Bone marrow stromal cells dictate lanosterol biosynthesis and ferroptosis of multiple myeloma.

Ferroptosis has been demonstrated a promising way to counteract chemoresistance of multiple myeloma (MM), however, roles and mechanism of bone marrow stromal cells (BMSCs) in regulating ferroptosis of MM cells remain elusive. Here, we uncovered that MM cells were more susceptible to ferroptotic induction under the interaction of BMSCs using in vitro and in vivo models. Mechanistically, BMSCs elevated the iron level in MM cells, thereby activating the steroid biosynthesis pathway, especially the production of lanosterol, a major source of reactive oxygen species (ROS) in MM cells. We discovered that direct coupling of CD40 ligand and CD40 receptor constituted the key signaling pathway governing lanosterol biosynthesis, and disruption of CD40/CD40L interaction using an anti-CD40 neutralizing antibody or conditional depletion of Cd40l in BMSCs successfully eliminated the iron level and lanosterol production of MM cells localized in the Vk*MYC Vk12653 or NSG mouse models. Our study deciphers the mechanism of BMSCs dictating ferroptosis of MM cells and highlights the therapeutic potential of non-apoptosis strategies for managing refractory or relapsed MM patients.

C-reactive protein impairs immune response of CD8+ T cells via FcγRIIb-p38MAPK-ROS axis in multiple myeloma.

BACKGROUND: C-reactive protein (CRP) is a prototypical acute phase protein in humans with the function of regulating immune cells. Serum CRP levels are elevated in multiple myeloma (MM), associated with MM cell proliferation and bone destruction. However, its direct effects on T lymphocytes in MM have not been elucidated. METHODS: Public data sets were used to explore the correlation of CRP levels with immune cell infiltration and cytotoxicity score of CD8+ T cells in MM. In vitro, repeated freeze-thaw myeloma cell lines were taken as tumor antigens to load dendritic cells (DCs) derived from HLA-A*0201-positive healthy donors. MM-specific cytotoxic T cells (MM-CTL) were obtained from T lymphocytes of the corresponding donors pulsed with these DCs. B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR)-T cells were manipulated by transfecting with lentivirus encoding an anti-BCMA single-chain variable fragment. Then T cells from healthy controls, MM-CTLs and BCMA CAR-T cells were exposed to CRP and analyzed for cell proliferation, cytotoxicity, immunophenotypes. CRP binding capacity to T cells before and after Fc gamma receptors IIb (FcγRIIb) blockage, p38 mitogen-activated protein kinase (MAPK) pathway and the downstream molecules were also detected. In vivo, both normal C57BL/6J mice and the Vk*MYC myeloma mouse models were applied to confirm the impact of CRP on T cells. RESULTS: CRP levels were negatively correlated with cell-infiltration and cytotoxicity score of CD8+ T cells in MM. In vitro experiments showed that CRP inhibited T-cell proliferation in a dose-dependent manner, impaired the cytotoxic activity and upregulated expression of senescent markers in CD8+ T cells. In vivo results validated the suppressive role of CRP in CD8+ T cells. CRP could bind to CD8+ T cells, mainly to the naïve T subset, while the binding was dramatically decreased by FcγRIIb blockage. Furthermore, CRP resulted in increased phosphorylation of p38 MAPK, elevated levels of reactive oxygen species and oxidized glutathione in CD8+ T cells. CONCLUSIONS: We found that CRP impaired immune response of CD8+ T cells via FcγRIIb-p38MAPK-ROS signaling pathway. The study casted new insights into the role of CRP in anti-myeloma immunity, providing implications for future immunotherapy in MM.

CXCL10 Recruitment of γδ T Cells into the Hypoxic Bone Marrow Environment Leads to IL17 Expression and Multiple Myeloma Progression.

