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Detailed genomic and epigenomic analyses of MECOM (the MDS1 and EVI1 complex locus) have revealed that inversion or translocation of chromosome 3 drives inv(3)/t(3;3) myeloid leukemias via structural rearrangement of an enhancer that upregulates transcription of EVI1. Here, we identify a novel, previously unannotated oncogenic RNA-splicing derived isoform of EVI1 that is frequently present in inv(3)/t(3;3) acute myeloid leukemia (AML) and directly contributes to leukemic transformation. This EVI1 isoform is generated by oncogenic mutations in the core RNA splicing factor SF3B1, which is mutated in >30% of inv(3)/t(3;3) myeloid neoplasm patients and thereby represents the single most commonly cooccurring genomic alteration in inv(3)/t(3;3) patients. SF3B1 mutations are statistically uniquely enriched in inv(3)/t(3;3) myeloid neoplasm patients and patient-derived cell lines compared with other forms of AML and promote mis-splicing of EVI1 generating an in-frame insertion of 6 amino acids at the 3′ end of the second zinc finger domain of EVI1. Expression of this EVI1 splice variant enhanced the self-renewal of hematopoietic stem cells, and introduction of mutant SF3B1 in mice bearing the humanized inv(3)(q21q26) allele resulted in generation of this novel EVI1 isoform in mice and hastened leukemogenesis in vivo. The mutant SF3B1 spliceosome depends upon an exonic splicing enhancer within EVI1 exon 13 to promote usage of a cryptic branch point and aberrant 3′ splice site within intron 12 resulting in the generation of this isoform. These data provide a mechanistic basis for the frequent cooccurrence of SF3B1 mutations as well as new insights into the pathogenesis of myeloid leukemias harboring inv(3)/t(3;3).

Chronic myelomonocytic leukemia (CMML) is a hematological malignancy characterized by uncontrolled proliferation of dysplastic myelomonocytes and frequent progression to acute myeloid leukemia (AML). We identified mutations in the Cbl gene, which encodes a negative regulator of cytokine signaling, in a subset of CMML patients. To investigate the contribution of mutant Cbl in CMML pathogenesis, we generated conditional knockin mice for Cbl that express wild-type Cbl in a steady state and inducibly express CblQ367P, a CMML-associated Cbl mutant. CblQ367P mice exhibited sustained proliferation of myelomonocytes, multilineage dysplasia, and splenomegaly, which are the hallmarks of CMML. The phosphatidylinositol 3-kinase (PI3K)-AKT and JAK-STAT pathways were constitutively activated in CblQ367P hematopoietic stem cells, which promoted cell cycle progression and enhanced chemokine-chemokine receptor activity. Gem, a gene encoding a GTPase that is upregulated by CblQ367P, enhanced hematopoietic stem cell activity and induced myeloid cell proliferation. In addition, Evi1, a gene encoding a transcription factor, was found to cooperate with CblQ367P and progress CMML to AML. Furthermore, targeted inhibition for the PI3K-AKT and JAK-STAT pathways efficiently suppressed the proliferative activity of CblQ367P-bearing CMML cells. Our findings provide insights into the molecular mechanisms underlying mutant Cbl-induced CMML and propose a possible molecular targeting therapy for mutant Cbl-carrying CMML patients.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

Additional sex combs-like 1 (ASXL1), an epigenetic modulator, is frequently mutated in myeloid neoplasms. Recent analyses of mutant ASXL1 conditional knockin (ASXL1-MT-KI) mice suggested that ASXL1-MT alone is insufficient for myeloid transformation. In our previous study, we used retrovirus-mediated insertional mutagenesis, which exhibited the susceptibility of ASXL1-MT-KI hematopoietic cells to transform into myeloid leukemia cells. In this screening, we identified the hematopoietically expressed homeobox (HHEX) gene as one of the common retrovirus integration sites. In this study, we investigated the potential cooperation between ASXL1-MT and HHEX in myeloid leukemogenesis. Expression of HHEX enhanced proliferation of ASXL1-MT-expressing HSPCs by inhibiting apoptosis and blocking differentiation, whereas it showed only modest effect in normal HSPCs. Moreover, ASXL1-MT and HHEX accelerated the development of RUNX1-ETO9a and FLT3-ITD leukemia. Conversely, HHEX depletion profoundly attenuated the colony-forming activity and leukemogenicity of ASXL1-MT-expressing leukemia cells. Mechanistically, we identified MYB and ETV5 as downstream targets for ASXL1-MT and HHEX by using transcriptome and chromatin immunoprecipitation-next-generation sequencing analyses. Moreover, we found that expression of ASXL1-MT enhanced the binding of HHEX to the promoter loci of MYB or ETV5 via reducing H2AK119ub. Depletion of MYB or ETV5 induced apoptosis or differentiation in ASXL1-MT-expressing leukemia cells, respectively. In addition, ectopic expression of MYB or ETV5 reversed the reduced colony-forming activity of HHEX-depleted ASXL1-MT-expressing leukemia cells. These findings indicate that the HHEX-MYB/ETV5 axis promotes myeloid transformation in ASXL1-mutated preleukemia cells.

