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Altered metabolism is a hallmark of both cell division and cancer. Chronic lymphocytic leukemia (CLL) cells circulate between peripheral blood (PB) and lymph nodes (LNs), where they receive proliferative and prosurvival signals from surrounding cells. However, insight into the metabolism of LN CLL and how this may relate to therapeutic response is lacking. To obtain insight into CLL LN metabolism, we applied a 2-tiered strategy. First, we sampled PB from 8 patients at baseline and after 3-month ibrutinib (IBR) treatment, which forces egress of CLL cells from LNs. Second, we applied in vitro B-cell receptor (BCR) or CD40 stimulation to mimic the LN microenvironment and performed metabolomic and transcriptomic analyses. The combined analyses indicated prominent changes in purine, glucose, and glutamate metabolism occurring in the LNs. CD40 signaling mostly regulated amino acid metabolism, tricarboxylic acid cycle (TCA), and energy production. BCR signaling preferably engaged glucose and glycerol metabolism and several biosynthesis routes. Pathway analyses demonstrated opposite effects of in vitro stimulation vs IBR treatment. In agreement, the metabolic regulator MYC and its target genes were induced after BCR/CD40 stimulation and suppressed by IBR. Next, 13C fluxomics performed on CD40/BCR-stimulated cells confirmed a strong contribution of glutamine as fuel for the TCA cycle, whereas glucose was mainly converted into lactate and ribose-5-phosphate. Finally, inhibition of glutamine import with V9302 attenuated CD40/BCR-induced resistance to venetoclax. Together, these data provide insight into crucial metabolic changes driven by the CLL LN microenvironment. The prominent use of amino acids as fuel for the TCA cycle suggests new therapeutic vulnerabilities.

Given the plasticity of hematopoietic stem and progenitor cells, multiple routes of differentiation must be blocked in the the pathogenesis of acute myeloid leukemia, the molecular basis of which is incompletely understood. We report that posttranscriptional repression of the transcription factor ARID3A by miR-125b is a key event in the pathogenesis of acute megakaryoblastic leukemia (AMKL). AMKL is frequently associated with trisomy 21 and GATA1 mutations (GATA1s), and children with Down syndrome are at a high risk of developing the disease. The results of our study showed that chromosome 21–encoded miR-125b synergizes with Gata1s to drive leukemogenesis in this context. Leveraging forward and reverse genetics, we uncovered Arid3a as the main miR-125b target behind this synergy. We demonstrated that, during normal hematopoiesis, this transcription factor promotes megakaryocytic differentiation in concert with GATA1 and mediates TGFβ-induced apoptosis and cell cycle arrest in complex with SMAD2/3. Although Gata1s mutations perturb erythroid differentiation and induce hyperproliferation of megakaryocytic progenitors, intact ARID3A expression assures their megakaryocytic differentiation and growth restriction. Upon knockdown, these tumor suppressive functions are revoked, causing a blockade of dual megakaryocytic/erythroid differentiation and subsequently of AMKL. Inversely, restoring ARID3A expression relieves the arrest of megakaryocytic differentiation in AMKL patient-derived xenografts. This work illustrates how mutations in lineage-determining transcription factors and perturbation of posttranscriptional gene regulation can interact to block multiple routes of hematopoietic differentiation and cause leukemia. In AMKL, surmounting this differentiation blockade through restoration of the tumor suppressor ARID3A represents a promising strategy for treating this lethal pediatric disease.