In multiple myeloma (MM), bone marrow stromal cells (BMSC) shape a unique niche within the bone marrow, promoting T-cell dysfunction and driving MM progression; however, the precise underlying mechanisms remain elusive. Here, we show that BMSC-mediated reprogramming of MM cells led to heightened production of CXCL10. CXCL10 orchestrated the recruitment of γδ T cells into the bone marrow, and this was observed in both the Vk*MYC and 5TGM1 mouse models of MM, as well as in patients experiencing refractory or relapsed MM. Furthermore, the dysfunctional γδ T cells in the MM bone marrow niche exhibited increased PD-1 expression and IL17 production. In the Vk*MYC mouse model, MM-associated bone lesions and mortality were markedly alleviated in Tcrd-/- mice, and MM disease progression could be rescued in these mice upon transplantation of γδ T cells expanded from wild-type mice, but not from Il17-/- mice. Mechanistically, the hypoxic microenvironment prevailing in the MM bone marrow niche stimulated the expression of steroid receptor coactivator 3 (SRC-3) in γδ T cells, which in turn interacted with the transcriptional factor RORγt, promoting Il17 transcription. Pharmacologic inhibition of SRC-3 utilizing SI-2 effectively suppressed Il17A expression in γδ T cells, leading to alleviation of MM progression in the murine models and enhancing the anti-multiple myeloma efficacy of bortezomib. Our results illuminated the bone marrow microenvironment's involvement in provoking γδ T-cell dysfunction throughout MM progression and suggest SRC-3 inhibition as a promising strategy to enhance the effectiveness of immunotherapies targeting γδ T cells.

Whole Blood-Derived circUSP10 Acts as a Diagnostic Biomarker in Patients With Early-Stage Non-Small-Cell Lung Cancer.

Accumulating evidence has indicated that differentially expressed noncoding circular RNAs (circRNAs) play essential roles in the occurrence and development of various types of cancer. Here, we aimed to identify and explore the diagnostic value of hsa_circ_0003026 (named circUSP10) in patients with early non-small-cell lung cancer (NSCLC). The differentially expressed circRNAs were screened from the microarray-based assay of human NSCLC tissues and their corresponding noncancerous tissues, and the candidate circRNAs were further verified in patients with NSCLC using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Circulating circUSP10 was isolated from whole blood of healthy people and patients with NSCLC and was detected by RT-qPCR. In addition, the diagnostic value of circUSP10 in early NSCLC was evaluated by receiver operating characteristic (ROC) curve analysis. We found that circUSP10 was upregulated in tumor tissues from patients with early NSCLC and associated with tumor size and tumor-node-metastasis (TNM) stage. Importantly, circUSP10 was obviously upregulated in the whole blood of patients with NSCLC. Additionally, whole blood-derived circUSP10 showed good diagnostic performance for screening early NSCLC and was relatively stable in blood under adverse conditions. These findings demonstrate that circUSP10 may act as a novel biomarker for the diagnosis of early-stage NSCLC, suggesting the potential of circUSP10 in RNA-based therapy for cancer.

Deacetylation induced nuclear condensation of HP1γ promotes multiple myeloma drug resistance.

Acquired chemoresistance to proteasome inhibitors is a major obstacle in managing multiple myeloma but key regulators and underlying mechanisms still remain to be explored. We find that high level of HP1γ is associated with low acetylation modification in the bortezomib-resistant myeloma cells using SILAC-based acetyl-proteomics assay, and higher HP1γ level is positively correlated with poorer outcomes in the clinic. Mechanistically, elevated HDAC1 in the bortezomib-resistant myeloma cells deacetylates HP1γ at lysine 5 and consequently alleviates the ubiquitin-mediated protein degradation, as well as the aberrant DNA repair capacity. HP1γ interacts with the MDC1 to induce DNA repair, and simultaneously the deacetylation modification and the interaction with MDC1 enhance the nuclear condensation of HP1γ protein and the chromatin accessibility of its target genes governing sensitivity to proteasome inhibitors, such as CD40, FOS and JUN. Thus, targeting HP1γ stability by using HDAC1 inhibitor re-sensitizes bortezomib-resistant myeloma cells to proteasome inhibitors treatment in vitro and in vivo. Our findings elucidate a previously unrecognized role of HP1γ in inducing drug resistance to proteasome inhibitors of myeloma cells and suggest that targeting HP1γ may be efficacious for overcoming drug resistance in refractory or relapsed multiple myeloma patients.

All-trans retinoic acid improves NSD2-mediated RARα phase separation and efficacy of anti-CD38 CAR T-cell therapy in multiple myeloma.

