ALBERT

Description: Ph-like acute lymphoblastic leukemia (also known as BCR-ABL1-like ALL) is a new disease entity of B-cell ALL (B-ALL) that exhibits a mRNA expression profile similar to that of Philadelphia chromosome-positive ALL (Ph+ ALL). Ph-like signature is presumably driven by kinase-activating gene alterations. Thus, both gene expression pattern and DNA mutational status should be assessed to make a definitive diagnosis for Ph-like ALL. A variety of approaches combining multiple methods, including RNA sequencing (RNA-seq), Taqman low-density array (LDA), fluorescence in situ hybridization (FISH) and targeted DNA sequencing, are being tested; however, such multi-omics approaches are available only in limited institutions. Since Ph-like ALL patients generally exhibit poor response to standard chemotherapy, and tyrosine kinase inhibitors (TKIs) may benefit them when used in a timely manner, a fast, accurate and generalizable diagnostic method is critically needed. In the present study, we have developed a nCounter-based diagnostic method for Ph-like ALL and validated it using a cohort of Japanese adult B-ALL cases. To identify genes that are uniquely expressed (or not expressed) in Ph+ B-ALL, we first obtained publicly-available gene expression datasets comprising 1146 B-ALL cases and identified 82 differentially-expressed genes in Ph+ ALL cases. We then assessed expression levels of those genes in an independent cohort using the nCounter, which enables fast, sensitive and accurate RNA detection. We also tested whether nCounter-based methods can detect fusion transcripts relevant for Ph-like ALL pathogenesis using probes targeting ABL1, ABL2, CSF1R, PDGFRB, and JAK2. We analyzed 123 samples (Ph+ = 42, Ph- = 81, age 16 to 67) obtained from newly-diagnosed adult B-ALL patients enrolled in two clinical trials conducted by the Fukuoka Blood and Marrow Transplantation Group (FBMTG) (Nagafuji et al. Eur J Haematol 2019). Unsupervised hierarchical clustering successfully stratified 123 cases into two disease clusters: Ph+ and Ph- subgroups. As expected, Ph+ subgroup included almost all Ph+ ALL cases (40 out of 42 cases), while 18 out of 81 Ph- ALL cases (22%) were categorized into the Ph+ subgroup. We defined these cases as Ph-like ALL. To validate the nCounter-based Ph-like ALL classification, we performed RNA-seq and target-capture DNA sequencing of all Ph- ALL cases. As expected, we detected kinase-activating fusions/rearrangements, including CRLF2 rearrangements (7 cases), PDGFRB fusions (3 cases), JAK2 fusions (2 cases), EPOR rearrangements (2 cases), ABL1 fusion (1 case), and FLT3 internal tandem duplication (1 case) in 16 Ph-like ALL cases, while no genetic alterations were detected in 2 cases. Fusion genes involving PDGFRB were consistently detected by nCounter (3/3); however, detection of those involving JAK2 (1/2) and ABL1 (0/1) were inconsistent. JAK2 and/or RAS mutations were detected in 5 of 7 Ph-like ALL cases harboring CRLF2 rearrangements. Of note, CRLF2 protein expression was detected by FACS in all CRLF2-rearranged cases. We next assessed significance of the Ph-like signature on clinical outcomes using a cohort of 40 Ph- ALL cases, in which minimal/measurable residual disease (MRD) status, assessed by IgH and/or TCR rearrangements, as well as clinical data were available (Nagafuji et al. Eur J Haematol 2019). Ph-like ALL cases exclusively exhibited MRD positivity after induction therapy as compared to non-Ph-like cases (p=0.04), indicative of the chemo-resistant nature of Ph-like ALL as previously reported (Roberts et al. N Engl J Med, 2014 and Roberts et al. J Clin Oncol, 2017). As expected, Ph-like ALL cases exhibited significantly poor disease-free survival compared with non-Ph-like ALL cases (p=0.04); however, no significant difference was evident in overall survival (p=0.62) presumably due to the fact that all MRD-positive cases were subjected to allo-HSCT after induction therapy. These data indicate that MRD-based therapy stratification could overcome chemo-resistant nature of Ph-like ALL. Our data suggest that nCounter-based diagnostic method is fast and accurate to identify Ph-like ALL. Since Ph-like signature generally dictates poor clinical outcomes, and upfront TKI therapy may improve them, our method could facilitate precision medicine in the treatment of Ph- B-ALL. Disclosures Akashi: Sumitomo Dainippon, Kyowa Kirin: Consultancy; Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding.

