Multifunctional nanoparticle-mediated SHARP1 knockdown in MLL-AF6 acute myeloid leukemia

Acute myeloid leukemia (AML) has an extremely poor prognosis and high relapse and fatality rates. We targeted SHARP1 using multifunctional small interfering RNA (siRNA) and bortezomib (BTZ)-loaded cRGD-guided PEGylated cationic liposomal nanoparticles to monitor their antileukemic activity in MLL-AF6 AML cells. Ecient siRNA/BTZ co-delivery by the nanoparticles signicantly inhibited cell viability, decreasing clonogenic growth of AML cells and stimulating robust apoptosis. We hypothesized that SHARP1 downregulation induced nonfunctional MLL-AF6, DOT1L, MEN1, and LEDGF fusion protein accumulation, preventing MLL-AF complex formation and downregulating RAS-GTP and Bcl-2, consequently triggering autophagy and apoptosis. The BTZ combination substantially augmented therapeutic synergy leading to enhanced autophagic and apoptotic events. Our ndings demonstrate a state-of-the-art biodegradable nanoplatform for siRNA/BTZ co-delivery with targeted SHARP1 knockdown, demonstrating a potential therapeutic option for MLL-AF6 AML. SHARP1 silencing in MLL-AF6 AML cells. These ndings indicate that animal useful data for the development of nanomedicines for AML treatment. We demonstrated the

