Tumor-specific Therapeutic Targets Research Articles

Mapping pediatric brain tumors to their origins in the developing cerebellum.

Distinguishing the cellular origins of childhood brain tumors is key for understanding tumor initiation and identifying lineage-restricted, tumor-specific therapeutic targets. Previous strategies to map the cell-of-origin typically involved comparing human tumors to murine embryonal tissues, which is potentially limited due to species-specific differences. The aim of this study was to unravel the cellular origins of the 3 most common pediatric brain tumors, ependymoma, pilocytic astrocytoma, and medulloblastoma, using a developing human cerebellar atlas. We used a single-nucleus atlas of the normal developing human cerebellum consisting of 176 645 cells as a reference for an in-depth comparison to 4416 bulk and single-cell transcriptome tumor datasets, using gene set variation analysis, correlation, and single-cell matching techniques. We find that the astroglial cerebellar lineage is potentially the origin for posterior fossa ependymomas. We propose that infratentorial pilocytic astrocytomas originate from the oligodendrocyte lineage and MHC II genes are specifically enriched in these tumors. We confirm that SHH and Group 3/4 medulloblastomas originate from the granule cell and unipolar brush cell lineages. Radiation-induced gliomas stem from cerebellar glial lineages and demonstrate distinct origins from the primary medulloblastoma. We identify tumor genes that are expressed in the cerebellar lineage of origin, and genes that are tumor specific; both gene sets represent promising therapeutic targets for future study. Based on our results, individual cells within a tumor may resemble different cell types along a restricted developmental lineage. Therefore, we suggest that tumors can arise from multiple cellular states along the cerebellar "lineage of origin."

Genetically engineered probiotics as catalytic glucose depriver for tumor starvation therapy.

Cancer cells predominantly adapt the frequent but less efficient glycolytic process to produce ATPs rather than the highly efficient oxidative phosphorylation pathway. Such a regulated metabolic pattern in cancer cells offers promising therapeutic opportunities to kill tumors by glucose depletion or glycolysis blockade. In addition, to guarantee tumor-specific therapeutic targets, effective tumor-homing, accumulation, and retention strategies toward tumor regions should be elaborately designed. In the present work, genetically engineered tumor-targeting microbes (transgenic microorganism EcM-GDH (Escherichia coli MG1655) expressing exogenous glucose dehydrogenase (GDH) have been constructed to competitively deprive tumors of glucose nutrition for metabolic intervention and starvation therapy. Our results show that the engineered EcM-GDH can effectively deplete glucose and trigger pro-death autophagy and p53-initiated apoptosis in colorectal tumor cells/tissues both in vitro and in vivo. The present design illuminates the promising prospects for genetically engineered microbes in metabolic intervention therapeutics against malignant tumors based on catalytically nutrient deprivation, establishing an attractive probiotic therapeutic strategy with high effectiveness and biocompatibility.

Alternative splicing downstream of EMT enhances phenotypic plasticity and malignant behavior in colon cancer.

Phenotypic plasticity allows carcinoma cells to transiently acquire the quasi-mesenchymal features necessary to detach from the primary mass and proceed along the invasion-metastasis cascade. A broad spectrum of epigenetic mechanisms is likely to cause the epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) transitions necessary to allow local dissemination and distant metastasis. Here, we report on the role played by alternative splicing (AS) in eliciting phenotypic plasticity in epithelial malignancies with focus on colon cancer. By taking advantage of the coexistence of subpopulations of fully epithelial (EpCAMhi) and quasi-mesenchymal and highly metastatic (EpCAMlo) cells in conventional human cancer cell lines, we here show that the differential expression of ESRP1 and other RNA-binding proteins (RBPs) downstream of the EMT master regulator ZEB1 alters the AS pattern of a broad spectrum of targets including CD44 and NUMB, thus resulting in the generation of specific isoforms functionally associated with increased invasion and metastasis. Additional functional and clinical validation studies indicate that both the newly identified RBPs and the CD44s and NUMB2/4 splicing isoforms promote local invasion and distant metastasis and are associated with poor survival in colon cancer. The systematic elucidation of the spectrum of EMT-related RBPs and AS targets in epithelial cancers, apart from the insights in the mechanisms underlying phenotypic plasticity, will lead to the identification of novel and tumor-specific therapeutic targets.

Mapping the cell-membrane proteome of the SKBR3/HER2+ cell line to the cancer hallmarks.

