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SUMMARYMesenchymal stem/stromal cells (MSCs) within the bone marrow microenvironment (BMME) support normal hematopoietic stem and progenitor cells (HSPCs). However, the heterogeneity of human MSCs has limited the understanding of their contribution to clonal dynamics and evolution to myelodysplastic syndromes (MDS). We combined three MSC cell surface markers, CD271, VCAM-1 (Vascular Cell Adhesion Molecule-1) and CD146, to isolate distinct subsets of human MSCs from bone marrow aspirates of healthy controls (Control BM). Based on transcriptional and functional analysis, CD271+CD106+CD146+(NGFR+/VCAM1+/MCAM+/Lin-;NVML) cells display stem cell characteristics, are compatible with murine BM- derived Leptin receptor positive MSCs and provide superior support for normal HSPCs. MSC subsets from 17 patients with MDS demonstrated shared transcriptional changes in spite of mutational heterogeneity in the MDS clones, with loss of preferential support of normal HSPCs by MDS-derived NVML cells. Our data provide a new approach to dissect microenvironment-dependent mechanisms regulating clonal dynamics and progression to MDS.

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by bone marrow (BM) failure and associated with aging. We have previously shown that reversal of BM microenvironment (BMME) dysfunction in MDS mitigates MDS associated marrow failure and delays progression to acute leukemia. However, the exact mechanisms driving BMME dysfunction in MDS remain unknown. We recently reported that interleukin-1 (IL1) Receptor Type1 (IL1R1) signaling is a driver in myeloid bias via disruption of BMME in aging. In addition, we have found that IL1R1 signaling is involved in disease progression of AML. Therefore, to assess the role of IL1R1 signaling in MDS associated BMME dysfunction and marrow failure, we employed an age appropriate murine transplant model for MDS utilizing NUP98-HOXD13 (NHD13) transgenic mice. Methods: BM cells (NHD13 transgenic or wild type (WT), 7 weeks) and competitor cells were transplanted into irradiated aged recipients (WT or IL1R1 KO, 60 weeks), and subsequently monitored for development of marrow failure. When marrow failure developed, mice were euthanized and peripheral blood, BM, BM extracellular fluid (BMEF), and collagenase-1 digested bone associated cells were analyzed including flow cytometry, colony forming units (CFU) assay, and cytokine analyses. Next, BM from NHD13 (8-10 weeks) and competitor cells were transplanted into lethally irradiated aged recipients (WT, 50-60 weeks). At onset of marrow failure, mice were treated with inhibitors of IL1/Toll-like receptor signaling (IL1R antagonist, MCC950, or IL1R-associated kinase 4 protein (IRAK4) inhibitor) for fourteen days, and then euthanized and analyzed as above. Finally, we evaluated cytokine profile in the BM serum from the patients with MDS and normal donors. Results: Transplant of NHD13 BM cells into aged IL1R1 wt recipients (NHD13→IL1R1 wt) was not associated with a significant difference in survival rates or levels of NHD13 engraftment compared to NHD13 into IL1R1 ko recipients (NHD13→IL1R1 ko). IL1R1 wt developed macrocytic anemia compared to IL1R1 ko recipient (Hb 11.3±0.57 v.s 13.1±0.42 g/dL, n=12 and 9, p Conclusions: Collectively, our findings demonstrate that IL1R1 signaling alters the BMME and contributes to the disease phenotype of MDS and that the effects of targeting IL1R1 pharmacologically have differing effects based on the modality of inhibition as well as the cell population. IL1R1 signaling can be a promising target to alleviate the complexity of MDS via improving inflammatory status in BMME. Disclosures No relevant conflicts of interest to declare.

