Nonpersistent Nanoarchitectures Enhance Concurrent Chemoradiotherapy in an Immunocompetent Orthotopic Model of HPV+ Head/Neck Carcinoma.

Cisplatin chemoradiotherapy (CRT) is the established standard of care for managing locally advanced human papillomavirus-positive head/neck carcinoma. The typically young patients may suffer serious and long-time side effects caused by the treatment, such as dysphagia, and hearing loss. Thus, ensuring a satisfactory post-treatment quality of life is paramount. One potential replacing approach to the classical CRT involves the combination of standard-dose radiotherapy and radiosensitizers such as noble metal nanoparticles (NPs). However, several concerns about size, shape, and biocompatibility limit the translation of metal nanomaterials to the clinical practice. Here, it is demonstrated that a new model of nonpersistent gold nanoarchitectures containing cisplatin (NAs-Cluster-CisPt) generates, in combination with radiotherapy, a significant in vivo tumor-reducing effect compared to the standard CRT, achieving a complete tumor clearance in 25% of the immunocompetent models that persist for 60 days. These findings, together with the negligible amount of metals recognized in the excretory organs, highlight that the concurrent administration of NAs-Cluster-CisPt and radiotherapy has the potential to overcome some clinical limitations associated to NP-based approaches while enhancing the treatment outcome with respect to standard CRT. Overall, despite further mechanistic investigations being essential, these data support the exploiting of nonpersistent metal-nanomaterial-mediated approaches for oral cancer management.

Biomarker Identification in Liver Cancers Using Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) Imaging: An Approach for Spatially Resolved Metabolomics.

Liver cancers are characterized by interindividual and intratumoral heterogeneity, which makes early diagnosis and the development of therapies challenging. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging is a potent and sensitive MS ionization technique for direct, unaltered 2D and 3D imaging of metabolites in complex biological samples. Indeed, DESI gently desorbs and ionizes analyte molecules from the sample surface using an electrospray source of highly charged aqueous spray droplets in ambient conditions. DESI-MS imaging of biological samples allows untargeted analysis and characterization of metabolites in liver cancers to identify new biomarkers of malignancy. In this chapter, we described a detailed protocol using liver cancer samples collected and stored for histopathology examination, either as frozen or as formalin-fixed, paraffin-embedded specimens. Such hepatocellular carcinoma samples can be subjected to DESI-MS analyses, illustrating the capacity of spatially resolved metabolomics to distinguish malignant lesions from adjacent normal liver tissue.

Molecular profiling of biliary tract cancers reveals distinct genomic landscapes between circulating and tissue tumor DNA.

Biliary tract cancers (BTCs) are heterogeneous malignancies with dismal prognosis due to tumor aggressiveness and poor response to limited current therapeutic options. Tumor exome profiling has allowed to successfully establish targeted therapeutic strategies in the clinical management of cholangiocarcinoma (CCA). Still, whether liquid biopsy profiling could inform on BTC biology and patient management is unknown. In order to test this and generate novel insight into BTC biology, we analyzed the molecular landscape of 128 CCA patients, using a 394-gene NGS panel (Foundation Medicine). Among them, 32 patients had matched circulating tumor (ct) DNA and tumor DNA samples, where both samples were profiled. In both tumor and liquid biopsies, we identified an increased frequency of alterations in genes involved in genome integrity or chromatin remodeling, including ARID1A (15%), PBRM1 (9%), and BAP1 (14%), which were validated using an in-house-developed immunohistochemistry panel. ctDNA and tumor DNA showed variable concordance, with a significant correlation in the total number of detected variants, but some heterogeneity in the detection of actionable mutations. FGFR2 mutations were more frequently identified in liquid biopsies, whereas KRAS alterations were mostly found in tumors. All IDH1 mutations detected in tumor DNA were also identified in liquid biopsies. These findings provide novel insights in the concordance between the tumor and liquid biopsies genomic landscape in a large cohort of patients with BTC and highlight the complementarity of both analyses when guiding therapeutic prescription.

Antitumoral effect of local injection of TLR-9 agonist emulsified in Lipiodol with systemic anti-PD-1 in a murine model of colorectal carcinoma.

