Exosomes in multiple myeloma: from bench to bedside.

Multiple myeloma (MM) remains an incurable plasma cell malignancy that develops in the bone marrow (BM). This BM is partially responsible for protecting the MM cells against current standard-of-care therapies and for accommodating MM-related symptoms such as bone resorption and immune suppression. Increasing evidence has implicated extracellular vesicles (EV), including exosomes in the different processes within the BM. Exosomes are <150-nm-sized vesicles secreted by different cell types including MM cells. These vesicles contain protein and RNA cargo that they deliver to the recipient cell. In this way, they have been implicated in MM-related processes including osteolysis, angiogenesis, immune suppression, and drug resistance. Targeting exosome secretion could therefore potentially block these different processes. In this review, we will summarize the current findings of exosome-related processes in the BM and describe not only the current treatment strategies to counter them but also how exosomes can be harnessed to deliver toxic payloads. Finally, an overview of the different clinical studies that investigate EV cargo as potential MM biomarkers in liquid biopsies will be discussed.

S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma.

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.

Targeting mTOR signaling pathways in multiple myeloma: biology and implication for therapy.

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.

Metformin confers sensitisation to syrosingopine in multiple myeloma cells by metabolic blockage and inhibition of protein synthesis.

Multiple myeloma (MM) remains an incurable haematological malignancy despite substantial advances in therapy. Hypoxic bone marrow induces metabolic rewiring in MM cells contributing to survival and drug resistance. Therefore, targeting metabolic pathways may offer an alternative treatment option. In this study, we repurpose two FDA-approved drugs, syrosingopine and metformin. Syrosingopine was used as a dual inhibitor of monocarboxylate transporter 1 and 4 (MCT1/4) and metformin as an inhibitor for oxidative phosphorylation (OXPHOS). Anti-tumour effects were evaluated for single agents and in combination therapy. Survival and expression data for MCT1/MCT4 were obtained from the Total Therapy 2, Mulligan, and Multiple Myeloma Research Foundation cohorts. Cell death, viability, and proliferation were measured using Annexin V/7-AAD, CellTiterGlo, and BrdU, respectively. Metabolic effects were assessed using Seahorse Glycolytic Rate assays and LactateGlo assays. Differential protein expression was determined using western blotting, and the SUnSET method was implemented to quantify protein synthesis. Finally, the syngeneic 5T33MMvv model was used for in vivo analysis. High-level expression of MCT1 and MCT4 both correlated with a significantly lower overall survival of patients. Lactate production as well as MCT1/MCT4 expression were significantly upregulated in hypoxia, confirming the Warburg effect in MM. Dual inhibition of MCT1/4 with syrosingopine resulted in intracellular lactate accumulation and reduced cell viability and proliferation. However, only at higher doses (>10 µm) was syrosingopine able to induce cell death. By contrast, combination treatment of syrosingopine with metformin was highly cytotoxic for MM cell lines and primary patient samples and resulted in a suppression of both glycolysis and OXPHOS. Moreover, pathway analysis revealed an upregulation of the energy sensor p-AMPKα and more downstream a reduction in protein synthesis. Finally, the combination treatment resulted in a significant reduction in tumour burden in vivo. This study proposes an alternative combination treatment for MM and provides insight into intracellular effects. © 2023 The Pathological Society of Great Britain and Ireland.

Targeting the ß2 -adrenergic receptor increases chemosensitivity in multiple myeloma by induction of apoptosis and modulating cancer cell metabolism.

While multi-drug combinations and continuous treatment have become standard for multiple myeloma, the disease remains incurable. Repurposing drugs that are currently used for other indications could provide a novel approach to improve the therapeutic efficacy of standard multiple myeloma treatments. Here, we assessed the anti-tumor effects of cardiac drugs called ß-blockers as a single agent and in combination with commonly used anti-myeloma therapies. Expression of the ß2 -adrenergic receptor correlated with poor survival outcomes in patients with multiple myeloma. Targeting the ß2 -adrenergic receptor (ß2 AR) using either selective or non-selective ß-blockers reduced multiple myeloma cell viability, and induced apoptosis and autophagy. Blockade of the ß2 AR modulated cancer cell metabolism by reducing the mitochondrial respiration as well as the glycolytic activity. These effects were not observed by blockade of ß1 -adrenergic receptors. Combining ß2 AR blockade with the chemotherapy drug melphalan or the proteasome inhibitor bortezomib significantly increased apoptosis in multiple myeloma cells. These data identify the therapeutic potential of ß2 AR-blockers as a complementary or additive approach in multiple myeloma treatment and support the future clinical evaluation of non-selective ß-blockers in a randomized controlled trial. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Dendritic Cell-Based Immunotherapy in Multiple Myeloma: Challenges, Opportunities, and Future Directions.

