ALBERT

Description: Abstract 1901 Poster Board I-924 Leukocyte alkaline phosphatase (LAP) is considered as a neutrophil activation marker. The level of LAP is quantitated as the LAP score. It is well known that patients with chronic myelogenous leukemia (CML) usually have low LAP scores, whereas those with BCR-ABL negative chronic myeloproliferative disorders (MPD) have elevated LAP scores. In CML patients, the premature release of granulocytes from the bone marrow into the peripheral blood is considered as the cause of low LAP scores. However, the reason for elevated LAP scores in BCR-ABL negative MPD patients has been unclear. An acquired JAK2V617F mutation is observed in most patients with BCR-ABL negative MPD. It has been shown that the JAK2V617F mutation induces constitutive activation of its downstream signaling pathways such as STAT3/STAT5, Ras/MAPK and PI3K pathways. We speculated that an elevated LAP score might be due to the activation of Jak2 downstream pathways through the JAK2V617F mutation. We analyzed LAP expression in BCR-ABL negative MPD patients. JAK2V617F homozygous patients had higher LAP expression than JAK2V617F heterozygous or negative patients. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of JAK2V617F positive patients. The myeloid cell line NB4 was transfected with the JAK2V617F mutation and a wild-type Jak2 using lentivirus vectors. It was observed that the JAK2V617F mutation, but not wild-type Jak2, enhanced cell proliferation. Then the LAP expression in NB4 cells was evaluated after these cells were differentiated by all-trans retinoic acid and granulocyte colony-stimulating factor. It was observed that the JAK2V617F mutation, but not wild-type Jak2, increased LAP expression. Next, we examined which of the Jak2 downstream pathways played a major role in increasing LAP expression and prompting cell proliferation. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, STAT3 siRNA and STAT5 siRNA, we demonstrated that the JAK2V617F mutation primarily used the STAT3 pathway to increase LAP expression. On the other hand, the JAK2V617F mutation used the STAT5, the Ras/MAPK and the PI3K pathways, but not the STAT3 pathway, to prompt proliferation of NB4 cells. In conclusion, we obtained direct evidence that the JAK2V617F mutation induced elevation of LAP scores via the STAT3 pathway, and prompted proliferation of NB4 cells via the STAT5, the Ras/MAPK and the PI3K pathways. Our findings showed the possibility that the JAK2V617F mutation might use specific downstream pathways depending on various phenotypic manifestations of BCR-ABL negative MPD. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2301 To evaluate stem cell potential of human cells in xenotransplant models, a variety of immunodeficient mouse lines have been developed. Depletion of lymphoid cells including T, B and NK cells by introducing with Il2rgnull, and SCID or RAGnull mutations is necessary to avoid rejection of human cells in these models. Interestingly, in mice having these immunodeficiencies, the NOD strain shows even better engraftment of human cells as compared to C57BL/6 or Balb/c strains. Recently, we found that in xenograft rejection, the innate phagocytic reaction of mouse macrophages could occur because murine signal regulatory protein alpha (mSIRPA) on macrophages cannot bind to human CD47 (hCD47). However, NOD-specific polymorphism of mSIRPA allows NOD-type SIRPA to bind hCD47, resulting in inhibition of phagocytic reaction against human cells in this strain at least in vitro. Here, we have established a new immunodeficient BRGS mouse line, C57BL/6.Rag2nullIl2rgnull mice with NOD-type SIRPA. To test the reconstitution activity of human hematopoiesis in vivo, we transplanted 5 × 103 CD34+CD38− human cord blood cells intrafemorally into C57BL/6.Rag2nullIl2rgnull (C57BL/6-RG), BRGS or NOD.Rag1nullIl2rgnull (NOD-RG) mice. At 8 weeks after transplantation, human CD45+ cells were not detectable in C57BL/6-RG mice in the bone marrow. Both BRGS and NOD-RG showed successful reconstitution, and their frequency of human CD45+ cells in the bone marrow was 59.9 % and 55.8% in average, respectively. The frequency of human CD45+ cells was maintained