Diverse Tumor Types Research Articles

Modulation of fatty acid metabolism and immune suppression are features of in vitro tumour sphere formation in ontogenetically distinct dog cancers

Non-adherent, 3-dimensional sphere formation is used as an in vitro surrogate to evaluate cellular potential for tumour initiation and self-renewal. To determine if a shared molecular program underlies the capacity for sphere formation by cells originating from diverse tumour types, we characterized molecular and functional properties of 10 independent cell lines derived from 3 ontogenetically distinct dog cancers: hemangiosarcoma, osteosarcoma and glial brain tumours. Genome-wide gene expression profiling identified tumour-of-origin-dependent patterns of adjustment to sphere formation in a uniform culture condition. However, expression of the stem/progenitor markers CD34 and CD117, resistance to cytotoxic drugs and dye efflux (side population assays) showed no association with these gene expression profiles. Instead, primary sphere-forming capacity was inversely correlated with the ability to reform secondary spheres, regardless of tumour ontogeny. Primary sphere formation seemed to be proportional to the number of pre-existing cells with sphere-forming capacity in the cell lines. Cell lines where secondary sphere formation was more proficient than primary sphere formation showed enrichment of genes involved in fatty acid synthesis and immunosuppressive cytokines. In contrast, cell lines where secondary sphere formation was approximately equivalent to or less proficient than primary sphere formation showed upregulation of CD40 and enrichment of genes involved in fatty acid oxidation. Our data suggest that in vitro sphere formation is associated with upregulation of gene clusters involved in metabolic and immunosuppressive functions, which might be necessary for self-renewal and for tumour initiation and/or tumour propagation in vivo.

Molecular and functional crosstalk between extracellular Hsp90 and ephrin A1 signaling.

The Eph receptor tyrosine kinase family member EphA2 plays a pivotal role in modulating cytoskeletal dynamics to control cancer cell motility and invasion. EphA2 is frequently upregulated in diverse solid tumors and has emerged as a viable druggable target. We previously reported that extracellular Hsp90 (eHsp90), a known pro-motility and invasive factor, collaborates with EphA2 to regulate tumor invasion in the absence of its cognate ephrin ligand. Here, we aimed to further define the molecular and functional relationship between EphA2 and eHsp90. Ligand dependent ephrin A1 signaling promotes RhoA activation and altered cell morphology to favor transient cell rounding, retraction, and diminished adhesion. Exposure of EphA2-expressing cancer cells to ligand herein revealed a unique role for eHsp90 as an effector of cytoskeletal remodeling. Notably, blockade of eHsp90 via either neutralizing antibodies or administration of cell-impermeable Hsp90-targeted small molecules significantly attenuated ligand dependent cell rounding in diverse tumor types. Although eHsp90 blockade did not appear to influence receptor internalization, downstream signaling events were augmented. In particular, eHsp90 activated a Src-RhoA axis to enhance ligand dependent cell rounding, retraction, and ECM detachment. Moreover, eHsp90 signaling via this axis stimulated activation of the myosin pathway, culminating in formation of an EphA2-myosin complex. Inhibition of either eHsp90 or Src was sufficient to impair ephrin A1 mediated Rho activation, activation of myosin intermediates, and EphA2-myosin complex formation. Collectively, our data support a paradigm whereby eHsp90 and EphA2 exhibit molecular crosstalk and functional cooperation within a ligand dependent context to orchestrate cytoskeletal events controlling cell morphology and attachment.

1O - Comprehensive Genomic Profiling (CGP) of 114,200 advanced cancers identifies recurrent Kinase Domain Duplications (KDD) and novel oncogenic fusions in diverse tumor types

1O - Comprehensive Genomic Profiling (CGP) of 114,200 advanced cancers identifies recurrent Kinase Domain Duplications (KDD) and novel oncogenic fusions in diverse tumor types

