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.

Repurposing Sulfasalazine as a Radiosensitizer in Hypoxic Human Colorectal Cancer.

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.

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.

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.

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.

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.

The Transfer of Sphingomyelinase Contributes to Drug Resistance in Multiple Myeloma.

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.