OGT and OGA gene-edited human induced pluripotent stem cells for dissecting the functional roles of O-GlcNAcylation in hematopoiesis.

Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor O-GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes O-GlcNAcase (OGA) and O-GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of OGA-depleted (OGAi) and OGT-depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34+ hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory. The established OGAi and OGTi hiPSCs exhibiting an increase or decrease in cellular O-GlcNAcylation concomitant with their loss of OGA and OGT, respectively, appeared normal in phenotype and karyotype, and retained pluripotency, although they may favor differentiation toward certain germ lineages. Upon hematopoietic differentiation through mesoderm induction and endothelial-to-hematopoietic transition, we found that OGA inhibition accelerates hiPSC commitment toward HSPCs and that disruption of O-GlcNAc homeostasis affects their commitment toward erythroid lineage. The differentiated HSPCs from all groups were capable of giving rise to all hematopoietic progenitors, thus confirming their functional characteristics. Altogether, the established single-cell clones of OGTi and OGAi hiPSCs represent a valuable platform for further dissecting the roles of O-GlcNAcylation in blood cell development at various stages and lineages of blood cells. The incomplete knockout of OGA and OGT in these hiPSCs makes them susceptible to additional manipulation, i.e., by small molecules, allowing the molecular dynamics studies of O-GlcNAcylation.

Flash nanoprecipitation assisted self-assembly of ionizable lipid nanoparticles for nucleic acid delivery.

Ionizable lipid nanoparticles (LNPs) have emerged as a powerful tool for the intracellular delivery of nucleic acids. Following the recent success of LNP-based siRNA therapeutics and mRNA vaccines, the use of ionizable lipids for nucleic acid delivery has tremendously increased. Here, we introduce a flash nanoprecipitation (FNP) approach using the confined impingement (CIJ) mixer to stably self-assemble ionizable LNPs. To validate this approach, we employed three clinically relevant LNP formulations containing SM102, ALC0315, and DLin-MC3-DMA as ionizable lipids. FNP-assembled LNPs showed >95% encapsulation efficiency of mRNA and siRNA payloads and particle sizes below 150 nm. SM102 or ALC0315 LNPs demonstrated efficient delivery of mRNA into immune cells in vitro and to lymphoid organs in vivo, whereas Dlin-MC3-DMA LNPs allowed effective intracellular siRNA delivery with great functional ability. The FNP technique could economically produce LNPs in smaller volumes that are highly suitable for the discovery phase.

Flash nanoprecipitation allows easy fabrication of pH-responsive acetalated dextran nanoparticles for intracellular release of payloads.

Acetalated dextran (Ac-Dex) nanoparticles are currently of immense interest due to their sharp pH-responsive nature and high biodegradability. Ac-Dex nanoparticles are often formulated through single- or double-emulsion methods utilizing polyvinyl alcohol as the stabilizer. The emulsion methods utilize toxic organic solvents such as dichloromethane or chloroform and require multi-step processing to form stable Ac-Dex nanoparticles. Here, we introduce a simple flash nanoprecipitation (FNP) approach that utilizes a confined impinging jet mixer and a non-toxic solvent, ethanol, to form Ac-Dex nanoparticles rapidly. Ac-Dex nanoparticles were stabilized using nonionic PEGylated surfactants, D-α-Tocopherol polyethylene glycol succinate (TPGS), or Pluronic (F-127). Ac-Dex nanoparticles formed using FNP were highly monodisperse and stably encapsulated a wide range of payloads, including hydrophobic, hydrophilic, and macromolecules. When lyophilized, Ac-Dex TPGS nanoparticles remained stable for at least one year with greater than 80% payload retention. Ac-Dex nanoparticles were non-toxic to cells and achieved intracellular release of payloads into the cytoplasm. In vivo studies demonstrated a predominant biodistribution of Ac-Dex TPGS nanoparticles in the liver, lungs, and spleen after intravenous administration. Taken together, the FNP technique allows easy fabrication and loading of Ac-Dex nanoparticles that can precisely release payloads into intracellular environments for diverse therapeutic applications. pH-responsive Acetalateddextran can be formulated using nonionic surfactants, such as TPGS or F-127, for intracellular release of payloads. Highly monodisperse and stable nanoparticles can be created through the simple, scalable flash nanoprecipitation technique, which utilizes a confined impingement jet mixer.

Musashi-2 in cancer-associated fibroblasts promotes non-small cell lung cancer metastasis through paracrine IL-6-driven epithelial-mesenchymal transition.

