Bone Marrow Cell Transplantation Research Articles

Allogeneic yet major histocompatibility complex-matched bone marrow transplantation in mice results in an impairment of osteoblasts and a significantly reduced trabecular bone

Secondary osteopenia following allogeneic bone marrow or stem cell transplantation (BMT or HSCT) is a significant source of morbidity in patients. It is believed to be caused by a number of factors related to the myeloablative conditioning and subsequent therapy regimen. We here aimed to investigate whether the allogeneic bone marrow by itself directly impacts on the bone mass of the patient. We thus performed syn- and allogeneic BMT between two inbred mouse strains, which share an identical major histocompatibility complex background yet differ in their bone phenotypes. BMT was well tolerated, yielded survival rates of 97% and allowed for a regular physiological development. However, allogeneic BMT led to a significant reduction of trabecular bone mass that was independent of strain, sex, immunosuppressive medication, complications resulting from graft versus host disease, underlying bone phenotype and numbers of osteoclasts. Instead, reduced trabecular bone mass correlated with reduced plasma levels of amino-terminal propeptide of type I collagen. Our results suggest that osteopenia following allogeneic BMT is significantly influenced by an impaired osteoblast activity that may stem from a lack of communication between the resident osteoblasts and an allogeneic bone marrow-derived cell type. Elucidating this incompatibility will open new approaches for the therapy of secondary osteopenia.

Abstract 1126: Potent inhibition of HCMV by modulating the cellular SNARE syntaxin 5

Abstract Human cytomegalovirus (HCMV), also referred to as human herpesvirus-5 (HHV-5), can cause serious and even fatal disease in immunocompromised individuals and newborns, namely individuals with AIDS, solid organ transplant recipients, chemotherapy patients and recipients of bone marrow and stem cell transplants. HCMV is a ubiquitous virus found throughout all geographic regions and socioeconomic groups, infecting greater than 50% of adults in industrialized countries and as many as 100% in developing countries. Although current therapeutics improves clinical outcomes, they are limited by toxicity, intravenous infusion and the development of resistance by the virus. Thus, there is a pressing need to develop novel therapeutics to prevent HCMV infections with concomitant organ toxicity. Formation of the cytoplasmic viral assembly compartment (cVAC) is an important step for efficient HCMV assembly. To do this, the virus must alter and repurpose the normal cellular balance of membrane and protein flux, a process that is not well understood. We have identified a compound Retro94, which potently inhibits production of infectious HCMV virions in cells by operating against a host cell process, rather than directly targeting the virus. The presence of Retro94 results in the severely impaired production of infectious virions, as great as 5 logs (99-99.99%). Here we discuss in vitro, in vivo, stability, and binding study of Retro94. Overall, our findings have identified Retro94, as novel agent that affects key cellular trafficking factors important for supporting HCMV infection. Citation Format: Dhimant H. Desai, Linda Cruz, Nicholas T. Streck, Trisha D. Desai, Aron Lukacher, Shantu G. Amin, Nicholas J. Buchkovich. Potent inhibition of HCMV by modulating the cellular SNARE syntaxin 5 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2017-1126

Combined Therapy with Shock Wave and Retrograde Bone Marrow-Derived Cell Transplantation for Osteochondral Lesions of the Talus

Multiple treatment strategies have been developed for osteochondral lesions (OCLs) of the talus. The purpose of this retrospective study was to assess retrograde autologous bone marrow cell (BMC) transplantation via core drilling (CD) combined with focused extracorporeal shock wave treatment (ESWT) in undisplaced OCL of the talus. A total of 69 patients with unilateral osteochondral lesions of the talus (Hepple grade I–III) were divided into two groups: 41 patients received combined therapy of ESWT and BMC transplantation (group A), while 28 were administered BMC transplantation alone (group B). The patients were followed up clinically and radiographically for a minimum of 2 years. Mean follow-up was 4.1 ± 2.8 years. AOFAS scores increased more significantly while pain intensity levels decreased in group A after treatment, compared with group B values (P < 0.001). In MRI follow-up, a more remarkable improvement of OCLs of the talus was observed in group A compared with group B (P = 0.040). Therefore, the combined technique reported here is a highly effective therapeutic option in OCLs of the talus with intact cartilage. It promotes patient recovery with pain control, and improves clinical outcome for more than 2 years after surgery.

Sphingosine-1-Phosphate Prevents Egress of Hematopoietic Stem Cells From Liver to Reduce Fibrosis

Background & AimsThere is growing interest in the use of bone marrow cells to treat liver fibrosis, however, little is known about their antifibrotic efficacy or the identity of their effector cell(s). Sphingosine-1-phosphate (S1P) mediates egress of immune cells from the lymphoid organs into the lymphatic vessels; we investigated its role in the response of hematopoietic stem cells (HSCs) to liver fibrosis in mice.MethodsPurified (c-kit+/sca1+/lin-) HSCs were infused repeatedly into mice undergoing fibrotic liver injury. Chronic liver injury was induced in BoyJ mice by injection of carbon tetrachloride (CCl4) or placement on a methionine-choline–deficient diet. Some mice were irradiated and given transplants of bone marrow cells from C57BL6 mice, with or without the S1P antagonist FTY720; we then studied HSC mobilization and localization. Migration of HSC lines was quantified in Transwell assays. Levels of S1P in liver, bone marrow, and lymph fluid were measured using an enzyme-linked immunosorbent assay. Liver tissues were collected and analyzed by immunohistochemical quantitative polymerase chain reaction and sphingosine kinase activity assays. We performed quantitative polymerase chain reaction analyses of the expression of sphingosine kinase 1 and 2, sphingosine-1-phosphate lyase 1, and sphingosine-1-phosphate phosphatase 1 in normal human liver and cirrhotic liver from patients with alcohol-related liver disease (n = 6).ResultsInfusions of HSCs into mice with liver injury reduced liver scarring based on picrosirius red staining (49.7% reduction in mice given HSCs vs control mice; P < .001), and hepatic hydroxyproline content (328 mg/g in mice given HSCs vs 428 mg/g in control mice; P < .01). HSC infusion also reduced hepatic expression of α-smooth muscle actin (0.19 ± 0.007-fold compared with controls; P < .0001) and collagen type I α 1 chain (0.29 ± 0.17-fold compared with controls; P < .0001). These antifibrotic effects were maintained with infusion of lymphoid progenitors that lack myeloid potential and were associated with increased numbers of recipient neutrophils and macrophages in liver. In studies of HSC cell lines, we found HSCs to recruit monocytes, and this process to require C-C motif chemokine receptor 2. In fibrotic liver tissue from mice and patients, hepatic S1P levels increased owing to increased hepatic sphingosine kinase-1 expression, which contributed to a reduced liver:lymph S1P gradient and limited HSC egress from the liver. Mice given the S1P antagonist (FTY720) with HSCs had increased hepatic retention of HSCs (1697 ± 247 cells in mice given FTY720 vs 982 ± 110 cells in controls; P < .05), and further reductions in fibrosis.ConclusionsIn studies of mice with chronic liver injury, we showed the antifibrotic effects of repeated infusions of purified HSCs. We found that HSCs promote recruitment of endogenous macrophages and neutrophils. Strategies to reduce SIP signaling and increase retention of HSCs in the liver could increase their antifibrotic activities and be developed for treatment of patients with liver fibrosis.

