A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma.

Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. Highlights: Plasma membrane enriched proteomics defines surfaceome of neuroblastomaMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancersDLK1 expression is driven by a super-enhancer DLK1 silencing in neuroblastoma cells results in cellular differentiation ADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models.

Evaluation of an EZH2 inhibitor in patient-derived orthotopic xenograft models of pediatric brain tumors alone and in combination with chemo- and radiation therapies.

Brain tumors are the leading cause of cancer-related death in children. Tazemetostat is an FDA-approved enhancer of zeste homolog (EZH2) inhibitor. To determine its role in difficult-to-treat pediatric brain tumors, we examined EZH2 levels in a panel of 22 PDOX models and confirmed EZH2 mRNA over-expression in 9 GBM (34.6 ± 12.7-fold) and 11 medulloblastoma models (6.2 ± 1.7 in group 3, 6.0 ± 2.4 in group 4) accompanied by elevated H3K27me3 expression. Therapeutic efficacy was evaluated in 4 models (1 GBM, 2 medulloblastomas and 1 ATRT) via systematically administered tazemetostat (250 and 400 mg/kg, gavaged, twice daily) alone and in combination with cisplatin (5 mg/kg, i.p., twice) and/or radiation (2 Gy/day × 5 days). Compared with the untreated controls, tazemetostat significantly (Pcorrected < 0.05) prolonged survival times in IC-L1115ATRT (101% at 400 mg/kg) and IC-2305GBM (32% at 250 mg/kg, 45% at 400 mg/kg) in a dose-dependent manner. The addition of tazemetostat with radiation was evaluated in 3 models, with only one [IC-1078MB (group 4)] showing a substantial, though not statistically significant, prolongation in survival compared to radiation treatment alone. Combining tazemetostat (250 mg/kg) with cisplatin was not superior to cisplatin alone in any model. Analysis of in vivo drug resistance detected predominance of EZH2-negative cells in the remnant PDOX tumors accompanied by decreased H3K27me2 and H3K27me3 expressions. These data supported the use of tazemetostat in a subset of pediatric brain tumors and suggests that EZH2-negative tumor cells may have caused therapy resistance and should be prioritized for the search of new therapeutic targets.

Initial in vivo testing of TPO-receptor agonist eltrombopag in osteosarcoma patient-derived xenograft models by the pediatric preclinical testing consortium.

Eltrombopag is a small molecule, thrombopoietin receptor agonist approved for the treatment of patients with aplastic anemia and chronic immune thrombocytopenia. It is also a polyvalent cation chelator and inhibits leukemia cell proliferation via reduction of intracellular iron. The in vivo efficacy of eltrombopag was tested against a panel of six Pediatric Preclinical Testing Consortium osteosarcoma xenografts at doses of 5 mg/kg/day (moderate dose) and 50 mg/kg/day (high dose). Eltrombopag, at moderate doses, failed to significantly improve event-free survival (EFS) in 6/6 models. At high doses, eltrombopag significantly prolonged EFS in 2/2 models, though the effect size was small. All models tested demonstrated progressive disease. While eltrombopag did not meaningfully inhibit osteosarcoma growth, it also did not stimulate tumor growth, suggesting it may be safely investigated as a supportive care agent to enhance platelet recovery post chemotherapy.

Dose-response effect of eribulin in preclinical models of osteosarcoma by the pediatric preclinical testing consortium.

The pediatric preclinical testing program previously demonstrated activity of eribulin in osteosarcoma patient-derived xenograft (PDX) models. The phase 2 trial in patients with relapsed osteosarcoma failed to meet response endpoints. Eribulin was evaluated in the original and an expanded set of PDX models and tested at multiple dose levels and schedules to evaluate dose-response. Maximal response was observed at the highest dose, consistent with prior results. The alternative schedule generated similar responses. We demonstrate steep dose-response for eribulin in osteosarcoma PDX models, implying that any deviation from achievement of effective concentrations may have a significant impact on activity.

OBI-3424, a Novel AKR1C3-Activated Prodrug, Exhibits Potent Efficacy against Preclinical Models of T-ALL.