The hallmarks of biological processes that underlie the development of cancer have been long recognized, yet, existing therapeutic treatments cannot prevent cancer from continuing to be one of the leading causes of death worldwide. This work was aimed at exploring the extent to which the cell-membrane proteins are implicated in triggering cancer hallmark processes, and assessing the ability to pinpoint tumor-specific therapeutic targets through a combined membrane proteome/cancer hallmark perspective. By using GO annotations, a database of human proteins associated broadly with ten cancer hallmarks was created. Cell-membrane cellular subfractions of SKBR3/HER2+ breast cancer cells, used as a model system, were analyzed by high resolution mass spectrometry, and high-quality proteins (FDR<3%) identified by at least two unique peptides were mapped to the cancer hallmark database. Over 1,400 experimentally detected cell-membrane or cell-membrane associated proteins, representing ~18% of the human cell-membrane proteome, could be matched to the hallmark database. Representative membrane constituents such as receptors, CDs, adhesion and transport proteins were distributed over the entire genome and present in every hallmark category. Sustained proliferative signaling/cell cycle, adhesion/tissue invasion, and evasion of immune destruction emerged as prevalent hallmarks represented by the membrane proteins. Construction of protein-protein interaction networks uncovered a high level of connectivity between the hallmark members, with some receptor (EGFR, ERBB2, FGFR, MTOR, CSF1R), antigen (CD44), and adhesion (MUC1) proteins being implicated in most hallmark categories. An illustrative subset of 138 hallmark proteins that included 42 oncogenes, 24 tumor suppressors, 9 oncogene/tumor suppressor, and 45 approved drug targets was subjected to a more in-depth analysis. The existing drug targets were implicated mainly in signaling processes. Network centrality analysis revealed that nodes with high degree, rather than betweenness, represent a good resource for informing the selection of putative novel drug targets. Through heavy involvement in supporting cancer hallmark processes, we show that the functionally diverse and networked landscape of cancer cell-membrane proteins fosters unique opportunities for guiding the development of novel therapeutic interventions, including multi-agent, immuno-oncology and precision medicine applications.

Dissecting the Oncogenic Roles of Keratin 17 in the Hallmarks of Cancer

There is an unmet need to identify and validate tumor-specific therapeutic targets to enable more effective treatments for cancer. Heterogeneity in patient clinical characteristics as well as biological and genetic features of tumors present major challenges for the optimization of therapeutic interventions, including the development of novel and more effective precision medicine. The expression of keratin 17 (K17) is a hallmark of the most aggressive forms of cancer across a wide range of anatomical sites and histological types. K17 correlates with shorter patient survival, predicts resistance to specific chemotherapeutic agents, and harbors functional domains that suggest it could be therapeutically targeted. Here, we explore the role of K17 in the hallmarks of cancer and summarize evidence to date for K17-mediated mechanisms involved in each hallmark, elucidating functional roles that warrant further investigation to guide the development of novel therapeutic strategies.

ROR1 targeting with the antibody-drug conjugate VLS-101 is effective in Richter syndrome patient–derived xenograft mouse models

AbstractRichter syndrome (RS) represents the transformation of chronic lymphocytic leukemia (CLL), typically to an aggressive lymphoma. Treatment options for RS are limited and the disease is often fatal. Receptor tyrosine kinase–like orphan receptor 1 (ROR1) is expressed on CLL cells and other cancers but not on healthy adult tissues, making it an attractive, tumor-specific therapeutic target. VLS-101 is being developed as an antibody-drug conjugate (ADC) for therapy of ROR1-expressing (ROR1+) cancers. VLS-101 comprises UC-961 (a humanized immunoglobulin G1 monoclonal antibody that binds an extracellular epitope of human ROR1), a maleimidocaproyl-valine-citrulline-para-aminobenzoate linker, and the antimicrotubule cytotoxin monomethyl auristatin E (MMAE). VLS-101 binding to ROR1 results in rapid cellular internalization and delivery of MMAE to induce tumor cell death. We studied 4 RS patient-derived xenografts (RS-PDXs) with varying levels of ROR1 expression (11%, 32%, 85%, and 99% of cells). VLS-101 showed no efficacy in the lowest-expressing RS-PDX but induced complete remissions in those with higher levels of ROR1 expression. Responses were maintained during the posttherapy period, particularly after higher VLS-101 doses. In systemic ROR1+ RS-PDXs, VLS-101 dramatically decreased tumor burden in all RS-colonized tissues and significantly prolonged survival. Animals showed no adverse effects or weight loss. Our results confirm ROR1 as a target in RS and demonstrate the therapeutic potential of using an ADC directed toward ROR1 for the treatment of hematological cancers. A phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with RS and other hematological malignancies.

Targeting ROR1 Using the Antibody Drug Conjugate Vls-101 in Aggressive Mantle Cell Lymphoma

