Immunotherapy has emerged as a promising strategy for multiple myeloma (MM), yet relapse remains frequent due to the immunosuppressive bone marrow (BM) microenvironment, characterized by T cell dysfunction and accumulation of immunosuppressive myeloid cells. The co-stimulatory receptor 4-1BB (CD137, TNFRSF9) can enhance T and NK cell effector functions, but its therapeutic utility in MM is not well established. Tasquinimod (TQ), a clinical-stage S100A9 inhibitor, offers a complementary approach by limiting the recruitment and activity of suppressive myeloid cells. 4-1BB expression was assessed during disease progression in MM mice and in newly diagnosed MM patients using single-cell RNA sequencing and flow cytometry. Therapeutic potential was evaluated in 5TGM1 tumor-bearing mice treated with two 4-1BB agonists, LOB12.3 (IgG1κ) and 3H3 (IgG2a), using isotype controls. The lead agonist was subsequently combined with TQ to investigate dual targeting of the immunosuppressive tumor microenvironment. Tumor burden was quantified via BM and spleen plasmacytosis and serum M-protein levels. Immune modulation was analyzed using multi-parameter flow cytometry. Statistical significance was determined using the Mann–Whitney U test or one-way ANOVA (p < 0.05). 4-1BB expression progressively increased on T and NK cells during tumor development in mice. In primary MM patient BM samples, ex vivo 4-1BB stimulation with urelumab enhanced effector responses, increasing IFN-γ+ and Granzyme B+ CD3+ T cells, alongside trends toward increased CD56+ NK cells and elevated IFN-γ+ NK cell activity. In vivo, 4-1BB agonist treatment promoted expansion of T cell subsets, with clone-specific effects: the IgG2a clone 3H3 significantly reduced M-protein levels and BM plasmacytosis, whereas the IgG1 clone LOB12.3 induced NK cell depletion and demonstrated limited anti-tumor activity. Combining 3H3 with TQ provided superior anti-myeloma efficacy, reducing BM plasmacytosis from 62.5

Adrenergic signaling regulates immune homeostasis through neuroendocrine pathways, but its role in hematologic malignancies remains poorly understood. Multiple myeloma (MM) originates in the bone marrow (BM), a highly innervated niche where neural, immune, and stromal signals are integrated. Whether adrenergic stimulation shapes BM immunity and influences myeloma progression is unclear. Using the 5T33MM mouse model, we investigated how adrenergic activation affects tumor growth and the BM immune landscape. Sustained adrenergic signaling was induced in vivo, while pharmacological β-adrenergic stimulation was used to assess effects on tumor-immune interactions. Immune composition and tumor burden were analyzed in BM and spleen, β2-adrenergic receptor expression was examined across murine and human immune populations, and ex vivo assays were performed using murine and patient-derived BM samples treated with adrenergic agonists alone or combined with relevant myeloma immunotherapies. Chronic restraint stress remodeled the BM immune compartment in MM-bearing mice, characterized by expansion of innate effector populations, including neutrophils and natural killer cells, and was associated with a significant reduction in BM tumor burden. These effects were compartment-restricted and not reflected in splenic tumor load or systemic disease markers. Pharmacological β-adrenergic stimulation reproduced these immune alterations and transiently delayed myeloma progression. In contrast, short-term β-adrenergic agonism ex vivo did not alter MM cell viability but consistently reduced the efficacy of MM immunotherapies. In summary, adrenergic signaling exerts context-dependent effects on myeloma progression, restraining tumor growth in vivo while impairing immunotherapy efficacy ex vivo, identifying a BM-specific neuroimmune axis relevant for cancer treatment.

The role of the sympathetic nervous system (SNS) in cancer biology has gained increasing attention, and its ability to affect immunotherapy is starting to become clearer. Extensive evidence shows that neuro-onco-immune interactions significantly influence tumor progression and the effectiveness of cancer treatments. Blocking SNS signaling, primarily through β-adrenergic receptors, enhances immune cell functions, by increasing CD8+ T-cell activation and cytokine production, while reducing immunosuppressive cell populations. This review explores the relationship between SNS signaling and cancer immunotherapy, emphasizing how SNS activation affects the efficacy of various immunotherapies, including immune modulators, immune checkpoint inhibitors, oncolytic virus therapy, therapeutic vaccines, and CAR-T cell therapies. We summarize retrospective studies investigating the use of β-blockers during immunotherapy, suggesting potential benefits for treatment outcomes of blocking SNS signaling in the tumor microenvironment. We examine ongoing clinical trials that evaluate the use of beta-blockers with immune checkpoint inhibitors, which aim to improve patient outcomes. While translational and preclinical studies provide ample evidence for targeting SNS signaling in cancer immunotherapy, clinical studies are only beginning to emerge. Ultimately, this review underscores the need for further research to better understand how SNS signaling can be targeted to optimize immunotherapy, paving the way for more effective treatment strategies.