In vivo longitudinal visualization of bone marrow engraftment process in mouse calvaria using two-photon microscopy

Intravital microscopy of mouse calvarial bone marrow (BM) is a powerful method for studying hematopoietic stem cells (HSCs) and the BM microenvironment at the cellular level. However, the current method used to access the mouse calvaria allows for only a few imaging times in the same mouse because of scar formation and inflammation induced by multiple surgeries. Longitudinal imaging of the BM may help better understand its microenvironment. In this study, a mouse calvarial window model was developed for longitudinal imaging that involves attaching a cover glass window onto the mouse calvaria and sealing the surrounding exposed area with cyanoacrylate glue and dental cement. The model was used for the longitudinal two-photon microscopy (TPM) imaging of the BM engraftment process. The same BM cavity sites were imaged multiple times over 4 weeks after BM transplantation (BMT). Temporal changes in the BM microenvironment, such as the reconstitution of transplanted BM cells and the recovery of vasculature, were observed and analysed qualitatively and quantitatively. Longitudinal intravital microscopy using the mouse calvarial window model was successfully demonstrated and may be useful for further BM studies.

Challenges associated with carrier screening in patients with history of bone marrow or stem cell transplant

Challenges associated with carrier screening in patients with history of bone marrow or stem cell transplant

RB inactivation in keratin 18 positive thymic epithelial cells promotes non-cell autonomous T cell hyperproliferation in genetically engineered mice

Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia in vivo. To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice (TgK18GT121; K18 mice). We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.

Societal implications of medical insurance coverage for imatinib as first-line treatment of chronic myeloid leukemia in China: a cost-effectiveness analysis

Objectives: Imatinib (Glivec) and nilotinib (Tasigna) have been covered by critical disease insurance in Jiangsu province of China since 2013, which changed local treatment patterns and outcomes of patients with chronic myeloid leukemia (CML). This study evaluated the long-term cost-effectiveness of insurance coverage with imatinib as the first-line treatment for patients with CML in China from a societal perspective.Methods: A decision-analytic model based on previously published and real-world evidence was applied to simulate and evaluate the lifetime clinical and economic outcomes associated with CML treatments before and after imatinib was covered by medical insurance. Incremental cost-effectiveness ratio (ICER) was calculated with both costs and quality-adjusted life years (QALYs) discounted at 3% annually. Different assumptions of treatment benefits and costs were taken to address uncertainties and were tested with sensitivity analyses.Results: In base case analysis, both cost and effectiveness of CML treatments increased after imatinib was covered by the medical insurance; on average, the incremental QALY and cost were 5.5 and ¥277,030 per patient in lifetime, respectively. The ICER of insurance coverage with imatinib was ¥50,641, which is less than the GDP per capita of China. Monte Carlo simulation resulted in the estimate of 100% probability that the insurance coverage of imatinib is cost-effective. Total cost was substantially saved at 5 years after patients initiated imatinib treatment with insurance coverage compared to no insurance coverage, the saved cost at 5 years was ¥99,565, which included the cost savings from both direct (e.g. cost of bone marrow or stem cell transplant) and indirect costs (e.g. productivity loss of patients and care-givers).Conclusions: The insurance coverage of imatinib is very cost-effective in China, according to the local cost and clinical data in Jiangsu province. More importantly, the insurance coverage of imatinib and nilotinib have changed the treatment patterns of CML patients, thus dramatically increasing life expectancy and quality-of-life (QoL) saving on productivity losses for both CML patients and their caregivers.

Further Characteristics of Pregnant Вone Marrow Cells Action on Rat Development during Early Pregnancy

There were early results that effect of single intravenous transplantation of pregnant rats Percoll derived mononuclear bone marrow cells (BMC) suspention of 4-5, 7-9 or 11-12 pregnant days to rats with the same date of pregnancy depends on gestation period of transplantation. The weight of 18th day fetuses after intravenous transplantation during 7-9 days was increased with significant difference in comparison with weight of normal fetuses. In case of intravenous BMC transplantation during placentation at 11-12 days of pregnancy the weight of fetuses was decreased, the weight of placentas was increased and survival of fetuses was disturbed. There are several explanations of increase of weight of fetuses after intravenous BMC transplantation within gastrulation including paracrine effect of allogenic transplanted cells on the DC development, the apoptosis and proliferation of embryonal cells and possible epigenetic action. The retardation of fetuses growth after intravenous BMC transplantation during placenta formation at pregnant 11-12 days may be explained by cytotoxic action of uNK cells for embryo. At the same time the sub-dermal BMC transplantation at 11 and 13 pregnant days stopped the embryotoxic action of uNK cells and increased the weight of fetuses and preserved the survival of embryos.

Successful Hematopoietic Stem Cell Transplantation Using an Immunosuppressive Conditioning Regimen in Ten Patients with Severe Congenital Neutropenia: A Single-Institute Experience

Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by severe chronic neutropenia, with absolute neutrophil counts below 0.5×109/L, and by recurrent bacterial infections from early infancy. Granulocyte colony-stimulating factor (G-CSF) is widely used for the treatment of neutropenia in patients with SCN. However, the long-term G-CSF therapy has a relative risk of developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The only curative treatment available for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning (RIC) regimens have been applied to the treatment of SCN patients without malignant transformation who have become G-CSF refractory. However, the optimal conditions of HSCT for SCN patients have not been established. In this study, we conducted bone marrow cell transplantations (BMT) in ten patients with SCN using an immunosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital.Ten patients with a total of 11 HSCT procedures in our institution (performed from 2007 to 2015) were enrolled in this study. Four of the ten patients had experienced engraftment failure of the initial HSCT and three of them were referred to our hospital for re-transplantation. Heterozygous mutation inthe ELANE gene was identified in nine of ten patients. These nine patients received BMT less than 10 years of age. All ten patients had recurrently experienced moderate to severe bacterial or fungal infection before HSCT and received temporal or regular administration of G-CSF. Bone marrow cells (BM) were obtained from five HLA-matched related (MRD), three HLA-matched unrelated (MUD), and three HLA-mismatched unrelated (7/8) donors (MMUD), respectively. The conditioning regimen basically consisted of fludarabine (100 to 125 mg/m2), cyclophosphamide (100 to 150 mg/kg), melphalan (70 to 90 mg/m2), total body irradiation (3 to 3.6 Gy), and/or anti-thymocyte globulin (10 to 12 mg/kg). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was successfully observed within 24 days of post-transplantation in all patients. All patients achieved complete chimerism at the time of engraftment. Two patients who underwent BMT from MRD and one patient who underwent BMT from MUD showed the gradual decrease of donor-derived cells. Donor lymphocyte infusion treatment successfully achieved the complete chimerism or stable mixed chimerism in these 3 patients. Although 3 patients experienced the acute GVHD (Grade I-II), the addition of glucocorticoids to tacrolimus prevented the extension of acute GVHD. Only one patient developed mild chronic GVHD presenting limited type of skin involvement. All patients are alive for 9 months to 9 years after HSCT with no signs of severe infections or transplantation-related morbidity.Our results demonstrate that BMT together with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. Although our results through the small number of cohort is limited to conclude, the BMT with the optimal donors may lead to the increased opportunity for lower risk of SCN patients especially at younger age as a curative treatment. The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. DisclosuresNo relevant conflicts of interest to declare.

Role of the Polycomb Methyltransferase Ezh1 in Myelodysplastic Syndrome Induced By Ezh2 Insufficiency

Ezh1 and Ezh2, the catalytic components of polycomb-repressive complex 2 (PRC2), negatively control gene expression by catalyzing mono, di, and tri-methylation of histone H3 at lysine 27 (H3K27me1/me2/me3). Loss-of-function mutations of EZH2, but not those of EZH1, have been found in patients with hematologic malignancies such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), and MDS/MPN overlap disorders.We previously demonstrated that hematopoietic cell-specific Ezh2 knockout mice (Ezh2Δ/Δ) developed hematologic malignancies including MDS and MDS/MPN. Although deletion of Ezh1, another enzymatic component of PRC2, (Ezh1-/-) did not significantly affect global H3K27me3 levels or hematopoiesis, deletion of both Ezh1 and Ezh2 in mice (Ezh1-/-Ezh2Δ/Δ) caused rapid exhaustion of hematopoietic stem cells (HSCs). Given that only Ezh1 and Ezh2 are known as enzymatic components of PRC2, we concluded that residual PRC2 enzymatic activity is required for HSC maintenance and development of hematologic malignancies in the setting of EZH2 insufficiency frequently observed in MDS. However, the role of Ezh1 in Ezh2-insufficient hematologic malignancies is still not fully understood since hematopoiesis could not be maintained in Ezh1-/-Ezh2Δ/Δ mice. Here we analyzed the impact of Ezh1 heterozygosity on Ezh2-null hematopoiesis (Ezh1+/-Ezh2Δ/Δ), in which PRC2 activity is mediated by a single allele of Ezh1, for better understanding of Ezh2-deficient hematologic malignancies.We first transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice with CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Ezh1+/-Ezh2Δ/Δ cells exhibited a lower repopulation capacity than Ezh2Δ/Δ but established persistent repopulation for at least 6 months after the deletion of Ezh2 while double knockout cells (Ezh1-/-Ezh2Δ/Δ) were outcompeted by competitor cells immediately. We next transplanted BM cells from Ezh1+/-Ezh2flox/flox CD45.2 mice without CD45.1 wild-type competitor cells into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen. Importantly, recipient mice reconstituted with Ezh1+/-Ezh2Δ/Δ cells exhibited MDS-like phenotypes including anemia and morphological myelodysplasia, which were more pronounced than those of Ezh2Δ/Δ mice. Ezh1+/-Ezh2Δ/Δ mice also showed more advanced hematological abnormalities such as erythroid differentiation block, increased apoptosis of erythroid cells, and extramedullary hematopoiesis in the spleen than Ezh2Δ/Δ mice did. These results suggest that Ezh1 heterozygosity promotes the development of myelodysplasia in the setting of Ezh2insufficiency.Next we examined the molecular mechanism by which the loss of Ezh1 promotes myelodysplasia. Western blot and ChIP-sequence analyses revealed that global levels of H3K27me3 were not significantly changed but H3K27me3 levels at promoter regions of the PRC2 target genes were obviously reduced by Ezh1 heterozygosity in Ezh2Δ/Δ HSPCs. As a consequence, PRC2 target genes were highly de-repressed in Ezh1+/-Ezh2Δ/Δ LSK HSPCs compared with Ezh2Δ/Δ HSPCs. Among these, several genes appeared to be associated with MDS such as S100A9, encoding an inflammatory protein implicated in dyserythropoiesis in MDS. Furthermore, gene set enrichment analysis showed that the genes highly expressed in myeloid cells were positively enriched by Ezh1 heterozygosity in Ezh2Δ/ΔHSPCs.These findings indicate that dosage of Ezh1 is critical in the maintenance of Ezh2-insufficient hematopoiesis as well as the progression of MDS with Ezh2 insufficiency. DisclosuresNo relevant conflicts of interest to declare.