Introduction: Local treatments of cancer, including transarterial chemoembolization, could enhance responses to systemic immune checkpoint inhibitors such as anti-PD-1 antibodies. Lipiodol, a radiopaque oil, is widely used for transarterial chemoembolization as a tumor-targeting drug carrier and could be used in emulsion with immunomodulators. This study aimed at evaluating the antitumoral effect of intra-tumoral injection of Lipiodol-immunomodulator emulsions combined with systemic anti-PD-1 therapy in a murine model of colorectal carcinoma. Method: Mice (male BALB/c) with anti-PD-1-resistant subcutaneous CT26 tumors were injected with immunomodulators, emulsified or not with Lipiodol (N=10-12/group). Results: The TLR-9 agonist CpG displayed antitumor effects, while Poly I:C and QS21 did not. The Lipiodol-CpG emulsion appeared to be stable and maintained CpG within tumors for a longer time. Repeated intra-tumoral injections, combined with anti-PD-1, induced responses towards the tumor as well as to a distant metastatic-like nodule. This treatment was associated with an increase in proliferative CD8+ T cells and of IFN-γ expression, a decrease in proliferative regulatory T cells but also, surprisingly, an increase in myeloid derived suppressor cells. Conclusions: Local administration of CpG emulsified with Lipiodol led to an effective antitumoral effect when combined to systemic anti-PD-1 therapy. Lipiodol, apart from its radiopaque properties, is an efficient drug-delivery system. The formulated oil-in-water emulsion allows efficient loading and control release of CpG, which induces favorable immune modifications in this murine tumor model.

De novo generation of the NPM-ALK fusion recapitulates the pleiotropic phenotypes of ALK+ ALCL pathogenesis and reveals the ROR2 receptor as target for tumor cells.

BACKGROUND: Anaplastic large cell lymphoma positive for ALK (ALK+ ALCL) is a rare type of non-Hodgkin lymphoma. This lymphoma is caused by chromosomal translocations involving the anaplastic lymphoma kinase gene (ALK). In this study, we aimed to identify mechanisms of transformation and therapeutic targets by generating a model of ALK+ ALCL lymphomagenesis ab initio with the specific NPM-ALK fusion. METHODS: We performed CRISPR/Cas9-mediated genome editing of the NPM-ALK chromosomal translocation in primary human activated T lymphocytes. RESULTS: Both CD4+ and CD8+ NPM-ALK-edited T lymphocytes showed rapid and reproducible competitive advantage in culture and led to in vivo disease development with nodal and extra-nodal features. Murine tumors displayed the phenotypic diversity observed in ALK+ ALCL patients, including CD4+ and CD8+ lymphomas. Assessment of transcriptome data from models and patients revealed global activation of the WNT signaling pathway, including both canonical and non-canonical pathways, during ALK+ ALCL lymphomagenesis. Specifically, we found that the WNT signaling cell surface receptor ROR2 represented a robust and genuine marker of all ALK+ ALCL patient tumor samples. CONCLUSIONS: In this study, ab initio modeling of the ALK+ ALCL chromosomal translocation in mature T lymphocytes enabled the identification of new therapeutic targets. As ROR2 targeting approaches for other cancers are under development (including lung and ovarian tumors), our findings suggest that ALK+ ALCL cases with resistance to current therapies may also benefit from ROR2 targeting strategies.

TGFß receptor inhibition unleashes interferon-ß production by tumor-associated macrophages and enhances radiotherapy efficacy.

