AC-73 and Syrosingopine Inhibit SARS-CoV-2 Entry into Megakaryocytes by Targeting CD147 and MCT4.

Coagulation disorders are described in COVID-19 and long COVID patients. In particular, SARS-CoV-2 infection in megakaryocytes, which are precursors of platelets involved in thrombotic events in COVID-19, long COVID and, in rare cases, in vaccinated individuals, requires further investigation, particularly with the emergence of new SARS-CoV-2 variants. CD147, involved in the regulation of inflammation and required to fight virus infection, can facilitate SARS-CoV-2 entry into megakaryocytes. MCT4, a co-binding protein of CD147 and a key player in the glycolytic metabolism, could also play a role in SARS-CoV-2 infection. Here, we investigated the susceptibility of megakaryocytes to SARS-CoV-2 infection via CD147 and MCT4. We performed infection of Dami cells and human CD34+ hematopoietic progenitor cells induced to megakaryocytic differentiation with SARS-CoV-2 pseudovirus in the presence of AC-73 and syrosingopine, respective inhibitors of CD147 and MCT4 and inducers of autophagy, a process essential in megakaryocyte differentiation. Both AC-73 and syrosingopine enhance autophagy during differentiation but only AC-73 enhances megakaryocytic maturation. Importantly, we found that AC-73 or syrosingopine significantly inhibits SARS-CoV-2 infection of megakaryocytes. Altogether, our data indicate AC-73 and syrosingopine as inhibitors of SARS-CoV-2 infection via CD147 and MCT4 that can be used to prevent SARS-CoV-2 binding and entry into megakaryocytes, which are precursors of platelets involved in COVID-19-associated coagulopathy.

Advances in Understanding the Links between Metabolism and Autophagy in Acute Myeloid Leukemia: From Biology to Therapeutic Targeting.

Autophagy is a highly conserved cellular degradation process that regulates cellular metabolism and homeostasis under normal and pathophysiological conditions. Autophagy and metabolism are linked in the hematopoietic system, playing a fundamental role in the self-renewal, survival, and differentiation of hematopoietic stem and progenitor cells, and in cell death, particularly affecting the cellular fate of the hematopoietic stem cell pool. In leukemia, autophagy sustains leukemic cell growth, contributes to survival of leukemic stem cells and chemotherapy resistance. The high frequency of disease relapse caused by relapse-initiating leukemic cells resistant to therapy occurs in acute myeloid leukemia (AML), and depends on the AML subtypes and treatments used. Targeting autophagy may represent a promising strategy to overcome therapeutic resistance in AML, for which prognosis remains poor. In this review, we illustrate the role of autophagy and the impact of its deregulation on the metabolism of normal and leukemic hematopoietic cells. We report updates on the contribution of autophagy to AML development and relapse, and the latest evidence indicating autophagy-related genes as potential prognostic predictors and drivers of AML. We review the recent advances in autophagy manipulation, combined with various anti-leukemia therapies, for an effective autophagy-targeted therapy for AML.

CD147 Targeting by AC-73 Induces Autophagy and Reduces Intestinal Fibrosis Associated with TNBS Chronic Colitis.

