siRNA in chronic lymphocytic leukaemia cells

International Journal of Pharmaceutics 574 (2020) 118895

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Effects of eEF1A1 targeting by aptamer/siRNA in chronic lymphocytic leukaemia cells

T

Barbara Dapasa, Gabriele Pozzatob, Sonia Zorzeta, Sara Capollaa, Macor Paoloa, Bruna Scaggiantea, Michela Coana, Chiara Guerraa, Chiara Gnanc, Valter Gatteid, ⁎ Fabrizio Zanconatib, Gabriele Grassia,b, a

Department of Life Sciences, University of Trieste, Via Giorgeri 1, 34127 Trieste, Italy Department of Medical, Surgical and Health Sciences, University of Trieste, Cattinara Hospital, Strada di Fiume, 447, 34149 Trieste, Italy c Institute for Maternal and Child Health - “IRCCS Burlo Garofolo”, Via dell'Istria, 65, 34137 Trieste, Italy d Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Via Franco Gallini, 2, 33081 Aviano, Italy b

A R T I C LE I N FO

A B S T R A C T

Keywords: Aptamer siRNA Eukaryotic elongation factor 1A1 protein Chronic lymphocytic leukemia

Background: The effectiveness of therapies for chronic lymphocytic leukemia (CLL), the most common leukemia in Western countries adults, can be improved via a deeper understanding of its molecular abnormalities. Whereas the isoforms of the eukaryotic elongation factor 1A (eEF1A1 and eEF1A2) are implicated in different tumors, no information are available in CLL. Methods: eEF1A1/eEF1A2 amounts were quantitated in the lymphocytes of 46 CLL patients vs normal control (real time PCR, western blotting). eEF1A1 role in CLL was investigated in a cellular (MEC-1) and animal model of CLL via its targeting by an aptamer (GT75) or a siRNA (siA1) delivered by electroporation (in vitro) or lipofection (in vivo). Results: eEF1A1/eEF1A2 were elevated in CLL lymphocytes vs control. eEF1A1 but not eEF1A2 levels were higher in patients which died during the study compared to those surviving. eEF1A1 targeting (GT75/siA1) resulted in MEC-1 viability reduction/autophagy stimulation and in vivo tumor growth down-regulation. Conclusions: The increase of eEF1A1 in dead vs surviving patients may confer to eEF1A1 the role of a prognostic marker for CLL and possibly of a therapeutic target, given its involvement in MEC-1 survival. Specific aptamer/ siRNA released by optimized delivery systems may allow the development of novel therapeutic options.

1. Introduction Chronic lymphocytic leukaemia (CLL), the most common form of leukaemia in adults in Western countries, has a median age at diagnosis of 72 years, with a higher incidence in males (1.7:1) (Hallek, 2017; Kipps et al., 2017; Zenz et al., 2010). CLL is characterized by the clonal expansion of B cells that accumulate in peripheral blood, bone marrow and lymphoid tissues mainly as a result of impaired apoptosis (Ferrer and Montserrat, 2018). The disease is characterized by a considerable heterogeneity in the clinical outcome of patients: while some of them live for decades without any therapy, others die within years of diagnosis despite multiple treatments (Parikh and Shanafelt, 2016). Recent advancements in the understanding of the biology of the disease has considerably improved the prognosis of patients due to the precise stratification into subgroups with distinctive clinical and biological features (Fischer and Hallek, 2017). However, despite the major

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advances in its therapy, therapeutic effectiveness can be further improved. Thus, a deeper understanding of the biologic and molecular aberrations contributing to the pathogenesis of CLL can significantly ameliorate our understanding of the disease thus improving patient managements and outcome. In the present study we evaluate the role in CLL of the elongation factor 1 A (eEF1A), a protein implicated in different forms of human tumours. eEF1A is involved in the elongation step of protein synthesis (Scaggiante et al., 2014). Two major isoforms of eEF1A proteins exist: the ubiquitous eEF1A1 and the tissue-specialized eEF1A2, whose expression is mostly confined to skeletal muscle, heart and nervous system. Beside the role in translation, considered the “canonical function” of eEF1As, both isoforms possess “non-canonical functions” essential for cellular homeostasis such as the regulation of cell cycle and apoptosis (Lamberti et al., 2004). Both eEF1A isoforms play a role in solid and hematologic human tumors, mainly due to the dysregulation

Corresponding author at: Department of Life Sciences, Cattinara University Hospital, Trieste University, Strada di Fiume 447, I-34149 Trieste, Italy. E-mail address: [email protected] (G. Grassi).

https://doi.org/10.1016/j.ijpharm.2019.118895 Received 18 August 2019; Received in revised form 13 November 2019; Accepted 18 November 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.

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Table 1 Patients molecular characterization. Patient number

ZAP-70

CD38

CD49d

Δ 11q22.3 (ATM)

trisomy 12 (CEP12)

Δ 13q14.3

Δ 17p (p53)

IGHV status

1 2 3 4 5 6 7 8 9 10 11 12 13 14* 15 16 17 18 19 20 21 22* 23 24 25 26* 27 28 29 30 31 32* 33 34 35 36 37 38* 39* 40 41 42 43 44 45 46

10 14 11 7 20 5 40 16 5 50 46 40 0 0 0 9 22 0 0 30 5 19 15 9 23 7 0 6 15 35 21 40 23 12 13 8 32 10 25 13 1 18 8 1 6 8

6 50 1 2 30 9 0 15 1 6 96 5 62 0 1 5 27 0 0 40 2 7 5 3 9 31 0 3 2 17 9 20 32 3 1 88 2 12 35 99 1 1< 1 4 10 30

