Despite significant advancements, multiple myeloma (MM) remains incurable, largely due to drug resistance. Our previous research has demonstrated that proline metabolism plays a role in MM progression and that inhibiting PYCR1, the final enzyme in proline synthesis, enhances bortezomib sensitivity in MM cells. Given the high expression of PYCR1 in bone marrow stromal cells (BMSCs), we sought to investigate the effects of PYCR1 inhibition in BMSCs and its indirect influence on MM cell metabolism and viability. Culturing MM cells in conditioned medium (CM) of PYCR1-silenced BMSC significantly impaired oxidative phosphorylation and sensitised MM cells to bortezomib. Analysis of the CM secretome revealed a reduction in activin A release. Proline and activin A supplementation were able to counteract MM sensitivity to bortezomib. Combination therapy of the PYCR1 inhibitor pargyline and bortezomib reduced tumour load in a 3D model and reduced serum activin A levels in 5TGM1-bearing mice. This study demonstrates the contribution of stromal cell metabolism to MM progression. Inhibiting PYCR1 in BMSCs leads to less activin A release, limits oxidative phosphorylation in MM cells and enhances bortezomib efficacy.

Introduction Multiple myeloma (MM) is a hematological cancer, characterized by the accumulation of monoclonal plasma cells in the bone marrow. It remains an incurable cancer due to drug resistance, wherein the bone marrow microenvironment plays a crucial role. We have previously shown that glutamine-to-proline conversion is upregulated in MM cells upon hypoxic culture. Moreover, inhibition of proline production by blocking its converting enzyme PYCR1 successfully reduced proliferation and viability in vitro. Importantly, PYCR1 inhibition combined with standard-of-care agent bortezomib decreased tumor load in vivo. As PYCR1 is also highly expressed in stromal cells and proline an important component of extracellular matrix proteins, we investigated whether PYCR1 targeting in stromal cells affects MM viability and its structural microenvironment. Material and methods PYCR1 expression was investigated by the use of microarray data from the Heidelberg/Montpellier cohort. For in vitro experiments, the human MM cell line OPM-2 and human stromal cell line HS-5 were used. CD138+ and CD138- fractions from primary patient samples were separated by MACS. To obtain primary stromal cells, the CD138- fraction was plated out in fresh medium. After 48h, the medium was refreshed and all adherent cells were further cultured and used as primary stromal cells. PYCR1 expression in HS-5 cells was reduced through siRNA. Hypoxic culture (<1% O2) was established through chambers. RNA expression was measured by RT-qPCR, while protein expression was measured by western blot. Viability was assessed by CellTiterGlo assay. Results Gene expression analysis shows high RNA expression of PYCR1 in healthy bone marrow plasma cells (BMPCs), plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and MM patients, but also in bone marrow stromal cells (BMSCs). PYCR1 expression was low or absent in other cell types, including T cells, osteoclasts, monocytic and granulocytic cells. On protein level, we also confirmed PYCR1 expression in MM cells (CD138+), CD138- fraction (including stromal cells) and cultured primary stromal cells. Moreover, PYCR1 expression increased upon hypoxic culture in primary stromal cells and stromal cell line HS-5. PYCR1 knockdown in stromal cell line HS-5 did not affect viability of the stromal cells, but its conditioned medium did reduce its protective effects when OPM-2 MM cells were treated with standard-of-care agent bortezomib. Further investigation revealed that knockdown of PYCR1 in HS-5 reduces RNA expression of structural proteins col1a1, col1a2, col3a1, ctgf and acta2 in HS-5. Conclusion PYCR1 is highly expressed in myeloma cells and stromal cells. PYCR1 inhibition in stromal cells reduces its proliferative effect on myeloma cells when combined with bortezomib. Preliminary data indicates a link between PYCR expression in stromal cells and rearrangement of extracellular matrix proteins. Citation Format: Inge Oudaert, Catharina Muylaert, Hatice Satilmis, Sylvia Faict, Kim De Veirman, Elke De Bruyne, Karin Vanderkerken, Eline Menu. PYCR1 inhibition in multiple myeloma-associated stroma limits tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2505.