Unraveling the Erythroid Function of Tfr2 in Beta-Thalassemia

Transferrin receptor 2 (TFR2), a liver activator of the iron hormone hepcidin mutated in hemochromatosis type 3 (Camaschella C. et al., Nat Genet 2000), is an erythropoietin (EPO) receptor partner in erythroid cells (Forejtnikovà H. et al., Blood 2010). We have recently shown that the loss of bone marrow (BM) Tfr2 increases erythroblast EPO sensitivity and mimics a condition of mild iron-deficiency in mice (Nai A. et al., Blood 2015). β-thalassemias are iron-loading anemias due to recessive mutations in the β-globin gene, characterized by severe ineffective erythropoiesis. Since several studies demonstrated that iron restriction ameliorates the β-thalassemia phenotype, we generated thalassemic mice (Hbbth3/+) with selective BM inactivation of Tfr2 (Tfr2BMKO/Hbbth3/+) through transplantation of BM cells (BMT) from Hbbth3/+ and Tfr2-/-/Hbbth3/+ mice into lethally irradiated wt recipients. Two months after BMT Tfr2BMKO/Hbbth3/+ mice have Hb levels higher than Hbbth3/+ (Hbbth3/+: 9.47±0.59 g/dL; Tfr2BMKO/Hbbth3/+: 11.22±0.47 g/dL; p<0.0001). In contrast, 4 months after BMT Tfr2BMKO/Hbbth3/+ mice have more severe anemia, ineffective and extramedullary erythropoiesis, increased iron in serum (TS) and liver (LIC), reduced spleen iron (SIC), high hepcidin levels, increased Epo-target gene (Epor, Bcl-xL, Erfe) expression in BM and high levels of serum Erfe. These results suggest that deleting Tfr2 in thalassemic erythroblasts increases their Epo sensitivity as occurs in normal ones. This effect is positive during BM repopulation, but transient: on a long-term a severe phenotype develops, similar to the one observed in thalassemia major models (Rivella S. et al., Blood 2003; Huo Y. et al., Blood 2009), likely induced by excessive EPO stimulation of ineffective immature erythropoiesis. We speculate thatin Tfr2BMKO/Hbbth3/+ mice the continuous production of Erfe, the hepcidin inhibitor released by the expanded marrow (Kautz L. et al., Nat Genet 2014), worsens iron-overload, further damaging the thalassemic erythropoiesis. In order to exclude any interference of BMT, we analyzed also Hbbth3/+, Tfr2+/-/Hbbth3/+ and Tfr2-/-/Hbbth3/+ mice, lacking germ-line Tfr2 at the heterozygous and homozygous state respectively. Preliminary data indicate that young Tfr2-/-/Hbbth3/+ mice have less severe anemia than Hbbth3/+, while Tfr2+/-/Hbbth3/+ animals have an intermediate phenotype, suggesting that Tfr2 haploinsufficiency may be sufficient to improve the ineffective erythropoiesis at short time point. Follow up analysis will test whether the phenotype of Tfr2-/-/Hbbth3/+ mice worsens with aging as occurs in transplanted animals. Interestingly we noticed that 2-month-old Tfr2-/-/Hbbth3/+ mice, beside higher Hb levels, have higher LIC than thalassemic littermates, with comparable SIC. This observation lead us to hypothesize that low SIC, rather than high LIC, contributes to the worsening of the phenotype in Tfr2BMKO/Hbbth3/+ mice 4 months after BMT, likely impairing the compensatory spleen erythropoiesis. To dissect causality between high LIC and low SIC we are challenging transplanted mice with iron-deficient diet to decrease LIC or with iron-dextran injection to increase SIC. Moreover, a group of animals sacrificed 2 months after BMT will help to elucidate the mechanism(s) of the early amelioration of anemia. Preliminary results confirm the Hb and red cells count increase in Tfr2BMKO/Hbbth3/+ as compared to Hbbth3/+ mice 2 months after BMT without increased spleen size, thus suggesting a real erythropoiesis improvement at this time point. Erythroid cells at the different stages of maturation sorted from these animals will be subjected to RNAseq analysis in comparison to cells isolated from animals at the time of phenotype worsening. The results are expected to define the pathway/s altered by the loss of Tfr2 in thalassemic erythroid cells and those responsible for the amelioration of the phenotype with the aim of identifying potential novel target/s for therapy of β-thalassemia. DisclosuresNo relevant conflicts of interest to declare.

An Altered Bone Marrow Vascular Niche Impacts Normal Hematopoiesis and Tilts the Balance Between Myelopoiesis and Lymphopoiesis

The bone marrow niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained to ensure their lifelong contribution to hematopoiesis. Recent evidence has highlighted the critical importance of the perivascular bone marrow (BM) niche as the key host and regulator of HSPCs. Bone marrow endothelial cells (BMECs) are major components of the vascular niche. While studies have shown that an alteration in a component of the niche can affect hematopoiesis and promote the development of myeloproliferative disorders/myelodysplastic syndromes, it remains unclear how altered BMECs can impact hematopoiesis. To this end, we have generated a Tamoxifen (TAM)-inducible Tie2-CreER/LSL-KRasG12D;tdTomato mouse model to introduce an oncogenic KRas mutation specifically in adult endothelial cells. The tdTomato reporter overlaps with the CD31 and vascular endothelial growth factor receptor 2 (VEGFR2) endothelial cell markers and shows no detectable leakage into the adult hematopoietic compartment. To evaluate changes in hematopoiesis, we performed complete blood counts at 12 weeks post TAM injection and found that the Tie2-CreER/LSL-KRasG12D mice (KRasG12D mice) had significantly more leukocytes (p=0.031) and neutrophils (p=0.002) than controls. Flow cytometry analysis confirmed that the KRasG12D mice had a significantly higher percentage of myeloid cells with concurrent decrease in lymphocyte percentage in the peripheral blood (p=0.016). At 16 weeks post TAM injection, a significant decrease in B cells could also be noted in the blood of KRasG12D mice (p=0.028). Compared to controls, the KRasG12D mice displayed splenomegaly (p=0.025) and their spleens had a higher percentage of myeloid cells (p=0.002). There was an increase in the common myeloid progenitor compartment in the spleen and a significant increase in the granulocyte macrophage progenitor compartment (p=0.014) of KRasG12D mice. These mice also had an increase in the short-term hematopoietic stem cell (ST-HSC) compartment both in the BM and spleen. Colony forming assays revealed that KRasG12D mice had a higher number of total colonies formed from BM (p=0.044), spleen (p=0.007) and blood cells (p=0.56). Genotyping PCR showed no KRasG12D activation in hematopoietic cells, confirming that the observed phenotypes were due to an effect in BMECs. To complement our native inducible mouse model, we transplanted BM cells from syngeneic BoyJ mice into lethally irradiated Tie2-CreER;KRasG12D or KRasWT recipients. The endothelial KRasG12D recipientsdied between 75-200 days post transplantation (p=0.0079) while the KRasWT recipients remained alive. The KRasG12D recipients also displayed splenomegaly (p=0.004). Competitive transplant studies with donor cells from KRasG12D or KRasWT mice with competitor cells from syngeneic mice (CD45.1) showed that BM cells from the KRasG12D mice (CD45.2) outcompeted cells from KRasWT mice with a significantly higher percentage of CD45.2 donor chimerism in all blood lineages examined. To uncover any molecular events underpinning these hematopoietic changes, we performed quantitative real-time polymerase chain reaction. Our preliminary experiments from total BM RNA of KRasG12D or KRasWT mice indicate that there is a significant increase in VEGFα and a decrease in transforming growth factor β in KRasG12D mice, accompanying the above noted increase in the ST-HSC population. Collectively, our data provide strong evidence that an abnormal vascular niche caused by oncogenic insults in BMECs can disrupt normal hematopoiesis and promote a myeloproliferative phenotype, thereby implicating abnormal BMECs as novel contributors to blood pathogenesis. Studies are underway to further assess the molecular contributions from the disrupted vascular niche and the resulting HSPCs. Uncovering the mechanism of how altered BMECs can remodel hematopoiesis holds the exciting promise of better therapeutic strategies. DisclosuresNo relevant conflicts of interest to declare.

