Immunomodulatory Factors Control the Fate of Melanoma Tumor Initiating Cells.

Melanoma is a highly heterogeneous tumor for which recent evidence supports a model of dynamic stemness. Melanoma cells might temporally acquire tumor-initiating properties or switch from a status of tumor-initiating cells (TICs) to a more differentiated one depending on the tumor context. However, factors driving these functional changes are still unknown. We focused on the role of cyto/chemokines in shaping TICs isolated directly from tumor specimens of two melanoma patients, namely Me14346S and Me15888S. We analyzed the secretion profile of TICs and of their corresponding melanoma differentiated cells and we tested the ability of cyto/chemokines to influence TIC self-renewal and differentiation. We found that TICs, grown in vitro as melanospheres, had a complex secretory profile as compared to their differentiated counterparts. Some factors, such as CCL-2 and IL-8, also produced by adherent melanoma cells and melanocytes did not influence TIC properties. Conversely, IL-6, released by differentiated cells, reduced TIC self-renewal and induced TIC differentiation while IL-10, produced by Me15888S, strongly promoted TIC self-renewal through paracrine/autocrine actions. Complete neutralization of IL-10 activity by gene silencing and antibody-mediated blocking of the IL-10Rα was required to sensitize Me15888S to IL-6-induced differentiation. For the first time these results show that functional heterogeneity of melanoma could be directly influenced by inflammatory and suppressive soluble factors, with IL-6 favoring TIC differentiation, and IL-10 supporting TIC self-renewal. Thus, understanding the tumor microenvironment (TME) role in modulating melanoma TIC phenotype is fundamental to identifying novel therapeutic targets to achieve long-lasting regression of metastatic melanoma. Stem Cells 2016;34:2449-2460.

Induction of death receptor 5 expression in tumor vasculature by perifosine restores the vascular disruption activity of TRAIL-expressing CD34(+) cells.

The proapoptotic death receptor 5 (DR5) expressed by tumor associated endothelial cells (TECs) mediates vascular disrupting effects of human CD34(+) cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL (+) cells) in mice. Indeed, lack of DR5 on TECs causes resistance to CD34-TRAIL (+) cells. By xenografting in nonobese diabetic/severe combined immunodeficient mice the TRAIL-resistant lymphoma cell line SU-DHL-4V, which generates tumors lacking endothelial DR5 expression, here we demonstrate for the first time that the Akt inhibitor perifosine induces in vivo DR5 expression on TECs, thereby overcoming tumor resistance to the vascular disruption activity of CD34-TRAIL (+) cells. In fact, CD34-TRAIL (+) cells combined with perifosine, but not CD34-TRAIL (+) cells alone, exerted marked antivascular effects and caused a threefold increase of hemorrhagic necrosis in SU-DHL-4V tumors. Consistent with lack of DR5 expression, CD34-TRAIL (+) cells failed to affect the growth of SU-DHL-4V tumors, but CD34-TRAIL (+) cells plus perifosine reduced tumor volumes by 60 % compared with controls. In view of future clinical studies using membrane-bound TRAIL, our results highlight a strategy to rescue patients with primary or acquired resistance due to the lack of DR5 expression in tumor vasculature.

Human CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand target both tumor cells and tumor vasculature.

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

Gene signatures of circulating breast cancer cell models are a source of novel molecular determinants of metastasis and improve circulating tumor cell detection in patients.

