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1.  SYK Is a Critical Regulator of FLT3 In Acute Myeloid Leukemia 
Cancer cell  2014;25(2):226-242.
SUMMARY
Cooperative dependencies between mutant oncoproteins and wild-type proteins are critical in cancer pathogenesis and therapy resistance. Although spleen tyrosine kinase (SYK) has been implicated in hematologic malignancies, it is rarely mutated. We used kinase activity profiling to identify collaborators of SYK in acute myeloid leukemia (AML) and determined that FMS-like tyrosine kinase 3 (FLT3) is transactivated by SYK via direct binding. Highly activated SYK is predominantly found in FLT3-ITD positive AML and cooperates with FLT3-ITD to activate MYC transcriptional programs. FLT3-ITD AML cells are more vulnerable to SYK suppression than FLT3 wild-type counterparts. In a FLT3-ITD in vivo model, SYK is indispensable for myeloproliferative disease (MPD) development, and SYK overexpression promotes overt transformation to AML and resistance to FLT3-ITD-targeted therapy.
SIGNIFICANCE
Although imatinib therapy has been paradigm shifting for treating patients with BCR-ABL-rearranged chronic myelogenous leukemia (CML), the application of targeted kinase inhibitors to treating AML has been a more complex undertaking. In this study, we identified an oncogenic partnership between the most commonly mutated kinase in AML, FLT3, and the cytoplasmic kinase SYK. SYK transactivates FLT3 by a direct physical interaction, is critical for the development of FLT3-ITD-induced myeloid neoplasia, and is more highly activated in primary human FLT3-ITD-positive AML. These studies also raise the possibility of SYK activation as a mechanism of resistance to FLT3 inhibitors, suggest FLT3 mutant AML as a subtype for SYK inhibitor testing, and nominate the clinical testing of SYK and FLT3 inhibitor combinations.
doi:10.1016/j.ccr.2014.01.022
PMCID: PMC4106711  PMID: 24525236
SYK; FLT3-ITD; AML; MYC; MPD; tyrosine kinase
2.  The small heat shock protein B8 (HSPB8) confers resistance to bortezomib by promoting autophagic removal of misfolded proteins in multiple myeloma cells 
Oncotarget  2014;5(15):6252-6266.
Velcade is one of the inescapable drug to treat patient suffering from multiple myeloma (MM) and resistance to this drug represents a major drawback for patients. However, the mechanisms underlying velcade resistance remain incompletely understood. We derived several U266 MM cell clones that resist to velcade. U266-resistant cells were resistant to velcade-induced cell death but exhibited a similar sensitivity to various proapoptotic stimuli. Careful analysis of proteosomal subunits and proteasome enzymatic activities showed that neither the composition nor the activity of the proteasome was affected in velcade-resistant cells. Elimination of velcade-induced poly-ubiquitinated proteins and protein aggregates was drastically stimulated in the resistant cells and correlated with increased cell survival. Inhibition of the lysosomal activity in velcade-resistant cells resulted in an increase of cell aggregates and decrease survival, indicating that aggregates are eliminated through lysosomal degradation. In addition, pangenomic profiling of velcade-sensitive and resistant cells showed that the small heat shock protein HSPB8 was overexpressed in resistant cells. Finally, gain and loss of function experiment demonstrated that HSPB8 is a key factor for velcade resistance. In conclusion, HSPB8 plays an important role for the elimination of aggregates in velcade-resistant cells that contributes to their enhanced survival.
PMCID: PMC4171627  PMID: 25051369
multiple myeloma; velcade; resistance; HSPB8; aggregates; autophagy
3.  Tumor suppressor function of miR-483-3p on squamous cell carcinomas due to its pro-apoptotic properties 
Cell Cycle  2013;12(14):2183-2193.