ROR1 is a transmembrane receptor with tightly controlled expression during embryonic development. While it is expressed on multiple tumor types it is not expressed in normal adult tissues. ROR1-positive B cell non-Hodgkin's lymphomas (B-NHL) include mantle cell lymphoma (MCL), and diffuse large B cell lymphoma. Given its unique expression pattern, ROR1 represents a tumor-specific therapeutic target with little or no normal tissue toxicity. VLS-101, utilizes the UC-961 anti-ROR1 antibody which is conjugated to monomethyl auristatin E (MMAE) via a cleavable linker. VLS-101/ROR1 complex induce internalization facilitates the release of a cytotoxic agent resulting in killing of ROR1-expressing cancer cells. With the rise of novel targeted therapies, resistance has been a major challenge among MCL patients. MCL is one of the most therapeutically resistant and aggressive forms of NHL-B, making it a challenging cancer to treat. The advancement of ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor, has been a beneficial change; however, ibrutinib-resistant MCL remains an urgent unmet clinical need. Furthermore, MCLs are either intrinsically resistant to ibrutinib, as well as to venetoclax (Bcl-2 inhibitor) and CAR T-cell therapy, or they may acquire resistance after a short response to these therapies. While many studies have looked into overcoming each of these different types of resistance, no studies have been conducted to overcome a multiple-resistance phenotype simultaneously. Therefore, identifying effective therapies that target these resistance mechanisms is crucial. In this study, we assessed the efficacy of VLS-101 in preclinical models of MCL with intrinsic or aquired resistance to ibrutinib, venetoclax, and CD19 CAR T therapies. By using MCL cell lines, primary patient samples and our established MCL patient-derived xenografts, we explored the expression of ROR1 in MCL and investigated the effects of in vitro and in vivo effects ROR1 targeting using VLS-101. To assess total and cell surface ROR1 expression we used flow cytometry (FACS), western blot analysis, and reverse-transcriptase-polymerase chain reaction (RT-PCR). Cell viability and apoptosis assays were performed to evaluate the in vitro efficacy of VLS-101 in MCL cell lines and primary patient MCL samples. To assess the apoptosis and cell cycle arrest in cells treated with VLS-101, we used annexin V/propidium iodide-staining followed by flow cytometry analyses. In vivo efficacy was tested using various PDX models with various multiple resistances. All 5 cell lines and all 9 PDX models of MCL tested showed high ROR1 mRNA and protein expression levels, whereas 3 out of 4 primary human samples expressed cell surface ROR1. VLS-101 treatment showed anti-MCL activity at the concentrations of 0.3-9 μg/ml in most of cell models tested, which correlated with a significant G2/M cell cycle arrest. Furthermore, VLS-101 induced a time- and dose-dependent apoptosis, as shown by increases in annexin V/propidium iodide-staining. VLS-101 treatment of ibrutinib-venetoclax dual-resistant ROR1+ PDX model resulted in significant regressions of the tumor bearing spleens and livers when compared to vehicle controls (p=0.0001 and p=0.002 respectively). In the ibrutinib-CD19 CAR T dual-resistant ROR1+ model, vehicle-treated animals showed s.c. tumor masses with a mean volume of 1440 mm3, whereas tumor masses were barely palpable (mean = 160 mm3) or had completely regressed in animals treated with VLS-101 at 1.0 mg/kg or 2.0 mg/kg, respectively. VLS-101 showed no adverse effects as monitored by animal clinical observations and weekly body weight measurements. Collectively, these results indicate that ROR1 expression on MCL cells can be utilized for selective targeting. These preclinical data document that the MMAE-containing ADC, VLS-101, can cause cell cycle arrest and induce apoptosis in vitro and safely induce tumor regressions in highly resistant in vivo PDX models of MCL derived from patient tumors. Importantly, our study revealed that even the heavyly pretreated tumors in the clinical setting may express targetable levels of ROR1 and that targeting ROR1 using ADC is a promising approach for the treatment of MCL. Building on these types of results, a Phase 1 clinical trial on VLS-101 (NCT03833180) is ongoing in patients with lymphoid cancers. Disclosures Jessen: VelosBio: Current Employment, Current equity holder in private company; eFFECTOR: Current equity holder in private company. Lannutti:VelosBio: Current Employment, Current equity holder in private company; Gilead Sciences: Current equity holder in publicly-traded company. Wang:Dava Oncology: Honoraria; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; Lu Daopei Medical Group: Honoraria; Molecular Templates: Research Funding; Oncternal: Consultancy, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; InnoCare: Consultancy; Pulse Biosciences: Consultancy; Juno: Consultancy, Research Funding; Beijing Medical Award Foundation: Honoraria; Nobel Insights: Consultancy; Verastem: Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OncLive: Honoraria; Guidepoint Global: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria.

Single-Cell Analyses Reveal Megakaryocyte-Biased Hematopoiesis in Myelofibrosis and Identify Mutant Clone-Specific Targets

SummaryMyelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage− hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.

Vls-101 Is a Novel Therapeutic Antibody-Drug Conjugate (ADC) Targeting Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1) in Richter's Syndrome (RS)

