Local delivery of hrBMP4 as an anticancer therapy in patients with recurrent glioblastoma: a first-in-human phase 1 dose escalation trial.

BACKGROUND: This Phase 1 study evaluates the intra- and peritumoral administration by convection enhanced delivery (CED) of human recombinant Bone Morphogenetic Protein 4 (hrBMP4) - an inhibitory regulator of cancer stem cells (CSCs) - in recurrent glioblastoma. METHODS: In a 3 + 3 dose escalation design, over four to six days, fifteen recurrent glioblastoma patients received, by CED, one of five doses of hrBMP4 ranging from 0·5 to 18 mg. Patients were followed by periodic physical, neurological, blood testing, magnetic resonance imaging (MRI) and quality of life evaluations. The primary objective of this first-in-human study was to determine the safety, dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) of hrBMP4. Secondary objectives were to assess potential efficacy and systemic exposure to hrBMP4 upon intracerebral infusion. RESULTS: Intra- and peritumoral infusion of hrBMP4 was safe and well-tolerated. We observed no serious adverse events related to this drug. Neither MTD nor DLT were reached. Three patients had increased hrBMP4 serum levels at the end of infusion, which normalized within 4 weeks, without sign of toxicity. One patient showed partial response and two patients a complete (local) tumor response, which was maintained until the most recent follow-up, 57 and 30 months post-hrBMP4. Tumor growth was inhibited in areas permeated by hrBMP4. CONCLUSION: Local delivery of hrBMP4 in and around recurring glioblastoma is safe and well-tolerated. Three patients responded to the treatment. A complete response and long-term survival occurred in two of them. This warrants further clinical studies on this novel treatment targeting glioblastoma CSCs. TRIAL REGISTRATION: ClinicaTrials.gov identifier: NCT02869243.

A functional role of Ephrin type-B receptor 6 (EPHB6) in T-cell acute lymphoblastic leukemia.

T-cell lymphoblastic acute leukemia (T-ALL) is an aggressive blood cancer, characterized by restricted cellular subsets with enriched leukemia initiating cells (LICs). Recently, Ephrin receptors (Eph) were described to be highly expressed in cancer stem cells. Here, using public RNA-Seq datasets of human T-ALL, we reported that EphB6 was the only member within the Eph family overexpressed in over 260 samples. We also found the highest level of EphB6 in a minor cell subpopulation within bulk tumors of patient-derived xenografts, obtained through the injection of primary patient biopsy material into immunocompromised NOD-Scid/IL2Rγc-/- (NSG) mice. Interestingly, this EphB6 positive (EphB6+) subset showed an enriched LIC activity after in vivo transplantation into NSG mice. Additionally, gene expression data at the single-cell level of primary patients' leukemic cells revealed that EphB6 + cells were significantly selected in minimal residual disease up to 30 days from the standard treatments and characterized by high levels of markers related to cell proliferation and poor clinical outcome, such as CCNB1 and KIF20A. Taken together, our data suggest that EphB6 supports LICs' maintenance and progression in T-ALL and, thus, targeting EphB6 + cells could be therapeutically relevant for the treatment of T-ALL patients.

Different states of stemness of glioblastoma stem cells sustain glioblastoma subtypes indicating novel clinical biomarkers and high-efficacy customized therapies.