BACKGROUND: Progression to stage IV disease remains the main cause of breast cancer-related deaths. Increasing knowledge on the hematogenous phase of metastasis is key for exploiting the entire window of opportunity to interfere with early dissemination and to achieve a more effective disease control. Recent evidence suggests that circulating tumor cells (CTCs) possess diverse adaptive mechanisms to survive in blood and eventually metastasize, encouraging research into CTC-directed therapies. METHODS: On the hypothesis that the distinguishing molecular features of CTCs reveal useful information on metastasis biology and disease outcome, we compared the transcriptome of CTCs, primary tumors, lymph-node and lung metastases of the MDA-MB-231 xenograft model, and assessed the biological role of a panel of selected genes, by in vitro and in vivo functional assays, and their clinical significance in M0 and M+ breast cancer patients. RESULTS: We found that hematogenous dissemination is governed by a transcriptional program and identified a CTC signature that includes 192 up-regulated genes, mainly related to cell plasticity and adaptation, and 282 down-regulated genes, involved in chromatin remodeling and transcription. Among genes up-regulated in CTCs, FADS3 was found to increases cell membrane fluidity and promote hematogenous diffusion and lung metastasis formation. TFF3 was observed to be associated with a subset of CTCs with epithelial-like features in the experimental model and in a cohort of 44 breast cancer patients, and to play a role in cell migration, invasion and blood-borne dissemination. The analysis of clinical samples with a panel of CTC-specific genes (ADPRHL1, ELF3, FCF1, TFF1 and TFF3) considerably improved CTC detection as compared with epithelial and tumor-associated markers both in M0 and stage IV patients, and CTC kinetics informed disease relapse in the neoadjuvant setting. CONCLUSIONS: Our findings provide evidence on the potential of a CTC-specific molecular profile as source of metastasis-relevant genes in breast cancer experimental models and in patients. Thanks to transcriptome analysis we generated a novel CTC signature in the MDA-MB-231 xenograft model, adding a new piece to the current knowledge on the key players that orchestrate tumor cell hematogenous dissemination and breast cancer metastasis, and expanding the list of CTC-related biomarkers for future validation studies.

Senescent Thyrocytes, Similarly to Thyroid Tumor Cells, Elicit M2-like Macrophage Polarization In Vivo.

Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.

Valproic acid enhances bosutinib cytotoxicity in colon cancer cells.

Unbalanced histone deacetylase (HDAC) hyperactivity is a common feature of tumor cells. Inhibition of HDAC activity is often associated with cancer cell growth impairment and death. Valproic acid (VPA) is a HDAC inhibitor used for the treatment of epilepsy. It has recently been recognized as a promising anticancer drug. We investigated the effects of VPA on growth and survival of colon cancer cells. VPA caused growth inhibition and programmed cell death that correlated with histone hyperacetylation. VPA modulated the expression of various factors involved in cell cycle control and apoptosis and induced caspase activation. Interestingly, VPA induced downregulation of c-Src and potentiated the cytotoxic effects of the c-Src inhibitor bosutinib, both in vitro and in vivo. The combination of sublethal doses of VPA and bosutinib led to massive apoptosis of colon cancer cells, irrespective of their genetic background. These results suggest that VPA may be employed as a positive modulator of bosutinib antitumor activity in colorectal cancer.

IFN-gamma enhances the antimyeloma activity of the fully human anti-human leukocyte antigen-DR monoclonal antibody 1D09C3.

To investigate the therapeutic activity of the fully human anti-HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138(+) cells, analyzed the capacity of IFN-gamma to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-gamma. CD138(+)HLA-DR(+) cells were detected in 31 of 60 patients, with 15 of 60 patients having >/=20% CD138(+)HLA-DR(+) cells (median, 50%; range, 23-100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-gamma restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-gamma/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P

The Detection and Morphological Analysis of Circulating Tumor and Host Cells in Breast Cancer Xenograft Models.

Hematogenous dissemination may occur early in breast cancer (BC). Experimental models could clarify mechanisms, but in their development, the heterogeneity of this neoplasia must be considered. Here, we describe circulating tumor cells (CTCs) and the metastatic behavior of several BC cell lines in xenografts. MDA-MB-231, BT-474, MDA-MB-453 and MDA-MB-468 cells were injected at the orthotopic level in immunocompromised mice. CTCs were isolated using a size-based method and identified by cytomorphological criteria. Metastases were detected by COX IV immunohistochemistry. CTCs were detected in 90% of animals in each model. In MDA-MB-231, CTCs were observed after 5 weeks from the injection and step wisely increased at later time points. In animals injected with less aggressive cell lines, the load of single CTCs (mean ± SD CTCs/mL: 1.8 ± 1.3 in BT-474, 122.2 ± 278.5 in MDA-MB-453, 3.4 ± 2.5 in MDA-MB468) and the frequency of CTC clusters (overall 38%) were lower compared to MDA-MB231 (946.9 ± 2882.1; 73%). All models had lung metastases, MDA-MB-453 and MDA-MB468 had ovarian foci too, whereas lymph nodal involvement was observed in MDA-MB231 and MDA-MB-468 only. Interestingly, CTCs showed morphological heterogeneity and were rarely associated to host cells. Orthotopic xenograft of BC cell lines offers valid models of hematogenous dissemination and a possible experimental setting to study CTC-blood microenvironment interactions.