ROR1 is a transmembrane receptor with tightly controlled expression during development. It is present on multiple tumor types but not on normal adult tissues. Hematological malignancies are often ROR1-positive, including chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and diffuse large B cell lymphoma (DLBCL). Given its unique pattern of expression, ROR1 represents a tumor-specific therapeutic target. The anti-ROR1 antibody, UC-961, is ahumanized IgG1 monoclonal antibody (mAb) that binds with high affinity to a specific extracellular epitope of human ROR1 receptor and can block Wnt5a-induced ROR1 signaling. Nonclinical studies document that UC-961 does not react with normal adult human tissues and selectively binds to tumor cells. Because of the antibody high specificity, rapid internalization, and trafficking to lysosomes, UC-961 appears ideally suited to serve as the targeting moiety for an anti-ROR1 ADC. Accordingly, we have developed VLS-101, a UC-961-linker-monomethyl auristatin E (MMAE) ADC that preserves the high-affinity binding and specificity of UC-961 and allows for ROR1-targeted intracellular release of MMAE. RS is an aggressive lymphoma, typically of DLBCL type, arising as transformation of CLL. Despite, progressive improvements in the therapy of CLL, very few effective treatment options exist for patients with RS. Using our recently established RS patient-derived xenografts (RS-PDXs), we explored the expression and signaling properties of ROR1 in RS and investigated the ex-vivo and in vivo effects of VLS-101. When assessed by flow cytometry (FACS), immunohistochemistry (IHC), and reverse-transcriptase-polymerase chain reaction (RT-PCR), 3 of 4 RS-PDXs showed ROR1 positivity (2 highly positive: 99% and 80% of cells; 1 medium/low positive: 25% of cells by FACS). The extent of ROR1 expression correlated among the 3 assays methods and was consistent with ROR1 expression data reported for non-RS DLBCL samples. When engaged by its ligand Wnt-5a, ROR1 activated downstream targets, Rac1 and RhoA, and induced phosphorylation of the p65 subunit of NF-kB and Jnk in RS cells. When cells purified from RS-PDX tumor masses were exposed to VLS-101 ex-vivo, the drug induced time- and dose-dependent apoptosis, as shown by increases in annexin V/propidium iodide and by Caspase-3 and PARP cleavage. VLS-101 efficacy was then assessed in vivo in both subcutaneous and systemic RS-PDX models. When palpable masses had formed in subcutaneous models, mice were randomly assigned to vehicle or VLS-101, bi-weekly treated for 3 consecutive weeks, and then compared for tumor growth and survival. In the 3 ROR1-expressing RS-PDX models, VLS-101 caused highly significant disease regressions. Complete regressions were observed even in RS-PDXs without universal ROR1 expression, suggesting a MMAE bystander effect. After treatment discontinuation, VLS-101-treated animals showed no tumor regrowth for several months and had significantly extended survival. Data were confirmed in systemic ROR1-expressing RS models in which tumor cells were intravenously injected and allowed to engraft for ~14 days before starting treatment. VLS-101 was administered with the same schedule adopted for the subcutaneous model. VLS-101 eliminated RS cells in bone marrow, peripheral blood, and spleen, increasing survival in VLS-101-treated animals relative to controls (approximately 20-50 days, depending on the RS-PDX model). Of note, no in vivo VLS-101 efficacy was observed in the ROR1-negative RS-PDX, confirming the specificity of VLS-101 targeting. VLS-101 showed no adverse effects on animal well-being or body weight. Collectively, these results indicate that ROR1 is expressed on RS cells where it can transduce pro-survival signals and can be diagnostically evaluated for selective targeting. Nonclinical data document that the MMAE-containing ADC, VLS-101, can cause RS-PDX apoptosis in vitro and can safely and selectively induce complete tumor regressions in in vivo models of RS derived from patient tumors with heavy prior clinical treatment and variable levels of ROR1 expression. Building on these types of results, a Phase 1 clinical trial of VLS-101 (NCT03833180) is ongoing in patients with lymphoid cancers. Disclosures Vaisitti: VelosBio Inc.: Research Funding; Verastem Inc: Research Funding. Jessen:VelosBio Inc.: Employment. Vo:VelosBio Inc: Employment. Ko:VelosBio Inc: Employment. Allan:Sunesis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics LLC, an AbbVie company: Consultancy; Acerta Pharma: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Verastem Oncology, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Bayer: Consultancy. Furman:Acerta Pharma: Consultancy; Pharmacyclics: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy; Genentech: Consultancy; Incyte: Consultancy; Oncotracker: Consultancy; Verastem: Consultancy; Abbvie: Consultancy; Sunesis: Consultancy; TG Therapeutics: Consultancy; Janssen: Consultancy. Miller:VelosBio Inc.: Employment. Lanutti:VelosBio Inc.: Employment. Deaglio:iTeos Therapeutics: Research Funding; Verastem Inc: Research Funding; VelosBio Inc.: Research Funding. OffLabel Disclosure: The drug used in this project is an anti-ROR1-toxin-conjugated antibody

Patient-derived cell line models revealed therapeutic targets and molecular mechanisms underlying disease progression of high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC) is the most frequent type of ovarian cancer. Most patients have primary response to platinum-based chemotherapy but frequently relapse, which leads to patient death. A lack of well documented and characterized patient-derived HGSOC cell lines is so far a major barrier to define tumor specific therapeutic targets and to study the molecular mechanisms underlying disease progression. We established 34 patient-derived HGSOC cell lines and characterized them at cellular and molecular level. Particularly, we demonstrated that a cancer-testis antigen PRAME and Estrogen Receptor could serve as therapeutic targets. Notably, data from the cell lines did not demonstrate acquired resistance due to tumor recurrence that matched with clinical observations. Finally, we presented that all HGSOC had no or very low CDKN1A (p21) expression due to loss of wild-type TP53, suggesting that loss of cell cycle control is the determinant for tumorigenesis and progression. In conclusion, patient-derived cell lines reveal that PRAME is a potential tumor specific therapeutic target in HGSOC and counteracting the down-regulation of p21 caused by loss of wild-type TP53 might be the key to impede disease progression.