97 0 1 90 0 0 0 1 1 0 91 0 60 0 1 0 14 0 0 0 0 3 0 2 99 0 0 0 26 1 5 99 3 0 2 4 0 2 0 89 0 9 3 2 0 0

0 0 93 27 0 0 0 0 0 0 0 0 0 94 0 0 72 0 0 0 0 0 0 0 0 0 0 0 0 86 44 0 0 0 0 0 0 0 0 85 0 0 0 0 0 0

0 68 0 0 0 0 0 0 0 0 0 0 0 5< 0 0 0 0 0 0 0 0 0 0 0 36 0 0 0 0 0 0 50 0 0 0 0 0 54 0 0 0 0 0 0 0

45 0 0 75 25 15 91 18 94 49 0 0 18 5< 0 0 0 19 0 38 0 0 80 85 0 0 0 61 20 82 56 89 0 0 60 0 38 92 0 0 68 0 76 65 55 14

8 0 0 0 0 0 0 0 0 0 64 0 0 5< 0 0 0 0 0 0 0 0 0 0 0 0 0 39 0 0 0 0 0 0 0 0 0 83 0 0 0 68 0 0 60 0

nd m nm m m m nm m m m nm m m nm m m nm nm m m m m m m m m nm m m nm nm m nm m m nm m m nm nm m m m m m m

The positivity of the biological markers in patients cells is given in %. * Dead patients; m = mutated; nm = non mutated.

repulsion from the negatively charged cell membrane. Moreover, their hydrophilic nature substantially prevents the crossing of the hydrophobic layer of the cell membrane. These aspects, together with others (Farra et al., 2018), oblige the use of appropriate delivery systems to exert their functions in the biological environment. However, the choice of the delivery system is not trivial and has to consider the target cells. The CLL cell line (MEC-1) we used here is known not to be easily transfected by lipofection in vitro, as we also experienced. Thus, we used electroporation that seems to provide better outcome in vitro (Gassner et al., 2018; Longo et al., 2008). However, for in vivo tests, we used lipofection. This choice was mainly due to the fact that electroporation application in vivo requires a sophisticated apparatus and a laborious set up of different parameters (Sersa et al., 2015). Moreover, we have previous positive experience of nucleic acid transfection in vivo by lipofection (Dereani et al., 2014). Here, we report the increase of the expression levels of eEF1A1 and eEF1A2 in a cohort of CLL patients compared to matched control. Notably, eEF1A1 but not eEF1A2 protein levels were more elevated in patients with a shorter Overall Survival (OS), referred to the study duration. Finally, both in vitro and in vivo, we show that eEF1A1

of their non-canonical functions (Scaggiante et al., 2014). In this regard, it has been shown that the differentiation of a human acute promyelocytic leukaemia cell line induced by the all-trans-retinoic acid was associated by a reduction of eEF1A1 protein levels (Harris et al., 2004). Moreover, we described the presence of a basic variant of eEF1A1 protein in acute lymphoblastic leukaemia cells but not in normal lymphocytes (Dapas et al., 2003; Scaggiante et al., 2013). Notably, the disappearance of the basic variant resulted in the reduction of tumour cell growth and aggressiveness. Despite the growing evidence of the involvement of eEF1A in cancer and in haematological tumour disorders, nothing is known in CLL. To study the functional role of eEF1A1 in CLL cells, we have explored the effects of its specific targeting by means of a specific aptamer or siRNA we previously developed (Dapas et al., 2002; Farra et al., 2017; Scaggiante et al., 2012; Scaggiante et al., 2006; Scaggiante et al., 2005; Scaggiante et al., 2013; Scaggiante et al., 2016). Due their chemical nature, aptamers and siRNAs have great difficulties to reach the target if administered in the naked form (Farra et al., 2019; Farra et al., 2018). In particular, due to their negative electric charge, derived from the phosphate groups of their backbone, they are subjected to the 2

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repetition T(GTTT)18GT (total length 75 nucleotides); this aptamer can bind eEF1A1 in a variety of human cells (Dapas et al., 2002; Scaggiante et al., 2012; Scaggiante et al., 2006; Scaggiante et al., 2005; Scaggiante et al., 2013; Scaggiante et al., 2016). As control a CT75 (cytosine-thymidine) sequence T(CTTT)18CT not able to bind eEF1A1 was used. As anti eEF1A1 siRNA we used siA1 (sense 5′-AUGCGGUGGCAUCGAC AAA-3′, antisense 5′-UUUGUCGAUGCCACCGCAU-3′) we previously showed to specifically target eEF1A1 (Farra et al., 2017); as control, we employed a siRNA directed against the mRNA of luciferase named siGL2, (sense 5′-CGUACGCGGAAUACUUCGA-3′, antisense 5′-UCGAAG UAUUCCGCGUACG-3′) (Farra et al., 2017)). MEC-1 cell line were electroporated by the Amaxa Nucleofector II device (AmaxaBiosystem, Cologne, Germany). It should be noted that in the AmaxaNucleofector II device it is not possible to select parameters like voltage, number and time of pulses. The instrument offers a list of programs with an alphanumeric code and every program is associated with one or several cell lines as a result of an optimization procedure from the producer. Before starting with experiments, we optimized the electroporation conditions of MEC-1 cell line using a combination of programs and solutions. Notably, the programs of the AmaxaNucleofector device are covered by patent pending rights owned by Lonza (Cologne, AG). The electroporation solution we used was the Ingenio electroporation solution (Mirius Bio LLC, WI, USA), compatible with AmaxaNucleofector II device; also in this case solution composition is covered by U.S. patent owned by Mirius Bio LLC (Madison, WI –USA). 1x106cells were resuspended in 100 μl of Ingenio electroporation solution (Mirius Bio LLC, WI, USA) and mixed with 2,5 μg of aptamer or siRNA. Twenty-four hours after electroporation (program U-013), 5x103 cells or 1x106 cells were seeded in complete medium in 96 well microtiter plates or 25-cm2 flasks, respectively. MEC-1 cells were processed for MTT assay, cell counts or collected for qRT-PCR and western blot analysis at different time point ranging from 2 to 6 days after electroporation. For uptake studies, FITC labelled siRNA/aptamer were used (same conditions as above reported). Following electroporation, the number of FITC positive MEC-1 cells was evaluated by fluorescence microscopy (Leica DM2000). Aptamer and siRNA stability was tested using 2.5 μg of each molecule, which was then incubated either with the electroporation solution alone or electroporation solution followed by electroporation under the same condition used for the delivery into the MEC-1. Subsequently, all the samples were loaded onto a 12% polyacrylamide gel and run at 12 V/cm in TBE 1X buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3). The aptamer and siRNA bands were then visualized by Stainall dye solution (Sigma-Aldrich) or by ethidium bromide (Sigma-Aldrich), respectively, and the bands photographed.