BACKGROUND: Transforming growth factor-beta (TGFß) can limit the efficacy of cancer treatments, including radiotherapy (RT), by inducing an immunosuppressive tumor environment. The association of TGFß with impaired T cell infiltration and antitumor immunity is known, but the mechanisms by which TGFß participates in immune cell exclusion and limits the efficacy of antitumor therapies warrant further investigations. METHODS: We used the clinically relevant TGFß receptor 2 (TGFßR2)-neutralizing antibody MT1 and the small molecule TGFßR1 inhibitor LY3200882 and evaluated their efficacy in combination with RT against murine orthotopic models of head and neck and lung cancer. RESULTS: We demonstrated that TGFß pathway inhibition strongly increased the efficacy of RT. TGFßR2 antibody upregulated interferon beta expression in tumor-associated macrophages within the irradiated tumors and favored T cell infiltration at the periphery and within the core of the tumor lesions. We highlighted that both the antitumor efficacy and the increased lymphocyte infiltration observed with the combination of MT1 and RT were dependent on type I interferon signaling. CONCLUSIONS: These data shed new light on the role of TGFß in limiting the efficacy of RT, identifying a novel mechanism involving the inhibition of macrophage-derived type I interferon production, and fostering the use of TGFßR inhibition in combination with RT in therapeutic strategies for the management of head and neck and lung cancer.

Therapeutic potential of the human endogenous retroviral envelope protein HEMO: a pan-cancer analysis.

Human endogenous retroviruses represent approximately 8% of our genome. Most of these sequences are defective except for a few genes such as the ancestral retroviral HEMO envelope gene (Human Endogenous MER34 ORF), recently characterized by our group. In this study, we characterized transcriptional activation of HEMO in primary tumors from The Cancer Genome Atlas (TCGA) and in metastatic tumors from a Gustave Roussy cohort. Pan-cancer detection of the HEMO protein in a series of patient samples validated these results. Differential gene expression analysis in various TCGA datasets revealed a link between HEMO expression and activation of Wnt/ß-catenin signaling, in particular in endometrial cancer. Studies on cell models led us to propose that the Wnt/ß-catenin pathway could act as an upstream regulator of this retroviral endogenous sequence in tumor condition. Characterization of transcriptomic profiles of both HEMOLow and HEMOHigh tumors suggested that activation of HEMO is negatively associated with immune response signatures. Taken together, these results highlight that HEMO, as an endogenous retroviral envelope protein specifically expressed in tumors, represents a promising tumor biomarker and therapeutic target.

Serial transplantation unmasks galectin-9 contribution to tumor immune escape in the MB49 murine model.

Mechanisms of tumor immune escape are quite diverse and require specific approaches for their exploration in syngeneic tumor models. In several human malignancies, galectin-9 (gal-9) is suspected to contribute to the immune escape. However, in contrast with what has been done for the infiltrating cells, the contribution of gal-9 produced by malignant cells has never been demonstrated in an animal model. Therefore, we derived isogenic clones-either positive or negative for gal-9-from the MB49 murine bladder carcinoma cell line. A progressive and consistent reduction of tumor growth was observed when gal-9-KO cells were subjected to serial transplantations into syngeneic mice. In contrast, tumor growth was unaffected during parallel serial transplantations into nude mice, thus linking tumor inhibition to the enhancement of the immune response against gal-9-KO tumors. This stronger immune response was at least in part explained by changing patterns of response to interferon-γ. One consistent change was a more abundant production of CXCL10, a major inflammatory factor whose production is often induced by interferon-γ. Overall, these observations demonstrate for the first time that serial transplantation into syngeneic mice can be a valuable experimental approach for the exploration of novel mechanisms of tumor immune escape.

MT2A is an early predictive biomarker of response to chemotherapy and a potential therapeutic target in osteosarcoma.

Osteosarcoma is the most prevalent primary bone malignancy in children and young adults. Resistance to chemotherapy remains a key challenge for effective treatment of patients with osteosarcoma. The aim of the present study was to investigate the preventive role of metallothionein-2A (MT2A) in response to cytotoxic effects of chemotherapy. A panel of human and murine osteosarcoma cell lines, modified for MT2A were evaluated for cell viability, and motility (wound healing assay). Cell-derived xenograft models were established in mice. FFPE tumour samples were assessed by IHC. In vitro experiments indicated a positive correlation between half-maximal inhibitory concentration (IC50) for drugs in clinical practice, and MT2A mRNA level. This reinforced our previously reported correlation between MT2A mRNA level in tumour samples at diagnosis and overall survival in patients with osteosarcoma. In addition, MT2A/MT2 silencing using shRNA strategy led to a marked reduction of IC50 values and to enhanced cytotoxic effect of chemotherapy on primary tumour. Our results show that MT2A level could be used as a predictive biomarker of resistance to chemotherapy, and provide with preclinical rational for MT2A targeting as a therapeutic strategy for enhancing anti-tumour treatment of innate chemo-resistant osteosarcoma cells.