BACKGROUND: Lung cancer, the most common cause of cancer-related mortality worldwide, is predominantly associated with advanced/metastatic disease. The interaction between tumor cells and cancer-associated fibroblasts (CAFs) in tumor microenvironment is known to be essential for regulating tumor progression and metastasis, but the underlying mechanisms, particularly the role of RNA-binding protein Musashi-2 (MSI2) in CAFs in promoting non-small cell lung cancer (NSCLC) invasiveness and metastatic spread, remain obscure. METHODS: Genomic and proteomic database analyses were performed to evaluate the potential clinical significance of MSI2 in NSCLC tumor and stromal clinical specimens. Molecular approaches were used to modify MSI2 in CAFs and determine its functional role in NSCLC cell motility in vitro using 2D and 3D models, and in metastasis in a xenograft mouse model using live-cell imaging. RESULTS: MSI2, both gene and protein, is upregulated in NSCLC tissues and is associated with poor prognosis and high metastatic risk in patients. Interestingly, MSI2 is also upregulated in NSCLC stroma and activated fibroblasts, including CAFs. Depletion of MSI2 in CAFs by CRISPR-Cas9 strongly inhibits NSCLC cell migration and invasion in vitro, and attenuates local and distant metastatic spread of NSCLC cells in vivo. The crosstalk between CAFs and NSCLC cells occurs via paracrine signaling, which is regulated by MSI2 in CAFs via IL-6. The secreted IL-6 promotes epithelial-mesenchymal transition in NSCLC cells, which drives metastasis. CONCLUSION: Our findings reveal for the first time that MSI2 in CAFs is important in CAF-mediated NSCLC cell invasiveness and metastasis via IL-6 paracrine signaling. Therefore, targeting the MSI2/IL-6 axis in CAFs could be effective in combating NSCLC metastasis.

Chemotherapy-induced PDL-1 expression in cancer-associated fibroblasts promotes chemoresistance in NSCLC.

OBJECTIVES: A cure for cancer is out of reach for most patients due to chemoresistance. Cancer-associated fibroblasts (CAFs) play a vital role in cancer chemoresistance, but detailed understanding of the process particularly in chemoresistant lung cancer is lacking. In this study, we investigated programmed death-ligand 1 (PDL-1) as a potential biomarker for CAF-induced chemoresistance and evaluated its role and the underlying mechanisms of chemoresistance in non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: A systemic search of gene expression profiles of multiple tissues in NSCLC was carried out to determine the expression intensities of traditional fibroblast biomarkers and CAF-secreted protumorigenic cytokines. PDL-1 expression in CAFs was analyzed by ELISA, Western blotting, and flow cytometry. Human cytokine array was used to identify specific cytokines secreted from CAFs. Role of PDL-1 in NSCLC chemoresistance was assessed using CRISPR/Cas9 knockdown and various functional assays including MTT, cell invasion, sphere formation, and cell apoptosis. In vivo experiments were conducted using a co-implantation xenograft mouse model with live cell imaging and immunohistochemistry. RESULTS: We demonstrated that chemotherapy-stimulated CAFs promoted tumorigenic and stem cell-like properties of NSCLC cells, which contribute to their chemoresistance. Subsequently, we revealed that PDL-1 expression is upregulated in chemotherapy-treated CAFs and is associated with poor prognosis. Silencing PDL-1 expression suppressed CAFs' ability to promote stem cell-like properties and invasiveness of lung cancer cells, favoring chemoresistance. Mechanistically, an upregulation of PDL-1 in chemotherapy-treated CAFs led to an increase in hepatocyte growth factor (HGF) secretion, which stimulates cancer progression, cell invasion, and stemness of lung cancer cells, while inhibiting apoptosis. CONCLUSION: Our results show that PDL-1-positive CAFs modulate stem cell-like properties of NSCLC cells by secreting elevated HGF, thereby promoting chemoresistance. Our finding supports PDL-1 in CAFs as a chemotherapy response biomarker and as a drug delivery and therapeutic target for chemoresistant NSCLC.

Inhibition of O-GlcNAcase Inhibits Hematopoietic and Leukemic Stem Cell Self-Renewal and Drives Dendritic Cell Differentiation via STAT3/5 Signaling.

Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown. Using small molecule inhibitors and the CRISPR/Cas9 system, we reveal for the first time that inhibition of either OGA or OGT, which subsequently caused an increase or decrease in cellular O-GlcNAcylation, inhibits the self-renewal and maintenance of CD34+ hematopoietic stem/progenitor cells (HSPCs) and leukemic stem/progenitor cells and drives normal and malignant myeloid differentiation. We further unveiled the distinct roles of OGA and OGT inhibition in lineage-specific differentiation. While OGT inhibition induces macrophage differentiation, OGA inhibition promotes the differentiation of both CD34+ HSPCs and AML cells into dendritic cells (DCs), in agreement with an upregulation of a multitude of genes involved in DC development and function and their ability to induce T-cell proliferation, via STAT3/5 signaling. Our novel findings provide significant basic knowledge that could be important in understanding AML pathogenesis and overcoming differentiation blockage-agnostic to the genetic background of AML. Additionally, the parallel findings in normal HSPCs may lay the groundwork for future cellular therapy as a means to improve the ex vivo differentiation of normal DCs and macrophages.