BACKGROUND AND AIMS: Intestinal fibrosis is a common complication of inflammatory bowel diseases. Medical treatment of intestinal fibrosis is an unmet therapeutic need. CD147 overexpression can induce myofibroblast differentiation associated with extracellular matrix deposition, favouring the development of fibrosis. To understand whether CD147 may promote intestinal fibrosis, we analysed its expression and blocked its function by using its specific inhibitor AC-73 [3-{2-[([1,1'-biphenyl]-4-ylmethyl) amino]-1-hydroxyethyl} phenol] in the murine TNBS [trinitrobenzenesulfonic acid]-chronic colitis model associated with intestinal fibrosis. METHODS: TNBS chronic colitis was induced by weekly intrarectal administration of escalating doses of TNBS. Ethanol-treated and untreated mice were used as controls. Separated groups of TNBS, ethanol-treated or untreated mice received AC-73 or vehicle administered intraperitoneally from day 21 to day 49. At day 49, mice were killed, and colons collected for histological analysis, protein and RNA extraction. CD147, α-SMA and activated TGF-ß1 protein levels, CD147/ERK/STAT3 signalling pathway and autophagy were assessed by Western blot, collagen and inflammatory/fibrogenic cytokines mRNA tissue content by quantitative PCR. RESULTS: In mice with chronic TNBS colitis, CD147 protein level increased during fibrosis development in colonic tissue, as compared to control mice. CD147 inhibition by AC-73 treatment reduced intestinal fibrosis, collagen and cytokine mRNA tissue content, without significant modulation of activated TGF-ß1 protein tissue content. AC-73 inhibited CD147/ERK1/2 and STAT3 signalling pathway activation and induced autophagy. CONCLUSIONS: CD147 is a potential new target for controlling intestinal fibrosis and its inhibitor, AC-73, might represent a potential new anti-fibrotic therapeutic option in IBD.

Targeting Lactate Metabolism by Inhibiting MCT1 or MCT4 Impairs Leukemic Cell Proliferation, Induces Two Different Related Death-Pathways and Increases Chemotherapeutic Sensitivity of Acute Myeloid Leukemia Cells.

Metabolism in acute myeloid leukemia (AML) cells is dependent primarily on oxidative phosphorylation. However, in order to sustain their high proliferation rate and metabolic demand, leukemic blasts use a number of metabolic strategies, including glycolytic metabolism. Understanding whether monocarboxylate transporters MCT1 and MCT4, which remove the excess of lactate produced by cancer cells, represent new hematological targets, and whether their respective inhibitors, AR-C155858 and syrosingopine, can be useful in leukemia therapy, may reveal a novel treatment strategy for patients with AML. We analyzed MCT1 and MCT4 expression and function in hematopoietic progenitor cells from healthy cord blood, in several leukemic cell lines and in primary leukemic blasts from patients with AML, and investigated the effects of AR-C155858 and syrosingopine, used alone or in combination with arabinosylcytosine, on leukemic cell proliferation. We found an inverse correlation between MCT1 and MCT4 expression levels in leukemic cells, and showed that MCT4 overexpression is associated with poor prognosis in AML patients. We also found that AR-C155858 and syrosingopine inhibit leukemic cell proliferation by activating two different cell-death related pathways, i.e., necrosis for AR-C155858 treatment and autophagy for syrosingopine, and showed that AR-C155858 and syrosingopine exert an anti-proliferative effect, additive to chemotherapy, by enhancing leukemic cells sensitivity to chemotherapeutic agents. Altogether, our study shows that inhibition of MCT1 or MCT4 impairs leukemic cell proliferation, suggesting that targeting lactate metabolism may be a new therapeutic strategy for AML, and points to MCT4 as a potential therapeutic target in AML patients and to syrosingopine as a new anti-proliferative drug and inducer of autophagy to be used in combination with conventional chemotherapeutic agents in AML treatment.

The small-molecule compound AC-73 targeting CD147 inhibits leukemic cell proliferation, induces autophagy and increases the chemotherapeutic sensitivity of acute myeloid leukemia cells.

CD147 is a transmembrane glycoprotein with multiple functions in human healthy tissues and diseases, in particular in cancer. Overexpression of CD147 correlates with biological functions that promote tumor progression and confers resistance to chemotherapeutic drugs. In contrast to solid tumors, the role of CD147 has not been extensively studied in leukemia. Understanding whether CD147 represents a new hematologic target and whether its inhibitor AC-73 may be used in leukemia therapy may reveal an alternative treatment strategy in patients with acute myeloid leukemia (AML). We analyzed CD147 expression and function in hematopoietic progenitor cells from normal cord blood, in several leukemic cell lines and in primary leukemic blasts obtained from patients with AML. We investigated the effects of AC-73, used alone or in combination with arabinosylcytosine (Ara-C) and arsenic trioxide (ATO), on leukemic cell proliferation. We demonstrated that CD147 overexpression promotes leukemic cell proliferation. We showed that AC-73 exhibits a potent growth inhibitory activity in leukemic cells, by inhibiting the ERK/STAT3 activation pathway and activating autophagy. We demonstrated that AC-73 exerts an anti-proliferative effect additive to chemotherapy by enhancing leukemic cell sensitivity to Ara-C-induced cytotoxicity or to ATO-induced autophagy. We also reported CD147 expression in the fraction of leukemic blasts expressing CD371, a marker of leukemic stem cells. Altogether, our study indicates CD147 as a novel potential target in the treatment of AML and AC-73 as an anti-proliferative drug and an inducer of autophagy in leukemic cells to use in combination with chemotherapeutic agents.

