Preclinical evaluation of the third-generation, bi-steric mechanistic target of rapamycin complex 1-selective inhibitor RMC-6272 in NF2-deficient models.

Background: NF2-associated meningiomas are progressive, highly morbid, and nonresponsive to chemotherapies, highlighting the need for improved treatments. We have established aberrant activation of the mechanistic target of rapamycin (mTOR) signaling in NF2-deficient tumors, leading to clinical trials with first- and second-generation mTOR inhibitors. However, results have been mixed, showing stabilized tumor growth without shrinkage offset by adverse side effects. To address these limitations, here we explored the potential of third-generation, bi-steric mTOR complex 1 (mTORC1) inhibitors using the preclinical tool compound RMC-6272. Methods: Employing human NF2-deficient meningioma lines, we compared mTOR inhibitors rapamycin (first-generation), INK128 (second-generation), and RMC-6272 (third-generation) using in vitro dose-response testing, cell-cycle analysis, and immunoblotting. Furthermore, the efficacy of RMC-6272 was assessed in NF2-null 3D-spheroid meningioma models, and its in vivo potential was evaluated in 2 orthotopic meningioma mouse models. Results: Treatment of meningioma cells revealed that, unlike rapamycin, RMC-6272 demonstrated superior growth inhibitory effects, cell-cycle arrest, and complete inhibition of phosphorylated 4E-BP1 (mTORC1 readout). Moreover, RMC-6272 had a longer retention time than INK128 and inhibited the expression of several eIF4E-sensitive targets on the protein level. RMC-6272 treatment of NF2 spheroids showed significant shrinkage in size as well as reduced proliferation. Furthermore, in vivo studies in mice revealed effective blockage of meningioma growth by RMC-6272, compared with vehicle controls. Conclusions: Our study in preclinical models of NF2 supports possible future clinical evaluation of third-generation, investigational mTORC1 inhibitors, such as RMC-5552, as a potential treatment strategy for NF2.

Translatome analysis of tuberous sclerosis complex 1 patient-derived neural progenitor cells reveals rapamycin-dependent and independent alterations.

BACKGROUND: Tuberous sclerosis complex (TSC) is an inherited neurocutaneous disorder caused by mutations in the TSC1 or TSC2 genes, with patients often exhibiting neurodevelopmental (ND) manifestations termed TSC-associated neuropsychiatric disorders (TAND) including autism spectrum disorder (ASD) and intellectual disability. Hamartin (TSC1) and tuberin (TSC2) proteins form a complex inhibiting mechanistic target of rapamycin complex 1 (mTORC1) signaling. Loss of TSC1 or TSC2 activates mTORC1 that, among several targets, controls protein synthesis by inhibiting translational repressor eIF4E-binding proteins. Using TSC1 patient-derived neural progenitor cells (NPCs), we recently reported early ND phenotypic changes, including increased cell proliferation and altered neurite outgrowth in TSC1-null NPCs, which were unaffected by the mTORC1 inhibitor rapamycin. METHODS: Here, we used polysome profiling, which quantifies changes in mRNA abundance and translational efficiencies at a transcriptome-wide level, to compare CRISPR-edited TSC1-null with CRISPR-corrected TSC1-WT NPCs generated from one TSC donor (one clone/genotype). To assess the relevance of identified gene expression alterations, we performed polysome profiling in postmortem brains from ASD donors and age-matched controls. We further compared effects on translation of a subset of transcripts and rescue of early ND phenotypes in NPCs following inhibition of mTORC1 using the allosteric inhibitor rapamycin versus a third-generation bi-steric, mTORC1-selective inhibitor RMC-6272. RESULTS: Polysome profiling of NPCs revealed numerous TSC1-associated alterations in mRNA translation that were largely recapitulated in human ASD brains. Moreover, although rapamycin treatment partially reversed the TSC1-associated alterations in mRNA translation, most genes related to neural activity/synaptic regulation or ASD were rapamycin-insensitive. In contrast, treatment with RMC-6272 inhibited rapamycin-insensitive translation and reversed TSC1-associated early ND phenotypes including proliferation and neurite outgrowth that were unaffected by rapamycin. CONCLUSIONS: Our work reveals ample mRNA translation alterations in TSC1 patient-derived NPCs that recapitulate mRNA translation in ASD brain samples. Further, suppression of TSC1-associated but rapamycin-insensitive translation and ND phenotypes by RMC-6272 unveils potential implications for more efficient targeting of mTORC1 as a superior treatment strategy for TAND.

