RARγ activation sensitizes human myeloma cells to carfilzomib treatment through the OAS-RNase L innate immune pathway.

Proteasome inhibitors (PIs) such as bortezomib (Btz) and carfilzomib (Cfz) are highly efficacious for patients with multiple myeloma (MM). However, relapses are frequent, and acquired resistance to PI treatment emerges in most patients. Here, we performed a high-throughput screen of 1855 Food and Drug Administration (FDA)-approved drugs and identified all-trans retinoic acid (ATRA), which alone has no antimyeloma effect, as a potent drug that enhanced MM sensitivity to Cfz-induced cytotoxicity and resensitized Cfz-resistant MM cells to Cfz in vitro. ATRA activated retinoic acid receptor (RAR)γ and interferon-ß response pathway, leading to upregulated expression of IRF1. IRF1 in turn initiated the transcription of OAS1, which synthesized 2-5A upon binding to double-stranded RNA (dsRNA) induced by Cfz and resulted in cellular RNA degradation by RNase L and cell death. Similar to ATRA, BMS961, a selective RARγ agonist, could also (re)sensitize MM cells to Cfz in vitro, and both ATRA and BMS961 significantly enhanced the therapeutic effects of Cfz in established MM in vivo. In support of these findings, analyses of large datasets of patients' gene profiling showed a strong and positive correlation between RARγ and OAS1 expression and patient's response to PI treatment. Thus, this study highlights the potential for RARγ agonists to sensitize and overcome MM resistance to Cfz treatment in patients.

Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism.

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

Oxidative stress enhances tumorigenicity and stem-like features via the activation of the Wnt/ß-catenin/MYC/Sox2 axis in ALK-positive anaplastic large-cell lymphoma.

BACKGROUND: The phenomenon that malignant cells can acquire stemness under specific stimuli, encompassed under the concept of cancer cell plasticity, has been well-described in epithelial malignancies. To our knowledge, cancer cell plasticity has not yet been described in hematopoietic cancers. To illustrate and study cancer cell plasticity in hematopoietic cancers, we employed an in-vitro experimental model of ALK-positive anaplastic large-cell lymphoma (ALK+ALCL) that is based on the phenotypic and functional dichotomy of these cells, with cells responsive to a Sox2 reporter (i.e. RR cells) being significantly more stem-like than those unresponsive to the reporter (i.e. RU cells). METHODS: H2O2 was employed to trigger oxidative stress. GFP expression and luciferase activity, readouts of the Sox2 reporter activity, were quantified by using flow cytometry and luciferase activity assay, respectively. Doxorubicin-resistance and clonogenicity were assessed by using the MTS, methylcellulose colony formation and limiting dilution assays. Western blotting and quantitative PCR were used to assess the expression of various members of the Wnt/ß-catenin pathway. Pull-down studies using a Sox2 binding consensus sequence were used to assess Sox2-DNA binding. Quercetin and 10074-G5 were used to inhibit ß-catenin and MYC, respectively. siRNA was used to downregulate Sox2. RESULTS: Under H2O2-induced oxidative stress, a substantial fraction of RU cells was found to convert to RR cells, as evidenced by their acquisition of GFP expression and luciferase activity. Compared to the native RU cells, converted RR cells had significantly higher levels of doxorubicin-resistance, clonogenicity and sphere formation. Converted RR cells were characterized by an upregulation of the Wnt/ß-catenin/MYC/Sox2 signaling axis, previously found to be the key regulator of the RU/RR dichotomy in ALK+ALCL. Furthermore, Sox2 was found to bind to DNA efficiently in converted RR cells but not RU cells, and this finding correlated with significant elevations of several Sox2 downstream targets such as WNT2B and BCL9. Lastly, inhibition of ß-catenin, MYC or Sox2 in RU cells significantly abrogated the H2O2-induced RU/RR conversion. CONCLUSIONS: We have demonstrated that cancer cell plasticity exists in ALK+ALCL, a type of hematopoietic cancer. In this cancer type, the Wnt/ß-catenin/MYC/Sox2 axis is an important regulator of cancer cell plasticity.