Successful treatment of ovarian cancer with apatinib combined with chemotherapy

Rationale:The standard treatment for ovarian cancer is chemotherapy with 2 drugs (taxanes and platinum drugs). However, the traditional combination of the 2 drugs has many adverse effects (AEs) and the cancer cells will quickly become resistant to the drugs. Apatinib is a small-molecule antiangiogenic agent which has shown promising therapeutic effects against diverse tumor types, but it still remains unknown whether apatinib has an antitumor effect in patients with ovarian cancer. Herein, we present a successfully treated case of ovarian cancer using chemotherapy and apatinib, in order to demonstrate the effectiveness of this new combined regimen in ovarian cancer.Patients concerns:A 51-year-old Chinese woman presented with ovarian cancer >4.5 years. The disease and the cancer antigen 125 (CA-125) had been controlled well by surgical treatment and following chemotherapy. However, the drugs could not control the disease anymore as the CA-125 level was significantly increasing.Diagnosis:Ovarian cancer.Interventions:The patient was treated with apatinib combined with epirubicin. Apatinib was administered orally, at an initial daily dose of 500 mg, and was then reduced to 250 mg qd after the appearance of intolerable hand–foot syndrome (HFS) and oral ulcer. Then, the oral ulcer disappeared and the HFS was controlled by dose adjustment, oral vitamin B6, and hand cream application.Outcomes:The CA-125 reverted to the normal value after treatment with the new regimen. Magnetic resonance imaging showed that the original tumor lesions had disappeared. Apatinib monotherapy as maintenance therapy was then used to successfully control the cancer with a complete response. Our study is the first, to our knowledge, to report the therapeutic effects of apatinib and epirubicin on ovarian cancer.Lessons:Apatinib combined with chemotherapy and apatinib monotherapy as maintenance therapy could be a new therapeutic strategy for ovarian cancer, especially adenocarcinomas.

Objective measurement and clinical significance of IDO1 protein in hormone receptor-positive breast cancer

BackgroundImmunostimulatory therapies targeting immune-suppressive pathways produce durable responses in advanced solid tumors. Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting oxidoreductase that catalyzes the degradation of tryptophan to kynurenine. IDO induces immune tolerance by downregulating CD8+ and effector CD4+ T cell responses. IDO1, the most active isoform, is expressed in diverse tumor types and can be targeted using small molecule inhibitors. We used an objective, in situ assay to measure IDO1 in a collection of hormone receptor-positive breast cancers (HR+ BC).MethodsIDO1 protein was measured using quantitative immunofluorescence in 362 stage I-III HR+ BC represented in tissue microarrays. IDO1 levels were determined in the tumor and stroma, and stratified using median cut-point. Associations between IDO1, clinico-pathological features and CD3+, CD8+, CD20+ and FOXP3 tumor-infiltrating lymphocytes were examined using χ2 and Mann-Whitney tests. Survival was studied using Kaplan-Meier estimator and a proportional hazards model. All tests were two-sided.ResultsIDO1 protein was observed in 76.2% of HR+ BC. There was no association between IDO1 and major clinico-pathological characteristics. Increased IDO1 correlated with decreased CD20+ infiltration (P = 0.0004) but not with CD3+, CD8+ or FOXP3 levels. Elevated IDO1 expression was associated with worse 20-year overall survival (log-rank P = 0.02, HR = 1.39, 95% C.I.: 1.05-1.82). IDO1 scores were independently associated with outcome in multivariable analysis.ConclusionsIDO1 protein is expressed in the majority of HR+ BC and is an independent negative prognostic marker. Additionally, IDO1 expression is negatively associated with tumor B-cell infiltration. Measurement of IDO1 has the potential to identify a population that might derive benefit from IDO1 blockade.