Glycosylation of the surface immunoglobulin (Ig) variable region is a remarkable follicular lymphoma–associated feature rarely seen in normal B cells. Here, we define a subset of diffuse large B-cell lymphomas (DLBCLs) that acquire N-glycosylation sites selectively in the Ig complementarity-determining regions (CDRs) of the antigen-binding sites. Mass spectrometry and X-ray crystallography demonstrate how the inserted glycans are stalled at oligomannose-type structures because they are buried in the CDR loops. Acquisition of sites occurs in ∼50% of germinal-center B-cell–like DLBCL (GCB-DLBCL), mainly of the genetic EZB subtype, irrespective of IGHV-D-J use. This markedly contrasts with the activated B-cell–like DLBCL Ig, which rarely has sites in the CDR and does not seem to acquire oligomannose-type structures. Acquisition of CDR-located acceptor sites associates with mutations of epigenetic regulators and BCL2 translocations, indicating an origin shared with follicular lymphoma. Within the EZB subtype, these sites are associated with more rapid disease progression and with significant gene set enrichment of the B-cell receptor, PI3K/AKT/MTORC1 pathway, glucose metabolism, and MYC signaling pathways, particularly in the fraction devoid of MYC translocations. The oligomannose-type glycans on the lymphoma cells interact with the candidate lectin dendritic cell–specific intercellular adhesion molecule 3 grabbing non-integrin (DC-SIGN), mediating low-level signals, and lectin-expressing cells form clusters with lymphoma cells. Both clustering and signaling are inhibited by antibodies specifically targeting the DC-SIGN carbohydrate recognition domain. Oligomannosylation of the tumor Ig is a posttranslational modification that readily identifies a distinct GCB-DLBCL category with more aggressive clinical behavior, and it could be a potential precise therapeutic target via antibody-mediated inhibition of the tumor Ig interaction with DC-SIGN–expressing M2-polarized macrophages.

Primary effusion lymphoma (PEL) is an aggressive malignancy with poor prognosis even under chemotherapy. Kaposi sarcoma–associated herpesvirus (KSHV), one of the human oncogenic viruses, is the principal causative agent. Currently, there is no specific treatment for PEL; therefore, developing new therapies is of great importance. Sphingolipid metabolism plays an important role in determining the fate of tumor cells. Our previous studies have demonstrated that there is a correlation between sphingolipid metabolism and KSHV+ tumor cell survival. To further develop sphingolipid metabolism-targeted therapy, after screening a series of newly synthesized ceramide analogs, here, we have identified compounds with effective anti-PEL activity. These compounds induce significant PEL apoptosis, cell-cycle arrest, and intracellular ceramide production through regulation of ceramide synthesizing or ceramide metabolizing enzymes and dramatically suppress tumor progression without visible toxicity in vivo. These new compounds also increase viral lytic gene expression in PEL cells. Our comparative transcriptomic analysis revealed their mechanisms of action for inducing PEL cell death and identified a subset of novel cellular genes, including AURKA and CDCA3, controlled by sphingolipid metabolism, and required for PEL survival with functional validation. These data provide the framework for the development of promising sphingolipid-based therapies against this virus-associated malignancy.

Epigenetic changes during B cell differentiation generates distinct DNA methylation signatures specific for B cell subsets, including memory B cells (MBCs) and plasma cells (PCs). Waldenström's macroglobulinemia (WM) is a complex B cell malignancy uniquely comprised of a mixture of lymphocytic and plasmacytic phenotypes. Here we integrated genome-wide DNA methylation, transcriptome, mutation and other phenotypic features of tumor cells from 35 MYD88-mutated WM patients in relation to normal plasma and B cell subsets. We discovered that WM patients naturally segregate into two groups according to DNA methylation patterns, related to normal MBC and PC profiles, and reminiscent of other memory and plasma cell-derived malignancies. Concurrent analysis of DNA methylation changes in normal and WM development were used to capture tumor-specific events, highlighting a selective reprogramming of enhancer regions in MBC-like WM and repressed and heterochromatic regions in PC-like WM. MBC-like WM hypomethylation was enriched in motifs belonging to PU.1, TCF3 and OCT2 transcription factors and involved elevated MYD88/TLR pathway activity. PC-like WM displayed marked global hypomethylation and selective overexpression of histone genes. Finally, WM subtypes exhibited differential genetic, phenotypic and clinical features. MBC-like WM harbored significantly more clonal CXCR4 mutations (P=0.015), deletion 13q (P=0.006), splenomegaly (P=0.02) and thrombocytopenia (P=0.004), while PC-like WM harbored more deletion 6q (P=0.012), gain 6p (P=0.033), had increased frequencies of IGHV3 genes (P=0.002), CD38 surface expression (P=4.1e-5), and plasmacytic differentiation features (P=0.008). Together our findings illustrate a novel approach to subclassify WM patients using patterns of DNA methylation and reveal divergent molecular signatures among WM patients.