Induction of m6A methylation in adipocyte exosomal LncRNAs mediates myeloma drug resistance.

BACKGROUND: Therapeutic resistance occurs in most patients with multiple myeloma (MM). One of the key mechanisms for MM drug resistance comes from the interaction between MM cells and adipocytes that inhibits drug-induced apoptosis in MM cells; MM cells reprogram adipocytes to morph into different characterizations, including exosomes, which are important for tumor-stroma cellular communication. However, the mechanism by which exosomes mediate the cellular machinery of the vicious cycle between MM cells and adipocytes remains unclear. METHODS: Adipocytes were either isolated from bone marrow aspirates of healthy donors or MM patients or derived from mesenchymal stem cells. Co-culturing normal adipocytes with MM cells was used to generate MM-associated adipocytes. Exosomes were collected from the culture medium of adipocytes. Annexin V-binding and TUNEL assays were performed to assess MM cell apoptosis. Methyltransferase activity assay and dot blotting were used to access the m6A methylation activity of methyltransferase like 7A (METTL7A). RIP, MeRIP-seq, and RNA-protein pull down for assessing the interaction between long non-cording RNAs (LncRNAs) and RNA binding proteins were performed. Adipocyte-specific enhancer of zeste homolog 2 (EZH2) knockout mice and MM-xenografted mice were used for evaluating MM therapeutic response in vivo. RESULTS: Exosomes collected from MM patient adipocytes protect MM cells from chemotherapy-induced apoptosis. Two LncRNAs in particular, LOC606724 and SNHG1, are significantly upregulated in MM cells after exposure to adipocyte exosomes. The raised LncRNA levels in MM cells are positively correlated to worse outcomes in patients, indicating their clinical relevancy in MM. The functional roles of adipocyte exosomal LOC606724 or SNHG1 in inhibition of MM cell apoptosis are determined by knockdown in adipocytes or overexpression in MM cells. We discovered the interactions between LncRNAs and RNA binding proteins and identified methyltransferase like 7A (METTL7A) as an RNA methyltransferase. MM cells promote LncRNA package into adipocyte exosomes through METTL7A-mediated LncRNA m6A methylation. Exposure of adipocytes to MM cells enhances METTL7A activity in m6A methylation through EZH2-mediated protein methylation. CONCLUSION: This study elucidates an unexplored mechanism of how adipocyte-rich microenvironment exacerbates MM therapeutic resistance and indicates a potential strategy to improve therapeutic efficacy by blocking this vicious exosome-mediated cycle.

Osteocyte CIITA aggravates osteolytic bone lesions in myeloma.

Osteolytic destruction is a hallmark of multiple myeloma, resulting from activation of osteoclast-mediated bone resorption and reduction of osteoblast-mediated bone formation. However, the molecular mechanisms underlying the differentiation and activity of osteoclasts and osteoblasts within a myelomatous microenvironment remain unclear. Here, we demonstrate that the osteocyte-expressed major histocompatibility complex class II transactivator (CIITA) contributes to myeloma-induced bone lesions. CIITA upregulates the secretion of osteolytic cytokines from osteocytes through acetylation at histone 3 lysine 14 in the promoter of TNFSF11 (encoding RANKL) and SOST (encoding sclerostin), leading to enhanced osteoclastogenesis and decreased osteoblastogenesis. In turn, myeloma cell-secreted 2-deoxy-D-ribose, the product of thymidine catalyzed by the function of thymidine phosphorylase, upregulates CIITA expression in osteocytes through the STAT1/IRF1 signaling pathway. Our work thus broadens the understanding of myeloma-induced osteolysis and indicates a potential strategy for disrupting tumor-osteocyte interaction to prevent or treat patients with myeloma bone disease.

Development of Traceable Rituximab-Modified PEO-Polyester Micelles by Postinsertion of PEG-phospholipids for Targeting of B-cell Lymphoma.