Targeted irradiation (TR) is widely used for tumor treatment in the clinic. TR benefits tumor therapy through direct effects as well as poorly understood systemic (abscopal) effects. Recent studies suggest that the systemic innate and acquired immune responses to TR contribute to elimination of tumor cells, but also cause systemic inflammation with prolonged tissue injury that may result in secondary malignancies. To elucidate and eventually target the mechanisms underlying these systemic effects of TR, we utilized a murine model using the small animal radiation research platform (SARRP). To define the dynamics of cytokine production and immune responses after TR, we administered local irradiation to a single tibia of 6-8 week old C57BL/6 male mice using a single dose of 15 Gy. We analyzed bone marrow (BM) and BM extracellular fluid (BMEF) from both the irradiated (TR) and non-irradiated, contralateral (CONT) tibiae at 2, 6, 48 hours, 1 and 3 weeks post-TR, performing phenotypic (flow cytometry) and cytokine analyses. As a tumor-bearing model, we utilized 3-4 weeks old C57BL/6 mice injected with Rhabdomyosarcoma (RMS) in one hind limb, and treated with (1) one dose i.p injection of 1mg/Kg Vincristine (Vin) as chemotherapy model, (2) 4.8GyX5times fractionated TR to the tumor area and (3) combination (TR+Vin) therapy. Analysis of peripheral blood (PB), BM, BMEF was performed 3 weeks after the final TR dose (n = 5-13 mice/time point). We found that multiple inflammatory cytokines and chemokines, such as IL-1b, IL-18, CCL2, CCL3, CXCL2, CXCL9, CXCL10 were upregulated from very early phase (2hrs) up to 48hrs in BMEF of the radiated tibiae. Consistent with the dynamics of these cytokines, we observed influx of myeloid cells in both TR and CONT side and expansion of T cells peaking at 6hrs in BM. At the same time of these immune responses, Norepinephrine (NE) was elevated in BMEF even in CONT side. In the tumor-bearing model of RMS, fractionated TR eliminated the tumor while systemically expanding CD8+ cytotoxic T cells and reducing neutrophils. Vin alone did not eliminate the tumor and was associated with systemic decrease of lymphoid cells and expansion of neutrophils. In Vin+TR, tumor control and CD8+ cell expansion were restored, with normalization of neutrophils. These data suggest that TR in the setting of tumor differentially activates lymphoid and myeloid cells. Since recent studies showed catecholamine production from myeloid cells may augment cytokine production in the setting of infection, we hypothesized that BM myeloid cells respond to radiation-induced cell damage by producing catecholamines that trigger a systemic inflammatory response after TR. To test this hypothesis, we utilized standard long-term bone marrow cultures (LT-BM) that reproduce three-dimensional BM structures with myeloid-skewing in vitro, and irradiated them to look at inflammatory changes induced by radiation at 2, 6 and 24hrs. In this experimental model, 5Gy of radiation led to the elevation of NE along with the production of chemokines CCL2, CCL3, CXCL2, CXCL9 mostly peaking at 6hrs in the cell culture supernatants. In contrast, these responses could not be reproduced in spleen cultures, which also had a much lower baseline NE production compared to LT-BMs. These data indicate that radiation induced-chemokine elevations might come from myeloid cells stimulated by NE, independent of systemic innervation. To define the contribution of catecholamines to cytokine production in LT-BM, we directly stimulated culture-LT-BM with NE and Isoproterenol, a pan beta stimulant. While both agents showed similar effect and increased CXCL2, CXCL9, CCL2 and CCL3 at 6hrs, they decreased CXCL10 level, suggesting that catecholamine mostly stimulate myeloid cells but rather inhibit lymphoid activation through chemokine production. Together, these data show that local irradiation initiates global immune responses, and identify local BM production of NE as its potential trigger. Blocking local catecholamine production in the bone marrow could therefore be a positive adjuvant to TR in tumor treatment by inhibiting unfavorable effects of radiation, such as chronic inflammation with systemic increases of neutrophils, while facilitating expansion and recruitment of the cytotoxic T cells which play an essential beneficial role in tumor immunity. Disclosures No relevant conflicts of interest to declare.