Immunotherapeutic approaches, including adoptive cell therapy, revolutionized treatment in multiple myeloma (MM). As dendritic cells (DCs) are professional antigen-presenting cells and key initiators of tumor-specific immune responses, DC-based immunotherapy represents an attractive therapeutic approach in cancer. The past years, various DC-based approaches, using particularly ex-vivo-generated monocyte-derived DCs, have been tested in preclinical and clinical MM studies. However, long-term and durable responses in MM patients were limited, potentially attributed to the source of monocyte-derived DCs and the immunosuppressive bone marrow microenvironment. In this review, we briefly summarize the DC development in the bone marrow niche and the phenotypical and functional characteristics of the major DC subsets. We address the known DC deficiencies in MM and give an overview of the DC-based vaccination protocols that were tested in MM patients. Lastly, we also provide strategies to improve the efficacy of DC vaccines using new, improved DC-based approaches and combination therapies for MM patients.

Expert review on soft-tissue plasmacytomas in multiple myeloma: definition, disease assessment and treatment considerations.

In this review, two types of soft-tissue involvement in multiple myeloma are defined: (i) extramedullary (EMD) with haematogenous spread involving only soft tissues and (ii) paraskeletal (PS) with tumour masses arising from skeletal lesions. The incidence of EMD and PS plasmacytomas at diagnosis ranges from 1·7% to 4·5% and 7% to 34·4% respectively. EMD disease is often associated with high-risk cytogenetics, resistance to therapy and worse prognosis than in PS involvement. In patients with PS involvement a proteasome inhibitor-based regimen may be the best option followed by autologous stem cell transplantation (ASCT) in transplant eligible patients. In patients with EMD disease who are not eligible for ASCT, a proteasome inhibitor-based regimen such as lenalidomide-bortezomib-dexamethasone (RVD) may be the best option, while for those eligible for high-dose therapy a myeloma/lymphoma-like regimen such as bortezomib, thalidomide and dexamethasone (VTD)-RVD/cisplatin, doxorubicin, cyclophosphamide and etoposide (PACE) followed by SCT should be considered. In both EMD and PS disease at relapse many strategies have been tried, but this remains a high-unmet need population.

A niche-dependent myeloid transcriptome signature defines dormant myeloma cells.

The era of targeted therapies has seen significant improvements in depth of response, progression-free survival, and overall survival for patients with multiple myeloma. Despite these improvements in clinical outcome, patients inevitably relapse and require further treatment. Drug-resistant dormant myeloma cells that reside in specific niches within the skeleton are considered a basis of disease relapse but remain elusive and difficult to study. Here, we developed a method to sequence the transcriptome of individual dormant myeloma cells from the bones of tumor-bearing mice. Our analyses show that dormant myeloma cells express a distinct transcriptome signature enriched for immune genes and, unexpectedly, genes associated with myeloid cell differentiation. These genes were switched on by coculture with osteoblastic cells. Targeting AXL, a gene highly expressed by dormant cells, using small-molecule inhibitors released cells from dormancy and promoted their proliferation. Analysis of the expression of AXL and coregulated genes in human cohorts showed that healthy human controls and patients with monoclonal gammopathy of uncertain significance expressed higher levels of the dormancy signature genes than patients with multiple myeloma. Furthermore, in patients with multiple myeloma, the expression of this myeloid transcriptome signature translated into a twofold increase in overall survival, indicating that this dormancy signature may be a marker of disease progression. Thus, engagement of myeloma cells with the osteoblastic niche induces expression of a suite of myeloid genes that predicts disease progression and that comprises potential drug targets to eradicate dormant myeloma cells.