Since a report of some 50 years ago describing refractory anemia associated with group C monosomy, monosomy 7 (−7) and interstitial deletions of chromosome 7 (del(7q)) have been established as one of the most frequent chromosomal aberrations found in essentially all types of myeloid tumors regardless of patient age and disease etiology. In the last century, researchers sought recessive myeloid tumor-suppressor genes by attempting to determine commonly deleted regions (CDRs) in del(7q) patients. However, these efforts were not successful. Today, tumor suppressors located in 7q are believed to act in a haploinsufficient fashion, and powerful new technologies such as microarray comparative genomic hybridization and high-throughput sequencing allow comprehensive searches throughout the genes encoded on 7q. Among those proposed as promising candidates, 4 have been validated by gene targeting in mouse models. SAMD9 (sterile α motif domain 9) and SAMD9L (SAMD9-like) encode related endosomal proteins, mutations of which cause hereditary diseases with strong propensity to infantile myelodysplastic syndrome (MDS) harboring monosomy 7. Because MDS develops in SAMD9L-deficient mice over their lifetime, SAMD9/SAMD9L are likely responsible for sporadic MDS with −7/del(7q) as the sole anomaly. EZH2 (enhancer of zeste homolog 2) and MLL3 (mixed lineage leukemia 3) encode histone-modifying enzymes; loss-of-function mutations of these are detected in some myeloid tumors at high frequencies. In contrast to SAMD9/SAMD9L, loss of EZH2 or MLL3 likely contributes to myeloid tumorigenesis in cooperation with additional specific gene alterations such as of TET2 or genes involved in the p53/Ras pathway, respectively. Distinctive roles with different significance of the loss of multiple responsible genes render the complex nature of myeloid tumors carrying −7/del(7q).

Background: Despite the aging society, few studies have evaluated risk factors for febrile neutropenia (FN) in the very elderly. This may be due to the difficulty of conducting a prospective study in the presence of comorbidities and a limited number of patients. We retrospectively analyzed risk factors for FN in the first cycle of rituximab-cyclophosphamide-doxorubicin-vincristine-prednisolone (R-CHOP) regimen in the very elderly aged ≥ 80 years using a nationwide inpatient database in Japan. Study Design and Methods: This study was a retrospective cohort study using the Diagnosis Procedure Combination database, a nationwide inpatient database in Japan. The database includes discharge abstracts and administrative claims data from >1200 acute-care hospitals and covers around 90% of all tertiary-care emergency hospitals in Japan. We identified patients aged ≥ 80 years old with newly diagnosed diffuse large B-cell lymphoma (DLBCL) who received the first cycle of R-CHOP regimen between July 2007 and March 2017 from the database. The initial dose intensity (IDI, %) was calculated as the average ratio of the actual dose to the 100% dose of cyclophosphamide and doxorubicin. IDI less than 80% was defined as reduced IDI. Multivariable logistic regression analysis fitted with a generalized estimating equation accounting for within-hospital clustering was performed to identify factors associated with the occurrence of FN. P Results: We identified 1,819 patients aged ≥ 80 years old with DLBCL who were newly diagnosed and treated with R-CHOP. The median age was 83 years (interquartile range: 82-85). A total of 73 (4%) patients received antimicrobial prophylaxis, and 270 (15%) received primary prophylaxis with G-CSF. Reduced IDI of R-CHOP regimen was performed in 1,444 (79%) patients, including 697 patients with 60-80% of IDI and 747 patients with 40-60% of IDI. FN occurred in 115 of the 1,819 patients (6.3%) with a median of 10 days (interquartile range: 8-12) after the administration of cyclophosphamide. Antimicrobial prophylaxis, primary prophylaxis with daily G-CSF, and pegfilgrastim were not significantly associated with a lower occurrence of FN in the very elderly patients (odds ratio 0.69 [95% confidence interval 0.30-2.21], 1.39 [0.73-2.64], 1.43 [0.73-2.81], respectively). Reduced IDI was significantly associated with a lower occurrence of FN (40-60%: odds ratio 0.54 [0.32-0.92]). Advanced-stage lymphoma (>stage 2) and placement of central venous catheter was significantly associated