BACKGROUND: Immunotherapies targeting CD38 have demonstrated salient efficacy in relapsed/refractory multiple myeloma (MM). However, loss of CD38 antigen and outgrowth of CD38 negative plasma cells have emerged as a major obstacle in clinics. All-trans retinoic acid (ATRA) has been reported to upregulate CD38 expression, but the mechanism and adaptive genetic background remain unexplored. METHODS: The efficacy of ATRA in upregulating CD38 expression in MM cells is evaluated by flow cytometry. The interaction between NSD2 and the RARα is analyzed by immunoprecipitation, and the nuclear condensation of RARα is evaluated under laser confocal microscope. A graft model of MM is established in NOD.Cg-PrkdcscidIl2rgtm1Wjl /SzJ mice, and the tumor burden is assessed by in vivo fluorescence imaging. RESULTS: We report that ATRA upregulates MM cells CD38 in a non-linear manner, which is t(4;14) translocation dependent, and t(4;14) translocation-induced NSD2 shows positive correlation with ATRA-induced level of, but not with basal level of CD38 expression. Mechanistically, NSD2 interacts with the ATRA receptor, RARα, and protects it from degradation. Meanwhile, NSD2 enhances the nuclear condensation of RARα and modifies the histone H3 dimethylation at lysine 36 on CD38 promoter. Knockdown of NSD2 attenuates the sensitization of MM against ATRA induced CD38 upregulation. Translationally, ATRA is prone to augment the efficacy of anti-CD38 CAR T cells in NSD2high MM cells in vitro and in vivo. CONCLUSION: This study elucidates a mechanism of ATRA in regulating CD38 expression and expands the clinical potential of ATRA in improving immunotherapies against CD38 in patients with MM.Cite Now.

Hypoxia induces chemoresistance to proteasome inhibitors through orchestrating deSUMOylation and ubiquitination of SRC-3 in multiple myeloma.

The bone marrow microenvironment in multiple myeloma (MM) is hypoxic and provides multi-advantages for the initiation of chemoresistance, but the underlying mechanisms and key regulators are still indistinct. In the current study, we found that hypoxia stimulus easily induced chemoresistance to proteasome inhibitors (PIs), and the steroid receptor coactivator 3 (SRC-3) expression was remarkably augmented at posttranslational level. Protein interactome analysis identified SENP1 as a key modifier of SRC-3 stability, as SENP1-mediated deSUMOylation attenuated the K11-linked polyubiquitination of SRC-3. SENP1 depletion in the SENP1fl/flCD19Cre/+ B cells showed impaired SRC3 stability, and knockdown of SENP1 in MM cells by CRISPR/cas9 sgRNA accelerated the degradation of SRC-3 and remarkably overcame the resistance to PIs. In the Vk*Myc and 5TGM1 mouse models as well as patient-derived xenograft (PDX) of myeloma, SENP1 inhibitor Momordin Ιc (Mc) increased the sensitivity to PIs in MM cells. Importantly, SENP1 level was positively correlated with SRC-3 level in the tissues from refractory/relapsed MM, as well as in xenograft tissues from mice treated with bortezomib and Mc. Taken together, our findings suggest that hypoxia-induced SENP1 is a crucial regulator of chemoresistance to PIs, and shed light on developing therapeutic strategies to overcome chemoresistance by using small molecules targeting SENP1 or SRC-3.

Posttranslational modification of Aurora A-NSD2 loop contributes to drug resistance in t(4;14) multiple myeloma.