targeting by two nucleic acid based molecule (Grassi et al., 2004; Grassi and Marini, 1996; Grassi et al., 2010) i.e. an aptamer and a siRNA we previously developed (Farra et al., 2017; Scaggiante et al., 2016), considerably down-regulates the expansion of MEC-1, a prototype cell line of CLL. Together our data suggest for eEF1A1 a relevant role in CLL biology possibly indicating this protein as a novel potential marker/ therapeutic target for CLL. 2. Materials and methods 2.1. Patients Forty-six patients (31 males, 15 females) affected by CLL were included in this study. All cases were enrolled between September 2014 and May 2018 at the Department of Clinical and Surgical Sciences, University of Trieste, Italy. According to the RAI classification there were 68%, 19%, 4%, 4% and 4% of the patients in Rai 0, I, II, III and IV, respectively. According to Binet classification, there were 94%, 0% and 6% in Binet A, B and C, respectively. Blood samples were not specifically obtained for the present study; they were performed due to clinician indication. Consent to the processing of data in an anonymous form for clinical research, epidemiology, and training (and with the aim of improving knowledge, treatment and prevention), in agreement with the Italian law art.81 D.lgs 196/2003, sez. D, was obtained at the diagnosis of cases by the hospital. The ethic commission of the University of Trieste, Italy, (n. 100, 18-10-19) approved the study. With regard to the inclusion-exclusion criteria, we selected patient with cytometric and FISH-based diagnosis of CLL according to the standard criteria of the International working group guide (WHO classification) (Hallek et al., 2008); exclusion criteria were the presence of an active bacterial infection, chronic infection by HBV, HVC, HIV and the presence of coexisting tumors. Patient molecular characterization is reported in Table 1. CLL patients did not significantly differ from the control with regard to the age (76 ± 8 and 71 ± 11, respectively, p = 0.3245). Twenty-six age matched healthy volunteers (16 males, 10 females) which underwent blood collection during routinely hematological controls, were used as control group. Written informed consent was obtained by each subject. Exclusion criteria were: presence of ongoing/ previous solid/hematologic tumors, acute inflammatory conditions, reduction of white blood cells below the threshold level. Inclusion criteria was based on the age falling within the range of that of CLL patients. 2.2. Lymphocytes isolation CD19 + B cells were isolated by negative selection from whole blood of CLL patients or healthy donors using RosetteSepTM B-cell isolation cocktail (Stem Cell Technologies, Vancouver, Canada) following manufacturer’s instructions. After incubation of blood with antibodies cocktail cross-link unwanted cells to erythrocytes, the B-cells enriched population were collected by density gradient centrifugation on Ficoll-Paque (GE Healthcare, Uppsala, Sweden) in the interface between plasma and Ficoll. Isolated cells were washed twice with phosphate-buffered saline (PBS) and collected for western blot or qRT-PCR experiments after cell count and Trypan blue cell viability test. The human B-CLL cell line MEC-1 was purchased from DSMZ (Braunschweig, Germany) and cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (EuroClone, Italy) at 37 °C in a humidified atmosphere containing 5% CO2.

2.4. UV crosslinking Three μg of total nuclear extracts were incubated with 2 ng of (γ-32P)ATP labelled GT75 oligonucleotide and 2000 fold molar excess of un-labelled oligonucleotide and poly(dI-dC) as competitor in a buffer containing 20 mM Hepes pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol (v/v) supplemented with proteases and phosphatases inhibitors, in a final volume of 20 μl. After 25 min incubation at room temperature, the samples were irradiated for 10 min at 302 nm utilizing a trans illuminator UV (BioRadLaboratories). Then, 3 μl Laemmli sample buffer were added and the mixture was boiled for 5 min. The denatured samples were then loaded and resolved on a 10% (w/v) SDS-polyacrylamide gel according to the Laemmli procedure. 2.5. eEF1A1 mRNA, protein level and cell viability determination

2.3. Electroporation Cell viability was evaluated by MTT test as described (Farra et al., 2011). For qRT-PCR, total RNA was extracted, quantified and the integrity evaluated as described (Baiz et al., 2009; Grassi et al., 1995). qRT-PCR conditions were previously reported (Grassi et al., 2007).