Abstract Introduction Immunotherapy has shown clinical promise in multiple myeloma (MM), but relapse remains common, largely due to the immunosuppressive bone marrow (BM) microenvironment, characterized by T cell exhaustion and the accumulation of myeloid-derived suppressor cells (MDSCs). Recent advances highlight the therapeutic promise of 4-1BB (CD137) agonist antibodies in enhancing T cell–mediated anti-tumor responses, though their efficacy in MM remains poorly defined. Investigating 4-1BB expression dynamics in a preclinical MM model informed our rationale for targeting this pathway. Tasquinimod (TasQ), a small-molecule immunomodulatory agent, currently evaluated in a phase Ib/IIa clinical trial in MM patients (NCT04405167), offers a complementary strategy. By inhibiting the S100A9 signaling pathway, TasQ interferes with the recruitment and function of MDSCs, resulting in a less suppressive tumor microenvironment (TME) which reestablishes the anti -tumor immunity. In this study, we assessed the therapeutic potential of two distinct 4-1BB agonist antibody clones in an immunocompetent preclinical MM model. Building on these findings, we further explored the combination of the most effective 4-1BB agonist with TasQ, aiming to overcome the immunosuppressive tumor microenvironment, and strengthen anti-myeloma immune activity. Methods 4-1BB expression was analyzed during disease progression in 5T33MM mice using single-cell RNA sequencing of spleen and BM, 2 myeloma-infiltrating organs. These findings were validated by flow cytometry in both 5T33MM and 5TGM1 models. Therapeutic potential was assessed by treating 5TGM1 tumor-bearing mice with two 4-1BB agonist clones—LOB12.3 (IgG1κ; n=7/group) and 3H3 (IgG2a; n=5/group), using clone-specific isotype controls. Beginning on day 3 post-tumor inoculation, mice received 100µg of antibody intraperitoneally twice weekly until end stage. In a follow-up experiment, the lead 4-1BB agonist was combined with TasQ, administered at 30mg/kg in drinking water, to evaluate the impact of dual targeting the immunosuppressive TME (n=11/group). Tumor burden was assessed by determining the percentage of plasmacytosis in BM and spleen through cytospin stainings, together with M-protein measurement by serum electrophoresis. Immunomodulating effects were investigated using multi-parameter flow cytometry. Statistical significance was determined using the Mann–Whitney U test or one-way ANOVA, with p<0.05 considered significant. Results 4-1BB was predominantly detected on T cells and natural killer (NK) cells, with its expression further increasing as the disease progressed. Treatment of 5TGM1 mice with 4-1BB agonists significantly increased the percentage of CD4+ and CD8+ T cells in the BM and spleen. Interestingly, treatment with clone LOB12.3 resulted in a significant reduction in NK cell percentages in both the BM and spleen, while clone 3H3 selectively reduced splenic NK cells. Therapeutically, clone 3H3 significantly decreased M-protein levels and BM plasmacytosis (p<0.01), while no significant effects were observed for clone LOB12.3. Treatment with the IgG2a-formatted 4-1BB agonist combined with TasQ led to a significant reduction in M-protein levels and BM plasmacytosis (p<0001). Plasmacytosis was 62.50% in the isotype control group, decreased to 36.18% with 4-1BB agonist, 37.64% with TasQ, and further reduced to 14.09% in the combination therapy group, highlighting the enhanced efficacy of dual treatment. These effects were mediated by increased granzyme B–mediated T and NK-cell activation and enhanced differentiation of effector T cells (CD44⁺CD62L⁻). While this therapy did not alter the frequency of dendritic cells (DCs) in the BM, it enhanced their maturation, as evidenced by increased CD86 expression—particularly on type 1 and type 2 conventional DC subsets. Conclusion In conclusion, our findings demonstrate that 4-1BB activation can enhance anti-tumor immunity in MM; however, the therapeutic efficacy is dependent on the specific agonist used. Notably, the IgG2a-formatted 4-1BB agonist showed superior activity, underscoring the importance of isotype selection in achieving optimal immunotherapeutic outcomes. Moreover, co-administration with TasQ enhanced therapeutic efficacy, supporting the potential benefit of a combinatorial approach. Further investigation is warranted to elucidate the mechanisms driving these responses and to refine 4-1BB-targeted strategies for effective clinical translation.