with the occurrence of FN (odds ratio 1.59 [1.02-2.49], 3.19 [1.81-5.62], respectively). Conclusion: The present nationwide study showed that IDI was reduced in most of the very elderly patients with DLBCL in a real-world clinical practice. The proportion of the very elderly patients who developed FN tended to be lower than those in previous studies with younger patients, which may be explained by reduced IDI in the very elderly. Whereas, prevention strategies with G-CSF for FN may be less effective in the very elderly. Disclosures Matsuda: Ono Pharmaceutical: Other: Lecture fee; Kyowa Kirin: Other: Lecture fee. Jo: Tsumura: Other: Lecture fee, Research Funding; AstraZeneca: Other: Lecture fee; Sanofi: Other: Lecture fee; Boehringer Ingelheim: Other: Lecture fee. Shimura: Eisai: Other: Lecture fee. Honda: Otsuka Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Takeda Pharmaceutical: Other: Lecture fee. Masamoto: MSD K.K.: Speakers Bureau; Eisai Co., Ltd.: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Takeda Pharmaceutical Company Limited.: Speakers Bureau; Chugai Pharmaceutical Company: Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Speakers Bureau; AbbVie GK: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau; SymBio Pharmaceuticals: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau. Yasunaga: Pfizer: Consultancy, Other: Lecture fee; Novartis: Consultancy, Other: Lecture fee; Boehringer Ingelheim: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Tsumura: Other: Lecture fee. Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau.

Primary cutaneous CD30+ T-cell lymphoproliferative disorders (CD30+ LPD) are the second most common cutaneous lymphomas. According to the World Health Organization (WHO), CD30+ LPD include primary cutaneous anaplastic large cell lymphoma (pcALCL) and lymphomatoid papulosis (LyP) as well as borderline lesions. pcALCL and LyP is thought to represent two ends of a spectrum of diseases that have different clinical presentations, clinical courses, and prognoses in their classic forms, but share the same histology of medium to large CD30+ atypical lymphoid cell infiltrates. Because the behavior of these entities is different clinically and prognostically, we aim to search for oncogenic genomic variants using whole exome sequencing (WES) that drive the development of LyP and pcALCL. Clinical information, pathology, immunohistochemistry, and T-cell rearrangements on six cases of LyP and five cases of pcALCL were reviewed to confirm the rendered diagnosis prior to WES of all specimens. All cases of CD30 LPD had recurrent mutations in at least one of the epigenetic modifying genes, with the most frequent mutations found in the mixed lineage methyltransferase family involved in the methylation of H3K4: CREBBP (27%), KMT2A (36%), KMT2D (36%), SETD2 (27%), and SMARCA4 (27%) (Figure). Within the JAK/STAT pathway, mutations of STAT3 were observed in 2 cases of pcALCL (n=2, 18%), STAT5B in one case of LyP (n=1, 9%) and JAK1 mutation in one case of LyP(n=1, 9%). Lastly, mutations were also identified in the T-cell signaling pathway. 3/5 cases of pcALCL demonstrated loss of function mutations in EOMES, a T-box transcription factor important in lymphocyte development. We had two cases of C-ALCL and one case of LyP with NOTCH1 mutations supporting the importance of this gene in T-cell lymphomas. TP53 mutations and copy number loss is more characteristic of aggressive lymphomas and these abnormalities were absent in pcALCL and LyP and may explain the indolent nature of CD30+ LPD. While the JAK/STAT pathway, T-signaling, and epigenetic alterations possibly play a role in the pathogenesis of these diseases, genes involved in cell-cycle control and apoptosis (ie. TP53) that are characteristic of more aggressive diseases were not identified. Extending this investigation to a larger number of samples will allow us to detect additional mutations that may help distinguish between CD30+ LPDs of LyP and pcALCL as well as other histologic mimics such as systemic ALCL and large cell transformation of MF. Figure 1 Figure 1. Disclosures Abdulla: Caris Life Licenses: Current Employment. Zain: Kiyoaw Kirin, Secura Bio, Seattle Genetics: Honoraria; Secura Bio, DaichiSankyo, Abbvie: Research Funding; Secura Bio, Ono , Legend, Kiyowa Kirin, Myeloid Therapeutics Verastem Daichi Sankyo: Consultancy.

