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Current treatment options for relapsed acute myeloid leukemia (AML) are limited and ineffective for the majority of patients. In AML, primitive leukemia stem cells (LSCs) and pre-leukemic populations are able to maintain the disease and drive relapse. Thus, therapies targeting LSC populations may increase the overall survival of AML patients. In this study, we aim to identify the drivers favoring LSC expansion following treatment and relapse and develop potential therapies for AML. The transcriptome analyses of 12 pairs of functionally defined LSC fractions at diagnosis and relapse revealed significant changes of IL-1 signaling in AML patients. We demonstrated that the protein expression levels of interleukin-1 receptor type I (IL1R1) and its complex member interleukin-1 receptor accessory protein (IL1RAP) were both up-regulated in human leukemia stem and progenitor cells (LSPCs) at diagnosis or in relapse compared to normal hematopoietic stem and progenitor cells (HSPCs). Knockdown of IL1R1 and IL1RAP suppressed the clonogenicity and engraftment growth of primary human AML cells but showed low impacts on HSPCs in the normal bone marrow. Additionally, knockout of IL1R1 in leukemia MLL-AF9 mice significantly reduced the LSC frequency and prolonged the overall survival rate. To target IL-1/TLR signaling in LSCs, we performed iterative structure-activity relationship (SAR) guided medicinal chemistry, in silico modeling and leukemia cell line reporter assays to screen and identify a novel interleukin-1 receptor-associated kinase 1/4 (IRAK1/4) inhibitor (termed UR241-2). UR241-2 robustly inhibits IL-1/TLR signaling in AML cells including the activation of NF-κB following IL-1 stimulation. UR241-2 repressed LSPC function as assessed by colony-forming unit assays in primary human AML cells at diagnosis and in relapse while minimally impacting normal HSPC function. Taken together, our findings demonstrate the important role of IL-1/TLR signaling in supporting AML LSC expansion following treatment and relapse and suggest that targeting IL-1/TLR signaling using the novel IRAK1/4 inhibitor, UR241-2, can target LSC function to improve patient outcomes in AML. Disclosures No relevant conflicts of interest to declare.

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.

Most cancers evolve over time as patients initially responsive to therapy acquire resistance to the same drugs at relapse. Cancer stem cells have been postulated to represent a therapy-refractory reservoir for relapse, but formal proof of this model is lacking. We prospectively characterized leukemia stem cell populations (LSCs) from a well-defined cohort of patients with acute myelogenous leukemia (AML) at diagnosis and relapse to assess the effect of the disease course on these critical populations. Leukemic samples were collected from patients with newly diagnosed AML before therapy and after relapse, and LSC frequency was assessed by limiting dilution analyses. LSC populations were identified using fluorescent-labeled cell sorting and transplantation into immunodeficient NOD/SCID/interleukin 2 receptor γ chain null mice. The surface antigen expression profiles of pretherapy and postrelapse LSCs were determined for published LSC markers. We demonstrate a 9- to 90-fold increase in LSC frequency between diagnosis and relapse. LSC activity at relapse was identified in populations of leukemic blasts that did not demonstrate this activity before treatment and relapse. In addition, we describe genetic instability and exceptional phenotypic changes that accompany the evolution of these new LSC populations. This study is the first to characterize the evolution of LSCs in vivo after chemotherapy, identifying a dramatic change in the physiology of primitive AML cells when the disease progresses. Taken together, these findings provide a new frame of reference by which to evaluate candidate AML therapies in which both disease control and the induction of more advanced forms of disease should be considered.