at least until 24 weeks after transplantation. Percentages of CD19+ B cells, CD33+ myeloid cells and CD34+CD38− cells that contain the majority of human hematopoietic stem cells (HSCs) were almost equal between the BRGS and the NOD-RG strains. In the spleen, the majority of human cells were CD19+ B cells expressed surface immunoglobulin light chain λ/κ, reflecting their normal maturation. In the thymus, CD4+CD8+ thymic precursors, and CD4+ and CD8+ single positive T cells were present, and they expressed surface T cell receptor (TCR)-ab or TCR-gd. These data show that replacement of the C57BL/6-Sirpa with the NOD-Sirpa is sufficient for the C57BL/6-RG strain to gain the human cell engraftment ability equal to the NOD-RG strain. In addition, the BRGS strain has normal complement activity, in contrast to the NOD strain that does not have C5, a component necessary for complement-dependent cytotoxic (CDC) activity. We injected 8 × 105 cells of Raji cells into BRGS or NOD-RG mice, and administered rituximab, an anti-CD20 antibody that has both CDC and ADCC activities, to test the in vivo efficacy of rituximab on transplanted Raji cells. After injection of rituximab, percentages of human CD45+ Raji cells were significantly decreased in BRGS mice (15.1 %), whereas percentages of Raji cells in NOD-RG mice were only slightly reduced by rituximab treatment (79.2 %). These data clearly show that the CDC activity of antibodies can operate in the BRGS strain. Collectively, this study formally proves that the excellent transplantability of human grafts in the NOD strain is explained simply by a single gene mutation, NOD-specific polymorphism of SIRPA, and that the BRGS strain should be very useful in future xenotransplant experiments of human stem cells. Disclosures: No relevant conflicts of interest to declare.

Description: Immunodeficient mice are used to reconstitute human hematopoiesis and analyze human hematopoietic function in vivo by xenotransplantation of hematopoietic stem cells (HSCs). It is well recognized that in addition to disruption of the lymphoid system, the strain background such as NOD is critical for efficient human cell engraftment in xenotransplantation models. We have reported that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows the mouse SIRPA to bind human CD47, preventing activation of recipient macrophages to engulf human hematopoietic cells (Takenaka et al., Nature Immunol., 2006). We then developed a C57BL/6.Rag2nullIl2rgnull mouse line harboring the NOD-type Sirpa, named the BRGS mouse. The engraftment efficiency of human HSCs in BRGS mice was equal to that of NOD-Rag2nullIl2rgnull mice. This BRGS strain should be useful for further genetic modification because its background is simplified (Yamauchi et al., Blood, 2013). One of the persistent problems with the current xenograft models is that reconstituted human hematopoiesis is B-lymphoid dominant, whereas the engraftment levels of human myeloid cells, including megakaryocyte/erythrocyte lineage cells are quite low. In mouse syngeneic and allogeneic transplantation, recipients bearing mutation in the receptor tyrosine kinase Kit can accept donor HSCs without irradiation preconditioning. These data suggest that the Kit mutation impairs endogenous murine HSC self-renewal and allows donor HSCs efficiently to access to niche space. To develop a more efficient xenograft model, we further introduced a loss-of-function KitWv into the BRGS strain to deteriorate self-renewal and reconstitution capability of host HSCs. Here we show that this new mouse strain, termed the BRGSK mouse, showed very efficient human cell engraftment including myeloid, erythroid and megakaryocyte lineage cells. CD34 + cells obtained from human umbilical cord blood were transplanted into sublethally-irradiated BRGSK and BRGS mice at the age of 6-8 weeks by intrafemoral injection. At 8-12 weeks after transplantation, the average human hematopoietic chimerism defined by human CD45+ cells was more than 90% in the BM and this chimerism was maintained over 24 weeks in homozygous BRGSK recipients. Furthermore, the chimerism of human CD33+ myeloid cells in the BM was significantly improved (~5.7% in BRGS