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.

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.

Hypoxic Bone Marrow Stromal Cells Secrete miR-140-5p and miR-28-3p That Target SPRED1 to Confer Drug Resistance in Multiple Myeloma.

Bone marrow stromal cell (BMSC)-derived small extracellular vesicles (sEV) promote drug resistance to bortezomib in multiple myeloma cells. Elucidating the components of BMSC sEV that induce drug resistance in multiple myeloma cells could help identify strategies to overcome resistance. Considering the hypoxic nature of the myeloma microenvironment, we explored the role of hypoxia in regulating BMSC sEV cargo and investigated whether hypoxia-driven sEV miRNAs contribute to the drug resistance in multiple myeloma cells. Hypoxia increased the release of sEVs from BMSCs, and these sEVs more strongly attenuated bortezomib sensitivity in multiple myeloma cells than sEVs from BMSCs under normoxic conditions. RNA sequencing revealed that significantly elevated levels of miR-140-5p and miR-28-3p were enclosed in hypoxic BMSC-derived sEVs. Both miR-140-5p and miR-28-3p conferred bortezomib resistance in multiple myeloma cells by synergistically targeting SPRED1, a member of the Sprouty protein family that regulates MAPK activation. SPRED1 inhibition reduced sensitivity to bortezomib in multiple myeloma cells through activating MAPK-related pathways and significantly promoted multiple myeloma bortezomib resistance and tumor growth in a mouse model. These findings shed light on the role of hypoxia-induced miRNAs shuttled in BMSC-derived sEVs to multiple myeloma cells in inducing drug resistance and identify the miR-140-5p/miR-28-3p/SPRED1/MAPK pathway as a potential targetable axis for treating multiple myeloma. SIGNIFICANCE: Hypoxia induces stromal cells to secrete extracellular vesicles with increased miR-140-5p and miR-28-3p that are transferred to multiple myeloma cells and drive drug resistance by increasing the MAPK signaling.

Targeting S100A9 protein affects mTOR-ER stress signaling and increases venetoclax sensitivity in Acute Myeloid Leukemia.

Acute Myeloid Leukemia (AML) is a heterogeneous disease with limited treatment options and a high demand for novel targeted therapies. Since myeloid-related protein S100A9 is abundantly expressed in AML, we aimed to unravel the therapeutic impact and underlying mechanisms of targeting both intracellular and extracellular S100A9 protein in AML cell lines and primary patient samples. S100A9 silencing in AML cell lines resulted in increased apoptosis and reduced AML cell viability and proliferation. These therapeutic effects were associated with a decrease in mTOR and endoplasmic reticulum stress signaling. Comparable results on AML cell proliferation and mTOR signaling could be observed using the clinically available S100A9 inhibitor tasquinimod. Interestingly, while siRNA-mediated targeting of S100A9 affected both extracellular acidification and mitochondrial metabolism, tasquinimod only affected the mitochondrial function of AML cells. Finally, we found that S100A9-targeting approaches could significantly increase venetoclax sensitivity in AML cells, which was associated with a downregulation of BCL-2 and c-MYC in the combination group compared to single agent therapy. This study identifies S100A9 as a novel molecular target to treat AML and supports the therapeutic evaluation of tasquinimod in venetoclax-based regimens for AML patients.

RNA-sequencing based first choice of treatment and determination of risk in multiple myeloma.