The objective of this work was to develop rituximab (RTX)-modified polymeric micelles for targeting of B-cell lymphoma cells, through postinsertion of RTX-poly(ethylene glycol)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (RTX-PEG-DSPE) into methoxy poly(ethylene oxide)-poly(ε-caprolactone) (PEO-PCL) or methoxy poly(ethylene oxide)-poly(ε-benzylcarboxylate-ε-caprolactone) (PEO-PBCL) micelles. Mixed micelles were made traceable by introducing Cy5.5 to RTX and conjugating Cy3 to propargyl moiety, end-capped PCL or PBCL. Successful adaptation of the postinsertion method for the formation of immunomicelles was evidenced by measurement of RTX levels on the micellar surface, purified from free RTX by size exclusion chromatography, using microBSA assay. A change in the micellar diameter, from 50-70 nm for PEO-PCL and PEO-PBCL micelles and 20 nm for PEG-DSPE micelles, to 80-95 nm for the mixed micellar population as well as the critical micellar concentration of mixed micelles provided further proof for the success of the postinsertion method applied here. Mixed micelles containing PCL or PBCL with a degree of polymerization of 22 (PCL22 and PBCL22) were thermodynamically and kinetically more stable than those with PCL15. Accordingly, RTX micelles containing PCL22 or PBCL22 showed a higher percentage of Cy3+/Cy5.5+ cell population in CD20+ KG-15 cells, than those with PCL15. The percentage of Cy3+/Cy5.5+ cell population drastically reduced in the presence of competing RTX for micelles containing PCL22 or PBCL22 cores, indicating the superiority of these structures for active targeting of CD20+ cells. No significant difference in the cytotoxicity of paclitaxel in RTX-micelles versus plain ones was observed, reflecting the noninternalizing function of CD20. The results show that traceable mixed micelles prepared through postinsertion of RTX-PEG-DSPE to PEO-PCL22 or PEO-PBCL22 micelles can be used for targeting and/or imaging of CD20+ B cell lymphoma cells. The postinsertion method can be adopted to prepare other PEO-poly(ester)-based immunomicelles for active targeting of other diseased cells.

NPM-ALK Is a Key Regulator of the Oncoprotein FOXM1 in ALK-Positive Anaplastic Large Cell Lymphoma.

Forkhead Box M1 (FOXM1) is an oncogenic transcription factor implicated in the pathogenesis of solid and hematologic cancers. In this study, we examined the significance of FOXM1 in NPM-ALK-positive anaplastic large cell lymphoma (NPM-ALK + ALCL), with a focus on how it interacts with NPM-ALK, which is a key oncogenic driver in these tumors. FOXM1 was expressed in NPM-ALK + ALCL cell lines (5/5), patient samples (21/21), and tumors arising in NPM-ALK transgenic mice (4/4). FOXM1 was localized in the nuclei and confirmed to be transcriptionally active. Inhibition of FOXM1 in two NPM-ALK + ALCL cells using shRNA and pharmalogic agent (thiostrepton) resulted in reductions in cell growth and soft-agar colony formation, which were associated with apoptosis and cell-cycle arrest. FOXM1 is functionally linked to NPM-ALK, as FOXM1 enhanced phosphorylation of the NPM-ALK/STAT3 axis. Conversely, DNA binding and transcriptional activity of FOXM1 was dependent on the expression of NPM-ALK. Further studies showed that this dependency hinges on the binding of FOXM1 to NPM1 that heterodimerizes with NPM-ALK, and the phosphorylation status of NPM-ALK. In conclusion, we identified FOXM1 as an important oncogenic protein in NPM-ALK+ ALCL. Our results exemplified that NPM-ALK exerts oncogenic effects in the nuclei and illustrated a novel role of NPM1 in NPM-ALK pathobiology.

Decoration of Anti-CD38 on Nanoparticles Carrying a STAT3 Inhibitor Can Improve the Therapeutic Efficacy Against Myeloma.

