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Acute myeloid leukemia (AML) is still incurable due to its heterogeneity and complexity of tumor microenvironment. It is imperative therefore to understand the molecular pathogenesis of AML and identify leukemia-associated biomarkers to formulate effective treatment strategies. Here, we systematically analyzed the clinical characters and natural killer (NK) cells portion in seventy newly-diagnosis (ND) AML patients. We found that the proportion of NK cells in the bone marrow of ND-AML patients could predict the prognosis of patients by analyzing the types and expression abundance of NK related ligands in tumor cells. Furthermore, MCL1 inhibitor but not BCL2 inhibitor combined with NK cell-based immunotherapy could effectively improve the therapeutic efficiency via inhibiting proliferation and inducing apoptosis of AML primary cells as well as cell lines in vitro. There results provide valuable insights that could help for exploring new therapeutic strategies for leukemia treatment. Supplementary Information The online version contains supplementary material available at 10.1186/s12943-020-01302-6.

The treatment of relapse or refractory multiple myeloma (RRMM) is still a big challenge in clinic. Recently, several clinical trials indicated that the XPO1 inhibitor, selinexor could significantly improve the remission rate in MM patients. However, the heterogeneous genetic background greatly influenced the efficiency of selinexor among MM. Here, we tried to characterized the biomarkers associated with selinexor sensitivity by analyzing gene expression data in MM patients. We found the cytogenetic background of selinexor sensitive MM patients was not limited to specific cytogenetic subtypes. In addition, by weighted gene co-expression network analysis (WGCNA), we obtained 10 key genes which showed a strong correlation with the selinexor sensitivity in MM patients. Notably, ABCC4 (MRP4) was the only gene which was both differentially expressed and proved to be clinical prognostic valuable (both for event-free survival and overall survival) in MM patients. We further validated the heterogenous expression of ABCC4 in MM cell lines and its value as a novel indicator for selinexor sensitivity. Moreover, immune infiltration analysis showed that ABCC4 expression had a significantly positive correlation with NK infiltration as well as immunotherapy target TIM-3 (HAVCR2) expression. Collectively, our findings indicated that ABCC4 might be a predictive biomarker of selinexor sensitivity in MM patients, which could be enhanced if combined with immunotherapy drugs such as TIM-3 inhibitor.

We explored the roles of adenylyl cyclases (ADCYs) in acute myeloid leukemia (AML). Expression ADCYs in AML and their effect on prognosis was analyzed using data from Oncomine, GEPIA and cBioPortal databases. Frequently altered neighbor genes (FANGs) of ADCYs were detected using the 3D Genome Browser, after which the functions of these FANGs were predicted using Metascape tools. Cell viability and apoptosis were assessed using CCK-8 and Annexin V-FITC/PI kits. Expression levels of ADCYs were higher in AML cells lines and in bone marrow-derived mononuclear cells from AML patients than in control cells, and were predictive of a poor prognosis. A total of 58 ADCY FANGs were identified from the topologically associating domains on the basis of the Hi-C data. Functional analysis of these FANGs revealed abnormal activation of the MAPK signaling pathway. Drug sensitivity tests showed that fasudil plus trametinib or sapanisertib had a synergistic effect suppressing AML cell viability and increasing apoptosis. These findings suggest that dysregulation of ADCY expression leads to altered signaling in the MAPK pathway in AML and that the ADCY expression profile may be predictive of prognosis in AML patients.

Myelodysplastic syndrome (MDS) is clonal disease featured by ineffective haematopoiesis and potential progression into acute myeloid leukaemia (AML). At present, the risk stratification and prognosis of MDS need to be further optimized. A prognostic model was constructed by the least absolute shrinkage and selection operator (LASSO) regression analysis for MDS patients based on the identified metabolic gene panel in training cohort, followed by external validation in an independent cohort. The patients with lower risk had better prognosis than patients with higher risk. The constructed model was verified as an independent prognostic factor for MDS patients with hazard ratios of 3.721 (1.814‐7.630) and 2.047 (1.013‐4.138) in the training cohort and validation cohort, respectively. The AUC of 3‐year overall survival was 0.846 and 0.743 in the training cohort and validation cohort, respectively. The high‐risk score was significantly related to other clinical prognostic characteristics, including higher bone marrow blast cells and lower absolute neutrophil count. Moreover, gene set enrichment analyses (GSEA) showed several significantly enriched pathways, with potential indication of the pathogenesis. In this study, we identified a novel stable metabolic panel, which might not only reveal the dysregulated metabolic microenvironment, but can be used to predict the prognosis of MDS.

