Rationale: AXL expression has been identified as a prognostic factor in acute myeloid leukemia (AML) and is detectable in approximately 50% of AML patients. In this study, we developed AXL-specific single domain antibodies (sdAbs), cross -reactive for both mouse and human AXL protein, to non -invasively image and treat AXL-expressing cancer cells. Methods: AXL-specific sdAbs were induced by immunizing an alpaca with mouse and human AXL proteins. SdAbs were characterized using ELISA, flow cytometry, surface plasmon resonance and the AlphaFold2 software. A lead compound was selected and labeled with 99mTc for evaluation as a diagnostic tool in mouse models of human (THP-1 cells) or mouse (C1498 cells) AML using SPECT/CT imaging. For therapeutic purposes, the lead compound was fused to a mouse IgG2a-Fc tail and in vitro functionality tests were performed including viability, apoptosis and proliferation assays in human AML cell lines and primary patient samples. Using these in vitro models, its anti -tumor effect was evaluated as a single agent, and in combination with standard of care agents venetoclax or cytarabine. Results: Based on its cell binding potential, cross -reactivity, nanomolar affinity and GAS6/AXL blocking capacity, we selected sdAb20 for further evaluation. Using SPECT/CT imaging, we observed tumor uptake of 99mTc-sdAb20 in mice with AXL-positive THP-1 or C1498 tumors. In THP-1 xenografts, an optimized protocol using pre -injection of cold sdAb20-Fc was required to maximize the tumor -to -background signal. Besides its diagnostic value, we observed a significant reduction in tumor cell proliferation and viability using sdAb20-Fc in vitro. Moreover, combining sdAb20-Fc and cytarabine synergistically induced apoptosis in human AML cell lines, while these effects were less clear when combined with venetoclax. Conclusions: Because of their diagnostic potential, sdAbs could be used to screen patients eligible for AXL-targeted therapy and to follow-up AXL expression during treatment and disease progression. When fused to an Fc-domain, sdAbs acquire additional therapeutic properties that can lead to a multidrug approach for the treatment of AXL-positive cancer patients.

Multiple Myeloma (MM), a cancer of terminally differentiated plasma cells, is the second most prevalent hematological malignancy and is incurable due to the inevitable development of drug resistance. Intense protein synthesis is a distinctive trait of MM cells, supporting the massive production of clonal immunoglobulins or free light chains. The mammalian target of rapamycin (mTOR) kinase is appreciated as a master regulator of vital cellular processes, including regulation of metabolism and protein synthesis, and can be found in two multiprotein complexes, mTORC1 and mTORC2. Dysregulation of these complexes is implicated in several types of cancer, including MM. Since mTOR has been shown to be aberrantly activated in a large portion of MM patients and to play a role in stimulating MM cell survival and resistance to several existing therapies, understanding the regulation and functions of the mTOR complexes is vital for the development of more effective therapeutic strategies. This review provides a general overview of the mTOR pathway, discussing key discoveries and recent insights related to the structure and regulation of mTOR complexes. Additionally, we highlight findings on the mechanisms by which mTOR is involved in protein synthesis and delve into mTOR-mediated processes occurring in MM. Finally, we summarize the progress and current challenges of drugs targeting mTOR complexes in MM.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is incurable because of the inevitable development of drug resistance. Methionine adenosyltransferase 2α (MAT2A) is the primary producer of the methyl donor S-adenosylmethionine (SAM) and several studies have documented MAT2A deregulation in different solid cancers. As the role of MAT2A in MM has not been investigated yet, the aim of this study was to clarify the potential role and underlying molecular mechanisms of MAT2A in MM, exploring new therapeutic options to overcome drug resistance. By analyzing publicly available gene expression profiling data, MAT2A was found to be more highly expressed in patient-derived myeloma cells than in normal bone marrow plasma cells. The expression of MAT2A correlated with an unfavorable prognosis in relapsed patients. MAT2A inhibition in MM cells led to a reduction in intracellular SAM levels, which resulted in impaired cell viability and proliferation, and induction of apoptosis. Further mechanistic investigation demonstrated that MAT2A inhibition inactivated the mTOR-4EBP1 pathway, accompanied by a decrease in protein synthesis. MAT2A targeting in vivo with the small molecule compound FIDAS-5 was able to significantly reduce tumor burden in the 5TGM1 model. Finally, we found that MAT2A inhibition can synergistically enhance the anti-MM effect of the standard-of-care agent bortezomib on both MM cell lines and primary human CD138+ MM cells. In summary, we demonstrate that MAT2A inhibition reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, our findings suggest that the MAT2A inhibitor FIDAS-5 could be a novel compound to improve bortezomib-based treatment of MM.