CXCR4-SDF-1 Signaling in Nestin+ Mesenchymal Stem Cell Is Required for HSC Maintenance during Homeostasis and Regeneration after Irradiation

Hematopoietic stem cells (HSC) reside in a complex microenvironment (niche) within the bone marrow (BM), where multiple populations of microenvironmental stromal cells regulate and finely tune their proliferation, differentiation and trafficking. Recent studies have shown that mesenchymal stem cells (MSC) are an essential component of the HSC niche. Intrinsic HSC CXCR4-SDF-1 signaling has been implicated in self-renewal and quiescence; however, the role of microenvironment CXCR4-SDF-1 signaling in supporting HSC function remains unclear. We previously demonstrated that microenvironmental stromal cell-derived CXCR4 is important for HSC recovery, as transplantation of wild-type HSC into CXCR4 deficient recipients showed reduced HSC engraftment. In this study, we now show that CXCR4-SDF-1 signaling in nestin+ MSC regulates HSC maintenance under normal homeostatic conditions and promotes hematopoietic regeneration after irradiation. Multivariate flow cytometry analysis of marrow stroma cells revealed that mouse BM MSCs identified as CD45-Ter119-CD31-Nestin+PDGFR+CD51+ express the CXCR4 receptor, which was confirmed by RT-PCR analysis. To investigate the role of MSC CXCR4 signaling in niche maintenance and support of HSC function, we utilized genetic mouse models, in which CXCR4 could be deleted in specific stromal cell types. Selective deletion of CXCR4 from nestin+ MSC in adult tamoxifen inducible nestin-cre CXCR4flox/flox mice resulted in reduced total MSC in BM (Control vs. Deleted: 647±128 vs. 209±51/femur, respectively, n=5, p&lt;0.05), which was associated with a significant reduction in Lineage-Sca-1+c-Kit+ (LSK) cells (Control vs. Deleted: 18,033±439 vs. 4523±358/femur, respectively n=5, p&lt;0.05). Selective CXCR4 deletion in nestin+ MSC also resulted in enhanced LSK cell egress to the peripheral circulation (Control vs. Deleted: 1022±106 vs. 2690±757/ml blood, respectively n=5, p&lt;0.05), with no detectable difference in HSC cell cycle or apoptosis. However, the repopulation ability of HSC obtained from mice where CXCR4 was deleted in nestin+ MSC was reduced by &gt;2 fold. In contrast, deletion of CXCR4 from osteoblasts using osteocalcin cre CXCR4flox/flox mice had no effect on HSC numbers in BM and blood.To investigate the role of nestin+ MSC CXCR4 signaling in BM niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or nestin+ MSC CXCR4 deleted recipients after lethal irradiation (950 rad) and analyzed HSC homing, niche recovery and hematopoietic reconstitution. Deletion of CXCR4 from nestin expressing MSC resulted in significantly reduced LSK cell homing at 16 hrs post transplantation (Control vs. Deleted: 8643±1371 vs. 3004±1044/ mouse, respectively, n=5, p&lt;0.05). Robust apoptosis and senescence after total body irradiation was observed in nestin expressing MSCs lacking CXCR4 expression. At 15 days post-transplantation, chimeric mice with nestin+ MSC lacking CXCR4 expression displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. In conclusion, our study suggests that CXCR4-SDF-1 signaling in nestin+ MSC is critical for the maintenance and retention of HSC in BM during homeostasis and promotes niche regeneration and hematopoietic recovery after transplantation. Furthermore, our data suggest the modulating CXCR4 signaling in the hematopoietic niche could be beneficial as a means to enhance HSC recovery following clinical hematopoietic transplantation or radiation/chemotherapy injury. Disclosures No relevant conflicts of interest to declare.

TRAF6 Is Essential for Maintaining Hematopoietic Stem Cell Homeostasis

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. DisclosuresNo relevant conflicts of interest to declare.

HDAC8 Regulates Long-Term Hematopoietic Stem Cell Quiescence and Maintenance

Long-term (LT) hematopoietic stem cells (HSC) are responsible for life-long production of mature blood cells of all lineages through tightly concerted cell fate decisions including quiescence, self-renewal, differentiation and apoptosis. Histone deacetylase 8 (HDAC8) is a member of class I HDAC enzymes that remove acetyl moieties from lysine residues on histones and a variety of non-histone proteins. Specifically, HDAC8 has been shown to modulate the acetylation cycle of cohesin complex protein SMC3. Loss-of-function mutations in HDAC8, located on the X chromosome q13, have been found in patients with Cornelia de Lange Syndrome (CdLS) and those with CdLS-like features. These HDAC8 mutations are associated with severely skewed X-inactivation (100% wild type allele) in the peripheral blood of female patients, possibly due to selection against the mutant alleles. However, the expression and function of HDAC8 in normal HSCs and hematopoiesis remain unknown. In this study, we show that Hdac8 is highly expressed in the phenotypic LT-HSC (Lin-cKit+Sca1+CD150+CD48-) population in adult mice. To determine the function of HDAC8 in adult hematopoiesis, we generated conditional Hdac8 deficient mice using the Mx1-Cre and a floxed Hdac8 allele (Mx1-Cre/Hdac8f/f(y)) andconfirmed that Hdac8 is successfully deleted by polyinosinic-polycytidylic acid [poly (I:C)] treatment. Phenotypic analysis of Hdac8 deficient mice showed increased LT-HSC population compared to similarly treated control mice. However, largely normal steady state hematopoietic profile was found in Hdac8 deficient mice at 6 weeks and 1 year after induction. To further track Hdac8-deleted cells, we generated Cre/Hdac8f/f(y) mice with a dual fluorescence Rosa26mT/mG (mT/mG) Cre reporter allele, which expresses dTomato prior to Cre induction and becomes GFP+ after Cre-mediated recombination. We assessed hematopoietic repopulation by transplanting bone marrow cells from Cre/Hdac8f/f(y)/mTmG+ mice (2 x 105) together with wild type support cells (2 x 105) into lethally irradiated CD45.1+ congenic recipients. Hdac8 deletion was induced by treating the recipients with 7 does (14 m□g/kg/dose) of poly (I:C). We found that Hdac8 deletion did not affect CD45.2+ or GFP+ donor-derived overall engraftment or lineage repopulation up to 16 weeks. There was also no change in the frequency or number of GFP+ donor-derived phenotypic LT-HSCs in the bone marrow. Serial transplantation was performed to further assess long-term repopulating activity of HSCs. Hdac8 deficient cells were significantly (p=0.019; n=3) compromised in multi-lineage repopulation in secondary transplant recipients. Except a modest reduction in Pre-GM, there was no change in the overall composition of Hdac8 deficient CD45.2+-derived populations. Upon tertiary transplantation, no donor engraftment was observed for Hdac8 deficient cells (0 out of 4) compared to 50% positive engraftment in control group (4 out of 8). These results indicate that HDAC8 is crucial for maintaining long-term serial-repopulating activity over time. Cell cycle analysis revealed that Hdac8 deficient LT-HSCs display reduced quiescence and increased cycling, consistent with the increased number of phenotypic LT-HSC seen in Hdac8 deleted mice. Therefore, we further tested the sensitivity of Hdac8 deficient mice to serial ablation with 5-fluorouracil (5FU), an S phase-specific cytotoxic chemotherapeutic agent. Impaired hematopoietic recovery and increased lethality (p<0.001; n=23) was seen in Hdac8 deficient mice treated with 5-FU (100 mg/kg) every 7 days, indicating that Hdac8 deletion renders hypersensitivity to serial ablation. There were significnatly less phenotypic LT-HSCs in Hdac8 deficient mice 6 days after 5-FU treatment (p<0.01; n=4). In parallel, we observed increased DNA strand beaks as indicated by γ-H2AX staining and comet assays (p<0.001; n>100 cells). Analysis of p53 activation, cell cycle regulators and DNA dmage response are ongoing. Collectively, our study indicates that HDAC8 plays a pivotal role in LT-HSC quiescence and maintenance. DisclosuresNo relevant conflicts of interest to declare.