Background: Immunotherapeutic targets in multiple myeloma (MM) have variable expression height and are partly expressed in subfractions of patients only. With increasing numbers of available compounds, strategies for appropriate choice of targets (combinations) are warranted. Simultaneously, risk assessment is advisable as patient's life expectancy varies between months and decades. Methods: We first assess feasibility of RNA-sequencing in a multicenter trial (GMMG-MM5, n=604 patients). Next, we use a clinical routine cohort of untreated symptomatic myeloma patients undergoing autologous stem cell transplantation (n=535, median follow-up (FU) 64 months) to perform RNA-sequencing, gene expression profiling (GEP), and iFISH by ten-probe panel on CD138-purified malignant plasma cells. We subsequently compare target expression to plasma cell precursors, MGUS (n=59), asymptomatic (n=142) and relapsed (n=69) myeloma patients, myeloma cell lines (n=26), and between longitudinal samples (MM vs. relapsed MM). Data are validated using the independent MMRF CoMMpass-cohort (n=767, FU 31 months). Results: RNA-sequencing is feasible in 90.8% of patients (GMMG-MM5). Actionable immune-oncological targets (n=19) can be divided in those expressed in all normal and >99% of MM-patients (CD38, SLAMF7, BCMA, GPRC5D, FCRH5, TACI, CD74, CD44, CD37, CD79B), those with expression loss in subfractions of MM-patients (BAFF-R [81.3%], CD19 [57.9%], CD20 [82.8%], CD22 [28.4%]), aberrantly expressed in MM (NY-ESO1/2 [12%], MUC1 [12.7%], CD30 [4.9%], mutated BRAF V600E/K [2.1%]), and resistance-conveying target-mutations e.g., against part but not all BCMA-directed treatments. Risk is assessable regarding proliferation, translated GEP- (UAMS70-, SKY92-, RS-score) and de novo (LfM-HRS) defined risk scores. LfM-HRS delineates three groups of 40%, 38%, and 22% of patients with 5-year and 12-year survival rates of 84% (49%), 67% (18%), and 32% (0%). R-ISS and RNA-sequencing identify partially overlapping patient populations, with R-ISS missing, e.g., 30% (22/72) of highly proliferative myeloma. Conclusion: RNA-sequencing based assessment of risk and targets for first choice treatment is possible in clinical routine.

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.

Tasquinimod suppresses tumor cell growth and bone resorption by targeting immunosuppressive myeloid cells and inhibiting c-MYC expression in multiple myeloma.

BACKGROUND: Immunotherapy emerged as a promising treatment option for multiple myeloma (MM) patients. However, therapeutic efficacy can be hampered by the presence of an immunosuppressive bone marrow microenvironment including myeloid cells. S100A9 was previously identified as a key regulator of myeloid cell accumulation and suppressive activity. Tasquinimod, a small molecule inhibitor of S100A9, is currently in a phase Ib/IIa clinical trial in MM patients (NCT04405167). We aimed to gain more insights into its mechanisms of action both on the myeloma cells and the immune microenvironment. METHODS: We analyzed the effects of tasquinimod on MM cell viability, cell proliferation and downstream signaling pathways in vitro using RNA sequencing, real-time PCR, western blot analysis and multiparameter flow cytometry. Myeloid cells and T cells were cocultured at different ratios to assess tasquinimod-mediated immunomodulatory effects. The in vivo impact on immune cells (myeloid cell subsets, macrophages, dendritic cells), tumor load, survival and bone disease were elucidated using immunocompetent 5TMM models. RESULTS: Tasquinimod treatment significantly decreased myeloma cell proliferation and colony formation in vitro, associated with an inhibition of c-MYC and increased p27 expression. Tasquinimod-mediated targeting of the myeloid cell population resulted in increased T cell proliferation and functionality in vitro. Notably, short-term tasquinimod therapy of 5TMM mice significantly increased the total CD11b+ cells and shifted this population toward a more immunostimulatory state, which resulted in less myeloid-mediated immunosuppression and increased T cell activation ex vivo. Tasquinimod significantly reduced the tumor load and increased the trabecular bone volume, which resulted in prolonged overall survival of MM-bearing mice in vivo. CONCLUSION: Our study provides novel insights in the dual therapeutic effects of the immunomodulator tasquinimod and fosters its evaluation in combination therapy trials for MM patients.

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.

The prognostic value and therapeutic targeting of myeloid-derived suppressor cells in hematological cancers.