BACKGROUND: Glioblastoma (GBM) is the most malignant among gliomas with an inevitable lethal outcome. The elucidation of the physiology and regulation of this tumor is mandatory to unravel novel target and effective therapeutics. Emerging concepts show that the minor subset of glioblastoma stem cells (GSCs) accounts for tumorigenicity, representing the true target for innovative therapies in GBM. METHODS: Here, we isolated and established functionally stable and steadily expanding GSCs lines from a large cohort of GBM patients. The molecular, functional and antigenic landscape of GBM tissues and their derivative GSCs was highlited in a side-by-side comprehensive genomic and transcriptomic characterization by ANOVA and Fisher's exact tests. GSCs' physio-pathological hallmarks were delineated by comparing over time in vitro and in vivo their expansion, self-renewal and tumorigenic ability with hierarchical linear models for repeated measurements and Kaplan-Meier method. Candidate biomarkers performance in discriminating GBM patients' classification emerged by classification tree and patients' survival analysis. RESULTS: Here, distinct biomarker signatures together with aberrant functional programs were shown to stratify GBM patients as well as their sibling GSCs population into TCGA clusters. Of importance, GSCs cells were demonstrated to fully resemble over time the molecular features of their patient of origin. Furthermore, we pointed out the existence of distinct GSCs subsets within GBM classification, inherently endowed with different self-renewal and tumorigenic potential. Particularly, classical GSCs were identified by more undifferentiated biological hallmarks, enhanced expansion and clonal capacity as compared to the more mature, relatively slow-propagating mesenchymal and proneural cells, likely endowed with a higher potential for infiltration either ex vivo or in vivo. Importantly, the combination of DCX and EGFR markers, selectively enriched among GSCs pools, almost exactly predicted GBM patients' clusters together with their survival and drug response. CONCLUSIONS: In this study we report that an inherent enrichment of distinct GSCs pools underpin the functional inter-cluster variances displayed by GBM patients. We uncover two selectively represented novel functional biomarkers capable of discriminating GBM patients' stratification, survival and drug response, setting the stage for the determination of patient-tailored diagnostic and prognostic strategies and, mostly, for the design of appropriate, patient-selective treatment protocols.

Effects of epidermal growth factor receptor blockade on ependymoma stem cells in vitro and in orthotopic mouse models.

Some lines of evidence suggest that tumors, including ependymoma, might arise from a subpopulation of cells, termed cancer stem cells (CSCs), with self-renewal and tumor-initiation properties. Given the strict dependence of CSCs on epidermal growth factor (EGF) through EGF receptor (EGFR), we investigated the effects of EGFR inhibitors in ependymoma-stem cells (SCs) in vitro and in orthotopic mouse models. We established two ependymoma-SC lines from two recurrent pediatric ependymoma. Both lines expressed markers of radial glia--the candidate SCs of ependymoma--and showed renewal ability, multipotency, and tumorigenicity after orthotopic implantation, despite markedly different expression of CD133 (94 vs. 6%). High phosphorylated-EGFR/EGFR ratio was detected, which decreased after differentiation. EGFR inhibitors (gefitinib and AEE788) reduced clonogenicity, proliferation and survival of ependymoma-SC lines dose-dependently, and blocked EGF-induced activation of EGFR, Akt and extracellular signal-regulated kinase 1/2. Overall, AEE788 was more effective than gefitinib. EGFR blockade as well as differentiation strongly reduced CD133 expression. However, ex vivo treatment with AEE788 did not impair orthotopic tumor engraftment, whereas ex vivo differentiation did, suggesting that CD133 does not absolutely segregate for tumorigenicity in ependymoma-SCs. Orally administered AEE788 prolonged survival of mice bearing ependymoma-SC-driven orthotopic xenografts from 56 to 63 days, close to statistical significance (log-rank p=0.06). Our study describes for the first time EGFR signaling in ependymoma-SCs and the effects of EGFR blockade in complementary in vitro and in vivo systems. The experimental models we developed can be used to further investigate the activity of EGFR inhibitors or other antineoplastic agents in this tumor.

Growth factor independence underpins a paroxysmal, aggressive Wnt5aHigh/EphA2Low phenotype in glioblastoma stem cells, conducive to experimental combinatorial therapy.