Aptamers and siRNA were purchase from Eurofins Genomics (Ebersberg, Germany). As aptamer, we used the GT75 sequence we previously developed and which contains GT (guanosine-thymidine) 3

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Normalized to GAPDH

3,0

A) Protein levels

*

2,5 2,0

§

1,5 1,0 0,5 0,0

eEF1A1 patients

eEF1A1 control

eEF1A2 patients

eEF1A2 control

P1

C1

P2

C2

P3

C3

P4

P1

C1

P2

C2

P3

C3

P4

EEF1A1 GAPDH

EEF1A2 GAPDH

Normalized to 28S

2,4

+

B) mRNA levels

2,0 1,6 1,2 0,8 0,4 0,0

Normalized to GAPDH

eEF1A1 patients 6 5

eEF1A1 control

eEF1A2 patients

eEF1A2 control

C) Protein levels dead vs suriving patients

#

4

Dead patients

3

Surviving patients

2 1 0

eEF1A1

eEF1A2

Fig. 1. Protein and mRNA levels of eEF1A1/eEF1A2 in patient B-lymphocytes. Following B-lymphocytes isolation, eEF1A1 and eEF1A2 were quantified by western blotting (data summarized in the upper part of A and also shown by representative blots in the lower part of A; P = patient; C = control) and real time PCR (B) in 46 CLL patients and 26 healthy control. (C) Comparison between the protein levels of eEF1A1/eEF1A2 in dead vs surviving patients. * p = 0,028 compared to control; § p = 0,0001 compared to control; + p = 0,0081 compared to control; # p = 0,0042 compared to surviving patients..

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3. Results

Protein extracts were prepared in RIPA Buffer (Sigma-Aldrich, St Louis, MO, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). The protein concentration was evaluated by BCA protein assay reagent (Pierce, Rockford, IL, USA). For western blot, 30 μg of proteins were resolved by 12% or 15% (w/v) SDS-polyacrilamide matrix gel and transferred in a 0.22 μM nitrocellulose membrane (Schleicher & Schuell, Keene, NH, USA). Rabbit monoclonal antibody anti-eEF1A1 (AbCam, Cambridge, UK), rabbit polyclonal antibody anti-eEF1A2 (Santa Cruz, CA, USA), rabbit polyclonal antibody anti-LC3B (microtubule associated protein 1 light chain 3 beta, GeneTex Inc., CA, USA) and rabbit polyclonal antibody anti-GAPDH (Santa Cruz) were used as primary antibodies. Blots were developed using a corresponding secondary horseradish peroxidase antibody (GeneTex Inc.,) and enhanced chemiluminescent detection substrate (Pierce, Rockford, IL, USA). GS-700 imaging densitometer and quantity-one software (Biorad, CA, USA) were used for protein bands quantification. PARP (Poly (ADP-ribose) polymerase) cleavage, were performed as previously described (Zanetti et al., 2008).

3.1. Expression level of eEF1A1 and eEF1A2 in CLL patients Following B-lymphocytes isolation, EF1A1 and eEF1A2 protein levels were evaluated by western blot in 46 CLL patients and in 26 control volunteers. Our data indicate a significant increase of the levels of both eEF1A1 (p = 0.028) and eEF1A2 (p = 0.0001) protein in CLL patients vs control (Fig. 1A). At the mRNA level, the significant difference between CLL patients and control was maintained for eEF1A1 (p = 0.0081) but not eEF1A2 (Fig. 1B). No significant correlations were observed among eEF1A1/eEF1A2 protein levels and either CLL patient age (suppl. mat. 1) or the percent of positivity to CLL biological markers (reported in Table 1). Similarly, no correlation was found between eEF1A1/eEF1A2 protein levels and the fact that patients required/non required therapy during the study. In contrast, for eEF1A1 but not for eEF1A2, we observed a significant increase (p = 0.0042) of the protein level in patients which died during the study compared to those surviving (Fig. 1C). Of note, dead patients did not differ in age compared to surviving patients (77 ± 10 vs 76 ± 7, p = 0.7). Finally, no significant differences were observed for the mRNA levels of both eEF1A1 and eEF1A2 in surviving compared to dead CLL patients (Suppl mat 2A).

2.6. Animal study Animal studies were carried out using female severe combined immunodeficiency (SCID) mice (4–6 weeks old). Animals were purchased from Charles River (Milan, Italy) and maintained under pathogen-free conditions. All the experimental procedures involving animals were conducted in compliance with the guidelines of the European (86/609/ EEC) and the Italian (D.L.116/92) laws, and were approved by both the Italian Ministry of Health and the Administration of the University Animal House (Prot. 42/2012). The SCID mice received subcutaneous injection of 1 × 107 MEC-1 cells into the right flank as previously described (Capolla et al., 2015). When the tumors reached an average volume of 250–300 mm3 (equivalent to mg), mice were randomly distributed in groups of 5 animals and the tumor masses were injected with aptamers, siRNA or saline solution (0.9% w/v of NaCl in distilled water). In particular, 300 µg of aptamer or siRNA (both diluted in 50 μl of saline) were added to 85 µg of Lipofectamine 2000 (Invitrogen, Milan, Italy) and then injected intratumorally. A similar approach was previously used in vivo for DNA delivery (Dereani et al., 2014; Durigutto et al., 2013). Tumor size was assessed every two/three days by caliper measurement; the tumor volume was calculated according to the formula: volume = Dxd2x π/6, where D and d are the long and the short diameters of the tumor mass, respectively. For ethical reasons, in the experiments aimed at the evaluation of tumor mass growth, animals were sacrificed when the tumor masses reached the weight of 1300–1400 mg. Aptamer and siRNA stability was tested using 2.5 μg of each molecule, which was then incubated with either saline or saline + lipofectamine under the same condition used for the delivery into the tumor mass in vivo. Before gel loading, the aptamer-siRNA were isolated from the complex aptamer-siRNA/saline + lipofectamine by the use of 1% v/v Triton X-100. Subsequently, all the samples were loaded onto a 12% polyacrylamide gel and run at 12 V/cm in TBE 1X buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3). The aptamer and siRNA bands were then visualized by Stainall dye solution (Sigma-Aldrich) or by ethidium bromide (Sigma-Aldrich), respectively, and the bands photographed.