vs. ~32.5% in BRGSK). Surprisingly, CD71- CD235a+ mature erythrocytes and CD41+ platelets were detected in the BM. All human erythroid series including mature erythrocytes, CD71+ CD235a- early erythroblasts and CD71+ CD235a+ late erythroblasts were clearly seen (~0.1% in BRGS vs. ~42.2% in BRGSK). Immunohistochemical staining showed that these human erythroid cells were positive for human hemoglobin subunit alpha. Confocal immunofluorescence imaging revealed that human erythroid cells formed large erythroid islands, independently from mouse erythroid islands in the sternal marrow of recipient BRGSK mice. A number of CD41+ megakaryocytes were scattered in the BM. The BRGSK mice also well supported terminal differentiation of myeloid cells such as neutrophils, eosinophils, basophils, mast cells, monocytes, and dendritic cells. Thus, reconstitution and maintenance of human myelo-erythropoiesis is significantly improved in the new BRGSK strain. The new BRGSK xenograft model should be a strong tool to investigate normal and malignant human hematopoiesis. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Description: [Introduction] Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma (PTCL) with a dismal prognosis. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment in ATL patients. Mogamulizumab, a humanized anti-CC chemokine receptor 4 (CCR4) monoclonal antibody, is a novel immunotherapeutic agent, effective in treating patients with PTCL such as ATL, PTCL-not specified, and cutaneous T-cell lymphoma. However, in allo-HSCT setting, we should be careful to use mogamulizumab because CCR4 is expressed in regulatory T cells: The mogamulizumab treatment may accelerate GVHD by eradicating regulatory T cells in allo-HSCT patients. Here, we retrospectively analyzed the effect of mogamulizumab on GVHD development in ATL patients treated with mogamulizumab prior to allo-HSCT. [Patients and Methods] Data from the Fukuoka Bone Marrow Transplantation Group were retrospectively analyzed after the approval of mogamulizumab use in Japan. [Results] A total of 24 patients with ATL received mogamulizumab prior to allo-HSCT between April 2012 and April 2015 in our group. The median age at allo-HSCT was 58.5 years (range, 32-72). The median intervals from the last administration of mogamulizumab to allo-HSCT were 25 days (range, 9-126). The median total dose of mogamulizumab was 3 mg/kg (range, 1-8 mg/kg). After treatment with mogamulizumab, 18 patients (75%) had achieved in remission (CR in 4 patients and PR in 14) at allo-HSCT. Ten patients received unrelated bone marrow, 5 received related peripheral blood, and 9 received cord blood as stem cell sources. Eleven patients were treated with full-intensity conditioning and 13 received reduced-intensity conditioning. Graft-versus-host disease (GVHD) prophylaxis consisted of calcineurin inhibitors (cyclosporine or tacrolimus) with short-term methotrexate in 14 patients and mycophenolate mofetil in 9. The cumulative incidence (CI) of acute GVHD at 100 days was 66.6% in grade 2-4 and 33.3% in grade 3-4. The involved organs of acute GVHD were skin in 14 patients, gut in 10, and liver in 4. Among 14 patients who developed grade 2-4 acute GVHD, 5 had severe fluid retention such as pleural effusion or ascites associated with GVHD. Chronic GVHD was observed in 6 patients, and 5 of them were extensive disease. The CI of transplant-related mortality (TRM) and relapse at 1-year were 53.2% (95%CI, 29.3-72.3%) and 29.6% (95%CI, 12.6-48.9%), respectively. The leading cause of death was GVHD (n = 7). The 1-year overall survival and progression-free survival were 19.2% (95%CI, 5.7-38.8%) and 17.2% (95%CI, 4.9-35.7%), respectively. [Discussion] Use of mogamulizumab prior to transplantation in allo-HSCT patients has a merit to decrease the burden of ATL cells. However, it was associated with an increase of TRM due to severe GVHD. Although most of ATL patients achieved better disease status at allo-HSCT through mogamulizumab and the survival rate was expected to be 50% based on the previous data, the survival in the present study was ~20%. These data suggest that mogamulizumab administered before transplantation may have retained until an early phase of post-transplantation, and the donor or host-derived regulatory T cells might be eliminated, allowing the GVHD T-cell clone to expand. Since mogalizumab is a potent anti-ATL agent, we need to develop new treatment protocols integrating mogalizumab at a suitable dose or administration timing, to minimize the unwanted GVHD development in future studies. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Description: Myeloproliferative neoplasms (MPNs) are chronic hematopoietic stem cell disorders characterized by overproduction of mature myeloid cells. Recently, somatic mutation of calreticulin (CALR) was frequently found in MPN patients who do not have JAK2 mutation. The CALR mutation in MPN patients usually resulted in loss-of-function of CALR, which may induce impairment of physiological phagocytotic pathway, because surface CALR plays a critical role for macrophages in recognition of low-density lipoprotein receptor-related protein 1 (LRP1) on the targets, mediating pro-phagocytic signals. We hypothesized that the non-functional CALR mutation renders cells resistant to phagocytosis, and impairs the “programmed cell removal” of progenitors or mature blood cells, resulting in accumulation of hematopoietic cells in MPNs. In 135 Japanese MPNs patients enrolled in this study, including polycythemia vera (PV), essential thrombocytosis (ET) or primary myelofibrosis (PMF), 34 patients (25.2%) had CALR mutations, and 80 (59.3%) patients had JAK2 V617F mutation, respectively. CALR mutations were heterozygous in all 34 patients (27 patients with ET, 7 with PMF). On the other hand, JAK2 V617F mutations were found in 26 patients with PV, 39 with ET, and 15 with PMF. The expression levels of pro-phagocytotic CALR were normal in these MPN patients. We then performed in vitro phagocytosis assay to test whether the heterozygous CALR mutation affects engulfment of blood cells by macrophages. Hematopoietic stem cells (HSCs), progenitor cell populations such as common myeloid progenitors (CMPs), megakaryocyte/erythroid progenitors (MEPs) and granulocyte/monocyte progenitors (GMPs), and mature myeloid cells were isolated and opsonized, and were co-cultured with activated macrophages for 2 hours. After the culture, we enumerate macrophages and engulfed cells to analyze phagocytosis index (number of engulfed cells/number of macrophages) (Kuriyama et al. Blood 2012). However, the phagocytosis index was not changed in any of purified hematopoietic cells, irrespective of the presence of CALR or JAK2 mutation. These results strongly suggest that heterozygous, non-functional CALR mutation, and gain-of-function JAK2 mutations should not affect the engulfment process for hematopoietic cells by macrophages. We then investigated the effect of CALR or JAK2 mutations on differentiation and proliferation of stem or progenitor cells in MPNs. We performed colony-forming cell assay of multipotent cells, such as HSCs and CMPs, and evaluated clonal burden of CALR and JAK2 mutations in colonies derived from these stem and progenitor cells. In vitro culture showed that HSCs and CMPs with CALR and JAK2 mutations gave rise to granulocyte/monocyte (GM) or megakaryocyte/erythroid (MegE)-related colonies, whose frequencies were almost identical to those in wild-type controls, suggesting that these mutations do not affect myelo-erythroid lineage commitment at the multipotent stem or progenitor stages. In contrast, when we cultured GMPs and MEPs, frequencies of colonies with CALR or JAK2 mutations were significantly higher as compared to those in HSCs or CMPs (P