Local immunomodulation combined to radiofrequency ablation results in a complete cure of local and distant colorectal carcinoma.

Radiofrequency ablation (RFA) of colorectal liver metastases activates a specific T-cell response that is ineffective in avoiding recurrence. Recently, local immunomodulation garnered interests as a way to improve the immune response. We were interested in improving the RFA immune response priming to propose a curative treatment of colorectal cancer (CRC) based on antitumor immunity. First, we demonstrated that the RFA did not increase the tumor infiltrating lymphocytes in secondary distant tumors of patients and in mice model and could not avoid relapse. Remarkably, RFA and in situ immunomodulation with GM-CSF-BCG hydrogel induced complete cure of microscopic secondary lesions in mice, related to a strong specific immune response. Then, we demonstrated that the immune escape of large secondary lesions was reversed by addition of the systemic PD-1 blockade to the in situ immunomodulation. The lack of an effective distant immune response in patients treated with RFA confirmed the relevance of this new combination strategy. Increasing the in situ priming response of radiofrequency ablation provides effective adjuvants to induce an abscopal effect. In the case of large lesions, synergy between PD1 blockade inhibitor, ineffective alone or after single RFA, with in situ immunomodulation, could lead to reconsideration of the use of checkpoint inhibition in metastatic MSS CRC.

Partial trisomy 21 contributes to T-cell malignancies induced by JAK3-activating mutations in murine models.

JAK3-activating mutations are commonly seen in chronic or acute hematologic malignancies affecting the myeloid, megakaryocytic, lymphoid, and natural killer (NK) cell compartment. Overexpression models of mutant JAK3 or pharmacologic inhibition of its kinase activity have highlighted the role that these constitutively activated mutants play in the T-cell, NK cell, and megakaryocytic lineages, but to date, the functional impact of JAK3 mutations at an endogenous level remains unknown. Here, we report a JAK3A572V knockin mouse model and demonstrate that activated JAK3 leads to a progressive and dose-dependent expansion of CD8+ T cells in the periphery before colonization of the bone marrow. This phenotype is dependent on the γc chain of cytokine receptors and presents several features of the human leukemic form of cutaneous T-cell lymphoma (L-CTCL), including skin involvements. We also showed that the JAK3A572V-positive malignant cells are transplantable and phenotypically heterogeneous in bone marrow transplantation assays. Interestingly, we revealed that activated JAK3 functionally cooperates with partial trisomy 21 in vivo to enhance the L-CTCL phenotype, ultimately leading to a lethal and fully penetrant disorder. Finally, we assessed the efficacy of JAK3 inhibition and showed that CTCL JAK3A572V-positive T cells are sensitive to tofacitinib, which provides additional preclinical insights into the use of JAK3 inhibitors in these disorders. Altogether, this JAK3A572V knockin model is a relevant new tool for testing the efficacy of JAK inhibitors in JAK3-related hematopoietic malignancies.

Establishment and characterization of in vivo orthotopic bioluminescent xenograft models from human osteosarcoma cell lines in Swiss nude and NSG mice.

