide RNAs (Bcor sgRNAs). Recipient mice transplanted with NP23 bone marrow or fetal liver cells that had been transduced with a Bcor sgRNA developed pro-B1 ALL, characterized by a B-1 progenitor immunophenotype, clonal Igh gene rearrangement, and Bcor indel mutation, whereas control recipients did not. Similar to a subset of human B-cell precursor ALL, the murine pro-B1 ALL had acquired somatic mutations in Jak kinase genes. JAK inhibitors (ruxolitinib and tofacitinib) inhibited the growth of pro-B1 ALL cell lines established from Bcor sgRNA/NP23 recipients at clinically achievable concentrations (100 nM). Our results demonstrate that Bcor mutations collaborate with NP23 to induce pro-B1 ALL, and that JAK inhibitors are potential therapies for pro-B1 ALL.

Thrombocytopenia-absent radii (TAR) syndrome, characterized by neonatal thrombocytopenia and bilateral radial aplasia with thumbs present, is typically caused by the inheritance of a 1q21.1 deletion and a single-nucelotide polymorphism in RBM8A on the nondeleted allele. We evaluated two siblings with TAR-like dysmorphology but lacking thrombocytopenia in infancy. Family NCI-107 participated in an IRB-approved cohort study and underwent comprehensive clinical and genomic evaluations, including aCGH, whole-exome, whole-genome, and targeted sequencing. Gene expression assays and electromobility shift assays (EMSAs) were performed to evaluate the variant of interest. The previously identified TAR-associated 1q21.1 deletion was present in the affected siblings and one healthy parent. Multiple sequencing approaches did not identify previously described TAR-associated SNPs or mutations in relevant genes. We discovered rs61746197 A > G heterozygosity in the parent without the deletion and apparent hemizygosity in both siblings. rs61746197 A > G overlaps a RelA-p65 binding motif, and EMSAs indicate the A allele has higher transcription factor binding efficiency than the G allele. Stimulation of K562 cells to induce megakaryocyte differentiation abrogated the shift of both reference and alternative probes. The 1q21.1 TAR-associated deletion in combination with the G variant of rs61746197 on the nondeleted allele is associated with a TAR-like phenotype. rs61746197 G could be a functional enhancer/repressor element, but more studies are required to identify the specific factor(s) responsible. Overall, our findings suggest a role of rs61746197 A > G and human disease in the setting of a 1q21.1 deletion on the other chromosome.

Purpose: Vandetanib is well-tolerated in patients with advanced medullary thyroid carcinoma (MTC). Long-term outcomes and mechanisms of MTC progression have not been reported previously. Experimental Design: We monitored toxicities and disease status in patients taking vandetanib for hereditary, advanced MTC. Tumor samples were analyzed for molecular mechanisms of disease progression. Results: Seventeen patients [8 male, age 13 (9–17)* years] enrolled; 16 had a RET p.Met918Thr germline mutation. The duration of vandetanib therapy was 6.1 (0.1–9.7+)* years with treatment ongoing in 9 patients. Best response was partial response in 10, stable disease in 6, and progressive disease in one patient. Duration of response was 7.4 (0.6–8.7+)* and 4.9 (0.6–7.8+)* years in patients with PR and SD, respectively. Six patients died 2.0 (0.4–5.7)* years after progression. Median progression-free survival (PFS) was 6.7 years [95% confidence interval (CI): 2.3 years–undefined] and 5-year overall survival (OS) was 88.2% (95% CI: 60.6%–96.9%). Of 16 patients with a RET p.Met918Thr mutation, progression-free survival was 6.7 years (95% CI: 3.1–undefined) and 5-year overall survival was 93.8% (95% CI: 63.2%–99.1%). No patients terminated treatment because of toxicity. DNA sequencing of tissue samples (n = 11) identified an increase in copy number alterations across the genome as a potential mechanism of drug resistance [*median (range)]. Conclusions: This study demonstrates that vandetanib is safe and results in sustained responses in children and adolescents with hereditary MTC. Our preliminary molecular data suggest that an increase in copy number abnormalities may be associated with tumor progression in hereditary MTC patients treated with vandetanib. Clin Cancer Res; 24(4); 753–65. ©2017 AACR.

Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are clinically disparate primary liver cancers with etiological and biological heterogeneity. We identified common molecular subtypes linked to similar prognosis among 199 Thai ICC and HCC patients through systems integration of genomics, transcriptomics, and metabolomics. While ICC and HCC share recurrently mutated genes, including TP53, ARID1A, and ARID2, mitotic checkpoint anomalies distinguish the C1 subtype with key drivers PLK1 and ECT2, whereas the C2 subtype is linked to obesity, T cell infiltration, and bile acid metabolism. These molecular subtypes are found in 582 Asian, but less so in 265 Caucasian patients. Thus, Asian ICC and HCC, while clinically treated as separate entities, share common molecular subtypes with similar actionable drivers to improve precision therapy.

The NCI-60 cell lines are the most frequently studied human tumor cell lines in cancer research. This panel has generated the most extensive cancer pharmacology database worldwide. In addition, these cell lines have been intensely investigated, providing a unique platform for hypothesis-driven research focused on enhancing our understanding of tumor biology. Here, we report a comprehensive analysis of coding variants in the NCI-60 panel of cell lines identified by whole exome sequencing, providing a list of possible cancer specific variants for the community. Furthermore, we identify pharmacogenomic correlations between specific variants in genes such as TP53, BRAF, ERBBs, and ATAD5 and anticancer agents such as nutlin, vemurafenib, erlotinib, and bleomycin showing one of many ways the data could be used to validate and generate novel hypotheses for further investigation. As new cancer genes are identified through large-scale sequencing studies, the data presented here for the NCI-60 will be an invaluable resource for identifying cell lines with mutations in such genes for hypothesis-driven research. To enhance the utility of the data for the greater research community, the genomic variants are freely available in different formats and from multiple sources including the CellMiner and Ingenuity websites. Cancer Res; 73(14); 4372–82. ©2013 AACR.

Context: Recent studies showed that transcription of the MYC gene is driven by the interaction of bromodomain and extraterminal domain (BET) proteins with acetylated histones on chromatin. JQ1, a potent inhibitor that effectively disrupts the interaction of BET proteins with acetylated histones, preferentially suppresses transcription of the MYC gene. We recently reported that JQ1 decreased thyroid tumor growth and improved survival in a mouse model of anaplastic thyroid cancer (ATC) by targeting MYC transcription. The role of MYC in human ATC and whether JQ1 can effectively target MYC as a treatment modality have not been elucidated. Objective: To understand the underlying molecular mechanisms of JQ1, we evaluated its efficacy in human ATC cell lines and xenograft models. Design: We determined the effects of JQ1 on proliferation and invasion in cell lines and xenograft tumors. We identified key regulators critical for JQ1-affected proliferation and invasion of tumor cells. Results: JQ1 markedly inhibited proliferation of four ATC cell lines by suppression of MYC and elevation of p21and p27 to decrease phosphorylated Rb and delay cell cycle progression from the G0/G1 phase to the S phase. JQ1 blocked cell invasion by attenuating epithelial-mesenchymal transition signals. These cell-based studies were further confirmed in xenograft studies in which the size and rate of tumor growth were inhibited by JQ1 via inhibition of p21-cyclin/cyclin-dependent kinase-Rb-E2F signaling. Conclusions: These results suggest targeting of the MYC protein could be a potential treatment modality for human ATC for which effective treatment options are limited.