ALBERT

Description: Abstract 2586 Introduction: Acute Myeloid Leukemia (AML) is a heterogeneous disorder arising from a clonal expansion of Leukemic Stem Cell (LSC). The characterization of LSC is crucial because it is resistant to conventional chemotherapy and is ultimately responsible for leukemic relapses. The LSC in AML is a phenotypically heterogeneous population (CD34+CD38-, CLL1 +, CD96 +…). In this sense, “Side Population” cells (SPHo342Low) are considered to be a type of stem cells that can self-renew and differentiate into tissues. SP are characterized by their ability to efflux the vital dye Hoechst 33342 through the drug ABCG2 pump. SPHo342Low cells have been described in many types of solid tumors and AML as potential LSC. The objective in this study is to analyze the frequency of SPHo342Low in AML, their phenotype and the possible prognostic impact on outcomes. Patients and Methods: Bone marrow samples (BM) obtained from 57 patients (median age 58 years, range: 4–82), diagnosed with AML between Mar-07 to Mar-12, were included. Distribution of cytogenetic risk groups was: Favorable (12.5%), Intermediate (60.7%) and Unfavorable (26.8%). NPM1mut was present in 11 cases and FLT3-ITD in 6 cases. Prior MDS was present in 10 cases. After achieving complete remission (CR) with conventional chemotherapy, allogeneic or autologous stem cell transplantation was performed in 17 and 12 patients respectively, according to individual risk and availability of donor. Eleven frail patients received as front-line, low intensity therapy with Azacytidine. We detected LSC, SPHo342Low in marrow MNCs obtained at diagnosis (N=40), at morphologic complete remission (CR) (N=21) or at relapsed / resistant (N=16) disease. For detection, 2×10(6) MNC/ml were resuspended in HBSS medium with 5 ug/ml of Ho342 dye and CD45-FITC, CD34-PE Mn-Abs, analyzing at least 1×105 viable cells in UV laser FACSVantage cytometer with the combination of filters BP 670/40 for emission in red and BP 450/30 for the blue emission. We verified SP region by inhibiting ABCG2 pump with Verapamil (50μM/mL). As controls we analyzed MNCs from BM aspirates from healthy donors (N=5). Results: In all BM samples from healthy donors, SPHo342Low population was detected accounting for 0.5% (range: 0.1 to 0.9%) and it was CD34negCD45neg phenotype in 80% of cases. SPHo342Low cells were detected in 23/40 cases (57.5%) of samples from AML diagnosis with a median of 0.08% (range 0.01–2.3%). Phenotype of SPHo342Low cells at diagnosis was CD34+CD45+/− in 36% of cases. The presence of SPHo342Low cells presented in AML at diagnosis did not statistically correlate with any prognostic clinical variables such as age, cytogenetic-molecular risk or prior MDS. Interestingly, the detection of LSC SPHo342Low at diagnosis was statistically associated to the presence of 〉0.1% of CD34+CD38- AML cells (P=0,03). In BM samples obtained from AML patients in CR, SPHo342Low cells were detected in 17/21 (81.0%) with a median of 0.17% (range: 0.1 to 0.76%), with a phenotype mostly CD34 negative. In BM samples obtained from AML patients in relapsed/refractory situation, SPHo342Low cells were detected in 14/16 (87.5%) with a median of 0.22% (range: 0.2 to 0.91%) with a phenotype of CD34+ CD45+/− in 33% of cases. Interestingly, patients who did not achieve CR, have a significantly higher percentage of SPHo342Low at diagnosis (0.42% vs. 0.06%, P = 0.044) as well as those who need more than one cycle to achieve CR (0.52% vs. 0.07%, P = 0.04). Moreover, for those patients achieving CR, persistence of Minimal Residual Disease (MRD+) was associated to a higher percentage of SPHo342Low at diagnosis (0.28% vs. 0.05%, P = 0.021). Likewise, Relapse-free survival (RFS) was significantly higher in AML patients lacking SPHo342Low at diagnosis (70 ± 18.2% vs. 43.3 ± 17.6%, P = 0.0324, Log rank test). Conclusions: Detection of LSC SPHo342Low+CD34+CD45+/− phenotype in AML at diagnosis is a common finding that is associated with increased resistance to achieve CR, clearance of MRD and lower RFS. During progression of disease this SPHo342Low+ population increases and maintains CD34+CD45neg phenotype. BM samples obtained from AML patients at CR were SPHo342Low+ CD34negCD45+/− phenotype which can be considered responsible for normal hematopoietic regeneration. Disclosures: No relevant conflicts of interest to declare.