218 - Tyrosine nitration regulates signaling pathways involved in Neurofibromatosis type 2-associated Schwannoma growth

Neurofibromatosis type 2 (NF2) is a genetic disorder of the nervous system caused by inactivation of the merlin tumor suppressor gene. NF2 patients develop bilateral vestibular schwannomas (VS) and other nervous system tumors throughout their life for which there is no effective drug treatment. Production of the oxidant peroxynitrite occurs in pathological conditions and leads to tyrosine (Tyr) nitration of proteins. Although Tyr nitration is found in multiple tumor types, its role in tumorigenesis is unknown. We discovered that VS and human merlin-deficient (MD) Schwann cells show significantly increased levels of peroxynitrite and Tyr nitration compared to normal Schwann cells. Notably, prevention of tyrosine nitration selectively decreases MD-Schwann cell survival. Our goal is to identify nitrated proteins that support VS growth as new potential tumor-specific therapeutic targets, enabling the development of safe pharmacological strategies for NF2 schwannomas. This is the first focused effort to determine the signaling pathways regulated by tyrosine nitration in pathological conditions. By performing immunoprecipitations using a polyclonal anti-nitrotyrosine antibody followed by mass spectrometry analysis and western blot, we identified a limited number of proteins endogenously nitrated in VS from NF2 patients, and in mouse and human MD-Schwann cells. A group of the identified proteins play a role in pathways that are dysregulated in NF2. Further, using complementary phospho-kinase arrays, we found that prevention of tyrosine nitration using urate and edaravone has an impact on several of these key signaling pathways. The identification of the specific nitrated proteins that promote schwannoma growth could provide exceptional novel targets for the treatment of NF2 and possibly other tumors of the nervous system as well. Supported by DoD, NRP, NIA W81XWH-17-1-0409 (to MCF).

USP7 Is a Tumor-Specific WNT Activator for APC-Mutated Colorectal Cancer by Mediating β-Catenin Deubiquitination

SummaryThe tumor suppressor gene adenomatous polyposis coli (APC) is mutated in most colorectal cancers (CRCs), resulting in constitutive Wnt activation. To understand the Wnt-activating mechanism of the APC mutation, we applied CRISPR/Cas9 technology to engineer various APC-truncated isogenic lines. We find that the β-catenin inhibitory domain (CID) in APC represents the threshold for pathological levels of Wnt activation and tumor transformation. Mechanistically, CID-deleted APC truncation promotes β-catenin deubiquitination through reverse binding of β-TrCP and USP7 to the destruction complex. USP7 depletion in APC-mutated CRC inhibits Wnt activation by restoring β-catenin ubiquitination, drives differentiation, and suppresses xenograft tumor growth. Finally, the Wnt-activating role of USP7 is specific to APC mutations; thus, it can be used as a tumor-specific therapeutic target for most CRCs.

Abstract 4771: The role of Hes1 in the normal and tumor stem cells of the intestine

Abstract Notch-Hes pathway plays a crucial role in the differentiation, proliferation, and tumorigenesis of the intestine. We previously reported that prenatal deletion of Hes1 reduces intestinal tumor proliferation; however, postnatal deletion of Hes1, which recapitulates Hes1-targeted tumor therapy, has not been thoroughly investigated. Here, we elucidated the novel role of Hes1 in normal stem cells (NSCs) and tumor stem cells (TSCs) of the adult intestine. First, we deleted Hes1 in Lgr5-expressing or Bmi1-expressing NSCs and performed lineage tracing using Lgr5CreERT2/+; Hes1flox/flox; Rosa26LacZ/+ and Bmi1CreER/+; Hes1flox/flox; Rosa26LacZ/+ mice. After Hes1 deletion, Lgr5-expressing or Bmi1-expressing NSCs gave rise to progeny cells, but could not self-renew. However, Hes1 deletion in NSCs did not disrupt intestinal homeostasis, because Hes1-positive NSCs were newly generated soon after Hes1-deleted NSCs were depleted. Next, we stabilized beta-catenin for intestinal tumor formation and deleted Hes1 in Lgr5-expressing cells using Lgr5CreERT2/+; Ctnnb1lox(ex3)/+; Hes1flox/flox mice. Tumor formation was dramatically suppressed, and the animal survival was significantly improved, suggesting that Hes1 is required for the initiation of intestinal tumor formation. Finally, we deleted Hes1 in Lgr5-expressing TSCs of established intestinal tumors using Lgr5CreERT2/+; Hes1flox/flox; ApcMin/+ mice. Of note, Hes1 deletion in TSCs of established intestinal tumors induced immediate apoptosis of TSCs and significantly reduced the number of tumors. qRT-PCR analysis of sorted Lgr5-expressing cells one day after Hes1 deletion revealed upregulation of pro-apoptotic genes and downregulation of anti-apoptotic genes in Hes1-deleted TSCs, but not in Hes1-deleted NSCs. We also deleted Hes1 in Dclk1-expressing TSCs of established intestinal tumors, and confirmed that, irrespective of stem cell markers analyzed, Hes1 deletion in TSCs leads to tumor regression by inducing immediate apoptosis in TSCs. Taken together, Hes1 is required in different ways for the maintenance of NSCs and TSCs: Hes1 deletion in NSCs does not perturb homeostasis, whereas Hes1 deletion in TSCs leads to tumor regression. Furthermore, Hes1 is necessary for the initiation of intestinal tumor formation. Therefore, we propose Hes1 is a novel tumor-specific therapeutic target. Citation Format: Norihiro Goto, Akihisa Fukuda, Tsutomu Chiba, Hiroshi Seno. The role of Hes1 in the normal and tumor stem cells of the intestine [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 4771. doi:10.1158/1538-7445.AM2017-4771