Background: Erdheim-Chester disease (ECD) is a type of systemic, non-Langerhans histiocytic disorder characterized by diffuse organ damage with infiltration of CD68-positive, CD163-positive, and CD1a-negative histiocytes. Although most patients have mutations associated with the hyperactivation of the MAPK pathway, including BRAF, MAP2K1, and NRAS, the remaining have no known driver mutations. So far, there is no specific target of treatment for them. To elucidate novel driver mutations and establish new treatment strategies in these patients, we performed whole-exome sequencing (WES) and whole-genome sequencing (WGS) using patient samples with ECD. In addition, we performed WES with multi-organ lesions in a patient with ECD to reveal the mutational profiles of each organ lesion. Methods: We performed a nationwide survey and collected clinical samples of ECD in Japan. We collected 22 samples of ECD lesions from 15 adult patients. All cases were pathologically proved. Twenty of 22 samples were formalin-fixed and paraffin-embedded tissue (FFPE) and were examined by WES. For WGS, DNA was extracted from 2 raw samples of ECD lesions. In addition, we collected multi-organ lesions in 3 patients. One patient developed myelodysplastic syndrome (MDS) and subsequent acute myeloid leukemia (AML). Therefore, we also performed WES with each bone marrow (BM) sample to reveal the relationship between ECD and these myeloid malignancies. We used 6 samples of peripheral blood, 1 BM sample, 1 skin sample, and 1 oral mucosa sample as normal controls. Result: We detected known driver mutations in seven of 15 cases (47%). Among them, BRAF V600E was detected in 5 cases (8 samples), MAP2K1 C121S in 1 case (1 sample), and NRAS Q61R in 1 case (2 samples) by WES and WGS. A mean of 69 nonsynonymous mutations per patient was identified in normal-tumor analysis (range, 2-491) and 188 in tumor-only analysis (range, 17-3598) of WES, and 3134 in normal-tumor analysis (range, 2588-3680) of WGS. The median variant allele frequency (VAF) for the 11 samples with known activating kinase mutations identified by WES and WGS was 14.4% (range, 6.3-34.7). We could not detect known driver mutations in the other 8 cases (53%). Therefore, to reveal novel driver mutations, we focused on these 8 cases. Notably, EPHA2 P786L, MYBPC3 D798N, TDRD5 P115L, and TCEAL4 F17L mutations are recurrently found in 2 out of the 8 cases, suggesting new driver mutations are contained in these. The VAFs of EPHA2 P786L were 9.1% and 9.3%, MYBPC3 D798N 5.5% and 11.9%, TDRD5 P115L 10.2% and 18.7%, and TCEAL4 F17L 7.5% and 9.0% in each case. We also analyzed multiple organ samples of a case with BRAF V600E mutation (5 samples of ECD lesions, BM with MDS, BM with AML, and skin as normal control). Interestingly, BRAF V600E mutation was identified in 3 samples (bone lesion, heart, and intestine) but was not in other samples (kidney lesion, dura matter, BM tumor, BM with MDS, and BM with AML), suggesting mutational profiles are different depending on the organ of the lesion. Conclusion: Our nation-wide analysis revealed that no well-known driver mutations were found in more than half of the ECD cases. We identified EPHA2 P786L, MYBPC3 D798N, TDRD5 P115L, and TCEAL4 F17L mutations as candidates of novel driver mutations in ECD. To elucidate the role of these mutations in pathogenesis of ECD, further functional analyses are warranted. In addition, we revealed heterogeneity of mutational profile of multi lesions in an ECD patient with BRAF V600E mutation. It is noteworthy that mutational profiles might be different depending on the organ of the lesion. Disclosures Honda: Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee; Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee. Matsuda: Kyowa Kirin: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee. Taoka: ONO PHARMACEUTICAL CO., LTD.: Other; Chugai Pharmaceutical Company: Other. Kurokawa: Teijin Limited: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau.