Epigenetic changes during B-cell differentiation generate distinct DNA methylation signatures specific for B-cell subsets, including memory B cells (MBCs) and plasma cells (PCs). Waldenström macroglobulinemia (WM) is a B-cell malignancy uniquely comprising a mixture of lymphocytic and plasmacytic phenotypes. Here, we integrated genome-wide DNA methylation, transcriptome, mutation, and phenotypic features of tumor cells from 35 MYD88-mutated WM patients in relation to normal plasma and B-cell subsets. Patients naturally segregate into 2 groups according to DNA methylation patterns, related to normal MBC and PC profiles, and reminiscent of other memory and PC-derived malignancies. Concurrent analysis of DNA methylation changes in normal and WM development captured tumor-specific events, highlighting a selective reprogramming of enhancer regions in MBC-like WM and repressed and heterochromatic regions in PC-like WM. MBC-like WM hypomethylation was enriched in motifs belonging to PU.1, TCF3, and OCT2 transcription factors and involved elevated MYD88/TLR pathway activity. PC-like WM displayed marked global hypomethylation and selective overexpression of histone genes. Finally, WM subtypes exhibited differential genetic, phenotypic, and clinical features. MBC-like WM harbored significantly more clonal CXCR4 mutations (P = .015), deletion 13q (P = .006), splenomegaly (P = .02), and thrombocytopenia (P = .004), whereas PC-like WM harbored more deletion 6q (P = .012), gain 6p (P = .033), had increased frequencies of IGHV3 genes (P = .002), CD38 expression (P = 4.1e-5), and plasmacytic differentiation features (P = .008). Together, our findings illustrate a novel approach to subclassify WM patients using DNA methylation and reveal divergent molecular signatures among WM patients.

Erdheim-Chester disease (ECD) is a clonal hematopoietic disorder characterized by the accumulation of foamy histiocytes within organs (in particular, frequent retroperitoneal involvement) and a high frequency of BRAFV600E mutations. Although ECD is not commonly recognized to have overt peripheral blood (PB) or bone marrow (BM) disease, we recently identified that ECD patients have a high frequency of a concomitant myeloid malignancy. We thus conducted a systematic clinical and molecular analysis of the BM from 120 ECD patients. Surprisingly, 42.5% of ECD patients (51 of 120) had clonal hematopoiesis whereas 15.8% of patients (19 of 120) developed an overt hematologic malignancy (nearly all of which were a myeloid neoplasm). The most frequently mutated genes in BM were TET2, ASXL1, DNMT3A, and NRAS. ECD patients with clonal hematopoiesis were more likely to be older (P < .0001), have retroperitoneal involvement (P = .02), and harbor a BRAFV600E mutation (P = .049) than those without clonal hematopoiesis. The presence of the TET2 mutation was associated with a BRAFV600E mutation in tissue ECD lesions (P = .0006) and TET2-mutant ECD patients were more likely to have vascular involvement than TET2 wild-type ECD patients. Clonal hematopoiesis mutations in ECD were detected in cells derived from CD34+CD38− BM progenitors and PB monocytes but less frequently present in PB B and T lymphocytes. These data identify a heretofore unrecognized high frequency of clonal hematopoiesis in ECD patients, reaffirm the development of additional high risk of myeloid neoplasms in ECD, and provide evidence of a BM-based precursor cell of origin for many patients with ECD.