The success of immunotherapeutic approaches in hematological cancers is partially hampered by the presence of an immunosuppressive microenvironment. Myeloid-derived suppressor cells (MDSC) are key components of this suppressive environment and are frequently associated with tumor cell survival and drug resistance. Based on their morphology and phenotype, MDSC are commonly subdivided into polymorphonuclear MDSC (PMN-MDSC or G-MDSC) and monocytic MDSC (M-MDSC), both characterized by their immunosuppressive function. The phenotype, function and prognostic value of MDSC in hematological cancers has been intensively studied; however, the therapeutic targeting of this cell population remains challenging and needs further investigation. In this review, we will summarize the prognostic value of MDSC and the different attempts to target MDSC (or subtypes of MDSC) in hematological cancers. We will discuss the benefits, challenges and opportunities of using MDSC-targeting approaches, aiming to enhance anti-tumor immune responses of currently used cellular and non-cellular immunotherapies.

Metabolic cross-talk within the bone marrow milieu: focus on multiple myeloma.

Cancer cells are well-known for their capacity to adapt their metabolism to their increasing energy demands which is necessary for tumor progression. This is no different for Multiple Myeloma (MM), a hematological cancer which develops in the bone marrow (BM), whereby the malignant plasma cells accumulate and impair normal BM functions. It has become clear that the hypoxic BM environment contributes to metabolic rewiring of the MM cells, including changes in metabolite levels, increased/decreased activity of metabolic enzymes and metabolic shifts. These adaptations will lead to a pro-tumoral environment stimulating MM growth and drug resistance In this review, we discuss the identified metabolic changes in MM and the BM microenvironment and summarize how these identified changes have been targeted (by inhibitors, genetic approaches or deprivation studies) in order to block MM progression and survival.

Aberrant DNA methylation in multiple myeloma: A major obstacle or an opportunity?

Drug resistance (DR) of cancer cells leading to relapse is a huge problem nowadays to achieve long-lasting cures for cancer patients. This also holds true for the incurable hematological malignancy multiple myeloma (MM), which is characterized by the accumulation of malignant plasma cells in the bone marrow (BM). Although new treatment approaches combining immunomodulatory drugs, corticosteroids, proteasome inhibitors, alkylating agents, and monoclonal antibodies have significantly improved median life expectancy, MM remains incurable due to the development of DR, with the underlying mechanisms remaining largely ill-defined. It is well-known that MM is a heterogeneous disease, encompassing both genetic and epigenetic aberrations. In normal circumstances, epigenetic modifications, including DNA methylation and posttranslational histone modifications, play an important role in proper chromatin structure and transcriptional regulation. However, in MM, numerous epigenetic defects or so-called 'epimutations' have been observed and this especially at the level of DNA methylation. These include genome-wide DNA hypomethylation, locus specific hypermethylation and somatic mutations, copy number variations and/or deregulated expression patterns in DNA methylation modifiers and regulators. The aberrant DNA methylation patterns lead to reduced gene expression of tumor suppressor genes, genomic instability, DR, disease progression, and high-risk disease. In addition, the frequency of somatic mutations in the DNA methylation modifiers seems increased in relapsed patients, again suggesting a role in DR and relapse. In this review, we discuss the recent advances in understanding the involvement of aberrant DNA methylation patterns and/or DNA methylation modifiers in MM development, progression, and relapse. In addition, we discuss their involvement in MM cell plasticity, driving myeloma cells to a cancer stem cell state characterized by a more immature and drug-resistant phenotype. Finally, we briefly touch upon the potential of DNA methyltransferase inhibitors to prevent relapse after treatment with the current standard of care agents and/or new, promising (immuno) therapies.

