Aberrant activation of the PI3K/mTOR pathway promotes resistance to sorafenib in AML.

Therapy directed against oncogenic FLT3 has been shown to induce response in patients with acute myeloid leukemia (AML), but these responses are almost always transient. To address the mechanism of FLT3 inhibitor resistance, we generated two resistant AML cell lines by sustained treatment with the FLT3 inhibitor sorafenib. Parental cell lines carry the FLT3-ITD (tandem duplication) mutation and are highly responsive to FLT3 inhibitors, whereas resistant cell lines display resistance to multiple FLT3 inhibitors. Sanger sequencing and protein mass-spectrometry did not identify any acquired mutations in FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, whereas it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer FLT3 dependent. Gene expression analysis of sensitive and resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway in the resistant phenotype, which was further supported by next-generation sequencing and phospho-specific-antibody array analysis. Furthermore, a selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation, and induced apoptosis in resistant cell lines. Gedatolisib significantly extended survival of mice in a sorafenib-resistant AML patient-derived xenograft model. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway in FLT3-ITD-dependent AML results in resistance to drugs targeting FLT3.

SYK regulates mTOR signaling in AML.

Spleen tyrosine kinase (SYK) was recently identified as a new target in acute myeloid leukemia (AML); however, its mechanistic role in this disease is poorly understood. Based on the known interaction between SYK and mammalian target of rapamycin (mTOR) signaling in lymphoma, we hypothesized that SYK may regulate mTOR signaling in AML. Both small-molecule inhibition of SYK and SYK-directed shRNA suppressed mTOR and its downstream signaling effectors, as well as its upstream activator, AKT. Moreover, the inhibition of multiple nodes of the phosphatidylinositol 3'-kinase (PI3K) signaling pathway enhanced the effects of SYK suppression on AML cell viability and differentiation. Evaluation of the collateral mitogen-activated protein kinase (MAPK) pathway revealed a heterogeneous response to SYK inhibition in AML with downregulation of MEK and extracellular signal-regulated kinase (ERK) phosphorylation in some AML cell lines but a paradoxical increase in MEK/ERK phosphorylation in RAS-mutated AML. These studies reveal SYK as a regulator of mTOR and MAPK signaling in AML and demonstrate that inhibition of PI3K pathway activity enhances the effects of SYK inhibition on AML cell viability and differentiation.

Sestrin2 integrates Akt and mTOR signaling to protect cells against energetic stress-induced death.

The phosphoinositide-3 kinase/Akt (PI3K/Akt) pathway has a central role in cancer cell metabolism and proliferation. More importantly, it is one of the cardinal pro-survival pathways mediating resistance to apoptosis. The role of Akt in response to an energetic stress is presently unclear. Here, we show that Sestrin2 (Sesn2), also known as Hi95, a p53 target gene that protects cells against oxidative and genotoxic stresses, participates in the protective role of Akt in response to an energetic stress induced by 2-deoxyglucose (2-DG). Sesn2 is upregulated in response to an energetic stress such as 2-DG and metformin, and mediates the inhibition of mammalian target of rapamycin (mTOR), the major cellular regulator of energy metabolism. The increase of Sesn2 is independent of p53 but requires the anti-apoptotic pathway, PI3K/Akt. Inhibition of Akt, as well as loss of Sesn2, sensitizes cells to 2-DG-induced apoptosis. In addition, the rescue of Sesn2 partially reverses the pro-apoptotic effects of 2-DG. In conclusion, we identify Sesn2 as a new energetic stress sensor, which appears to be protective against energetic stress-induced apoptosis that integrates the pro-survival function of Akt and the negative regulation of mTOR.

The caspase 6 derived N-terminal fragment of DJ-1 promotes apoptosis via increased ROS production.

In pathological conditions, the amount of DJ-1 determines whether a cell can survive or engage a cell death program. This is exemplified in epithelial cancers, in which DJ-1 expression is increased, while autosomal recessive early onset Parkinson's disease mutations of DJ-1 generally lead to decreased stability and expression of the protein. We have shown previously that DJ-1 is cleaved by caspase-6 during induction of apoptosis. We demonstrate here that the N-terminal cleaved fragment of DJ-1 (DJ-1 Nt) is specifically expressed in the nucleus and promotes apoptosis in SH-SY5Y neuroblastoma cell lines. In addition, overexpression of DJ-1 Nt in different cell lines leads to a loss of clonogenic potential and sensitizes to staurosporin and 1-methyl-4-phenylpyridinium (MPP+)-mediated caspase activation and apoptosis. Importantly, inhibition of endogenous DJ-1 expression with sh-RNA or DJ-1 deficiency mimics the effect of DJ-1 Nt on cell growth and apoptosis. Moreover, overexpression of DJ-1 Nt increases reactive oxygen species (ROS) production, and sensitizes to MPP+-mediated apoptosis and DJ-1 oxidation. Finally, specific exclusion of DJ-1 Nt from the nucleus abrogates its pro-apoptotic effect. Taken together, our findings identify an original pathway by which generation of a nuclear fragment of DJ-1 through caspase 6-mediated cleavage induces ROS-dependent amplification of apoptosis.