Description: Cell differentiation is achieved by sequential gene expression. Late differentiation marker genes are already regulated at the chromatin level prior to differentiation in embryonic stem cells and many cell line models. Therefore, we hypothesized that ‘stem-ness’ of hematopoietic stem/progenitor cells (HSPCs) are programmed by epigenetic mechanisms and attempted to reveal the molecular mechanisms in hematopoietic gene expression. Histone H3 molecule, which is one of the most basic components of chromatin, has at least three variants: H3.1, H3.2, and H3.3. Previous studies have shown that one of the H3 variants, H3.3, was consistent with open chromatin structure. Here we found that the incorporation of histone variant H3.3 initiates on hematopoietic genes in HSPCs prior to differentiation. HSPC fractions were purified from C57BL/6J mouse bone marrow, and chromatin immunoprecipitation sequencing (ChIPSeq) analysis was performed using newly-established monoclonal antibodies that specifically recognize endogenous H3.3. Although previous conventional studies have demonstrated that H3.3 deposition dominantly occurred in the “gene body”, our sensitive ChIPSeq analysis revealed that more than half of the H3.3 existed in the inter-genic regions around hematopoietic genes. The region of H3.3 incorporation changed during differentiation, i.e., virtually all genes were marked with H3.3 in embryonic stem cells, while all hematopoietic genes were marked with H3.3 in LSK, and more lineage specific genes were marked when cells are differentiated. Furthermore, our analysis visualized that within the regions incorporated with H3.3, transcriptionally active regions marked by H3K4me3 and repressed regions marked by H3K27me3 are mutually exclusive. These data suggest that in hematopoietic differentiation, H3.3 incorporation initiates around relatively wide ranges of hematopoietic genes, and then either of active or repressive histone modification sequentially occurs. Interestingly, in leukemic cells, such selective H3.3 incorporation appeared to be disorganized. To identify factors that induce H3.3 incorporation defect in leukemic cells, we used a public database provided by the ENCODE project. We have constructed a system to manage all these datasets and to comprehensively explore the factors closely related to H3.3. Interestingly, correlations of our H3.3 ChIPSeq data with the ENCODE transcription factors’ binding site data were significantly different between analyses of AML and normal cells. By this approach, we identified hematopoietic transcription factors such as CEBPB and YY1 were associated with impaired H3.3 incorporation in AML. In addition, by comparing these transcription factors and single nucleotide variants (SNVs) obtained from Exome-Sequence, we found links between these transcription factors and particular SNVs in common pathways. These data suggest that this H3.3 ChIPSeq analysis should also be useful to extract oncogenic variants from many SNVs obtained by conventional Exome-Sequence analysis. Disclosures: No relevant conflicts of interest to declare.