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 μ m ) 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.

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 β‐blockers as a single agent and in combination with commonly used anti‐myeloma therapies. Expression of the β 2 ‐adrenergic receptor correlated with poor survival outcomes in patients with multiple myeloma. Targeting the β 2 ‐adrenergic receptor (β 2 AR) using either selective or non‐selective β‐blockers reduced multiple myeloma cell viability, and induced apoptosis and autophagy. Blockade of the β 2 AR modulated cancer cell metabolism by reducing the mitochondrial respiration as well as the glycolytic activity. These effects were not observed by blockade of β 1 ‐adrenergic receptors. Combining β 2 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 β 2 AR‐blockers as a complementary or additive approach in multiple myeloma treatment and support the future clinical evaluation of non‐selective β‐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.

xCT overexpression in cancer cells has been linked to tumor growth, metastasis and treatment resistance. Sulfasalazine (SSZ), an FDA-approved drug for the treatment of rheumatoid sarthritis, and inflammatory bowel diseases, has anticancer properties via inhibition of xCT, leading to the disruption of redox homeostasis. Since reactive oxygen species (ROS) are pivotal for the efficacy of radiotherapy (RT), elevated levels of ROS are associated with improved RT outcomes. In this study, the influence of SSZ treatment on the radiosensitivity of human colorectal cancer (CRC) cells was investigated. Our principal finding in human HCT116 and DLD-1 cells was that SSZ enhances the radiosensitivity of hypoxic CRC cells but does not alter the intrinsic radiosensitivity. The radiosensitizing effect was attributed to the depletion of glutathione and thioredoxin reductase levels. In turn, the reduction leads to excessive levels of ROS, increased DNA damage, and ferroptosis induction. Confirmation of these findings was performed in 3D models and in DLD-1 xenografts. Taken together, this study is a stepping stone for applying SSZ as a radiosensitizer in the clinic and confirms that xCT in cancer cells is a valid radiobiological target.

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.

Background: Multiple myeloma (MM) is a cancer characterized by the accumulation of malignant plasma cells in the bone marrow (BM). The BM is an immune suppressive environment, which hampers the efficacy of modern immune therapeutic strategies. Immune suppression is partially orchestrated by the hypoxic nature of the BM. Due to this hypoxia, cancer cells are pushed towards aerobic glycolysis, during which pyruvate is converted to lactate. Lactate can be imported and exported by monocarboxylate transporters (MCTs). Lactate in the BM milieu contributes to an immune suppressive environment by several mechanisms including driving differentiation and attraction of myeloid derived suppressor cell (MDSC). Aims: In this study, we aimed to evaluate the effects of MCT inhibition on MM cell viability and the immune suppressive character of the BM. Methods: Correlation of MCT-expression with survival was investigated using the Mulligan cohort. In vitro, several MM cell lines (LP-1, RPMI-8226, 5TGM1) were used. Syrosingopine was used as dual inhibitor of MCT1 and MCT4, AZD0095 as MCT4 inhibitor and AZD3965 as MCT1 inhibitor. The effect on apoptosis was quantified by AnnexinV/7-AAD staining. For MDSC differentiation, BM was isolated from naïve mice. CD11b+ cells were selected by MACS and cultured with conditioned medium (CM). CM was prepared from 5TGM1 cells treated with DMSO or syrosingopine, for 48 hours in hypoxia (1% O2). The effect on MDSC differentiation was evaluated by western blotting. Results: High expression of MCT1/MCT4 genes correlated with a significant lower overall survival in MM patients. Increased expression of MCT1 and MCT4 was observed in MM cell lines after 48 hours of hypoxia. Syrosingopine treatment resulted in intracellular lactate accumulation but did not induce apoptosis. Neither did single inhibition of MCT1 or MCT4. MM cells could however be sensitized to syrosingopine by treatment with metformin, a complex I inhibitor, resulting in apoptosis. AZD0095 and AZD3965 were less potent to induce cell death in combination with metformin, compared to dual MCT inhibition. The effects of the combination treatment on metabolism and cellular pathways are currently investigated. Extrinsic effects of export inhibition were evaluated by using CM of MM cells treated with syrosingopine on MDSCs. Both lactate and MM-CM promoted MDSC differentiation in vitro, while CM deprived of lactate resulted in a clear decrease of differentiation markers such as p-stat3 and arginase. This indicates a less suppressive status of the MDSC’s. Summary/Conclusion: Dual inhibition of MCT1 and MCT4 is not sufficient to induce apoptosis in MM cells; however, they can be sensitized to syrosingopine with metformin. Blocked lactate export from MM cells resulted in impaired MDSC differentiation. Targeting lactate metabolism could be a new approach to hamper MM survival and create a less suppressive immune environment. Arne Van der Vreken and Inge Oudaert contributed equally to this work.