BACKGROUND: Glioblastoma multiforme (GBM) is an incurable tumor, with a median survival rate of only 14-15 months. Along with heterogeneity and unregulated growth, a central matter in dealing with GBMs is cell invasiveness. Thus, improving prognosis requires finding new agents to inhibit key multiple pathways, even simultaneously. A subset of GBM stem-like cells (GSCs) may account for tumorigenicity, representing, through their pathways, the proper cellular target in the therapeutics of glioblastomas. GSCs cells are routinely enriched and expanded due to continuous exposure to specific growth factors, which might alter some of their intrinsic characteristic and hide therapeutically relevant traits. METHODS: By removing exogenous growth factors stimulation, here we isolated and characterized a subset of GSCs with a "mitogen-independent" phenotype (I-GSCs) from patient's tumor specimens. Differential side-by-side comparative functional and molecular analyses were performed either in vitro or in vivo on these cells versus their classical growth factor (GF)-dependent counterpart (D-GSCs) as well as their tissue of origin. This was performed to pinpoint the inherent GSCs' critical regulators, with particular emphasis on those involved in spreading and tumorigenic potential. Transcriptomic fingerprints were pointed out by ANOVA with Benjamini-Hochberg False Discovery Rate (FDR) and association of copy number alterations or somatic mutations was determined by comparing each subgroup with a two-tailed Fisher's exact test. The combined effects of interacting in vitro and in vivo with two emerging GSCs' key regulators, such as Wnt5a and EphA2, were then predicted under in vivo experimental settings that are conducive to clinical applications. In vivo comparisons were carried out in mouse-human xenografts GBM model by a hierarchical linear model for repeated measurements and Dunnett's multiple comparison test with the distribution of survival compared by Kaplan-Meier method. RESULTS: Here, we assessed that a subset of GSCs from high-grade gliomas is self-sufficient in the activation of regulatory growth signaling. Furthermore, while constitutively present within the same GBM tissue, these GF-independent GSCs cells were endowed with a distinctive functional and molecular repertoire, defined by highly aggressive Wnt5aHigh/EphA2Low profile, as opposed to Wnt5aLow/EphA2High expression in sibling D-GSCs. Regardless of their GBM subtype of origin, I-GSCs, are endowed with a raised in vivo tumorigenic potential than matched D-GSCs, which were fast-growing ex-vivo but less lethal and invasive in vivo. Also, the malignant I-GSCs' transcriptomic fingerprint faithfully mirrored the original tumor, bringing into evidence key regulators of invasiveness, angiogenesis and immuno-modulators, which became candidates for glioma diagnostic/prognostic markers and therapeutic targets. Particularly, simultaneously counteracting the activity of the tissue invasive mediator Wnt5a and EphA2 tyrosine kinase receptor addictively hindered GSCs' tumorigenic and invasive ability, thus increasing survival. CONCLUSION: We show how the preservation of a mitogen-independent phenotype in GSCs plays a central role in determining the exacerbated tumorigenic and high mobility features distinctive of GBM. The exploitation of the I-GSCs' peculiar features shown here offers new ways to identify novel, GSCs-specific effectors, whose modulation can be used in order to identify novel, potential molecular therapeutic targets. Furthermore, we show how the combined use of PepA, the anti-Wnt5a drug, and of ephrinA1-Fc to can hinder GSCs' lethality in a clinically relevant xenogeneic in vivo model thus being conducive to perspective, novel combinatorial clinical application.

FADD-calmodulin interaction: a novel player in cell cycle regulation.

Analyses of knockout and mutant transgenic mice as well as in vitro studies demonstrated a complex role of FADD in the regulation of cell fate. FADD is involved in death receptor induced apoptosis, cell cycle progression and cell proliferation. In a search for mechanisms that might regulate FADD functions, we identified, upon the screening of a lambda-phage cDNA library, calmodulin (CaM) as a novel FADD interacting protein. CaM is a key mediator of signals by the secondary messenger calcium and it is an essential regulator of cell cycle progression and cell survival. Here, we describe the identification and characterization of two calcium dependent CaM binding sites in the alpha helices 8-9 and 10-11 of FADD. Phosphorylation of human FADD at the C-terminal serine 194, by casein kinase I alpha (CKIalpha), has been shown to regulate FADD-dependent non-apoptotic activities. Remarkably, we showed that both FADD and CaM are CKIalpha substrates and that in synchronized HeLa cells, FADD, CaM and CKIalpha co-localize at the mitotic spindle in metaphase and anaphase. Moreover, complementation experiments in Jurkat FADD-/- T cells indicated that: a) cells expressing FADD mutants in the CaM binding sites are protected from Taxol-induced G2/M cell cycle arrest; b) FADD/CaM interaction is not required for Fas receptor-mediated apoptosis although Fas and CaM might compete for binding to FADD. We suggest that the interplay of FADD, CaM and CKIalpha may have an important role in the regulation of cell fate.