PURPOSE: OBI-3424 is a highly selective prodrug that is converted by aldo-keto reductase family 1 member C3 (AKR1C3) to a potent DNA-alkylating agent. OBI-3424 has entered clinical testing for hepatocellular carcinoma and castrate-resistant prostate cancer, and it represents a potentially novel treatment for acute lymphoblastic leukemia (ALL). EXPERIMENTAL DESIGN: We assessed AKR1C3 expression by RNA-Seq and immunoblotting, and evaluated the in vitro cytotoxicity of OBI-3424. We investigated the pharmacokinetics of OBI-3424 in mice and nonhuman primates, and assessed the in vivo efficacy of OBI-3424 against a large panel of patient-derived xenografts (PDX). RESULTS: AKR1C3 mRNA expression was significantly higher in primary T-lineage ALL (T-ALL; n = 264) than B-lineage ALL (B-ALL; n = 1,740; P < 0.0001), and OBI-3424 exerted potent cytotoxicity against T-ALL cell lines and PDXs. In vivo, OBI-3424 significantly prolonged the event-free survival (EFS) of nine of nine ALL PDXs by 17.1-77.8 days (treated/control values 2.5-14.0), and disease regression was observed in eight of nine PDXs. A significant reduction (P < 0.0001) in bone marrow infiltration at day 28 was observed in four of six evaluable T-ALL PDXs. The importance of AKR1C3 in the in vivo response to OBI-3424 was verified using a B-ALL PDX that had been lentivirally transduced to stably overexpress AKR1C3. OBI-3424 combined with nelarabine resulted in prolongation of mouse EFS compared with each single agent alone in two T-ALL PDXs. CONCLUSIONS: OBI-3424 exerted profound in vivo efficacy against T-ALL PDXs derived predominantly from aggressive and fatal disease, and therefore may represent a novel treatment for aggressive and chemoresistant T-ALL in an AKR1C3 biomarker-driven clinical trial.

Broad Spectrum Activity of the Checkpoint Kinase 1 Inhibitor Prexasertib as a Single Agent or Chemopotentiator Across a Range of Preclinical Pediatric Tumor Models.

PURPOSE: Checkpoint kinase 1 (CHK1) inhibitors potentiate the DNA-damaging effects of cytotoxic therapies and/or promote elevated levels of replication stress, leading to tumor cell death. Prexasertib (LY2606368) is a CHK1 small-molecule inhibitor under clinical evaluation in multiple adult and pediatric cancers. In this study, prexasertib was tested in a large panel of preclinical models of pediatric solid malignancies alone or in combination with chemotherapy. EXPERIMENTAL DESIGN: DNA damage and changes in cell signaling following in vitro prexasertib treatment in pediatric sarcoma cell lines were analyzed by Western blot and high content imaging. Antitumor activity of prexasertib as a single agent or in combination with different chemotherapies was explored in cell line-derived (CDX) and patient-derived xenograft (PDX) mouse models representing nine different pediatric cancer histologies. RESULTS: Pediatric sarcoma cell lines were highly sensitive to prexasertib treatment in vitro, resulting in activation of the DNA damage response. Two PDX models of desmoplastic small round cell tumor and one malignant rhabdoid tumor CDX model responded to prexasertib with complete regression. Prexasertib monotherapy also elicited robust responses in mouse models of rhabdomyosarcoma. Concurrent administration with chemotherapy was sufficient to overcome innate resistance or prevent acquired resistance to prexasertib in preclinical models of neuroblastoma, osteosarcoma, and Ewing sarcoma, or alveolar rhabdomyosarcoma, respectively. CONCLUSIONS: Prexasertib has significant antitumor effects as a monotherapy or in combination with chemotherapy in multiple preclinical models of pediatric cancer. These findings support further investigation of prexasertib in pediatric malignancies.

Combining targeted agents with modern radiotherapy in soft tissue sarcomas.

Improved understanding of soft-tissue sarcoma (STS) biology has led to better distinction and subtyping of these diseases with the hope of exploiting the molecular characteristics of each subtype to develop appropriately targeted treatment regimens. In the care of patients with extremity STS, adjunctive radiation therapy (RT) is used to facilitate limb and function, preserving surgeries while maintaining five-year local control above 85%. In contrast, for STS originating from nonextremity anatomical sites, the rate of local recurrence is much higher (five-year local control is approximately 50%) and a major cause of death and morbidity in these patients. Incorporating novel technological advancements to administer accurate RT in combination with novel radiosensitizing agents could potentially improve local control and overall survival. RT efficacy in STS can be increased by modulating biological pathways such as angiogenesis, cell cycle regulation, cell survival signaling, and cancer-host immune interactions. Previous experiences, advancements, ongoing research, and current clinical trials combining RT with agents modulating one or more of the above pathways are reviewed. The standard clinical management of patients with STS with pretreatment biopsy, neoadjuvant treatment, and primary surgery provides an opportune disease model for interrogating translational hypotheses. The purpose of this review is to outline a strategic vision for clinical translation of preclinical findings and to identify appropriate targeted agents to combine with radiotherapy in the treatment of STS from different sites and/or different histology subtypes.