Background: Traditional prognostic classification of myelodysplastic syndromes (MDS) is based on scoring systems such as IPSS, WPSS, and IPSS-R. The scoring system lacks several important pieces of information in the patient's condition, especially age, gender, and performance status（PS).Therefore, prognosis prediction by scoring system is not accurate enough because it does not reflect the actual patient situation such as age and PS. Now, we demonstrate the use of a more accurate and intelligent machine learning-based model, and it is desirable to construct a more comprehensive prognostic system suitable for actual real worlds data in clinical practice. Methods: We analyzed clinical data set of 333 patients with MDS diagnosed at the University of Tokyo Hospital from April 1996 to March 2020 using machine learning. Now, we created two novel prediction models for MDS using machine learning. 1) A novel prognostic prediction system was constructed by machine learning of the patient information including age, gender, PS, white blood counts, neutrophil, hemoglobin level, platelet counts, blast counts in bone marrow and peripheral blood, chromosomal abnormalities and treatment. 70% of the patient information in the dataset was analyzed as training data, and the remaining 30% was evaluated as test data. The cases were randomly assigned to the training test datasets. First, we used the training dataset to build a prediction system to link clinical information with subsequent leukemogenesis and mortality. Next, we evaluated the performance including the AUC and accuracy of the system on the test data. 2) To assess the benefit of drugs administered for the treatment of MDS, the effectiveness of drugs must be compared among patients with a similar level of risk. To this end, we devised a leukemogenesis index, in which we classified MDS patients into two groups: low risk (index 0.5). We compared the mortality of these leukemogenesis risk-matched subgroups between the therapeutic agent and non-therapeutic agent. The outcome of patients in the leukemogenesis risk-matched subgroups demonstrated the real-world effectiveness of the therapeutic agents. Results: We first attempted to find the optimal algorithm for machine learning out of 15 candidate algorithms by using the PyCaret, a machine learning library. It was determined that CatBoost performed the best in terms of an AUC (Area Under the Curve) and accuracy. Using CatBoost algorithm, the prediction performances of our algorithm for mortality with MDS patients yielded an AUC score of 0.73 and an accuracy score of 0.75. The AUC of this novel system with machine learning was 0.75, which showed an improvement of 16% in performance compared to the AUC of 0.59 for the traditional IPSS system. The prediction performances for leukemogenesis yielded an AUC score of 0.73 and an accuracy score of 0.76. To assess the effectiveness of administered therapeutic agents, it is necessary to examine the effects of agents among MDS patients in subgroups of uniform predicted risk at the time of presentation when drug treatment is considered. To this end, we devised an evaluation method for leukemogenesis risk-matched analysis using the risk index. We compared the mortality rates of these leukemogenesis risk-matched subgroups between the treatment groups of Aza (azacytidine) and the other-treatment groups in non-transplantable patients. In the low-risk group, the Kaplan-Meier estimates of mortality by 1 year were 85 % in the Aza treatment group and same as 85 % in the other treatment group. In the high-risk group, the Kaplan-Meier estimates of mortality by 1 year were 64 % in the Aza treatment group and 42 % in the other treatment group. Due to the 60 cases in high-risk group, the difference did not reach significance. Conclusion: In summary, we have developed novel comprehensive prediction models of leukemogenesis and prognosis for MDS patients with high accuracy using machine learning. We also developed a novel system, "leukemogenesis risk-matched analysis," to infer the real-world effectiveness of Aza stratifying MDS patients based on leukemogenesis risk as assessed by machine learning according to the patient information at the time of initial diagnosis. In addition, the leukemogenesis risk index can be used to distinguish between high-risk and low-risk patients, which is useful in supporting the management of treatment. Disclosures Honda: Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee. Kurokawa: AbbVie GK: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; MSD K.K.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau.