Research progress of fatty acid synthase in cancer

Fatty acid synthase (FAS) can be regarded as a tumor-specific therapeutic target. High expression of FAS is found in many tumor tissues, and inhibition of its expression can induce tumor cell death. Recent researches has shown that FAS can be used not only as the target of tumor therapy, but also as a tumor marker. The serum FAS is higher in cancer patients. High expression of FAS is associated with tumor treatment tolerance, and some new inhibitors for FAS show good anti-cancer effects. Key words: Cancer; Fatty acid synthase; Resistance mechanism

Abstract POSTER-THER-1432: Intraperitoneal delivery of the disintegrin vicrostatin (VCN) effectively limits ovarian cancer growth and progression

Abstract Although prevention and early detection are the ultimate goals for ovarian cancer, the fact cannot be ignored that in 2014 21,980 women will be diagnosed with ovarian cancer in the U.S. 75% of these women will be confronted with a high stage tumor and a five-year survival rate of 45.5% (American Cancer Society, Atlanta). For these patients, improved treatment for residual disease following surgery, reduced complications during therapy as well as a decreased risk of cancer recurrence, would reduce mortality and improve their quality of life significantly. After surgery and first-line chemotherapy, 50% to 75% of responders will relapse within approximately 18 months and require further systemic therapy. Approximately 50% of women die due to carcinomatosis. Exfoliated ovarian tumor cells are carried via peritoneal fluid to secondary sites in the abdominal cavity, where they attach, invade the submesothelial connective tissue and proliferate to create peritoneal micrometastasis. These cancer cells in effusions are not amenable to surgical removal, failure of their eradication is one of the main causes for unsuccessful treatment and recurrence. The strategy of localized intraperitoneal (IP) chemotherapy targets these cells in particular and is therefore highly effective in preventing recurrence. According to the Cochrane database, IP chemotherapy successfully increased overall survival and progression free-survival, resulting in a 21.6% decrease in the risk of death. Therefore, the U.S. National Cancer Institute recommended consideration of IP chemotherapy for patients with advanced stage III ovarian cancer after optimal surgical debulking. Integrins are heterodimeric receptors that evolved to mediate complex cell-ECM interactions. These interactions regulate the ability of cells to mechanically sense their environment by integrating multiple signaling pathways initiated by extracellular cues with the cell’s cytoskeleton. Integrins (including αvβ3, αvβ5, α5β1 and αvβ6) have important roles in OC cell attachment, survival, migration, invasion and angiogenesis. However, the precise roles played by different integrin subunits in various aspects of tumor progression and why some integrins appear to be especially supportive of tumor progression are still not fully understood. Due to their pivotal roles in OC biology, integrins represent an attractive target for a novel form of OC therapy. Disintegrins are among the most potent soluble ligands of integrins representing a class of cysteine-rich polypeptides originally isolated from snake venom, many of which contain a cyclic-Arg-Gly-Asp (c-RGD) motif. These polypeptides hold a significant translational potential as anti-cancer therapeutic agents based on their high affinity interaction with integrins as well as their excellent pharmacological properties. However, these polypeptides are extremely difficult to produce recombinantly. Previously the Markland group reported that a sequence-engineered RGD-disintegrin (called vicrostatin or VCN) can be reliably produced in large quantity. Through multiple integrin ligation (i.e., αvβ3, αvβ5 and α5β1), VCN targets both endothelial and OC cells. The use of small RGD peptides or multimeric cyclic RGD peptides for targeting of integrins for both therapy and imaging of tumors has been widely reported but there is a distinct advantage to the use of disintegrins such as VCN. VCN not only acts as an antagonist (in a manner identical to RGD peptides), VCN also elicits signaling responses through direct interaction with integrins. Importantly, integrin αvβ3 is expressed at very low level on epithelial cells and mature endothelial cells, but is overexpressed on the endothelial cells of the tumor neovasculature and on tumor cells. Therefore, this integrin presents an attractive and tumor specific therapeutic target for rapidly growing solid tumors. In the present studies we delivered VCN formulated in a novel carboxymethyl cellulose (CMC) polyethyleneoxide (PEO) gel (Oxiplex™) through IP delivery. In studies described here we show that IP VCN delivery via Oxiplex™, following implantation of SKOV-3 sphereoids and confirmation of tumor growth by bioluminescent imaging, significantly reduced tumor growth and dissemination. In addition we have evaluated the dose of VCN delivered and the timing of the administrations and shown that a clinically relevant dosing schedule can be achieved. Citation Format: S. Swenson, R. Minea, J. Moore, M. Mikhail, Z. Kirakosyan &amp; Francis S. Markland Jr. Intraperitoneal delivery of the disintegrin vicrostatin (VCN) effectively limits ovarian cancer growth and progression [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1432.

The evolving genomic landscape of myeloproliferative neoplasms.