Completely Non-Myeloablative/Non-Lymphoablative Conditioning for BMT/HSCT Using Anti-Ckit Immunotoxins

Bone marrow/hematopoietic stem cell transplantation (BMT/HSCT) holds the remarkable ability to correct any blood or immune disease. Unfortunately, despite the tremendous potential of this procedure, BMT remains fairly limited in part due to the severe risks associated with the toxic conditioning regimens, such as irradiation and chemotherapy that are currently employed to enable donor HSC engraftment. Although significant work has been done to dose reduce the amount of these preparative agents, patients still experience many side effects including neutropenia/infections, anemia, mucositis, infertility, organ damage and secondary malignancies. Complete elimination of these toxic conditioning regimens could dramatically improve the safety profile of BMT and expand the potential applications to include many more non-malignant hematologic disorders, a wide variety of autoimmune disorders including diabetes, as well as facilitate solid organ tolerance.We have previously shown that competition with host HSC limits donor HSC engraftment, and that in immunocompromised hosts antagonistic anti-ckit monoclonal antibodies deplete host HSC and are an effective and safe alternative conditioning approach (Czechowicz, Science 2007). However, this modality of conditioning is not effective in hosts with competent immune systems. To further understand efficacy of antagonistic anti-ckit conditioning, we tested its functionality in multiple strains of immunocompromised mice and show that inhibition of SCF signaling is not sufficient to deplete host HSC in mouse strains with competent B-cells or T-cells, and that the addition of these cells interferes with the ability of antagonistic anti-ckit antibodies to effectively condition. In an attempt to overcome this hurdle, wildtype mice were immune-depleted with a variety of regimens but none enabled antagonistic anti-ckit conditioning in the immunocompetent setting.To strengthen the potency of anti-ckit mAbs we linked them to protein synthesis toxins, which when internalized by host HSC led to their rapid decline in vitro and in vivo. Administration of anti-ckit-saporin to wild-type mice resulted in >99% depletion of host HSC (Ckit+Lin-Sca1+CD150+CD48-), and lack of residual host HSC activity in the bone marrow was confirmed by CFC assays and competitive transplantation into lethally irradiated recipients. Interestingly, although ckit is expressed by a majority of HSPC, LT-HSC were most significantly affected and no cellularity changes in the bone marrow were observed. Uniquely this regimen was entirely non-peripheral blood ablative unlike other more broadly targeted conditioning regimens such as CD45 immunotoxins (Palchaudhuri, Nat Biotech 2016), and treated animals did not experience any significant depletion of myeloid, lymphoid, or erythroid cells. [Display omitted] Treatment with anti-ckit-saporin effectively conditioned wild-type animals and near complete donor granulocyte chimerism was rapidly achieved post transplantation of whole bone marrow cells (99.54 ± 0.35 % vs. 6.79 ± 0.57 %, p<0.001), a >25-fold increase compared to unconditioned controls. Similarly, anti-ckit-saporin conditioning enable efficient engraftment of FACS purified donor HSC (Ckit+Lin-Sca1+CD150+CD48-). In both settings, donor HSC chimerism matched donor granulocyte chimerism further confirming replacement of host HSC. Importantly, host immunity was entirely intact in these animals throughout, with slower recalibration of the longer-lived immune cells given the lack of their direct depletion. [Display omitted] This work sets the stage for redefining the way BMT/HSCT is performed, as it opens up the possibility for entirely safe, quick and easy transplantation that potentially could be done in the outpatient setting with no perturbation to host immunity. Extrapolation of these methods to humans may enable efficient yet gentle conditioning regimens for transplantation, which is especially exciting in the gene-therapy settings where no immune suppression is required, allowing for simple, safe and curative treatment of a wide magnitude of grievous blood and immune diseases ranging from sickle cell to hemophilia to HIV. As multiple anti-ckit mAbs are currently in development and being tested in clinical trials, such an approach may be rapidly translatable to patients. DisclosuresCzechowicz:Third Rock Ventures: Consultancy; Global Blood Therapeutics: Equity Ownership; Editas Medicines: Equity Ownership, Patents & Royalties; Decibel Therapeutics: Equity Ownership; Magenta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; Forty Seven Inc: Patents & Royalties. Palchaudhuri:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoggatt:Magenta Therapeutics: Consultancy, Equity Ownership, Research Funding. Scadden:Teva: Consultancy; Apotex: Consultancy; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Dr. Reddy’s: Consultancy; GlaxoSmithKline: Research Funding; Fate Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Bone Therapeutics: Consultancy. Rossi:Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Intellia Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Moderna Therapeutics: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