Background: Children with sickle cell disease (SCD) are at increased risk of cerebrovascular events such as stroke, silent infarcts and neurocognitive impairment. The role of Transcranial Doppler ultrasound scanning (TCD) to identify sickle cell anemia (SCA) children at high risk of stroke is well established. Adam et al in 1998 recommended those with abnormal cerebrovascular flow velocities are offered prophylactic blood transfusion therapy to prevent stroke between ages 2 to 16 years. Therefore, TCD screening for stroke prevention in now is a mandatory in all guidelines for the management of children with SCA. However, there is still no uniform implementation of the program globally and in European countries (Rees 2016). Moreover, the information available on the quality of the TCD screening is limited to educational experiences in a few countries (Inusa 2019) but to evaluation of stroke prevention programs has been performed in Europe. As more disease modifying therapies become available for children with SCD, it is mandatory to know TCD availability, screening practices, and real-world data on stroke prevention in Europe. EuroBloodNet is the European Reference Network (ERN) on rare Hematological Disorders, one of the 24 ERN established by the European Union to improve care of patients with rare disorders in Europe. EuroBloodNet's main goal is to improve the healthcare and overall quality of life of patients with a Rare Hematological Disease by: 1) Improving equal access to highly specialized healthcare delivery through a multidisciplinary patient centered approach; 2) Enhancing the best practices in prevention, diagnosis and safe clinical care across Europe based on promotion of evidence based guidelines. We wanted to assess the state of the art of TCD screening and stroke prevention programs in European Expert Centers. Methods: An online survey was developed by SCD experts in 5 European countries and sent to all Representatives of the Health Care Providers (HCP) and the Red Cell Disorder representatives in each HCP within the EuroBloodNet network, as well as to National Representatives of Scientific Societies within European Countries. Items in the survey are listed in Table 1. Results 81 hematologists or pediatricians from 77 centers in 16 European countries responded to the survey (14/16 in Western Europe); 39/77 (51%) were EuroBloodNet Expert centers, 14/77 (18%) were under evaluation as being recognized; 67/77 specified their expertise: 24% were pediatric, 3% adult, 58% both; 12 centers had >200 patients in the age range 1-16 years. 36% Physicians reported not having a dedicated TCD/TCDi service for children with SCD so exams had to be performed by cardiologists (10%), general radiologists (28%), TCD is not performed (31%), or patients have to be sent in another center (31%). 74% reported requesting annual TCD for their patients, but to the question "What percentage of your patients receives annual TCD" only 28% confirmed that all their patients managed to actually receive annual TCD, due to lack of trained staff (43%), lack of TCD instruments (11%), refusal of patients due to logistical difficulties (22%) (i.e TCD in another city), lack of funds for dedicated staff or equipment (11%), or other reasons. Only 74% of hematologists were aware of the protocol in use at their center by the staff performing TCD; the STOP criteria were applied by 64% of the physicians, mainly due non evaluation of the Internal Carotid Artery. The extracranial part of the carotid artery was evaluated only in 30% of the respondents. In case of abnormal/conditional TCD results, the approach varies and is not uniform across centers. Conclusions Our data show that less than 30% of children with SCD followed in European Centers receive annual TCD according to recognized guidelines. This first multinational European survey allowed the identification of issues related to the lack of access to TCD, lack of trained staff, lack of adequate protocols for implementation of TCD and treatment afterwards, which will need to be addressed through dedicated actions. Figure 1 Figure 1. Disclosures Casale: Novartis Farma SpA: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mañú Pereira: Novartis: Research Funding; Agios Pharmaceuticals: Research Funding. de Montalembert: Vertex: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Colombatti: Global Blood Therapeutics: Research Funding; Addmedica: Consultancy; Forma Therapeutics: Consultancy; Novartis: Consultancy; NovoNordisk: Consultancy; BlueBirdBio: Consultancy; Global Blood Therapeutics: Consultancy; BlueBirdBio: Research Funding.