A novel E-cadherin/SOX9 axis regulates cancer stem cells in multiple myeloma by activating Akt and MAPK pathways.

Cancer stem cells (CSCs) have been identified in multiple myeloma (MM) and are widely regarded as a key driver of MM initiation and progression. E-cadherin, in addition to its established role as a marker for epithelial-mesenchymal transition, also plays critical roles in controlling the aggressive behaviors of various tumor cells. Here, we show that depletion of E-cadherin in MM cells remarkably inhibited cell proliferation and cell cycle progression, in part through the decreased prosurvival CD138 and Bcl-2 and the inactivated Akt and MAPK pathways. CSC features, including the ability of the cells to form clonogenic colonies indicative of self-renewal and side population, were greatly suppressed upon the depletion of E-cadherin and subsequent loss of SOX9 stem-cell factor. We further provide evidence that SOX9 is a downstream target of E-cadherin-mediated CSC growth and self-renewal-ectopic re-expression of SOX9 in E-cadherin-depleted cells rescued its inhibitory effects on CSC-like properties and survival signaling. Collectively, our findings unveil a novel regulatory mechanism of MM CSCs via the E-cadherin/SOX9 axis, which could be important in understanding the long-term cell survival and outgrowth that leads to relapsed/refractory MM.

O-GlcNAcylation homeostasis controlled by calcium influx channels regulates multiple myeloma dissemination.

BACKGROUND: Multiple myeloma (MM) cell motility is a critical step during MM dissemination throughout the body, but how it is regulated remains largely unknown. As hypercalcemia is an important clinical feature of MM, high calcium (Ca2+) and altered Ca2+ signaling could be a key contributing factor to the pathological process. METHODS: Bioinformatics analyses were employed to assess the clinical significance of Ca2+ influx channels in clinical specimens of smoldering and symptomatic MM. Functional and regulatory roles of influx channels and downstream signaling in MM cell migration and invasion were conducted and experimental MM dissemination was examined in a xenograft mouse model using in vivo live imaging and engraftment analysis. RESULTS: Inhibition of TRPM7, ORAI1, and STIM1 influx channels, which are highly expressed in MM patients, and subsequent blockage of Ca2+ influx by CRISPR/Cas9 and small molecule inhibitors, effectively inhibit MM cell migration and invasion, and attenuate the experimental MM dissemination. Mechanistic studies reveal a nutrient sensor O-GlcNAcylation as a downstream regulator of Ca2+ influx that specifically targets cell adhesion molecules. Hyper-O-GlcNAcylation following the inhibition of Ca2+ influx channels induces integrin α4 and integrin ß7 downregulation via ubiquitin-proteasomal degradation and represses the aggressive MM phenotype. CONCLUSIONS: Our findings unveil a novel regulatory mechanism of MM cell motility via Ca2+ influx/O-GlcNAcylation axis that directly targets integrin α4 and integrin ß7, providing mechanistic insights into the pathogenesis and progression of MM and demonstrating potential predictive biomarkers and therapeutic targets for advanced MM.

A novel TRPM7/O-GlcNAc axis mediates tumour cell motility and metastasis by stabilising c-Myc and caveolin-1 in lung carcinoma.

BACKGROUND: Calcium is an essential signal transduction element that has been associated with aggressive behaviours in several cancers. Cell motility is a prerequisite for metastasis, the major cause of lung cancer death, yet its association with calcium signalling and underlying regulatory axis remains an unexplored area. METHODS: Bioinformatics database analyses were employed to assess correlations between calcium influx channels and clinical outcomes in non-small cell lung cancer (NSCLC). Functional and regulatory roles of influx channels in cell migration and invasion were conducted and experimental lung metastasis was examined using in vivo live imaging. RESULTS: High expression of TRPM7 channel correlates well with the low survival rate of patients and high metastatic potential. Inhibition of TRPM7 suppresses cell motility in various NSCLC cell lines and patient-derived primary cells and attenuates experimental lung metastases. Mechanistically, TRPM7 acts upstream of O-GlcNAcylation, a post-translational modification and a crucial sensor for metabolic changes. We reveal for the first time that caveolin-1 and c-Myc are favourable molecular targets of TRPM7/O-GlcNAc that regulates NSCLC motility. O-GlcNAcylation of caveolin-1 and c-Myc promotes protein stability by interfering with their ubiquitination and proteasomal degradation. CONCLUSIONS: TRPM7/O-GlcNAc axis represents a potential novel target for lung cancer therapy that may overcome metastasis.