Improving Gemcitabine Sensitivity in Pancreatic Cancer Cells by Restoring miRNA-217 Levels.

Chemoresistance is a major problem in the therapeutic management of pancreatic cancer, concurring to poor clinical outcome. A number of mechanisms have been proposed to explain resistance to gemcitabine, a standard of care for this malignancy, among which is included aberrant miRNA expression. In the current study, we investigated the role of miR-217, which is strongly down-regulated in cancerous, compared to normal, pancreatic tissues or cells, in sensitizing human pancreatic cancer cell lines to this drug. The low expression of miR-217 in pancreatic cancer patients was confirmed in two gene expression datasets (GSE41372 and GSE60980), and the prognostic value of two target genes (ANLN and TRPS1), was estimated on clinical data from the Tumor Cancer Genome Atlas (TCGA). Transfecting miR-217 mimic in pancreatic cancer cells reduced viability, enhanced apoptosis, and affected cell cycle by promoting a S phase arrest in gemcitabine-treated cells. Moreover, in drug-exposed cells subjected to miR-217 forced expression, a down-regulation for several genes involved in cancer drug resistance was observed, many of which are cell cycle regulators, such as CCND1, CCNE1, CDK2, CDKN1A, CDKN1B, while others, such as ARNT, BRCA1, BRCA2, ELK1, EGFR, ERBB4, and RARA are involved in proliferation and cell cycle progression. Our results support the notion that miR-217 enhances pancreatic cancer sensitivity to gemcitabine, mainly impairing cell cycle progression.

High Levels of Prebiotic Resistant Starch in Diet Modulate a Specific Pattern of miRNAs Expression Profile Associated to a Better Overall Survival in Pancreatic Cancer.

Dietary patterns are well known risk factors involved in cancer initiation, progression, and in cancer protection. Previous in vitro and in vivo studies underline the link between a diet rich in resistant starch (RS) and slowing of tumor growth and gene expression in pancreatic cancer xenograft mice. The aim of this study was to investigate the impact of a diet rich in resistant starch on miRNAs and miRNAs-target genes expression profile and on biological processes and pathways, that play a critical role in pancreatic tumors of xenografted mice. miRNA expression profiles on tumor tissues displayed 19 miRNAs as dysregulated in mice fed with RS diet as compared to those fed with control diet and differentially expressed miRNA-target genes were predicted by integrating (our data) with a public human pancreatic cancer gene expression dataset (GSE16515). Functional and pathway enrichment analyses unveiled that miRNAs involved in RS diet are critical regulators of genes that control tumor growth and cell migration and metastasis, inflammatory response, and, as expected, synthesis of carbohydrate and glucose metabolism disorder. Mostly, overall survival analysis with clinical data from TCGA (n = 175) displayed that almost four miRNAs (miRNA-375, miRNA-148a-3p, miRNA-125a-5p, and miRNA-200a-3p) upregulated in tumors from mice fed with RS were a predictor of good prognosis for pancreatic cancer patients. These findings contribute to the understanding of the potential mechanisms through which resistant starch may affect cancer progression, suggesting also a possible integrative approach for enhancing the efficacy of existing cancer treatments.

BRAFV600E mutation impinges on gut microbial markers defining novel biomarkers for serrated colorectal cancer effective therapies.