Resistance to anthracycline and cytarabine based conventional chemotherapy often occurs and results in extremely poor prognosis in patients with acute myeloid leukemia (AML). Although chemotherapy resistance is the most critical clinical problem, the mechanisms by which AML confers resistance to conventional chemotherapy are not yet fully understood. In this study, we investigated the key mechanisms of chemotherapy resistance through single cell RNA-sequencing analysis using paired bone marrow AML cells longitudinally collected from two AML-MRC patients at diagnosis and relapse after anthracycline-based chemotherapy. AML blasts were sorted by CD45/SSC gating and subjected to single cell RNA-seq analysis. Single cell RNA-seq was performed using 10x Genomics' Chromium System. Mean estimated number of cells per sample was 3.403 (2,731-4,200) and median detected genes per cell ranged 3,030 to 3,918 among four samples. Data collected from paired samples were combined in following analysis. Transcriptome based clustering following UMAP dimensionality reduction distinguished 5 and 9 cluster groups in each paired sample. Chemotherapy sensitive cluster groups dominant at diagnosis and chemotherapy resistant cluster groups dominant at relapse were clearly divided. In each paired sample, a few AML cells at diagnosis were allocated to chemotherapy resistant cluster groups. This suggested that transcriptionally identifiable less frequent cells resistant to chemotherapy existed at diagnosis and may expand during and/or after chemotherapy maintaining its transcriptional features. Next, to determine whether these transcriptional features are correlated with DNA mutation profiles, we labeled DNA mutation status to each cell and compared frequencies of mutation. As far as we detected, AML recurrent mutations such as DNMT3A R882C and TP53 missense mutation were not related to chemotherapy resistant cluster groups, although this method was relatively limited by the nature of RNA-seq-based mutation detection. Then we sought to determine transcriptional features of resistant clones. Gene set enrichment analysis identified some gene groups such as E2F signaling pathway, MYC signaling pathway, hedgehog signaling pathway and TNFA signaling pathway as transcriptional signatures related to emergence after chemotherapy. Analysis of known hematopoietic differentiation gene signatures showed distinct differentiation profiles in each cluster groups, whereas resistant cluster groups were not necessarily related to hematopoietic stem cell signatures. Intrapatient variations of transcriptional signatures among the resistant cluster groups were detected, which indicated that accurate detection of transcriptional features related to chemotherapy resistance may be difficult by using bulk RNA-seq method. As for other cluster groups which were not dominant both at diagnosis and relapse, these cluster groups hardly changed its frequencies between at diagnosis and relapse, which suggested less proliferative leukemia cells persisted during chemotherapy and have various transcriptional features although whether these persisting cells contribute to relapse was unclear. Since enriched transcriptional signatures in resistant cluster groups were not consistent between the two patients, further analysis using samples collected from more patients would be needed to determine common critical chemotherapy resistant transcriptional signature. In conclusion, our analysis suggested that a transcriptionally identifiable small fraction of cells showing gene signatures related to chemotherapy resistance at diagnosis may expand during chemotherapy and revealed intrapatient transcriptional complexity of response to chemotherapy, which cannot be uncovered by bulk RNA-sequencing. Disclosures Honda: Takeda Pharmaceutical: Other: Lecture fee; Otsuka Pharmaceutical: Other: Lecture fee; Chugai Pharmaceutical: Other: Lecture fee; Ono Pharmaceutical: Other: Lecture fee; Jansen Pharmaceutical: Other: Lecture fee; Nippon Shinyaku: Other: Lecture fee. Kurokawa: MSD K.K.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Company.: Research Funding, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding, Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding, Speakers Bureau; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Chugai Pharmaceutical Company: Research Funding, Speakers Bureau; AbbVie GK: Research Funding, Speakers Bureau.

Dyskeratosis congenita (DC) is a short telomere syndrome with bone marrow failure (BMF), mucocutaneous fragility and predisposition to malignancy due to inherited mutations in telomere associated genes. The genetic cause remains unknown in 10-20% of DC patients. Here, we describe a new DC candidate gene, RPA1, which encodes the largest subunit of the Replication Protein A (RPA) complex, a single stranded DNA (ssDNA) binding protein essential for DNA replication, damage repair and telomere maintenance. The patient presented at the age of 5 years with pancytopenia and hypocellular marrow with morphology resembling refractory cytopenia of childhood, mucocutaneous triad and a congenital retinal anomaly. Further workup revealed telomeres in blood below 1st percentile, normal female karyotype and a negative chromosomal breakage test. After excluding known genetic causes for DC, family whole exome sequencing identified a de novoRPA1 c.718G>A (p.E240K) mutation with a predicted pathogenic CADD score of 23 and absent in >140,000 gnomAD population controls. The mutation had 50% allelic frequency in fibroblasts in comparison to a reduced mutation burden of 27% in bone marrow (BM) cells. Using bulk and single cell sequencing of BM, we identified 2 somatic compensatory events that likely explain patient's stable hematologic phenotype over a period of 20 years: i) an acquired RPA1 p.E579X mutation (in cis with p.E240K) was found at 10% frequency, which resulted in RNA degradation of the mutated allele as confirmed by RNA sequencing; ii) a founder clone with uniparental isodisomy on chromosome 17p encompassing RPA1 locus causing loss of germline E240K mutation. Analysis of serial BM samples using SNP arrays and deep sequencing demonstrated UPD17p clonal expansion concurrent with a decline of germline p.E240K and increase of acquired p.E579X mutation. This is consistent with a toxic, likely gain-of-function (GOF) effect of germline p.E240K mutation leading to acquired loss-of-function (LOF) escape mechanisms. RPA1 is essential to all DNA metabolism transactions that encounter ssDNA, including binding to telomeric 3' overhangs during late S-phase to unfold G-quadruplexes and facilitate telomerase activity. Several RPA1 genetic variants impart telomere shortening and an S-phase defect in yeast and human cell lines. However, the molecular mechanisms are not well understood and thus far, RPA1 gene has not been linked to human disease. In an iPSC-based disease model with homozygous RPA1 p.E240K knock-in, we discovered severe telomere shortening in RPA1 mutant iPSCs and iPSC derived hematopoietic progenitors (HPs) compared to WT counterparts. Furthermore, decreased erythroid differentiation capacity was noted in RPA1-mutant HPs. Because the germline p.E240K mutation is located in DNA binding domain A of RPA1, we performed electromobility shift assay to assess the DNA binding affinity of mutant RPA1 protein. The p.E240K heterotrimeric RPA complex shows increased ssDNA binding compared to wild type RPA. In summary, we describe the first human disease manifesting as short telomere syndrome secondary to a de novoRPA1 mutation with likely GOF effect, which forces the development of multiple revertant mosaic events, similar to the newly described SAMD9/SAMD9L disorders. Our studies also have implications for the mechanistic understanding of the role of RPA1 in telomere maintenance and hematopoiesis. Disclosures Niemeyer: Celgene: Consultancy; Novartis: Consultancy.