3.2. Effect of eEF1A1 targeting by the GT75 aptamer in a CLL model cell line The observation that patients that died during the study had more elevated eEF1A1 levels compared to those surviving, suggests a contribution of elevated eEF1A1 levels to CLL progression. Thus, to explore a possible functional involvement of eEF1A1 in CLL, eEF1A1 was targeted by the GT75 aptamer we previously developed (Dapas et al., 2002; Scaggiante et al., 2012; Scaggiante et al., 2006; Scaggiante et al., 2005; Scaggiante et al., 2013; Scaggiante et al., 2016). The cell line MEC-1, a commonly used cell model for B- CLL (Capolla et al., 2016), was employed to explore the effects of GT75. Before testing GT75 effects in living MEC-1, we proved its ability to target eEF1A1 in a cell free test using an U.V. crosslinking assay. For this purpose, 32P labelled GT75 was mixed with total protein nuclear extract of MEC-1. The specificity of interaction was obtained by the high ionic strength of the incubation medium and by the addition of 2000 fold-molar excess of poly(dI-dC) unlabeled competitor. The binding of GT75 with the target was then made stable (covalent) by UV irradiation, in order to prevent any unbinding during gel running. The pattern of binding was then compared to that of the hepatocellular carcinoma cell line JHH6, where GT75 is known to bind to eEF1A1 (Scaggiante et al., 2016). As reported in Fig. 2A, a complex near to 85 KDa was detected, compatible with the combination of the molecular weight of eEF1A protein with that of the 75 long aptamer GT75. After demonstrating that GT75 can bind to eEF1A1 in vitro, the aptamer was delivered to living MEC-1 by electroporation, a technique suited for this kind of cells (Gassner et al., 2018; Longo et al., 2008). Compared to the control CT75 aptamer, GT75 significantly (p < 0.0012) down-regulated MEC-1 viability in a time dependent fashion (Fig. 2B). Moreover, it also reduced (p = 0.048) MEC-1 number compared to CT75 (Fig. 2C). Finally, GT75 did not result in any significant decrease of both protein (Fig. 2D) and mRNA of eEF1A1 (Suppl. mat 2B), in agreement with our previous experience in HCC cell lines (Scaggiante et al., 2016).

2.7. Statistic

3.3. Effect of eEF1A1 targeting by a siRNA in a CLL model cell line

P values were calculated by the GraphPad InStat tools (GraphPad Software, Inc., La Jolla, CA, USA) using the unpaired t test with or without Welch correction and the Mann-Whitney Test, Wilcoxon matched-pairs signed-ranks test, as appropriate. When appropriated, the paired t test was used. P values < 0.05 were considered statistically significant.

To further proving the relevant role of eEF1A1 in MEC-1 vitality, eEF1A1 was targeted by a specific siRNA (siA1) we previously developed (Farra et al., 2017). Our data indicate that also with siA1, delivered by electroporation, MEC-1 vitality (p = 0.0001) and cell number 5

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JHH6

B) Mec-1 vitality -117 - 85 - 48 - 34 - 26

% Normalized to the average of CT75 treated cells

MEC-1

A) 32P-GT75 biniding to eEF1A in protein extract

NTC CT75 GT75

160 140 120 100 80 60

*

*

2

3

*

40 20

1

4

5

Days 1600000

C) Mec-1 cell number (day 3)

Cell number

1400000 1200000 1000000

6

7

Fig. 2. GT75 binding to eEF1A1 in vitro and effects on MEC-1 viability and cell number. A) UV crosslinking: 32P labelled GT75 was mixed with total protein nuclear extracts of MEC-1 and of the hepatocellular carcinoma cell line JHH6, where GT75 is known to bind to eEF1A1; the arrow indicates the size of the expected complex. B) Effects of GT75 on MEC-1 viability measured by MTT (electroporation). C) Effects of GT75 on MEC-1 cell number (electroporation). D) Following electroporation of GT75 into MEC-1, the effects on eEF1A1 protein were measured by western blotting (representative blot). NTC = cells electroporated only by the electroporation solution; GT75 = cells electroporated by the GT75 aptamer; CT75 = cells electroporated by the control aptamer CT75. * p < 0,0012 compared to CT75 treated cells, n = 10; § p < 0,048 compared to CT75 treated cells, n = 9. Data are shown as mean ± SEM.

§

800000 600000 400000 200000 0

GT75

CT75

D) eEF1A1 protein level following aptamer treatment EEF1A1 GAPDH

both GT75 and siA1. Together these data indicate the possibility to compare aptamer and siRNAs effects in MEC-1 following electroporation.

(p = 0.0127) were remarkably down-regulated in a time dependent manner (Fig. 3A and B, respectively) compared to cell treated by a control siRNA (siGL2). This suggests for eEF1A1 a relevant role in maintaining MEC-1 viability. Finally, siA1 treatment significantly decreased the levels of both protein (p = 0.047) and mRNA (p = 0.027) (Fig. 3C and D, respectively) in agreement with our previous studies in HCC cell lines (Farra et al., 2017).