Highlights of This Issue

Using liquid chromatography/mass spectrometry (LC/MS)-based metabolomics and the MEK inhibitor RO4987655, 21 plasma metabolite markers of MEK inhibition in two sensitive BRAF mutant, human melanoma xenograft models were identified. Of these, it was found that on-treatment changes in the concentrations of 15 metabolites significantly predicted objective responses in patients with advanced melanoma treated with RO4987655. In patients with progressive disease, it was demonstrated that propionyl carnitine and amino acid levels increased; while in those patients that responded, phosphatidylcholines and sphingomyelin concentrations decreased. In addition, the pre-treatment levels of seven plasma metabolites (phosphatidylcholines and sphingomyelins) had significant prognostic value.Given the lack of meaningful clinical efficacy of pharmacological agents targeting the IGF pathway when applied in unselected cancer patients, the identification of selection biomarkers is of crucial importance. Sanderson and colleagues report that autocrine IGF2 overexpression hyperactivates the IGF pathway in colorectal cancer (CRC) and other cancers and predicts for sensitivity to the IGF1R/INSR inhibitor BI 885578, either as monotherapy or in combination with VEGF-pathway inhibition. Coupled with the identification of substantial subsets of IGF2-overexpressing patients in diverse tumor types, these findings support the assessment of IGF2 expression for patient selection in future trials of IGF pathway inhibitors.Molecularly targeted drugs have yielded significant therapeutic advances in oncogene-driven non-small cell lung cancer (NSCLC), but most patients eventually develop acquired resistance. Here, Shien and colleagues have identified that the IL6 family cytokine oncostatin-M (OSM) induced a switch to the epithelial-to-mesenchymal phenotype and protected cells from targeted drug-induced apoptosis in OSM receptors (OSMRs)/JAK1/STAT3-dependent manner, and co-targeting inhibition of the oncogenic pathway and JAK1 revered resistance to targeted drugs. These results suggest that the OSMRs/JAK1/STAT3 axis contributes to resistance to targeted drugs in oncogene-driven NSCLC cells, implying that this pathway could be a therapeutic target.OPCML, a GPI anchored tumor-suppressor protein is a negative regulator of receptor tyrosine kinases (RTK), is inactivated by somatic methylation in many cancer types. Here, Zanini and colleagues report that restoration of OPCML in deficient cells leads to sensitization to anti-HER RTK inhibitors. OPCML interacts with HER2 and not EGFR, thereby disrupting formation of HER2-EGFR heterodimers leading to better response to both lapatinib and erlotinib in HER2-expressing cancer cell lines. High OPCML expression is associated with better clinical response to lapatinib therapy in breast cancer patients. These results suggest that OPCML/RTK inhibitor co-therapy could be a valuable sensitizing approach to RTK inhibitors.

Mucinous cystadenocarcinoma of the breast: the challenge of diagnosing a rare entity.

Mucinous cystadenocarcinoma is an extremely rare variant of primary breast tumor which is histologically similar to mucinous cystadenocarcinoma of the ovary and pancreas. Herein we report a case of a 63 years old woman diagnosed with diverse histological types of non-synchronous rare primary breast tumors, a medullary carcinoma of the right breast and a mucinous cystadenocarcinoma of the left breast. Macroscopically the neoplasm appeared multilocular filled with mucoid material. Under light microscopy the cystic areas were lined by columnar cells with abundant intracellular and extracellular mucin. Solid areas were composed of tall columnar cells with intracellular mucin. Moderate to marked atypia was noticed and tumor cells stained positive for cytokeratin 7 and negative for cytokeratin 20. Moreover tumor cells displayed a basal like immunophenotype expressed as followed: ER negative, PR negative, HER-2 negative, cytokeratin (CK5/6) positive and EGFR positive.

1700O - Genomic profiling of 114,200 advanced cancers identifies recurrent kinase domain duplications (KDD) and oncogenic rearrangements (RE) across diverse tumor types

1700O - Genomic profiling of 114,200 advanced cancers identifies recurrent kinase domain duplications (KDD) and oncogenic rearrangements (RE) across diverse tumor types

EGFR-targeted therapies in the post-genomic era.

Over 90% of head and neck cancers overexpress the epidermal growth factor receptor (EGFR). In diverse tumor types, EGFR overexpression has been associated with poorer prognosis and outcomes. Therapies targeting EGFR include monoclonal antibodies, tyrosine kinase inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, and antisense gene therapy. Few EGFR-targeted therapeutics are approved for clinical use. The monoclonal antibody cetuximab is a Food and Drug Administration (FDA)-approved EGFR-targeted therapy, yet has exhibited modest benefit in clinical trials. The humanized monoclonal antibody nimotuzumab is also approved for head and neck cancers in Cuba, Argentina, Colombia, Peru, India, Ukraine, Ivory Coast, and Gabon in addition to nasopharyngeal cancers in China. Few other EGFR-targeted therapeutics for head and neck cancers have led to as significant responses as seen in lung carcinomas, for instance. Recent genome sequencing of head and neck tumors has helped identify patient subgroups with improved response to EGFR inhibitors, for example, cetuximab in patients with the KRAS-variant and the tyrosine kinase inhibitor erlotinib for tumors harboring MAPK1E322K mutations. Genome sequencing has furthermore broadened our understanding of dysregulated pathways, holding the potential to enhance the benefit derived from therapies targeting EGFR.

Assessing Tumor-infiltrating Lymphocytes in Solid Tumors: A Practical Review for Pathologists and Proposal for a Standardized Method From the International Immunooncology Biomarkers Working Group: Part 1: Assessing the Host Immune Response, TILs in Invasive Breast Carcinoma and Ductal Carcinoma In Situ, Metastatic Tumor Deposits and Areas for Further Research.