BACKGROUND: t(4;14)(p16;q32) cytogenetic abnormality renders high level of histone methyltransferase NSD2 in multiple myeloma (MM) patients, and predicts poor clinical prognosis, but mechanisms of NSD2 in promoting chemoresistance have not been well elucidated. METHODS: An epigenetics compound library containing 181 compounds was used to screen inhibitors possessing a prior synergistic effect with bortezomib (BTZ) in vitro. Molecular biology techniques were applied to uncover underlying mechanisms. Transcriptome profile assay was performed by RNA-seq. NSG mouse-based xenograft model and intra-bone model were applied to qualify the synergistic effect in vivo. RESULTS: We identified an Aurora kinase A inhibitor (MLN8237) possessed a significant synergistic effect with BTZ on t(4;14) positive MM cells. Aurora A protein level positively correlated with NSD2 level, and gain- and loss-of-functions of Aurora A correspondingly altered NSD2 protein and H3K36me2 levels. Mechanistically, Aurora A phosphorylated NSD2 at S56 residue to protect the protein from cleavage and degradation, thus methylation of Aurora A and phosphorylation of NSD2 bilaterally formed a positive regulating loop. Transcriptome profile assay of MM cells with AURKA depletion identified IL6R, STC2 and TCEA2 as the downstream target genes responsible for BTZ-resistance (BR). Clinically, higher expressions of these genes correlated with poorer outcomes of MM patients. Combined administration of MLN8237 and BTZ significantly suppressed tumour growth in LP-1 cells derived xenografts, and remarkably alleviated bone lesion in femurs of NSG mice. CONCLUSIONS: Aurora A phosphorylates NSD2 at S56 residue to enhance NSD2 methyltransferase activity and form a positive regulating loop in promoting MM chemoresistance, thus pharmacologically targeting Aurora A sensitizes t(4;14) positive MM to the proteasome inhibitors treatment. Our study uncovers a previously unknown reason of MM patients with t(4;14) engendering chemoresistance, and provides a theoretical basis for developing new treatment strategy for MM patients with different genomic backgrounds.

Epigenomic reprogramming via HRP2-MINA dictates response to proteasome inhibitors in multiple myeloma with t(4;14) translocation.

The chromosomal t(4;14) (p16;q32) translocation drives high expression of histone methyltransferase nuclear SET domain-containing 2 (NSD2) and plays vital roles in multiple myeloma (MM) evolution and progression. However, the mechanisms of NSD2-driven epigenomic alterations in chemoresistance to proteasome inhibitors (PIs) are not fully understood. Using a CRISPR/Cas9 sgRNA library in a bone marrow-bearing MM model, we found that hepatoma-derived growth factor 2 (HRP2) was a suppressor of chemoresistance to PIs and that its downregulation correlated with a poor response and worse outcomes in the clinic. We observed suppression of HRP2 in bortezomib-resistant MM cells, and knockdown of HRP2 induced a marked tolerance to PIs. Moreover, knockdown of HRP2 augmented H3K27me3 levels, consequentially intensifying transcriptome alterations promoting cell survival and restriction of ER stress. Mechanistically, HRP2 recognized H3K36me2 and recruited the histone demethylase MYC-induced nuclear antigen (MINA) to remove H3K27me3. Tazemetostat, a highly selective epigenetic inhibitor that reduces H3K27me3 levels, synergistically sensitized the anti-MM effects of bortezomib both in vitro and in vivo. Collectively, these results provide a better understanding of the origin of chemoresistance in patients with MM with the t(4;14) translocation and a rationale for managing patients with MM who have different genomic backgrounds.

Circulating circTOLLIP serves as a diagnostic biomarker for liquid biopsy in non-small cell lung cancer.

BACKGROUND: Circular RNAs (CircRNAs) have been found to possess vital functions in tumorigenesis of various cancer types, including non-small cell lung cancer (NSCLC). The aim of this study was to identify and explore the diagnostic values of the newly found Toll interacting protein (TOLLIP)-derived circRNA (circTOLLIP) for liquid biopsy in NSCLC. METHODS: RNase R and actinomycin D assays were conducted to confirm the existence and stability of circTOLLIP. RT-qPCR was performed to identify the expression levels of circTOLLIP in NSCLC tumor tissues, whole blood, and cell lines. The diagnostic values were evaluated by receiver operating characteristic (ROC) curve analysis. RESULTS: CircTOLLIP was screened as a candidate biomarker and was found to be significantly down-regulated in both NSCLC tissues and cell lines. Interestingly, circulating circTOLLIP was also lower-expressed in the whole blood of patients with NSCLC compared to that of patients with benign lung disease and healthy controls. Importantly, the circulating circTOLLIP represented better diagnostic values in comparison to the traditional tumor markers (NSE, CYFR21-1, and CA72-4), and showed higher stability even though the whole blood was exposed to various tough conditions. CONCLUSIONS: Our findings indicate that circTOLLIP can be used as a non-invasive biomarker to distinguish early-stage NSCLC from benign lung diseases and from healthy controls, suggesting the potential application of circTOLLIP for liquid biopsy in NSCLC.

DKK1 activates noncanonical NF-κB signaling via IL-6-induced CKAP4 receptor in multiple myeloma.