In vitro evidence suggests that the bone marrow microenvironment (BMME) is altered in myelodysplastic syndromes (MDSs). Here, we study the BMME in MDS in vivo using a transgenic murine model of MDS with hematopoietic expression of the translocation product NUP98-HOXD13 (NHD13). This model exhibits a prolonged period of cytopenias prior to transformation to leukemia and is therefore ideal to interrogate the role of the BMME in MDS. In this model, hematopoietic stem and progenitor cells (HSPCs) were decreased in NHD13 mice by flow cytometric analysis. The reduction in the total phenotypic HSPC pool in NHD13 mice was confirmed functionally with transplantation assays. Marrow microenvironmental cellular components of the NHD13 BMME were found to be abnormal, including increases in endothelial cells and in dysfunctional mesenchymal and osteoblastic populations, whereas megakaryocytes were decreased. Both CC chemokine ligand 3 and vascular endothelial growth factor, previously shown to be increased in human MDS, were increased in NHD13 mice. To assess whether the BMME contributes to disease progression in NHD13 mice, we performed transplantation of NHD13 marrow into NHD13 mice or their wild-type (WT) littermates. WT recipients as compared with NHD13 recipients of NHD13 marrow had a lower rate of the combined outcome of progression to leukemia and death. Moreover, hematopoietic function was superior in a WT BMME as compared with an NHD13 BMME. Our data therefore demonstrate a contributory role of the BMME to disease progression in MDS and support a therapeutic strategy whereby manipulation of the MDS microenvironment may improve hematopoietic function and overall survival.

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.

Background Acute Myelogenous Leukemia (AML) evolves as many patients who are responsive to therapy upfront are resistant to the same agents when applied at relapse. We previously reported the results of our prospective efforts to formally assess the evolution of the leukemia stem cell (LSC) population(s) during patients' clinical courses. We identified a 9-90 fold increase in LSC activity and greatly increased phenotypic diversity of the LSC population. To identify the potential mechanisms underlying these changes we further characterized functionally-defined LSC populations from paired diagnosis and relapse samples. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy as well as normal donors. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Transcriptional profiling of highly enriched LSC populations from seven patients was performed using ABI TaqMan® Low Density Array (TLDA) qPCR analyses following pre-amplification using a novel 153 gene expression platform. Protein expression levels of interleukin-1 receptor accessory protein (IL1RAP) on bulk leukemia cells and LSC populations from 25 patients were assessed by flow cytometry. The impact of loss of IL1RAP was assessed using lentiviral based shRNA targeting all IL1RAP isoforms followed by assessment of proliferation, apoptosis, colony forming unit (CFU) activity and NSG engraftment capacity in human cell lines as well as in primary patient samples. Downstream signaling events for IL1RAP were probed using a small molecule inhibitor approach. Results While the majority of the LSC populations' gene expression profile remained stable, twelve genes were differentially expressed between pre-treatment and relapsed LSC populations including IL1RAP. Flow cytometric analyses confirmed that IL1RAP is overexpressed on both bulk leukemia populations as well as LSC populations at diagnosis and relapse in comparison to normal hematopoietic stem cell (HSC) populations. Targeting ILRAP1 using shRNA in both cell lines and primary AML samples resulted in impaired proliferation, increased apoptosis, a marked loss of CFU capacity and impaired NSG engraftment. IL1 signaling is known to involve both the MAPkinase and NFKappB pathways. To determine which pathways are involved in IL1RAP mediated LSC survival, we performed a small molecule inhibitor screen targeting elements in both signaling cascades. Established inhibitors of the NFKappaB pathway resulted in loss in loss of leukemic cell function while MAPK signaling inhibition had minimal to no effect. Conclusions We identified IL1RAP as being overexpressed in both bulk leukemia and functionally defined LSC populations from pre-treatment and relapsed AML samples. Loss of IL1RAP was associated with a marked decline in LSC function. Preliminary studies support a primary role for the NF Kappa B pathway in LSC function. Our findings support a critical role for IL1RAP in LSC function and support its development as a target for AML therapy in both the upfront and relapse setting. Disclosures Wang: Immunogen: Research Funding. Calvi:Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