Proteasomal pathway inhibition as a potential therapy for NF2-associated meningioma and schwannoma.

BACKGROUND: Neurofibromatosis 2 (NF2) is an inherited disorder caused by bi-allelic inactivation of the NF2 tumor suppressor gene. NF2-associated tumors, including schwannoma and meningioma, are resistant to chemotherapy, often recurring despite surgery and/or radiation, and have generally shown cytostatic response to signal transduction pathway inhibitors, highlighting the need for improved cytotoxic therapies. METHODS: Leveraging data from our previous high-throughput drug screening in NF2 preclinical models, we identified a class of compounds targeting the ubiquitin-proteasome pathway (UPP), and undertook studies using candidate UPP inhibitors, ixazomib/MLN9708, pevonedistat/MLN4924, and TAK-243/MLN7243. Employing human primary and immortalized meningioma (MN) cell lines, CRISPR-modified Schwann cells (SCs), and mouse Nf2-/- SCs, we performed dose response testing, flow cytometry-based Annexin V and cell cycle analyses, and RNA-sequencing to identify potential underlying mechanisms of apoptosis. In vivo efficacy was also assessed in orthotopic NF2-deficient meningioma and schwannoma tumor models. RESULTS: Testing of three UPP inhibitors demonstrated potent reduction in cell viability and induction of apoptosis for ixazomib or TAK-243, but not pevonedistat. In vitro analyses revealed that ixazomib or TAK-243 downregulates expression of c-KIT and PDGFRα, as well as the E3 ubiquitin ligase SKP2 while upregulating genes associated with endoplasmic reticulum stress-mediated activation of the unfolded protein response (UPR). In vivo treatment of mouse models revealed delayed tumor growth, suggesting a therapeutic potential. CONCLUSIONS: This study demonstrates the efficacy of proteasomal pathway inhibitors in meningioma and schwannoma preclinical models and lays the groundwork for use of these drugs as a promising novel treatment strategy for NF2 patients.

Generation of biologically active recombinant human OCT4 protein from E. coli.

Octamer-binding transcription factor 4 (OCT4) is vital for early embryonic development and is a master regulator of pluripotency in embryonic stem cells. Notably, OCT4 is a key reprogramming factor to derive induced pluripotent stem cells, which have tremendous prospects in regenerative medicine. In the current study, we report heterologous expression and purification of human OCT4 in E. coli to produce pure recombinant protein under native conditions. To achieve this, the 1083 bp coding sequence of the human OCT4 gene was codon-optimized for heterologous expression in E. coli. The codon-optimized sequence was fused with fusion tags, namely a cell-penetrating peptide sequence for intracellular delivery, a nuclear localization sequence for intranuclear delivery, and a His-tag for affinity purification. Subsequently, the codon-optimized sequence and the fusion tags were cloned in the protein expression vector, pET28a(+), and transformed into E. coli strain BL21(DE3) for expression. The recombinant OCT4 protein was purified from the soluble fraction under native conditions using immobilized metal ion affinity chromatography in a facile manner, and its identity was confirmed by Western blotting and mass spectrometry. Furthermore, the secondary structure of the recombinant protein was analyzed using far ultraviolet circular dichroism spectroscopy, which confirmed that the purified fusion protein maintained a secondary structure conformation, and it predominantly composed of α-helices. Next, the recombinant OCT4 protein was applied to human cells, and was found that it was able to enter the cells and translocate to the nucleus. Furthermore, the biological activity of the transduced OCT4 protein was also demonstrated on human cells. This recombinant tool can substitute for genetic and viral forms of OCT4 to enable the derivation of integration-free pluripotent cells. It can also be used to elucidate its biological role in various cellular processes and diseases and for structural and biochemical studies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02758-z.

