Rosa G Lopez-Reyes

Grégoire Quinet

Maria Gonzalez-Santamarta

Clément Larrue

Jean-Emmanuel Sarry

UbiCARE Manuel S Rodriguez
email: manuel.rodriguez@itav.fr

D R Manuel

S Rodriguez

Inhibition of the proteasome and proteaphagy enhances apoptosis in FLT3-ITD-driven acute myeloid leukemia

Keywords: AML, acute myeloid leukaemia, Atg, autophagy, ALS, autophagy-lysosome system, BafA, Bafilomycin A, Bz, Bortezomib, FLT3, fms-like tyrosine Proteaphagy, Bortezomib, Ubiquitin, AML, FLT3-ITD, Leukaemia

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Introduction

Acute myeloid leukaemia (AML) is a clonal disorder that affects hematopoietic stem cells or myeloid progenitors. It is characterized by an accumulation of immature leukemic cells in the bone marrow and peripheral blood and leads to bone marrow failure [START_REF] Horton | Recent advances in acute myeloid leukemia stem cell biology[END_REF]. AML is a heterogeneous disease with a variety of distinct genetic alterations [START_REF] Staudt | Targeting Oncogenic Signaling in Mutant FLT3 Acute Myeloid Leukemia: The Path to Least Resistance[END_REF]. One of the most common mutations occurs in the gene encoding fms-like tyrosine kinase 3 (FLT3) [START_REF] Pratz | Will FLT3 Inhibitors Fulfill Their Promise in AML?[END_REF]. This type III receptor tyrosine kinase regulates the normal growth and differentiation of hematopoietic cells via the activation of multiple signalling including Akt, MAPK and Stat5 [START_REF] Takahashi | Downstream molecular pathways of FLT3 in the pathogenesis of acute myeloid leukemia: biology and therapeutic implications[END_REF][START_REF] Choudhary | Signal transduction of oncogenic Flt3[END_REF]. FLT3 cooperates with other recurrent molecular abnormalities to induce acute leukaemia in preclinical models. Internal tandem duplication (ITD) mutations in the FLT3 gene are found in approximately 30% of AML patients and are associated with a poor clinical outcome. Previous studies demonstrated that AML cells expressing FLT3-ITD are more sensitive to the proteasome inhibitor (PI) Bortezomib (Bz), than FLT3 wild-type cells. This cytotoxicity is mediated by autophagy [START_REF] Larrue | Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells[END_REF]. Furthermore, the genetic inhibition of early autophagy steps or of autophagosome formation block FLT3-ITD, Stat5 and Akt degradation induced by Bz, [START_REF] Larrue | Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells[END_REF].

The proteasome has been envisioned as a promising target for the development of anticancer

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therapeutic drugs [START_REF] Liu | Chapter 13 -Proteasome Inhibitors as Sensitizing Agents for Cancer Chemotherapy[END_REF]. The 26S proteasome is a large multi-subunit protease (1500-2000 kDa), formed by the 20S proteolytic core, and one or two 19S regulatory particles [START_REF] Finley | Gates, Channels, and Switches: Elements of the Proteasome Machine[END_REF]. Three proteolytically active subunits integrate the 20S core: β1 with a caspase-like activity, β2 with a trypsin-like activity, and β5 with a chymotrypsin-like activity. β5 is the primary target for most PIs that reached a clinical phase [START_REF] Finley | Gates, Channels, and Switches: Elements of the Proteasome Machine[END_REF][START_REF] Orlowski | Catalytic activities of the 20 S proteasome, a multicatalytic proteinase complex[END_REF]. Cancer therapy also targets the Autophagy-Lysosome System (ALS), another proteolytic process that is responsible for the bulk degradation of cytoplasmic components. Amino acid deficiency activates autophagy by regulating signalling cascades controlling this proteolytic pathway [START_REF] Mejlvang | Starvation induces rapid degradation of selective autophagy receptors by endosomal microautophagy[END_REF]. During selective autophagy, phagophores engulf cytoplasmic material, and then fuse to form double-membrane autophagosomes. Cargo recruitment occurs through a family of autophagy receptors including p62, OPTN or NBR1 that are often used as markers for autophagy activation together with the ubiquitin-like molecule Atg8 [START_REF] Rogov | Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy[END_REF]. The autophagy (Atg) machinery was first identified in yeast and equivalent molecules reported in mammalian cells [START_REF] Rogov | Interactions between autophagy receptors and ubiquitin-like proteins form the molecular basis for selective autophagy[END_REF][START_REF] Kraft | Selective autophagy: ubiquitin-mediated recognition and beyond[END_REF][START_REF] Mizushima | The Role of Atg Proteins in Autophagosome Formation[END_REF]. Distinct autophagy events drive the degradation of organelles or aggregated proteins such as mitophagy (mitochondria), aggrephagy (protein aggregates) or proteasome (proteaphagy), to name a few [START_REF] Mancias | Mechanisms of selective autophagy in normal physiology and cancer[END_REF].

Many of the current autophagy inhibitors act at a late stage of the system, such as the V-ATPase inhibitor Bafilomycin A (BafA). BafA inhibits autophagy in a non-selective way, by neutralizing acidic pH of lysosomal hydrolases which drive autophagic degradation [START_REF] Juhász | Interpretation of bafilomycin, pH neutralizing or protease inhibitor treatments in autophagic flux experiments: novel considerations[END_REF]. Verteporfin (VT) is an FDA-approved drug that was identified in a screen for chemicals that prevent autophagosome formation [START_REF] Donohue | Inhibition of autophagosome formation by the benzoporphyrin derivative verteporfin[END_REF]. Unlike BafA, VT inhibits autophagy at an early stage, and does not allow autophagosome accumulation [START_REF] Donohue | Inhibition of autophagosome formation by the benzoporphyrin derivative verteporfin[END_REF]. A better understanding of the proteolytic regulation mechanism and interplay will allow exploring alternative/combined treatments to tackle cancer development and/or drug resistance.

Here we aimed to better understand the proteolytic crosstalk connecting proteasome with autophagy after Bz treatment in FLT3-ITD positive MOLM-14 AML cells. Using a chemical approach to block autophagy at distinct levels together with ubiquitin traps (known as TUBEs [START_REF] Hjerpe | Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities[END_REF]), immunoprecipitation (IP) and immunofluorescence, we found that proteaphagy was activated after Bz treatment. Although proteaphagy is a process preserved in distinct species [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF][START_REF] Marshall | Autophagic Turnover of Inactive 26S Proteasomes in Yeast Is Directed by the Ubiquitin Receptor Cue5 and the Hsp42 Chaperone[END_REF][START_REF] Cohen-Kaplan | p62and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome[END_REF], we found that the presence of FLT3-ITD predisposes MOLM-14 cells to activate it.

Material and Methods

Cell lines

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Human myeloid leukaemia cell lines MOLM-14 and OCI-AML-3 were purchased from the ATCC collection. AML cell lines were maintained in RPMI supplemented with 10% foetal calf serum in the presence of 100 U/mL of penicillin and 100 µg/ml of streptomycin. Cells were incubated at 37°C with 5% CO 2 [START_REF] Larrue | Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells[END_REF] . To facilitate autophagy analysis, 2% calf serum was used during chemical inhibitors treatment for a maximum time of 8 hours.