Metallothionein 1G acts as an oncosupressor in papillary thyroid carcinoma.

The molecular pathogenesis of tumors arising from the thyroid follicular epithelial cells, including papillary (PTC) and follicular thyroid carcinoma (FTC), is only partially understood, and the role of tumor suppressor genes has not yet been assessed. The metallothionein (MT) gene family encodes a class of metal-binding proteins involved in several cellular processes, and their expression is often deregulated in human tumors. Recently, downregulation of MT gene expression in PTC has been reported, suggesting a possible oncosuppressor role of this gene family in the pathogenesis of thyroid tumors. To further explore this possibility, we performed expression and functional studies. Analysis of microarray data of thyroid tumors of different histologic types showed that several MT genes were downregulated with respect to normal tissue. The microarray data were corroborated by quantitative PCR experiments, showing downregulation of MTs in PTC and FTC, but to a greater extent in papillary carcinoma. The expression of MTs was also investigated at the protein level by immunohistochemistry; the results were consistent with the microarray data, showing general downregulation in tumor samples, which was more evident in PTC. The functional consequence of MT downregulation was addressed employing an experimental model made of the PTC-derived K1 cell line in which MT1G expression is repressed by promoter methylation. Restoration of MT1G expression by cDNA transfection affected growth rate and in vivo tumorigenicity of K1 cells, indicating an oncosuppressor role for MT1G in thyroid papillary tumorigenesis.

Characterization of compound 584, an Abl kinase inhibitor with lasting effects.

BACKGROUND: Resistance to imatinib is an important clinical issue in the treatment of Philadelphia chromosome-positive leukemias which is being tackled by the development of new, more potent drugs, such as the dual Src/Abl tyrosine kinase inhibitors dasatinib and bosutinib and the imatinib analog nilotinib. In the current study we describe the design, synthesis and biological properties of an imatinib analog with a chlorine-substituted benzamide, namely compound 584 (cmp-584). DESIGN AND METHODS: To increase the potency, we rationally designed cmp-584, a compound with enhanced shape complementarity with the kinase domain of Abl. cmp-584 was synthesized and characterized in vitro against a panel of 67 serine/threonine and tyrosine kinases using radioactive and enzyme-linked immunosorbent kinase assays. We studied inhibitory cellular activity using Bcr/Abl-positive human cell lines, murine transfectants in proliferation experiments, and a murine xenotrans-planted model. Kinase assays on isolated Bcr/Abl protein were also performed. Finally, we used a wash-out approach on whole cells to study the binding kinetics of the inhibitor. RESULTS: cmp-584 showed potent anti-Abl activity both on recombinant protein (IC(50): 8 nM) and in cell-based assays (IC(50): 0.1-10 nM). The drug maintained inhibitory activity against platelet-derived growth factor receptors and c-KIT and was also active against Lyn (IC(50): 301 nM). No other kinase of the panel was inhibited at nanomolar doses. cmp-584 was 20- to 300-fold more active than imatinib in cells. This superior activity was evident in intact cells, in which full-length Bcr-Abl is present. In vivo experiments confirmed the activity of cmp-584. Wash-out experiments showed that short exposure to the drug impaired cell proliferation and Bcr-Abl phosphorylation for a substantially longer period of time than imatinib. CONCLUSIONS: The present results suggest a slower off-rate (dissociation rate) of cmp-584 compared to imatinib as an explanation for the increased cellular activity of the former.

In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor, against imatinib-resistant Bcr-Abl+ neoplastic cells.