BACKGROUND:The plasma cell malignancy multiple myeloma (MM) remains incurable due to the inevitable development of drug resistance (DR). Epigenetic modifiers are frequently mutated or deregulated in MM patients, contributing to MM progression and relapse. Overexpression of the de novo DNA methyltransferase 3B (DNMT3B) in MM has been reported, correlating with poor prognosis. However, its exact role in MM cell biology and relapse remains elusive. METHODS:To evaluate the basal expression and prognostic value of DNMT3B mRNA in terms of overall survival the publicly available gene expression profiling datasets GSE2658, GSE9782, GSE4581, E-MTAB-372, E-TABM-1088 and E-TABM-937 were used. Both the DNMT3B selective inhibitor Nanaomycin A and genetic knockdown using a doxycycline inducible shRNA against DNMT3B were used to target DNMT3B. Viability and apoptosis were assessed using respectively a CellTiter-Glo assay and AnnexinV/7AAD stainings. Cell proliferation was measured by BrdU incorporation and cell cycle analysis, while the clonogenic capacity was evaluated by a colony formation assay. Finally, RNA-seq was performed upon genetic knockdown. RESULTS:Here, we show that DNMT3B is significantly increased in the relapsed setting and high DNMT3B levels are strongly correlating with disease progression and high-risk disease, irrespective of the treatment. Targeting DNMT3B using either genetic inhibition or the selective inhibitor Nanaomycin A strongly impaired MM cell growth, survival and clonogenicity. Moreover, Nanaomycin A reduced viability of primary MM cells from newly diagnosed and relapsed patients. Mechanistic studies revealed that DNMT3B inhibition mainly affects cell cycle and stemness-related transcriptional programs. Notably, DNMT3B depletion affected the stability of the master cell cycle regulator MYC, thereby reducing c-MYC levels and cell viability both in parental and c-MYC overexpressing cells. Finally, Nanaomycin A (re)sensitized MM cells to bortezomib, melphalan and anti-CD38 monoclonal antibodies (daratumumab, isatuximab). CONCLUSION:Collectively, our findings uncover DNMT3B as a targetable vulnerability in high-risk patients with high DNMT3B/MYC levels.

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.

The long-term effectiveness of immunotherapies against Multiple Myeloma (MM) remains elusive, demonstrated by the inevitable relapse in patients. This underscores the urgent need for an in-depth analysis of the MM tumor-immune microenvironment (TME). Hereto, a representative immunocompetent MM mouse model can offer a valuable approach to study the dynamic changes within the MM-TME and to uncover potential resistance mechanisms hampering effective and durable therapeutic strategies in MM. We generated a comprehensive single-cell RNA-sequencing atlas of the MM-TME in bone marrow and spleen encompassing different stages of disease, using the immunocompetent 5T33MM mouse model. Through comparative analysis, we correlated our murine dataset with the pathogenesis in MM patients by reanalyzing publicly available datasets of human bone marrow samples across various disease stages. Using flow cytometry, we validated the dynamic changes upon disease progression in the 5T33MM model. Furthermore, interesting target populations, as well as the immune-boosting anti-CD40 agonist (αCD40) therapy were tested ex vivo on murine and human primary samples and in vivo using the 5T33MM model. In this study, we identified the heterogenous and dynamic changes within the TME of murine and human MM. We found that the MM-TME was characterized by an increase in T cells, accompanied with an exhausted phenotype. Although neutrophils appeared to be rather innocuous at early disease stages, they acquired a pro-tumorigenic phenotype during MM progression. Moreover, conventional dendritic cells (cDCs) showed a less activated phenotype in MM, underscoring the potential of immune-boosting therapies such as αCD40 therapy. Importantly, we provided the first pre-clinical evaluation of αCD40 therapy and demonstrated successful induction of cDC- and T-cell activation, accompanied by a significant short-term anti-tumor response. This resource provides a comprehensive and detailed immune atlas of the evolution in human and murine MM disease progression. Our findings can contribute to immune-based patient stratification and facilitate the development of novel and durable (immune) therapeutic strategies in MM.

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.

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.

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.

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.

S266from primary patient samples provide an environment that allows evaluation of different study conditions and its impact not just on the patient's own myeloma cells, but their immune compartment as well.Additional studies to assess its application in T-cell redirecting therapy and other novel immunotherapies using this model is warranted to correlate with clinical outcomes.