Alox5 Functions As Both Tumor Suppressor and Drug Sensitizer in AML

IntroductionAcute myeloid leukemia (AML) is a heterogeneous disease characterized by the malignant expansion of dysfunctional myeloid progenitors and the suppression of normal hematopoiesis. Despite of the intensive efforts in developing novel therapeutic strategies in the past decades, the standard chemotherapy, i.e., the "5+3" regimen, is still the main approach for treating AML, and resistance to the standard chemotherapy remains the major cause of relapse and therapeutic failure. Therefore, it is crucial to understand the mechanisms underlying AML pathogenesis and chemotherapy resistance, and thus identify new therapeutic targets.The arachidonate5-lipoxygenase gene (Alox5) is known to be involved in various physiological and pathological processes, including oxidative stress response, inflammation and cancer. Previous reports show that loss of Alox5 impairs the function of leukemic stem cells in BCR-ABL-induced chronic myelogenous leukemia (CML), as well as the long-term self-renewal of hematopoietic cells of AML with RUNX1-ETO9a, MLL-AF9 and PML-RARA. To our surprise, our Affymetrix microarray-based, genome-wide gene expression profiling of primary AML patient samples reveals that Alox5 is expressed at a particularly lower level in certain AML subtypes (e.g., MLL-rearranged AML) that are associated with poor prognosis than in AML subtypes (e.g., t(8;21) and inv(16) AMLs) that are associated with favorable prognosis. The aim of this study is to identify the influence and mechanism of Alox5 overexpression in AML pathogenesis and therapeutic response.MethodsTo assess the potential effect of Aloxon leukemic cell viability and drug response/resistance, AML cell lines with stable ectopic expression of Alox5 or control plasmids induced by lentivirus were treated with DMSO control, doxorubicin (DOX) or cytarabine (Ara-C). Cell viability and proliferation assays, i.e., MTT assays, were performed. To determine the influence of Alox5 on the transformation capacity, viability and drug response of mouse BM progenitor cells bearing MLL-AF9, colony-forming/replating assay (CFA) and MTT assay were conducted. To evaluate the effect of Alox5 on AML progression and chemotherapeutic response in vivo, we first carried out primary bone marrow transplantation (BMT) by use of hematopoietic progenitor cells retrovirally co-infected with MLL-AF9 and Alox5 or empty vector as primary donors, and then applied the leukemic blast cells isolated from the primary leukemic recipients as donor cells in a secondary BMT followed by "5+3" chemotherapy.Gene Set Enrichment Analysis (GSEA) was used to analyze the signal pathway enrichments in the BMT samples based on RNA sequencing results.ResultsIn AML cell lines, lentivirus induced overexpression of Alox5 resulted in a moderate repression of cell viability, while significantly sensitizing the cells to DOX and Ara-C treatment. In mouse BM progenitor cells with MLL-AF9, forced expression of Alox5 remarkably enhanced the inhibitory effect of DOX and Ara-C on the colony forming capacity and cell viability.Secondary BMT assay results showed that overexpression of Alox5 inhibited MLL-AF9-induced AML progression (MLL-AF9+Alox5+Ctrl, with medium survival of 44.5 days; MLL-AF9+Ctrl, with medium survival of 39.5 days; P=0.001) in vivo. The "5+3" chemotherapy alone indeed improved survival as compared with MLL-AF9+Ctrl (MLL-AF9+DOX/Ara-C, with medium survival of 50 days; P<0.001). The therapeutic effect of DOX+Ara-C treatment was remarkably enhanced by Alox5 overexpression, and the medium survival was prolonged to >200 days (MLL-AF9+Alox5+DOX/Ara-C) (P<0.001) (Fig. 1A).RNA-seqand GSEA suggested that overexpressing Alox5 activates the E2F signaling and oxidative stress response, while repressing the K-ras and Stat pathways (Fig. 1B).ConclusionsOur results suggest that while forced expression of Alox5 shows a moderate tumor suppressor role in the progression of MLL-AF9-mediated AML, it significantly enhanced the effect of the standard "5+3" chemotherapy both in vitro and in vivo. The potential mechanism is through targeting E2F, oxidative stress response, K-ras and Stat signaling pathways, which were all known to be closely associated with tumorigenesis and drug response/resistance. Our study indicates the restoration of Alox5 in combination with the classic chemotherapy as a potential novel therapeutic strategy in treating AML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Time Points, Dynamics and Clinical Impact of Minimal Residual Disease (MRD) Monitoring in AML with NPM1 mutation: A Study on 428 Patients

Abstract Introduction: Investigation of minimal residual disease (MRD) using NPM1 as a target has been proven to be of importance in AML. Guidelines for best schedules and implication on clinical use need to be defined. Aims: To better define the clinical impact and to suggest strategies for MRD monitoring in AML with NPM1 mutation. Patients and Methods: Between 2005 and 2015 we investigated 428 AML patients (pts) with NPM1 mutation at diagnosis and at a minimum of 2 follow-up time points. All pts had to achieve at least once a complete molecular remission (CMR) to be considered for this study. Sensitivity for MRD detection was at least 1:10,000. The median age of the cohort was 57 years (range: 18-85 yrs) and comprised of 198 males and 230 females. 3,039 samples (median number of samples per pts: 7, range: 2-35) were studied during course of disease. Molecular techniques applied included gene scan, sequencing and quantitative real-time PCR at diagnosis and quantitative real-time PCR during follow-up. Median time between 2 investigations was 2.8 months (mo; range: 0.3-71.0 mo). All pts were treated with standard protocols according to genotype and age. Allogeneic bone marrow or stem cell transplantation was performed in 136 pts (31.8%). Results: NPM1 type A mutation was the most frequent mutation type (317/428, 74.1%), followed by type B and D (36/428, 8.4% and 23/428, 5.4%), respectively. 25 other NPM1 types occurred at frequencies between 0.2 and 3.7%, in total demonstrating the expected distribution of NPM1 mutation types in an adult AML cohort. Subgroups of these pts were analyzed for FLT3-ITD (n=421) and mutations in DNMT3A (n=236). 122/421 (29%) pts showed a FLT3-ITD. In 96/236 (41%) DNMT3A was mutated. Further in 33/235 (14%) both genes were mutated. 103/235 (44%) screened for all three genes had a sole NPM1 mutation. All sole NPM1 mutated study pts achieved the CMR after a median of 4.1 mo (range: 1.0-8.6 mo). The presence of an additional DNMT3A mutation (CMR after a median of 4.4 mo, range 1.0-8.7) or a FLT3-ITD (CMR after a median of 2.7 mo, range 1.0-8.7) or both mutations (CMR after a median of 4.1 mo, range 1.1-7.9 mo) had no influence on time to achieve CMR. After achievement of CMR an increase of NPM1 ratio was detected in 185/428 (43%) pts. The median time to loss of CMR was 5.1 mo (range: 0.4-88 mo). In more detail, 42/185 of these patients also had FLT3-ITD, 53/109 had DNMT3A mutations and 13/109 had mutations in both genes. Patients with a DNMT3A mutation showed more often loss of CMR (40/60, 67%), while FLT3-ITD and FLT3-ITD/DNMT3A mutated patients showed no significant influence on loss of CMR ratio (46% and 48%, respectively) maybe due to number of cases. In 152/185 molecular relapses further follow up samples after loss of CMR were available. The median time between detected loss of CMR and the next follow-up sample was 2.0 mo. Due to treatment intervention 46/152 patients achieved a second CMR and 27/152 a decrease in NPM1 ratio. However, in 79/152 a further increase leading to clinical relapse was observed. The increase after loss of CMR was in median 13-fold between first and second sample after CMR was lost. Importantly, keeping periods between two MRD samplings at an interval of 3 mo allowed the detection of nearly all cases of first relapse at the molecular level. Addressing the sensitivity levels of the assays applied to bone marrow (BM) versus peripheral blood (pB) samples showed a 1.6 fold higher sensitivity for BM samples (median copies of reference gene, 14,628 vs 9,363). Due to the comparable sensitivities pB can be investigated until a first increase on the molecular level is detectable, followed by BM sampling for confirmation 4 weeks later. Conclusions: 1) NPM1 has proven to be a good marker for MRD monitoring in AML. 2) Time to CMR is short with a median of 4.1 mo. 3) An increase of NPM1 in all cases is followed by relapse after a median of 5.1 mo, if no treatment intervention has been initiated before. 4) Time intervals for MRD should be no longer than 3 mo, pB can be used. 5) Transplantation should already be planned after first molecular increase is detected. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Worseg:MLL Munich Leukemia Laboratory: Employment. Perglerová:MLL2 s.r.o: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