Our understanding of the genetic basis of the Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) has moved forward at a staggering pace over the last decade. With the discoveries of underlying mutations in JAK2, MPL, and, most recently, calreticulin (CALR), that together account for ∼90% of patients with MPNs, these conditions are now among the best characterized of hematological malignancies. While JAK-STAT pathway activation has been shown to be central to the pathogenesis of the MPN phenotype, the mechanism by which mutant CALR alters cellular function to result in myeloid proliferation remains unclear. Other mutations in several epigenetic modifiers, such as ASXL1, DNMT3a, TET2, EZH2, IDH1, and IDH2, as well as in genes involved in mRNA splicing, such as SF3B1 and U2AF2, have also been described in recent years in patients with MPNs, and evidence is emerging as to how these may be contributing to disease biology. From a therapeutic perspective, the discovery of aberrations in JAK2 has rapidly translated into the successful clinical use of JAK inhibitors in MPNs. Mutant calreticulin has the potential to be a tumor-specific therapeutic target because the mutations generate a novel protein C-terminus. In this chapter, we detail the genomic alterations that underlie MPNs, with a focus on the recent discovery of mutations in CALR, and explore the clinical and biological relevance of the altered genomic landscape in MPNs.

GE-33 * A COMPREHENSIVE ASSESSMENT OF VIRAL TRANSCRIPTS IN DNA- AND RNA-SEQ DATASETS FROM HIGH-GRADE GLIOMAS REVEALS NO ASSOCIATION

Glioblastoma multiforme (GBM) is a devastating disease with poor survival rates. Despite years of research, there has been little change in the survival rate or median survival time. HCMV (human cytomegalovirus) was reported in GBM over a decade ago and this finding has the potential to increase our understanding of the disease and it offers a potential tumor specific therapeutic target. Because of this promise, there is a fair amount of time, energy and money being directed towards understanding and utilizing this connection for eventual therapeutic purposes. Despite these ongoing efforts and the time lapsed since the discovery of HCMV in GBM, the association remains controversial. Here we utilized both RNA and DNA sequence data from high-grade gliomas from the Cancer Genome Atlas to assess the veracity of the association between GBM and HCMV (or other infectious agents). Raw sequence reads were aligned to a reference genome containing a human genome (hg19) and a library of virus sequences known to infect humans. Alignments were performed using Spliced Transcripts Alignments to a Reference (STAR). In 56 out of 56 GBM RNA-seq datasets no known viruses were identified. Virome analysis of the DNA-seq datasets demonstrated 40 out of 40 datasets with human herpesvirus 7 ranging from 32 to 10,845 reads. However, upon further analysis, it was determined that these herpesvirus 7 matching reads have low complexity and are consistent with mapping to human telomeric repeats. Therefore, no known viruses were identified at the DNA or RNA levels. This analysis raises the possibility that viruses may not be associated with gliomas as previously reported.

Abstract LB-111: Neuronal Pentraxin 2: a novel tumor-specific molecular target that mediates clear cell renal cell carcinoma malignancy

Abstract Introduction: Renal cell carcinoma (RCC) is the most common malignancy of the kidney, responsible for 13,000 deaths in the United States annually. Patients who present with the clear cell (ccRCC) variant of this disease demonstrate a worse prognosis as compared to other subtypes. Due to its asymptomatic nature, patients often do not demonstrate signs of illness until the disease has progressed to an advanced stage. It is estimated that 30% of patients present with metastatic disease at initial diagnosis, and 20-30% of patients with localized cases treated surgically relapse with metastatic disease likely due to the presence of undetectable micrometastases. The treatment for metastatic disease is limited due to its profound resistance against chemotherapy, radiation, and current FDA approved targeted therapies. Despite advances in treatment strategies for patients with metastatic RCC, there remains a need for identification of tumor-specific therapeutic targets. Additionally, elucidation of key signatures which contribute to the development of metastatic disease is paramount. Our group has identified secreted neuronal pentraxin 2 (NPTX2) as a novel oncogenic factor that is highly overexpressed specifically in ccRCC at all stages of disease, and facilitates tumor cell invasion through its interaction with the ionotropic glutamate receptor 4 subunit (GluR4). Methods: NPTX2 expression was evaluated by Affymetrix gene array and immunohistochemistry analysis of patient tumor tissue compared to matched normal samples. Nextbio meta-analysis was used to evaluate NPTX2 expression patterns among RCC subtypes and disease staging. Lentiviral mediated knock-down of NPTX2 and GluR4 was performed in representative ccRCC cell lines and resulting changes in proliferation, viability, morphology, and invasion were evaluated. NPTX2-GluR4 interactions were evaluated via immunoprecipitation, immunofluorescence, and intracellular calcium assays. Results: NPTX2 mRNA and protein is consistently elevated in the clear cell variant of RCC at all stages of disease, and is important for tumor cell viability. We correlate NPTX2 expression with an invasive phenotype, and demonstrate that over-expression of NPTX2 in tumor cells leads to actin cytoskeletal remodeling and increased invasion. We identify GluR4 as a mediator for NPTX2 activity which leads to increased intracellular calcium influx in tumor cells. Conclusions: NPTX2 is a tumor-specific factor that is consistently over-expressed in ccRCC. We further identify GluR4 as a downstream mediator of NPTX2 activity, whereby it modulates intracellular calcium levels, actin cytoskeletal remodeling, and tumor cell invasion. Inhibition of NPTX2 or GluR4 expression via shRNA leads to decreased tumor cell proliferation and invasion, and induces programed cell death. We propose NPTX2 as a new candidate for targeted therapy not previously described in cancer, whose inhibition may demonstrate a clinical benefit in patients suffering from metastatic ccRCC. Citation Format: Christina A. von Roemeling, Derek C. Radisky, Laura A. Marlow, Simon J. Cooper, Stefan K. Grebe, Panagiotis Z. Anastasiadis, Han W. Tun, John A. Copland. Neuronal Pentraxin 2: a novel tumor-specific molecular target that mediates clear cell renal cell carcinoma malignancy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-111. doi:10.1158/1538-7445.AM2014-LB-111