Here, we exhibit the rst preclinical experimental research for SHARP1-based AML therapy using therapeutic siRNA nanodelivery (Fig. 1a,b). We show that SHARP1 is an MLL-AF6-dependent leukemogenic driver, consistent with multifunctional bioengineered nanoparticle activity in SHARP1 downregulation, revealing a potential approach for human MLL-AF6 AML treatment.
We used confocal microscopy to evaluate Lipo-siRNA-BTZ-PEG-cRGD uptake by ML-2 cells (see Supplementary Fig. 2a). ML-2 cells were transfected with ATTO 550 red-labeled Lipo-siRNA-BTZ-PEG-cRGD, which was observed in the cytoplasm after 4 h of incubation. The comprehensive cellular uptake study showed aggregated and dense ATTO 550 red-labeled Lipo-siRNA-BTZ-PEG-cRGD surrounding the nucleus. Moreover, 3D images ( Supplementary Fig. 2b) revealed a noteworthy boost of ATTO 550 redlabeled Lipo-siRNA-BTZ-PEG-cRGD. Flow cytometry with uorescence intensity quanti cation further con rmed the intracellular uptake e ciency of Lipo-siRNA-BTZ-PEG-cRGD (Supplementary Fig. 3 and 4). Furthermore, the visible colocalization between cell nuclei (blue) and ATTO 550-labeled Lipo-siRNA-BTZ-PEG-cRGD (red) showed effective nanoparticle transfection in ML-2 cells (Supplementary Fig. 2c and Supplementary Fig. 5). Notably, lipid bilayer fusion, endocytosis, drug conjugation, and facilitated diffusion of a lipofectamine-based formulation 19 with further surface modi cation by PEGylation allowed targeting of the α v β 3 integrin ligand 20 . These mechanisms were used to allow Lipo-siRNA-BTZ-PEG-cRGD to deliver cargo to ML-2 cells, elucidating how multifunctional bioengineered nanoparticles improve therapeutic e ciency and safety by optimizing delivery in MLL-AF6 AML. Naked siRNA cannot passively diffuse across cell membranes owing to charge instability, high molecular weight, water solubility, and intracellular enzyme degradation 21 . Peptide-guided PEGylated cationic nanoliposomes are effective for siRNA delivery 22 ; we showed this strategy is effective for SHARP1 targeting. Consistently, our cellular uptake results demonstrated that Lipo-siRNA-BTZ-PEG-cRGD undergoes αvβ3 receptor-mediated endocytosis.
To investigate the roles of SHARP1 downregulation-induced autophagy in MLL-AF6 AML, we studied the mRNA expression of MLL-AF6 regulation and DOT1L oncogenic genes affected by SHARP1 knockdown in ML-2 cells. Mechanistically, DOT1L and MLL-AF6 are oncogenic proteins that directly regulate SHARP1 expression. It has been reported that downregulation of SHARP1 does not affect the expression of DOT1L gene 8 . Therefore, it was suggested that SHARP1 together with the inhibition of DOT1L may be a favorable treatment modality for MLL-AF6 AML 9 , since it is known that downregulation of DOT1L can inhibit the regulation of MLL-AF6 AML 26,27 . However, so far, there are no reports regarding the downregulation of SHARP1 affecting DOT1L and MLL-AF6 regulation. Here, we hypothesized that DOT1L/MLL-AF6 expression would be suppressed by multifunctional SHARP1-targeted Lipo-siRNA-BTZ-PEG-cRGD in MLL-AF6 AML cells. The mRNA expression of DOT1L and MLL-AF6 were profoundly downregulated in Lipo-siRNA-BTZ-and Lipo-siRNA-BTZ-PEG-cRGD-treated cells, while those treated with naked siRNA and Lipo-BTZ-PEG-cRGD were not affected at p < 0.05 (Fig. 3a,b). Lipo-siRNA-BTZ-PEG-cRGD inhibited DOT1L and MLL-AF6 the most (approximately 80% and 50%, respectively), emphasizing induction of autophagy and indicating a new oncogenic role of SHARP1 in MLL-AF6 and DOT1L functions in MLL-AF6 AML cells. The MLL-AF6-driven transcriptional machinery correlating with SHARP1related genes for MLL-AF6 AML growth and maintenance are shown in Fig. 3c. Therefore, three fundamental regulatory mechanisms initiated the autophagic signals that eradicated MLL-AF6 AML cells. First, BTZ combination disrupted the ubiquitin-proteasome signaling pathway, causing accumulation of unfolded ubiquitin, triggering autophagy 28 . Second, selective SHARP1 inhibition, which results in the breakdown of oncogenic fusion proteins responsible for DOT1L-dependent MLL-AF6-MEN1-LEDGF complex synthesis crucial for leukemogenesis, was signi cantly abrogated, strengthening autophagy 29 . Eventually, MLL-AF6 downregulation resulted in considerable RAS-GTP pathway interference. RAS-GTP pathway is necessary for promoting cancer cell survival through binding RAS-GTP selectively to RALB-GTP for overexpressing Bcl-2 30 . Our nanoparticles primarily interfered with this important oncogenic pathway, leading to autophagic signals. Thus, Lipo-siRNA-BTZ-PEG-cRGD nanoparticles may be RAS inhibitors, potentiating MLL-AF6 AML therapy. Based on these ndings, we plotted an ML-2 cell growth curve (Supplementary Fig. 10) to measure cell proliferation, which was strikingly attenuated upon Lipo-siRNA-BTZ-PEG-cRGD treatment. Manipulating autophagy may provide powerful evidence for the effect of Lipo-siRNA-BTZ-PEG-cRGD as a multifunctional targeted therapy in MLL-AF6 AML cells. We provided a comprehensive in vitro study on new multifunctional bioengineered smart nanoparticles using wellcharacterized cRGD-conjugated thiolated PEG (NHS-PEG6-maleimide) to effectively co-deliver siRNA/BTZ for targeted SHARP1 silencing in MLL-AF6 AML cells. These ndings indicate that animal experiments may provide useful data for the development of nanomedicines for AML treatment. We demonstrated the effects of SHARP1 downregulation on DOT1L and MLL-AF6 expression levels and highlighted a new vital oncogenic role of SHARP1 in MLL-AF6 AML growth and maintenance. Lipo-siRNA-BTZ-PEG-cRGD are multifunctional particles that reveal versatile regulatory mechanisms, including SHARP1 silencing, MLL-AF6/DOT1L inhibition, p53 activation, RAS suppression, proteasome inhibition, and autophagy/apoptosis induction. This approach should open new avenues for applying smart biocompatible re-engineered nanostructures in in vivo studies and further clinical translation to produce advanced MLL-AF6 AMLtargeting therapeutics.