Description: Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase downstream of Toll-like receptors (TLR), is required for mediating signals in response to foreign pathogens and stress molecules, and is implicated in the pathogenesis of MDS and AML. Although TLRs are expressed on normal HSC and TRAF6 is implicated in malignant HSC function, the normal physiological role of TRAF6 in HSC homeostasis and during hematopoiesis remains unknown. We find that TRAF6 is expressed in human and mouse HSPC (LT-HSC, ST-HSC, and MPP) at comparable or elevated levels relative to mature myeloid and lymphoid cells. To understand the role of TRAF6 in HSPC homeostasis, we generated hematopoietic-specific and inducible TRAF6 deleted mice by crossing Traf6-floxed with Vav-Cre (Traf6-HscKO) or Mx1-Cre (Traf6-iKO after PolyIC treatment) mice, respectively. Traf6-HscKO mice are born smaller and become moribund shortly after birth. Examination of peripheral blood (PB) and bone marrow (BM) revealed a significant expansion of myeloid cells and reduction of lymphoid cells. Moreover, moribund mice developed splenomegaly and extramedullary hematopoiesis. To determine whether the observed phenotype could be driven by loss of TRAF6 in mature myeloid cells, we generated mice in which TRAF6 is only deleted in myeloid cells by crossing Traf6-floxed with LysM-Cre mice (Traf6-MyKO). Interestingly, Traf6-MyKO mice did not develop myeloid expansion in the PB, BM, or spleen, indicating that TRAF6 plays a role in normal HSPC function. To determine the cell-intrinsic role of TRAF6 in hematopoiesis, we transplanted BM cells from Traf6-HscKO mice into lethally-irradiated recipient mice. The recipient mice with Traf6-HscKO BM cells similarly displayed myeloid-biased hematopoiesis in PB, BM, and spleens. Strikingly, LT-HSCs from Traf6-HscKO mice were significantly reduced in the BM of recipient mice. To exclude a possible effect of myeloid cells on the reduction in LT-HSC, we examined BM HSPC from Traf6-MyKO mice. Consistent with a role of TRAF6 in normal HSC function, the LT-HSC proportions and numbers were not affected in Traf6-MyKO mice. We next examined the functional consequences of deleting TRAF6 in HSC by performing competitive BM transplantation assays. Although initial homing to the BM was comparable between WT and Traf6-HscKO cells, the donor-derived chimerism of Traf6-HscKO cells was significantly reduced for myeloid and lymphoid populations 1 month post transplantation, and declined to below 5% after 4 months as compared with control mice. In addition, donor-derived HSC, HPC, and total BM cell chimerism of Traf6-HscKO cells was dramatically reduced. To examine the effects of TRAF6 deletion on HSC function after BM engraftment has been achieved, competitive BMT were performed with BM cells from Traf6-iKO mice. Upon deletion of Traf6 (PolyIC treatment 2 months post transplantation), total PB and BM chimerism, and chimerism of Traf6-deleted LT-HSC and HPC dramatically declined. Collectively, these findings indicate that TRAF6 is essential for normal HSPC function and homeostasis. To understand the function of TRAF6 in HSPC, HSC-enriched Lin-Sca1+Kit+(LSK) BM cells were isolated and examined for gene expression changes by RNA-sequencing. Genes directly implicated in cell cycle control were among the most differentially expressed in Traf6-deficient HSPC. Particularly, the cyclin-dependent kinase inhibitors (CDKIs) p21, p27 and p57 were significantly down-regulated in Traf6-deficient LSK cells as compared to normal LSK cells. CDKIs are negative regulators of cell cycle progression and involved in maintaining HSC quiescence. Consistent with the observed reduction in CDKI genes, LT-HSC and HPC (LSK) from Traf6-HscKO mice were less quiescent (lower proportion of G0 cells) and more actively cycling (higher proportion of G1/S/G2/M cells). Despite the established requirement of TRAF6 in myeloid and lymphoid cells during infection, our study uncovers a critical role of TRAF6 during normal HSC function and homeostasis. Our findings suggest that TRAF6 is a novel hematopoietic-requisite factor for maintaining HSC quiescence and controlling myeloid-biased differentiation. These findings reinforce the importance of innate immune pathway gene dosage and signaling requirements in normal and malignant HSPC. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 5115 INTRODUCTION Allogeneic stem cell transplantation (allo-SCT) is a potentially curative treatment option for patients with hematological disorders. Alloreactive donor-derived T lymphocytes exert a beneficial graft-versus-leukemia (GVL) effect through the recognition of leukemia-restricted (or preferentially expressed) antigens as Wilms tumor protein (WT1), survivin (SURV) or proteinase (PR1). Currently research in transplant immunology focuses in enhancing GVL while preventing the deleterious graft-versus-host disease (GVHD) that could be achieved by manipulating donor-derived antigen-specific T-populations. In this study we tested the presence of peripheral blood leukemia-associated antigen-specific CD8+ T-lymphocytes during post allo-SCT follow-up. PATIENTS AND METHODS Forty-three consecutive HLA*0201 patients (homo or heterozygotous) undergoing conventional myeloablative (n=24) or non-myeloablative (n=19) allo-SCT as treatment of hematological disorders were included. Allogeneic donor was an HLA-identical sibling in 26 cases (60.5%) and unrelated in 17 cases (39.5%). Hematopoietic stem cell source included mobilized peripheral blood (n=20), bone marrow (n=18) and umbilical cord blood (n=5). As GVHD prophylaxis regimens Cyclosporine plus Methotrexate (n=20) or Cyclosporine plus Mofetil micofenolate (n=23) were employed. In addition, 22 patients received rabbit antithymocyte globulin at 6-8mg/kg. At last follow-up four patients had relapsed 9-14 months after allo-SCT. We sought for leukemia-antigen specific CD8 lymphocytes in peripheral blood samples drawn within a median of 7 months (range 2-38) when lymphocyte recovery had occurred and complete donor chimerism was achieved. We used four color multiparametric flow cytometry in a FACSCanto II acquiring at least 5 ×105 viable (Propidium Iodide low) lymphoid gated events, stained with MnAbs: CD8-FITC and CD3PE/APC MnAb. To identify leukemia-antigen specific CD8 lymphocytes we used class I HLA pentamers 0201 APC or PE conjugated (Proimmune, London, UK) against the following nonapeptides: Proteinase 1: VLQELNVTV (169-177) WT1: RMFPNAPYL (126-134) and SURV: ELTLGEFLKL (95-104). As positive staining control we used CMV pp65: NLVPMVATV (495-503) and as negative controls we used irrelevant nonapeptide and peripheral blood samples from patients lacking HLA* 0201 genotype. RESULTS Detection of donor-derived CD8+ lymphocytes against CMV pp65 occurred in 61% of recruited patients with a median percentage of 0.1% (range 0.03-13 over CD3+CD8+ events). Likewise, it was possible to detect CD8+ lymphocytes specific for PR1, WT1 and SURV in 65.2%, 47.8% y 39.1% of recruited patients respectively. Median percentage of PR1 and WT1 leukemia-antigen specific lymphocytes was 0.1% (range 0.04-1% over CD3+CD8+ events) and for WT1 0.1% (range: 0.01-0.2%). Detection of leukemia-antigen specific CD8+ lymphocytes was not significantly associated with clinical variables such as conditioning regimen (conventional or non-myeloablative), Disease status at transplant, donor type (sibling or unrelated), ATG use or HLA-disparity degree. The presence of WT1 specific CD8+ lymphocytes was significantly more frequent in patients undergoing allo-SCT for lymphoid hematological malignancy (p=0.04). By contrast, the presence of circulating anti-PR1 specific CD8+ lymphocytes was not more frequently found in patients undergoing allo-SCT for myeloid malignancies. Of note, none of the four patients who eventually relapsed harbored circulating leukemia-specific CD8+ lymphocytes. CONCLUSIONS Multiparametric flow cytometry is a useful tool to detect and quantify rare donor-derived CD8+ lymphocytes specific for leukemia-associated antigens as PR1, WT1 or SURV. The presence of these populations in peripheral blood is not associated to conventional clinical variables and in our series anti-WT1 CD8+ lymphocytes were more frequently detected in patients receiving allo-SCT for lymphoid malingnacies. By contrast, larger series are needed to assess if the lack of these leukemia-associated antigen-specific CD8 lymphocytes in peripheral blood could identify patients in a higher risk of relapse. Financial support This study was supported by a grant of Conserjeria de Salud, Junta de Andalucia 2006/0355. J. Serrano López is a post-doc fellow from Fundación Española de Hematología y Hemoterapia Disclosures No relevant conflicts of interest to declare.