Abstract 5340: Bioinformatic analyses approaches for personalized oncogenomics

Abstract The Personalized Oncogenomics initiative at the British Columbia Cancer Agency aims to identify tumor-specific therapeutic targets in cancer patients with late stage disease who have failed standard therapy. Comprehensive profiling of individual patients' tumor(s) at the DNA and RNA level allows for characterization of altered pathways and hence identification of therapeutics designed to specifically target them. Data for each individual study included whole genome and transcriptome sequence of the fresh biopsy and whole genome sequence of patient's blood. When tissues were available, transcriptome of a matched normal sample and genome of the commonly formalin-fixed paraffin-embedded primary tumor were also sequenced. All sequencing experiments were performed on Illumina machines. Genomic data was examined, depending on the case, for germline and/or somatic mutations. These included single nucleotide variants, small insertions and deletions and copy number variations. All sequence data were assembled de novo in order to identify rearrangements causing gene fusions; transcriptome data also revealed allelic expression of variants and provided a profile for the entire transcribed genome. Differential abundance estimation was run against a rich repository of publicly available data from The Cancer Genome Atlas project and transcriptome datasets available in-house. The variants and pathways were then mapped to drug databases as well as clinical trial records. This was followed by an extensive literature search for evidence of drug combinations, drug-drug interactions and efficacy of a drug for a particular cancer type, especially those not recognized as the approved disease group for the drug under consideration. The project has sequenced 50 patients; the average length of time between acquiring the biopsy and delivering a report to clinical oncologists was 37 days. Bioinformatic analysis of the sequence data led to identification of informative or actionable targets in up to 80% of cases. The findings were not restricted to target identification but also led to change of diagnosis, treatment and characterization of tumor evolution. De novo assembly of the data in a non-small cell lung cancer patient led to the identification of the well-characterized EML4-ALK oncogenic fusion which had been missed through the use of clinically approved fluorescence in situ hybridization test. Analysis of two separate malignant masses in another patient revealed two divergent and unique tumors. Two different therapeutic were prescribed in order to target these; this led to the disappearance of both tumors and disease stabilization for 7 months. Through the design of an efficient and automated bioinformatics pipeline, individual patient's tumor specimen(s) were profiled in a clinically relevant time frame. This in turn enabled the delivery of targeted therapies and disease stabilization in patients who had no remaining standard therapeutic options. Citation Format: Katayoon Kasaian, Yaoqing Shen, Sreeja Leelakumari, Peter Eirew, Yvonne Y. Li, Erin Pleasance, Richard Corbett, Karen L. Mungall, Jacquie Schein, Andrew J. Mungall, Yongjun Zhao, Richard A. Moore, Stephen Yip, Karen Gelmon, Howard Lim, Daniel Renouf, Robyn Roscoe, Yussanne Ma, Marco A. Marra, Janessa Laskin, Steven JM Jones. Bioinformatic analyses approaches for personalized oncogenomics. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5340. doi:10.1158/1538-7445.AM2014-5340

Assessment and prognostic significance of the epidermal growth factor receptor vIII mutation in glioblastoma patients treated with concurrent and adjuvant temozolomide radiochemotherapy

The epidermal growth factor receptor vIII mutant (EGFRvIII) is found in ~50% of all EGFR-amplified glioblastomas and constitutes a tumor-specific therapeutic target. To assess molecular testing approaches and the prognostic role of EGFRvIII in patients treated according to current standards of care, we compared different EGFRvIII detection methods and correlated EGFRvIII status with outcome in a prospective patient cohort of the German Glioma Network. In total, 184 newly diagnosed glioblastoma patients were investigated for EGFR amplification and for expression of EGFR and EGFRvIII by immunohistochemistry. Further, the EGFRvIII status was additionally studied by multiplex ligation-dependent probe amplification (MLPA) analysis and reverse transcription-PCR (RT-PCR). Immunohistochemistry demonstrated EGFRvIII in 34 of 184 patients (18%). RT-PCR or MLPA analysis detected four additional EGFRvIII-positive patients. Overall, RT-PCR and immunohistochemistry were more sensitive for EGFRvIII detection than MLPA. EGFRvIII status was not associated with progression-free and overall survival. EGFRvIII also had no prognostic significance in the subgroup of patients who were free from progression after concomitant radiochemotherapy and thus would be eligible for the ongoing ACT IV EGFRvIII vaccination trial. Age, extent of resection and O⁶-methylguanine DNA methyltransferase (MGMT) promoter methylation status appeared to be less prognostic in EGFRvIII-positive patients. Thus, EGFRvIII positivity is not a major negative prognostic factor in glioblastoma patients treated according to current standards of care. Data from phase II EGFRvIII-targeted vaccination trials compare favorably with the present contemporary results, supporting the further exploration of EGVRvIII vaccination in newly diagnosed glioblastoma.