The forkhead box C1 (FOXC1) transcription factor is downregulated in acute promyelocytic leukemia.

Forkhead box (FOX) genes encode transcription factors, which regulate embryogenesis and play an important role in hematopoietic differentiation and in mesenchymal niche maintenance. Overexpression of the family member FOXC1 has been reported in solid tumors and acute myeloid leukemia (AML). We studied FOXC1 expression and function in acute promyelocytic leukemia (APL) and normal hematopoietic progenitors. FOXC1 mRNA and protein levels were significantly lower in primary marrow samples from 27 APL patients, as compared to samples obtained from 27 patients with other AML subtypes, and 5 normal CD34+ hematopoietic cells. FOXC1 expression significantly increased in APL samples at the time of remission following consolidation treatment. In cell lines overexpressing PML-RARA, and in the NB4 t(15;17)-positive cell line, FOXC1 expression was lower than in other non-APL cell lines, and increased following treatment with all-trans retinoic acid (ATRA), due to functional binding of ATRA to the FOXC1 promoter region. Reduced FOXC1 expression was also associated to DNA hypermethylation of the +354 to +568 FOXC1 region, both in primary APL, and in NB4 cells. Treatment of NB4 cells with decitabine demethylated FOXC1 and upregulated its expression. Our findings indicate that FOXC1 is consistently repressed in APL due to hypermethylation and the presence of the PML-RARA rearrangement. A potential role of hypomethylating treatment in advanced APL remains to be established.

Endothelial progenitor cells in hematologic malignancies.

Studies carried out in the last years have improved the understanding of the cellular and molecular mechanisms controlling angiogenesis during adult life in normal and pathological conditions. Some of these studies have led to the identification of some progenitor cells that sustain angiogenesis through indirect, paracrine mechanisms (hematopoietic angiogenic cells) and through direct mechanisms, i.e., through their capacity to generate a progeny of phenotypically and functionally competent endothelial cells [endothelial colony forming cells (ECFCs)]. The contribution of these progenitors to angiogenetic processes under physiological and pathological conditions is intensively investigated. Angiogenetic mechanisms are stimulated in various hematological malignancies, including chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myelodysplastic syndromes and multiple myeloma, resulting in an increased angiogenesis that contributes to disease progression. In some of these conditions there is preliminary evidence that some endothelial cells could derive from the malignant clone, thus leading to the speculation that the leukemic cell derives from the malignant transformation of a hemangioblastic progenitor, i.e., of a cell capable of differentiation to the hematopoietic and to the endothelial cell lineages. Our understanding of the mechanisms underlying increased angiogenesis in these malignancies not only contributed to a better knowledge of the mechanisms responsible for tumor progression, but also offered the way for the discovery of new therapeutic targets.

PML-RAR alpha induces the downmodulation of HHEX: a key event responsible for the induction of an angiogenetic response.