Multiple myeloma (MM) cells derive proliferative signals from the bone marrow (BM) microenvironment via exosomal crosstalk. Therapeutic strategies targeting this crosstalk are still lacking. Bortezomib resistance in MM cells is linked to elevated expression of xCT (the subunit of system Xc-). Extracellular glutamate released by system Xc- can bind to glutamate metabotropic receptor (GRM) 3, thereby upregulating Rab27-dependent vesicular trafficking. Since Rab27 is also involved in exosome biogenesis, we aimed to investigate the role of system Xc- in exosomal communication between BM stromal cells (BMSCs) and MM cells. We observed that expression of xCT and GRMs was increased after bortezomib treatment in both BMSCs and MM cells. Secretion of glutamate and exosomes was simultaneously enhanced which could be countered by inhibition of system Xc- or GRMs. Moreover, glutamate supplementation increased exosome secretion by increasing expression of Alix, TSG101, Rab27a/b and VAMP7. Importantly, the system Xc- inhibitor sulfasalazine reduced BMSC-induced resistance to bortezomib in MM cells in vitro and enhanced its anti-MM effects in vivo. These findings suggest that system Xc- plays an important role within the BM and could be a potential target in MM.

BACKGROUND:Assessing cytotoxicity is fundamental to studying natural killer (NK) cell function. Various radioactive and non-radioactive cytotoxicity assays measuring target cell death have been developed. Among these methods, the most commonly used 51 Chromium-release assay (CRA) and flow cytometry-based cytotoxicity assays (FCCs) are the major representatives. Nonetheless, several drawbacks, including dye leakage and the potential effects of prior labeling on cells, curb the broad applicability of the FCCs. METHODS:Here, we report a rapid FCC for quantifying target cell death after co-incubation with NK cells. In this assay, after 4 hours of NK cell-target cell co-incubation, fluorochrome-conjugated CD2 antibody was used to identify NK cells, and SYTOX Green and Annexin V-FITC were further used to detect target cell death in CD2-negative population. In parallel, both CRA and FCC assay using CFSE/ 7-AAD were performed to validate the reproducibility and replicability. RESULTS:We observed that CD2 is exclusively positive on NK cells other than the most common hematological target tumor cells, such as K562, HL60, MOLM13, Raji, NCI-H929, rpmi8226, MM.1S, and KMS11. Assessment of target cell death using the CD2-based FCC shows a significantly higher percent specific lysis of the target cells compared to the standard CRA and the FCC assay using CFSE and 7-AAD. CONCLUSIONS:We demonstrated that this CD2-based FCC is a fast, simple, and reliable method for evaluating NK cell cytotoxicity.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into a variety of cell types. Bortezomib, the first approved proteasome inhibitor used for the treatment of multiple myeloma (MM), has been shown to induce osteoblast differentiation, making it beneficial for myeloma bone disease. In the present study, we aimed to investigate the effects and underlying mechanisms of bortezomib on the cell cycle during osteogenic differentiation. We confirmed that low doses of bortezomib can induce MSCs towards osteogenic differentiation, but high doses are toxic. In the course of bortezomib-induced osteogenic differentiation, we observed cell cycle exit characterized by G0 /G1 phase cell cycle arrest with a significant reduction in cell proliferation. Additionally, we found that the cell cycle exit was tightly related to the induction of the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 . Notably, we further demonstrated that the up-regulation of p21Cip1 and p27Kip1 is transcriptionally dependent on the bortezomib-activated ER stress signalling branch Ire1α/Xbp1s. Taken together, these findings reveal an intracellular pathway that integrates proteasome inhibition, osteogenic differentiation and the cell cycle through activation of the ER stress signalling branch Ire1α/Xbp1s.

Proteasome inhibitor bortezomib is one of the most effective drugs currently available for the treatment of multiple myeloma (MM). However, the intrinsic and acquired resistance to bortezomib can limit its effectiveness. The activation of heat shock response has been characterized as a potential resistance mechanism protecting MM cells from bortezomib-induced cell death. In this study, in response to bortezomib therapy, we discovered that HSP70 is one of the most substantially upregulated heat shock proteins. In order to further explore approaches to sensitizing bortezomib-based treatment for MM, we investigated whether targeting HSP70 using a specific inhibitor VER-155008 combined with bortezomib could overcome the acquired resistance in MM. We found that HSP70 inhibitor VER-155008 alone significantly decreased MM cell viability. Moreover, the combination of VER-155008 and bortezomib synergistically induced MM cell apoptosis markedly in vitro. Notably, the combined treatment was found to increase the cleavage of PARP, an early marker of chemotherapy-induced apoptosis. Importantly, the reduction of anti-apoptotic Bcl-2 family member Bcl-2, Bcl-xL, and Mcl-1 and the induction of pro-apoptotic Bcl-2 family member BH3-only protein NOXA and Bim were confirmed to be tightly associated with the synergism. Finally, the ER stress marker CHOP (CCAAT-enhancer binding protein homologous protein), which can cause transcriptional activation of genes involved in cell apoptosis, was markedly induced by both VER-155008 and bortezomib. Taken together, our finding of a strong synergistic interaction between VER-155008 and bortezomib may support for combination therapy in MM patients in the future.