Antibodies and reagents

Antibodies anti-LC3B, anti-Erk 1/2, anti-Stat5, anti-Akt, anti-Ubiquitin (P4D1), anti-PSMB5, were purchased from Cell Signalling Technology (Beverly, MA, USA). Anti-FLT3, anti-p62/SQSTM1 and anti-Beta 2 were obtained from Santa Cruz Biotechnology (Dallas, Texas, USA), anti-PSMA6 from Invitrogen. Anti-PSMB6 and anti-Rpn10 were purchased from Enzo, anti-PSMD3 from Thermofisher and anti-GAPDH from Sigma.

Bortezomib and Chloroquine were purchased from Sigma-Aldrich and Bafilomycin A from InvivoGen, and Verteporfin from Sigma Aldrich.

Western blot analysis

Proteins were resolved using 8 to 12% PAGE and electro-transferred to PVDF membranes.

Membranes were then blocked with 5% skimmed powdered milk in TBS, or 5% bovine serum albumin in TBS. Membranes were immunostained with appropriate antibodies and horseradish peroxidase-conjugated secondary antibodies and visualized with enhanced chemiluminescence system.

Apoptosis assay

Cells line were cultured in RPMI 5%, treated at different times with Bortezomib, Bafilomycin A and Verteporfin. Then 5x10 5 cells were washed with PBS and resuspended in 100 µL of Annexin-V binding buffer. Two microliters of Annexin-V-FITC were added at room temperature. All samples were analysed by a fluorescent-activated cell sorter FACS Calibur flow cytometer [START_REF] Larrue | Proteasome inhibitors induce FLT3-ITD degradation through autophagy in AML cells[END_REF].

TUBES capture

TUBEs were produced according to Hjerpe et al., [START_REF] Hjerpe | Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities[END_REF][START_REF] Aillet | Isolation of ubiquitylated proteins using tandem ubiquitin-binding entities[END_REF]. 20 millions of cells were used for each condition (TUBE p62, TUBE HHR23 or GST Control). Cells were resuspended in 500 µL of TUBE lysis buffer, kept 10 minutes at 4ºC and spin down at 15500 g. A fraction of this supernatant was diluted in 3X boiling buffer (50 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, bromophenol blue, 10% β-mercaptoethanol) and considered as Input. Supernatant was transferred to the glutathione-agarose beads with TUBEs or GST control, and samples were incubated overnight in rotation at 4ºC. On the next day, samples were spin down at 500g at 4ºC, a fraction

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recollected, diluted in BB and considered as Flow-throw (FT). Beads were washed 3 times with 1 mL of PBS Tween 0.05%. Beads were resuspended in 100 µL of BB and boiled 5 minutes before protein electrophoresis.

Antibody crosslinking

Thirty microliters per point of magnetic beads protein A (Milipore) were washed with PBS and equilibrated in binding buffer (50mM Tris pH 8.5, 150mM NaCl, + 0.5% NP40), using a magnetic holder. Antibodies were added to beads and rotated overnight. On the next day 10-20 µL of supernatant were kept to control antibody binding. Beads were washed twice with PBS and once with 500 µL of coupling buffer (200mM Borate, 3M NaCl pH9). Fifty millimolar DMP were added to coupling buffer, and samples were rotated during 30 minutes with this crosslinking solution (CS). Supernatant was discarded, replaced with fresh CS and incubated at 4ºC during 30 minutes. Beads were washed twice with coupling buffer before blocking with 20 mM ethanolamine pH 8.2. Supernatant was discarded and replaced by fresh ethanolamine and incubated for an hour. Beads were washed twice with PBS. Non-coupled antibodies were removed with 2 washes of 1M NaCl/binding buffer. A PBS equilibration was done before washing 3 times with 200 mM glycine pH 2.5. Beads were blocked with 0.1% BSA in binding buffer during 90 minutes. Magnetic beads were equilibrated in binding buffer and kept in PBS until used.

A fraction of these beads (10-20 µL) were analyzed by electrophoresis followed by Coomassie blue staining to compare antibodies before and after crosslinking.

Immunoprecipitation in presence of TUBEs

Twenty millions of cells were spin down at 300g/10min, the dry pellet was resuspended in 500 L of TUBE lysis buffer including 100 g of TUBE p62 or TUBE HHR23 [START_REF] Hjerpe | Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities[END_REF]. Cell lysates were homogenized with 40 strokes at 4ºC using a dounce homogenizer. The whole sample was centrifuged at 200g/5min and supernatant was recovered for immunoprecipitation. A fraction (1/20) of the supernatant was considered as Input. The crosslinked antibody was incubated with cell lysates in rotation during 1h30 at 4ºC. Samples were disposed in magnetic holder to separate bound from unbound material. Proteins unbound to crosslinked antibodies were considered as flow-through (FT) fraction. Magnetic beads were washed with PBS/tween 0.05% 3-5 times and resuspended in 30 µL BB 3x to be analyzed by Western blot.

Statistical analysis

Data from 4 independent experiments were reported as the mean ± standard error of the mean.

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Results

Proteaphagy activation after proteasome inhibition in FLT3-ITD mutant driven MOLM-14 leukemic cells

To mechanistically understand the regulation of the crosstalk between UPS and ALS in leukaemia, we investigated the role of proteaphagy which is known to be activated after proteasome inhibition [START_REF] Waite | Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particles[END_REF]. The impact of Bz was assessed in MOLM-14 cells with the FLT3-ITD mutation and compared with the FLT3 wild-type cell line OCI-AML3 (Fig. 1). Bz treatment does not always promote an obvious degradation of proteasome subunits since proteolysis can be compensated by the novo synthesis of these subunits as previously reported [START_REF] Meiners | Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes[END_REF][START_REF] Welk | Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes[END_REF]. For this reason, proteaphagy was evaluated by the degradation of 20S and 19S proteasomal subunits after Bz treatment and their accumulation with autophagy inhibitors. BafA treatment resulted in the accumulation of autophagy markers p62 and LC3B in the presence or absence of Bz, indicating that autophagy was activated under these experimental conditions in both cell lines (Fig. 1A). Nevertheless, lipidated forms of LC3B were only observed after BafA treatment in MOLM-14 but not in OCI-AML3. The low levels of apoptosis observed after 8h of individual or combined Bz/BafA treatment excluded the possibility that differences could be due to massive death of MOLM-14 cells (Fig. S1). Our results showed a modest but consistent Bz-mediated degradation of 20S proteasome subunits 6s and 5 and 19S subunits Rpn1 and Rpn3 that was blocked by BafA in MOLM-14 (Fig. 1B andC).

However, the combination of BafA with Bz did not significantly accumulate proteasome subunits in OCI-AML3 as it was the case in MOLM-14 cells (Fig. 1B and C low panels), suggesting that a predisposition for degradation of 26S proteasome could be linked to the presence of FLT3-ITD.

These results were also confirmed by immunofluorescence where proteasomes subunit 2 or α2 colocalized with autophagosomes (Atg8 equivalent LC3B or p62 staining respectively) after Bz/BafA treatment of MOLM-14 cells (Fig. 2A andB). This Bz-induced degradation of proteasome subunits is blocked by BafA, indicating that proteaphagy mediated these proteolytic events.