Description: Peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS) is the largest subgroup with dismal prognosis in peripheral T-cell lymphomas (PTCL) that do not fit into any of the specifically defined categories in the World Health Organization classification, and it comprises over 25% of PTCL. PTCL-NOS is sometimes referred to as the gwaste-basketh category because of the lack of specific features, and therefore contains heterogeneous disease entities. Its heterogeneity represents a major obstacle to elucidation of pathogenesis, development of novel therapeutic targets and establishment of standardized therapeutic strategies and results in its poor prognosis. The stratification strategy of PTCL-NOS still remains controversial, although previous studies tried to reclassify PTCL-NOS from the viewpoint of gCell-of-Originh of tumor cells (e.g. T helper 1/2 cells, regulatory T cells) by utilizing histopathologic methods or gene expression profiling. These situations prompted us to investigate microenvironment profile of PTCL-NOS and to develop the novel stratification strategy. For this purpose, we analyzed the expression levels of 800 of tumor microenvironment-related genes including the phenotypic markers, transcription factors and functional molecules of microenvironment cells in formalin fixed paraffin embedded (FFPE) samples derived from 33 PTCL-NOS patients utilizing nCounter system, which is novel RNA counting system based on a digital molecular barcoding technology and enables accurate quantification of genes with low expression levels. Thirty-three patients who were diagnosed as PTCL-NOS between 1993 and 2011 were included in this study. The median follow-up time was 796 days (range, 15-2973). The overall survival rate at 1 year after diagnosis (1yr OS) was 40%, and it was comparable to previous reports. Unsupervised hierarchical clustering analysis revealed three distinct clusters based on the gene expression pattern of the microenvironment-related genes: B-cell type (42.4 %), M2 macrophage type (24.2 %), and others (33.3 %) (Figure 1). On the other hand, the expression patterns of the major T-cell subset-associated genes were complicated and diverse depending on patients, and failed to form any distinct clusters. B-cell-related genes, such as CD19, CD79B and PAX5 were highly expressed in the B-cell type, and the M2 macrophage-related genes, such as CD68, CD163, CD14 and MARCO were highly expressed in the M2 macrophage type. More importantly, these microenvironment-based subgroups, B-cell type and M2 macrophage type, represent prognostically favorable and unfavorable subgroups, respectively (1yr OS: B-cell type, 84.8%; M2 macrophage type, 16.7%; others, 56.2%; log rank test, p = 0.01) (Figure 2). Interestingly, genes associated with immune checkpoint, such as PD-L1, TIM-3 and IDO-1, were highly expressed in the M2 macrophage type than the others (PD-L1, p = 0.02; TIM-3, p = 0.05; IDO-1, p 〈 0.01), indicating the existence of specific subgroup of PTCL-NOS patients as anticipated good-responder for immunotherapeutic agents including immune checkpoint inhibitors. Collectively, our findings strongly suggest that microenvironment immune signature, not gCell-of-Originh of tumor cells, could provide novel prognostic stratification method and therapeutic strategy of PTCL-NOS. Disclosures Akashi: Asahi Kasei Pharma Corporation: Research Funding; Shionogi & Co., Ltd: Research Funding; Astellas Pharma: Research Funding; Celgene: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Sunitomo Dainippon Pharma: Consultancy; Bristol Meyers Squibb: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding.

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: In human hematopoiesis, the megakaryocyte (Meg) lineage is known to diverge from bipotent megakaryocytic/erythroid progenitor (MEP; CD34+ CD38+ IL3Ra- CD45RA-), which resides downstream of common myeloid progenitor (CMP; CD34+ CD38+ IL3Ra+ CD45RA-). However, the definition of unipotent Meg progenitor (MegP) is still controversial and, in the hierarchical map, where Meg potential mainly resides remains unclear. Here, we report prospective isolation of unipotent MegP, which is endowed with much more robust Meg potential than MEP, and emerges directly from CMP bypassing MEP. Because CD41 is known as a Meg lineage-specific marker, we first searched for the CD41-expressing cell population in the CD34 positive stem/progenitor fraction of healthy individuals. A fraction (7.9 ± 3.2 %) of CMPs expressed CD41, but none of the other CD34+ subpopulation including hematopoietic stem cells (HSCs; CD34+ CD38- CD45RA-), MEPs and granulocyte-macrophage progenitors (GMPs; CD34+ CD38+ IL3Ra+ CD45RA+) expressed significant levels of CD41. Purified CD41+ CMPs retained myeloblast-like immature morphological characteristics with large and round nucleus and basophilic cytoplasm, closely resembling those of CD41- CMPs and more immature HSCs. Interestingly, cells with immature 4N-nuclei were exclusively found in CD41+ CMPs (about 1%), indicating that a minor fraction of this population is initiating endomitosis and polyploidization process, an important characteristic of megakaryoblasts. A conventional In vitro culture condition with fetal bovine serum and a myeloerythroid cytokine cocktail revealed that CD41+ CMPs completely lacked erythroid or granulocyte/monocyte (GM) lineage potentials, whereas CD41- CMPs generated all types of myeloid colonies. These unexpected observations led us to hypothesize that CD41+ CMPs were committed to the Meg lineage. We