Introduction: Acute Myeloid Leukemia (AML) patients who are refractory to induction chemotherapy (RF) have a dismal prognosis. Properties of AML cell populations that cause the refractory phenotype are poorly understood. It is postulated that the leukemia stem cell (LSC) pool promotes chemotherapy resistance in AML; yet whether differences exist in this pool as a function of eventual remission status, and thus may account for induction failure, is unknown. Our group has recently found that in AML patients who initially achieve complete remission (CR) and then relapse, the LSC pool evolves to become larger and more immunophenotypically diverse at the time of relapse [Blood 2013; 122(21): abstract #883]. Since relapsed AML patients are often refractory to salvage chemotherapy, we postulated that diagnostic specimens from RF AML patients would have enlarged, immunophenotypically-diverse LSC pools relative to AML patients achieving CR, implicating these properties in the refractory phenotype. Defining unique properties of LSCs in RF AML might provide greater insight into the mechanisms responsible for the treatment-resistant phenotype. Methods: RF AML patients were defined by having ≥ 6% leukemic blasts in their bone marrow or peripheral blood after 1 or 2 cycles of anthracycline/cytarabine-based induction chemotherapy. All studies were conducted with pre-treatment AML specimens on approved IRB protocols at the University of Rochester Medical Center. LSC frequency was determined by limiting dilution analysis and xenotransplantation into sublethally irradiated NOD scid gamma (NSG) mice. To determine immunophenotypically-defined cell populations harboring functional LSC activity, AML specimens were sorted into four distinct populations defined by CD34 and CD38 staining. The presence of LSC activity in the four resultant populations (CD34+/CD38-, CD34+/CD38+, CD34-/CD38+, and CD34-/CD38-) was determined by the ability of each sorted population to engraft NSG mice in primary and secondary transplantation experiments. RNA-seq analysis was conducted on LSC-enriched cell populations. Results: Specimens from RF AML patients were more likely to engraft NSG mice relative to those from AML patients achieving CR (6/6 vs. 13/29 in RF vs. CR patients, respectively; P=0.02). The LSC pool from six RF AML patients was studied in detail. LSC frequency ranged from 1/19 to 1/326,123. Four of the 6 RF specimens harbored LSC frequencies 6- to 7,700-fold higher than those previously reported in primary AML [Sarry et al. J Clin Invest 2011; 121(1384-395]. In 5/6 RF specimens, LSCs were present in more than one immunophenotypically-defined cell population. Relative to specimens from AML patients achieving CR, specimens from RF patients were more likely to harbor LSCs in both CD34+ and CD34- populations (P=0.04). Treatment of NSG mice xenografted with specimens from RF patients with anthracycline/cytarabine-based chemotherapy failed to eradicate disease from engrafted mice; thus, therapeutic outcome of NSG mice engrafted with specimens from RF patients resembles that seen in patients. To identify genes potentially driving the refractory LSC phenotype, we compared gene expression profiles of CD34+ cells from RF patients to those from AML patients achieving long-term remission and to those from normal donors. Ninety-nine genes were uniquely deregulated in RF LSCs, including numerous Homeobox transcription factors and components of the Wnt and Hedgehog signaling pathways, all implicated in the maintenance of stemness. Pathway analysis revealed that amino acid metabolic pathways critical to other treatment-resistant cancers and molecules involved in IL-1 signaling and implicated in metastasis of solid tumors are dysregulated in primary refractory LSCs. Conclusions: This study is the first to systematically analyze the LSC pool in primary refractory AML. Similar to our findings in relapsed AML, RF AML patients harbor enlarged, phenotypically diverse LSC pools relative to AML patients achieving CR. Challenge of leukemic mice with an anthracycline/cytarabine treatment regimen mimics the effect seen in patients, facilitating the development of patient-derived xenograft models of RF AML. We identify a refractory LSC gene signature, opening the door to future mechanistic investigations and novel therapeutic approaches for this AML patient population in great need. Disclosures Calvi: Fate Therapeutics: Patents & Royalties. Becker:Millenium: Research Funding.

In vitro data provide evidence of an altered bone marrow microenvironment (BMME) in the myelodysplastic syndromes (MDS). To assess the role of the BMME in MDS in vivo, we used a well-established transgenic murine model with expression of the translocation product Nup98-HOXD13 (NHD13) in hematopoietic cells that leads to development of an MDS phenotype, fully penetrant by 5 months of age. In order to assess whether the BMME contributes to diminished hematopoiesis as a feature of MDS, we transplanted marrow from 5-month-old NHD13 mice and normal competitor marrow into irradiated NHD13 mice and their wild type (WT) littermates. Serial analysis of peripheral blood (PB) indicated engraftment of NHD13 marrow was improved in WT recipients relative to NHD13 recipients (2-way ANOVA, WT vs. NHD13: p Disclosures Calvi: Fate Therapeutics: Patents & Royalties.