Proteasome inhibitors, such as bortezomib (BTZ), represent the key elements in chemotherapy regimens for multiple myeloma (MM), whereas acquired chemoresistance and ultimately relapse remain a major obstacle. In the current study, we screened differently expressed cytokines in bortezomib-resistant MM cells and found that Dickkopf-1 (DKK1) level was remarkably augmented, whereas CD138 level was significantly suppressed. DKK1 in vitro specifically enhanced the resistance of myeloma cells to bortezomib treatment, and excessive DKK1 drove CD138 downregulation via inhibition of canonical Wnt signaling. Notably, DKK1 mainly induced drug resistance in MM cells via the receptor of CKAP4. Mechanistically, CKAP4 transduced DKK1 signal and evoked NF-κB pathway through recruiting and preventing cullin associated and neddylation dissociated 1 from hampering the assembly of E3 ligase-mediated ubiquitination of IκBα. In addition, we found that interleukin-6 (IL-6) stimulated CKAP4 expression to generate drug resistance, and disturbance of DKK1-CKAP4 axis improved sensitivity to BTZ treatment of MM and attenuated bone destruction in a mouse model. Collectively, our study revealed the previously unidentified role of DKK1 in myeloma drug resistance via Wnt signaling dependent and independent manners, and clarified the importance of antagonism of DKK1-IL-6 loop in bone marrow microenvironment.

Targeting NSD2-mediated SRC-3 liquid-liquid phase separation sensitizes bortezomib treatment in multiple myeloma.

Development of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid-liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

Hypoxia-induced CREB cooperates MMSET to modify chromatin and promote DKK1 expression in multiple myeloma.

Myeloma cells produce excessive levels of dickkopf-1 (DKK1), which mediates the inhibition of Wnt signaling in osteoblasts, leading to multiple myeloma (MM) bone disease. Nevertheless, the precise mechanisms underlying DKK1 overexpression in myeloma remain incompletely understood. Herein, we provide evidence that hypoxia promotes DKK1 expression in myeloma cells. Under hypoxic conditions, p38 kinase phosphorylated cAMP-responsive element-binding protein (CREB) and drove its nuclear import to activate DKK1 transcription. In addition, high levels of DKK1 were associated with the presence of focal bone lesions in patients with t(4;14) MM, overexpressing the histone methyltransferase MMSET, which was identified as a downstream target gene of hypoxia-inducible factor (HIF)-1α. Furthermore, we found that CREB could recruit MMSET, leading to the stabilization of HIF-1α protein and the increased dimethylation of histone H3 at lysine 36 on the DKK1 promoter. Knockdown of CREB in myeloma cells alleviated the suppression of osteoblastogenesis by myeloma-secreted DKK1 in vitro. Combined treatment with a CREB inhibitor and the hypoxia-activated prodrug TH-302 (evofosfamide) significantly reduced MM-induced bone destruction in vivo. Taken together, our findings reveal that hypoxia and a cytogenetic abnormality regulate DKK1 expression in myeloma cells, and provide an additional rationale for the development of therapeutic strategies that interrupt DKK1 to cure MM.

Circular RNAs: Regulatory functions in respiratory tract cancers.

Circular RNAs (circRNAs) are a class of single-stranded RNAs having a covalently closed loop structure generated from back-splicing of pre-mRNA. These novel RNAs are characterized by high stability, abundance and conservation. Accumulating evidence has revealed that circRNAs are intimately associated with the pathogenesis, development and progression of multiple human diseases, including respiratory tract cancers. CircRNAs may serve as oncogenes or tumor suppressors to influence cell proliferation, differentiation, apoptosis, invasion and metastasis. CircRNAs may act as microRNA (miRNA) sponges, interact with RNA-binding proteins (RBPs), regulate gene transcription and/or translate into mini-peptides or proteins. In this review, we discuss recent progress in understanding the pathologic roles of circRNAs in respiratory tract cancers, such as nasopharyngeal carcinoma, laryngeal squamous cell carcinoma, and especially lung adenocarcinoma. We further discuss the diagnostic, therapeutic and prognostic roles as potential biomarkers in respiratory tract cancers, providing insight into the possibilities of applying circRNAs as therapeutic targets and biomarkers in precision oncology.