BACKGROUND: Recent studies indicate that angiogenesis is important in the pathogenesis of acute myeloid leukemias (AMLs). Among the various AMLs, the bone marrow angiogenetic response is particularly pronounced in acute promyelocytic leukemia (APL). However, the molecular mechanisms responsible for this angiogenetic response are largely unknown. In the present study, we have explored the role of HHEX, a homeodomain transcription factor, as a possible mediator of the pro-angiogenetic response observed in APL. This transcription factor seems to represent an ideal candidate for this biologic function because it is targeted by PML-RARα, is capable of interaction with PML and PML-RARα, and acts as a regulator of the angiogenetic response. METHODS: We used various cellular systems of APL, including primary APL cells and leukemic cells engineered to express PML-RARα, to explore the role of the PML-RARα fusion protein on HHEX expression. Molecular and biochemical techniques have been used to investigate the mechanisms through which PML-RARα downmodulates HHEX and the functional consequences of this downmodulation at the level of the expression of various angiogenetic genes, cell proliferation and differentiation. RESULTS: Our results show that HHEX expression is clearly downmodulated in APL and that this effect is directly mediated by a repressive targeting of the HHEX gene promoter by PML-RARα. Studies carried out in primary APL cells and in a cell line model of APL with inducible PML-RARα expression directly support the view that this fusion protein through HHEX downmodulation stimulates the expression of various genes involved in angiogenesis and inhibits cell differentiation. CONCLUSIONS: Our data suggest that HHEX downmodulation by PML-RARα is a key event during APL pathogenesis.

Differential hypoxic regulation of the microRNA-146a/CXCR4 pathway in normal and leukemic monocytic cells: impact on response to chemotherapy.

High expression of the chemokine receptor 4, CXCR4, associated with a negative prognosis in acute myeloid leukemia, is related to hypoxia. Because CXCR4 expression is under the post-transcriptional control of microRNA-146a in normal and leukemic monocytic cells, we first investigated the impact of hypoxia on microRNA-146a and CXCR4 expression during monocytopoiesis and in acute monocytic leukemia. We then analyzed the effects of hypoxia on drug sensitivity of CXCR4-expressing leukemic cells. We found that microRNA-146a is a target of hypoxia-inducible factor-1α or -2α in relation to the stage of monocytopoiesis and the level of hypoxia, and demonstrated the regulation of the microRNA-146a/CXCR4 pathway by hypoxia in monocytes derived from CD34(+) cells. Thus, in myeloid leukemic cell lines, hypoxia-mediated control of the microRNA-146a/CXCR4 pathway depends only on the capacity of hypoxia-inducible factor-1α to up-regulate microRNA-146a, which in turn decreases CXCR4 expression. However, at variance with normal monocytic cells and leukemic cell lines, in acute monocytic leukemia overexpressing CXCR4, hypoxia up-modulates microRNA-146a but fails to down-modulate CXCR4 expression. We then investigated the effect of hypoxia on the response of leukemic cells to chemotherapy alone or in combination with stromal-derived factor-1α. We found that hypoxia increases stromal-derived factor-1α-induced survival of leukemic cells by decreasing their sensitivity to anti-leukemic drugs. Altogether, our results demonstrate that hypoxia-mediated regulation of microRNA-146a, which controls CXCR4 expression in monocytic cells, is lost in acute monocytic leukemia, thus contributing to maintaining CXCR4 overexpression and protecting the cells from anti-leukemic drugs in the hypoxic bone marrow microenvironment.

Salinomycin potentiates the cytotoxic effects of TRAIL on glioblastoma cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to exhibit therapeutic activity in cancer. However, many tumors remain resistant to treatment with TRAIL. Therefore, small molecules that potentiate the cytotoxic effects of TRAIL could be used for combinatorial therapy. Here we found that the ionophore antibiotic salinomycin acts in synergism with TRAIL, enhancing TRAIL-induced apoptosis in glioma cells. Treatment with low doses of salinomycin in combination with TRAIL augmented the activation of caspase-3 and increased TRAIL-R2 cell surface expression. TRAIL-R2 upmodulation was required for mediating the stimulatory effect of salinomycin on TRAIL-mediated apoptosis, since it was abrogated by siRNA-mediated TRAIL-R2 knockdown. Salinomycin in synergism with TRAIL exerts a marked anti-tumor effect in nude mice xenografted with human glioblastoma cells. Our results suggest that the combination of TRAIL and salinomycin may be a useful tool to overcome TRAIL resistance in glioma cells and may represent a potential drug for treatment of these tumors. Importantly, salinomycin+TRAIL were able to induce cell death of well-defined glioblastoma stem-like lines.