The frequent alteration of miRNA expression in many cancers, together with our recent reports showing a robust accumulation of miR-483-3p at the final stage of skin wound healing, and targeting of CDC25A leading to an arrest of keratinocyte proliferation, led us to hypothesize that miR-483-3p could also be endowed with antitumoral properties. We tested that hypothesis by documenting the in vitro and in vivo impacts of miR-483-3p in squamous cell carcinoma (SCC) cells. miR-483-3p sensitized SCC cells to serum deprivation- and drug-induced apoptosis, thus exerting potent tumor suppressor activities. Its pro-apoptotic activity was mediated by a direct targeting of several anti-apoptotic genes, such as API5, BIRC5, and RAN. Interestingly, an in vivo delivery of miR-483-3p into subcutaneous SCC xenografts significantly hampered tumor growth. This effect was explained by an inhibition of cell proliferation and an increase of apoptosis. This argues for its further use as an adjuvant in the many instances of cancers characterized by a downregulation of miR-483-3p.
doi:10.4161/cc.25330
PMCID: PMC3755068  PMID: 24067364
microRNA; tumor suppressor; apoptosis; miR-483-3p; epidermis
4.  In Vivo RNA Interference Screening Identifies a Leukemia-Specific Dependence on Integrin Beta 3 Signaling 
Cancer cell  2013;24(1):45-58.
SUMMARY
We used an in vivo short hairpin RNA (shRNA) screening approach to identify genes that are essential for MLL-AF9 acute myeloid leukemia (AML). We found that Integrin Beta 3 (Itgb3) is essential for murine leukemia cells in vivo, and for human leukemia cells in xenotransplantation studies. In leukemia cells, Itgb3 knockdown impaired homing, downregulated LSC transcriptional programs, and induced differentiation via the intracellular kinase, Syk. In contrast, loss of Itgb3 in normal HSPCs did not affect engraftment, reconstitution, or differentiation. Finally, we confirmed that Itgb3 is dispensable for normal hematopoiesis and required for leukemogenesis using an Itgb3 knockout mouse model. Our results establish the significance of the Itgb3 signaling pathway as a potential therapeutic target in AML.
doi:10.1016/j.ccr.2013.05.004
PMCID: PMC3746037  PMID: 23770013
5.  SYK Regulates mTOR Signaling in AML 
Leukemia  2013;27(11):2118-2128.
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 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 PI3K signaling pathway enhanced the effects of SYK suppression on AML cell viability and differentiation. Evaluation of the collateral MAPK pathway revealed a heterogeneous response to SYK inhibition in AML with down-regulation of MEK and 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.
doi:10.1038/leu.2013.89
PMCID: PMC4028963  PMID: 23535559
AML; SYK; mTOR; differentiation therapy; RPS6; 4E-BP1; MAPK
6.  Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition 
Cancer discovery  2013;3(3):308-323.
Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically-defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis demonstrated downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knock-down phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in three in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.
doi:10.1158/2159-8290.CD-12-0418
PMCID: PMC3672953  PMID: 23430699
BET bromodomain inhibitor; JQ1; MYCN; neuroblastoma; BRD4
7.  Targeting MYCN in Neuroblastoma by BET Bromodomain Inhibition 
Cancer discovery  2013;3(3):308-323.
Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically-defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis demonstrated downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knock-down phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in three in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma.
Significance
Biomarkers of response to small-molecule inhibitors of BET bromodomains, a new compound class with promising anti-cancer activity, have been lacking. Here, we reveal MYCN amplification as a strong genetic predictor of sensitivity to BET bromodomain inhibitors, demonstrate a mechanistic rationale for this finding, and provide a translational framework for clinical trial development of BET bromodomain inhibitors for pediatric patients with MYCN-amplified neuroblastoma.
doi:10.1158/2159-8290.CD-12-0418
PMCID: PMC3672953  PMID: 23430699
BET bromodomain inhibitor; JQ1; MYCN; neuroblastoma; BRD4
8.  The anti-apoptotic Bcl-B protein inhibits BECN1-dependent autophagic cell death 
Autophagy  2012;8(4):637-649.