3.5. Effect of eEF1A1 targeting in a mouse model of CLL To confirm the role of eEF1A1, we explored the effects of GT75 and siA1 in a mouse model of B-cell malignancy developed using MEC-1 cells (Capolla et al., 2015). For these tests, we could have employed, in analogy to the experiments performed in cultured MEC-1, electroporation as delivery strategy. However, due to the reasons exposed in the introduction, lipofection was preferred. Following one single injection of GT75 complexed with lipofectamine, we observed a significant (p < 0.003) slowing down of tumor mass growth compared to non-treated and control treated (CT75) animals (Fig. 5A). Notably, also siA1 could significantly (p < 0.014) slow down tumor mass growth (Fig. 5B) thus confirming the in vitro results. Remarkably, GT75 also significantly (p < 0.014) prolonged animal survival (Fig. 5C) compared to control.

3.4. Stability of siA1/aptamer following electroporation and transduction efficacy To compare the effects of GT75 vs siA1, we verified any possible deleterious effects of electroporation on GT75/siA1 molecular integrity. We first tested GT75/siA1 integrity following the incubation with the electroporation solutions under the same condition employed for the electroporation tests (see material and methods). As reported in Fig. 4AB, the incubation of GT75/siA1 with the electroporation solution did not significantly affect molecules integrity. Moreover, also the electric pulse of the electroporation had no major deleterious effects on the integrity of GT75/siA1. Notably, the similar stability of active (GT75/ siA1) and control (CT75/siGL2) molecules indicate the possibility to compare the effects of active vs control molecules. Finally, electroporation allowed a similar transduction efficiency (Suppl. mat 3A) for

3.6. Stability of siA1 and aptamer following lipofection To compare the effects of GT75 vs siA1 in vivo, we verified any 6

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A) MEC-1 vitality

120 100 80

^

60

20

siGL2 siA1

2

3

4

5

6

Days

B) MEC-1 cell number (day 3)

1200000 1000000 Cell number

NTC

^

40

+

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Fig. 3. siA1 effects on MEC-1 viability and cell number. A) Effects of siA1 on MEC-1 viability measured by MTT (electroporation). B) Effects of siA1 on MEC-1 cell number (electroporation). C) Effects of siA1 on the protein level of eEF1A1 evaluated by western blotting (data are summarized on the left and are also shown by a representative blot on the right). D) Effects of siA1 on the mRNA level of eEF1A1 evaluated by real time PCR. NTC = cells electroporated only by the electroporation solution; siA1 = cells electroporated with the siRNA against eEF1A1, siGl2 = cells electroporated with the control siRNA. .^ p = 0,0001 compared to siGL2 treated cells, n = 10; + p = 0,0127 compared to siGL2 treated cells, n = 6. °p = 0,047 compared to siGL2 treated cells, n = 6; & p = 0,027 compared to siGL2 treated cells, n = 4. Data are shown as mean ± SEM.

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on the reduced death rate of the tumor cells (Kipps et al., 2017; Zenz et al., 2010). Thus, we explored the effects of eEF1A1 targeting on the two major cell death pathways, i.e. apoptosis and autophagy. Whereas we could not detect any evident sign of apoptosis (as evaluated by PARP cleavage, Suppl. mat 3B), we observed a significant (p = 0.0238) processing of LC3B, a hallmark of autophagy (Levy et al., 2017) (Fig. 7). Autophagy induction was detected both after GT75 or siA1 delivery to MEC-1; this suggests that the effects of eEF1A1 targeting are independent from the targeting molecule, rather they depend on the target per se.

possible deleterious effects of lipofection on GT75/siA1 molecular integrity. We also evaluated the effects of the saline solution in which GT75/siA1 were suspended before complexation with lipofectamine and injection in mice. Neither lipofectamine nor saline solution significantly affected molecules integrity as demonstrated following Triton X-100 extraction of the samples (Fig. 6A, B and Fig. 6C, D). These observations indicate the possibility to compare aptamer and siRNAs effects in the in vivo model used. Moreover, the similar stability (Fig. 6A, B, C and D) of active (GT75/siA1) and control (CT75/siGL2) molecules indicate the possibility to compare the effects of active vs control molecules.

4. Discussion 3.7. eEF1A1 targeting promotes autophagy in MEC-1 Whereas eEF1A1 and eEF1A2 have been implicated in the development/progression of many human cancers, no information are

Accumulation of clonal B cell in CLL patient seems to depend mainly 7

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A) Aptamer stability and electroporation