Osteoprogenitor cells (OPCs) are marrow microenvironmental cells known to modulate hematopoietic stem and progenitor cells (HSPCs). Specifically, OPCs regulate HSPCs in response to Parathyroid hormone (PTH) treatment in murine models. However, the role of OPCs in human HSPC regulation and whether human OPCs can be manipulated is poorly understood. Niche stimulation is an appealing strategy to aid in the treatment of hematopoietic dysfunction. Myelodysplastic syndromes (MDS) are clonal disorders with ineffective hematopoiesis resulting in cytopenias and risk of transformation to acute leukemia (AML). In mouse models, disruption of the osteolineage cells can contribute to initiation of ineffective hematopoiesis with phenotypic features of MDS. Our long term goal is to utilize microenvironmental stimulation as a therapeutic tool to improve hematopoietic disorders. We hypothesized that human cells isolated from the marrow fraction containing spicules harbor HSPC supportive cells, which can be manipulated to improve HSPC support. Moreover we hypothesized that OPC number and function is impaired by dysplasia-initiated microenvironmental disruption as a potential mechanism for reduced support of HSPCs and ineffective hematopoiesis. Our objective was to isolate human bone marrow spicule associated cells (SACs) and define their ability to support HSPCs, determine the impact of PTH treatment of SAC/HSPCs interactions and characterize dysplasia-induced osteolineage changes in human MDS and AML bone marrow. To achieve this objective, we used normal as well as MDS/AML patient-derived OPCs using a mouse-human co-culture system. Human bone marrow SACs isolated by collagenase digestion were either used for co-culture, analyzed with flow cytometry or cultured in mineralization media in limited dilutions. To assess the potential impact of PTH on human OPC interaction with HSPCs, we developed a 7 day co-culture of human bone marrow SACs treated with either vehicle or PTH, with mouse Lineage- Sca1+ c-Kit+ (LSK) hematopoietic progenitor cells. At the end of the co-culture, all cells present were used for competitive transplantation. Transplant experiments demonstrated that PTH treatment of the human bone marrow SACs leads to improved function of the co-cultured LSK cells as demonstrated by significantly improved engraftment of the LSK cells after transplant into irradiated C57/bl6 recipient mice when sampled at pre-specified time points over a 20-week period (N=12, 2-way ANOVA; p < 0.05). Flow cytometry analysis showed that mature (Lin- CD31- CD146+ CD105-) and immature osteolineage (Lin- CD31- CD146+ CD105+) cells were present in SACs and more abundant compared to within BMMCs (1% vs 0.1% and 0.24% vs 0.12% for the same patient). Notably, the putative HSC-supportive MSC pool was increased in SACs vs BMMCs (0.052% vs 0.019%). The presence of OPCs was functionally confirmed using colony forming unit osteoblasts (CFU-OBs). CFU-OB frequency was calculated using L-Calc TM (StemCell technologies). Among normal donors the frequency of CFU-OBs was low in marrow donors >50 years old compared to These data demonstrate that human SACs contain HSPC-supportive cells which can be stimulated to improve HSPC function. Human SACs comprise MSCs and osteolineage cells including osteoprogenitor cells. Aging decreases OPC pools in SACs. Our data in our small sample also suggest that dysplastic bone marrow microenvironment may negatively impact OPCs, which may in turn decrease OPC support of HPSCs. PTH treatment in our in-vitro model shows the potential to improve the interaction between the OPCs and HSPCs, resulting in amelioration of HSC function. Together these data suggest a strategy where targeting the MDS microenvironment may add to the currently available treatment modalities. Disclosures Calvi: Fate Therapeutics: Patents & Royalties.