Role of p62 and ubiquitin in Bz-induced autophagy

To analyse the role of p62 in the Bz-induced autophagic degradation of the proteasomes and

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FLT3-ITD, the interaction of p62 with proteasome subunits and FLT3-ITD was investigated. IP experiments using a specific p62 antibody were performed using MOLM-14 cells treated or not with Bz/BafA. Taking advantage of the protective effects of TUBEs that block the action of proteases, accumulate ubiquitylated proteins and interact with multiple chain types [START_REF] Hjerpe | Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities[END_REF][START_REF] Altun | Activity-based chemical proteomics accelerates inhibitor development for deubiquitylating enzymes[END_REF], IPs were done in the presence or absence of TUBEs based in the UBA domain of HHR23 (TUBE-HHR23) or the UBA domain of p62 (TUBE-p62) (Fig. 3A). In the absence of TUBEs, p62 was immunoprecipitated without treatment and Bz/BafA reduced the level of precipitated p62.

However, in the presence of both TUBEs, p62 was protected from proteasome and/or autophagy mediated degradation under Bz/BafA conditions (Fig. 3A). Compared to the situation without any TUBEs or TUBE-HHR23, the presence of TUBE-p62 allows a better coimmunoprecipitation of p62-bound proteasome subunits 2 and but not RPN1. High molecular weight forms most likely representing ubiquitylated forms of RPN1 were better immunoprecipitated in the presence of TUBE-HHR23 (Fig. 3A). Consistent with these observations, FLT3-ITD was also protected by TUBE-p62 but putative ubiquitylated forms of this protein were better immunoprecipitated in the presence of TUBE-HHR23.

Since ubiquitin is a major coordinator of UPS and ALS, we investigated its role in the crosstalk of these pathways induced after Bz and BafA treatments. In particular, we were interested in investigating whether the high molecular weight forms of FLT3 co-immunoprecipitated with p62 corresponded to ubiquitylated FLT3. TUBE-HHR23 was used to capture total ubiquitylated proteins from MOLM-14 cells treated or not with individual and combined Bz/BafA treatment (Fig. 3B). Total ubiquitylated proteins were efficiently trapped by TUBE-HHR23. BafA treatment alone did not significantly enrich ubiquitylated proteins captured by TUBE-HHR23 compared to Bz or Bz/BafA. Proteasome subunits 5 and Rpn1 are captured by TUBE-HHR23 (Fig. 3B). The p62 receptor was also captured under the same conditions but the combination of Bz/BafA enriched this protein compared to Bz alone (Fig. 3B). Interestingly, ubiquitylated forms of FLT3-ITD were captured by TUBE-HHR23 under all conditions but best enriched when Bz/BafA treatment was used (Fig. 3B).

p62 drives proteaphagy and autophagic degradation of FLT3-ITD

To further assess the role of the autophagy receptor p62 in proteaphagy, VT was used to treat MOLM-14 cells and Western blot analyses were performed to detect distinct proteasome subunits.

High molecular weight forms of p62 were detected after VT treatment (Fig. 4A). Interestingly, the lipidated form of LC3B decreases after VT treatment, indicating that this drug reduced the

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FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. autophagy flux. Bz-mediated degradation of 20S or 19S subunits was blocked by VT even if the Bz-mediated degradation of 19S subunits was more prominent than 20S subunits (Fig. 4B andC).

The colocalization of p62/α 2 was significantly reduced with VT or Bz/VT indicating that blocking of p62 could inhibit proteaphagy through a mechanism distinct from BafA (Fig. 4D). To investigate if the high molecular weight forms of p62 observed in Fig 4A were aggregated and/or ubiquitylated, IP were performed with p62 antibodies in the presence or absence of TUBE-p62 (Fig 4E ) and TUBE-capture of ubiquitylated proteins (Fig. 4F). Interestingly, the aggregated forms of p62 generated after the Bz/VT treatment do not interact with the lipidated form of LC3B indicating that this treatment negatively affected this interaction (Fig. 4E). The lipidated forms of LCB3 were also reduced in the input fraction indicating that VT stops the autophagy flux as it was observed in Figure 4A. In order to explore the ubiquitylation status of p62 after VT treatment we captured ubiquitylated proteins using TUBE-p62 (Figure 4F). VT has a negative impact on the total ubiquitylated proteins as it can be observed in the input fraction. Nevertheless, the levels of ubiquitylation could be recovered when VT was combined with Bz. These results were also observed in the TUBE capture of total ubiquitylated proteins. Under these experimental conditions, p62 increased its ubiquitylated levels after Bz treatment and decreased when cells were treated with VT (Figure 4F). Altogether these evidences indicated that after VT treatment, aggregated forms of p62 are accumulated and these forms are less ubiquitylated. The reduction of the interaction p62/lipidated LC3B indicate that this p62 was not integrated into autophagosomes after Bz/VT treatment. In a similar manner, VT reduced the localization of p62 with proteasome subunits, supporting the notion that proteaphagy is hampered by this treatment (Figure 4D). Thus, these data show that p62 plays an important role in proteaphagy observed in MOLM-14 cells.

Inhibition of UPS and ALS pathways enhances apoptosis of FLT3-ITD cells

Individual or combined treatments were used to investigate if Bz-induced degradation of FLT3-ITD was blocked by VT in MOLM-14 cells (Fig 5A). Our results indicate that FLT3-ITD and

FLT3 were degraded up to 25% after Bz treatment in MOLM-14 cells (Fig 5A). FLT3-ITD degradation was blocked by VT while high molecular weight forms of FLT3 were formed under the same conditions in MOLM-14 suggesting a ubiquitin driven autophagy-mediated proteolysis event (Fig. 5A). Interestingly, in OCI-AML3 cells, under the same experimental conditions FLT3 was not degraded by Bz and VT treatment rather accumulated high mobility forms of this protein (Fig. 5B). The double Bz/VT treatment does not significantly accumulate FLT3 in OCI-AML3

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(Fig. 5B), supporting the notion that proteolytic pathways are not efficiently activated in these cells.

Finally, we investigated the consequences of accumulated proteasome and FLT3-ITD after inhibition of UPS and ALS pathways in MOLM-14 cells and compared to OCI-AML3 cells.

Apoptosis was measured by analysing Annexin-V positive cells after single agent or combined treatments (Fig. 5). Bz-induced cell death was always kept below 50% to differentiate positive or negative effects of BafA on the Bz treatments. To improve the efficiency of autophagy inhibition, cells were pre-treated 8h with BafA before adding Bz for an additional 16h at the indicated doses (Fig. 5C andD). Our results showed that the toxicity of the individual BafA treatment was around 20% (MOLM-14) or below 5% (OCI-AML3) and both drugs efficiently cooperated to enhance the cell killing effect on MOLM-14 (Fig. 5C) but this was not in the case in OCI-AML3 (Fig. 5D).

Apoptosis induced by VT also improved the results observed with Bz alone in MOLM-14 cells (Fig. 5E) but not in OCI-AML3 cells (Fig. 5F). In this case, Bz and VT were simultaneously added to cell cultures to work below the VT IC50. This results in an additional 8h of Bz compared to Fig. 5A, explaining the higher apoptosis observed with Bz treatment only. Cooperative effects observed with the double Bz/VT treatment were statistically significant in MOLM-14 cells but not in OCI-AML3 (Fig. 5E and5F). Altogether, our results show that the inhibition of both proteolytic pathways markedly enhances apoptosis levels in MOLM-14 that express FLT3-ITD (Fig. 6) but not in OCI-AML3 that express WT FLT3.