The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma.

BACKGROUND: The aggressive B-cell non-Hodgkin lymphomas diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are characterised by a high proliferation rate. The anaphase-promoting complex/cyclosome (APC/C) and its co-activators Cdc20 and Cdh1 represent an important checkpoint in mitosis. Here, the role of the APC/C and its co-activators is examined in DLBCL and MCL. METHODS: The expression and prognostic value of Cdc20 and Cdh1 was investigated using GEP data and immunohistochemistry. Moreover, the therapeutic potential of APC/C targeting was evaluated using the small-molecule inhibitor proTAME and the underlying mechanisms of action were investigated by western blot. RESULTS: We demonstrated that Cdc20 is highly expressed in DLBCL and aggressive MCL, correlating with a poor prognosis in DLBCL. ProTAME induced a prolonged metaphase, resulting in accumulation of the APC/C-Cdc20 substrate cyclin B1, inactivation/degradation of Bcl-2 and Bcl-xL and caspase-dependent apoptosis. In addition, proTAME strongly enhanced the anti-lymphoma effect of the clinically relevant agents doxorubicin and venetoclax. CONCLUSION: We identified for the first time APC/C as a new, promising target in DLBCL and MCL. Moreover, we provide evidence that Cdc20 might be a novel, independent prognostic factor in DLBCL and MCL.

Inhibiting the osteocyte-specific protein sclerostin increases bone mass and fracture resistance in multiple myeloma.

Multiple myeloma (MM) is a plasma cell cancer that develops in the skeleton causing profound bone destruction and fractures. The bone disease is mediated by increased osteoclastic bone resorption and suppressed bone formation. Bisphosphonates used for treatment inhibit bone resorption and prevent bone loss but fail to influence bone formation and do not replace lost bone, so patients continue to fracture. Stimulating bone formation to increase bone mass and fracture resistance is a priority; however, targeting tumor-derived modulators of bone formation has had limited success. Sclerostin is an osteocyte-specific Wnt antagonist that inhibits bone formation. We hypothesized that inhibiting sclerostin would prevent development of bone disease and increase resistance to fracture in MM. Sclerostin was expressed in osteocytes from bones from naive and myeloma-bearing mice. In contrast, sclerostin was not expressed by plasma cells from 630 patients with myeloma or 54 myeloma cell lines. Mice injected with 5TGM1-eGFP, 5T2MM, or MM1.S myeloma cells demonstrated significant bone loss, which was associated with a decrease in fracture resistance in the vertebrae. Treatment with anti-sclerostin antibody increased osteoblast numbers and bone formation rate but did not inhibit bone resorption or reduce tumor burden. Treatment with anti-sclerostin antibody prevented myeloma-induced bone loss, reduced osteolytic bone lesions, and increased fracture resistance. Treatment with anti-sclerostin antibody and zoledronic acid combined increased bone mass and fracture resistance when compared with treatment with zoledronic acid alone. This study defines a therapeutic strategy superior to the current standard of care that will reduce fractures for patients with MM.

DNMTi/HDACi combined epigenetic targeted treatment induces reprogramming of myeloma cells in the direction of normal plasma cells.

BACKGROUND: Multiple myeloma (MM) is the second most common hematologic malignancy. Aberrant epigenetic modifications have been reported in MM and could be promising therapeutic targets. As response rates are overall limited but deep responses occur, it is important to identify those patients who could indeed benefit from epigenetic-targeted therapy. METHODS: Since HDACi and DNMTi combination have potential therapeutic value in MM, we aimed to build a GEP-based score that could be useful to design future epigenetic-targeted combination trials. In addition, we investigated the changes in GEP upon HDACi/DNMTi treatment. RESULTS: We report a new gene expression-based score to predict MM cell sensitivity to the combination of DNMTi/HDACi. A high Combo score in MM patients identified a group with a worse overall survival but a higher sensitivity of their MM cells to DNMTi/HDACi therapy compared to a low Combo score. In addition, treatment with DNMTi/HDACi downregulated IRF4 and MYC expression and appeared to induce a mature BMPC plasma cell gene expression profile in myeloma cell lines. CONCLUSION: In conclusion, we developed a score for the prediction of primary MM cell sensitivity to DNMTi/HDACi and found that this combination could be beneficial in high-risk patients by targeting proliferation and inducing maturation.