STAT3 is an oncoprotein which has been shown to contribute to drug resistance in multiple myeloma (MM). Nonetheless, the clinical utility of STAT3 inhibitors in treating MM has been limited, partly related to some of their pharmacologic properties. To overcome these challenges, our group had previously packaged STAT3 inhibitors using a novel formulation of nanoparticles (NP) and found encouraging results. In this study, we aimed to further improve the pharmacologic properties of these NP by decorating them with monoclonal anti-CD38 antibodies. NP loaded with S3I-1757 (a STAT3 inhibitor), labeled as S3I-NP, were generated. S3I-NP decorated with anti-CD38 (labeled as CD38-S3I-NP) were found to have a similar nanoparticular size, drug encapsulation, and loading as S3I-NP. The release of S3I-1757 at 24 h was also similar between the two formulations. Using Cy5.5 labeling of the NP, we found that the decoration of anti-CD38 on these NP significantly increased the cellular uptake by two MM cell lines (p < 0.001). Accordingly, CD38-S3I-NP showed a significantly lower inhibitory concentration at 50% (IC50) compared to S3I-NP in two IL6-stimulated MM cell lines (p < 0.001). In a xenograft mouse model, CD38-S3I-NP significantly reduced the tumor size by 4-fold compared to S3I-NP on day 12 after drug administration (p = 0.006). The efficacy of CD38-S3I-NP in suppressing STAT3 phosphorylation in the xenografts was confirmed by using immunocytochemistry and Western blot analysis. In conclusion, our study suggests that the decoration of anti-CD38 on NP loaded with STAT3 inhibitors can further improve their therapeutic effects against MM.

Constitutive Activation of STAT3 in Myeloma Cells Cultured in a Three-Dimensional, Reconstructed Bone Marrow Model.

Malignant cells cultured in three-dimensional (3D) models have been found to be phenotypically and biochemically different from their counterparts cultured conventionally. Since most of these studies employed solid tumor types, how 3D culture affects multiple myeloma (MM) cells is not well understood. Here, we compared MM cells (U266 and RPMI8226) in a 3D culture model with those in conventional culture. While the conventionally cultured cells were present in single cells or small clusters, MM-3D cells grew in large spheroids. We discovered that STAT3 was the pathway that was more activated in 3D in both cell lines. The active form of STAT3 (phospho-STAT3 or pSTAT3), which was absent in MM cells cultured conventionally, became detectable after 1⁻2 days in 3D culture. This elevated pSTAT3 level was dependent on the 3D environment, since it disappeared after transferring to conventional culture. STAT3 inhibition using a pharmacological agent, Stattic, significantly decreased the cell viability of MM cells and sensitized them to bortezomib in 3D culture. Using an oligonucleotide array, we found that 3D culture significantly increased the expression of several known STAT3 downstream genes implicated in oncogenesis. Since most primary MM tumors are naturally STAT3-active, studies of MM in 3D culture can generate results that are more representative of the disease.

Micellar nano-carriers for the delivery of STAT3 dimerization inhibitors to melanoma.

The objective of this research was to develop polymeric micellar formulations of inhibitors of signal transducer and activator of transcription 3 (STAT3) dimerization, i.e., S3I-1757 and S3I-201, and evaluate the activity of successful formulations in B16-F10 melanoma, a STAT3 hyperactive cancer model, in vitro and in vivo. STAT3 inhibitory agents were encapsulated in methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO114-b-PCL22) and methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (PEO114-b-PBCL20) micelles using co-solvent evaporation. Polymeric micelles of S3I-1757 showed high encapsulation efficiency (>88%), slow release profile (<32% release in 24 h) under physiological conditions, and a desirable average diameter for tumor targeting (33-54 nm). The same formulations showed low encapsulation efficiencies and rapid drug release for S3I-201. Further studies evidenced the delivery of functional S3I-1757 by polymeric micelles to B16-F10 melanoma cells, leading to a dose-dependent inhibition of cell growth and vascular endothelial growth factor (VEGF) production comparable with that of free drug. Encapsulation of S3I-1757 in polymeric micelles significantly reduced its cytotoxicity in normal bone marrow-derived dendritic cells (DCs). Micelles of S3I-1757 were able to significantly improve the function of B16-F10 tumor-exposed immunosuppressed DCs in the production of IL-12, an indication for functionality in the induction of cell-mediated immune response. In a B16-F10 melanoma mouse model, S3I-1757 micelles inhibited tumor growth and enhanced the survival of tumor-bearing mice more than free S3I-1757. Our findings show that both PCL- and PBCL-based polymeric micelles have potential for the solubilization and delivery of S3I-1757, a potent STAT3 inhibitory agent.