Description: Primary myelofibrosis (PMF) is a clonal stem cell disorder, characterized by deregulated proliferation of myelofibroblasts. Pathogenesis of PMF has been intensively investigated by analyzing clonality of the disease mainly by X-linked gene inactivation assays. These studies suggested that hematopoiesis in PMF is clonal, whereas fibroblasts are polyclonal not belonging to the PMF clone but they secondarily proliferate in response to cytokines produced by malignant megakaryocytes or monocytes. However, we have previously reported that PMF-initiating cells exist within the circulating CD34+CD38- fraction expressing CD45, whose phenotype is analogous to normal hematopoietic stem cells (HSCs), and this population can reconstitute myelofibrosis after xenotransplantation into NOD/SCID/IL2rg-null mouse newborns (Saito et al. ASH Annual Meeting 2007). In this assay, human PMF HSCs reconstitute PMF-like disease in mouse after transplantation, and strikingly the vast majority of myelofibroblasts were of human origin on FISH analysis, suggesting that myelofibroblasts in PMF can be generated directly from circulating PMF stem cells. We here tried to test this hypothesis by directly analyzing human PMF samples. We performed clonal analyses of fibrotic tissues in PMF patients at the single cell level by utilizing JAK2 V617F mutation as a clonal marker. Nine patients of PMF with JAK2 V617F mutation were enrolled in this study. The fibrotic bone marrow tissues were stained with vimentin, CD34 and KP1 to identify fibroblast-like cells, endothelial cells and myeloid cells, respectively. Single cells of fibrotic tissues were sampled by a laser micro-dissection system, and CD34+CD38-CD45+ HSCs were purified from the blood by FACS. Genomic DNA from single sampled cells was tested for JAK2 V617F mutation. This extensive analysis showed that ∼20-40% of single HSCs and granulocyte/monocyte progenitors (GMPs) had JAK2 V617F. Similar percentages of single vimentin+ fibroblast-like cells, CD34+ endothelial cells and KP-1+ myeloid cells had JAK2 V617F in each patient. Furthermore, each population also constituted similar percentages of heterozygous and homozygous JAK2 V617F as HSCs and GMPs did in each case. Because JAK2 V617F exists in each cell component of myelofibrotic tissues, whose frequency was almost equal to that of PMF HSCs, this study strongly suggests that the majority of myelofibrotic tissue cells belong to the PMF clone, and are differentiated directly from PMF HSCs. These data are compatible with our previous xenotransplantation data. Finally, it is also suggested that JAK2 V617F signaling is not necessary for PMF development. The new understanding of PMF pathophysiology shown here by our experiments might be useful to develop new treatment strategies for human PMF in future studies. Disclosures: Miyamoto: Kyushu University Hospital: Employment.

Description: Introduction: Combination of proteasome inhibitor (PI) bortezomib and immunomodulatory drugs (IMiDs), lenalidomide or thalidomide, have revealed superior outcomes in multiple myeloma (MM) over their single use. PI and IMiDs possess their direct anti-tumor activity as well as exert the indirect pleiotropic biological effects by modulating immune response, cytokine production, cell adhesion molecule, anti-apoptotic factors, cell cycle regulators. We recently found that circulating immature granulocytes and erythrocytes/megakaryocytes frequently appeared in peripheral blood (PB) in patients treated with bortezomib and IMiDs. In this study, we tested whether this effect is derived from PI, IMiDs or combination, and tried to understand its underlying mechanism to further understand their combinatory effects to normal hematopoiesis. Patients & Methods: 47 patients aged 37 - 81 years with newly diagnosed or relapsed/refractory MM were enrolled into this study. PB or bone marrow (BM) samples were collected from 7 patients treated with bortezomib/lenalidomide/dexamethasone (VRD), 15 with bortezomib/thalidomide/dexamethasone (VTD), 6 with bortezomib/dexamethasone (BD), 19 with lenalidomide/dexamethasone (RD) regimen. Clonogenic progenitor assay (CFU-C) was performed using PB samples. Change in expression level of adhesion molecules on mobilized immature cells was analyzed with flowcytometric (FCM) analysis. We counted 250 differential WBC in May-Giemsa-stained PB smears under the microscope every other day after start of chemotherapy. Result: In PB analysis, a significant fraction of immature myeloid or erythro-megakaryocyte cells was morphologically documented in 16 out of 22 (73%) patients treated with VTD/VRD, but never in the patients treated with BD or RD (n=22). During the time course, circulating immature hematopoietic cells gradually increased after the commencement of VRD or VTD therapy, and