Bcl-2 family members are key modulators of apoptosis that have recently been shown to also regulate autophagy. It has been previously reported that Bcl-2 and Bcl-XL bind and inhibit BECN1, an essential mediator of autophagy. Bcl-B is an anti-apoptotic member of the Bcl-2 family that possesses the four BH (Bcl-2 homology) domains (BH1, BH2, BH3 and BH4) and a predicted C-terminal trans-membrane domain. Although the anti-apoptotic properties of Bcl-B are well characterized, its physiological function remains to be established. In the present study, we first established that Bcl-B interacts with the BH3 domain of BECN1. We also showed that Bcl-B overexpression reduces autophagy triggered by a variety of pro-autophagic stimuli. This impairment of autophagy was closely related to the capacity of Bcl-B to bind to BECN1. Importantly, we have demonstrated that Bcl-B knockdown triggers autophagic cell death and sensitizes cells to amino acid starvation. The cell death induced by Bcl-B knockdown was partially dependent on components of the autophagy machinery (LC3; BECN1; ATG5). These findings reveal a new role of Bcl-B in the regulation of autophagy.
doi:10.4161/auto.19084
PMCID: PMC3405843  PMID: 22498477
BECN1; Bcl-B; amino acid starvation; apoptosis; autophagy
9.  All tyrosine kinase inhibitor-resistant chronic myelogenous cells are highly sensitive to Ponatinib 
Oncotarget  2012;3(12):1557-1565.
The advent of tyrosine kinase inhibitor (TKI) therapy has considerably improved the survival of patients suffering chronic myelogenous leukemia (CML). Indeed, inhibition of BCR-ABL by imatinib, dasatinib or nilotinib triggers durable responses in most patients suffering from this disease. Moreover, resistance to imatinib due to kinase domain mutations can be generally circumvented using dasatinib or nilotinib, but the multi-resistant T315I mutation that is insensitive to these TKIs, remains to date a major clinical problem. In this line, ponatinib (AP24534) has emerged as a promising therapeutic option in patients with all kinds of BCR-ABL mutations, especially the T315I one. However and surprisingly, the effect of ponatinib has not been extensively studied on imatinib-resistant CML cell lines. Therefore, in the present study, we used several CML cell lines with different mechanisms of resistance to TKI to evaluate the effect of ponatinib on cell viability, apoptosis and signaling. Our results show that ponatinib is highly effective on both sensitive and resistant CML cell lines, whatever the mode of resistance and also on BaF3 murine B cells carrying native BCR-ABL or T315I mutation. We conclude that ponatinib could be effectively used for all types of TKI-resistant patients.
PMCID: PMC3681494  PMID: 23238683
CML; BCR ABL; TKI; Resistance; Ponatinib
10.  When autophagy meets cancer through p62/SQSTM1 
Although p62/SQSTM1 was initially identified as an essential mediator of NFκB signaling, several recent studies have also highlighted its important role at the crossroad between the mTOR or MAPK signaling pathways and selective autophagy. The p62 structure containing important interaction domains attests to the ability of this protein to regulate and modulate the activation of these signaling pathways during tumor formation and propagation. The second very important function of this protein is to act as a molecular adaptor between the autophagic machinery and its substrates. Consequently, p62 is degraded following an increase in autophagic flux for which this protein currently serves as an indicator. However, the measurement of p62 expression strictly as a marker of autophagic flux is still controversial and can be misinterpreted mainly because this protein is subject to complex regulation at both the transcriptional and post-translational levels. Finally, because p62 is an autophagic substrate, it acts as a molecular link between cancer and autophagy by conferring a high level of selectivity through the degradation of important signaling molecules.
PMCID: PMC3410580  PMID: 22860231
Paget’s disease; mTOR; NFκB; NRF2; MAPK; Atg; ROS; ubiquitin; protein aggregates; oxidative stress
11.  BCL2L10 is a predictive factor for resistance to Azacitidine in MDS and AML patients 
Oncotarget  2012;3(4):490-501.
Azacitidine is the leading compound to treat patients suffering myelodysplastic syndrome (MDS) or AML with less than 30% of blasts, but a majority of patients is primary refractory or rapidly relapses under treatment. These patients have a drastically reduced life expectancy as compared to sensitive patients. Therefore identifying predictive factors for AZA resistance is of great interest to propose alternative therapeutic strategies for non-responsive patients. We generated AZA-resistant myeloid cell line (SKM1-R) that exhibited increased expression of BCL2L10 an anti-apoptotic Bcl-2 family member. Importantly, BCL2L10 knockdown sensitized SKM1-R cells to AZA effect suggesting that increased BCL2L10 expression is linked to AZA resistance in SKM1-R. We next established in 77 MDS patients that resistance to AZA is significantly correlated with the percentage of MDS or AML cells expressing BCL2L10. In addition, we showed that the proportion of BCL2L10 positive bone marrow cells can predict overall survival in MDS or AML patients. We propose a convenient assay in which the percentage of BCL2L10 expressing cells as assessed by flow cytometry is predictive of whether or not a patient will become resistant to AZA. Therefore, systematic determination of BCL2L10 expression could be of great interest in newly diagnosed and AZA-treated MDS patients.