A positive feedback loop involving the Wnt/ß-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subpopulation in ALK-positive anaplastic large cell lymphoma.

BACKGROUND: We have previously described the existence of two phenotypically distinct cell subsets in ALK-positive anaplastic large cell lymphoma (ALK + ALCL) based on their differential responsiveness to a Sox2 reporter (SRR2), with reporter-responsive (RR) cells being more tumorigenic and chemoresistant than reporter-unresponsive (RU) cells. However, the regulator(s) of RU/RR dichotomy are not identified. In this study, we aim to delineate the key regulator(s) of RU/RR dichotomy. METHODS: JASPER motif match analysis was used to identify the putative factors binding to SRR2 sequence. SRR2 probe pull-down assay and quantitate real-time PCR were performed to analyze the regulation of Sox2 transcriptional activity by MYC. Methylcellulose colony formation assay, chemoresistance to doxorubicin and mouse xenograft study were performed to investigate the biological functions of MYC. PCR array and western blotting were executed to study related signaling pathways that regulate MYC expression. Immunofluorescence and immunohistochemistry assay were initiated to evaluate the expression of MYC and its correlation with its regulator by chi-square test analysis in human primary tumor cells. RESULTS: We identified MYC as a potential regulator of RU/RR dichotomy. In support of its role, MYC was highly expressed in RR cells compared to RU cells, and inhibition of MYC substantially decreased the Sox2/SRR2 binding, Sox2 transcriptional activity, chemoresistance, and methylcellulose colony formation. In contrast, enforced expression of MYC in RU cells conferred the RR phenotype. The Wnt/ß-catenin pathway, a positive regulator of MYC, was highly active in RR but not RU cells. While inhibition of this pathway in RR cells substantially decreased MYC expression and SRR2 reporter activity, experimental activation of this pathway led to the opposite effects in RU cells. Collectively, our results support a model in which a positive feedback loop involving Wnt/ß-catenin/MYC and Sox2 contributes to the RR phenotype. In a mouse xenograft model, RU cells stably transfected with MYC showed upregulation of the Wnt/ß-catenin/MYC/Sox2 axis and increased tumorigenecity. Correlating with these findings, there was a significant correlation between the expression of active ß-catenin and MYC in ALK + ALCL primary tumor cells. CONCLUSIONS: A positive feedback loop involving the Wnt/ß-catenin/MYC/Sox2 axis defines a highly tumorigenic cell subset in ALK + ALCL.

The use of cellular thermal shift assay (CETSA) to study Crizotinib resistance in ALK-expressing human cancers.

Various forms of oncogenic ALK proteins have been identified in various types of human cancers. While Crizotinib, an ALK inhibitor, has been found to be therapeutically useful against a subset of ALK(+) tumours, clinical resistance to this drug has been well recognized and the mechanism of this phenomenon is incompletely understood. Using the cellular thermal shift assay (CETSA), we measured the Crizotinib-ALK binding in a panel of ALK(+) cell lines, and correlated the findings with the ALK structure and its interactions with specific binding proteins. The Crizotinib IC50 significantly correlated with Crizotinib-ALK binding. The suboptimal Crizotinib-ALK binding in Crizotinib-resistant cells is not due to the cell-specific environment, since transfection of NPM-ALK into these cells revealed substantial Crizotinib-NPM-ALK binding. Interestingly, we found that the resistant cells expressed higher protein level of ß-catenin and siRNA knockdown restored Crizotinib-ALK binding (correlated with a significant lowering of IC50). Computational analysis of the crystal structures suggests that ß-catenin exerts steric hindrance to the Crizotinib-ALK binding. In conclusion, the Crizotinib-ALK binding measurable by CETSA is useful in predicting Crizotinib sensitivity, and Crizotinib-ALK binding is in turn dictated by the structure of ALK and some of its binding partners.