Soluble expression, purification, and secondary structure determination of human MESP1 transcription factor.

Transcription factor MESP1 is a crucial factor regulating cardiac, hematopoietic, and skeletal myogenic development. Besides, it also contributes to the generation of functional cardiomyocytes. Here, we report the soluble expression and purification of the full-length human MESP1 protein from the heterologous system, which can be delivered into the target mammalian cells. To generate this biological macromolecule, we cloned its codon-optimized gene sequence fused to a nuclear localization sequence, a cell-penetrating peptide, and a His-tag into the protein expression vector and expressed in the bacterial system (E. coli strain BL21(DE3)). Subsequently, we have screened and identified the optimal expression parameters to obtain this recombinant fusion protein in soluble form from E. coli and examined its expression concerning the placement of fusion tags at either terminal. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Notably, this purified fusion protein has maintained its secondary structure after purification, primarily comprising α-helices and random coils. This molecular tool can potentially replace its genetic and viral forms in the cardiac reprogramming of fibroblasts to induce a cardiac transcriptional profile in an integration-free manner and elucidating its role in various biological processes and diseases. KEY POINTS: • Screening of the suitable gene construct was performed and identified. • Screening of optimal expression conditions was performed and identified. • Native purification of recombinant human MESP1 protein from E. coli was performed. • Recombinant MESP1 protein has retained its secondary structure after purification.

Synthesis of functionalized silk-coated chitosan-gold nanoparticles and microparticles for target-directed delivery of antitumor agents.

Chemically modified biopolymers derived nanomaterials have shown great potential in drug delivery and live-cell imaging. We have developed two materials, doxorubicin-loaded chitosan-gold nanoparticles and beads, both embedded with functionalized silk fibroin. Nanoparticles with size 8 ± 3 nm were synthesized using chitosan as reducing and stabilizing agent. Beads with 900-1000 µm size were formulated by the ionic gelation technique. Both the materials were coated with functionalized silk fibroin for targeted and sustained drug release properties. The coated materials showed retarded drug release compared to the uncoated ones. The cytotoxicity was assessed in HeLa cell lines, which demonstrated a maximum dose-dependent decrease in cell viability for the cells treated with folate conjugated silk fibroin coated nanoparticles. The live-cell imaging of the nanoparticles unveiled the increased cellular uptake of the coated materials by seven folds than the uncoated ones. Thus, functionalized silk coated materials can be effective drug delivery tools for targeted and sustained drug release.

Generation of cell-permeant recombinant human transcription factor GATA4 from E. coli.

Transcription factor GATA4 is expressed during early embryogenesis and is vital for proper development. In addition, it is a crucial reprogramming factor for deriving functional cardiomyocytes and was recently identified as a tumor suppressor protein in various cancers. To generate a safe and effective molecular tool that can potentially be used in a cell reprogramming process and as an anti-cancer agent, we have identified optimal expression parameters to obtain soluble expression of human GATA4 in E. coli and purified the same to homogeneity under native conditions using immobilized metal ion affinity chromatography. The identity of GATA4 protein was confirmed using western blotting and mass spectrometry. Using circular dichroism spectroscopy, it was demonstrated that the purified recombinant protein has maintained its secondary structure, primarily comprising of random coils and α-helices. Subsequently, this purified recombinant protein was applied to human cells and was found that it was non-toxic and able to enter the cells as well as translocate to the nucleus. Prospectively, this cell- and nuclear-permeant molecular tool is suitable for cell reprogramming experiments and can be a safe and effective therapeutic agent for cancer therapy.

Generation of a Recombinant Stem Cell-Specific Human SOX2 Protein from Escherichia coli Under Native Conditions.