Assessment of tumor-infiltrating lymphocytes (TILs) in histopathologic specimens can provide important prognostic information in diverse solid tumor types, and may also be of value in predicting response to treatments. However, implementation as a routine clinical biomarker has not yet been achieved. As successful use of immune checkpoint inhibitors and other forms of immunotherapy become a clinical reality, the need for widely applicable, accessible, and reliable immunooncology biomarkers is clear. In part 1 of this review we briefly discuss the host immune response to tumors and different approaches to TIL assessment. We propose a standardized methodology to assess TILs in solid tumors on hematoxylin and eosin sections, in both primary and metastatic settings, based on the International Immuno-Oncology Biomarker Working Group guidelines for TIL assessment in invasive breast carcinoma. A review of the literature regarding the value of TIL assessment in different solid tumor types follows in part 2. The method we propose is reproducible, affordable, easily applied, and has demonstrated prognostic and predictive significance in invasive breast carcinoma. This standardized methodology may be used as a reference against which other methods are compared, and should be evaluated for clinical validity and utility. Standardization of TIL assessment will help to improve consistency and reproducibility in this field, enrich both the quality and quantity of comparable evidence, and help to thoroughly evaluate the utility of TILs assessment in this era of immunotherapy.

Role of Type I and II Interferons in Colorectal Cancer and Melanoma.

Cancer can be considered an aberrant organ with a hierarchical composition of different cell populations. The tumor microenvironment, including the immune cells and related cytokines, is crucial during all the steps of tumor development. In particular, type I and II interferons (IFNs) are involved in a plethora of mechanisms that regulate immune responses in cancer, thus balancing immune escape versus immune surveillance. IFNs are involved in both the direct and indirect regulation of cancer cell proliferation and metastatic potential. The mutational background of genes involved in IFNs signaling could serve as a prognostic biomarker and a powerful tool to screen cancer patients eligible for checkpoint blocking therapies. We herewith describe the latest findings regarding the contribution of IFNs in colorectal cancer and melanoma by researching their dual role as either tumor promoter or suppressor, in diverse tumor types, and microenvironmental context. We are reporting the most innovative and promising approaches of IFN-based therapies that have achieved considerable outcomes in clinical oncology practice and explain the possible mechanisms responsible for their failure.

Citrate Suppresses Tumor Growth in Multiple Models through Inhibition of Glycolysis, the Tricarboxylic Acid Cycle and the IGF-1R Pathway

In this study we have tested the efficacy of citrate therapy in various cancer models. We found that citrate administration inhibited A549 lung cancer growth and additional benefit accrued in combination with cisplatin. Interestingly, citrate regressed Ras-driven lung tumors. Further studies indicated that citrate induced tumor cell differentiation. Additionally, citrate treated tumor samples showed significantly higher infiltrating T-cells and increased blood levels of numerous cytokines. Moreover, we found that citrate inhibited IGF-1R phosphorylation. In vitro studies suggested that citrate treatment inhibited AKT phosphorylation, activated PTEN and increased expression of p-eIF2a. We also found that p-eIF2a was decreased when PTEN was depleted. These data suggest that citrate acts on the IGF-1R-AKT-PTEN-eIF2a pathway. Additionally, metabolic profiling suggested that both glycolysis and the tricarboxylic acid cycle were suppressed in a similar manner in vitro in tumor cells and in vivo but only in tumor tissue. We reproduced many of these observations in an inducible Her2/Neu-driven breast cancer model and in syngeneic pancreatic tumor (Pan02) xenografts. Our data suggests that citrate can inhibit tumor growth in diverse tumor types and via multiple mechanisms. Dietary supplementation with citrate may be beneficial as a cancer therapy.

A comprehensive genomic pan-cancer classification using The Cancer Genome Atlas gene expression data