Myelodysplastic syndrome (MDS) is a hematological clonal stem cell disease. Recurrent splicing factors mutations are reported in 50% of MDS. Interestingly, mutations in the splicing factor gene SF3B1 are over-represented in MDS with ring sideroblasts (MDS-RS), co-occurring in up to 90% of patients. In MDS-RS, anemia is the major clinical manifestation. Erythropoiesis stimulating agents (ESAs) are used to treat anemia; however, the overall response rates are 20% to 40% with a duration of response of 18-24 months. New therapeutic options are needed to improve response to ESAs treatment and delay red blood cell transfusion, which are associated with acute myeloid leukemia progression and increase in morbidity. Mutations in SF3B1 modify the recognition pattern of the 3' splice site and lead to subsequent mis-splicing of its targets. To identify critical mis-splicing events involved in the erythroid differentiation blockage, we performed splicing analysis on RNA sequencing generated from hematopoietic stem/progenitor cells undergoing differentiation. Three MDS primary samples harboring SF3B1 mutations and three age-matched healthy donors cultured under normoxia and hypoxia conditions were initially used for the analysis. High depth RNA sequencing and differential splicing analyses using rMATS identified 2,845 mis-spliced events including 200 shared between hypoxia and normoxia conditions. Here, using a cohort of 42 MDS samples, we report the mis-splicing of the coenzyme A synthase (COASY) transcript. Heme synthesis relies on succinyl-CoA synthesis, and its production itself depends on the availability of cellular CoA. We thus hypothesised that COASY mis-splicing is a key driver of ineffective erythropoiesis in MDS-RS patients. In primary hematopoietic cells, COASY is upregulated during erythroid differentiation and its silencing in CD34 + cells severely impedes the generation of mature erythroid cells CD71 - CD235a + and causes disruption in heme production. Functional characterisations of the CRISPR-CAS9 edited K562 SF3B1K700E and the SF3B1-mutated HNT-34 cell lines confirmed that COASY mis-splicing impairs COASY protein synthesis that ultimately results in 60% loss of the protein. Metabolomic analysis showed that COASY mis-splicing depletes cells in CoA and succinyl-CoA metabolites, however this phenotype can be rescued by supplementation with vitamin B5, a CoA precursor. Consequently, we showed in vitro that saturating the 40% of remaining COASY enzyme with vitamin B5 or supplementing medium with its downstream by-product, succinyl-CoA, improved erythropoietic differentiation in MDS SF3B1mut patients. In summary, our results for the first time show that SF3B1 mutations induce coenzyme A synthase (COASY) transcript mis-splicing, that consequently leads to measurable defects in metabolites essential for heme biosynthesis. Our report reveals a novel critical role of COASY in regulating normal bone marrow erythropoiesis through control of succinyl-coA during human erythroid differentiation. Remarkably, partial loss of the coenzyme A synthase in MDS-RS patients leads to disruption in the erythroid lineage as well as heme deficiency, that can be rescued by exogenous treatment with vitamin B5 or succinyl-CoA. Therefore, vitamin B5 could represent a very attractive agent to combine with existing treatments in order to increase erythroid maturation and delay red blood cell transfusion dependency in MDS-RS patients. Graphical representation: SF3B1 mutant causes mis-splicing in COASY that results in loss of protein. Deficiency in COASY triggers a downregulation of succinyl-CoA that is involved in the rate limiting step of heme synthesis. Heme deficiency subsequently impairs erythroid differentiation. Treatment of MDS SF3B1 mutant cells with vitamin B5 (precursor of CoA), or succinyl-CoA, rescues erythroid differentiation. Figure 1 Figure 1. Disclosures Platzbecker: Geron: Honoraria; Takeda: Honoraria; Janssen: Honoraria; Celgene/BMS: Honoraria; Novartis: Honoraria; AbbVie: Honoraria. Wiseman: Bristol Myers Squibb: Consultancy; Novartis: Consultancy; StemLine: Consultancy; Takeda: Consultancy; Astex: Research Funding. Gribben: Abbvie: Honoraria; AZ: Honoraria, Research Funding; BMS: Honoraria; Gilead/Kite: Honoraria; Janssen: Honoraria, Research Funding; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutis: Honoraria.