Resistance to imatinib represents an important scientific and clinical issue in chronic myelogenous leukemia. In the present study, the effects of the novel inhibitor SKI-606 on various models of resistance to imatinib were studied. SKI-606 proved to be an active inhibitor of Bcr-Abl in several chronic myelogenous leukemia cell lines and transfectants, with IC(50) values in the low nanomolar range, 1 to 2 logs lower than those obtained with imatinib. Cells expressing activated forms of KIT or platelet-derived growth factor receptor (PDGFR), two additional targets of imatinib, were unaffected by SKI-606, whereas activity was found against PIM2. SKI-606 retained activity in cells where resistance to imatinib was caused by BCR-ABL gene amplification and in three of four Bcr-Abl point mutants tested. In vivo experiments confirmed SKI-606 activity in models where resistance was not caused by mutations as well as in cells carrying the Y253F, E255K, and D276G mutations. Modeling considerations attribute the superior activity of SKI-606 to its ability to bind a conformation of Bcr-Abl different from imatinib.

The anti-human leukocyte antigen-DR monoclonal antibody 1D09C3 activates the mitochondrial cell death pathway and exerts a potent antitumor activity in lymphoma-bearing nonobese diabetic/severe combined immunodeficient mice.

The fully human anti-HLA-DR antibody 1D09C3 has been shown to delay lymphoma cell growth in severe combined immunodeficient (SCID) mice. The present study was aimed at (a) investigating the mechanism(s) of 1D09C3-induced cell death and (b) further exploring the therapeutic efficacy of 1D09C3 in nonobese diabetic (NOD)/SCID mice. The chronic lymphocytic leukemia cell line JVM-2 and the mantle cell lymphoma cell line GRANTA-519 were used. Generation of reactive oxygen species (ROS) and mitochondrial membrane depolarization were measured by flow cytometry following cell incubation with dihydroethidium and TMRE, respectively. Western blot analysis was used to detect c-Jun-NH(2)-kinase (JNK) phosphorylation and apoptosis-inducing factor (AIF). NOD/SCID mice were used to investigate the activity of 1D09C3 in early- or advanced-stage tumor xenografts. In vitro, 1D09C3-induced cell death involves a cascade of events, including ROS increase, JNK activation, mitochondrial membrane depolarization, and AIF release from mitochondria. Inhibition of JNK activity significantly reduced 1D09C3-induced apoptosis, indicating that 1D09C3 activity involves activation of the kinase. In vivo, 1D09C3 induces long-term disease-free survival in a significant proportion of tumor-bearing mice treated at an early stage of disease. Treatment of mice bearing advanced-stage lymphoma results in a highly significant prolongation of survival. These data show that 1D09C3 (a) exerts a potent antitumor effect by activating ROS-dependent, JNK-driven cell death, (b) cures the great majority of mice treated at an early-stage of disease, and (c) significantly prolongs survival of mice with advanced-stage disease.

CD52 antigen expressed by malignant plasma cells can be targeted by alemtuzumab in vivo in NOD/SCID mice.

OBJECTIVE: To explore new treatments specifically targeting malignant plasma cells (PCs), we examined CD52 antigen expression on primary PCs as well as multiple myeloma (MM) cell lines, and investigated in vivo the antimyeloma activity of alemtuzumab. MATERIALS AND METHODS: PCs were enriched from the marrow of MM patients (n = 39) according to CD138 expression and then analyzed by 3-color flow cytometry and quantitative PCR. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. RESULTS: CD52 expression revealed a substantial heterogeneity in terms of both percentage of positive cells and fluorescence intensity, with 25/39 (64%) MM patients showing >or=30% CD138(+) PCs expressing the CD52 antigen (mean = 79%; range, 33-100%). Similarly to primary cells, cell lines showed heterogeneous CD52 expression. Expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. In vivo, alemtuzumab treatment significantly increased the median survival of animals with an early- (64 vs 77 days, p

Sodium 4-Carboxymethoxyimino-(4-HPR) a Novel Water-Soluble Derivative of 4-Oxo-4-HPR Endowed with In Vivo Anticancer Activity on Solid Tumors.