BackgroundThe Cancer Genome Atlas (TCGA) has generated comprehensive molecular profiles. We aim to identify a set of genes whose expression patterns can distinguish diverse tumor types. Those features may serve as biomarkers for tumor diagnosis and drug development.MethodsUsing RNA-seq expression data, we undertook a pan-cancer classification of 9,096 TCGA tumor samples representing 31 tumor types. We randomly assigned 75% of samples into training and 25% into testing, proportionally allocating samples from each tumor type.ResultsWe could correctly classify more than 90% of the test set samples. Accuracies were high for all but three of the 31 tumor types, in particular, for READ (rectum adenocarcinoma) which was largely indistinguishable from COAD (colon adenocarcinoma). We also carried out pan-cancer classification, separately for males and females, on 23 sex non-specific tumor types (those unrelated to reproductive organs). Results from these gender-specific analyses largely recapitulated results when gender was ignored. Remarkably, more than 80% of the 100 most discriminative genes selected from each gender separately overlapped. Genes that were differentially expressed between genders included BNC1, FAT2, FOXA1, and HOXA11. FOXA1 has been shown to play a role for sexual dimorphism in liver cancer. The differentially discriminative genes we identified might be important for the gender differences in tumor incidence and survival.ConclusionsWe were able to identify many sets of 20 genes that could correctly classify more than 90% of the samples from 31 different tumor types using TCGA RNA-seq data. This accuracy is remarkable given the number of the tumor types and the total number of samples involved. We achieved similar results when we analyzed 23 non-sex-specific tumor types separately for males and females. We regard the frequency with which a gene appeared in those sets as measuring its importance for tumor classification. One third of the 50 most frequently appearing genes were pseudogenes; the degree of enrichment may be indicative of their importance in tumor classification. Lastly, we identified a few genes that might play a role in sexual dimorphism in certain cancers.

Abstract 4827: Establishing a platform for the generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiave

Abstract Cancer Organoids are discrete multicellular structures that recapitulate tumor microanatomy (1). These reagents can be generated by extended culture of partially or fully dissociated tumor samples in three-dimensional matrices. By maintaining tumor and accessory cells in an appropriate context, they provide a biosimilar platform for studying disease pathogenesis and cellular pharmacology (2). Similarly, cancer organoid culture is useful for propagating slow growing tumors or those requiring heterotypic cell-cell interactions. Here, preliminary data will be presented regarding generation of organoids from diverse tumor types as part of the NCI patient-derived models (PDM) initiative. This initiative aims to develop a national repository of patient-derived cancer models (PDMs) consisting of clinically annotated patient-derived xenografts (PDXs) and patient-derived tumor cell cultures (PDCs) prepared from primary and metastatic tumors (3). A standardized panel of different organoid media formulations was constructed to optimize culture conditions for disease subsets. Using this approach, organoids were generated for colon, prostate, pancreatic, breast, melanoma, NSCLC, and bladder tumors. Although some samples were refractory to organoid generation, in several instances, samples that failed to generate 2D cultures thrived as organoids. A further finding was that direct implantation of organoid cultures was an efficient means of generating xenografts. Indeed, work will be presented detailing the exact number of organoids required to establish xenograft tumors. Protocols were developed for routine culture, passaging and long-term storage in liquid nitrogen. Similarly, organoids were amenable to characterization by FACS analysis, ICC/IHC and qRT-PCR to evaluate metrics such as tumor type, histological similarity with patient tumor, cell viability, percentage stroma and whether mouse cells persist in PDX-derived organoids. In summary, growth, expansion, analysis and storage of tumor organoids is feasible for a wide range of tumor types. Importantly, for certain samples, generation of cancer organoids appears to be a useful intermediary step for subsequent PDX model and 2D culture generation. Funded by NCI Contract No. HHSN261200800001E.

Abstract LB-105: Characterization of total mutational burden in the GENIE cohort: Small and large panels can provide TMB information but to varying degrees