Preclinical models of breast cancer have established mechanistic links between psychological stress and cancer progression. However, epidemiological evidence linking stress and cancer is equivocal. We tested the impact of stress exposure in female mice expressing the mouse mammary tumor virus polyoma middle-T antigen (MMTV-PyMT), a spontaneous model of mammary adenocarcinoma that mimics metastatic hormone receptor–positive human breast cancer development. MMTV-PyMT mice were socially isolated at 6 to 7 weeks of age during premalignant hyperplasia. To increase the potency of the stressor, singly housed mice were exposed to acute restraint stress (2 hours per day for 3 consecutive days) at 8 to 9 weeks of age during early carcinoma. Exposure to this dual stressor activated both major stress pathways, the sympathetic nervous system and hypothalamic-pituitary-adrenal axis throughout malignant transformation. Stressor exposure reduced mammary tumor burden in association with increased tumor cleaved caspase-3 expression, indicative of increased cell apoptosis. Stress exposure transiently increased tumor vascular endothelial growth factor and reduced tumor interleukin-6, but no other significant alterations in immune/inflammation-associated chemokines and cytokines or changes in myeloid cell populations were detected in tumors. No stress-induced change in second-harmonic generation-emitting collagen, indicative of a switch to a metastasis-promoting tumor extracellular matrix, was detected. Systemic indicators of slowed tumor progression included reduced myeloid-derived suppressor cell (MDSC) frequency in lung and spleen, and decreased transforming growth factor β (TGF-β) content in circulating exosomes, nanometer-sized particles associated with tumor progression. Chronic β-adrenergic receptor (β-AR) blockade with nadolol abrogated stress-induced alterations in tumor burden and cleaved caspase-3 expression, lung MDSC frequency, and exosomal TGF-β content. Despite the evidence for reduced tumor growth, metastatic lesions in the lung were not altered by stress exposure. Unexpectedly, β-blockade in nonstressed mice increased lung metastatic lesions and splenic MDSC frequency, suggesting that in MMTV-PyMT mice, β-AR activation also inhibits tumor progression in the absence of stress exposure. Together, these results suggest stress exposure can act through β-AR signaling to slow primary tumor growth in MMTV-PyMT mice.

The chemokine CCL3 is frequently overexpressed in malignancies and overexpression leads to microenvironmental dysfunction. In murine models of chronic myelogenous leukemia (CML), CCL3 is critical for the maintenance of a leukemia stem cell population, and leukemia progression. With CCL3 implicated as a potentially viable therapeutic target, it is important to carefully characterize its role in normal hematopoietic homeostasis. CCL3−/− mice were used to evaluate the role of CCL3 in regulating hematopoietic stem and progenitor cell (HSPC) populations. CCL3−/− mice had loss of mature myeloid populations, while myeloid progenitors and HSPCs were increased, and microenvironmental populations were unchanged. These data show that CCL3 promotes myeloid lineage differentiation and the size of the HSPC pool independent of the supportive bone marrow microenvironment. Our results demonstrate a previously unrecognized role of CCL3 in the maintenance of homeostatic hematopoiesis that should be evaluated when targeting CCL3 signaling for the treatment of hematologic malignancy.