4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR), an active polar metabolite of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR), was shown to exert promising antitumor activity through at least two independent mechanisms of action. Specifically, differently from 4-HPR and other retinoids, 4-oxo-4-HPR targets microtubules and inhibits tubulin polymerization causing mitotic arrest and on the other hand, analogously to the parent drug, it induces apoptosis through the activation of a signaling cascade involving the generation of reactive oxygen species (ROS). However, the potential in vivo use of 4-oxo-4-HPR is impaired by its poor solubility. By chemical modification of 4-oxo-4-HPR, a new class of compounds with improved solubility and in vivo bioavailability was obtained. We demonstrated here that, among them, the most promising molecule, sodium 4-carboxymethoxyimino-(4-HPR), was endowed with in vitro antitumor efficacy and entirely preserved the double mechanism of action of the parent drug in cancer cells of different histotypes. In fact, the retinoid induced the activation of the apoptotic cascade related to the generation of ROS through endoplasmic reticulum stress response and upregulation of phospho c-Jun N-terminal kinases and PLAcental Bone morphogenetic protein, leading to cell death through caspase-3 cleavage. Otherwise, sodium 4-carboxymethoxyimino-(4-HPR) caused a marked mitotic arrest coupled with multipolar spindle formation and tubulin depolymerization. To assess the compound antitumor activity, in vivo experiments were performed in three mouse xenograft models (ovarian and breast cancers and mesothelioma). The in vivo results demonstrated that retinoid administration as single agent significantly increased the survival in ovarian cancer xenografts, induced a statistically significant decrease in tumor growth in breast cancer xenografts, and caused a 30% reduction in tumor growth in a mesothelioma mouse model. Even though further studies investigating sodium 4-carboxymethoxyimino-(4-HPR) toxicity and in vitro and in vivo activities in combination with other drugs are required, the double mechanism of action of the retinoid coupled with its in vivo antitumor efficacy and potential low toxicity suggest a promising therapeutic potential for the compound in different solid tumors.

Targeting COPZ1 non-oncogene addiction counteracts the viability of thyroid tumor cells.

Thyroid carcinoma is generally associated with good prognosis, but no effective treatments are currently available for aggressive forms not cured by standard therapy. To find novel therapeutic targets for this tumor type, we had previously performed a siRNA-based functional screening to identify genes essential for sustaining the oncogenic phenotype of thyroid tumor cells, but not required to the same extent for the viability of normal cells (non-oncogene addiction paradigm). Among those, we found the coatomer protein complex ζ1 (COPZ1) gene, which is involved in intracellular traffic, autophagy and lipid homeostasis. In this paper, we investigated the mechanisms through which COPZ1 depletion leads to thyroid tumor cell death. We showed that siRNA-mediated COPZ1 depletion causes abortive autophagy, endoplasmic reticulum stress, unfolded protein response and apoptosis. Interestingly, we observed that mouse tumor xenografts, locally treated with siRNA targeting COPZ1, showed a significant reduction of tumor growth. On the whole, we demonstrated for the first time the crucial role of COPZ1 in the viability of thyroid tumor cells, suggesting that it may be considered an attractive target for novel therapeutic approaches for thyroid cancer.

Antitumor activity of human CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of transformed cells while sparing normal cells. To enhance the therapeutic index of soluble (s)TRAIL, we used CD34+ cells transduced with a replication-deficient adenovirus encoding the human TRAIL gene (CD34-TRAIL+) for the systemic delivery of membrane-bound (m)TRAIL to lymphoid tumors. CD34-TRAIL+ cells were evaluated for their activity in vitro and in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice xenografted with sTRAIL-sensitive and -resistant tumors. In vitro, coculturing CD34-TRAIL+ cells with sTRAIL-sensitive or -resistant lymphoma cell lines induced significant levels of caspase-dependent tumor cell death. In vivo, CD34-TRAIL+ cells significantly increased the survival of NOD/SCID mice bearing sTRAIL-sensitive or -resistant lymphoid tumors at an early or advanced stage of disease. No obvious toxicity was observed on administration of CD34-TRAIL+ cells. Histological analysis revealed high-level expression of the agonistic receptor TRAIL-R2 by tumor endothelial cells, and efficient tumor homing of transduced cells. Injection of CD34-TRAIL+ cells resulted in extensive damage of tumor vasculature followed by hemorrhagic necrosis exhibiting a perivascular distribution. These results show that CD34-TRAIL+ cells might be an efficient vehicle for mTRAIL delivery to tumors, where they exert a potent antitumor effect possibly mediated by both direct tumor cell killing and indirect vascular-disrupting mechanisms.