Toward a drug development path that targets metastatic progression in osteosarcoma.

Despite successful primary tumor treatment, the development of pulmonary metastasis continues to be the most common cause of mortality in patients with osteosarcoma. A conventional drug development path requiring drugs to induce regression of established lesions has not led to improvements for patients with osteosarcoma in more than 30 years. On the basis of our growing understanding of metastasis biology, it is now reasonable and essential that we focus on developing therapeutics that target metastatic progression. To advance this agenda, a meeting of key opinion leaders and experts in the metastasis and osteosarcoma communities was convened in Bethesda, Maryland. The goal of this meeting was to provide a "Perspective" that would establish a preclinical translational path that could support the early evaluation of potential therapeutic agents that uniquely target the metastatic phenotype. Although focused on osteosarcoma, the need for this perspective is shared among many cancer types. The consensus achieved from the meeting included the following: the biology of metastatic progression is associated with metastasis-specific targets/processes that may not influence grossly detectable lesions; targeting of metastasis-specific processes is feasible; rigorous preclinical data are needed to support translation of metastasis-specific agents into human trials where regression of measurable disease is not an expected outcome; preclinical data should include an understanding of mechanism of action, validation of pharmacodynamic markers of effective exposure and response, the use of several murine models of effectiveness, and where feasible the inclusion of the dog with naturally occurring osteosarcoma to define the activity of new drugs in the micrometastatic disease setting.

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors.

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy.

Testing of the topoisomerase 1 inhibitor Genz-644282 by the pediatric preclinical testing program.

BACKGROUND: Genz-644282 is a novel non-camptothecin topoisomerase I poison that is in clinical development. PROCEDURES: Genz-644282 was tested against the PPTP in vitro panel (0.1 nM to 1 µM), and in vivo using three times per week × 2 schedule repeated at day 21 at its maximum tolerated dose (MTD) of 4 mg/kg. Subsequently Genz-644282 was tested at 4, 3, 2, and 1 mg/kg in 3 models to assess the dose-response relationship. mRNA gene signatures predictive for Genz-644282 response in vitro were applied to select 15 tumor models that were evaluated prospectively. RESULTS: In vitro, Genz-644282 demonstrated potent cytotoxic activity with a median IC(50) of 1.2 nM (range 0.2-21.9 nM). In vivo, Genz-644282 at its MTD (4 mg/kg) induced maintained complete responses (MCR) in 6/6 evaluable solid tumor models. At 2 mg/kg Genz-644282 induced CR or MCR in 3/3 tumor models relatively insensitive to topotecan, but there were no objective responses at 1 mg/kg. Further testing at 2 mg/kg showed that Genz-644282 induced objective regressions in 7 of 17 (41%) models. There was a significant correlation between predictive response scores based on Affymetrix U133Plus2 baseline tumor expression profiles and the observed in vivo responses to Genz-644282. CONCLUSIONS: Genz-644282 was highly active within a narrow dose range (2-4 mg/kg), typical of other topoisomerase I poisons. As with other topoisomerase I poisons, how accurately these data will translate to clinical activity will depend upon the drug exposures that can be achieved in children treated with this agent.

The chemosensitizing activity of inhibitors of glucosylceramide synthase is mediated primarily through modulation of P-gp function.

Glucosylceramide synthase (GCS) is a key enzyme engaged in the biosynthesis of glycosphingolipids and in regulating ceramide metabolism. Studies exploring alterations in GCS activity suggest that the glycolase may have a role in chemosensitizing tumor cells to various cancer drugs. The chemosensitizing effect of inhibitors of GCS (e.g. PDMP and selected analogues) has been observed with a variety of tumor cells leading to the proposal that the sensitizing activity of GCS inhibitors is primarily through increases in intracellular ceramide leading to induction of apoptosis. The current study examined the chemosensitizing activity of the novel GCS inhibitor, Genz-123346 in cell culture. Exposure of cells to Genz-123346 and to other GCS inhibitors at non-toxic concentrations can enhance the killing of tumor cells by cytotoxic anti-cancer agents. This activity was unrelated to lowering intracellular glycosphingolipid levels. Genz-123346 and a few other GCS inhibitors are substrates for multi-drug resistance efflux pumps such as P-gp (ABCB1, gP-170). In cell lines selected to over-express P-gp or which endogenously express P-gp, chemosensitization by Genz-123346 was primarily due to the effects on P-gp function. RNA interference studies using siRNA or shRNA confirmed that lowering GCS expression in tumor cells did not affect their responsiveness to commonly used cytotoxic drugs.