Phase I Clinical Trial Evaluating Abatacept in Patient with Steroid-Refractory Chronic Graft Versus Host Disease

Introduction: Chronic graft versus host disease (cGVHD) remains a major source of morbidity and mortality following allogeneic transplantation. While corticosteroids remain first line therapy for cGVHD, they are associated with significant toxicity, and a substantial proportion of patients fail to completely respond. Treatments for steroid-refractory cGVHD are limited. While the pathophysiology of chronic GVHD is complex, activated T cells play a critical role, driven by allo-antigen stimulation. As such, inhibition of T cell activation via blockade of co-stimulation has potential as a therapeutic target in cGVHD. Abatacept is a recombinant fusion protein consisting of the extracellular domain of human CTLA-4 and a fragment of the Fc domain of human IgG1 that has been modified to prevent complement fixation and antibody-dependent cellular cytotoxicity. Abatacept is the first drug in a class of agents termed "selective co-stimulation modulators." The CTLA-4 moiety of Abatacept binds specifically to CD80 and CD86 and down-modulates the CD28-mediated co-stimulation of T cells. We conducted a phase I clinical trial was conducted to evaluate the safety, clinical and immune effects of Abatacept in patients with steroid-refractory cGVHD.Methods: The study followed a 3+3 design with two escalating doses of Abatacept to determine the maximum tolerated dose (MTD): 3 mg/kg and 10 mg/kg. Dose-limiting toxicities (DLTs) were defined as Grade 3 or 4 toxicities judged to be probably or definitely related to Abatacept. Infection was not considered a DLT. Abatacept was administered for a total of 6 doses. Doses 1-3 were administered at two-week intervals. One month following Dose 3, Abatacept was given at four-week intervals for three doses (Doses 4-6). Inclusion criteria included recipients of allogeneic bone marrow or stem cell transplantation with myeloablative or reduced intensity conditioning, with cGVHD defined by NIH consensus criteria. Patients must have had treatment with ≥ 0.5 mg/kg/day of prednisone for at least 4 weeks. Patients with active malignant disease relapse or other active malignancy and patients with uncontrolled infection were excluded. Peripheral blood was drawn prior to each dose of Abatacept and following completion of therapy to assess the effect of treatment on circulating T cells. PD-1 expression on circulating T cells, and T cell expression of interferon gamma versus IL-10 was assessed by multichannel FACS analysis.Results: 17 subjects were treated. Three patients were treated at a dose of 3 mg/kg without DLT. Three evaluable patients completed treatment on cohort 2, at a dose of 10mg/kg without DLT. A forth participant withdrew consent following one dose of treatment and therefore is not evaluable. Ten patients were treated on an expansion cohort at a dose of 10mg/kg. We observed one grade 4 pulmonary infection, and three grade 3 pulmonary infections which resolved. Other Abatacept related adverse events included grade 2 gastritis (n=1), grade 2 pain (n=1), and grade 1 diarrhea (n=2), fatigue (n=2), rash (n=1), and skin pain (n=1). Of the 16 evaluable patients, 7 (44%) achieved a clinical partial response as defined by improvement of two disease systems based on the 2011 NIH consensus criteria. Abatacept resulted in a 51.3% reduction in prednisone usage in clinical responders with a mean baseline dose of 27mg compared to a mean dose of 14mg 1 month following the 6th dose of Abatacept (p = 0.01). PD-1 expression on circulating CD4+ and CD8+ T cells increased from a mean of 3.4% and 2.7% respectively at baseline to a mean of 8.9% and 7.6% respectively at one month following the 6thdose of Abatacept in clinical responders (n=3; p<0.05). In contrast, no change in T cell expression of PD-1 was observed in non-responders. A shift from Th1 to Th2 cytokine secretion was observed in clinical responders, with a mean 2.8 fold decrease in interferon gamma and a mean 2.5 fold increase in IL-10 secretion by circulating T cell populations (n=4).Conclusion: Abatacept is well-tolerated in the treatment of steroid-refractory cGVHD. Abatacept resulted in the improvement in NIH cGVHD scores in 44% of patients with steroid-refractory GVHD with a significant decrease in prednisone dose. An increase in PD-1 expression and a skewing toward Th2 cytokines was observed in clinical responders. Based on this promising data, a phase II trial is being initiated. DisclosuresSoiffer:GentiumSpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Arnason:Gilead: Consultancy. Avigan:Astex: Research Funding; DCPrime: Research Funding. Rosenblatt:Astex: Research Funding; BMS: Research Funding; DCPrime: Research Funding.