Jill-Patrice Cassuto

Resveratrol Promotes Autophagic Cell Death in Chronic Myelogenous Leukemia Cells via JNK-Mediated p62/SQSTM1 Expression and AMPK Activation

Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress, cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62), but its role in the regulation of autophagy is controversial. Here, we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK, thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly, p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV, and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy.

A survey of the signaling pathways involved in megakaryocytic differentiation of the human K562 leukemia cell line by molecular and c-DNA array analysis.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.

Imatinib mesylate-resistant human chronic myelogenous leukemia cell lines exhibit high sensitivity to the phytoalexin resveratrol

Imatinib is successfully used in the treat ment of chronic myelogenous leukemia (CML), and the main mechanisms of resistance in refractory patients are now partially understood. In the present study, we investigated the mechanism of action of resveratrol in imatinib‐sensitive (IM‐S) and ‐resistant (IM‐R) CML cell lines. Resveratrol induced loss of viability and apopto sis in IM‐S and IM‐R in a time‐ and dose‐dependent fashion. Inhibition of cell viability was detected for concentrations of resveratrol as low as 5 μ,M, and the IC50 values for viability, clonogenic assays, apoptosis, and erythroid differentiation were in the 10–25 μ,M range. The effect of imatinib and resveratrol was additive in IM‐S but not in IM‐R clones in which the resveratrol effect was already maximal. The effect of resveratrol on apoptosis was partially rescued by zVAD‐ fmk, suggesting a caspase‐independent contribution. Resveratrol action was independent of BCR‐ABL ex pression and phosphorylation, and in agreement was additive to BCR‐ABL silencing. Finally, phytoalexin inhibited the growth of BaF3 cells expressing mutant BCR‐ABL proteins found in resistant patients, including the multiresistant T315I mutation. Our findings show that resveratrol induces apoptosis, caspase‐inde‐ pendent death, and differentiation that collectively contribute to the specific elimination of CML cells. Resveratrol should provide therapeutic benefits in IM‐R patients and in other hematopoietic malignancies.— Puissant, A., Grosso, S., Jacquel, A., Belhacene, N., Colosetti, P., Cassuto, J.‐P., Auberger, P. Imatinib mesylate‐resistant human chronic myelogenous leuke mia cell lines exhibit high sensitivity to the phytoalexin resveratrol. FASEB J. 22, 1894–1904 (2008)

Autophagy is an important event for megakaryocytic differentiation of the chronic myelogenous leukemia K562 cell line

Autophagy is a highly conserved catabolic process for the elimination and recycling of organelles and macromolecules, characterized by the formation of double membrane vesicles called autophagosomes. To date, the function of autophagy in cell differentiation is poorly documented. Here, we investigated the possibility that megakaryocytic differentiation of the Chronic Myelogenous Leukemia (CML) cell line K562, a process known to be accompanied by accumulation of vacuoles inside the cells, might involve autophagy. We show using various complementary approaches that the combination of the phorbol ester PMA and the p38MAPK inhibitor SB202190 (SB) which engaged a majority of K562 cells towards the megakaryocytic lineage also triggered vacuolization and autophagy. The combination of PMA+SB appears to induces both increase in autophagic fluxes and an autophagic degradation blockage. Induction of autophagy was accompanied also by increased expression of Beclin-1 and p62/SQSTM1 and was found to precede the onset of megakaryocytic differentiation. Moreover, knock-down of LC3 and Beclin-1 by specific siRNAs impaired PMA+SB-mediated vacuolization, LC3-II accumulation and megakaryocytic differentiation, as well. To the best of our knowledge, this is the first description that induction of autophagy is involved in megakaryocytic differentiation of K562 CML cells.

Visceral leishmaniasis: a new opportunistic infection in hematopoietic stem-cell-transplanted patients

Visceral leishmaniasis: a new opportunistic infection in hematopoietic stem-cell-transplanted patients

Persistent Activation of the Fyn/ERK Kinase Signaling Axis Mediates Imatinib Resistance in Chronic Myelogenous Leukemia Cells through Upregulation of Intracellular SPARC

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization, growth factor availability, cell adhesion, differentiation, and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study, we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)-resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity, whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably, we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly, SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings, increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together, our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC.

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.

Azacitidine-resistant SKM1 myeloid cells are defective for AZA-induced mitochondrial apoptosis and autophagy

Azacitidine (AZA) is the current treatment for patients with high-risk myelodysplastic syndrome, but resistance is a common feature of AZA-treated patients. To investigate the mechanisms associated with AZA resistance in vitro, we generated AZA-resistant SKM1 myeloid cells, called hereafter AZA-R. AZA-R cells exhibit impaired mitochondrial membrane permeabilization and caspase activation in response to AZA compared to their AZA-sensitive (AZA-S) counterpart. AZA induced LC3-II accumulation and cathepsin B activity in AZA-S cells, two hallmarks of autophagy. AZA-R cells displayed increased basal autophagy but are resistant to AZA-mediated autophagy. Inhibition of autophagy using LC3 siRNA revealed that autophagy is protective in AZA-S cells and AZA-R cells in basal conditions. By contrast, AZA-R cells exhibited impaired autophagy in response to AZA. Collectively, our findings indicate that AZA promotes apoptosis and autophagy in SKM1 cells, and that AZA-R cells are resistant to both apoptosis and autophagy induced by AZA.

Mechanism of action of the multikinase inhibitor Foretinib

Mitotic catastrophe (MC) is induced when stressed cells enter prematurely or inappropriately into mitosis and can be caused by ionizing radiation and anticancer drugs. Foretinib is a multikinase inhibitor whose mechanism of action is incompletely understood. We investigated here the effect of Foretinib on chronic myelogenous leukemia (CML) cell lines either sensitive (IM-S) or resistant (IM-R) to the tyrosine kinase inhibitor Imatinib. Foretinib decreased viability and clonogenic potential of IM-S and IM-R CML cells as well. Foretinib-treated cells exhibited increased size, spindle assembly checkpoint anomalies and enhanced ploidy that collectively evoked mitotic catastrophe (MC). Accordingly, Foretinib-stimulated CML cells displayed decreased expression of Cdk1, Cyclin B1 and Plk1. In addition, Foretinib triggered caspase-2 activation that precedes mitochondrial membrane permeabilization. Accordingly, z-VAD-fmk and a caspase-2 siRNA abolished Foretinib-mediated cell death but failed to affect MC, indicating that Foretinib-mediated apoptosis and MC are two independent events. Anisomycin, a JNK activator, impaired Foretinib-induced MC and inhibition or knockdown of JNK phenotyped its effect on MC. Moreover, we found that Foretinib acted as a potent inhibitor of JNK. Importantly, Foretinib exhibited no or very little effect on normal peripheral blood mononuclear cells, monocytes or melanocytes cells but efficiently inhibited the clonogenic potential of CD34+ cell from CML patients. Collectively, our data show that the multikinase inhibitor Foretinib induces MC in CML cells and other cell lines via JNK-dependent inhibition of Plk1 expression and triggered apoptosis by a caspase 2-mediated mechanism. This unusual mechanism of action may have important implications for the treatment of cancer.

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 (LynΔN) 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 LynΔN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynΔN or its catalytically inactive counterpart LynΔNKD. 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 LynΔN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynΔN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynΔN was suppressed by two inhibitors of Lyn activity. LynΔN 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.