Characterization
The sizes and zeta potentials of different treatment nanostructures were measured by dynamic light scattering using a Malvern Nano ZS90 Zetasizer (Malvern Instruments, Malvern, UK). Transmission electron microscopy (TEM) was performed using a 1200 EX transmission electron microscope (JEOL Ltd., Akishima, Japan). The morphologies of siRNA/BTZ-loaded cRGD-tagged PEGylated cationic nanoliposomes (Lipo-siRNA-BTZ-PEG-cRGD) were studied by scanning electron microscopy (SEM) using a JSM-7200F scanning electron microscope (JEOL Ltd.). Nanoparticle characterization by DLS measurements showed optimal physicochemical properties evidenced by hydrodynamic diameter and zeta potential values. TEM ndings indicated e cient siRNA encapsulation by the PEGylated modi ed surface conjugated with the targeted cRGD ligand. Furthermore, the narrow size distribution and regular spherical structure of Lipo-siRNA-BTZ-PEG-cRGD demonstrated by SEM indicated assembly and morphology.

Cell culture
The current study was performed on a human ML-2 cell line (ACC 15) obtained from DSMZ (Braunschweig, Germany). The cells were cultured in RPMI-1640 (Gibco) supplemented with 10% heatinactivated fetal bovine serum (FBS, Gibco) and maintained at 37 °C in a 5% CO 2 atmosphere.

Delivery study
Silencer Select pre-designed siRNA targeting SHARP1 was obtained from Invitrogen (antisense sequence, 5′-UAUACAAAGAGGAAUAGUCCA-3′; sense sequence, 5′-GACUAUUCCUCUUUGUAUATT-3′). ML-2 cells were transfected with naked siRNA and the indicated structured nanoparticles and investigated for SHARP1 knockdown e ciency by western blotting and qPCR at 2 d or 3 d post transfection. Intracellular uptake ML-2 cells were seeded in 35-mm glass-bottom dishes (Corning) (4 × 10 4 cells per well) 1 d prior to transfection. Lipo-siRNA-BTZ-PEG-cRGD nanoparticles were prepared with ATTO-550 (Sigma-Aldrich) uorophore (red) was used to track intracellular location of nanoparticles. After a 4-h incubation at 37 °C, cells were xed with 4% paraformaldehyde in PBS (Sigma-Aldrich) for 15 min and directly stained with Hoechst 33342 (Molecular Probes, Eugene, OR, USA) and Concanavalin A-FITC (Sigma-Aldrich) for nuclei (blue) and cell membrane (green) labeling, respectively. Cells were imaged using a TiE-A1R confocal laser scanning microscope (Nikon, Tokyo, Japan). The Z series images and 3D snapshots of cells were taken using Ni-E Z Drive for Z-stack mode measurements. Image data were analyzed using Nikon imaging software (NIS-Elements Viewer 4.50). For ow cytometric analysis, ML-2 cells were seeded in 96-well plates and harvested after 4 h using ATTO-550 (Sigma-Aldrich) uorophore (red), followed by ow cytometry on FACS CantoΙΙ (BD Biosciences, Franklin, NJ, USA).