目的 观察蛋白酶体抑制剂硼替佐米(bortezomib)对于小鼠MC3T3-E1细胞系成骨分化、增殖和凋亡的影响.方法 用不同浓度梯度的硼替佐米作用于培养的MC3T3-E1细胞,利用茜素红染色检测成骨分化,CCK-8法检测细胞增殖,流式细胞术分析细胞周期和凋亡,Western blot分析细胞周期相关蛋白变化.结果 ①硼替佐米剂量依赖性地抑制MC3T3 E1细胞的增殖活力[IC50=(7.37±0.34) nmol/L];②低浓度硼替佐米能够诱导MC3T3-E1细胞发生成骨分化;③高浓度硼替佐米对于MC3T3-E1细胞表现出明显的毒性,诱导细胞凋亡发生;④低浓度硼替佐米所诱导的MC3T3-E1成骨分化进程中,伴随有明显的G0/G1期细胞周期阻滞.Western blot检测发现,G0/G1期细胞周期阻滞与细胞周期素依赖性激酶CDK2和CDK4表达水平降低,以及细胞周期蛋白内质网应激活化引起的细胞周期抑制蛋白p21Cip1和p27Kip1的表达上调有关.结论 低剂量蛋白酶体抑制剂硼替佐米能够诱导成骨前体细胞MC3T3-E1发生成骨分化,并引起G0/G1期细胞周期阻滞介导的增殖抑制.

Osteosarcoma is a primary malignant bone tumor, characterized by high therapeutic resistance and poor outcomes, due to unclear pathological mechanisms. It has been shown recently that the platelet-derived growth factor (PDGF)/platelet-derived growth factor receptor (PDGFR) pathway is closely associated with the pathogenesis of osteosarcoma. Hypoxia is a critical hallmark of tumor microenvironment that promotes the malignant phenotype in many solid tumors and a fundamental impediment to effective tumor therapy. In this study, we confirmed that hypoxia is an important feature of osteosarcoma, validated by the positive immunohistochemistry staining of hypoxia marker hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase IX (CAIX) in osteosarcoma tissue samples. More importantly, we discovered that hypoxia could transcriptionally upregulate the expression of both PDGF-BB and PDGFR-β in osteosarcoma cells in vitro. Likewise, we also established that hypoxia-induced PDGF-BB is strongly related to the enhanced cell proliferation and migration, by activating AKT, ERK1/2, and STAT3 signaling pathways. Notably, when using an antibody to block the autocrine of PDGF-BB, cell proliferation and migration were partially aborted in hypoxia. Collectively, we demonstrated that the hypoxia-activated PDGF-BB/PDGFR-β axis plays essential roles in osteosarcoma progression. These findings may shed light on the molecular pathogenesis of osteosarcoma, and provide a novel strategy for osteosarcoma treatment by combinational targeting hypoxia and PDGF-BB/PDGFR signaling.