then cultured them at a serum-free culture condition that is highly-optimized for Meg differentiation. In this condition, the majority of single CD41+ CMPs gave rise to pure megakaryocyte colonies, without forming any other myeloerythroid colonies. In addition, in cDNA microarray analysis, CD41+ CMPs possessed a distinct gene expression profile with up-regulation of Meg lineage-specific genes related to megakaryocyte development, platelet production, platelet activation and aggregation, and down-regulation of erythroid and GM lineage-affiliated genes, reflecting their lineage potential. Therefore, we defined the CD41+ CMP as a human MegP. The Meg colony-producing potential of MegPs was almost thirty­­ times as strong as that of MEPs on per-cell-basis (672 ± 232 vs. 20 ± 6.2 colonies per 1000 cells) and MegP-derived Meg colonies accounted for more than ninety percent of those from whole CD34+ CD38+ progenitor populations, indicating that this population is the major source of megakaryocytes and platelets production rather than MEP. The MegP population was found in 7 day culture of CD41- CMPs but not of MEPs, suggesting that MegPs are downstream of CMPs, but are independent of MEP. More importantly, MegP pool dramatically expanded in essential thrombocythemia (ET) patients’ bone marrow, constituting three times larger part of CMP than that in normal BM (24.2 ± 8.6 % vs. 7.9 ± 3.2 % of CMP). We are currently further analyzing ET patients’ bone marrow to obtain data regarding V617F JAK2 mutation frequency, for example. Thus, the newly-identified MegP robustly contributes to physiological and pathological human megakaryopoiesis, thus could be a therapeutic target in various thrombopoietic disorders. Disclosures No relevant conflicts of interest to declare.

Description: Human interleukin-3 receptor alpha (IL-3Ra, CD123), which promotes the proliferation and differentiation of hematopoietic cells, is highly expressed in myeloid malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We newly generated KHK2823, a non-fucosylated fully human IgG1 monoclonal antibody against human IL-3Ra, by utilizing the POTELLIGENT® technology. Here, we describe the in vitro and in vivo preclinical efficacy and safety of KHK2823, as well as its pharmacodynamic (PD) profile. At first, we explored that KHK2823 bound to various hematological malignant cells and leukemic stem cells. The cells from AML and MDS bone marrows were found to be bound by KHK2823. A significant part of bone marrow cells derived from B-cell acute lymphoblastic leukemia (B-ALL) patients was also bound by KHK2823. KHK2823 bound to soluble human IL-3Ra protein with a sub-nanomolar dissociation constant (KD), and recognized CD34+ CD38+ (leukemic blast) and/or CD34+ CD38- (leukemic stem cell) cells in patients with AML/MDS, as well as AML cell lines, thereby obtaining a high antibody-dependent cellular cytotoxic activity without complement-dependent cytotoxicity. Interestingly, KHK2823 did not interfere with the binding of IL-3 to IL-3R. The lack of a receptor-ligand interaction may conserve the IL-3 signal, which plays an important role in normal hematopoiesis. In a tumor model xenografting the human AML cell line MOLM-13 on nude rats, KHK2823 significantly suppressed the tumor growth at doses of 0.1 and 1 mg/kg (Figure 1). The PD and toxicity profiles of KHK2823 were assessed in cynomolgus monkeys administered at doses ranging from 0.1 to 100 mg/kg by i.v. infusion, once weekly for 4 weeks. KHK2823 was generally well tolerated in monkeys, even at 100 mg/kg. The number of IL-3Ra-positive cells in the peripheral blood of cynomolgus monkeys decreased in all groups receiving KHK2823, which suggest KHK2823 could exert its depletion activity of IL-3Ra-positive cells in human (Figure 2). Currently, the safety and tolerability of KHK2823 is being investigated in patients with AML or MDS in a Phase 1 study (NCT02181699, https://clinicaltrials.gov/ct2/show/NCT02181699). This is the first non-randomized, open-label, dose escalation clinical study to investigate the safety, PK, immunogenicity and PD of repeated doses of KHK2823. In summary, KHK2823 was confirmed to bind to AML, MDS and B-ALL cells as the IL-3Ra in accordance with other publications. KHK2823 was also found to eliminate AML cells in vitro and also suppressed the AML tumor growth in the in vivo model. In addition, the number of IL-3Ra-positive cells in cynomolgus monkeys decreased following i.v. infusion of 0.1mg/kg KHK2823 with a tolerable safety profile, even at a dose of 100 mg/kg. Taken together, KHK2823 may therefore be a promising anti-IL-3Ra therapeutic drug for the treatment of AML. Figure 1. Antitumor activity of KHK2823 in a tumor xenograft nude rat model Figure 1. Antitumor activity of KHK2823 in a tumor xenograft nude rat model Figure 2. PD profile of KHK2823 in cynomolgus monkeys Figure 2. PD profile of KHK2823 in cynomolgus monkeys Disclosures Akiyama: Kyowa Hakko Kirin Co., Ltd.: Employment. Takayanagi:Kyowa Hakko Kirin Co., Ltd.: Employment. Maekawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Shimabe:Kyowa Hakko Kirin Co., Ltd.: Employment. Nishikawa:Kyowa Hakko Kirin Co., Ltd.: Employment. Yamawaki:Kyowa Hakko Kirin Co., Ltd: Employment. Iijima:Kyowa Hakko Kirin Co., Ltd: Employment. Hiura:Kyowa Hakko Kirin Co., Ltd.: Employment. Takahashi:Kyowa Hakko Kirin Co., Ltd.: Employment. Akashi:Asahi Kasei: 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; Celgene: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau. Tawara:Kyowa Hakko Kirin Co., Ltd: Employment.