Inhibition of the Protein Arginine Methyltransferase PRMT5 in High-Risk Multiple Myeloma as a Novel Treatment Approach.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Subsets of patients have high-risk features linked with dismal outcome. Therefore, the need for effective therapeutic options remains high. Here, we used bio-informatic tools to identify novel targets involved in DNA repair and epigenetics and which are associated with high-risk myeloma. The prognostic significance of the target genes was analyzed using publicly available gene expression data of MM patients (TT2/3 and HM cohorts). Hence, protein arginine methyltransferase 5 (PRMT5) was identified as a promising target. Druggability was assessed in OPM2, JJN3, AMO1 and XG7 human myeloma cell lines using the PRMT5-inhibitor EPZ015938. EPZ015938 strongly reduced the total symmetric-dimethyl arginine levels in all cell lines and lead to decreased cellular growth, supported by cell line dependent changes in cell cycle distribution. At later time points, apoptosis occurred, as evidenced by increased AnnexinV-positivity and cleavage of PARP and caspases. Transcriptome analysis revealed a role for PRMT5 in regulating alternative splicing, nonsense-mediated decay, DNA repair and PI3K/mTOR-signaling, irrespective of the cell line type. PRMT5 inhibition reduced the expression of upstream DNA repair kinases ATM and ATR, which may in part explain our observation that EPZ015938 and the DNA-alkylating agent, melphalan, have combinatory effects. Of interest, using a low-dose of mTOR-inhibitor, we observed that cell viability was partially rescued from the effects of EPZ015938, indicating a role for mTOR-related pathways in the anti-myeloma activity of EPZ015938. Moreover, PRMT5 was shown to be involved in splicing regulation of MMSET and SLAMF7, known genes of importance in MM disease. As such, we broaden the understanding of the exact role of PRMT5 in MM disease and further underline its use as a possible therapeutic target.

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.

System Xc- inhibition blocks bone marrow-multiple myeloma exosomal crosstalk, thereby countering bortezomib resistance.

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.

Pyrroline-5-Carboxylate Reductase 1: a novel target for sensitizing multiple myeloma cells to bortezomib by inhibition of PRAS40-mediated protein synthesis.

BACKGROUND: Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both MM cell survival and drug resistance. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are two mitochondrial enzymes that facilitate the last step in the glutamine-to-proline conversion. Overexpression of PYCR1 is involved in progression of several cancers, however, its' role in hematological cancers is unknown. In this study, we investigated whether PYCR affects MM viability, proliferation and response to bortezomib. METHODS: Correlation of PYCR1/2 with overall survival was investigated in the MMRF CoMMpass trial (653 patients). OPM-2 and RPMI-8226 MM cell lines were used to perform in vitro experiments. RPMI-8226 cells were supplemented with 13C-glutamine for 48 h in both normoxia and hypoxia (< 1% O2, by chamber) to perform a tracer study. PYCR1 was inhibited by siRNA or the small molecule inhibitor pargyline. Apoptosis was measured using Annexin V and 7-AAD staining, viability by CellTiterGlo assay and proliferation by BrdU incorporation. Differential protein expression was evaluated using Western Blot. The SUnSET method was used to measure protein synthesis. All in vitro experiments were performed in hypoxic conditions. RESULTS: We found that PYCR1 and PYCR2 mRNA expression correlated with an inferior overall survival. MM cells from relapsed/refractory patients express significantly higher levels of PYCR1 mRNA. In line with the strong expression of PYCR1, we performed a tracer study in RPMI-8226 cells, which revealed an increased conversion of 13C-glutamine to proline in hypoxia. PYCR1 inhibition reduced MM viability and proliferation and increased apoptosis. Mechanistically, we found that PYCR1 silencing reduced protein levels of p-PRAS40, p-mTOR, p-p70, p-S6, p-4EBP1 and p-eIF4E levels, suggesting a decrease in protein synthesis, which we also confirmed in vitro. Pargyline and siPYCR1 increased bortezomib-mediated apoptosis. Finally, combination therapy of pargyline with bortezomib reduced viability in CD138+ MM cells and reduced tumor burden in the murine 5TGM1 model compared to single agents. CONCLUSIONS: This study identifies PYCR1 as a novel target in bortezomib-based combination therapies for MM.

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.

Targeting coenzyme Q10 synthesis overcomes bortezomib resistance in multiple myeloma.