The dual mTORC1 and mTORC2 inhibitor AZD8055 has anti-tumor activity in acute myeloid leukemia.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34+ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.

Metformin inhibits melanoma development through autophagy and apoptosis mechanisms.

Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24 h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72 h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96 h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.

SPARC functions as an anti-stress factor by inactivating p53 through Akt-mediated MDM2 phosphorylation to promote melanoma cell survival.

Aberrant expression of Secreted Protein Acidic and Rich in Cysteine (SPARC)/osteonectin has been associated with an invasive tumor cell phenotype and poor outcome in human melanomas. Although it is known that SPARC controls melanoma tumorigenesis, the precise role of SPARC in melanoma cell survival is still unclear. Here, we show that SPARC has a cell-autonomous survival activity, which requires Akt-dependent regulation of p53. Suppression of SPARC by RNA interference in several human melanoma cells and xenografted A375 tumors triggers apoptotic cell death through the mitochondrial intrinsic pathway and activation of caspase-3. Cell death induced by depletion of SPARC is dependent on p53 and induction of Bax, and results in the generation of ROS. Stabilization of p53 in SPARC-depleted cells is associated with a decrease in Akt-mediated activating phosphorylation of MDM2. Inhibition of Akt signaling pathway is important for the observed changes as overexpression of constitutively active Akt protects cells against apoptosis induced by SPARC depletion. Conversely, increased expression of SPARC stimulates Akt and MDM2 phosphorylation, thus facilitating p53 degradation. Finally, we show that overexpression of SPARC renders cells more resistant to the p53-mediated cytotoxic effects of the DNA-damaging drug actinomycin-D. Our study indicates that SPARC functions through activation of Akt and MDM2 to limit p53 levels and that acquired expression of SPARC during melanoma development would confer survival advantages through suppression of p53-dependent apoptotic pathways.

Ciglitazone negatively regulates CXCL1 signaling through MITF to suppress melanoma growth.

We have previously demonstrated that the thiazolidinedione ciglitazone inhibited, independently of PPARγ activation, melanoma cell growth. Further investigations now show that ciglitazone effects are mediated through the regulation of secreted factors. Q-PCR screening of several genes involved in melanoma biology reveals that ciglitazone inhibits expression of the CXCL1 chemokine gene. CXCL1 is overexpressed in melanoma and contributes to tumorigenicity. We show that ciglitazone induces a diminution of CXCL1 level in different human melanoma cell lines. This effect is mediated by the downregulation of microphthalmia-associated transcription factor, MITF, the master gene in melanocyte differentiation and involved in melanoma development. Further, recombinant CXCL1 protein is sufficient to abrogate thiazolidinedione effects such as apoptosis induction, whereas extinction of the CXCL1 pathway mimics phenotypic changes observed in response to ciglitazone. Finally, inhibition of human melanoma tumor development in nude mice treated with ciglitazone is associated with a strong decrease in MITF and CXCL1 levels. Our results show that anti-melanoma effects of thiazolidinediones involve an inhibition of the MITF/CXCL1 axis and highlight the key role of this specific pathway in melanoma malignancy.

Cathepsin B release after imatinib-mediated lysosomal membrane permeabilization triggers BCR-ABL cleavage and elimination of chronic myelogenous leukemia cells.