Description: A small fraction of hematopoietic stem and progenitor cells (HSPCs) is cyclically released into the bloodstream from bone marrow (BM). The existence of HSCP-lymphatic-blood circulation with the potential to give rise to extramedullary tissue-resident myeloid cells has been described. However, the functional role and underlying mechanisms of this circulation are not known. We hypothesized that inflammation may be a mediator of HSPC mobilization to lymph. In order to determine if inflammation was related to increased circulation of HSPCs through lymph, we analyzed the HSPC content in lymph nodes (LN) of patients with lymphadenitis. Analysis of CFU and side population (SP+) cell content confirmed that LNs from patients with reactive lymphadenitis and patients with inflammatory Hodgkin's disease contained ~2-fold higher HSPCs than in patients diagnosed of non-Hodgkin's lymphoma. To identify the pattern and mechanisms that regulate HSPCs during inflammation. We analyzed the content of HSPCs in blood (PB), BM, LN, thoracic duct (TD) and other organs after systemic administration of lipopolysaccharide (LPS) and compared with the normal circadian migratory rhythms in control animals (PBS). Mobilization of BM myeloid progenitors (mostly granulo-macrophage progenitors-GMPs, but not stem cells), to LN and TD peaked as early as three hours after LPS administration, and followed a very different pattern than the mobilization kinetics in PB. By using 3D reconstruction of confocal microscopy imaging of complete LN from lymphatic endothelium reporter mice (Lyve1Cre-eGFP), we localized all myeloid progenitors in the mantle zone of secondary follicles. We note that they did not enter the follicle germ centers. To determine the molecular mechanisms at play to induce HSPC mobilization to the LN upon LPS administration, we analyzed whether the recruitment of HSPCs in LN required Traf6, a mediator downstream of the LPS/TLR4 signaling pathway. Inducible deficiency of Traf6 driven by Mx1-Cre recombinase completely abolished the migration of myeloid progenitors to LN, but not to PB, induced by LPS as compared with their Mx1-Cre;Wt littermates indicating that the mechanism controlling HSPC recruitment to the LN was distinct from the one controlling HSPC migration to PB. Using a combination of in vivo and in vitro assays, we found that Traf6 activity regulates HSPC migration in a non-cell autonomous manner that depends on its expression in a small (~1%) fraction of non-progenitor LN myeloid cells expressing high levels of Ccl19. In vivo neutralization of Ccr7, the receptor for Ccl19, results in abrogation of myeloid progenitor mobilization to LN. GMPs migrating to LN were biased to differentiate into dendritic cells within 7 days post-migration. Using Cx3cr1-GFP transgenic mice as macrophage-dendritic cell progenitor (MDP) reporters, we found that the vast majority of GMPs recruited to the LN were MDPs, and thus already poised to DC differentiation. Finally, a combination of genetic and pharmacological approaches revealed that Traf6 signaling effects are NFkB independent but dependent upon Irak1/4, Ucb13 and IKKβ resulting in SNAP23 phosphorylation and exocytosis of pre-formed cytokines from LN myeloid cells. This study identifies the cellular and molecular basis of inflammation dependent migration of DC progenitors and suggests that the mobilization of HSPCs from BM to LN results in homeostatic replenishment of highly specialized antigen presenting cells. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2645 Poster Board II-621 BACKGROUND AND AIMS: The PI3/Akt pathway has been implicated in the pathogenesis of a wide variety of cancers including Acute Myeloid Leukemia (AML). Activated Akt is known to function as an essential survival factor by inhibiting apoptosis through its ability to phosphorylate several targets including Bad, FoxO transcription factors, Raf-1, caspase-9 and inhibitor of apoptosis protein family (IAPs). Survivin is a member of IAP regulating both apoptosis and cell cycle progression. Survivin binds to several structural components of the mitotic apparatus and can block apoptosis by inhibiting caspases 9, 3 and 7. Recently the importance of PI3/Akt/survivin pathway in solid neoplasias such as breast or prostate cancer has been highlighted but its role in AML remains unknown. In this work we analyzed the PI3/Akt/survivin pathway in human AML. METHODS: Bone marrow samples obtained at diagnosis of 68 AML consecutive patients and K562, MV4-11 and HL-60 cell lines were included. Patients Median age was 62 years (range 8–89). Median leukocyte count was 10.3×109/L (range 0.8–285). FAB subtypes were: M0=8, M1=20, M2=13, M3=8, M4=11, M5=7 and M6=1 with the following cytogenetic findings: t(15;17)=8, t(8;21)=2, complex karyotype=8, 11q23=2, normal karyotype=36 and others=12. There were 11.3% patients