reached to a peak on a median of 12 days (range, 4 - 24 days), which was documented after 2nd or 3rd administration of bortezomib in the most patients. In these patients, a mean peak percentage of immature cells per WBC was 2.6% (0 - 6.5%) during VRD/VTD therapy. We also performed FCM analyses to confirm mobilization of immature hematopoietic progenitors in peripherally during VRD/VTD therapy. Number of mobilized CD34+ cells was statistically higher in the VRD/VTD group than that in BD/RD group (0.070% vs. 0.017%, p=0.041). We extended an analysis of change in expression of adhesion molecules on mobilized immature cells in PB mononuclear cells, since down-regulation of these molecules resulted in release of stem/progenitor cells from marrow and mobilization into the circulation. We found a significant decrease in mean fluorescence intensity (MFI) of CXCR-4 expressed on PB CD34+ cells in VRD/VTD group compared with that in BD/RD groups (11.1 vs 15.2, p=0.033). Number of CFU-cells (CFU-C) increased after 2nd or 3rd administration of bortezomib during VRD/VTD therapy compared to that before therapy (Figure), which was coincided with the appearance of immature cells in PB. A number of CFU-C derived from 100,000 PB mononuclear cells was increased up to 45 in VRD/VTD group compared to 11 in BD/RD group (p=0.021). Discussion: In this study, we unexpectedly found a transient mobilization of the immature hematopoietic progenitors in the majority of patients who received concurrently PI and IMiDs, but not in the patients treated with either alone. IMiDs promote degradation of IKZF1, which regulate GATA-1 and PU.1, critical transcription factors for erythroid and granuloid lineage, resulting in a suppression of erythropoiesis and granulocyte maturation arrest with accumulation of immature granulocyte progenitors in the BM. In contrast, PI downregulate expressions such as CXCL-12/CXCR-4 and VLA-4/VCAM-1 by which hematopoietic progenitors anchor to BM microenvironments. Therefore, we hypothesize that accumulated immature progenitors in marrow by treatment with IMiDs, can be quickly released into peripherally by concurrent administration of bortezomib. It is still unknown whether the mechanism of mobilization effect is linked to their anti-myeloma effect. Thus, our observations provided the unexpected biological effects of these drugs. Combinatory use of new drugs should be carefully evaluated to maximize their benefit or prevent possible adverse events. Figure 1. Figure 1. Disclosures Akashi: Asahi Kasei: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Chugai: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Novartis Pharma K.K.: Consultancy, Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding, Speakers Bureau.

Description: Hemophagocytic lymphohistiocytosis (HLH) is characterized by deregulated engulfment of hematopoietic stem cells (HSCs) by BM macrophages, which are activated presumably by systemic inflammatory hypercytokinemia. In the present study, we show that the pathogenesis of HLH involves impairment of the antiphagocytic system operated by an interaction between surface CD47 and signal regulatory protein α (SIRPA). In HLH patients, changes in expression levels and HLH-specific polymorphism of SIRPA were not found. In contrast, the expression of surface CD47 was down-regulated specifically in HSCs in association with exacerbation of HLH, but not in healthy subjects. The number of BM HSCs in HLH patients was reduced to approximately 20% of that of healthy controls and macrophages from normal donors aggressively engulfed HSCs purified from HLH patients, but not those from healthy controls in vitro. Furthermore, in response to inflammatory cytokines, normal HSCs, but not progenitors or mature blood cells, down-regulated CD47 sufficiently to be engulfed by macrophages. The expression of prophagocytic calreticulin was kept suppressed at the HSC stage in both HLH patients and healthy controls, even in the presence of inflammatory cytokines. These data suggest that the CD47-SIRPA antiphagocytic system plays a key role in the maintenance of HSCs and that its disruption by HSC-specific CD47 down-regulation might be critical for HLH development.