Discussion

Multiple mechanisms of interplay between the UPS and ALS have been documented over the last ten years. Here we identified proteaphagy as part of the selective autophagic events, which are activated after Bz treatment in FLT3-ITD positive leukemic cells. Our data showed that this tyrosine kinase translocation facilitates the Bz-mediated proteolysis of 20S and 19S subunits and their colocalization within autophagosomes. FLT3-ITD can potentially predispose to proteaphagy due to its capacity to activate multiple signalling cascades that have an impact on autophagy activation. Among these pathways are the phosphatidylinositol-3 kinase (PI3K) [START_REF] Piddock | The Role of PI3K Isoforms in Regulating Bone Marrow Microenvironment Signaling Focusing on Acute Myeloid Leukemia and Multiple Myeloma[END_REF][START_REF] Nepstad | The PI3K-Akt-mTOR Signaling Pathway in Human Acute Myeloid Leukemia (AML) Cells[END_REF], Akt [START_REF] Brandts | Constitutive activation of Akt by Flt3 internal tandem duplications is necessary for increased survival, proliferation, and myeloid transformation[END_REF], mammalian target of rapamycin (mTOR) [START_REF] Tabe | Inhibition of mTOR kinase as a therapeutic target for acute myeloid leukemia[END_REF], RAS, and extracellular signal-related kinase (Erk), MAPK and Stat5 [START_REF] Choudhary | Signal transduction of oncogenic Flt3[END_REF][START_REF] Spiekermann | Overexpression and constitutive activation of FLT3 induces STAT5 activation in primary acute myeloid leukemia blast cells[END_REF]. It remains to be demonstrated if any or various of these signalling pathways have a positive or negative impact on proteaphagy.

Proteaphagy is a complicated process to analyse since not all proteasomes are directly concerned

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FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. by this type of degradation. Nuclear proteasomes will not be immediately affected since proteaphagy is a cytoplasmic event. According to the literature, only 20-50% of the proteasomes are regulated by proteaphagy depending on the time, intensity and type of stimuli in distinct biologic models [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF][START_REF] Marshall | Autophagic Turnover of Inactive 26S Proteasomes in Yeast Is Directed by the Ubiquitin Receptor Cue5 and the Hsp42 Chaperone[END_REF][START_REF] Cohen-Kaplan | p62and ubiquitin-dependent stress-induced autophagy of the mammalian 26S proteasome[END_REF]. Our results demonstrate that the autophagy receptor p62 is implicated in the proteaphagy activated by Bz in MOLM-14 cells. However, our data do not exclude the participation of other autophagy receptors in this process [START_REF] Mancias | Mechanisms of selective autophagy in normal physiology and cancer[END_REF]. For instance, both the ubiquitin receptor Cue5 and the chaperon Hsp40 [START_REF] Marshall | Autophagic Turnover of Inactive 26S Proteasomes in Yeast Is Directed by the Ubiquitin Receptor Cue5 and the Hsp42 Chaperone[END_REF] or the proteasome subunit RPN10 [START_REF] Marshall | Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis[END_REF], respectively mediate proteaphagy in Saccharomyces cerevisiae or Arabidopsis thaliana. Nevertheless, the use of VT in leukemic cells showed that the inactivation of p62 stops Bz-induced proteaphagy supporting a major role of p62 in this process. Interestingly, VT favours the formation of high molecular weight aggregates of p62 [START_REF] Konstantinou | Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity[END_REF], which are also observed with FLT3-ITD with the same treatment. However, other effects have been reported for VT including the activation of ROS that could affect other processes [START_REF] Lefaki | Redox regulation of proteasome function[END_REF][START_REF] Finkel | Signal transduction by reactive oxygen species[END_REF] making difficult to attribute VT effects only to its action on p62.

The IP/protection assays [START_REF] Hjerpe | Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities[END_REF] revealed that TUBE-p62 protects p62, RPN1, 2 or FLT3-ITD from Bz-driven degradation blocked with BafA in MOLM14 cells. TUBE-HHR23 also protects these factors but to a lesser extent than TUBE-p62. However, TUBE-HHR23 better accumulates ubiquitylated forms of RPN1 or FLT3-ITD. Similar IP experiments performed with Bz/VT in MOLM-14 showed that high molecular weight forms of p62 did not interact with the lipidated form of LC3B, indicating that p62 was not integrated into autophagosomes under those conditions. This could be associated to the reduction of total ubiquitylated forms observed after VT treatment that might have a negative impact in ubiquitin-regulated events.

The ubiquitin proteome captured with TUBE-HHR23 includes several proteins implicated in proteaphagy such as p62, RPN1 or 5 after Bz or Bz/BafA treatments but not with BafA alone, indicating that this autophagy inhibitor does not accumulate ubiquitylated forms of these factors in the absence of proteasome inhibition. TUBE-p62 captures significantly less ubiquitylated proteins than TUBE-HHR23 and specific ubiquitylated forms can only be seen when overexposing.

Nonetheless, in this way we found that while Bz accumulated ubiquitylated forms of p62, VT reduces these forms most likely interfering with the integration of this receptor into autophagosomes. The use of both TUBEs to investigate UPS and ALS regulated events has the interest to keep all possibilities open to find which pathway will be playing a role under distinct experimental settings. While TUBE-HHR23 recognises virtually all types of chains [START_REF] Hjerpe | Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities[END_REF][START_REF] Altun | proteomics accelerates inhibitor development for deubiquitylating enzymes[END_REF][START_REF] Xolalpa | Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs[END_REF] UBA domain of p62 recognises mainly K63 ubiquitin chains explaining the observed differences [START_REF] Long | Ubiquitin Recognition by the Ubiquitin-associated Domain of p62 Involves a Novel Conformational Switch[END_REF][START_REF] Cabe | PB1 and UBA domains of p62 are essential for aggresome-like induced structure formation[END_REF].

Altogether our data indicate that when both proteolytic pathways are blocked, accumulation of cellular factors occurs due to the functional absence of these degradation machineries (Fig. 6). This contributes to enhance apoptosis in MOLM-14 but not OCI-AML3 cells under the same experimental conditions. In conclusion, targeting protein homeostasis could be an alternative to improve current treatments of FLT3-ITD AML cells. Although the crosstalk of these complex proteolytic mechanisms remains to be fully elucidated, our results open new possibilities for the treatment of this AML phenotype. or both drugs and cells were lysed as reported [START_REF] Hjerpe | Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities[END_REF]. Ubiquitylated proteins were captured using TUBEs-HHR23, TUBEs-p62 or GST control (A). Captured proteins were resolved is SDS-PAGE and immunoblotted with the indicated antibodies. Input and flow through (FT) fractions were also analysed with anti-ub antibody. GAPDH was used as loading control. (B) MOLM-14 cells were treated or not with Bz/BafA under the same conditions than "A". p62-bound proteins were captured by IP with a specific p62 antibody in the presence or absence of TUBE-HHR23 or TUBE-p62. Precipitated material was analysed by Western-blot with the indicated antibodies.

Figure legends

Input fraction was analysed for the indicated proteins.

  Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism 4 software. P0.05 was regarded as significant. *, **, ***, **** correspond to P0.05, P0.01, P0.001 and P0.0001 respectively.

  , the Accepted Article FEBS Open Bio (2020) © 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Fig. 1 .

 1 Fig. 1. Bz-driven proteaphagy is enhanced in FLT3-ITD phenotype. MOLM-14 (FLT3-ITD+/-) or OCI-AML3 (FLT3-WT) cells were treated 8h with Bz 10 nM, and 20 nM Bafilomycin. Total cell lysates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies recognizing the autophagy receptor p62 (A), proteasome core subunits α 6 and 5 (B) or 19S subunits Rpn1 and Rpn3 (C). Protein expression levels were quantified by densitometry analysis (Image J software). Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism 4 software. P0.05 was regarded as significant. *, **, ***, **** correspond to P0.05, P0.01, P0.001, P0.0001 respectively, the data are the s.e.m, n=4.

Fig. 2 .

 2 Fig. 2. Colocalization of proteasome and autophagy markers after Bz and autophagy inhibitors treatment in FLT3-ITD AML cells. Indirect immunofluorescence staining LC3B/β 2 (A) or p62/ α 2 (B) positive structures in MOLM-14 cells treated 8h with Bz 10 nM and 20 nM BafA. Images were captured by confocal microscopy. Scale bar 10 m Immunofluorescence images were quantified from 3 replicates. Statistical analyses were performed using unpaired twotailed Student t-tests with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively, the data are the s.e.m.

Fig. 3 .

 3 Fig. 3. Ubiquitin role in proteaphagy and degradation of FLT3-ITD under proteasome inhibition conditions. MOLM-14 cells were treated or not during 8h with Bz 10 nM, BafA 20 nMAccepted Article

Fig. 4 .

 4 Fig. 4. p62 drives proteaphagy in FLT3-ITD AML cells. MOLM-14 cells were treated 8h with Bz 10 nM and VT 1µM. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against autophagy markers LCB3 and p62 (A), proteasome core subunits 6 and 5 (B) and 19 subunits Rpn1 and Rpn3 (C). Protein expression levels were quantified by densitometry analysis (Image J software). (D) Indirect immunofluorescence staining of p62/ α 2 positive structures in MOLM-14 cells treated 8h with Bz 10 nM and 1 µM VT. Images were captured by confocal microscopy. Scale bar 10 m. Immunofluorescence images were quantified from 3 replicates. Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism 6 software. P<0.05 was regarded as significant. *, **, ***, **** correspond to P<0.05, P<0.01, P<0.001, P<0.0001 respectively, the data are the s.e.m. n=5. (E) Immunoprecipitation of p62 from MOLM-14 cells treated or not with Bz 10nM and VT 1M. Experiments were performed in the presence or absence of TUBE-p62. Precipitated material was analysed by Western blot with specific p62 and LC3B antibodies. Input fraction was analysed with the indicated antibodies. (F) Capture of ubiquitylated proteins using TUBE-p62 trap. GST was used as negative control. Captured proteins were analysed by Western blot with anti-ubiquitin or p62 antibodies. Input and flow through (FT) fractions were analysed with the indicated antibodies.

Fig. 5 .

 5 Fig. 5. Proteasome and autophagy inhibitors cooperate to improve apoptosis of FLT3-ITD expressing cells. MOLM-14 (A) and OCI-AML3 (B) cells were treated 8h with Bz 10 nM and VT 1µM. Total cell lysates were resolved by SDS-PAGE and immunoblotted with antibodies against FLT3 (A and B). Protein expression levels were quantified by densitometry analysis (Image J software). Statistical analyses were performed using unpaired two-tailed Student t-tests with Prism Accepted Article

Fig. 6 .

 6 Fig. 6. UPS and ALS crosstalk under proteasome and/or autophagy inhibition. 1) Under basal unstimulated conditions, turnover of important cellular factor is ensured by equilibrated proteolytic pathways 2) Inhibition of proteasome directs crucial cellular factors to ALS for degradation. 3) Autophagy inhibition drives the degradation of some cellular factors to proteasome-mediated degradation 4) when both proteolytic pathways are impaired, both UPS and ALS contribute to accumulate cellular factors and increase apoptosis in FLT3-ITD positive cells.

Acknowledgments

We thank Clémence Coutelle-Rebut for the proofreading of this manuscript. MSR and MGS are funded by UbiCODE a network supported by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 765445. MSR also receives funding from the Institut National du Cancer, France (PLBIO16-251), LASSERLAB-EUROPE under the grant No 654148, CONACyT-SRE (Mexico) under the grant No 0280365 and the Occitanie region, France, through the REPERE and Prematuration programs. GQ and RGLR are respectively fellows from The French Ministry of Higher Education and Research and CONACYT (Mexico).

Data Accessibility

Data will be available from the corresponding author upon reasonable request.

Author Contributions

RGLP, MSR, JES conceived and designed the project, RGLP, GQ and MGS acquired the data, RGLP, GQ, MGS and CL analysed and interpreted the data, MSR and RGLP wrote the paper, all authors contributed to proofread this manuscript.

Conflict of interest

Authors declare no conflict of interest.

Supporting Information

Supplementary


Antoine Pegat
email: antoine.pegat@chu-lyon.fr

William Boisseau

Thierry Maisonobe

Rabab Debs

Timothée Lenglet

Dimitri Psimaras

Arièle Azoulay-Cayla

Emmanuel Fournier

Karine Viala

Motor chronic inflammatory demyelinating polyneuropathy ( CIDP ) in 17 patients: Clinical characteristics, electrophysiological study, and response to treatment

Keywords: motor CIDP, pure motor CIDP, motor predominant CIDP, corticosteroid, cancer 44 45

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Chronic inflammatory demyelinating polyneuropathy (CIDP) is a treatable autoimmune acquired neuropathy with heterogeneous presentation. [START_REF] Vallat | Chronic inflammatory demyelinating polyradiculoneuropathy: diagnostic and therapeutic challenges for a treatable condition[END_REF][START_REF] Rabin | Chronic inflammatory demyelinating polyradiculoneuropathy: search for factors associated with treatment dependence or successful withdrawal[END_REF] Motor CIDP is one form of atypical CIDP which is described by the joint task force of the EFNS/PNS. [START_REF] Van Den Bergh | European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of chronic inflammatory demyelinating polyradiculoneuropathy: Report of a joint task force of the European Federation of Neurological Societies and the Peripher: EFNS/PNS CIDP guideline -first revision[END_REF] However, the clinical characteristics, electrophysiological features, and response to treatment of patients with motor CIDP have been poorly reported given the rarity of this CIDP subtype (only 1-10% of patients with CIDP). [START_REF] Gorson | Chronic inflammatory demyelinating polyneuropathy: clinical features and response to treatment in 67 consecutive patients with and without a monoclonal gammopathy[END_REF][START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Mahdi-Rogers | Epidemiology of chronic inflammatory neuropathies in southeast England[END_REF][START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF][START_REF] Ikeda | Clinicopathological characteristics of subtypes of chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Nobile-Orazio | Chronic inflammatory demyelinating polyradiculoneuropathy and variants: where we are and where we should go[END_REF][START_REF] Querol | Long-term outcome in chronic inflammatory demyelinating polyneuropathy patients treated with intravenous immunoglobulin: A retrospective study: Long-Term Effectiveness of IVIg in CIDP[END_REF] An early age at onset is probably more frequent compared to other types of CIDP [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF] and clinical course can be progressive or relapsing-remitting. [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] Electrophysiological studies show frequent conduction blocks (CB). [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] Finally, intravenous immunoglobulins (IVIg) treatment should be the first choice and corticosteroids have been shown to potentially lead to deterioration. [START_REF] Van Den Bergh | European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of chronic inflammatory demyelinating polyradiculoneuropathy: Report of a joint task force of the European Federation of Neurological Societies and the Peripher: EFNS/PNS CIDP guideline -first revision[END_REF][START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] However, the definition of motor CIDP is not precise according to the EFNS/PNS 2010 guidelines and it remains unclear whether diagnosis of motor CIDP should only be based on clinical findings or whether the presence of normal sensory nerve conduction is required. [START_REF] Nobile-Orazio | Chronic inflammatory demyelinating polyradiculoneuropathy and variants: where we are and where we should go[END_REF] The aims of the present study were therefore to describe the clinical and electrophysiological characteristics, as well as the response to treatment of a cohort of patients with motor CIDP.