PMCID: PMC3380582  PMID: 22577154
MDS; Azacitidine; resistance; BCL2L10; prognosis
12.  Acadesine Kills Chronic Myelogenous Leukemia (CML) Cells through PKC-Dependent Induction of Autophagic Cell Death 
PLoS ONE  2009;4(11):e7889.
CML is an hematopoietic stem cell disease characterized by the t(9;22) (q34;q11) translocation encoding the oncoprotein p210BCR-ABL. The effect of acadesine (AICAR, 5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside) a compound with known antileukemic effect on B cell chronic lymphoblastic leukemia (B-CLL) was investigated in different CML cell lines. Acadesine triggered loss of cell metabolism in K562, LAMA-84 and JURL-MK1 and was also effective in killing imatinib-resistant K562 cells and Ba/F3 cells carrying the T315I-BCR-ABL mutation. The anti-leukemic effect of acadesine did not involve apoptosis but required rather induction of autophagic cell death. AMPK knock-down by Sh-RNA failed to prevent the effect of acadesine, indicating an AMPK-independent mechanism. The effect of acadesine was abrogated by GF109203X and Ro-32-0432, both inhibitor of classical and new PKCs and accordingly, acadesine triggered relocation and activation of several PKC isoforms in K562 cells. In addition, this compound exhibited a potent anti-leukemic effect in clonogenic assays of CML cells in methyl cellulose and in a xenograft model of K562 cells in nude mice. In conclusion, our work identifies an original and unexpected mechanism by which acadesine triggers autophagic cell death through PKC activation. Therefore, in addition to its promising effects in B-CLL, acadesine might also be beneficial for Imatinib-resistant CML patients.
doi:10.1371/journal.pone.0007889
PMCID: PMC2775681  PMID: 19924252
13.  Dual Role of Sp3 Transcription Factor as an Inducer of Apoptosis and a Marker of Tumour Aggressiveness 
PLoS ONE  2009;4(2):e4478.
Background
The ambiguous role of transcription factor Sp3 for tumour progression is still debated since it was described as a transcriptional repressor or activator. Here we tried to decipher the molecular mechanisms implicated in Sp3 accumulation observed in aggressive tumours.
Methodology
We generated normal and tumour cell lines conditionally expressing Sp3. Cell growth was analyzed in vitro and after inoculation in nude mice. Apoptosis was assessed by pan- caspase activity assays, by counting fragmented nuclei and by determination of caspase 9 cleavage. Gene expression was determined by quantitative PCR. Cleavage by different caspases was performed after in vitro translation of the Sp3 cDNA in the presence of [S35] labelled methionine. Different tumour cell lines and head and neck tumour samples were tested for the presence of Sp3 by western blots. Correlation between Sp3 expression and overall survival has been statistically determined.
Principal Findings
Conditional over-expression of Sp3 induces apoptosis and modifies expression of genes implicated in the regulation of cell cycle and pro and anti apoptotic genes. Sp3 over-expression strongly reduces the development of tumours in nude mice confirming its pro-apoptotic potential in vivo. However, cells can survive to apoptosis through selective Sp3 cleavage by caspase. Sp3 induction in established tumours resulted in transient regression then progression. Progression coincides with re-accumulation of the full length form of Sp3. Sp3 is over-expressed in tumour cell lines of different origins. The presence of high levels of the full-length form of Sp3 indicates a poor prognosis for overall survival of patients with head and neck tumours.
Conclusions
Full length Sp3 accumulation highlights bypass of tumour cell apoptotic capacities and is indicative of head and neck tumours aggressiveness.
doi:10.1371/journal.pone.0004478
PMCID: PMC2636865  PMID: 19212434

Results 1-13 (13)