The stem cell-specific SOX2 transcription factor is critical for early embryonic development and the maintenance of embryonic and neural stem cell identity. It is also crucial for the generation of induced pluripotent and neural stem cells, thus providing immense prospect in patient-specific therapies. Here, we report soluble expression and purification of human SOX2 protein under native conditions from a bacterial system. To generate this macromolecule, we codon-optimized the protein-coding sequence and fused it to a nuclear localization signal, a protein transduction domain, and a His-tag. This was then cloned into a protein expression vector and was expressed in Escherichia coli. Subsequently, we have screened and identified the optimal expression conditions to obtain recombinant fusion protein in a soluble form and studied its expression concerning the position of fusion tags at either terminal. Furthermore, we purified two versions of recombinant SOX2 fusion proteins to homogeneity under native conditions and demonstrated that they maintained their secondary structure. This molecular tool can substitute genetic and viral forms of SOX2 to facilitate the derivation of integration-free induced pluripotent and neural stem cells. Furthermore, it can be used in elucidating its role in stem cells, various cellular processes and diseases, and for structural and biochemical studies.

Unfolding transmembrane TNFα dynamics in cancer therapeutics.

Cytokines are a group of glycoprotein signaling mediators, which play essential roles in maintaining several complex physiological functions of our body. TNFα is such a pleiotropic cytokine, which involves maintaining a plethora of immune responses. Initially, TNFα is synthesized as a 26 kDa full-length transmembrane form, which is enzymatically cleaved to produce the soluble circulating 17 kDa TNFα. Although the anti-cancer potential of soluble TNFα was discovered more than a century back, its dual ability to promote tumor, posed a major hindrance in finding its acceptance as a proper anti-cancer molecule. In contrast, the membrane-tethered tmTNFα holds the potential of tumor regression without initiating cell proliferation. The membrane-tethered form of TNFα is the physiological precursor of soluble TNFα that remains biologically active and is capable of initiating signaling cascades after binding with the TNFα receptors- TNFR I and TNFR II. In this review, we emphasize on the basic biology and molecular aspects of tmTNFα for its anti-cancer potential.

Deciphering insights of novel recombinant tmTNFα in cell growth inhibition.

Soluble TNFα, a member of TNF superfamily attributes dual roles in apoptosis and cell proliferation whereas its precursor transmembrane TNFα (tmTNFα) has potential for tumor reduction without initiating proliferation. In this perspective, we recombinantly expressed functional tmTNFα and explored its potential in cell growth inhibition. While structural characterizations of purified tmTNFα revealed integrity of the protein, cell viability assays demonstrated significant antiproliferative effect on HepG2 (IC50: 36 nM) and HeLa (IC50: 23 nM) cells. Mechanistic insights into mode of cell death unveiled G1 arrest in HepG2 and G2/M arrest in HeLa cells accompanied with disruption of mitochondrial membrane potential and activation of executioner caspases. Subsequent, flow cytometry based assays resulted confirmatory evidence of apoptosis after treatment with the recombinant protein. Additionally, effect of the recombinant protein on 3D tumor spheroids was explored, which rendered reduction in tumor size due to cell death as evident from confocal microscopy studies. Effectiveness of the tmTNFα in 2D monolayer as well as in complex 3D spheroids demonstrate the therapeutic significance of the protein, featuring recombinant tmTNFα as an attractive option for cancer therapeutics in days to come.

Optimality of poly-vinyl alcohol/starch/glycerol/citric acid in wound dressing applicable composite films.

This work addresses response surface methodology (RSM) design based investigations to obtain optimality of quaternary formulations [variant macromolecular concentrations of starch (St, 5-10 w/w%), polyvinyl alcohol (PVA, 5-10 w/w%), citric acid (CA, 15-40 wt%) and glycerol (Gl, 15-40 wt%)] associated to wound dressing films. Appropriate combinations of the swelling index (SI), weight loss (WL%) during 27 days, tensile strength (TS) and percentage elongation (%E) have been considered during such studies. The optimized composition was achieved through RSM optimization and exhibited very good water absorption (300.5% SI) and flexibility (87.5%E), and acceptable in-vitro degradation (51.4% WL) and TS (5 MPa) values, which are significantly better than reported data. Further, the film constitution indicated amplified antibacterial effectiveness against both Gram-positive (Listeria monocytogenes) and Gram-negative (Escherichia coli) bacteria and enhanced cell growth (145.5%) to thereby infer upon the potential associated with its application as a viable wound dressing film.