Introduction: asciminib is a first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor that potently inhibits aberrant kinase activity of the BCR-ABL1 oncoprotein via allosteric binding. asciminib has shown high efficacy profile in heavily pretreated Chronic Myeloid Leukemia (CML) patients with an adequate safety profile in phase I and III clinical trials. However, data from the use of asciminib in real life setting are still scarce. Methods: We gathered real-life retrospective data from 49 patients with BCR-ABL1 positive CML treated with asciminib (mean dose: 40 mg twice daily) between October 2018 and July 2021 at 33 institutions. The indication of asciminib was made according to the criterion of the attending physician and the drug was granted by Novartis under a controlled access program. Molecular biology tests were performed according to ELN guidelines and BCR-ABL/ABL ratios were expressed as % IS in all centers. Treatment responses were calculated with the patients at risk at each specific time points. For the event free survival (EFS), the events were treatment discontinuation due to any reason, progression or death. Data collection followed the local regulations for observational studies. Results: Median time on asciminib was 11,69 months for the entire cohort. Patients' characteristics are displayed on Table 1. Most patients were heavily pretreated with at least 3 prior TKI lines in 45 patients (91,83%), 18 of them receiving prior Ponatinib. Switch to asciminib occurred due to intolerance in 32 patients and due to resistance in the remaining 17. Fifteen patients (30,61%) harbored mutations in BCR-ABL1 (3 with a T315 mutation). Regarding efficacy (Table 2), probability of reaching or maintaining previous responses were 94%, 45% and 21% for complete hematological response (CHR), complete cytogenetic response (CCyR) and major molecular response (MMR), respectively. Considering probabilities of improving previous response, rates were 40%, 42% and 33% for the same parameters. Probabilities to obtain CCyR and MMR in resistant and intolerant patients were 29% (4/14) vs 55% (6/11) and 27% (4/15) vs 52% (11/21), respectively. Amid the patients previously treated with Ponatinib, probabilities of reaching or maintaining previous response were 53% (9/17) and 35% (6/17) for CCyR and MMR respectively, and 30% (3/10), 23% (3/13) displayed improvement of response. Regarding responses in patients with mutations, 39% (5/13) achieved or maintained CCyR and 31% (4/13) MMR; whereas 20% (2/10) and 18% (2/11) improved such responses. Of the three patients with T315I mutation, one discontinued due to progression to advanced stages, and the rest maintained the previous response. With a median follow-up of 11,69 months, the estimated EFS was 80% (figure 1). In terms of safety (Table 3), the most frequent extra-hematological adverse events (AE) were: fatigue (16,2%), joint pain (13,5%) and nausea (8,1%), most of them grade 1-2. Grade 3-4 AE were observed in 10% of patient (fatigue (2), cholestasis enzyme elevation (1), hypertension (1), pancreatitis (1) and pericardial effusion (1)). Thrombocytopenia was shown as the most frequent AE (16,3%), with 6% of patients suffering from grade 3-4. Dose reduction was required in 15 patients (30,6%). After a median follow up of 51 weeks, 73,5% of the patients remained on treatment. Only fourteen patients discontinued treatment due to progression or loss of efficacy, whereas 6% of patients discontinuing treatment due to intolerance. Conclusions: The results presented are in line with the data obtained in clinical trials, positioning asciminib as a potential safe and efficacious treatment for CML patients with failure to several TKI lines. Figure 1 Figure 1. Disclosures Sanchez-Guijo: Novartis: Consultancy, Honoraria, Research Funding; Celgene/Bristol-Myers-Squibb,: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Takeda: Honoraria, Research Funding; Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Garcia Gutierrez: BMS: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding.