Abstract Introduction: Total mutation burden (TMB) correlates with response to immune checkpoint inhibition. AACR Project Genomics Evidence Neoplasia Information Exchange (GENIE) recently released clinical sequencing data for ~19000 cases from 8 institutions. We determined the ability of diverse sequencing panels to assess TMB. Methods: De-identified sequencing data from eight international academic cancer centers were used to investigate the per sample non-silent total somatic mutational burden (TMB) per 1Mb sequenced for all submitted samples. Summary: We first limited TMB characterization to three sites with sequencing data from large gene panels (sampling greater than approximately 1Mb) comprising 14472 samples. The distribution of the TMB normalized per Mb sequenced demonstrated a wide range of mutational rates both within and between tumor types (Table 1). The other five sites utilized smaller targeted sequencing panels. We then restricted the large panel data to the footprint of the smaller targeted panels and compared the number of mutations in the footprint of the small panels to the TMB of each sample from the larger panel data. We found that samples with >5 mutations in the footprint of the smaller targeted panels almost always had a high TMB (94%). In contrast, samples with no mutations in the footprint of the small panels almost never had a high TMB (2%). However, in samples with 1-5 mutations (which represented 2/3 of the samples) the footprint of the smaller targeted panels was not predictive of TMB. Conclusions: While whole exome analyses remain the gold standard for determination of total mutational burden, large sequencing panels (covering greater than approximately 1Mb) can be used to estimate TMB in diverse tumor types. Smaller targeted panels can also predict TMB for a subset of samples, with either 0 or >5 mutations, but this approach appropriately characterizes only just under a third of cases. The majority of cases need the increased genomic sampling of the larger gene panels (or whole exome analyses) to accurately quantify TMB. In summary, while the detection of high mutational burden cases by smaller targeted panel data is quite specific it lacks sensitivity. [A. S. B. and T. S. contributed equally to this work.] Table 1Mutations per MbsMedianSpectrum[0,1)[1,10)>10nMelanoma7.804%54%42%408Skin Cancer, Non-Melanoma7.6521%32%47%182Bladder Cancer7.585%60%35%587Colorectal Cancer6.632%81%18%1170Small Cell Lung Cancer6.635%69%26%104Endometrial Cancer6.222%72%26%446Non-Small Cell Lung Cancer6.187%70%23%2069Cancer of Unknown Primary5.307%74%19%255Non-Hodgkin Lymphoma4.7811%71%18%188Cervical Cancer4.789%79%13%80Esophagogastric Cancer4.749%82%9%440Head and Neck Cancer4.309%77%14%300Breast Cancer3.868%86%6%1739Ovarian Cancer3.868%88%4%603Leukemia3.8615%84%1%349Glioma3.798%86%6%858 Table 2.Mutations per Mbs (based on full panel)[0,1)[1,10)>10n# Mutations (based on footprint of amplicon hotspot panels)033%65%2%4521[1-5]3%81%15%9806>50%6%94%145 Citation Format: Alexander S. Baras, Thomas Stricker, on behalf of the AACR Project GENIE Consortium. Characterization of total mutational burden in the GENIE cohort: Small and large panels can provide TMB information but to varying degrees [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-105. doi:10.1158/1538-7445.AM2017-LB-105

Abstract 3768: CAR T cells specific for CD19 can be redirected to kill CD19 negative tumors

Abstract Introduction. Remarkable progress has been made in the treatment of relapsed/refractory Acute Lymphocytic Leukemia and Non-Hodgkin Lymphoma with CAR-CD19 T cells. In contrast, progress against CD19-negative hematological cancers and solid tumors has been limited. Intensive efforts to optimize cellular therapeutics for better efficacy include provision of cytokine support and countering immunosuppression. However, lack of sufficient antigen is a significant additional hurdle that CAR-T therapeutics for solid tumors must overcome. We present a novel strategy to utilize CD19 for sustained antigen presentation in order to promote cellular therapeutic expansion, efficacy and persistence. The strategy, called IMPACT (Integrated Modules oPtimize Adoptive Cell Therapy), employs a methodology that is modular in design and and can be applied to diverse antigens and tumor types, yet retains the well-established advantages of CAR T cells directed to CD19. Experimental Procedures. A 3rd generation CAR-T was constructed in a lentiviral vector using the FMC63 anti-CD19 scFv for antigen recognition. A second expression cassette, cloned downstream of the CAR-T cassette, was constructed using a secretion signal sequence and exons 1 - 4 of the human CD19 protein linked in frame to a trastuzumab-derived Vh/Vl sequence. This integrated-gene module thus encodes a fusion protein (FP): CD19-anti-HER2-scFv. Results. ELISA and flow cytometry analyses were used to demonstrate bridging between HER2+ cells and the FMC63 mAb via the CD19 domain of the FP. ELISA analyses further demonstrated wild type binding of the 2 FP domains (i.e. CD19/FMC63 and the scFv/HER2). In vitro cytotoxicity studies showed robust redirected killing of HER2+ (CD19-) tumor cells by CAR19 T cells that is mediated by the secreted FP. The efficiency of cytotoxicity was enhanced by the presence of CD19+ B cells. A tumor xenograft model was used to demonstrate effective killing in vivo when HER2+ BT474 cells were injected in one flank and CD19+ RAMOS cell were injected in the opposite flank, but not when HER2+ cell were injected alone. Thus, in vivo, redirected killing required CD19+ cells for expansion and persistence sufficient to promote anti-HER2 cytotoxicity. Conclusions. A CD19-anti-HER2-scFv FP was able to redirect the cytotoxicity of CAR19 T cells in order to kill HER2+ tumor cells. In vivo modeling demonstrated that CAR-T cell expansion by CD19 recognition enabled efficient HER2+ cell cytotoxicity. The IMPACT technology is modular, allowing for FPs having diverse protein domains paired with diverse, distinct scFvs to allow CAR T cells to CD19 or other targets to be used to kill any tumor. We have further developed the technology to address antigens of utility for hematopoietic and solid tumors, including BCMA, ROR1, and PD-L1. The technology is broadly applicable and synergistic with existing cellular therapies. Citation Format: Christine Ambrose, Lihe Su, Lan Wu, Roy R. Lobb, Paul D. Rennert. CAR T cells specific for CD19 can be redirected to kill CD19 negative tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3768. doi:10.1158/1538-7445.AM2017-3768