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 13 C-glutamine for 48 h in both normoxia and hypoxia (< 1% O 2 , 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 13 C-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.

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.

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:Malignant cells can adapt their lipid metabolism to attain higher rates of proliferation, and resist chemotherapy‐induced apoptosis.Aims:In this study, we aimed to identify the changes in lipid metabolism in multiple myeloma (MM) patients, and to further investigate the role of this altered metabolism in vitro.Methods:We performed a lipidomics analysis to compare plasma from MM patients to healthy controls. Based on these results, lipid spike‐in assays enabled us to evaluate effects on MM cell proliferation. Sphingomyelinase (SMase) content of MM cells and their exosomes was measured, before and after treatment with standard‐of‐care drugs, using an AmplexRed assay. We inhibited both neutral and acid SMase (ASM) in MM cells to evaluate effects on drug efficacy. Finally, we investigated whether MM exosomes rich in ASM are able to induce drug resistance in MM cells low in ASM.Results:Lipidomics analysis revealed that several ceramide species were significantly more present in MM patient plasma, while sphingomyelin seemed to be decreased. When adding ceramide in vitro to MM cells, we observed higher viability and proliferation. The increased ceramide/sphingomyelin ratio in plasma lead us to believe that the sphingomyelinase enzyme, which converts sphingomyelin into ceramide, was upregulated in MM cells. This was confirmed in BM samples, comparing CD138+ MM cells to the CD138‐ fraction. In the MM cell lines JJN3, OPM2, LP1 and U266, we observed an increased expression of acid and total SMase when treating these cells with melphalan or bortezomib for 24 h. Furthermore, we investigated whether MM exosomes were involved in the secretion and transfer of acid SMase. Interestingly, amitriptyline, an inhibitor of acid SMase, often prescribed in MM patients for neuropathic pain, increased the efficacy of both drugs in vitro, and also induced apoptosis in primary MM cells as a single agent.Summary/Conclusion:SMase is increased in primary MM cells and cell lines upon treatment with melphalan and bortezomib, while inhibiting this expression using amitriptyline leads to a higher efficacy of drug therapy.

Multiple myeloma (MM) is well-known for the development of drug resistance, leading to relapse. Therefore, finding novel treatment strategies remains necessary. By performing a lipidomics assay on MM patient plasma, we aimed to identify new targets. We observed a dysregulation in the sphingolipid metabolism, with the upregulation of several ceramides and downregulation of sphingomyelin. This imbalance suggests an increase in sphingomyelinase, the enzyme responsible for hydrolyzing sphingomyelin into ceramide. We confirmed the upregulation of acid sphingomyelinase (ASM) in primary MM cells. Furthermore, we observed an increase in ASM expression in MM cell lines treated with melphalan or bortezomib, as well as in their exosomes. Exosomes high in ASM content were able to transfer the drug-resistant phenotype to chemosensitive cells, hereby suggesting a tumor-protective role for ASM. Finally, inhibition of ASM by amitriptyline improved drug sensitivity in MM cell lines and primary MM cells. In summary, this study is the first to analyze differences in plasma lipid composition of MM patients and match the observed differences to an upregulation of ASM. Moreover, we demonstrate that amitriptyline is able to inhibit ASM and increase sensitivity to anti-myeloma drugs. This study, therefore, provides a rational to include ASM-targeting-drugs in combination strategies in myeloma patients.