Cancer stem cells (CSCs) are characterized by self‐renewal and ‐differential potential as compared to common cancer cells and play an important role in the development and therapeutic resistance of liver hepatocellular carcinoma (LIHC). However, the specific pathogenesis of LIHC stem cells is still unclear, and the genes involved in the stemness of LIHC stem cells are currently unknown. In this study, we investigated novel biomarkers associated with LIHC and explored the expression characteristics of stem cell‐related genes in LIHC. We found that mRNA expression‐based stemness index (mRNAsi) was significantly overexpressed in liver cancer tissues. Further, mRNAsi expression in LIHC increased with the tumor pathological grade, with grade 4 tumors harboring the greatest stem cell features. Upon establishing mRNAsi scores based on mRNA expression of every gene, we found an association with poor overall survival in LIHC. Moreover, modules of interest were determined based on weighted gene co‐expression network analysis (WGCNA) inclusion criteria, and three significant modules (red, green, and brown) and 21 key genes (DCN, ECM1, HAND2, PTGIS, SFRP1, SRPX, COLEC10, GRP182, ADAMTS7, CD200, CDH11, COL8A1, FAP, LZTS1, MAP1B, NAV1, NOTCH3, OLFML2A, PRR16, TMEM119, and VCAN) were identified. Functional analysis of these 21 genes demonstrated their enrichment in pathways involved in angiogenesis, negative regulation of DNA‐binding transcription factor activity, apoptosis, and autophagy. Causal relationship with proteins indicated that the Wnt, Notch, and Hypoxia pathways are closely related to LIHC tumorigenesis. To our knowledge, this is the first report of a novel CSC biomarker, mRNAsi, to predict the prognosis of LIHC. Further, we identified 21 key genes through mRNA expression network analysis, which could be potential therapeutic targets to inhibit the stemness of cancer cells in LIHC., We first reported a novel biomarker mRNAsi of stem cell index to predict the prognosis of LIHC through network analysis of the expression levels of LIHC. 21 key genes were found played important roles in LIHC stem cells. These genes could be the potential therapeutic targets for inhibiting the stemness of LIHC.

Aiming to develop an effective catalytic process for utilizing biomass, conversion of corncob into furfural was firstly performed using tin-loaded bentonite (Sn-BTN) as solid acid catalyst. Corncob was pretreated with Sn-BTN (3.5 wt%) for producing furfural at 53.3% yield in methyl isobutyl ketone-water (5:5, v:v; pH 1.0) biphasic media within 0.5 h at 170 °C. Furthermore, an efficient Sn-BTN recycling was achieved after 5 cycles of repeated use. Finally, furoic acid was obtained at 53.3% yield from corncob via sequential catalysis with solid acid Sn-BTN and Brevibacterium lutescens whole cells harboring furfural-oxidizing activity in methyl isobutyl ketone-H2O (5:5, v:v) media was developed under the relatively mild conditions. Clearly, this hybrid strategy for the synthesis of furoic acid from biomass was successfully developed via tandem catalysis of corncob with Tin-loaded bentonite and Brevibacterium lutescens whole-cell.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, with acute respiratory failure as the most significant symptom, has led to a global pandemic. Angiotensin-converting enzyme 2 (ACE2) is considered as the most important receptor of SARS-CoV-2 and wildly expressed in human tissues. Whereas, the extremely low expression of ACE2 in lung could hardly interpret the severe symptom of pneumonia in COVID-19 patients. Here we profiled two SARS-CoV-2 infection related genes, the transmembrane serine protease 2 (TMPRSS2) and the interferon-inducible transmembrane protein 3 (IFITM3), in human tissues and organs. Consistent with the expression and distribution of ACE2, TMPRSS2 was also highly expressed in digestive, urinary and reproductive systems, but low expressed in lung. Notably, the anti-virus protein IFITM3 also expressed much lower in lung than other tissues, which might be related to the severe lung symptoms of COVID-19. In addition, the low expression of IFITM3 in immune cells suggested that SARS-CoV-2 might attack lymphocytes and induce the cytokine release syndrome (CRS). Furthermore, cancer patients were considered as more susceptible to SARS-CoV-2 infection. Our data supposed that fourteen types of tumors might have different susceptibility to the virus according to ACE2, TMPRSS2 and IFITM3 expression patterns. Interestingly the prognosis of six types of cancers including breast carcinoma (BRCA), lung adenocarcinoma (LUAD), uterine corpus endometrial carcinoma (UCEC), renal clear cell carcinoma (KIRC), prostate adenocarcinoma (PRAD), and hepatocellular carcinoma (LIHC) were closely related to these gene expressions. Our study explored the expression and distribution profiles of two potential novel molecules that might participate in SARS-CoV-2 infection and involved in immunity, which may provide a functional basis for preventing infection of SARS-CoV-2.