Reactive oxygen species mediate cancer stem-like cells and determine bortezomib sensitivity via Mcl-1 and Zeb-1 in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive, incurable non-Hodgkin B-cell lymphoma with good initial response to therapy then subsequently relapse. Cancer stem cells (CSCs) are considered to be an underlying cause of these inevitable drug resistance and tumor regrowth, but how CSCs are regulated is largely unknown. We demonstrate here for the first time the existence of CSC-like subpopulations that are modulated by reactive oxygen species (ROS) in MCL cell lines and patient-derived primary cells in an inverse correlation with bortezomib (BTZ) sensitivity. Using various known donors and inhibitors of cellular superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH), we unveil their distinct roles in the regulation of CSC-like subpopulations and thus MCL response to BTZ. O2- inhibits CSC-like cells and sensitizes BTZ-induced apoptosis, whereas H2O2 conversely enriches CSC-like cells and protects against apoptosis and OH has minimal effects. We further observed that an anti-apoptotic Mcl-1 and a transcription factor Zeb-1 are favorable targets of O2- and H2O2, respectively. Using small molecule inhibition, ectopic expression and CRISPR/Cas9-mediated gene manipulation, we verified the roles of Mcl-1 and Zeb-1 in CSC and apoptosis regulation by O2- and H2O2. Our findings provide a novel mechanistic insight into the significance of redox status of MCL cells in determining their drug response via CSC-like subpopulations, which are imperative to a better understanding of therapeutic resistance and relapse.

Inhibition of O-GlcNAcase Sensitizes Apoptosis and Reverses Bortezomib Resistance in Mantle Cell Lymphoma through Modification of Truncated Bid.

Aberrant energy metabolism represents a hallmark of cancer and contributes to numerous aggressive behaviors of cancer cells, including cell death and survival. Despite the poor prognosis of mantle cell lymphoma (MCL), due to the inevitable development of drug resistance, metabolic reprograming of MCL cells remains an unexplored area. Posttranslational modification of proteins via O-GlcNAcylation is an ideal sensor for nutritional changes mediated by O-GlcNAc transferase (OGT) and is removed by O-GlcNAcase (OGA). Using various small-molecule inhibitors of OGT and OGA, we found for the first time that O-GlcNAcylation potentiates MCL response to bortezomib. CRISPR interference of MGEA5 (encoding OGA) validated the apoptosis sensitization by O-GlcNAcylation and OGA inhibition. To identify the potential clinical candidates, we tested MCL response to drug-like OGA inhibitor, ketoconazole, and verified that it exerts similar sensitizing effect on bortezomib-induced apoptosis. Investigations into the underlying molecular mechanisms reveal that bortezomib and ketoconazole act in concert to cause the accumulation of truncated Bid (tBid). Not only does ketoconazole potentiate tBid induction, but also increases tBid stability through O-GlcNAcylation that interferes with tBid ubiquitination and proteasomal degradation. Remarkably, ketoconazole strongly enhances bortezomib-induced apoptosis in de novo bortezomib-resistant MCL cells and in patient-derived primary cells with minimal cytotoxic effect on normal peripheral blood mononuclear cells and hepatocytes, suggesting its potential utility as a safe and effective adjuvant for MCL. Together, our findings provide novel evidence that combination of bortezomib and ketoconazole or other OGA inhibitors may present a promising strategy for the treatment of drug-resistant MCL. Mol Cancer Ther; 17(2); 484-96. ©2017 AACR.

Hyper-O-GlcNAcylation induces cisplatin resistance via regulation of p53 and c-Myc in human lung carcinoma.

Aberrant metabolism in hexosamine biosynthetic pathway (HBP) has been observed in several cancers, affecting cellular signaling and tumor progression. However, the role of O-GlcNAcylation, a post-translational modification through HBP flux, in apoptosis remains unclear. Here, we found that hyper-O-GlcNAcylation in lung carcinoma cells by O-GlcNAcase inhibition renders the cells to apoptosis resistance to cisplatin (CDDP). Profiling of various key regulatory proteins revealed an implication of either p53 or c-Myc in the apoptosis regulation by O-GlcNAcylation, independent of p53 status. Using co-immunoprecipitation and correlation analyses, we found that O-GlcNAcylation of p53 under certain cellular contexts, i.e. high p53 activation, promotes its ubiquitin-mediated proteasomal degradation, resulting in a gain of oncogenic and anti-apoptotic functions. By contrast, O-GlcNAcylation of c-Myc inhibits its ubiquitination and subsequent proteasomal degradation. Gene manipulation studies revealed that O-GlcNAcylation of p53/c-Myc is in part a regulator of CDDP-induced apoptosis. Accordingly, we classified CDDP resistance by hyper-O-GlcNAcylation in lung carcinoma cells as either p53 or c-Myc dependence based on their molecular targets. Together, our findings provide novel mechanisms for the regulation of lung cancer cell apoptosis that could be important in understanding clinical drug resistance and suggest O-GlcNAcylation as a potential target for cancer therapy.