Materials and methods

Patients

Data were extracted from a prospective single center (Pitié-Salpêtrière hospital, Paris, France) database which includes all consecutive patients with CIDP between January 1 st 2008 and October 31 st 2018. Detailed material and methods have been reported elsewhere. [START_REF] Viala | A current view of the diagnosis, clinical variants, response to treatment and prognosis of chronic inflammatory demyelinating polyradiculoneuropathy[END_REF] Patients with definite or probable CIDP according to the EFNS/PNS 2010 guidelines, [START_REF] Van Den Bergh | European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of chronic inflammatory demyelinating polyradiculoneuropathy: Report of a joint task force of the European Federation of Neurological Societies and the Peripher: EFNS/PNS CIDP guideline -first revision[END_REF] having a motor CIDP with motor signs (weakness, cramps, fasciculations, motor cranial nerve palsy), in a symmetric or asymmetric polyneuropathic distribution [START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF] with no sensory symptoms or signs at diagnosis (including normal light touch, pinprick, temperature, vibratory sensations, normal gait, and negative Romberg sign), were included. Patients with focal (involvement of the brachial or lumbosacral plexus or of one or more peripheral nerves in one upper or lower limb), multifocal acquired demyelinating sensory and motor neuropathy (MADSAM)/Lewis-Sumner syndrome, [START_REF] Van Den Bergh | European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of chronic inflammatory demyelinating polyradiculoneuropathy: Report of a joint task force of the European Federation of Neurological Societies and the Peripher: EFNS/PNS CIDP guideline -first revision[END_REF] or multifocal motor neuropathy (MMN) [START_REF]Joint Task Force of the EFNS and the PNS. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of multifocal motor neuropathy. Report of a Joint Task Force of the European Federation of Neurological Societies and the Peripheral Nerve Society -first revis[END_REF] were excluded.

Patients with motor CIDP were classified into 2 groups according to sensory explorations upon electrodiagnostic examination (EDX): patients with normal sensory conduction studies were classified as having pure motor CIDP (PM-CIDP). Patients with at least 2 sensory nerves with sensory nerve action potential (SNAP) amplitude under the lower limit of normal value of the EDX laboratory were classified as having motor predominant CIDP (MPred-CIDP).

Data collection

Disease course and response to treatment as well as laboratory workup and nerve biopsy were retrospectively collected from medical files. Antiganglioside antibodies including anti-GM1, -GM2, -GM3, -GD1a, -GD1b, -GT1b and GQ1b antibodies of both IgM and IgG isotypes were detected by using immunodot assay.

Asymmetrical weakness was defined by a difference of 1 medical research council (MRC) grade if strength was MRC > 3, and 2 MRC grades if strength was MRC ≤ 3, as is used in MMN. [START_REF]Joint Task Force of the EFNS and the PNS. European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of multifocal motor neuropathy. Report of a Joint Task Force of the European Federation of Neurological Societies and the Peripheral Nerve Society -first revis[END_REF] The course of the disease was classified as either progressive or relapsing-remitting (defined as 2 episodes with intervening remission and relapse unrelated to a change in treatment). A positive response to treatment was defined as an improvement by at least 2 MRC grades in any muscle and/or by 1 point improvement in the overall neuropathy limitations scale (ONLS). A treatmentdependent patient was one who needed regular administration of his/her treatment to maintain its benefit and to avoid the risk of rapid relapse.

Neurophysiological investigations

EDX was performed -as previously described - [START_REF] Viala | Follow-up study and response to treatment in 23 patients with Lewis-Sumner syndrome[END_REF] by an experienced electromyographer using a Viking Nicolet electromyograph. Demyelinating features (including definite and probable partial motor conduction block, CB) on motor nerves were defined according to the EFNS/PNS 2010 guidelines. [START_REF] Van Den Bergh | European Federation of Neurological Societies/Peripheral Nerve Society Guideline on management of chronic inflammatory demyelinating polyradiculoneuropathy: Report of a joint task force of the European Federation of Neurological Societies and the Peripher: EFNS/PNS CIDP guideline -first revision[END_REF] The first EDX performed at the Pitié-Salpêtrière Hospital for each patient was considered in the present study. An inversed sensory ratio was defined by a median/sural or ulnar/sural or radial/sural SNAP amplitude ratio <1, without median or ulnar nerves with entrapment. Somatosensory evoked potentials (SSEP) were performed for certain patients.

Proximal conduction was suggestive of demyelination if it showed abnormal conduction in the N8-N22 segment or N18-N22 conduction time. [START_REF] Koutlidis | Segmental somatosensory-evoked potentials as a diagnostic tool in chronic inflammatory demyelinating polyneuropathies, and other sensory neuropathies[END_REF]

Statistical analyses

Continuous variables were compared using Student's test or Wilcoxon rank sum test, and discrete variables using Chi-squared test or Fisher's exact test. The level of significance was set at p<0.05.

All calculations were performed using the statistical software R* (R core team, 2017).

Ethics statement

Clinical data were obtained in accordance with ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. All patients were informed and gave their consent. For this retrospective study, no authorization from an ethics committee was required.

Results

Among 700 patients with CIDP in the database, 17 (prevalence of 2%) were included in the present study: 7 had PM-CIDP and 10 MPred-CIDP. All but 1 patient were classified as having definite CIDP (Patient #3 with PM-CIDP had probable CIDP; Table 1).

Clinical features

There were 5 women and 12 men (Table 1). The median age at onset was 48 years (range: 13-76 years), 6/17 patients (35%) were less than 30 years old at onset. In most patients, onset was chronic (12/16 patients [75%]). The initial symptom was lower limb (LL) weakness in 10/16 patients (59%, Table 1). At the more severe disease stage, the majority of patients had 4-limb weakness (15/16 patients [94%], Table 1). The median ONLS at the more severe disease stage was 4 points (range: 3-10) and 3 patients used a single crutch (patient #2, #12, and #13). Median ONLS at last follow-up was 3 (range: 0-5); 4 patients had a ONLS score of 0 or 1. An associated disease was found in 8/17 patients (47%), including 4/17 patients (24%) presenting with cancer (Table 1). In 2 patients (patient #7 and #8), cancer was diagnosed during CIDP investigations (B-cell lymphoma, and epidermoid carcinoma of lung) and in 2 others, it was diagnosed 1 year (patient #11 with palate cancer) and 11 years (patient #5 with generalized cancer) after CIDP diagnosis.