Abstract 2801: Identification of oncogenic/metastatic driver genes that cooperate with p53 or p53/Rb-loss to induce triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBCs) is an aggressive subtype with poor prognosis; drug-resistant metastases are common and highly lethal for which identification of therapeutic targets is of major medical interest. Previously we demonstrated that deletion of p53 and Rb in mouse mammary epithelium induced TNBC-like tumors but with limited capacity to metastasize. Therefore, we decided to employ sleeping beauty (SB) mutagenesis system that can identify oncogenic networks responsible to drive primary and metastatic tumors. In order to do this, we generated mice in which p53 (p53f/f) or p53 plus Rb (p53f/f:Rbf/f ) are deleted and the SB transposon is mobilized from chromosome 9 as well as controls in which both p53 and Rb are wild type. Forty primary tumors and forty metastases from each cohort will be harvested to identify common integration sites followed by deep sequencing. Then, the top 5-10 candidate oncogenes/tumor suppressors will be validated to determine whether their loss/activation would accelerate tumorigenesis/metastasis. H&E staining of mammary tumors collected so far from different genotypes revealed histologically diverse tumor types which includes but not limited to poorly differentiated tumors with areas of mesenchymal/spindle-like cells, acinar adenocarcinomas, and well differentiated tumors. All mice analyzed developed multiple mammary tumors in more than one mammary gland with average latency of 108, 158 and 170 days for p53/Rb deleted (N=7), p53 deleted (N=12), and wild type mice (N=2), respectively (p < 0.05). However, we did not always observe macro metastases in mice with primary mammary tumors and we decided to perform primary tumor survival surgery when tumors are oversized and let the mice live longer to develop metastases. With this technique, 3 out of 4 mice that underwent tumor removal surgery developed lung metastases when sacrificed. In total, lung metastases were observed in 5 out of 28 mice analyzed so far with/without surgery (18%) which histologically resemble their primary tumors. Since removing tumors from multiple glands is challenging, we decided to take a transplantation approach of the primary mammary epithelial cells of mid-pregnancy (13-16 dpc) p53f/f:Rbf/f:SB+ females and injecting them into the mammary glands of 3-4 week old NSG mice. So far, four NSG mice developed mammary tumors with average latency of 176 days. These tumors resemble spindle-shape histology. SB mutagenesis can promote metastasis in mouse models with limited dissemination potential such as the model proposed herein. The proposed experiments will identify novel oncogenic events that cooperate with p53-loss or combined p53/Rb-loss to induce primary and metastatic TNBCs which may serve as therapeutic targets for this aggressive disease. I have generated 3 sets of cohort of SB mutagenesis to identify these oncogenic events which are currently being analyzed in details. Citation Format: Ronak Ghanbari Azarnier, Agatha Zuchelkowski, Philip.E Chung, Zhe Jiang, Eldad Zacksenhaus. Identification of oncogenic/metastatic driver genes that cooperate with p53 or p53/Rb-loss to induce triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2801. doi:10.1158/1538-7445.AM2017-2801

ATF3 Repression of BCL-XL Determines Apoptotic Sensitivity to HDAC Inhibitors across Tumor Types.