Osteosarcoma is one of the most common primary bone tumors in childhood and adolescence. Metastases occurrence at diagnosis or during disease evolution is the main therapeutic challenge. New drug evaluation to improve patient survival requires the development of various preclinical models mimicking at best the complexity of the disease and its metastatic potential. We describe here the development and characteristics of two orthotopic bioluminescent (Luc/mKate2) cell-derived xenograft (CDX) models, Saos-2-B-Luc/mKate2-CDX and HOS-Luc/mKate2-CDX, in different immune (nude and NSG mouse strains) and bone (intratibial and paratibial with periosteum activation) contexts. IVIS SpectrumCT system allowed both longitudinal computed tomography (CT) and bioluminescence real-time follow-up of primary tumor growth and metastatic spread, which was confirmed by histology. The murine immune context influenced tumor engraftment, primary tumor growth, and metastatic spread to lungs, bone, and spleen (an unusual localization in humans). Engraftment in NSG mice was found superior to that found in nude mice and intratibial bone environment more favorable to engraftment compared to paratibial injection. The genetic background of the two CDX models also led to distinct primary tumor behavior observed on CT scan. Saos-2-B-Luc/mKate2-CDX showed osteocondensed, HOS-Luc/mKate2-CDX osteolytic morphology. Bioluminescence defined a faster growth of the primary tumor and metastases in Saos-2-B-Luc/mKate2-CDX than in HOS-Luc/mKate2-CDX. The early detection of primary tumor growth and metastatic spread by bioluminescence allows an improved exploration of osteosarcoma disease at tumor progression, and metastatic spread, as well as the evaluations of anticancer treatments. Our orthotopic models with metastatic spread bring complementary information to other types of existing osteosarcoma models.

Combined therapy of colon carcinomas with an oncolytic adenovirus and valproic acid.

The anti-tumor potential of oncolytic adenoviruses (CRAds) has been demonstrated in preclinical and clinical studies. While these agents failed to eradicate tumors when used as a monotherapy, they may be more effective if combined with conventional treatments such as radiotherapy or chemotherapy. This study seeks to evaluate the combination of a CRAd bearing a ∆24 deletion in E1A with valproic acid (VPA), a histone deacetylase inhibitor, for the treatment of human colon carcinomas. This combination led to a strong inhibition of cell growth both in vitro and in vivo compared to treatment with CRAd or VPA alone. This effect did not stem from a better CRAd replication and production in the presence of VPA. Inhibition of cell proliferation and cell death were induced by the combined treatment. Moreover, whereas cells treated only with CRAd displayed a polyploidy (> 4N population), this phenotype was increased in cells treated with both CRAd and VPA. In addition, the increase in polyploidy triggered by combined treatment with CRAd and VPA was associated with the enhancement of H2AX phosphorylation (γH2AX), a hallmark of DNA damage, but also with a decrease of several DNA repair proteins. Finally, viral replication (or E1A expression) was shown to play a key role in the observed effects since no enhancement of polyploidy nor increase in γH2AX were found following cell treatment with a replication-deficient Ad and VPA. Taken together, our results suggest that CRAd and VPA could be used in combination for the treatment of colon carcinomas.

HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors.

Capture of retroviral envelope genes is likely to have played a role in the emergence of placental mammals, with evidence for multiple, reiterated, and independent capture events occurring in mammals, and be responsible for the diversity of present day placental structures. Here, we uncover a full-length endogenous retrovirus envelope protein, dubbed HEMO [human endogenous MER34 (medium-reiteration-frequency-family-34) ORF], with unprecedented characteristics, because it is actively shed in the blood circulation in humans via specific cleavage of the precursor envelope protein upstream of the transmembrane domain. At variance with previously identified retroviral envelope genes, its encoding gene is found to be transcribed from a unique CpG-rich promoter not related to a retroviral LTR, with sites of expression including the placenta as well as other tissues and rather unexpectedly, stem cells as well as reprogrammed induced pluripotent stem cells (iPSCs), where the protein can also be detected. We provide evidence that the associated retroviral capture event most probably occurred >100 Mya before the split of Laurasiatheria and Euarchontoglires, with the identified retroviral envelope gene encoding a full-length protein in all simians under purifying selection and with similar shedding capacity. Finally, a comprehensive screen of the expression of the gene discloses high transcript levels in several tumor tissues, such as germ cell, breast, and ovarian tumors, with in the latter case, evidence for a histotype dependence and specific protein expression in clear-cell carcinoma. Altogether, the identified protein could constitute a "stemness marker" of the normal cell and a possible target for immunotherapeutic approaches in tumors.

Tetraspanin 8 (TSPAN 8) as a potential target for radio-immunotherapy of colorectal cancer.