Background: Multicentric Castleman's disease (MCD) is a rare lymphoproliferative disease accompanying various symptoms and multi-organ dysfunctions. Although anti-interleukin-6 monoclonal antibodies, tocilizumab and siltuximab are recommended as a therapeutic option, these treatments require patients to visit hospitals at least once a month for many years, and the drug costs are quite high. In Japan, where tocilizumab is the only biological agent covered by the medical insurance, glucocorticoid monotherapy is still an important treatment option. In fact, a part of MCD patients initially treated with glucocorticoids could be successfully controlled without adding or switching to other agents. Considering that emergence of neutralizing anti-drug antibodies which reduced therapeutic efficacy could also be a clinical problem in MCD, there would be a rationale for reserving newer agents for future relapse by using glucocorticoids as first-line treatment for potential responders. Here, we retrospectively analyzed clinical characteristics of MCD patients treated in our institution to explore factors predicting who could be successfully treated with glucocorticoid monotherapy. Methods: We retrospectively reviewed histologically confirmed Castleman's disease patients who visited the Department of Hematology and Oncology of the University of Tokyo Hospital from January 2000 to March 2020. Based on the diagnostic criteria of Castleman Disease Collaborative Network (CDCN), MCD was diagnosed when enlarged lymph nodes were present in more than 2 lymph node stations and laboratory and/or clinical diagnostic criteria were met. Unicentric Castleman's disease (UCD) was diagnosed when only one single lymph node region was involved. MCD patients who were initially treated with prednisolone (PSL) were divided into two groups: patients who continued PSL until the latest follow-up (PSL-only group) and patients who received subsequent treatment (Second-line group). Results: 8 patients with UCD and 27 patients with MCD were included in our analysis. With a median follow-up of 5.2 years, 21 MCD patients underwent first-line treatment (PSL, n=18; tocilizumab, n=3). Compared to patients who did not receive any treatment, patients who underwent treatment had marginally higher levels of CRP (7.15 mg/dl vs 4.17 mg/dl, respectively; p=0.066) and significantly lower levels of hemoglobin (9.5 g/dl vs 12.5 g/dl, respectively; p=0.036). Seven out of 18 patients initially treated with PSL had received subsequent treatments (tocilizumab, n=6; rituximab, n=1). Among the PSL-only group, biochemical responses at the latest follow-up could be assessed for 10 in 11 patients: two in complete response, five in partial response, two in stable disease, and one in progressive disease, based on the response criteria of CDCN. Compared to PSL-only group, patients classified into second-line group had higher levels of CRP (11.88 mg/dl vs 6.24 mg/dl, respectively; p=0.024) and lower hemoglobin levels (6.4 g/dl vs9.4 g/dl, respectively; p=0.033). Patients whose CRP levels were lower than 12 mg/dl before starting PSL had significantly longer time to next treatment (TTNT) (median not reached vs 0.88 years; HR, 0.078 [95%CI: 0.013-0.48], p=0.00044). Similarly, patients whose hemoglobin levels were more than 8 g/dl had marginally longer TTNT (median not reached vs 2.63 years; HR, 0.22 [95%CI: 0.040-1.17], p=0.054). In addition, patients with either Hb < 8 g/dl or CRP ≧12 mg/dl had significantly shorter TTNT (median not reached vs 2.12 years; HR, 0.090 [95%CI: 0.010-0.77], p=0.0074). The median PSL dose at the latest follow-up in PSL-only group was 3 mg per day [interquartile range: 0-5 mg per day], which would be comparably safe considering previous reports indicating that PSL dose of less than 5 mg per day was associated with lower incidence of adverse events. Conclusion: Out data suggest that glucocorticoid monotherapy has a good potential to induce sustained disease control for MCD patients with higher Hb or lower CRP levels. Furthermore, PSL doses could be tapered to safer doses among patients who could continue PSL until the latest follow-up. In contrast, the efficacy of glucocorticoids for MCD patients with lower Hb or higher CRP levels were limited. Such patients would be good candidates for novel agents such as tocilizumab or rituximab. Disclosures Honda: Nippon Shinyaku Co., Ltd.: Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau. Nakahara:Eisai Co., Ltd.: Honoraria; Astellas Pharma Inc.: Honoraria; Bristol-Myers Squibb Company: Honoraria. Kurokawa:Chugai: Consultancy, Research Funding, Speakers Bureau; Sanwa-Kagaku: Consultancy; Pfizer: Research Funding; Otsuka: Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; Teijin: Research Funding; Eisai: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Bioverativ Japan: Consultancy; Shire Plc: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; MSD: Consultancy, Research Funding, Speakers Bureau.