Radiotherapy is used in the treatment of ~50% of tumors. We and others have reported long-term suppression of hematopoietic stem and progenitor cells (HSPCs) in the setting of total body irradiation; however, it has been shown that even relatively small irradiation volumes can result in systemic adverse events, such as myeloablation and secondary malignancies. The mechanisms underlying these effects are unclear. We hypothesize that localized radiation may activate a systemic inflammatory response that can acutely alter HSPCs and bone marrow microenvironment (BMME) components, including marrow stromal cells (MSCs), thereby contributing to late effects. We therefore established a murine model of targeted irradiation (TR) using a small animal radiation research platform (SARRP). Methods: We administered local irradiation to a single tibia of 6-8 week old C57BL/6 male mice using a single dose of 15 Gy. Subsequently, we analyzed peripheral blood, BM, BM extracellular fluid (BMEF), collagenase-1 digested bone associated cells of both the irradiated (TR) and non-irradiated, contralateral (CONT) tibiae at 2, 6, 48 hours, 1 and 3 weeks post-TR, performing phenotypic (flow cytometry) and cytokine analyses. For all studies, n = 10-13 mice/time point. Results: In the TR tibia at 2 hours, although total cell numbers were unchanged, there was a significant upregulation of inflammatory cytokines (interleukin 1β (IL1b), IL18), chemokines (CXCL2, CXCL10, CCL2, CCL3) and macrophage colony stimulating factor (M-CSF). Of note, most of these changes normalized by 48 hours (M-CSF at 1 week). Changes in mediator expression were followed, at 6 hours post-TR, by significant increases in macrophage (macs) numbers, including CD206 phagocytic macs, neutrophils (PMNs) and cytotoxic lymphocytes, including CD8+ cells expressing CXCR3+, the receptor for CXCL9 and CXCL10. Interestingly, similar to the TR tibia, CXCL2 expression was also increased significantly in the CONT at 2 hours, followed (6 hours) by significant increases in macs and CD8+ cells, suggesting a systemic or abscopal effect. With respect to the effects of radiation on HSPCs, by 6 hours, most of the stem and progenitor cell (HSPC) populations in the TR marrow were significantly decreased; the decrease in long-term-HSCs was delayed until 48 hours post-TR. All populations remained severely depleted until 3 weeks post RT, demonstrating a rapid and sustained effect of TR on all HSPCs within the irradiation volume. In comparison, in the CONT tibia at 6 hours, CD41+ HSCs were expanded; this is consistent with previous demonstrations that CD41+ LT-HSCs expand with inflammatory signals and suggests that TR-induced signals induced a systemic impact on the non-irradiated HSPCs. By 1 week post-radiation, short term-HSCs were significantly decreased in the CONT marrow, likely due to mobilization since CFU-Cs were correspondingly significantly increased in the circulation. Finally, MSCs, previously shown to support HSCs, were found to be significantly increased in the TR tibia starting at 6 hours and peaking at 48 hours post-radiation. Surprisingly, MSCs were also expanded in the CONT marrow at 48 hours; this expansion was likely associated with the increased CXCL12 levels seen in both TR and CONT marrow, although the CXCL12 levels were higher in the irradiated tibia. Taken together, these changes indicate TR-induced global disruption of the HSC niche. Furthermore, in addition to the transient effects of localized irradiation, we observed a second wave of inflammatory signals, including a significant increase in CCL3, at 1 week post-TR and increased IL1b in the CONT marrow at 3 weeks, changes that may have contributed to the sustained loss of HSPC populations. Conclusions: We present the effects of local irradiation on global hematopoiesis, showing that, in addition to the anticipated acute local changes in the irradiated bone marrow, TR-induced persistent and, more importantly, systemic inflammation. We believe that using this murine model will allow us to dissect the contribution of direct (local) and indirect (systemic) responses to radiation on treatment effects, such as marrow failure and secondary malignancies. Disclosures No relevant conflicts of interest to declare.