Myelodysplastic Syndromes (MDS) are a group of clonal disorders characterized by ineffective hematopoiesis. Recently, data have emerged supporting a role of the bone marrow microenvironment (BMME ) in the initiation of MDS. We and others have previously shown that cells within the BMME play a central role in normal regulation of hematopoietic stem and progenitor cells (HSPCs). To determine if the HSPC compartment in MDS is defective and also if HSPC function in MDS is regulated by the BMME, we studied a transgenic murine model that expresses the Nup98/HOXD13 (NHD13) translocation product. As was previously reported, these mice develop ineffective hematopoiesis resulting in progressive cytopenias with dysmorphic cells, a phenotype similar to that of human MDS. We investigated the composition of the HSPC pool in these transgenic (TG) mice at 20 to 22 weeks from birth, a time when an MDS phenotype was evident but acute leukemia had not yet developed. Immunophenotypic analysis by flow cytometry on marrow cells from TG and wild type (WT) age-matched littermates demonstrated a severe defect in the TG HSPC pool, with a severe decline in Lin-Sca1+cKit+CD48-CD150+ long-term HSCs (WT vs. TG: 3.5 ± 1.2 x 104 vs 4.4 ± 3.4 x102, p =0.0025) and in Lin-Sca1+cKit+Flt3+Thy1.1- multipotent progenitors (WT vs. TG: 5.6 ± 1 x 105 vs 2.1 ± 0.4 x 104, p Disclosures: No relevant conflicts of interest to declare.

Acute Myelogenous Leukemia (AML) is a disease that clinically evolves over time as many patients who are responsive to therapy upfront acquire resistance to the same agents when applied in the relapse setting. The stem cell model for AML has been invoked to explain primary resistance to standard therapy; the leukemia stem cell (LSC) population representing a therapy-refractory reservoir for relapse. There have been no prospective efforts to formally assess the evolution of the LSC population during patients’ clinical course. We performed a prospective characterization of specimens from a well-defined cohort of patients with AML at diagnosis and relapse to assess the frequency and phenotype of functionally defined LSCs. Methods Primary bone marrow and peripheral blood samples were collected on IRB approved protocols from patients with newly diagnosed AML undergoing induction therapy. Twenty-five patients who relapsed after achieving a complete remission were selected for further study. Screening studies identified seven patients whose pre-therapy samples demonstrated sustained engraftment of NSG mice following transplantation. Pre-therapy and post-relapse LSC frequencies were assessed using xenotransplantation limiting dilution analyses (LDA). We assessed the frequencies of CD45RA, CD32, TIM-3, CD96, CD47, and CD97 expressing populations that have been previously published to possess LSC activity. Functionally validated pre-therapy and post-relapse LSC populations were identified using fluorescent labeled cell sorting and NSG xenotransplantation. LSC activity was confirmed for each population using secondary xenotransplantation. Gene expression analysis of highly enriched LSC populations from pre-therapy and post-relapse samples was performed using ABI TILDA qPCR analyses following pre-amplification. Results We demonstrated by LDA an 8 to 42-fold increase in LSC frequency between diagnosis and relapse in paired primary patient samples. The increase in LSC activity was not associated with an increase in frequency for phenotypically-defined populations previously reported to possess LSC activity. Rather, we found that LSC activity expanded at relapse to immunophenotypic populations of leukemic cells that did not possess LSC activity prior to treatment. Moreover, in all patients, the number of phenotypically distinct LSC populations (as defined by CD34 and CD38 or CD32 and CD38) detectable at relapse was dramatically expanded. Further, while the majority of the LSC populations’ gene expression profile remained stable between diagnosis and relapse, a subset of genes were enriched in defined LSC populations at relapse including IL3-receptor alpha and IL1-RAP, both previously demonstrated to play a role in LSC biology. Conclusions This study is the first to characterize the natural evolution of LSCs in vivo following treatment and relapse. We demonstrate an increase in LSC activity and greatly increased phenotypic diversity of the LSC population, suggesting a loss of hierarchical organization following relapse. These findings demonstrate that treatment of AML patients with conventional chemotherapy regimens can promote quantitative and qualitative expansion of the LSC compartment. Further, the data indicate that surface antigen immune-phenotype is not predictive of function in relapse and suggest a major limitation to efforts targeting specific surface antigens in the relapse setting. Understanding the mechanisms by which LSC expansion occurs and how to target it will likely improve our currently poor treatment options for patients who relapse. Disclosures: Becker: Millenium: Research Funding.