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.

Description: Key Points GVHD mediates donor T-cell infiltration and apoptosis of the ovarian follicle cells, leading to ovarian insufficiency and infertility. Ovarian insufficiency and infertility are independent of conditioning, and pharmacologic GVHD prophylaxis preserves fertility.

Description: Abstract 1193 Cell differentiation is achieved by sequential gene expression. Late differentiation marker genes are already regulated at the chromatin level prior to differentiation, even in pluripotent stem cells. Therefore, we hypothesized that “stem-ness” in hematopoiesis is already programmed in hematopoietic stem/progenitor cells (HSPCs) and attempted to reveal the molecular mechanisms of epigenetic regulations in hematopoietic gene expression. Histone H3 molecule, which is one of the most basic component of chromatin, has at least three variants:H3.1, H3.2, and H3.3. In previous study, it is known that one of H3 variants H3.3 was consistent with open chromatin structure. Only limited organisms which have complex differentiation mechanisms such as mammals have all three variants, suggesting that these histone variants significantly correlate with differentiation. Therefore, we focused on these H3 variants incorporation patterns in HSPCs, and found that the deposition of histone variant H3.3, that is a marker of open chromatin regions, specifically occurred on hematopoietic genes in HSPCs prior to differentiation. HSPC fractions were purified from C57BL/6J mouse bone marrow, and chromatin immunoprecipitation sequence analyses were performed utilizing monoclonal antibodies that specifically recognize H3.3. Although previous studies demonstrated that H3.3 deposition dominantly occurred in the “gene body”, our informatics analysis revealed that more than half of H3.3 existed in the inter-genic regions around hematopoietic genes. The region of H3.3 incorporation changed during differentiation, i.e., almost all of genes were marked with H3.3 in embryonic stem cells, while almost all of hematopoietic genes were marked with H3.3 in LSK, and more lineage specific genes were marked with H3.3 when the differentiation occurred. To explore the factor dependency of the deposition of H3.3, we examined H3.3 incorporation around binding sites of more than 20 different types of transcription factors. The binding sites of Oct3/4, Klf4, Sox2, and Myc, also known as iPS factors, were strongly correlated with H3.3 incorporation sites in embryonic stem cells, while in HSPCs, the incorporated regions of H3.3 significantly co-localized with the binding sites of several hematopoietic key transcription factors such as Scl and Runx1. When we knocked down Oct3/4 in embryonic stem cells, these H3.3 incorporations and differentiation marker genes expressions were diminished, on the other hand, when we knocked down H3.3 in embryonic stem cells, well-ordered differentiation marker genes expressions were suppressed. These data suggest that H3.3 incorporations by key transcription factors are the essences of the “stem-ness”. Interestingly, in leukemic cells, this selective H3.3 incorporation was not observed, suggesting that H3.3 incorporation could reflect the orderly progression of normal hematopoiesis. In conclusion, we found that the incorporation of H3.3 is the earliest epigenetic event involved in determining hematopoietic cell fate. Disclosures: No relevant conflicts of interest to declare.