Autocrine role of angiopoietins during megakaryocytic differentiation.

The tyrosine kinase Tie-2 and its ligands Angiopoietins (Angs) transduce critical signals for angiogenesis in endothelial cells. This receptor and Ang-1 are coexpressed in hematopoietic stem cells and in a subset of megakaryocytes, though a possible role of angiopoietins in megakaryocytic differentiation/proliferation remains to be demonstrated. To investigate a possible effect of Ang-1/Ang-2 on megakaryocytic proliferation/differentiation we have used both normal CD34(+) cells induced to megakaryocytic differentiation and the UT7 cells engineered to express the thrombopoietin receptor (TPOR, also known as c-mpl, UT7/mpl). Our results indicate that Ang-1/Ang-2 may have a role in megakaryopoiesis. Particularly, Ang-2 is predominantly produced and released by immature normal megakaryocytic cells and by undifferentiated UT7/mpl cells and slightly stimulated TPO-induced cell proliferation. Ang-1 production is markedly induced during megakaryocytic differentiation/maturation and potentiated TPO-driven megakaryocytic differentiation. Blocking endogenously released angiopoietins partially inhibited megakaryocytic differentiation, particularly for that concerns the process of polyploidization. According to these data it is suggested that an autocrine angiopoietin/Tie-2 loop controls megakaryocytic proliferation and differentiation.

A small molecule SMAC mimic LBW242 potentiates TRAIL- and anticancer drug-mediated cell death of ovarian cancer cells.

BACKGROUND: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (SMAC) has been described to sensitize for apoptosis. We have explored the pro-apoptotic activity of LBW242, a mimic of SMAC/DIABLO, on ovarian cancer cell lines (A2780 cells and its chemoresistant derivative A2780/ADR, SKOV3 and HEY cells) and in primary ovarian cancer cells. The effects of LBW242 on ovarian cancer cell lines and primary ovarian cancer cells was determined by cell proliferation, apoptosis and biochemical assays. PRINCIPAL FINDINGS: LBW242 added alone elicited only a moderate pro-apoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or anticancer drugs in inducing apoptosis of both ovarian cancer cell lines and primary ovarian cancer cells. Mechanistic studies show that LBW242-induced apoptosis in ovarian cancer cells is associated with activation of caspase-8. In line with this mechanism, c-FLIP overexpression inhibits LBW242-mediated apoptosis. CONCLUSION: LBW242 sensitizes ovarian cancer cells to the antitumor effects of TRAIL and anticancer drugs commonly used in clinic. These observations suggest that the SMAC/DIABLO mimic LBW242 could be of value for the development of experimental strategies for treatment of ovarian cancer.

CDDO-Im is a stimulator of megakaryocytic differentiation.

Although the triterpene CDDO and its potent derivatives, CDDO-Im and CDDO-Me, are now in phase I/II studies in the treatment of some pathological conditions, their effects on normal hematopoiesis are not known. In the present study we provide evidence that CDDO-Im exerts in vitro a potent inhibitory effect on erythroid cell proliferation and survival and a stimulatory action on megakaryocytic differentiation. The effect of CDDO-Im on erythroid and megakaryocytic differentiation was evaluated both on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid (E) or megakaryocytic (Mk) differentiation and on erythroleukemic cell lines HEL and TF1. The inhibitory effect of CDDO-Im on erythroid cell survival and proliferation is mainly related to a reduced GATA-1 expression. This conclusion is supported by the observation that GATA-1 overexpressing TF1 cells are partially protected from the inhibitory effect of CDDO-Im on cell proliferation and survival. The stimulatory effect of CDDO-Im on normal megakaryopoiesis is seemingly related to upmodulation of GATA2 expression and induction of mitogen-activated protein kinases ERK1/2.

Mechanism of human Hb switching: a possible role of the kit receptor/miR 221-222 complex.