Disease course and response to treatment

The median follow-up duration was 4 years (47 months, range: 1-153 months). During follow-up, 2 patients (patients #5 and #11) died due to cancer complications and 1 was lost to follow-up (patient #8). Clinical course was progressive in most patients (12/17 patients; 71%) and relapsing-remitting in 5/17 patients (29%, Table 2). Spontaneous improvement occurred at the beginning of disease in 3/17 patients (18%; patient #6, #14 and #17). Among those concerned, 12/16 patients (75%) had a positive response to IVIg, 4/5 (80%) to corticosteroids (including 3/3 with MPred-CIDP and 1/2 with PM-CIDP; the patient with PM-CIDP who did not respond to corticosteroids did not show clinical deterioration), and 2/5 (40%) to plasma exchange (PE; patient #7 had a lymphoma treatment (splenectomy) associated with PE, Table 2). Only 4/13 patients (31%) experienced successful treatment discontinuation with a median duration of 12 months (range: 7-42, Table 2).

Paraclinical exams

Neurophysiological investigations

In 11/17 patients (65%), EDX was performed before the onset of immunomodulatory treatment. Among all 17 patients, 15 (88%) had definite CIDP (Table 3). The median interval between first clinical signs and EDX was 24 months (range: 3-72 months). The median number of motor nerves tested was 8 (range: 5-8) and sensory nerves tested was 8 (range: 5-10).

The 2 most frequent demyelinating features on motor nerves were F wave abnormalities (prolongation or absence) and CB; F wave abnormalities were found in 15/17 patients (88%) representing 53/100 nerves (53%) and multifocal CB in 14/17 patients (82%) representing 58/95 nerves (61%, Table 3). The median number of CB per patient was 4 (range: 0-8). In total, 34/64 CB were definite (53%) and 30/64 were probable (47%). Among the 64 CB, 26 (41%) were located in the ulnar nerve (Table 3). In the 37 nerves without CB, 21 had abnormal F waves (57%). Prolonged DL and reduced CV were rare and found in 3/17 (18%) and 6/17 (35%) patients respectively (Table 3, mostly on nerves with CB).

The median number of reduced distal compound muscle action potential (CMAP) amplitude per patient was 4 (range: 0-8). Fibrillation potentials or positive sharp waves at rest were found in 11/17 patients (65%). A total of 5/17 (29%) patients had fasciculations in 1 muscle, and 1 patient (patient #11) had fasciculations in 4 muscles (Table 3).

Among patients with MPred-CIDP, the median number of abnormal SNAP amplitude was 4.5 (range: 2-9), 9/10 patients (90%) had an inversed sensory ratio.

Five patients (all were MPred-CIDP) had data for SSEP and 3/5 (60%) had proximal conduction abnormality suggestive of demyelination.

Other paraclinical exams

Two patients (patients #11 and #14,) had IgM anti-GM1 antibodies (patient #11 had associated anti-GQ1b antibodies) and 1 patient had IgM anti-GM2 antibodies (patients # 4, Table 1). The mean CSF protein concentration was 73 mg/dl (range: 0.36-150 mg/dl) and CSF protein was mildly elevated (>50 mg/dl) in 11/14 patients (79%; Table 1). Nerve biopsy was performed in 3 patients (all had MPred-CIDP) and found absence of inflammatory infiltrate in 3 patients and evidence of demyelination or remyelination in 2 patients.

Comparison of PM-CIDP and MPred-CIDP patients

During follow-up, 4/10 (40%) patients with MPred-CIDP developed mild sensory symptoms, none of those with PM-CIDP did so (p < 0.001, Table 4). These sensory symptoms consisted in tingling in extremities which appereared with a median delay of 18 months (range: 8-216 months), but none had gait or limb ataxia nor hypoesthesia (patients #14 and #16 : tingling in 4 extremities, patients #15 and #17: tingling limited to hands). Median follow-up duration was similar for patients with PM-CIDP (47 months, range: 1-81) and patients with MPred-CIDP (44 symptoms was 57 months (range: 39-153). The median ONLS at last follow-up in patients with PM-CIDP was 4 (range: 2-5) compared to 2 (range: 0-4) for those with MPred-CIDP (p = 0.03); 4 patients with MPred-CIDP had an ONLS of 0 or 1 while none of those with PM-CIDP did so (Table 4). Concerning the response to treatments, response to IVIg was not significantly different between PM-CIDP and MPred-CIDP patients (p=1, Table 4). Among the patients treated by corticosteroids, 1/2 PM-CIDP patients responded compared to 3/3 MPred-CIDP patients (p=0.4; Table 4).

Discussion

The present study shows that motor forms of CIDP can be considered as variants of CIDP that present with specific clinical, electrophysiological, and response to treatment courses.

More precisely, the present study confirms that motor CIDP seem to be (i) rare; [START_REF] Gorson | Chronic inflammatory demyelinating polyneuropathy: clinical features and response to treatment in 67 consecutive patients with and without a monoclonal gammopathy[END_REF][START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Mahdi-Rogers | Epidemiology of chronic inflammatory neuropathies in southeast England[END_REF][START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF][START_REF] Ikeda | Clinicopathological characteristics of subtypes of chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Nobile-Orazio | Chronic inflammatory demyelinating polyradiculoneuropathy and variants: where we are and where we should go[END_REF][START_REF] Querol | Long-term outcome in chronic inflammatory demyelinating polyneuropathy patients treated with intravenous immunoglobulin: A retrospective study: Long-Term Effectiveness of IVIg in CIDP[END_REF] (ii) tend to occur more often in males; [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Ikeda | Clinicopathological characteristics of subtypes of chronic inflammatory demyelinating polyradiculoneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] (iii) at a young age at onset; [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF] (iv) present with frequent and multifocal CB [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] and F wave abnormalities [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] on EDX; and (v) respond well to IVIg. [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] Other potential features of motor CIDP are highlighted herein. First, a high proportion of patients (almost half of them) presented with associated diseases. Three patients were diagnosed with cancer, either during CIDP investigations or in the first year of evolution. It is difficult to conclude on a possible link between cancer and motor CIDP given that tumors were diverse and not necessarily known to be associated with paraneoplastic neurologic disorders. Demyelinating neuropathy with predominantly motor form has been reported in association with lymphoma. [START_REF] Viala | Neuropathy in lymphoma: a relationship between the pattern of neuropathy, type of lymphoma and prognosis[END_REF] Melanoma, lung or esophageal cancer, have also been described in motor CIDP. [START_REF] Rajabally | Chronic inflammatory demyelinating polyneuropathy and malignancy: A systematic review: CIDP and Malignancy: a Review[END_REF][START_REF] Mostoufizadeh | A Case of Paraneoplastic Demyelinating Motor Polyneuropathy[END_REF] In a previous cohort of CIDP patients (not only motor CIDP), our group reported a concurrent illness in only one quarter of patients. [START_REF] Viala | A current view of the diagnosis, clinical variants, response to treatment and prognosis of chronic inflammatory demyelinating polyradiculoneuropathy[END_REF] Therefore, thorough investigations aiming at identifying diseases associated with motor CIDP are required. Second, in agreement with two previous studies, [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF] a majority of patients herein experienced a progressive clinical course. Conversely, all patients from the studies by Kimura et al. [START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] and Sabatelli et al. [START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF] presented with a relapsingremitting course. Finally, despite the use of various immunomodulatory treatments, the majority of patients experienced a poor clinical outcome: only a small quarter of patient had an acceptable ONLS score at 0 or 1 at the end of follow-up. This is discordant with the results from Kimura et al. who reported that patients with motor CIDP experienced very good outcome, better than patients with other CIDP types. [START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF] From an electrophysiological point of view, the finding of recurrent and multifocal CB in almost all nerves in which abnormal CV and DL were found indicates that CB are the cause of these demyelinating parameters and therefore should not be considered alone. Furthermore, in this context and with an objective of diagnosis, it is of note that the ulnar nerve was the most affected by CB, but also that CB can be present only in proximal segments. Conversely to CV and DL, F wave abnormalities were found both in nerves with and without CB, which should also be kept in mind during diagnostic workup.

