Combination of Bortezomib and Mitotic Inhibitors Down-Modulate Bcr-Abl and Efficiently Eliminates Tyrosine-Kinase Inhibitor Sensitive and Resistant Bcr-Abl-Positive Leukemic Cells

Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases

edem response to thapsigargin

S4 Fig. edem response to thapsigargin. (A) Quantitative RT-PCR measurements of edem mRNA levels in WT young animals under non stress (NS) and treatment with 5 μM thapsigargin (TG), (n = 3 independent experiments). (B) Percentage of eggs that developed into L4 larvae after 3 days on 10 μM thapsigargin. Each strain was scored in three independent experiments in triplicates. (C) Quantification of WT (EV) or RNAi-treated day one adults carrying Phsp-4::GFP transgene, treated or not with 5 μM thapsigargin. The worms were kept on RNAi plates for one generation before exposure to ER stress treatments. Values represent mean fluorescence/µm2 x 1000. The red bars indicate the average ±SEM. (D) Analysis of xbp-1 spliced/unspliced ratio from young adults of indicated strains. Total RNA was isolated, reverse transcribed and used for PCR analysis of xbp-1 splicing forms.Peer reviewe

Primers used in this study. Targeting EDEM protects against ER stress and improves development and survival in C. elegans

S2 Table. Primers used in this study.Peer reviewe

Targeting EDEM protects against ER stress and improves development and survival in C. elegans.

EDEM-1, EDEM-2 and EDEM-3 are key players for the quality control of newly synthesized proteins in the endoplasmic reticulum (ER) by accelerating disposal and degradation of misfolded proteins through ER Associated Degradation (ERAD). Although many previous studies reported the role of individual ERAD components especially in cell-based systems, still little is known about the consequences of ERAD dysfunction under physiological and ER stress conditions in the context of a multicellular organism. Here we report the first individual and combined characterization and functional interplay of EDEM proteins in Caenorhabditis elegans using single, double, and triple mutant combinations. We found that EDEM-2 has a major role in the clearance of misfolded proteins from ER under physiological conditions, whereas EDEM-1 and EDEM-3 roles become prominent under acute ER stress. In contrast to SEL-1 loss, the loss of EDEMs in an intact organism induces only a modest ER stress under physiological conditions. In addition, chronic impairment of EDEM functioning attenuated both XBP-1 activation and up-regulation of the stress chaperone GRP78/BiP, in response to acute ER stress. We also show that pre-conditioning to EDEM loss in acute ER stress restores ER homeostasis and promotes survival by activating ER hormesis. We propose a novel role for EDEM in fine-tuning the ER stress responsiveness that affects ER homeostasis and survival

Raw numerical data of all the figures. Targeting EDEM protects against ER stress and improves development and survival in C. elegans

S3 Table. Raw numerical data of all the figures.Peer reviewe

EDEM depletion mitigates ER stress

S3 Fig. EDEM depletion mitigates ER stress. (A) Representative confocal images of WT(EV) control or indicated RNAi depleted young adults carrying the Phsp-4::GFP transgene under non ER-stress conditions (upper panel), treatment with 5 μg/ml tunicamycin for 12h (middle panel), and heat shock, and 5 μM thapsigargin treatment (lower panel). TM-tunicamycin, HS-heat shock, TG-thapsigargin. The worms were outlined for better visualization. Images were obtained using the same confocal settings. Exposure adjustments were uniformly applied. Scale bar: 50 μm. (B) Quantitative RT-PCR measurements of Phsp-4::mRNA levels in indicated strains under non stress and treatment with 5 μg/ml tunicamycin (n = 3 independent experiments). (C) Quantitative RT-PCR measurements of Phsp-4::mRNA levels in indicated strains under non stress and heat stress treatment (n = 3 independent experiments). In (B) and (C) quantifications were normalized relative to WT non stress conditions. ****P<0.0001, ns, not significant. Quantifications were normalized relative to WT non stress conditions. (D) Representative confocal images of WT control and edem-2 mutants carrying the PC12C8.1::GFP transgene under non ER-stress conditions. The left panels show fluorescence images and the right panels DIC images.Peer reviewe

Immunoblot analysis of Pedem::GFP expression in young animals

S1 Fig. Immunoblot analysis of Pedem::GFP expression in young animals. (A) Quantitative real-time PCR (qPCR) of edem-3 mRNA. WT and edem-3 mutant worms treated with empty vector or edem-3 RNAi; expression were normalized to that of cdc-42 and pmp-3. (B) Total protein lysates derived from WT Pedem::GFP transgenic animals subjected to the indicated treatment were separated by SDS-PAGE and immunoblotted with anti-GFP polyclonal antisera; tubulin was used as loading control. NS- non-treated, TM- tunicamycin, HS-heat stress, OS- osmotic stress. The histograms show the densitometry values of Pedem::GFP bands normalized to the value of of NS condition (n=3 ± SEM, t test), *P<0.05; **P<0.01; ***P<0.001; ns, not significant. (C) RNAi downregulation of edem triggered accumulation of CPL-1* in intestinal cells. To overrule a significant contribution of autofluorescent stress granules to the GFP fluorescence, images captured with Diode laser were included. Scale bar: 20 μm.Peer reviewe

EDEM-1 and EDEM-2 are required for CPL-1* degradation

S2 Fig. EDEM-1 and EDEM-2 are required for CPL-1* degradation (A) Fluorescence intensities of WT animals carrying the CPL-1* transgene treated with indicated concentrations of kifunensine. Values in scatter gram represent mean fluorescence/µm2 x 1000 in arbitrary units (AU). The red bars indicate the average ±SEM. ****P<0.0001; one way ANOVA with Dunnett post test. (B) Immunoblot analysis of CPL-1* degradation in edem-1 and edem-2 mutants carrying the rescuing EDEM-1::mCherry and EDEM-2::mCherry transgenes, respectively. Total protein lysates derived from edem-1 and edem-2 mutants carrying the CPL-1* and rescuing transgenes were separated by SDS-PAGE and immunoblotted with anti-GFP polyclonal antisera. Tubulin was used as loading control. (C) Histogram showing the densitometry values of the bands presented in (B) normalized to the value of WT condition (n=3 ± SEM, one way ANOVA with Fisher test), *P<0.05; **P<0.01).Peer reviewe

Strains used in this study. Targeting EDEM protects against ER stress and improves development and survival in C. elegans

S1 Table. Strains used in this study.Peer reviewe

Tyrosinase Degradation Is Prevented when EDEM1 Lacks the Intrinsically Disordered Region

<div><p>EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121–598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40–119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.</p> </div