Purpose: Histone deacetylase inhibitors (HDACi) are epigenome-targeting small molecules approved for the treatment of cutaneous T-cell lymphoma and multiple myeloma. They have also demonstrated clinical activity in acute myelogenous leukemia, non-small cell lung cancer, and estrogen receptor-positive breast cancer, and trials are underway assessing their activity in combination regimens including immunotherapy. However, there is currently no clear strategy to reliably predict HDACi sensitivity. In colon cancer cells, apoptotic sensitivity to HDACi is associated with transcriptional induction of multiple immediate-early (IE) genes. Here, we examined whether this transcriptional response predicts HDACi sensitivity across tumor type and investigated the mechanism by which it triggers apoptosis.Experimental Design: Fifty cancer cell lines from diverse tumor types were screened to establish the correlation between apoptotic sensitivity, induction of IE genes, and components of the intrinsic apoptotic pathway.Results: We show that sensitivity to HDACi across tumor types is predicted by induction of the IE genes FOS, JUN, and ATF3, but that only ATF3 is required for HDACi-induced apoptosis. We further demonstrate that the proapoptotic function of ATF3 is mediated through direct transcriptional repression of the prosurvival factor BCL-XL (BCL2L1) These findings provided the rationale for dual inhibition of HDAC and BCL-XL, which we show strongly cooperate to overcome inherent resistance to HDACi across diverse tumor cell types.Conclusions: These findings explain the heterogeneous responses of tumor cells to HDACi-induced apoptosis and suggest a framework for predicting response and expanding their therapeutic use in multiple cancer types. Clin Cancer Res; 23(18); 5573-84. ©2017 AACR.

The Role of CtBP1 in Oncogenic Processes and Its Potential as a Therapeutic Target.

Transcriptional corepressor proteins have emerged as an important facet of cancer etiology. These corepressor proteins are often altered by loss- or gain-of-function mutations, leading to transcriptional imbalance. Thus, research directed at expanding our current understanding of transcriptional corepressors could impact the future development of new cancer diagnostics, prognostics, and therapies. In this review, our current understanding of the CtBP corepressors, and their role in both development and disease, is discussed in detail. Importantly, the role of CtBP1 overexpression in adult tissues in promoting the progression of multiple cancer types through their ability to modulate the transcription of developmental genes ectopically is explored. CtBP1 overexpression is known to be protumorigenic and affects the regulation of gene networks associated with "cancer hallmarks" and malignant behavior, including increased cell survival, proliferation, migration, invasion, and the epithelial-mesenchymal transition. As a transcriptional regulator of broad developmental processes capable of promoting malignant growth in adult tissues, therapeutically targeting the CtBP1 corepressor has the potential to be an effective method for the treatment of diverse tumor types. Although efforts to develop CtBP1 inhibitors are still in the early stages, the current progress and the future perspectives of therapeutically targeting this transcriptional corepressor are also discussed. Mol Cancer Ther; 16(6); 981-90. ©2017 AACR.

Evaluation of the prognostic value of the OncoMasTR RNA panel in diverse tumor types.

e23201 Background: Master transcriptional regulators (MTRs) are genes that are highly connected with many other genes and which can contribute to a specific cellular phenotype. The OncoMasTR panel, a set of 10 proliferation-linked MTRs and the key regulator of senescence CDKN2A, was identified in lymph node-negative breast cancer patients using publicly available data (Lanigan et al FEBS 2015 10.1111/febs.13354). Using two prognostic gene signatures and an experimentally defined ‘proliferative’ signature, highly concordant results for the top ranking sets of MTRs were found. One advantage of this process was the distillation of gene signatures into a discrete, mechanistically anchored biomarker set shown to be prognostically relevant, particularly in ER-positive patients, and with an ability to discriminate patients into low versus high risk of recurrence. Our aim here was to determine if the OncoMasTR panel was similarly useful in multiple other cancer types using publicly available transcriptomic data. Methods: The Gene Expression Omnibus was queried for datasets with relevant overall-, disease free- and/or recurrence free-survival information alongside microarray gene expression data. Survival analysis was conducted on 34 datasets using the R statistical environment package ‘survival’. Patient samples were scored based on median or tertile expression which were summed to give a final OncoMasTR RNA score, used to divide the patient samples into two groups (low and high risk) for analysis. Results: Using all 2,047 possible combinations of genes, a total of 23 datasets had significant prognostic associations. Following appropriate correction via multiple testing, this was reduced to 4 datasets representing bladder, lung and colorectal cancers, and multiple myeloma. Hazard ratios for the top 100 combinations in each data set were 1.8 – 2.1, 1.6 – 1.9, 1.1 – 1.3, 1.3 – 1.5 (standard error ±0.2, 0.1, 0.2, 0.1) respectively. All genes were found in the top ten combinations in all 4 datasets, and no specific combination was found to be prevalent across datasets, indicating that every MTR in the OncoMasTR panel contributes to its utility. Conclusions: The OncoMasTR panel is potentially prognostically useful in multiple cancer types.