Introduction: For chronic lymphocytic leukemia (CLL), especially in the lymph node (LN) setting where cells receive proliferative and pro-survival signals, in-depth studies of altered metabolism and its relationship with therapeutic responses are still lacking. Venetoclax, a BCL-2 inhibitor currently in wide clinical use for CLL, has shown high efficiency yet emerging resistance is a growing clinical problem. In cell line models, induced resistance to Venetoclax was accompanied by profound metabolic changes 1. This is in accordance with our earlier findings on metabolic and apoptotic changes that CLL cells undergo within the LN environment 2. In the current study, we performed RNA sequencing and applied fluxomics with 13C 6-glucose and 13C 5-glutamine to investigate in detail the metabolic routes in LN CLL. This led to studies to manipulate glutamine metabolism in a venetoclax resistance model. Methods: Peripheral blood (PB) samples from CLL patients were in vitro stimulated for 24 hrs by CD40 or B cell receptor (BCR), which are two potential key signals in LN. RNAseq analysis was compared with microarray data of paired PB/LN patient samples 3. For fluxomics, CLL cells were cultured for 2 hrs in medium containing either 5 mM 13C 6-glucose or 1 mM 13C 5-glutamine. Incorporation of 13C in metabolic intermediates was analyzed by LC-MS. For glutamine blockade, CLL cells were stimulated in presence of specific inhibitors of glutamine/glutamate metabolism or amino acid transporters. Cells were then treated with venetoclax, and viability was measured. Results: Gene expression profiles demonstrated that CLL cells obtained from LN tissue as well as after in vitro CD40 or BCR stimulation showed increased expression of gene sets involved in glycolysis, oxidative phosphorylation / citric acid cycle (OXPHOS/TCA) and amino acid metabolism as well as Myc activation. This confirmed that in vitro stimulation can be used to model the CLL LN setting. For unstimulated PB CLL cells, fluxomics data demonstrated low uptake of either glucose or glutamine, with 13C labelling close to zero for most metabolites. In contrast, both CD40 or BCR stimulation increased the uptake and utilization of glucose and glutamine. 13C labelling from glucose was detected in all glycolytic intermediates analyzed in both CD40- and BCR-stimulated CLL cells. Glucose was catalyzed to lactate and also partly converted to acetyl-CoA, which entered the TCA cycle. Additionally, labelling from glucose was also increased in several metabolites of the pentose phosphate pathway (PPP) suggesting it entered nucleotide synthetic routes. Compared to glucose, the contribution of glutamine was much higher in the TCA cycle in both BCR and CD40-stimulated cells. All intermediates of the TCA cycle were highly enriched with 13C from glutamine (Figure 1A). Combined, these data revealed that glutamine is the key metabolite to fuel the TCA cycle in LN CLL cells, and prompted us to study effects of glutamine blockade in conditions of Venetoclax resistance. It was found that venetoclax resistance induced by CD40 or BCR stimulation was clearly attenuated by glutamine uptake inhibition. CLL cells became re-sensitized to Venetoclax in both CD40- or BCR-stimulated samples, with an approximate 100-fold shift in IC50 (Figure 1B). Conclusions: Our study highlights the role of glutamine, in addition to glucose, in the metabolic reprogramming that CLL cells undergo in the LN (Figure 1C). These processes show potential for therapeutic targeting. Inhibition of glutamine import could contribute to dampen tumor microenvironment-induced Venetoclax resistance. References 1. Guièze, R. et al. Mitochondrial Reprogramming Underlies Resistance to BCL-2 Inhibition in Lymphoid Malignancies. Cancer Cell 36, (2019). 2. Chen, Z. et al. Effects of Ibrutinib on Metabolic Alterations and Micro-Environmental Signalling in Chronic Lymphocytic Leukaemia. Blood 136, (2020). 3. Herishanu, Y. et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-κB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 117, 563-574 (2011). Figure 1 Figure 1. Disclosures Forconi: AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Roche: Honoraria; Novartis: Honoraria; Gilead: Research Funding. van der Windt: Genmab: Current Employment. Kater: Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding; Genmab, LAVA: Other: Ad Board, Steering Committee.