Tumour-associated macrophage-mediated survival of myeloma cells through STAT3 activation.

Overcoming drug resistance is one of the greatest challenges in the treatment of multiple myeloma (MM). The interaction of myeloma cells with the bone marrow (BM) microenvironment is a major factor contributing to drug resistance. Tumour-associated macrophages (TAMs) with different polarization states constitute an important component of this microenvironment. Previous studies have revealed a role of TAMs in MM cell survival and drug resistance; however, the impact of macrophage polarization (anti-tumoural 'M1' versus pro-tumoural 'M2'-like phenotype) in this process has not yet been described. Here, the presence of TAMs was confirmed in BM sections from MM patients, both at diagnosis and relapse, with two M2 markers, CD163 and CD206. By following different TAM subpopulations during disease progression in the syngeneic murine 5T33MM model, we demonstrated a decrease in the number of inflammatory monocytes and an increase in the number of M2-oriented TAMs in BM. Co-culture experiments demonstrated that macrophages provide a survival benefit to myeloma cells that is maintained after treatment with several classes of anti-myeloma agent (melphalan and bortezomib); the greatest effect was observed with M2-polarized macrophages. The pro-survival effect was associated with activation of the STAT3 pathway in 5T33MM cells, less cleavage of caspase-3, and thus less apoptosis. AZD1480, an ATP-competitive JAK2 inhibitor, abrogated the observed TAM-mediated MM cell survival, and partially inhibited resistance to bortezomib. Despite having only a small quantitative impact on myeloid cells in vivo, AZD1480 treatment alone and in combination with bortezomib significantly reduced tumour load. In conclusion, M2 TAMs are present in the MM microenvironment, and contribute to MM cell survival and protection from drug-induced apoptosis. As a result of TAM-induced activation of the STAT3 pathway, 5T33MM cells are sensitized to AZD1480 treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bone marrow stromal cell-derived exosomes as communicators in drug resistance in multiple myeloma cells.

The interplay between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells performs a crucial role in MM pathogenesis by secreting growth factors, cytokines, and extracellular vesicles. Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and they mediate local cell-to-cell communication by transferring mRNAs, miRNAs, and proteins. Although BMSC-induced growth and drug resistance of MM cells has been studied, the role of BMSC-derived exosomes in this action remains unclear. Here we investigate the effect of BMSC-derived exosomes on the viability, proliferation, survival, migration, and drug resistance of MM cells, using the murine 5T33MM model and human MM samples. BMSCs and MM cells could mutually exchange exosomes carrying certain cytokines. Both naive and 5T33 BMSC-derived exosomes increased MM cell growth and induced drug resistance to bortezomib. BMSC-derived exosomes also influenced the activation of several survival relevant pathways, including c-Jun N-terminal kinase, p38, p53, and Akt. Exosomes obtained from normal donor and MM patient BMSCs also induced survival and drug resistance of human MM cells. Taken together, our results demonstrate the involvement of exosome-mediated communication in BMSC-induced proliferation, migration, survival, and drug resistance of MM cells.

The myeloma stem cell concept, revisited: from phenomenology to operational terms.

