Shi, Jie

Cancer cells have altered energy demand due to their increased proliferative capacity. In Waldenstrom Macroglobulinemia (WM), an indolent yet incurable B-cell lymphoma, the homeostasis of the bone marrow (BM) environment is disturbed due to the infiltration of the lymphoplamacytic cells that continuously produce monoclonal IgM. An alteration in energy demand could skew the balance of key proteins and metabolites towards a permissive niche for WM tumor cells growth, and unfavorably effect on the immune response against tumor cells. Therefore, the aim of our study was to identify how changes in certain metabolites and/or proteins could contribute to the pathobiology of WM and whether the cytokine and chemokine composition of the BM microenvironment play a role in such changes. WM patient's samples including BM plasma, peripheral blood serum and BM cells (n=101) as well as equivalent normal counterparts (n=86) were collected and used for metabolomics analysis. Comprehensive targeted metabolomics analysis was performed using Capillary Electrophoresis Time-of-Flight Mass spectrometry (CE-TOFMS), CE-triple quadrupole mass spectrometry (CE-qQqMS) and Liquid Chromatography (LC-TOFMS). Normal and WM peripheral blood serums samples were also used for untargeted proteomics analysis using a fully automated proteomic technology platform that includes an Agilent 1200 Series Quaternary HPLC system connected to a Q Exactive Plus mass spectrometer. Real-time PCR analysis was performed to detect the gene expression of the relevant metabolite transporters located on the cell membrane. BM cells from control and WM patients' samples were used for flow cytometry analysis. IHC was used to detect the proteins on the BM tissues. Principal Component Analysis (PCA) and Hierarchal Clustering Analysis (HCA) on both metabolomics and proteomics data identified two distinct clusters for disease and normal samples, indicating that there are differentially expressed proteins and metabolites in WM versus normal samples. Furthermore, pathway analysis showed that the majority of the altered metabolites were the members of the glutathione (GSH) metabolism pathway. This finding was further validated not only by data obtained from metabolomics analysis of BM cells and BM plasma, but also by proteomic data WM patients serum, implying that GSH metabolism is key to the biology of WM. Moreover, stimulation of WM cell lines by IL-6 and IL-21, cytokines involved in inducing WM cell proliferation and IgM secretion, resulted in increased gene expression of the transporters mediating uptake of metabolites required for GSH synthesis, including SLC7A11, 4F2HC and LAT1, indicating that cytokines in the WM BM could modulate GSH metabolism. In addition, IHC staining of the BM tissues as well as flow cytometry analysis of patients' lymphoplasmacytic cells identified glutathione peroxidase as one of the major proteins modulating GSH metabolism in WM. In summary, our data highlight a central role for GSH metabolism in WM disease biology and indicate that intervening with the metabolic processes could be a potential therapeutic strategy for patients with WM. Disclosures Ansell: AI Therapeutics: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Bristol Myers Squibb: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials.

Myeloid derived suppressor cells (MDSC) are a heterogeneous population of undifferentiated myeloid cells that are expanded and activated in pathological conditions and have the ability to potently suppress T-cell function and thereby contribute to immunosuppression and tumor progression. While there have been studies showing a role for MDSC in a variety of hematological malignancies, no data is available indicating that MDSCs contribute to the tumor progression in Waldenstrom's Macroglobulinemia (WM), an indolent lymphoma characterized by bone marrow (BM) infiltration of lymphoplasmacytic (LPL) cells and increased secretion of monoclonal IgM. In previous work, we have found increased GM-CSF and reduced arginine and cysteine in the BM microenvironment in WM. We hypothesized that this was due to the presence and activity of MDSCs in WM. BM aspirates from WM patients (n=17) were therefore processed to isolate LPL (CD19+/CD138+) cells from the rest of the BM cells (CD19-/CD138-). Sorted (CD19-/CD138-) cells from BM of patients with WM were studied with flow cytometry. Using a sequential gating strategy (lack of lineage markers, low levels of HLADR, CD33+, CD11b+) we identified a population of MDSCs that were then subdivided using CD14 and CD15 expression into total-, monocytic-, or granulocytic- MDSCs (m-MDSC, g-MDSC). We also analyzed unsorted BM cells using cytometry by time-of-flight (CyTOF) in order to further identify and phenotypically characterize the BM MDSC population in a group of WM patients with smoldering (asymptomatic) disease, symptomatic disease, or in remission post-treatment. BM samples from normal subjects were used as a control. Flow cytometry data showed significant higher numbers of MDSC subsets expressing PD-L1 and Arginase1 in WM patients when compared to the normal samples. BM cells from WM patients (n=18) then were compared to controls (n=11), and the absolute number of the total MDSC (p=0.05), m-MDSC (p=0.002), g-MDSC (p=0.02) was increased in WM specimens. When MDSCs from WM or normal monocytes from healthy controls were co-cultured with activated T-cells, the proliferation of activated T-cells in the presence of MDSCs from WM patients was impaired compared to controls, confirming the suppressive role of MDSCs. We then performed high dimensional analysis of the total BM MDSC cells using t-SNEand identified phenotypically distinct MDSC cell populations in the BM that were differentially present when healthy controls were compared to patients with smoldering WM or those with WM needing treatment. Specifically, WM patients needing treatment had increased numbers of a distinct MDSC population that was highly positive for CD163, and CD138. Moreover, conventional markers denoting m-MDSC and g-MDSC, such as CD14 and CD15, were highly expressed in all populations and their pattern of expression did not specifically define the MDSC subtypes, indicating that high dimensional phenotyping further details the MDSC sub-compartments beyond the conventional categorization of MDSC using conventional cytometry. In summary, we find that MDSCs are increased in the BM of WM patients compared to controls. MDSCs expressing CD163 and CD138 increase when WM patients become symptomatic and require therapy. Furthermore, MDSCs in the BM of WM patients suppress T-cell function and likely contribute to progression of the disease. MDSCs in the BM therefore present a therapeutic target that should be explored in WM patients. Disclosures Ansell: Bristol Myers Squibb: Other: research funding for clinical trials; Merck: Other: research funding for clinical trials; AI Therapeutics: Other: research funding for clinical trials; Affimed: Other: research funding for clinical trials; Takeda: Other: research funding for clinical trials; Pfizer: Other: research funding for clinical trials; Regeneron: Other: research funding for clinical trials; Seattle Genetics: Other: research funding for clinical trials.