Despite recent advances, there is still a lack of treatment options for patients with high-risk multiple myeloma (MM), warranting the need for novel therapeutic targets. Monopolar spindle 1 (Mps1), also known as TTK protein kinase, is localized at the kinetochores and centromeres essential for the mitotic spindle checkpoint and centrosome duplication. In cancer cells, high levels of Mps1 help to support aneuploidy, a common malignant trait. In fact, high MPS1 expression correlates to unfavorable prognosis in colon and breast cancers. Here, we report the importance of Mps1 in MM and perform functional characterization of a novel Mps1 inhibitor, OSU-13. To assess the relevance of Mps1 in MM prognosis, we analyzed the CoMMpass database and examined the association of MPS1 expression with clinical outcome and genetic alterations in MM patients (n=769). Elevated MPS1 expression correlates with decreased overall survival (p-value = 0.0001) and decreased event-free survival (p-value < 0.0001). In addition, MPS1 expression is higher in high-risk MM with specific genetic alterations, such as deletion of 17p (p-value = 0.0003), Myc translocation t(8;14) (p-value = 0.02), and gain of 1q21 (p-value = 0.0001). We therefore compared MPS1 expression in eight different MM cell lines and primary CD138 - and CD138 + cells isolated from bone marrow (BM) of four recently diagnosed, untreated MM patients. Gene expression levels assessed by quantitative qPCR were normalized to 18S mRNA internal control, and relative quantification was performed using the ΔΔCt method. MPS1 expression was higher in all MM cell lines compared to the average expression level in primary CD138 + BM plasma cells, with a minimum of 3.2-fold increase in L363 and a maximum of 16.2-fold increase in MM1S. In addition, MPS1 expression was 2-fold higher in CD138 + MM cells compared to CD138 - counterparts from the BM of the same patient, suggesting its selectivity as a target. Next, we characterized the effects of OSU-13, a novel Mps1 inhibitor. In an Mps1 target engagement assay (NanoBRET™) in HEK293 cells, we measured the relative levels of OSU-13-mediated inhibition of Mps1-NanoLuc binding to a fluorescent tracer in comparison to MM clinical candidates. OSU-13 showed EC 50=10 nM, almost 10-fold lower than other agents tested. Endpoint measurement by MTT (3-[4,5- dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate cytotoxicity of OSU-13. MM cell lines were treated with increasing doses of OSU-13 for 72h and cell viability was measured. OPM-2 was the most sensitive cell line (IC 50 = 610 nM), followed by H929 (IC 50 = 1440 nM), RPMI-8226 (IC 50 = 4196 nM), and U266 (IC 50 = 5071 nM). To investigate the kinetics and the mechanisms used by OSU-13 to cause cell death, we treated H929 and OPM-2 cell lines with 500 nM of OSU-13 for 24h, 48h, 72h, 96h, and 120h and assessed both Annexin V-FITC and Zombie-Aqua staining by flow cytometry. Treatment with OSU-13 caused an increase in Annexin V staining as early as 24h in H929 (2.1-fold) and OPM-2 (1.6-fold). Maximum increase of apoptotic cells (Annexin V single positive) was observed after 72h, with a 5-fold increase in OPM-2 and 8.6-fold increase in H929 cell line. Cell death peaked at 120h, with Zombie staining 74% of OPM-2 and 60% of H929 cells. The activity of caspases 3/7 was further assessed in H929 myeloma cells at five time points after treatment with different concentrations of OSU-13. Caspases 3/7 activation started after 48h with 500nM OSU-13, and peaked after 72h post-treatment with the same dose, in agreement with the Annexin staining kinetics results. Finally, we evaluated the in vivo effect of OSU-13 in tumor growth in a H929 MM subcutaneous xenograft model in CB.17 SCID mice. Mice were treated with 5 mg/kg or 10 mg/kg daily dose of OSU-13 per oral gavage for 21 days. Treatment with 10 mg/kg daily dose of OSU-13 produced a 22% tumor growth delay (p Collectively, we show here that elevated MPS1/TTK expression correlates with poorer prognosis in MM, and OSU-13, a novel Mps1/TTK inhibitor, induces apoptosis and cell death in MM cell lines, and decreases tumor growth in mice containing H929 cell xenografts. Together, our findings reveal that targeting the mitotic checkpoint using OSU-13 is a potential novel strategy, particularly for high risk MM. Disclosures No relevant conflicts of interest to declare.