The concept of the myeloma stem cell may have important therapeutic implications, yet its demonstration has been hampered by a lack of consistency in terms and definitions. Here, we summarize the current documentation and propose single-cell in vitro studies for future translational studies. By the classical approach, a CD19-/CD45low/-/CD38high/CD138+ malignant plasma cell, but not the CD19+/CD38low/- memory B cell compartment, is enriched for tumorigenic cells that initiate myeloma in xenografted immunodeficient mice, supporting that myeloma stem cells are present in the malignant PC compartment. Using a new approach, analysis of c-DNA libraries from CD19+/CD27+/CD38- single cells has identified clonotypic memory B cell, suggested to be the cell of origin. This is consistent with multiple myeloma being a multistep hierarchical process before or during clinical presentation. We anticipate that further characterization will require single cell geno- and phenotyping combined with clonogenic assays. To implement such technologies, we propose a revision of the concept of a myeloma stem cell by including operational in vitro assays to describe the cellular components of origin, initiation, maintenance, and evolution of multiple myeloma. These terms are in accordance with recent (2012) consensus statements on the definitions, assays, and nomenclature of cancer stem cells, which is technically precise without completely abolishing established terminology. We expect that this operational model will be useful for future reporting of parameters used to identify and characterize the multiple myeloma stem cells. We strongly recommend that these parameters include validated standard technologies, reproducible assays, and, most importantly, supervised prospective sampling of selected biomaterial which reflects clinical stages, disease spectrum, and therapeutic outcome. This framework is key to the characterization of the cellular architecture of multiple myeloma and its use in precision medicine.

Stimulation of invariant natural killer T cells by α-Galactosylceramide activates the JAK-STAT pathway in endothelial cells and reduces angiogenesis in the 5T33 multiple myeloma model.

Tumour pathogenesis in multiple myeloma (MM) correlates with a high vascular index. Therefore, targeting angiogenesis is an important therapeutic tool to reduce MM progression. This study aimed to investigate the role of invariant natural killer T (iNKT) cells in angiogenesis and the mechanisms behind the stimulation by α-Galactosylceramide (α-GalCer). We have previously found that α-GalCer could increase the survival of 5T33MM mice and here we demonstrate that α-GalCer reduces the microvessel density. We performed both in vivo and in vitro angiogenic assays to confirm this observation. We found that conditioned medium of α-GalCer stimulated iNKT cells reduced neovascularization in the chick chorioallantoic membrane and in matrigel plug assays. Moreover, we observed a reduction in proliferation, migration and network formation and an induction of apoptosis upon exposure of murine endothelial cell lines to this conditioned medium. We furthermore observed that the JAK-STAT signaling pathway was highly activated in endothelial cells in response to stimulated iNKT cells, indicating the possible role of IFN-γ in the anti-angiogenic process. In conclusion, these results highlight the possibility of recruiting iNKT cells to target MM and angiogenesis. This gives a rationale for combining immunotherapy with conventional anti-tumour treatments in view of increasing their therapeutic potential.

Activation of ATF4 mediates unwanted Mcl-1 accumulation by proteasome inhibition.

Myeloid cell leukemia-1 (Mcl-1) protein is an anti-apoptotic Bcl-2 family protein that plays essential roles in multiple myeloma (MM) survival and drug resistance. In MM, it has been demonstrated that proteasome inhibition can trigger the accumulation of Mcl-1, which has been shown to confer MM cell resistance to bortezomib-induced lethality. However, the mechanisms involved in this unwanted Mcl-1 accumulation are still unclear. The aim of the present study was to determine whether the unwanted Mcl-1 accumulation could be induced by the unfolded protein response (UPR) and to elucidate the role of the endoplasmic reticulum stress response in regulating Mcl-1 expression. Using quantitative RT-PCR and Western blot, we found that the translation of activating transcription factor-4 (ATF4), an important effector of the UPR, was also greatly enhanced by proteasome inhibition. ChIP analysis further revealed that bortezomib stimulated binding of ATF4 to a regulatory site (at position -332 to -324) at the promoter of the Mcl-1 gene. Knocking down ATF4 was paralleled by down-regulation of Mcl-1 induction by bortezomib and significantly increased bortezomib-induced apoptosis. These data identify the UPR and, more specifically, its ATF4 branch as an important mechanism mediating up-regulation of Mcl-1 by proteasome inhibition.

Hypoxia promotes dissemination of multiple myeloma through acquisition of epithelial to mesenchymal transition-like features.