Introduction: Myelodysplastic syndromes (MDS) are genetically diverse and heterogeneous diseases characterized by dysplasia and cytopenias and a dismal prognosis of ˂ 50% overall survival at 5-years. In vitro and in vivo experimental data have shown that the bone marrow microenvironment (BMME) may play a role in disease progression and, importantly, in murine models, transplantation of MDS to a healthy BMME was shown to mitigate disease. Mesenchymal Stem and Progenitor Cells (MSPCs), as part of the BMME, give rise to multiple non-hematopoietic progenitor cells and provide essential support to hematopoietic stem cells. MSPCs have also been shown to be functionally altered in patients with myeloid neoplasias. Murine studies have demonstrated distinct roles for specific subsets of bone marrow mesenchymal cells in myeloid malignancies. We hypothesized that specific subsets of human bone marrow MSPCs will play differential roles in the pathogenesis of MDS. Using flow-cytometry, high-throughput sequencing and gene set enrichment analysis (GSEA), we characterized human MSPCs in the bone marrow aspirates from patients with MDS and normal healthy controls (NBM). Methods: Mononuclear cells were isolated by iliac crest bone marrow aspiration from 10-healthy donors and 14-patients at the University of Rochester Medical Center. Within the non-hematopoietic (CD45-, CD235-), non-endothelial (CD31-) bone marrow compartment, we enriched and isolated 3 distinct-subpopulations of MSPCs based on cell-surface expression of CD271/NGFR, CD106/VCAM-1 and CD146/MCAM. Populations were defined as follows: CD271+/CD146- (CD271+), CD271+/CD146+/CD106+ (DPCD106+), and CD271+/CD146+/CD106- (DPCD106-). RNA-seq analysis was performed on each subpopulation to define transcriptional signatures (TS) and gene set enrichment patterns. Statistically significant differentially expressed genes (DEGs) were defined by fold-change ≥ ±1 and p-value ˂0.05. Results: We first set out to define differences in the TS and interrogate the function of MSPC populations in NBM. Principle component analysis (PCA) demonstrated the highest variance between the DPCD106+ and the CD271+ populations. The number of DEGs were also highest between the DPCD106+ and CD271+ populations (n=3,619 genes). GSEA identified 745 and 336 gene sets with positive enrichment in the DPCD106+ and CD271+ group, respectively, and illustrated that the DPCD106+ population was significantly enriched in gene sets involved in early embryonic developmental and "stem-like" pathways whereas the CD271+ population was enriched in cell cycling, DNA and chromosomal organization. In the setting of MDS, the mean relative frequency of MDS CD271+ nearly tripled (0.4230/0.1445; p-value 0.045). Compared to NBM, MDS DPCD106+ cells had the highest variance by PCA and the highest number of DEGs (n=560). GSEA identified 19-gene sets with significant enrichment in the MDS DPCD106+ group and, intriguingly, 12 (63%) were identical to gene sets enriched in the CD271+ group. Furthermore, of the 560 DEGs in the MDS DPCD106+ MSPCs, 300 were upregulated and, of those, 160 (53%) were identical to upregulated genes in the CD271+ NBM group including the acquisition of a proliferative signature. Altogether, this data suggests a switch in the TS of theDPCD106+ population in the setting of MDS. Importantly, this TS clustered MDS DPCD106+ from NBM, regardless of MDS risk category. Conclusion: We successfully characterized 3 subtypes of MSPCs in NBM and MDS. In NBM, we demonstrate that cell surface expression of CD271, CD146 and CD106 defined the most stem-like TS within the non-hematopoietic, non-endothelial bone marrow compartment. In the setting of MDS, the increase in population frequency of the CD271+ cells and the concomitant transcriptomic aberrations observed in the MDS derived DPCD106+ population support the hypothesis that specific MSPC populations have differential roles in MDS pathogenesis. Further, we identify a TS that discriminates MDS derived MSPCs from NBM irrespective of MDS-risk category. This suggests that alterations within specific MSPC populations may represent a unifying pathway in disease pathogenesis despite heterogeneity and genetic drivers intrinsic to the MDS clone. Thus, targeting the BMME represents a potentially novel therapeutic strategy aimed at mitigating disease and restoring normal hematopoiesis in patients with MDS. Disclosures Liesveld: Onconova: Other: Data safety monitoring board; Abbvie: Membership on an entity's Board of Directors or advisory committees. Scadden:Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Fate Therapeutics: Consultancy, Equity Ownership; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fog Pharma: Consultancy; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The non-invasive, in vivo measurement of microvascular blood flow has the potential to enhance breast cancer therapy monitoring. Here, longitudinal blood flow of 4T1 murine breast cancer (N=125) under chemotherapy was quantified with diffuse correlation spectroscopy based on layer models. Six different treatment regimens involving doxorubicin, cyclophosphamide, and paclitaxel at clinically relevant doses were investigated. Treatments with cyclophosphamide increased blood flow as early as 3 days after administration, whereas paclitaxel induced a transient blood flow decrease at 1 day after administration. Early blood flow changes correlated strongly with the treatment outcome and distinguished treated from untreated mice individually for effective treatments.

A significant difference between a treated and a control group of mice with breast cancer were observed in hemodynamic parameters quantified with diffuse optics at day 3 after cyclophosphamide treatment.