Thus, it is conceivable that eEF1A1 overexpression sustains tumor Bcell viability contributing to the maintenance/progression of the disease. To prove the functional role of eEF1A1 in the model cell line (MEC1), we have used an aptamer (GT75), able to bind eEF1A1 in the liver cancers cells (Scaggiante et al., 2016). To verify that GT75 was able to target eEF1A1 also in MEC-1, we have performed an UV crosslinking assay. By this test, we could prove that GT75 binds eEF1A1 in MEC-1 (Fig. 2A). This is a pivotal information to ensure that the effects exerted by GT75 in cultured cells and in the animal model can be considered related to its ability to bind eEF1A1. The present data together with those of our previous work (Scaggiante et al., 2016) indicate that GT75 can target eEF1A1 in a variety of human cell lines (MEC-1, JHH6, HepG2, HuH7) thus showing its specific binding ability. In line with our previous experiments in HCC cells (Farra et al., 2017; Scaggiante et al., 2016) siA1 (Fig. 3C and D) but not GT75 (Fig. 2D and Suppl. mat 2B) induces the reduction of both protein and mRNA levels of the target eEF1A1. This proves, in our experimental setting, the proper mechanisms of action of the two molecules, siA1 inducing the degradation of the target mRNA and GT75 most likely interacting with the target protein altering its functions. Despite the different mechanisms of action, both molecules induce very similar quantitative effects in vitro on cell viability (Figs. 2 and 3). However, in vivo, siA1 seems to be somewhat less effective than GT75 (compare Fig. 5B with 5A). Whereas the difference may depend on several factors, the RNA structure of siA1 may render it less resistant against nucleases compared to the DNA structure of GT75 (Grassi et al., 2010). Moreover, as siA1 is 21 nt long while GT75 is 75 nucleotide long, it is possible that the effects of degrading enzymes occur faster for the shorter siA1 compared to the longer GT75. In this regard, we have evidences that a shorter GT75 version (i.e. GT27, 27 nucleotides long) retains the ability to target eEF1A1 (Scaggiante et al., 2006). This implies that even when partially degraded, GT75 can maintain its activity. In contrast, it is known that also a modest shortening of siRNA length impairs the activity (Scaggiante et al., 2011). In vitro and in vivo we used two different transduction methods: electroporation and lipofection. As mentioned in the introduction, this depends on the poor transduction ability of lipofectamine in cultured MEC-1 (15% of the cells on average, our unpublished results). Despite this, lipofection-mediated delivery of GT75 in vivo resulted in a remarkable down regulation of tumor growth. A possible explanation for this apparent discrepancy vitro/vivo may reside in the fact that in vivo the GT75/lipofectamine complexes stayed in contact with the target cells for days after injection. In contrast, only few hours in vitro were possible, due to the unspecific toxicity exerted by lipofectamine (our unpublished observation). Thus, whereas lipofection may be less effective, the longer exposure time to the target cells may have compensated the lower transduction efficacy. An alternative to lipofection in vivo could have been electroporation, a physical procedure able to induce a short, intense electric field creating reversible pores in the cell membrane (Campana et al., 2019). This, in turn, allows the inflow through the cell membrane of different molecules, including aptamer and siRNA. While electroporation can be easily applied in in vitro studies, for in vivo applications, its use is far more complex. Indeed, electrical parameters must be adjusted in relation not only to the specific nucleic acid molecule transduced but also to the target tissue (Sersa et al., 2015). Thus, electric pulses duration, shape and amplitude, have to be optimized. Moreover, a specific and sophisticated apparatus is required to deliver the electric pulse to the diseased tissue. These aspects, together with our previous positive experience of nucleic acid transfection in vivo (Dereani et al., 2014), prompted us to use lipofection as a delivery system for in vivo tests. When we consider a possible future targeting in vivo of eEF1A1, by either GT75 or siA1, the issue of side effects should be carefully evaluated. In this regards, the use of targeted delivery system has to be always considered (Scarabel et al., 2017). Despite this, at least for

B) SiRNA stability and electroporation

Fig. 4. Aptamer and siRNA stability in electroporation solution. Aptamers (A) and siRNAs (B) were incubated with the electroporation solution. An aliquot was also subjected to electroporation under the same condition undertaken for the electroporation delivery to MEC-1 cells. Thereafter aptamer and siRNA have been loaded on a non-denaturing polyacrylamide gel (12%), stained by Stainall (GT75-CT75) or ethidium bromide (siA1-siGl2) and photographed. GT75: active aptamer; CT75: control aptamer; siA1: siRNA against eEF1A1; siGl2: control siRNA. Sol E = solution of electroporation; Electro: molecules that underwent electroporation.

available with regard to CLL. Here, we observe an increase of the protein levels both for eEF1A1 and eEF1A2 (Fig. 1A) in lymphocytes of CLL patient compared to those from healthy volunteers. Thus, for the first time, our observation shows a dis-regulation in the expression of these two proteins in CLL. Notably, we did not find any correlation with the main molecular signature for CLL (Table 1), suggesting that eEF1A1/2 may be considered as potential novel independent diagnostic marker for CLL. Notably, the mechanism responsible for the increase of the protein levels seems to be different: for eEF1A1 but not for eEF1A2, we observe an increase in the mRNA levels in CLL tumor cells compared to control (Fig. 1B). This suggests that eEF1A1 protein increase may derive from the augmented expression; in contrast, for eEF1A2 it is more likely that enhanced protein stability (half-life) is responsible for the increased level. In this regards, it is known that pathological posttranslational modification of eEF1A1/2 can determine increased protein stability (Scaggiante et al., 2014). With regard to the prognostic value, eEF1A1 seems to be of more interest. Indeed, eEF1A1 levels but not that of eEF1A2 are more elevated in patients that died, compared to those surviving (Fig. 1C). This suggests that high level of eEF1A1 may have the potential to be considered a negative prognostic factor in CLL. However, the small number of patients analyzed in this pilot study and the limited number of patients, which have been died, requires further studies in a more expanded cohort of CLL patients to reach a conclusive statement in this regard. Interestingly, the data obtained in in vitro and in vivo tests support a functional role for eEF1A1 in the maintenance/progression of the disease. Indeed, eEF1A1 targeting results both in vitro (Figs. 2 and 3) and in vivo (Fig. 5) in a significant down-regulation of MEC-1 growth. 8

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Fig. 5. Effects on tumor growth following eEF1A1 targeting by either GT75 or siA1. GT75 (A) or siA1 (B) were injected once (day 0) into the tumor mass generated following MEC-1 implantation into the right flank of severe combined immunodeficiency mice. The effects on tumor mass growth were followed over time. *p < 0.003 compared to NTC or CT75. °p < 0.014 compared to NTC or siGL2. GT75 = animals treated by the aptamer GT75, n = 4 animals; CT75 animals treated by the control aptamer CT75, n = 4 animals; NTC = animals treated by saline solution, n = 5 animals; siA1 = animals treated by the siRNA against eEF1A1, n = 3 animals; siGl2 = animal treated by the control siRNA, n = 3 animals; NTC = animals treated by saline solution, n = 5 animals. Data are shown as mean ± SEM. C) Effects of eEF1A1 targeting by GT75 on animal survival. GT75 = animals treated by the aptamer GT75, n = 4 animals; CT75 animals treated by the control aptamer CT75, n = 3 animals; NTC = animals treated by saline solution, n = 6 animals. GT75 curve is statistically different from NTC (p = 0.0038) and CT75 treated animals (p = 0.014).