The spread of multiple myeloma (MM) involves (re)circulation into the peripheral blood and (re)entrance or homing of MM cells into new sites of the BM. Hypoxia in solid tumors was shown to promote metastasis through activation of proteins involved in the epithelial-mesenchymal transition (EMT) process. We hypothesized that MM-associated hypoxic conditions activate EMT-related proteins and promote metastasis of MM cells. In the present study, we have shown that hypoxia activates EMT-related machinery in MM cells, decreases the expression of E-cadherin, and, consequently, decreases the adhesion of MM cells to the BM and enhances egress of MM cells to the circulation. In parallel, hypoxia increased the expression of CXCR4, consequently increasing the migration and homing of circulating MM cells to new BM niches. Further studies to manipulate hypoxia to regulate tumor dissemination as a therapeutic strategy are warranted.

AXL-specific single domain antibodies show diagnostic potential and anti-tumor activity in Acute Myeloid Leukemia.

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.

Bone marrow-derived mesenchymal stromal cells are attracted by multiple myeloma cell-produced chemokine CCL25 and favor myeloma cell growth in vitro and in vivo.

Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells that are predominantly localized in the bone marrow (BM). Mesenchymal stromal cells (MSCs) give rise to most BM stromal cells that interact with MM cells. However, the direct involvement of MSCs in the pathophysiology of MM has not been well addressed. In this study, in vitro and in vivo migration assays revealed that MSCs have tropism toward MM cells, and CCL25 was identified as a major MM cell-produced chemoattractant for MSCs. By coculture experiments, we found that MSCs favor the proliferation of stroma-dependent MM cells through soluble factors and cell to cell contact, which was confirmed by intrafemoral coengraftment experiments. We also demonstrated that MSCs protected MM cells against spontaneous and Bortezomib-induced apoptosis. The tumor-promoting effect of MSCs correlated with their capacity to enhance AKT and ERK activities in MM cells, accompanied with increased expression of CyclinD2, CDK4, and Bcl-XL and decreased cleaved caspase-3 and poly(ADP-ribose) polymerase expression. In turn, MM cells upregulated interleukin-6 (IL-6), IL-10, insulin growth factor-1, vascular endothelial growth factor, and dickkopf homolog 1 expression in MSCs. Finally, infusion of in vitro-expanded murine MSCs in 5T33MM mice resulted in a significantly shorter survival. MSC infusion is a promising way to support hematopoietic recovery and to control graft versus host disease in patients after allogeneic hematopoietic stem cell transplantation. However, our data suggest that MSC-based cytotherapy has a potential risk for MM disease progression or relapse and should be considered with caution in MM patients.

Effect of the HDAC inhibitor vorinostat on the osteogenic differentiation of mesenchymal stem cells in vitro and bone formation in vivo.

AIM: Vorinostat, a histone deacetylase (HDAC) inhibitor currently in a clinical phase III trial for multiple myeloma (MM) patients, has been reported to cause bone loss. The purpose of this study was to test whether, and to what extent, vorinostat influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro and bone formation in vivo. METHODS: Bone marrow-derived MSCs were prepared from both normal donors and MM patients. The MSCs were cultured in an osteogenic differentiation induction medium to induce osteogenic differentiation, which was evaluated by alkaline phosphatase (ALP) staining, Alizarin Red S staining and the mRNA expression of osteogenic markers. Naïve mice were administered vorinostat (100 mg/kg, ip) every other day for 3 weeks. After the mice were sacrificed, bone formation was assessed based on serum osteocalcin level and histomorphometric analysis. RESULTS: Vorinostat inhibited the viability of hMSCs in a concentration-dependent manner (the IC50 value was 15.57 µmol/L). The low concentration of vorinostat (1 µmol/L) did not significantly increase apoptosis in hMSCs, whereas pronounced apoptosis was observed following exposure to higher concentrations of vorinostat (10 and 50 µmol/L). In bone marrow-derived hMSCs from both normal donors and MM patients, vorinostat (1 µmol/L) significantly increased ALP activity, mRNA expression of osteogenic markers, and matrix mineralization. These effects were associated with upregulation of the bone-specifying transcription factor Runx2 and with the epigenetic alterations during normal hMSCs osteogenic differentiation. Importantly, the mice treated with vorinostat did not show any bone loss in response to the optimized treatment regimen. CONCLUSION: Vorinostat, known as a potent anti-myeloma drug, stimulates MSC osteogenesis in vitro. With the optimized treatment regimen, any decrease in bone formation was not observed in vivo.