BACKGROUND: Colorectal cancer (CRC) harboring BRAFV600E mutation exhibits low response to conventional therapy and poorest prognosis. Due to the emerging correlation between gut microbiota and CRC carcinogenesis, we investigated in serrated BRAFV600E cases the existence of a peculiar fecal microbial fingerprint and specific bacterial markers, which might represent a tool for the development of more effective clinical strategies. METHODS: By injecting human CRC stem-like cells isolated from BRAFV600E patients in immunocompromised mice, we described a new xenogeneic model of this subtype of CRC. By performing bacterial 16S rRNA sequencing, the fecal microbiota profile was then investigated either in CRC-carrying mice or in a cohort of human CRC subjects. The microbial communities' functional profile was also predicted. Data were compared with Mann-Whitney U, Welch's t-test for unequal variances and Kruskal-Wallis test with Benjamini-Hochberg false discovery rate (FDR) correction, extracted as potential BRAF class biomarkers and selected as model features. The obtained mean test prediction scores were subjected to Receiver Operating characteristic (ROC) analysis. To discriminate the BRAF status, a Random Forest classifier (RF) was employed. RESULTS: A specific microbial signature distinctive for BRAF status emerged, being the BRAF-mutated cases closer to healthy controls than BRAF wild-type counterpart. In agreement, a considerable score of correlation was also pointed out between bacteria abundance from BRAF-mutated cases and the level of markers distinctive of BRAFV600E pathway, including those involved in inflammation, innate immune response and epithelial-mesenchymal transition. We provide evidence that two candidate bacterial markers, Prevotella enoeca and Ruthenibacterium lactatiformans, more abundant in BRAFV600E and BRAF wild-type subjects respectively, emerged as single factors with the best performance in distinguishing BRAF status (AUROC = 0.72 and 0.74, respectively, 95% confidence interval). Furthermore, the combination of the 10 differentially represented microorganisms between the two groups improved performance in discriminating serrated CRC driven by BRAF mutation from BRAF wild-type CRC cases (AUROC = 0.85, 95% confidence interval, 0.69-1.01). CONCLUSION: Overall, our results suggest that BRAFV600E mutation itself drives a distinctive gut microbiota signature and provide predictive CRC-associated bacterial biomarkers able to discriminate BRAF status in CRC patients and, thus, useful to devise non-invasive patient-selective diagnostic strategies and patient-tailored optimized therapies.

Drug Delivery in an Orthotopic Tumor Stem Cell-Based Model of Human Glioblastoma.

Grade IV gliomas, also known as glioblastoma multiforme (GBM), are incurable, lethal brain tumors, whose average life expectancy is around 15 months. There is a desperate need for a better understanding of the basic biology of these tumors, in order to devise novel, more specific and effective therapeutics. The handling of GBM represents a daunting challenge to clinicians, also considering the few therapeutic options available, none of which can significantly alter the inevitable lethal outcome of these tumors. Hence, the development of effective therapies would greatly benefit from the availability of in vivo GBM models that can reliably mimic the characteristics of malignant cells and the features of the human disease. Candidate new drugs have to be tested in these in vivo models by adopting settings concerning direct intra-brain delivery in order to define their overall therapeutic efficacy under clinical-like conditions. Here, we describe local intracranial delivery of drugs by osmotic mini-pumps.

Stemness underpinning all steps of human colorectal cancer defines the core of effective therapeutic strategies.

BACKGROUND: Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. METHODS AND FINDINGS: We perfected a cell system of stable, primary lines from human CRC showing that they possess the full complement of ex- and in-vivo, in xenogeneic models, characteristics of CRC stem cells (CCSCs). Here we show how tumor-initiating, CCSCs cells can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). INTERPRETATION: Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, cCCSCs and mCCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. FUND: This work was financially supported by grants from "Ministero della Salute Italiano"(GR-2011-02351534, RC1703IC36 and RC1803IC35) to Elena Binda and from "Associazione Italiana Cancro" (IG-14368) Angelo L. Vescovi. None of the above funders have any role in study design, data collection, data analysis, interpretation, writing the project.