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progression (Wojnacki and Galli, 2018). Notably, data are arising with regard to the potential therapeutic relevance of autophagy stimulation in CLL. For example, the increase of the autophagy markers BECN1 and ATG5 has been associated with a more favorable prognosis and longer treatment free survival (Kong et al., 2018). Moreover, many currently used drugs, such as fludarabine, dexamethasone and idelalisib activate autophagy (Mahoney et al., 2012). Thus, based on the above consideration and on our data, eEF1A1 targeting has, in principle, the potential to be of therapeutic benefit in CLL.

GT75, we have previous evidences that this aptamer does not substantially affect normal lymphocytes (Dapas et al., 2003; Scaggiante et al., 2013; Scaggiante et al., 1998). This feature makes in principle GT75 an ideal molecule for the specific functional targeting of tumor Bcell. Autophagy (Levy et al., 2017) is a process by which cytoplasmic components are delivered into the lysosome for degradation. Notably, autophagy has been proposed as an alternative mechanism ruling cell death. Our data indicate that eEF1A1 targeting by both GT75 and siA1 results in the activation of autophagy (Fig. 7). A direct link between eEF1A1 and autophagy has not been fully demonstrated so far (Scaggiante et al., 2014). However, the multiple protein interactions implicated in the non-canonical functions may be at the basis of eEF1A1/autophagy link. In particular, eEF1A1 is known to form complex’s with actin (Novosylna et al., 2017) which in turn is related to autophagy since its filamentous form is requested for autophagy

5. Conclusion In this work, we show the increase of eEF1A1 and eEF1A2 expression in CLL patient cells compared to healthy control, opening the possibility to consider these two proteins as novel diagnostic markers in CLL. Notably, for eEF1A1, we observed increased levels in patients with 9

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A) Effetycs of saline on aptamer stablity

B) Effetycs of saline on siRNA stablity

C) Effetycs of lipofectamine on aptamer stablity

D) Effetycs of lipofectamine on siRNA stablity

LC3B

Fig. 6. Aptamer and siRNA stability in lipofection solution. Aptamers (A) and siRNAs (B) were incubated with the saline solution utilized for molecules resuspension before complexation with lipofectamine and injection in mice. An aliquot from samples in (A) and (B) was also combined with lipofectamine (C, aptamer; D, siRNA) under the same condition undertaken for the lipofection delivery to tumor mass in vivo. Thereafter, aptamer and siRNA were extracted from the lipofectamine complex by Triton X-100. Aptamer and siRNA where then loaded on a nondenaturing polyacrylamide gel (12%), stained by Stainall (GT75-CT75) or ethidium bromide (siA1-siGl2) and photographed. GT75: active aptamer; CT75: control aptamer; siA1: siRNA against eEF1A1; siGl2: control siRNA.

reduced overall survival, possibly suggesting a role as a negative prognostic marker. This observation together with the involvement of eEF1A1 in MEC-1 survival open perspectives for the development of novel therapeutic approaches for CLL based on eEF1A1 targeting by biological molecules such as aptamers and/or siRNAs. Obviously, this goal goes together with the selection of an appropriate delivery system able to protect the therapeutic nucleic acid molecule and to drive it specifically to the clonal B cell.

LC3B-I LC3B-II

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Author contributions Gabriele Grassi, Gabriele Pozzato and Bruna Scaggiante have conceptualized the work. Barbara Dapas has designed the experimental procedures and performed most of the in vitro experiments including the analysis of samples from patients. Michela Coan and Fabrizio Zanconati contributed to eEF1A1/2 protein and mRNA quantification. Chiara Guerra and Chiara Gnan focused on electroporation optimization. Gabriele Pozzato and Valter Gattei provided the patient data and samples. Sonia Zorzet, Sara Capolla and Macor Paolo took care about animal experiments. Bruna Scaggiante developed the UV cross-linking assay. Gabriele Grassi, Gabriele Pozzato and Bruna Scaggiante interpreted the data. Gabriele Grassi drafted the manuscript, which was then improved with major and minor suggestions by all the other authors.

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Declaration of Competing Interest

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CT75

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 7. Effects of eEF1A1 targeting by either GT75 or siA1 on cell autophagy. Three days after either GT75 or siA1 electroporation in MEC-1, the induction of cell autophagy was monitored evaluating the two forms of the autophagy marker LC3B: LC3B-I = cytosolic form of LC3B; LC3BII = phosphatidylethanolamine LC3B conjugate form recruited to autophagosomal membranes. NTC = cells electroporated by the electroporation solution only; siA1 = cells electroporated by the siRNA against eEF1A1; siGl2 = cells electroporated by the control siRNA; GT75 = cells electroporated by the GT75 aptamer; CT75 = cells electroporated by the control aptamer CT75. * p = 0,0238 compared to siGL2 and NTC, n = 4; § p = 0,0238 compared to CT75 and NTC, n = 4; Data are shown as mean ± SEM.

Acknowledgements The work was supported by research grant 2013 of “Associazione Italiana contro le leucemie, linfoma e mieloma (AIL)”, Trieste, Italy and by Associazione Italiana Ricerca sul Cancro (AIRC). We wish to thank prof Olaf Heidenreich, Prinses Máxima Centrum voor kinderoncologie, Heidelberglaan 25, UTRECHT, Nederland, for helpful discussion. 10

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Appendix A. Supplementary material

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