No consensual definition is available concerning the diagnosis of pure motor CIDP. The EFNS/PNS guidelines, which provide a more precise definition of motor CIDP, are currently under review. In the present study a clinical definition of motor CIDP, at diagnosis, was used and patients were included independently of sensory conduction abnormality upon EDX. Thus, 2 groups were identified herein, patients without sensitive nerve abnormalities (PM-CIDP) and those with (MPred-CIDP), as assessed by EDX. There was no significant difference in terms of clinical and paraclinical features between the 2 groups, except for outcome (patients with PM-CIDP had poorer outcome), and the development of sensory symptoms during follow-up (all patients who developed subtle sensory signs had MPred-CIDP). Some of the MPred-CIDP patients who developed mild sensory symptoms did not however develop ataxia or hypoesthesia as in common CIDP, and remained motor predominant. It is possible that these patients have an intermediate and milder form, which lies on the spectrum between the motor and typical forms. Donnedu et al. had previously reported the possibility that atypical CIDP, including motor CIDP, evolved towards typical CIDP. [START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF] The higher proportion of patients (more than half of patients) moving towards a typical form of CIDP observed by the authors could be explained in part by the longer median follow-up (6 years as opposed to 4 years herein). [START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF] Taken together, for a patient with a motor form, the existence of a sensory involvement upon EDX might be a predictive factor for moving towards a typical CIDP. In a similar way, patients with other atypical CIDP forms (pure sensory CIDP, MADSAM/Lewis-Sumner, distal acquired demyelinating symmetric neuropathy [DADS]) can evolve into typical CIDP. [START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF][START_REF] Viala | Follow-up study and response to treatment in 23 patients with Lewis-Sumner syndrome[END_REF][START_REF] Van Dijk | Development of weakness in patients with chronic inflammatory demyelinating polyneuropathy and only sensory symptoms at presentation: a long-term follow-up study[END_REF] Finally, pure motor CIDP has been frequently reported as a steroid unresponsive pathology. [START_REF] Busby | Chronic dysimmune neuropathy[END_REF][START_REF] Sabatelli | Pure motor chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Kimura | Motor-dominant chronic inflammatory demyelinating polyneuropathy[END_REF][START_REF] Molenaar | Pulsed high dose dexamethasone treatment in chronic inflammatory demyelinating polyneuropathy: a pilot study[END_REF] In the present study however, the majority of patients who underwent corticosteroid treatment showed improvements, but these concerned mainly patients with MPred-CIDP group. This result is in line with the study by Doneddu et al. which reported a beneficial effect of corticosteroids in 3/7 patients, but exclusively in patients with MPred-CIDP. [START_REF] Doneddu | Atypical CIDP: diagnostic criteria, progression and treatment response. Data from the Italian CIDP Database[END_REF] Using these recent data, corticosteroids might be considered as a therapeutic option in resistant IVIg motor CIDP patients, at least for patients with abnormal sensory conduction. More data are needed to rule on the efficiency of corticosteroid in PM-CIDP patients.

The current body of data indicates that 2 main alternative diagnoses of neuropathy could be considered in the context of motor deficits: MMN and motor neuron disease. Motor CIDP and MMN share several features: pure motor involvement, young age at onset, good response to IVIg therapy, possible antiganglioside antibodies, and multiple CB on EDX. However, they differ on 3 major aspects: motor CIDP is associated with diffuse weakness, more diffuse electrophysiological damage, and a possible improvement using corticosteroids. However, some MMN patients may develop a more diffuse distal and proximal weakness later in the disease course indicating that a continuum may exist between MMN and motor CIDP. The association of pure motor deficit, amyotrophy, and sometimes diffuse cramps and fasciculation (as in patient #11), suggests motor neuron disease. Motor neuron disease and motor CIDP can share common EDX abnormalities, for instance, reduced distal CMAP, fibrillation, fasciculations, and CB; the latter are reported transitory in patients with rapidly progressive motor neuron disease. [START_REF] Echaniz-Laguna | A study of three patients with amyotrophic lateral sclerosis and a polyneuropathy resembling CIDP[END_REF][START_REF] Rajabally | Chronic inflammatory demyelinating polyneuropathy-like disorder associated with amyotrophic lateral sclerosis[END_REF][START_REF] Ahdab | Rapidly progressive amyotrophic lateral sclerosis initially masquerading as a demyelinating neuropathy[END_REF] The present study has several limitations inherent to its retrospective observational design and the fact that it is based on a small number of patients because of the rarity of the disease. Furthermore, due to our expert center status, the first EDX was performed after immunomodulatory treatment initiation in a third of patients which could affect results. Finally, after initial investigations and treatment introduction, some patients were redirected to their local center which explains a shorter follow-up period.

To conclude, beyond the previously reported features of motor CIDP including its low prevalence, good response to IVIg, and frequent CB and F wave abnormalities upon EDX, the present study found a progressive clinical course in the majority of patients and frequent associated diseases. In contrast to MPred-CIDP patients, PM-CIDP patients seem to have poorer outcome and did not develop sensory symptoms during follow-up. Corticosteroids might be considered as a therapeutic option in resistant IVIg patients with MPred-CIDP but more data are needed to rule on the efficiency of corticosteroids in PM-CIDP patients.

Table 3 :

 3 Electroneuromyography data

					Demyelinating features on motor nerves		Topography of CB
	Patient Category	Electro-		Prolonged	Reduced	Abnormal	Number	Median	Ulnar	Com-	Tibial	Number of	Number of	Fibrillation	Fascicu-
		(PM/MPred-CIDP)	diagnostic criteria		DL	CV	F waves	of CB			mon fibular		motor nerve with	sensory nerves	or PSW at rest	lation at rest
			(*,	in									abnormal	with
			months)										CMAP	abnormal
													amplitude	SNAP
														amplitude
	1	PM-CIDP	Definite (48)	+	-	-	5	2 (D)	3 (2D, I)			7	0	+	-

Acknowledgments:

We thank Philip Robinson and Véréna Landel (DRCI, Hospices Civils de Lyon) for help in manuscript preparation.