BACKGROUND: The human hemoglobin switch (HbF-->HbA) takes place in the peri/post-natal period. In adult life, however, the residual HbF (<1%) may be partially reactivated by chemical inducers and/or cytokines such as the kit ligand (KL). MicroRNAs (miRs) play a pivotal role in normal hematopoiesis: downmodulation of miR-221/222 stimulates human erythropoietic proliferation through upmodulation of the kit receptor. DESIGN AND METHODS: We have explored the possible role of kit/KL in perinatal Hb switching by evaluating: i) the expression levels of both kit and kit ligand on CD34(+) cells and in plasma isolated from pre-, mid- and full-term cord blood samples; ii) the reactivation of HbF synthesis in KL-treated unilineage erythroid cell cultures; iii) the functional role of miR-221/222 in HbF production. RESULTS: In perinatal life, kit expression showed a gradual decline directly correlated to the decrease of HbF (from 80-90% to <30%). Moreover, in full-term cord blood erythroid cultures, kit ligand induced a marked increase of HbF (up to 80%) specifically abrogated by addition of the kit inhibitor imatinib, thus reversing the Hb switch. MiR-221/222 expression exhibited rising levels during peri/post-natal development. In functional studies, overexpression of these miRs in cord blood progenitors caused a remarkable decrease in kit expression, erythroblast proliferation and HbF content, whereas their suppression induced opposite effects. CONCLUSIONS: Our studies indicate that human perinatal Hb switching is under control of the kit receptor/miR 221-222 complex. We do not exclude, however, that other mechanisms (i.e. glucocorticoids and the HbF inhibitor BCL11A) may also contribute to the peri/post-natal Hb switch.

Primary ovarian cancer cells are sensitive to the proaptotic effects of proteasome inhibitors.

Resistance of tumors to cell death signals poses a complex clinical problem. In the present study, we have explored the capacity of proteasome inhibitors to induce cell death of ovarian cancer cells. We explored the sensitivity of primary ovarian cancer cells to a combination of bortezomib (also known as PS-341), a proteasome inhibitor and TRAIL, a death ligand, or mapatumumab or lexatumumab, TRAIL-R1 or TRAIL-R2 targeting agonist monoclonal antibodies, respectively. The results of our study showed that the large majority of primary ovarian cancers are clearly sensitive to the pro-apoptotic action of bortezomib, whose effects are potentiated by the concomitant addition of TRAIL or mapatumumab or lexatumumab. Interestingly, both cisplatin and paclitaxel-chemosensitive and chemoresistant ovarian tumors are equally sensitive to the cytotoxic effect of bortezomib. Bortezomib, combined with TRAIL or TRAIL-R1 or TRAIL-R2 agonist monoclonal antibodies may be a useful treatment for refractory ovarian cancer.

High sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide.

In the present study we have explored the sensitivity of ovarian cancer cells to the synthetic triterpenoid CDDO-Imidazolide (CDDO-Im). For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/ADR and A2780/CISP, OVCAR3, SKOV3 and HEY cancer cell lines and primary ovarian cancer cells, providing evidence that: (i) the majority of these cell lines are highly sensitive to the pro-apoptotic effects induced by CDDO-Im; (ii) TRAIL, added alone exerted only a weak proapoptotic, but clearly potentiated the cytotoxic effect elicited by CDDO-Im; (iii) the apoptotic effect induced by CDDO-Im involves GSH depletion, c-FLIP downmodulation and caspase-8 activation; (iv) CDDO-Im inhibits STAT3 activation and CDDO-Im sensitivity is inversely related to the level of constitutive STAT3 activation. Importantly, studies on primary ovarian cancer cells have shown that these cells are sensitive to the pro-apoptotic effects of CDDO-Im. These observations support the experimental use of synthetic triterpenoids in the treatment of ovarian cancer.

Colocalization of the VEGF-R2 and the common IL-3/GM-CSF receptor beta chain to lipid rafts leads to enhanced p38 activation.