Tetraspanin 8 (TSPAN8) overexpression is correlated with poor prognosis in human colorectal cancer (CRC). A murine mAb Ts29.2 specific for human TSPAN8 provided significant efficiency for immunotherapy in CRC pre-clinical models. We therefore evaluate the feasability of targeting TSPAN8 in CRC with radiolabeled Ts29.2. Staining of tissue micro-arrays with Ts29.2 revealed that TSPAN8 espression was restricted to a few human healthy tissues. DOTA-Ts29.2 was radiolabeled with 111In or 177Lu with radiochemical purities >95%, specific activity ranging from 300 to 600 MBq/mg, and radioimmunoreactive fractions >80%. The biodistribution of [111In]DOTA-Ts29.2 in nude mice bearing HT29 or SW480 CRC xenografts showed a high specificity of tumor localization with high tumor/blood ratios (HT29: 4.3; SW480-TSPAN8: 3.9 at 72h and 120h post injection respectively). Tumor-specific absorbed dose calculations for [177Lu]DOTA-Ts29.2 was 1.89 Gy/MBq, establishing the feasibility of using radioimmunotherapy of CRC with this radiolabeled antibody. A significant inhibition of tumor growth in HT29 tumor-bearing mice treated with [177Lu]DOTA-Ts29.2 was observed compared to control groups. Ex vivo experiments revealed specific DNA double strand breaks associated with cell apoptosis in [177Lu]DOTA-Ts29.2 treated tumors compared to controls. Overall, we provide a proof-of-concept for the use of [111In/177Lu]DOTA-Ts29.2 that specifically target in vivo aggressive TSPAN8-positive cells in CRC.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations.

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate-dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation-positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation-positive advanced hematologic malignancies.Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478-93. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Shih et al., p. 494This article is highlighted in the In This Issue feature, p. 443.

Dual inhibition using cabozantinib overcomes HGF/MET signaling mediated resistance to pan-VEGFR inhibition in orthotopic and metastatic neuroblastoma tumors.

MET is expressed on neuroblastoma cells and may trigger tumor growth, neoangiogenesis and metastasis. MET upregulation further represents an escape mechanism to various anticancer treatments including VEGF signaling inhibitors. We developed in vitro a resistance model to pan-VEGFR inhibition and explored the simultaneous inhibition of VEGFR and MET in neuroblastoma models in vitro and in vivo using cabozantinib, an inhibitor of the tyrosine kinases including VEGFR2, MET, AXL and RET. Resistance in IGR-N91-Luc neuroblastoma cells under continuous in vitro exposure pressure to VEGFR1-3 inhibition using axitinib was associated with HGF and p-ERK overexpression. Cabozantinib exhibited anti-proliferative effects in neuroblastoma cells and reduced cell migration in vitro as measured by phase-contrast with IncuCyte system. In vivo, an enhanced number of animals with IGR-N91-Luc metastases was noted following axitinib treatment as compared to control animals. Orally administered cabozantinib per gavage at 30 and 60 mg/kg/day significantly inhibited tumor growth of orthotopic adrenal IGR-N91-Luc and metastatic IMR-32-Luc xenografts. Antitumor activity was associated with decreased vascularization, inhibition of p-SRC and induction of apoptotic cell death. Activation of the HGF-mediated MET pathway is involved in escape to selective VEGFR inhibition in neuroblastoma suggesting combined inhibition of MET and VEGFR signaling to reduce secondary resistance and enhanced invasiveness.

Regorafenib: Antitumor Activity upon Mono and Combination Therapy in Preclinical Pediatric Malignancy Models.