During the development of drug resistance, multiple myeloma (MM) cells undergo changes to their metabolism. However, how these metabolic changes can be exploited to improve treatment efficacy is not known. Here we demonstrate that targeting coenzyme Q10 (CoQ) biosynthesis through the mevalonate pathway works in synergy with the proteasome inhibitor bortezomib (BTZ) in MM. We show that gene expression signatures relating to the mitochondrial tricarboxylic acid (TCA) cycle and electron transport chain (ETC) predispose to clinical BTZ resistance and poor prognosis in MM patients. Mechanistically, BTZ-resistant cells show increased activity of glutamine-driven TCA cycle and oxidative phosphorylation, together with an increased vulnerability towards ETC inhibition. Moreover, BTZ resistance is accompanied by high levels of the mitochondrial electron carrier CoQ, while the mevalonate pathway inhibitor simvastatin increases cell death and decreases CoQ levels, specifically in BTZ-resistant cells. Both in vitro and in vivo, simvastatin enhances the effect of bortezomib treatment. Our study links CoQ synthesis to drug resistance in MM and provides a novel avenue for improving BTZ responses through statin-induced inhibition of mitochondrial metabolism.

Single-cell analysis at the protein level delineates intracellular signaling dynamic during hematopoiesis.

BACKGROUND: Hematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry. RESULTS: With fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling. CONCLUSIONS: These comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

A distinct metabolic response characterizes sensitivity to EZH2 inhibition in multiple myeloma.

Multiple myeloma (MM) is a heterogeneous haematological disease that remains clinically challenging. Increased activity of the epigenetic silencer EZH2 is a common feature in patients with poor prognosis. Previous findings have demonstrated that metabolic profiles can be sensitive markers for response to treatment in cancer. While EZH2 inhibition (EZH2i) has proven efficient in inducing cell death in a number of human MM cell lines, we hereby identified a subset of cell lines that despite a global loss of H3K27me3, remains viable after EZH2i. By coupling liquid chromatography-mass spectrometry with gene and miRNA expression profiling, we found that sensitivity to EZH2i correlated with distinct metabolic signatures resulting from a dysregulation of genes involved in methionine cycling. Specifically, EZH2i resulted in a miRNA-mediated downregulation of methionine cycling-associated genes in responsive cells. This induced metabolite accumulation and DNA damage, leading to G2 arrest and apoptosis. Altogether, we unveiled that sensitivity to EZH2i in human MM cell lines is associated with a specific metabolic and gene expression profile post-treatment.

Targeting phosphoglycerate dehydrogenase in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is a hematological malignancy characterized by the clonal expansion of plasma cells in the bone marrow. To date, this disease is still incurable and novel therapeutic approaches are required. Phosphoglycerate dehydrogenase (PHGDH) is the first and rate-limiting enzyme in the de novo serine synthesis pathway, and it has been attributed to bortezomib-resistance in MM. METHODS: Two different PHGDH inhibitors, CBR5884 and NCT-503, were tested against human myeloma cell lines, primary MM cells from patients, and peripheral blood mononuclear cells isolated from healthy donors. The PHGDH inhibitors were then tested in combination with proteasome inhibitors in different MM cell lines, including proteasome-resistant cell lines. Furthermore, we confirmed the effects of PHGDH inhibition through knocking down PHGDH and the effect of NCT-503 in vivo in the 5T33MM mouse model. RESULTS: All the tested myeloma cell lines expressed PHGDH and were sensitive to doses of NCT-503 that were tolerated by peripheral blood mononuclear cells isolated from healthy donors. Upon testing bortezomib in combination with NCT-503, we noticed a clear synergy in several HMCLs. The sensitivity to bortezomib also increased after PHGDH knockdown, mimicking the effect of NCT-503 treatment. Interestingly, targeting PHGDH reduced the intracellular redox capacity of the cells. Furthermore, combination treatment with NCT-503 and bortezomib exhibited a therapeutic advantage in vivo. CONCLUSIONS: Our study shows the therapeutic potential of targeting PHGDH in MM, and suggest it as a way to overcome the resistance to proteasome inhibitors.