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.

Abstract Introduction Immunotherapy has revolutionized cancer treatment and significantly affected the management of Multiple Myeloma (MM) patients. Unfortunately, these immunotherapeutic approaches are hampered by the presence of a suppressive bone marrow microenvironment including myeloid derived suppressor cells and tumor associated macrophages. Tasquinimod (TasQ), an immunomodulatory compound, is currently in phase Ib/IIa for relapsed/refractory MM patients (NCT04405167). TasQ blocks the interaction between S100A9 and its receptors, which is associated with reduced MDSC accumulation. In this study, we investigated TasQ-mediated direct and indirect effects on MM cell growth, bone disease and immunomodulation in vitro and in vivo using human myeloma cell lines and the immunocompetent 5TMM models. Material and methods In vitro, murine (5T33vt, 5TGM1) and human (JJN3, LP1, OPM2, and RPMI8226) MM cell lines were cultured at different concentrations of TasQ. Cell proliferation was assessed by BrdU staining using flow cytometry. C-Myc and pSTAT3 expression were analyzed by western blot. In vitro T cell proliferation experiments were performed using MACS-sorted CD11b + cells and CFSE-labeled T cells from naïve mice. Cells were cocultured for 72h in the presence of MM conditioned medium (5T33MMvt CM) with CD3/CD28 microbeads, followed by flow cytometry to assess T cell proliferation. For in vivo experiments, we used the 5T33 (aggressive) and 5TGM1 (moderate) MM models. On the second day after tumor cell injection, the mice were randomly assigned to the treatment group and the control group. The treatment group received 30 mg/kg of TasQ in drinking water for 35 days (5TGM1) and 21 days (5T33). Anti-tumor and immunomodulating effects were analyzed by flow cytometry (e.g. tumor cells, myeloid subsets, CD4/CD8 + T cells), qRT-PCR, western blot and serum ELISA (interferon-gamma). Effects on osteogenesis in the 5TGM1 model was investigated by Micro-CT. Statistical differences were assessed by Mann-Whitney U test and One-way ANOVA with p<0.05 considered as statistically significant. Results TasQ-treatment of murine and human myeloma cell lines (HMCL), at concentrations of 10-25uM, significantly reduced MM cell proliferation after 24h and 48h in vitro (n=3, p<0.05). In addition, a downregulation in c-Myc expression could be observed 6h after treatment of human MM cell lines (n=3). In vitro, TasQ significantly increased T cell proliferation in co-culture experiments with T cells and myeloid cells in 5T33MMvt CM (n=3, p<0.05). Using the immunocompetent 5TGM1 and 5T33MM model, we investigated direct and indirect anti-tumor effects of TasQ. We found that TasQ significantly reduced tumor load in the bone marrow of 5TGM1 (n=10/group, p=0.0012) and 5T33MM mice (n=10/group, p=0.0106) compared to vehicle-treated control mice. Using flow cytometry, we could not observe a difference in the percentage of CD4 + and CD8 + T cells. However, a significant upregulation in serum interferon-gamma could be observed in the 5T33MM mice (p=0.0284). While the percentage of CD11b + cells in the TasQ-treated group was significantly increased (p<0.05), the percentage of monocytic myeloid cells (CD11b +Ly6G -) was significantly reduced in both models (p<0.05). qRT-PCR results showed that the expression of IL-10 was downregulated in purified CD11b + myeloid cells (p<0.05). Consistent with the in vitro data, we observed a decrease in the protein expression of c-Myc in purified MM cells obtained from TasQ-treated mice compared to control mice. Micro-CT analysis of femurs demonstrated a significant increase in the percentage BV/TV (ratio of bone material volume over tissue volume) and trabeculae number (p<0.0001) in TasQ-treated 5TGM1 mice compared to untreated mice. Conclusion TasQ has pleiotropic effects on the MM cells and its surrounding bone marrow microenvironment. It affects MM cell growth by decreasing c-Myc expression. In addition, TasQ targets the immunosuppressive monocytic myeloid cell population and increases serum interferon-gamma levels, indicative for immune cell activation. Moreover, it stimulates osteogenesis in vivo. Taken together, all these data provide evidence for the therapeutic benefits of TasQ as an anti-MM therapy for patients. Disclosures Törngren: Active Biotech: Current Employment. Eriksson: Active Biotech: Current Employment. De Veirman: Active Biotech AB: Research Funding.