Description: Progress has been made in deciphering molecular mechanisms underlying AML pathogenesis due in part to near-complete understanding of AML genomes. However, AML is yet a devastating disease with a long-term survival rate of less than 30%, underscoring an urgent need for the development of additional therapeutic modalities. To identify novel targets for AML therapy, we performed genome-wide CRISPR-Cas9 dropout screens employing two mouse AML cell lines (CALM/AF10 and MLL/AF9), followed by a second screen in vivo. These two cell lines, which we established, harbor wild-type (WT) Trp53with normal karyotype, enabling us to interpret screening results more easily due to a "clean" genetic background. We then validated our findings using human AML cell lines and patient-derived xenograft (PDX) models (Yamauchi et al. Cancer Cell 2018). In the current study, we assessed the screening results furtherusing MAGeCK MLE program (Li et al. Genome Biology 2015)and the DepMap (https://depmap.org/), a publicly available genome-wide CRISPR-Cas9 screen datasets of cancer cell lines including 15 human AML cell lines. We show that PAICS (Phosphoribosylaminoimidazole carboxylase), which encodes an enzyme involved in de novo purine biosynthesis, is a molecule essential for AML cell survival. MRT252040, a newly-developed PAICS inhibitor (PAICSi), efficiently killed AML cell lines with different genetic backgrounds and significantly prolonged survival of AML PDX models. Furthermore, we investigated the mechanism action of PAICSi employing additional functional screens: CRISPR-Cas9 mutagenesis scan of all Paicscoding exons and a genome-wide CRISPR/Cas9 dropout screen in the presence of PAICSi. Read counts for each Paics-targeted single-guide RNA (sgRNA) significantly decreased in vitro (AML cell lines) and in vivo (mouse AML model). We then assessed the functional significance of PAICS inhibition in AML cell survival via shRNA-mediated PAICSknockdown. AML cells expressing PAICS shRNA exhibited a proliferative disadvantage compared to non-transduced cells or those expressing scrambled shRNA, indicating a toxic effect of PAICS depletion in AML cells. We next asked whether inhibition of PAICS enzymatic activity affects AML cell proliferation and/or apoptosis using PAICSi. We assessed AML growth rate, cell cycle status and apoptosis and found that inhibition of PAICS enzymatic activity delays AML cell proliferation via inducing cell cycle arrest and apoptosis. As expected, CRISPR-Cas9 mutagenesis scan showed that sgRNAs targeting the exonic regions relevant to PAICS enzymatic activity were significantly decreased after the 16-day incubation. We next performed genome-wide CRISPR-Cas9 screens in the presence of PAICSi, followed by second screens using a small-scale sgRNA library containing 8-10 sgRNAs per candidate gene.We identified genes potentially involved in PAICSi resistance as well as those whose loss are synthetic lethal to PAICS inhibition. X-box-binding protein 1 (Xbp1) was among the top hits in the genes relevant to PAICSi resistance genes, and sgRNAs targeting Xbp1significantly enriched in the presence of PAICSi. In contrast, sgRNAs targeting Slc43a3or Hprt, both of which are implicated in the purine salvage pathway, were significantly dropped-out, indicating that PAICSi-mediated anti-leukemia effects can be enhanced upon concurrentinhibition of the purine salvage pathway. Finally, we explored potential anti-leukemia effects of PAICSi in vivo using AML PDX models established from two human AML lines. PAICSi exhibited anti-leukemic activity, as evidenced by the lower leukemia burden in peripheral blood and bone marrow of PAICSi-treated mice. They survived significantly longer than vehicle-treated mice, indicative of therapeutic efficacy of PAICSimonotherapy against AML in vivo. In summary, we identified PAICS as an essential gene for AML cell survival. We propose that pharmacological targeting of the de-novo purine synthesis pathway via PAICSi is a potential therapeutic strategy for AML therapy. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.