Inhibition of RET tyrosine kinase by SU5416.

Thyroid neoplasia is frequently associated with rearranged during transfection (RET) proto-oncogene mutations that cause hyperactivation of RET kinase activity. Selective inhibition of RET-mediated signaling should lead to an efficacious therapy. SU5416 is a potent inhibitor of vascular endothelial cell growth factor receptor, c-Kit, and FLT-3 receptor tyrosine kinases presently used in clinical trials. We found that SU5416 inhibits RET with similar potency, both in cell-free assays and in cells, thus causing proliferation arrest in oncogenic RET-transfected cells and in papillary thyroid carcinoma (PTC) cells expressing the RET/PTC1 oncogene, but not in RET-negative control cells. SU5416 inhibited RET-mediated signaling through the extracellular signal regulated kinase (ERK) and JNK pathways. In addition, we show that a naturally occurring MEN2 mutation at codon 804 confers resistance to SU5416, but not to the related compound SU4984. We provide a possible explanation to these results by using molecular docking. Finally, SU5416 was also assessed against an array of 52 tyrosine and serine/threonine kinases.

Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral blood progenitor cells in mice.

Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (P < or = 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined administration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P < or = 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a significant increase (P < or = 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 10(5) rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P < or = 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

Water-soluble derivatives of 4-oxo-N-(4-hydroxyphenyl) retinamide: synthesis and biological activity.

A novel series of 4-oxo-N-(4-hydroxyphenyl) retinamide (4-oxo-4-HPR) derivatives were synthesized with the aim of increasing the poor solubility of the parent compound in biological fluids, while maintaining the cytotoxic activity and the dual mechanism of action. The most promising compound 13a showed antiproliferative/apoptotic activity. The analysis of its mechanism of action revealed that it retained the particular characteristic of 4-oxo-4-HPR which is able to induce cell cycle arrest during the mitotic phase, coupled with the formation of aberrant mitotic spindles.

Primary cross-resistance to BRAFV600E-, MEK1/2- and PI3K/mTOR-specific inhibitors in BRAF-mutant melanoma cells counteracted by dual pathway blockade.

Intrinsic cross-resistance to inhibition of different signaling pathways may hamper development of combinatorial treatments in melanoma, but the relative frequency of this phenotype and the strategies to overcome this hurdle remain poorly understood. Among 49 BRAF-mutant melanoma cell lines from patients not previously treated with target therapy, 21 (42.9%) showed strong primary resistance (IC50 > 1 µM) to a BRAFV600E inhibitor. Most of the BRAF-inhibitor-resistant cell lines showed also strong or intermediate cross-resistance to MEK1/2- and to PI3K/mTOR-specific inhibitors. Primary cross-resistance was confirmed in an independent set of 23 BRAF-mutant short-term melanoma cell cultures. MEK1/2 and PI3K/mTOR co-targeting was the most effective approach, compared to BRAF and PI3K/mTOR dual blockade, to counteract primary resistance to BRAF inhibition and the cross-resistant phenotype. This was shown by extensive drug interaction analysis, tumor growth inhibition assays in-vivo, p-ERK and p-AKT inhibition, promotion of melanoma apoptosis, apoptosis-related protein modulation, activation of effector caspases and selective modulation of genes involved in melanoma drug resistance and belonging to the ERK/MAPK and PI3K/AKT canonical pathways. Compared to co-targeting of mutant BRAF and PI3K/mTOR, the association of a MEK1/2 and a PI3K/mTOR inhibitor was more effective in the activation of Bax and of caspase-3 and in the induction of caspase-dependent melanoma apoptosis. Furthermore Bax silencing reduced the latter effects. These results suggest that intrinsic resistance to BRAF inhibition is frequently associated with primary cross-resistance to MEK and PI3K/mTOR blockade in BRAF-mutant melanoma and provide pre-clinical evidence for a combinatorial approach to counteract this phenotype.