Novel SEC61G-EGFR Fusion Gene in Pediatric Ependymomas Discovered by Clonal Expansion of Stem Cells in Absence of Exogenous Mitogens.

The basis for molecular and cellular heterogeneity in ependymomas of the central nervous system is not understood. This study suggests a basis for this phenomenon in the selection for mitogen-independent (MI) stem-like cells with impaired proliferation but increased intracranial tumorigenicity. MI ependymoma cell lines created by selection for EGF/FGF2-independent proliferation exhibited constitutive activation of EGFR, AKT, and STAT3 and sensitization to the antiproliferative effects of EGFR tyrosine kinase inhibitors (TKI). One highly tumorigenic MI line harbored membrane-bound, constitutively active, truncated EGFR. Two EGFR mutants (ΔN566 and ΔN599) were identified as products of intrachromosomal rearrangements fusing the 3' coding portion of the EGFR gene to the 5'-UTR of the SEC61G, yielding products lacking the entire extracellular ligand-binding domain of the receptor while retaining the transmembrane and tyrosine kinase domains. EGFR TKI efficiently targeted ΔN566/ΔN599-mutant-mediated signaling and prolonged the survival of mice bearing intracranial xenografts of MI cells harboring these mutations. RT-PCR sequencing of 16 childhood ependymoma samples identified SEC61G-EGFR chimeric mRNAs in one infratentorial ependymoma WHO III, arguing that this fusion occurs in a small proportion of these tumors. Our findings demonstrate how in vitro culture selections applied to genetically heterogeneous tumors can help identify focal mutations that are potentially pharmaceutically actionable in rare cancers. Cancer Res; 77(21); 5860-72. ©2017 AACR.

Wnt5a Drives an Invasive Phenotype in Human Glioblastoma Stem-like Cells.

Brain invasion by glioblastoma determines prognosis, recurrence, and lethality in patients, but no master factor coordinating the invasive properties of glioblastoma has been identified. Here we report evidence favoring such a role for the noncanonical WNT family member Wnt5a. We found the most invasive gliomas to be characterized by Wnt5a overexpression, which correlated with poor prognosis and also discriminated infiltrating mesenchymal glioblastoma from poorly motile proneural and classical glioblastoma. Indeed, Wnt5a overexpression associated with tumor-promoting stem-like characteristics (TPC) in defining the character of highly infiltrating mesenchymal glioblastoma cells (Wnt5aHigh). Inhibiting Wnt5a in mesenchymal glioblastoma TPC suppressed their infiltrating capability. Conversely, enforcing high levels of Wnt5a activated an infiltrative, mesenchymal-like program in classical glioblastoma TPC and Wnt5aLow mesenchymal TPC. In intracranial mouse xenograft models of glioblastoma, inhibiting Wnt5a activity blocked brain invasion and increased host survival. Overall, our results highlight Wnt5a as a master regulator of brain invasion, specifically TPC, and they provide a therapeutic rationale to target it in patients with glioblastoma. Cancer Res; 77(4); 996-1007. ©2016 AACR.

N-terminal and C-terminal domains of calmodulin mediate FADD and TRADD interaction.

FADD (Fas-associated death domain) and TRADD (Tumor Necrosis Factor Receptor 1-associated death domain) proteins are important regulators of cell fate in mammalian cells. They are both involved in death receptors mediated signaling pathways and have been linked to the Toll-like receptor family and innate immunity. Here we identify and characterize by database search analysis, mutagenesis and calmodulin (CaM) pull-down assays a calcium-dependent CaM binding site in the α-helices 1-2 of TRADD death domain. We also show that oxidation of CaM methionines drastically reduces CaM affinity for FADD and TRADD suggesting that oxidation might regulate CaM-FADD and CaM-TRADD interactions. Finally, using Met-to-Leu CaM mutants and binding assays we show that both the N- and C-terminal domains of CaM are important for binding.