with FLT3-ITD and 16.9% with NPM1 mutation. Cytoplasmic and nuclear Proteins were harvested with Q-proteome cell compartment (Qiaqen) and protein concentration assayed using Protein Assay Kit (Bio-Rad). Total Akt, Akt-pSer473 and survivin proteins were detected by Western Blot and were visualized by enhanced chemiluminescence (ECL-Plus, GE Healthcare) in Chemigenius-2 and quantified using Gene-Tools software. Cell cycle analysis was assessed by double Hoechst 33342- Pyronin Y staining and flow cytometry in FACSvantage. Inhibition experiments were done using Ly294002 at 25 μM, and Wortmaninn at 250 nM for 12 hours. RESULTS: In our series p-Ser473Akt was detected in 56% of AML marrow samples (with high levels expression in 27%) and in all cell lines. In cytoplasmic protein extracts, Survivin WT and 2B isoform were detected in 45% and 45.2% of patients respectively. All three leukemic cell lines showed only Survivin 2B expression. Interestingly, there was strong statistical correlation between the levels of p-Ser473Akt with cytoplasmic Survivin (P=.01). Inhibitors of PI3K/Akt pathway LY294002 and Wortmaninn both decreased in vitro p-Ser473Akt expression but only the irreversible action of Wortmaninn caused a marked dowregulation of cytoplasmic survivin. Meaningfully, lack of cytoplasmic Survivin was associated with an increased proportion of cells in Go cell cycle phase (11.1 % vs. 3.6%, P=.04). Moreover, cytoplasmic Survivin WT localization and high p-Ser473 Akt levels, were both significantly correlated with less unfavourable FAB leukemia subtype and cytogenetic risk (P

Description: A better understanding of how signaling pathways govern cell fate is fundamental to advances in cancer development and treatment. The initialization of different tumors and their maintenance are caused by the deregulation of different signaling pathways and cancer stem cell maintenance. Quiescent stem cells are resistant to conventional chemotherapeutic treatments and, consequently, are responsible for disease relapse. In this review we focus on the conserved Hedgehog (Hh) signaling pathway which is involved in regulating the cell cycle of hematopoietic and leukemic stem cells. Thus, we examine the role of the Hh signaling pathway in normal and leukemic stem cells and dissect its role in acute myeloid leukemia. We explain not only the connection between illness and the signaling pathway but also evaluate innovative therapeutic approaches that could affect the outcome of patients with acute myeloid leukemia. We found that many aspects of the Hedgehog signaling pathway remain unknown. The role of Hh has only been proven in embryo and hematopoietic stem cell development. Further research is needed to elucidate the role of GLI transcription factors for therapeutic targeting. Glasdegib, an SMO inhibitor, has shown clinical activity in acute myeloid leukemia; however, its mechanism of action is not clear.

Description: Innate immune cellular effectors are actively consumed during systemic inflammation but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to regional lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NFkB independent, Traf6/IkB-kinase/SNAP23 activation dependent, and is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205+/CD172a+ conventional dendritic cells type 2 (cDC2) and upregulation of BM myeloid progenitor Ccr7 signaling. Mature myeloid Traf6 signaling is anti-inflammatory and necessary for lymph node (LN) myeloid cell development. This report unveils the existence and the mechanistic basis of a very early direct traffic of myeloid progenitors from BM to lymphatics during inflammation.

Description: Millions of patients suffer yearly from bone fractures and disorders such as osteoporosis or cancer, which constitute the most common causes of severe long-term pain and physical disabilities. The intrinsic capacity of bone to repair the damaged bone allows normal healing of most small bone injuries. However, larger bone defects or more complex diseases require additional stimulation to fully heal. In this context, the traditional routes to address bone disorders present several associated drawbacks concerning their efficacy and cost-effectiveness. Thus, alternative therapies become necessary to overcome these limitations. In recent decades, bone tissue engineering has emerged as a promising interdisciplinary strategy to mimic environments specifically designed to facilitate bone tissue regeneration. Approaches developed to date aim at three essential factors: osteoconductive scaffolds, osteoinduction through growth factors, and cells with osteogenic capability. This review addresses the biological basis of bone and its remodeling process, providing an overview of the bone tissue engineering strategies developed to date and describing the mechanisms that underlie cell–biomaterial interactions.