Previous studies suggested an important role for vascular endothelial growth factor (VEGF) and its receptors in postnatal haemopoiesis. However, it is unclear how VEGF receptor (VEGFR) signalling could interact with that issued from the activation of haematopoietic growth factor receptors. To elucidate this point we explored VEGF-R2 and granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) membrane localization and cell signalling in TF1-KDR cells (TF1 leukaemic cells that overexpress VEGF-R2/KDR). Activation of either GM-CSFR or VEGF-R2 was shown to determine the migration of both receptor elements (VEGF-R2 and the common beta-chain of the GM-CSFR) to lipid rafts. The study of receptor phosphorylation showed that GM-CSF induced the phosphorylation of its own receptor and the transphosphorylation of VEGF-R2; on the other hand, VEGF triggered the phosphorylation of its receptor and transphosphorylated the beta-chain of the GM-CSFR. Co-stimulation of TF1-KDR cells with both GM-CSF and VEGF-A resulted in massive migration of both the common GM-CSFR beta-chain and VEGF-R2 to lipid rafts and sustained p38 mitogen-activated protein kinase activation. Disruption of lipid rafts inhibited the capacity of both GM-CSF and VEGF-A to activate p38. Experiments with specific p38 inhibitors showed that p38 activation was required to sustain the VEGF- and GM-CSF-dependent proliferation of TF1-KDR and the survival of primary acute myeloid leukaemia blasts.

Expression of Tie-2 and other receptors for endothelial growth factors in acute myeloid leukemias is associated with monocytic features of leukemic blasts.

We investigated the expression of Tie-2 in primary blasts from 111 patients with acute myeloid leukemia (AML) to evaluate a possible linkage between the expression of this receptor and the immunophenotypic and biologic properties of leukemic blasts. Tie-2 was expressed at moderate and high levels in 39 and 23 of 111 AMLs, respectively. The analysis of the immunophenotype clearly showed that Tie-2 expression in AML was associated with monocytic features. Interestingly, Tie-2 expression on AML blasts was associated with concomitant expression of other receptors for endothelial growth factors, such as vascular endothelial growth factor receptor 1 (VEGF-R1), -R2, and -R3. Tie-2(+) AMLs were characterized by high blast cell counts at diagnosis, a high frequency of Flt3 mutations, and increased Flt3 expression. The survival of Tie-2(+) AMLs is sustained through an autocrine pattern involving Angiopoietin-1 and Tie-2, as suggested by experiments showing induction of apoptosis in Tie-2(+) AMLs by agents preventing the binding of angiopoietins to Tie-2. Finally, the in vitro growth of Tie-2(+) AMLs in endothelial culture medium supplemented with VEGF and angiopoietins resulted in their partial endothelial differentiation. These observations suggest that Tie-2(+) AMLs pertain to a mixed monocytic/endothelial lineage, derived from the malignant transformation of the normal counterpart represented by monocytic cells expressing endothelial markers. The autocrine angiopoietin/Tie-2 axis may represent a promising therapeutic target to improve the outcome of patients with monocytic AML. Disclosure of potential conflicts of interest is found at the end of this article.

A small molecule Smac mimic potentiates TRAIL-mediated cell death of ovarian cancer cells.

OBJECTIVES: Ovarian cancer remains a leading cause of death in women and development of new therapies is essential. Second mitochondria derived activator of caspase (Smac) has been described to sensitize for apoptosis. We have explored the proapoptotic activity of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines (A2780 cells and its chemoresistant derivatives A2780/ADR and A2780/DDP), cancer cell lines and in primary ovarian cancer cells. METHODS: The effects of a small molecule mimic of Smac/DIABLO on ovarian cancer cell lines and primary ovarian cancer cells were determined by cell proliferation, apoptosis and biochemical assays. RESULTS: This compound added alone elicited only a weak proapoptotic effect; however, it strongly synergizes with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAILR2 antibody (Lexatumumab) in inducing apoptosis of ovarian cancer cells. CONCLUSIONS: These observations suggest that small molecule mimic of Smac/DIABLO could be useful for the development of experimental strategies aiming to treat ovarian cancer. Interestingly, in addition to its well known proapoptotic effects, Smac/DIABLO elicited a significant increase of pro-caspase-3 levels.

Proteasome inhibitors sensitize ovarian cancer cells to TRAIL induced apoptosis.

In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.