Transmembrane TNFα-Expressed Macrophage Membrane-Coated Chitosan Nanoparticles as Cancer Therapeutics.

Transmembrane TNFα, a crucial signaling cytokine, holds anticell proliferative potential. Successful delivery of this intact transmembrane protein to the target site is quite intriguing. Amidst numerous nanocarriers, a novel class of new generation macrophage membrane-coated nanocarriers is endowed with innate tumor homing abilities and inherent capacity of escaping body's defense machinery. In this perspective, a novel therapeutic module has been fabricated by coating a nontoxic, biodegradable chitosan nanoparticle core with engineered macrophage membrane-tethered TNFα. Herein, the expression of membrane-bound TNFα was induced by challenging phorbol 12-myristate 13-acetate-differentiated THP-1 cells with bacterial lipopolysaccharide. Subsequently, the as-synthesized chitosan nanoparticle core was coated with a TNFα-expressed macrophage membrane through an extrusion process. While transmission electron microscopy imaging, sodium dodecyl sulphate polyacrylamide gel electrophoresis, and western blotting results demonstrated successful coating of the chitosan nanoparticles with the TNFα-induced membrane, the cell viability assays on several cancer cells such as-HeLa, MDA-MB-231, and MCF-7 revealed significant innate anticell proliferative potential of these membrane-coated nanoparticles. Additionally, evaluation of expression of several interleukins after treatment demonstrated excellent biocompatibility of the membrane-coated nanoparticles. The fabricated nanoparticles also demonstrated a dose-dependent cell death in tumor spheroids, which was further corroborated with calcein AM/propidium iodide dual staining results. Translation of the therapeutic efficacy of the synthesized nanoparticles from monolayers to tumor spheroids augments its potential in cancer therapy.

Integration of a Nonsteroidal Anti-Inflammatory Drug with Luminescent Copper for in Vivo Cancer Therapy in a Mouse Model.

A facile, process of fabrication of a luminescent bovine serum albumin-copper nanocluster (BSA-CuNC) customized ibuprofen nanodrug (BSA-CuNC-Ibf), encapsulating the ibuprofen was developed. Ibuprofen, which is commonly used to treat inflammation, was utilized here as a model drug. The formation of BSA-CuNC initiated by encapsulation of the Cu ions within the protein moiety followed by gradual reduction of the Cu ions by certain amino acid residues like tyrosine and tryptophan at alkaline pH resulted in the formation of BSA-CuNC within the protein template. Heat treatment and lowering the pH fitted the ibuprofen in the center by hydrogen bonding, hydrophobic and electrostatic interactions, and resulted in the formation of nanoparticles. The nanodrug (BSA-CuNC-Ibf) thus formed was characterized by transmission electron microscopy (TEM), dynamic and static light scattering (DLS), and zeta potential. The spherical shaped nanodrug has a hydrodynamic diameter of about 100.4 ± 28.9 nm. The encapsulation efficiency was found to be 94% which corresponds to 1880 µg/mL of ibuprofen in the BSA-CuNC-Ibf nanodrug. The as synthesized BSA-CuNC-Ibf exhibited cytotoxicity on both human cervical cancer cells (HeLa) and human lung cancer cells (A549). The present nanodrug when explored for its tumor preventive role on Daltons lymphoma ascites (DLA) bearing Swiss albino mice, exemplified sizable inhibition of tumor growth by reactive oxygen species mediated apoptosis and by modulating prostaglandin (PGE2) levels. It also inhibited metastasis of the cancer cells, thus enhancing the life expectancy of the mice.