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM). CD147, known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. With regard to MM, it was recently documented that the aberrantly elevated expression of CD147 has been tightly correlated with MM cell colonization and proliferation. During malignant transformation, many glycoproteins undergo a wide range of glycosylation alterations, especially increased sialylation, have been associated with malignant transformation and metastasis. However, it is still unclear the mechanisms of regulating CD147 and its glycosylation in MM. In this study, we found that CD147 and its sialylation can be up-regulated by interleukin-6 (IL-6), which is derived from either autocrine or paracrine sources and plays an essential role in the malignant progression of MM. When serum-starved MM cell lines RPMI8226, MM1.S and NCI-H929 were stimulated with 20ng/mL of IL-6, we found that the expression of CD147 on MM cell membrane was significantly upregulated, as determined by both flow cytometric analysis and Western blotting. Importantly, the results of Western blotting also clearly show that CD147 is a glycoprotein with higher molecular weight bands than core CD147. By using PNGase F and sialidase to digest the protein samples, we further demonstrated that CD147 is typical N-linked glycoprotein with high level of sialylation. Moreover, we found that the expression of α-2,3 and α-2,6 sialic acid on the cell surface were significantly upregulated by IL-6 analyzed by flow cytometry using MAA and SNA lectin binding assay. Next, we investigated the signaling pathway involved in the IL-6 mediated upregulation of CD147 and its sialylation in MM cells. By using Real-Time PCR, we confirmed that CD147 was transcriptionally upregulated by IL-6. Simultaneously, several sialyltransferases like ST3GAL3, ST3GAL6 and ST6GAL1 were also transcriptionally upregulated by IL-6. By using Western blotting, we further confirmed that IL-6 can activate the JAK/STAT3 signaling pathway by inducing the phosphorylation of STAT3 at Tyrosine 705 in MM cells. Further bioinformatics and ChIP analysis demonstrated that the existing of transcription factor STAT3 binding sites in the promoter of CD147 and sialyltransferase (ST3GAL3, ST3GAL6 and ST6GAL1) genes.To further validate the role of JAK/STAT3 in regulating CD147 and its sialylation, we used a specific STAT3 inhibitor Cryptotanshinone to treat the MM cells and found that the expression of CD147 and its sialylation in MM cells was accordingly decreased. Furthermore, we validated the roles of high level of CD147 and its sialylation in MM biology by knocking-down CD147 or inhibiting its sialylation using 3Fax-Peracetyl Neu5Ac, which is a specific sialyltransferase inhibitor. We found that both siRNA mediated knock-down of CD147 and sialylation inhibition did not affect the proliferation in RPMI8226 and MM1.S cells, measured by EDU incorporation assay using flow cytometry. However, knock-down of CD147 and sialylation inhibition were found to significantly reduce the adhesion to BMSCs and HUVECs, and the migration to BMSC-conditioned media in vitro. Finally, to further validate the influence of IL-6 and the related sialyltransferase genes in MM patients, we assessed the effect of expression on survival of patients using GEP data from the CoMMpass trial (n=664), and noted a significantly reduced PFS (progression-free survival) for patients with high levels of expression of IL-6 and ST3GAL6 (P <0.0001). Taken together, our data provide evidence that IL-6 can up-regulate CD147 expression and its sialylation in a STAT3-dependent manner, and offer a compelling rationale for exploring this axis as a therapeutic target for MM.

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation of plasma cells in the bone marrow (BM). CD147, also known as extracellular matrix metalloproteinase inducer (EMMPRIN), is a type I transmembrane glycoprotein that belongs to the immunoglobulin superfamily. CD147 is expressed in a variety of tissues, and is involved in a number of physiological and pathological processes. With regard to MM, it was recently documented that the aberrantly elevated expression of CD147 has been tightly correlated with MM cell colonization and proliferation. However, it is still unclear what mechanism is involved in the dysregulation of CD147 expression in MM. In this study, we found that CD147 can be up-regulated by interleukin-6 (IL-6), which is derived from either autocrine or paracrine sources and plays an essential role in the malignant progression of MM. When serum-starved MM cell lines RPMI8226, MM1.S and NCIH929 were stimulated with 20ng/mL of IL-6 for 24 hours, we found that the expression of CD147 on MM cell membrane was significantly up-regulated, as determined by flow cytometric analysis. Moreover, when using the IL-6 autocrine MM cell line U266, CD147 level was found to be continuously increased in serum-starved MM cells in a time-dependent manner. Interestingly, the increase of CD147 in U266 cells was found to be abrogated when using neutralizing antibody to block the secreted IL-6. Next, we investigated the molecular mechanisms involved in the IL-6-mediated upregulation of CD147 in MM cells. We confirmed that IL-6 can activate the JAK/STAT3 signaling pathway, inducing the phosphorylation of STAT3 at Tyrosine 705 in RPMI8226 and MM1.S cells. Further studies using ChIP analysis demonstrated that there is a STAT3 binding site at the position −659 to −650 in the promoter of the CD147 gene. In addition, when inhibiting of STAT3 phosphorylation with a specific inhibitor S3I-201, the CD147 expression in MM cells was accordingly decreased. Finally, we found that siRNA mediated knock-down of CD147 in RPMI8226 and MM1.S cells can decrease the proliferation and migration of MM cells induced by IL-6 and Cyclophilin A (CypA). In conclusion, our data provide evidence that IL-6 can up-regulate CD147 expression in a STAT3-dependent manner, and offer a compelling rationale for exploring this axis as a therapeutic target for MM.