DNMT3A mutations are frequently identified in acute myeloid leukemia (AML) and indicate poor prognosis. Previously, we found that the hotspot mutation DNMT3A R882H could upregulate CDK1 and induce AML in conditional knock-in mice. However, the mechanism by which CDK1 is involved in leukemogenesis of DNMT3A mutation-related AML, and whether CDK1 could be a therapeutic target, remains unclear. In this study, using fluorescence resonance energy transfer and immunoprecipitation analysis, we discovered that increased CDK1 could compete with EZH2 to bind to the PHD-like motif of DNMT3A, which may disturb the protein interaction between EZH2 and DNMT3A. Knockdown of CDK1 in OCI-AML3 cells with DNMT3A mutation markedly inhibited proliferation and induced apoptosis. CDK1 selective inhibitor CGP74514A (CGP) and the pan-CDK inhibitor flavopiridol (FLA) arrested OCI-AML3 cells in the G2/M phase, and induced cell apoptosis. CGP significantly increased CD163-positive cells. Moreover, the combined application of CDK1 inhibitor and traditional chemotherapy drugs synergistically inhibited proliferation and induced apoptosis of OCI-AML3 cells. In conclusion, this study highlights CDK1 overexpression as a pathogenic factor and a potential therapeutic target for DNMT3A mutation-related AML.

Background Treatment of hemophiliacs with inhibitors remains challenging, and new treatments are in urgent need. Coagulation factor X plays a critical role in the downstream of blood coagulation cascade, which could serve as a bypassing agent for hemophilia therapy. Base on platelet‐targeted gene therapy for hemophilia by our and other groups, we hypothesized that activated factor X (FXa) targeted stored in platelets might be effective in treating hemophilia A (HA) and B (HB) with or without inhibitors. Methods To achieve the storage of FXa in platelets, we constructed a FXa precursor and used the integrin αIIb promoter to control the targeted expression of FXa precursor in platelets. The expression cassette (2bFXa) was carried by lentivirus and introduced into mouse hematopoietic stem and progenitor cells (HSPCs), which were then transplanted into HA and HB mice. FXa expression and storage in platelets was examined in vitro and in vivo. We evaluated the therapeutic efficacy of platelet‐stored FXa by tail bleeding assays and the thrombelastography. In addition, thrombotic risk was assessed in the recipient mice and the lipopolysaccharide induced inflammation mice. Results By transplanting 2bFXa lentivirus‐transduced HSPCs into HA and HB mice, FXa was observed stably stored in platelet α‐granules, the stored FXa is releasable and functional upon platelet activation. The platelet‐stored FXa can significantly ameliorate bleeding phenotype in HA and HB mice as well as the mice with inhibitors. Meanwhile, no FXa leakage in plasma and no signs of increased risk of hypercoagulability were found in transplantation recipients and lipopolysaccharide induced septicemia recipients. Conclusions Our proof‐of‐principle data indicated that target expression of the FXa precursor to platelets can generate a storage pool of FXa in platelet α‐granules, the platelet‐stored FXa is effective in treating HA and HB with inhibitors, suggesting that this could be a novel choice for hemophilia patients with inhibitors., Target expression of the FXa precursor to platelets can generate a storage pool of FXa in platelet α‐granules. The platelet‐delivered FXa is effective in ameliorating the hemorrhagic phenotype of both HA and HB murine models, even in the presence of inhibitors. No sign of hypercoagulability was found in platelet FXa‐containing mice under either steady or pathological status.