Imatinib is the leading compound to treat patients with chronic myelogenous leukemia (CML) but the exact mechanism of its anti-leukemic effect is incompletely elucidated. Through inhibition of BCR-ABL, Imatinib blocks several downstream pathways and induces apoptosis of BCR-ABL positive cells. In this study, we analyzed further the mode of action of Imatinib in different appropriate cellular models of CML either sensitive or resistant to Imatinib and in CD34+ cells from CML patients. Pharmacological or short hairpin RNA-mediated inhibition of BCR-ABL triggers lysosomal membrane permeabilization (LMP) that culminates in activation and redistribution of Cathepsin B (CB) into the cytoplasm of CML cells, in which it triggers directly BCR-ABL degradation. Pharmacological inhibition of CB by CA-074Me or small interfering RNA-mediated knock-down of CB partly protects K562 cells from Imatinib-induced cell death and CB overexpression sensitizes these cells to Imatinib killing. Strikingly, Imatinib-triggered LMP, CB activation and BCR-ABL cleavage in CD34+ cells from CML patients and inhibition of CB confers protection against cell death in clonogenic assays of CD34+ primary cells from CML patients. Hence, we describe an original pathway by which Imatinib participates to the elimination of CML cells through LMP and CB-mediated specific degradation of BCR-ABL.

Inhibition of imatinib-mediated apoptosis by the caspase-cleaved form of the tyrosine kinase Lyn in chronic myelogenous leukemia cells.

Once cleaved by caspases, the Lyn tyrosine kinase (LynDeltaN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynDeltaN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynDeltaN or its catalytically inactive counterpart LynDeltaNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynDeltaN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynDeltaN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynDeltaN was suppressed by two inhibitors of Lyn activity. LynDeltaN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR-ABL pathway.

Apoptosis and erythroid differentiation triggered by Bcr-Abl inhibitors in CML cell lines are fully distinguishable processes that exhibit different sensitivity to caspase inhibition.

Imatinib targets the Bcr-Abl oncogene that causes chronic myelogenous leukemia (CML) in humans. Recently, we demonstrated that besides triggering apoptosis in K562 cells, imatinib also mediated their erythroid differentiation. Although both events appear to proceed concomitantly, it is not known at present whether or not imatinib-induced apoptosis and differentiation are interdependent processes. Hence, we investigated the requirements for Bcr-Abl inhibitor-mediated apoptosis and erythroid differentiation in several established and engineered CML cell lines. Imatinib triggered apoptosis and erythroid differentiation of different CML cell lines, but only apoptosis exhibited sensitivity to ZVAD-fmk inhibition. Conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor, SB202190, significantly slowed down erythroid differentiation without affecting caspase activation. Furthermore, imatinib and PD166326, another Bcr-Abl inhibitory molecule, triggered erythroid differentiation of K562 cell clones, nevertheless resistant to Bcr-Abl inhibitor-induced apoptosis. Finally, short hairpin RNA inhibitor (shRNAi) silencing of caspase 3 efficiently inhibited caspase activity but had no effect on erythroid differentiation, whereas silencing of Bcr-Abl mimicked imatinib or PD166326 treatment, leading to increased apoptosis and erythroid differentiation of K562 cells. Taken together, our findings not only demonstrate that Bcr-Abl inhibitor-mediated apoptosis and differentiation are fully distinguishable events, but also that caspases are dispensable for erythroid differentiation of established CML cell lines.

[Epithelioid hemangioendothelioma: disease course in 3 cases]. / Hémangioendotheéliome épithélioïde: évolution dans 3 cas.

INTRODUCTION: Epithelioid hemangioendothelioma is an uncommon vascular neoplasm, one of intermediate-grade malignancy. Cutaneous epithelioid hemangioendothelioma is rare and often associated with multiple site involvement. CASES: Three patients with cutaneous EHE without systemic involvement in one case, with skin, liver and lung tumoral lesions in two others cases are reported. One patient is in complete remission after one year. The second patient is stable without treatment after ten years. In the third patient, alpha interferon given for one year don't produce effective results and the lesions do not progress without treatment after eight years of follow-up. DISCUSSION: Cutaneous presentation of EHE is quite variable. After the diagnosis is done, systemic involvement must be detected specially in bone, liver or lung. Metastatic spread or mulitcentric origin of the tumor are a matter of controversy. The pathobiologic behavior of EHE is not clearly recognized. Little data are available regarding the results of treatment with alpha interferon.

[Acquired localized epidermolysis bullosa. A case with scalp involvement and immunoelectron microscopic study]. / Epidermolyse bulleuse acquise localisée. Un cas avec atteinte du cuir chevelu étudié en immunomicroscopie électronique.

The case of a 59-year old man who had a clinical Brunsting-Perry pemphigoid localized to the scalp is described. Direct immunoelectron microscopy demonstrated IgG and C3 immune deposits in the anchoring fibril zone, as in epidermolysis bullosa acquisita. No circulating antibodies were detected by Western immunoblotting on epidermal and dermal extracts. These findings show that Brunsting-Perry pemphigoid is not an immunological entity and that it widens the clinical spectrum of epidermolysis bullosa acquisita.