Western blot analysis
Cells were treated with naked siRNA or the indicated treatments for 3 d, and further cultured for 2 d before lysis. The cells were suspended in a radioimmunoprecipitation (RIPA) lysis buffer (Santa Cruz Biotechnology, Dallas, TX, USA) for whole cell lysis. Proteins were separated by SDS-PAGE and blotted onto PVD membrane (Millipore). Images were captured and chemiluminescent signals were analyzed using ImageQuant LAS 4010 (GE Healthcare, Chicago, IL, USA). Western blot experiments were performed using the following antibodies: anti-SHARP1 (sc-373763, 1:1000 working dilution, overnight shaking incubation at 4 °C), Cruz Marker molecular weight standards (sc-2035), and β-actin (sc-47778, 1:5000 working dilution, overnight shaking incubation at 4 °C) from Santa Cruz Biotechnology and a secondary horseradish peroxidase (HRP)-conjugated antibody from Abcam (ab205718, 1:2000 working dilution, incubation at 25 °C for 1 h).
Quantitative PCR RNA was extracted using an RNeasy kit (QIAGEN, Hilden, Germany) and reverse-transcribed using the QuantiTech Reverse Transcription kit (QIAGEN). PCR was performed using SYBR Green JumpStart Taq Ready Mix (Sigma-Aldrich) and quantitatively assessed on a Mx3000P (Agilent Technologies). For each sample, transcript levels of tested genes were normalized to GAPDH using the 2 -∆∆CT method. The highest expression was arbitrarily set to 1 and expressions in the other samples were normalized to this value. All experiments were performed in triplicate. PCR was performed using cDNA and primer sequences listed in Supplementary Table 2.
Cytotoxicity assay and colony forming assay For cytotoxicity assay, 1 × 10 4 cells were seeded in 96-well plates one day prior to transfection, incubated at 37 °C for 2 d, and, on the third day, subjected to CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. Colony formation assay was performed by plating 1,000 cells per well on a 6-well plate, transfected, and incubated for 7 d.
The colonies were xed with methanol:acetic acid 3:1 (v/v) and stained with 0.5% crystal violet in 20% methanol for 15 min.

Cell apoptosis study
Cell apoptosis was evaluated using an Annexin V-FITC apoptosis detection kit following the manufacturer's protocol (Thermo Fisher Scienti c) and analyzed with a FACS CantoΙΙ ow cytometer (BD Biosciences). For uorescence imaging, ML-2 cells were seeded into 35-mm glass-bottom dishes (Iwaki AGC Techno Glass, Japan) and treated with naked siRNA and the indicated nanoparticles for 24 h. After two washes with PBS, the cells were stained with 5 µL of Annexin V-FITC and PI and incubated at 25 °C in the dark for 15 min. For nuclei staining, stained cells were counterstained with 4,6-Diamidino-2phenylindole (DAPI; D1306, Invitrogen) and visualized using an inverted uorescence microscope (Keyence BZ-9000, Osaka, Japan). incubated with a goat anti-rabbit IgG secondary antibody conjugated to Alexa Fluor 568 (Thermo Fisher Scienti c; A-11036, 4 µg/mL) in staining buffer for 1 h. For nuclei staining, stained cells were counterstained with DAPI (D1306, Invitrogen) for 1 h before visualization by TiE-A1R confocal laser scanning microscope (Nikon). Image data were analyzed by Nikon imaging software (NIS-Elements Viewer 4.50), and the number of anti-SHARP1-positive cells was counted using MATLAB R2020b software (MathWorks, Natick, MA, USA).
Live cell imaging and cell growth assay For phase contrast, ML-2 cells were seeded into 96-well plates (~40,000 cells/well), incubated for 4 h, and then were treated with Lipo-siRNA-BTZ-PEG-cRGD. After washing twice with PBS, the cells were suspended in fresh media and monitored using the IncuCyte ZOOM (Essen BioScience, Ann Arbor, MI, USA) acquiring images at 30 min intervals for 48 h. For the uorescence-based technique, cells were seeded into 96-well plates (~40,000 cells/well), incubated for 4 h, and then treated with Lipo-siRNA-BTZ-PEG-cRGD. After two PBS washes, the cells were stained with IncuCyte NucLight Rapid Red Reagent for nuclear labeling (Essen Bioscience) and monitored using the IncuCyte ZOOM (Essen BioScience) acquiring images at 1 h intervals for 48 h. To assess cell growth, transfected cells were seeded in 96-well plate (5,000 cells/well) and imaged every 1 h using IncuCyte ZOOM (Essen Bioscience). The con uence was analyzed by the IncuCyte ZOOM 2016A software (Essen Bioscience).