As suggested by an increasing amount of evidence, there is alternative splicing (AS) modification within malignancy, which is related to cancer occurrence and development. AS within acute myeloid leukemia (AML) has not yet been systematically analyzed yet. This study analyzed the transcriptomic profiling and corresponding clinical data from AML cases based on The Cancer Genome Atlas (TCGA). In addition, the prediction model, along with the splicing network, was used to analyze the prognosis for AML patients according to the seven different AS event types. Among the 34,984 AS events across the 8830 genes, 2896 AS events were detected among 1905 genes, showing marked correlation with the overall survival of patients. The risk scoring model based on all AS event types was the most efficient in identifying the prognosis for AML patients. Meanwhile, the area under the curve at 1-, 3-, 5-year were 0.852, 0.935, 0.955, respectively. At the same time, the splicing regulating network, which was constituted by 21 splicing factor genes as well as 32 AS events related to survival, was characterized. In conclusion, our predictive model constructed based on the AS events accurately predicts the survival for AML patients. In addition, the network between AS events and splicing factor is established, which may serve as a potential mechanism.

Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all- trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5′-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I–binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 ( TRIM25 ) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I–mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I–induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.

Background The new coronavirus pneumonia (NCP) is now causing a severe public health emergency. The novel coronavirus 2019 (2019-nCoV) infected individuals by binding human angiotensin converting enzyme II (ACE2) receptor. ACE2 is widely expressed in multiple organs including respiratory, cardiovascular, digestive and urinary systems in healthy individuals. These tissues with high expression level of ACE2 seemed to be more vulnerable to SARS-CoV-2 infection. Recently, it has been reported that patients with tumors were likely to be more susceptible to SARS-CoV-2 infection and indicated poor prognosis. Methods The tissue atlas database and the blood atlas were used to analyze the distribution of ACE2 in human tissues or organs of cancers and normal samples. Starbase dataset was applied to predict the prognosis of cancers according to expression level of ACE2. Results In this study, we demonstrated a landscape profiling analysis on expression level of ACE2 in pan-cancers and showed the risky of different type of cancers to SARS-CoV-2 according to the expression level of ACE2. In addition, we found that ACE2 was both differential expression and related to the prognosis only in liver hepatocellular carcinoma (LIHC). Relative high expression of ACE2 indicated a favorable prognosis in LIHC, but they might be more susceptible to SARS-CoV-2. Conclusions We indeed emphasized that LIHC patients with high expression level of ACE2 should be more cautious of the virus infection. Our study might provide a potential clue for preventing infection of SARS-CoV-2 in cancers.

Background: SARS-COV-2-induced corona virus disease 2019 (COVID-19) broke out in China at the end of 2019 ACE2 was considered as the receptor of SARS-COV-2 and