The multikinase inhibitor regorafenib (BAY 73-4506) exerts both anti-angiogenic and anti-tumorigenic activity in adult solid malignancies mainly advanced colorectal cancer and gastrointestinal stromal tumors. We intended to explore preclinically the potential of regorafenib against solid pediatric malignancies alone and in combination with anticancer agents to guide the pediatric development plan. In vitro effects on cell proliferation were screened against 33 solid tumor cell lines of the Innovative Therapies for Children with Cancer (ITCC) panel covering five pediatric solid malignancies. Regorafenib inhibited cell proliferation with a mean half maximal growth inhibition of 12.5 µmol/L (range 0.7 µmol/L to 28 µmol/L). In vivo, regorafenib was evaluated alone at 10 or 30 mg/kg/d or in combination with radiation, irinotecan or the mitogen-activated protein kinase kinase (MEK) inhibitor refametinib against various tumor types, including patient-derived brain tumor models with an amplified platelet-derived growth factor receptor A (PDGFRA) gene. Regorafenib alone significantly inhibited tumor growth in all xenografts derived from nervous system and connective tissue tumors. Enhanced effects were observed when regorafenib was combined with irradiation and irinotecan against PDGFRA amplified IGRG93 glioma and IGRM57 medulloblastoma respectively, resulting in 100% tumor regressions. Antitumor activity was associated with decreased tumor vascularization, inhibition of PDGFR signaling, and induction of apoptotic cell death. Our work demonstrates that regorafenib exhibits significant antitumor activity in a wide spectrum of preclinical pediatric models through inhibition of angiogenesis and induction of apoptosis. Furthermore, radio- and chemosensitizing effects were observed with DNA damaging agents in PDGFR amplified tumors.

Level of RUNX1 activity is critical for leukemic predisposition but not for thrombocytopenia.

To explore how RUNX1 mutations predispose to leukemia, we generated induced pluripotent stem cells (iPSCs) from 2 pedigrees with germline RUNX1 mutations. The first, carrying a missense R174Q mutation, which acts as a dominant-negative mutant, is associated with thrombocytopenia and leukemia, and the second, carrying a monoallelic gene deletion inducing a haploinsufficiency, presents only as thrombocytopenia. Hematopoietic differentiation of these iPSC clones demonstrated profound defects in erythropoiesis and megakaryopoiesis and deregulated expression of RUNX1 targets. iPSC clones from patients with the R174Q mutation specifically generated an increased amount of granulomonocytes, a phenotype reproduced by an 80% RUNX1 knockdown in the H9 human embryonic stem cell line, and a genomic instability. This phenotype, found only with a lower dosage of RUNX1, may account for development of leukemia in patients. Altogether, RUNX1 dosage could explain the differential phenotype according to RUNX1 mutations, with a haploinsufficiency leading to thrombocytopenia alone in a majority of cases whereas a more complete gene deletion predisposes to leukemia.

Effect of an anti-human Co-029/tspan8 mouse monoclonal antibody on tumor growth in a nude mouse model.

New therapeutic agents are needed in digestive tract tumors. Co-029/tspan8 is a tetraspanin frequently expressed on human colorectal tumors, In this work, we report the effects of the monoclonal antibody Ts29.2, targeting Co-029/tspan8, on colorectal tumor cells in vitro and after implantation in nude mice. HT29, Isreco1 and SW480 colorectal tumor cell lines were used for this study. HT29 has a strong endogenous expression of Co-029/tspan8, whereas Isreco1 cells don't express Co-029/tspan8 and SW480 has only a weak expression. Isreco1 and SW480 were transduced to express Co-029/tspan8 at the same level as HT29. In order to check the specificity of the effect of monoclonal antibody Ts29.2, low Co-029/tspan8 expressing SW480 cells were injected simultaneously with transduced cells in the back, on the left and right sides of the mice. With an early treatment, Ts29.2 mAb inhibited growth of tumors expressing Co-029/tspan8 up to 70%, whereas a delayed treatment was less efficient. No effect of the antibody on cell proliferation or apoptosis induction was detected in vitro. No increase of activated caspase 3 labeling was observed in vivo and areas occupied by vessels were not significantly different between treated mice and controls. This suggests that the action of Ts29.2 is linked neither to cellular toxicity nor to the inhibition of the previously reported angiogenic properties of Co-029/tspan8. An inhibition of cell proliferation in vivo is demonstrated by a reduction of the mitotic index in HT29 tumors of Ts29.2 treated mice. The discrepancy between in vitro and in vivo data on cell proliferation suggests that the binding of Ts29.2 to tumor cells may modify their response to signals issued from the microenvironment. Given the restricted pattern of tissue expression of the tetraspanin Co-029/tspan8, these preliminary results put forth for consideration the antibody targeting of this tetraspanin in further investigations for therapeutic applications.