Combinations of PARP, hedgehog and HDAC inhibitors with standard drugs.

Objective methods for data analysis for the assessment of additivity, synergy, and antagonism that can be applied rigorously to cell-based and in vivo preclinical data have been defined. The Combination Index method has been most widely applied. This brief report discusses poly(ADP-ribose) polymerase (PARP) inhibitors, hedgehog inhibitors, and histone deacetylase inhibitors as examples of experimental agents used in combinations where additivity and synergy have been assessed. PARP, hedgehog, and HDAC inhibitors have demonstrated single agent activity in a limited spectrum of malignancies. Expanded application of these compounds may come from combination regimens. Several PARP inhibitors appear to be additive to synergistic with standard agents in cell culture; however, no determinations for additivity/synergy have been applied with these compounds in vivo. Hedgehog inhibitors have demonstrated interesting activity in some preclinical models and have been studied in vivo with gemcitabine. However, no analyses for additivity were conducted in these studies. Numerous studies have demonstrated additivity/synergy with several HDAC inhibitors and standard anticancer drugs in cell culture. Clinical trials with combination regimens are on-going with compounds from each of these classes based primarily upon limited cell culture data. Thus, there is still a distance to go in making optimal use of preclinical models to aid in the selection of combination regimens worthy of clinical trial.

Netrin-4 regulates angiogenic responses and tumor cell growth.

Netrin-4 is a 628 amino acid basement membrane component that promotes neurite elongation at low concentrations but inhibits neurite extension at high concentrations. There is a growing body of literature suggesting that several molecules, including netrins, are regulators of both neuronal and vascular growth. It is believed that molecules that guide neural growth and development are also involved in regulating morphogenesis of the vascular tree. Further, netrins have recently been implicated in controlling epithelial cell branching morphogenesis in the breast, lung and pancreas. Characterization of purified netrin-4 in in vitro angiogenesis assays demonstrated that netrin-4 markedly inhibits HMVEC migration and tube formation. Moreover, netrin-4 inhibits proliferation of a variety of human tumor cells in vitro. Netrin-4 has only modest effects on proliferation of endothelial and other non-transformed cells. Netrin-4 treatment results in phosphorylation changes of proteins that are known to control cell growth. Specifically, Phospho-Akt-1, Phospho-Jnk-2, and Phospho-c-Jun are reduced in tumor cells that have been treated with netrin-4. Together, these data suggest a potential role for netrin-4 in regulating tumor growth.

Tumor models for efficacy determination.

The first in vivo tumor models were developed in the mid-1960s. These models were mouse leukemia models grown as ascites. The growth pattern was like that of bacteria in vivo and therefore it was possible to apply similar mathematics of growth and response to these tumors as had been worked out for bacteria. Since the development of the murine leukemia models, investigators have devoted a large effort to modeling solid tumors in mice. There are now a variety of models including syngeneic mouse tumors and human tumor xenografts grown as s.c. nodules, syngeneic mouse tumors and human tumor xenografts grown in orthotopic sites, models of disseminated disease, "labeled" tumor models that can be visualized using varied technologies, and transgenic tumor models. Each of these types of models has advantages and disadvantages to the "drug hunter" searching for improved treatments.

Combination of antiangiogenic therapy with other anticancer therapies: results, challenges, and open questions.

Angiogenesis is necessary for tumor growth. Drug discovery efforts have identified several potential therapeutic targets on endothelial cells and selective inhibitors capable of slowing tumor growth or producing tumor regression by blocking angiogenesis in in vivo tumor models. Certain antiangiogenic therapeutics increase the activity of cytotoxic anticancer treatments in preclinical models. More than 75 antiangiogenic compounds have entered clinical trials. Most of the early clinical testing was conducted in patients with advanced disease resistant to standard therapies. Several phase III trials have been undertaken to compare the efficacy of standard chemotherapy versus the same in combination with an experimental angiogenesis inhibitor. Preliminary results of the clinical studies suggest that single-agent antiangiogenic therapy is poorly active in advanced tumors. Although some of the results of combination trials are controversial, recent positive outcomes with an antivascular endothelial growth factor antibody combined with chemotherapy as front-line therapy of metastatic colorectal cancer have renewed enthusiasm for this therapeutic strategy. This article presents an overview of experimental and clinical studies of combined therapy with antiangiogenic agents and highlights the challenges related to the appropriate strategies for selection of the patients, study design, and choice of proper end points for preclinical and clinical studies using these agents.