Affinity regulation of integrin αIIbβ3 for fibrinogen by inside-out signaling plays a critical role in hemostasis. Calcium and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) was identified as a Rap1-activating molecule, and its role in inside-out αIIbβ3 activation was established in CalDAG-GEFI-deficient mice. However, little information regarding CalDAG-GEFI in human platelets is available. Here, we report a 16-year-old girl with CalDAG-GEFI deficiency who has been suffering from severe bleeding tendency. Although talin and kindlin-3 were normally detected, CalDAG-GEFI was undetectable in her platelets by western blotting. Genetic analysis revealed compound heterozygous CalDAG-GEFI mutations, Lys309X and Leu360del, which were responsible for CalDAG-GEFI deficiency. The functional analysis demonstrated impaired αIIbβ3 activation by various agonists except for phorbol myristate acetate, normal calcium mobilization, and impaired Rap1 activation, which were consistent with the phenotype of CalDAG-GEFI-deficient mice. Despite substantial αIIbβ3 activation at high agonist concentrations, she had severe bleeding tendency. Further functional analysis demonstrated markedly delayed αIIbβ3 activation velocity and decreased shear-induced thrombus formation. Contrary to CalDAG-GEFI-deficient mice, which showed integrin-dependent neutrophil functional abnormality, neutrophil β2 integrin activation was not impaired in the patient. Our results demonstrate the critical role of CalDAG-GEFI in rapid αIIbβ3 activation of human platelets.

Karyotype is an important prognostic factor in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL), but the underlying pharmacogenomics remain unknown. Asparaginase is an integral component in current chemotherapy for childhood BCP-ALL. Asparaginase therapy depletes serum asparagine. Normal hematopoietic cells can produce asparagine by asparagine synthetase (ASNS) activity, but ALL cells are unable to synthesize adequate amounts of asparagine. The ASNS gene has a typical CpG island in its promoter. Thus, methylation of the ASNS CpG island could be one of the epigenetic mechanisms for ASNS gene silencing in BCP-ALL. To gain deep insights into the pharmacogenomics of asparaginase therapy, we investigated the association of ASNS methylation status with asparaginase sensitivity. The ASNS CpG island is largely unmethylated in normal hematopoietic cells, but it is allele-specifically methylated in BCP-ALL cells. The ASNS gene is located at 7q21, an evolutionally conserved imprinted gene cluster. ASNS methylation in childhood BCP-ALL is associated with an aberrant methylation of the imprinted gene cluster at 7q21. Aberrant methylation of mouse Asns and a syntenic imprinted gene cluster is also confirmed in leukemic spleen samples from ETV6-RUNX1 knockin mice. In 3 childhood BCP-ALL cohorts, ASNS is highly methylated in BCP-ALL patients with favorable karyotypes but is mostly unmethylated in BCP-ALL patients with poor prognostic karyotypes. Higher ASNS methylation is associated with higher L-asparaginase sensitivity in BCP-ALL through lower ASNS gene and protein expression levels. These observations demonstrate that silencing of the ASNS gene as a result of aberrant imprinting is a pharmacogenetic mechanism for the leukemia-specific activity of asparaginase therapy in BCP-ALL.