Background: Management of CML with TKIs is constrained by treatment resistance, which portends a poor prognosis particularly in pts failing 2 nd-generation TKIs. Cells with BCR-ABL1 T315I mutations are insensitive to 1 st- and 2 nd -generation TKIs, and compound BCR-ABL1 mutations complicate management with all TKIs (including 3 rd-generation ponatinib). Olverembatinib is a novel, potent, 3 rd-generation, orally active BCR-ABL1 TKI with promising activity against CML , largely irrespective of genotype and has a preliminary favorable safety profile. Methods: HQP1351-CC201 and HQP1351-CC202 are Chinese open, single-arm, multicenter phase 2 trials evaluating the safety and efficacy of olverembatinib in adults with TKI-resistant (BCR-ABL1 T351-mutated) CML-CP and CML-CP, respectively. Olverembatinib was administered at 40 mg orally on alternate days for 28-day cycles. The primary outcome measure is major cytogenetic response (MCyR) and major hematologic response (MaHR) by the end of Cycle 12 in CML-CP and CML-AP, respectively. Secondary study endpoints include : complete CyR (CCyR), complete hematologic response (CHR), major molecular response (MMR), progression-free survival (PFS), overall survival (OS), and safety, including treatment-related adverse events (TRAEs) and serious AEs (SAEs). Results: Baseline characteristics Study CC201 （CML-CP ） On the study cutoff date of August 25,2020, 41 pts were enrolled, of whom 32 (78%) completed ≥ 12 cycles and 21 (51.2%) were male. The median (range) follow-up was 13 (3.1-16.3) months, age was 47 (22-70) years, and interval from CML diagnosis to first olverembatinib dose was 5.31 (0.6-23.2) years. In all, 32 (78.1%) pts had received ≥ 2 prior TKIs and 9 pts withdrew because of progressive disease (PD), intolerance, or consent withdrawal before Cycle 12. Study CC202 （CML-AP ） On the cut-off date of July 27, 2020, 23 pts were enrolled, of whom 14 (61%) had completed ≥ 12 cycles and 18 (78.3%) were male. The median (range) follow-up was 13.5 (1.4-15.2) months, age was 41 (21-74) years, and interval from CML diagnosis to first olverembatinib dose was 4.96 (0.4-10.2) years. In all, 18 (78.3%) pts had received ≥ 2 prior TKIs, and 11 pts withdrew because of PD or intolerance before Cycle 12. Efficacy Study CC201 （CML-CP ） After ≥ 12 treatment cycles in pts without responses at baseline, all 31 (100%) experienced CHR (10 other pts had CHR at baseline); 31/41 (75.6%) MCyR; 28/41 (68.3%) CCyR; and 23/41 (56.1%) MMR (Figure 1). The median time to CHR was 1 (95% CI = 1.0-1.9) month, the median time to MCyR was 2.8 (95% CI = 2.8-5.6) months, and the median time to MMR was 6.5 (95% CI = 2.8 to not reached [NR]) months. At 12 months, the PFS rate was 89.3% (95% CI = 73.9%-95.8%), and the OS was 100% (95% CI = 100%-100%). Study CC202 （CML-AP ） After ≥ 12 treatment cycles in pts without responses at baseline, 17/23 (73.9%) experienced MaHR (65.2% CHR and 8.7% no evidence of leukemia [NEL]); 12/23 (52.2%) MCyR; 11/23 (47.8%) CCyR; and 9/23 (39.1%) MMR (Figure 1). The median time to MaHR was 2.8 (95% CI = 1.0-4.7) months, the median time to MCyR was 5.6 (95% CI = 2.00-NR) months, and the median time to MMR was 13.1 (95% CI = 5.6-NR) months. At 12 months, the PFS rate was 74.1% (95% CI = 48.2%-88.4%), and the OS was 91.3% (95% CI = 69.5%-97.8%). Safety Study CC201 （CML-CP ） Frequent TRAEs (all grades; grade 3-4; SAEs) included thrombocytopenia (70.7%; 48.8%; 7.3%), followed by anemia (61%; 26.8%; 2.4%), leukopenia (43.9%; 17.1%; 0), and neutropenia (36.6%; 19.5%; 0). Common nonhematologic TRAEs (all grades; G3-4) included skin pigmentation (56.1%, 0%) and elevations in creatine kinase (51.2%, 14.6%), ALT (39%, 2.4%) and AST (34.1%, 0) (Table 1). No deaths occurred. Study CC202 （CML-AP ） Common TRAEs (all grades; G3-4; SAEs) included thrombocytopenia (73.9%; 56.5%; 17.4%), anemia (60.9%; 34.8%; 13.0%), leukopenia (56.5%; 30.4%; 0), and neutropenia (26.1%; 21.7%; 0). Common nonhematologic AEs included skin pigmentation (69.6%), hypocalcemia (52.2%), proteinuria (47.8%), hypertriglyceridemia (56.5%), hyperphosphatemia (47.8%), hyperuricemia (21.7%), and arthralgia (34.8%), of which most were grade 1-2 (Table 2). Conclusions: Olverembatinib was efficacious and well tolerated when administered as monotherapy in pts with TKI-resistant CP-CML and AP-CML and the BCR-ABL1 T315Imutation. Internal study identifiers: HQP1351-CC201-CC202. ClinicalTrials.gov identifiers: NCT03883087 and NCT03883100. Figure 1 Figure 1. Disclosures Chen: Ascentage Pharma (Suzhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company. Niu: Ascentage Pharma (Suzhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company. Men: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company. Yang: Ascentage Pharma (Suzhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding.

Multiple myeloma (MM) is a malignant hematological cancer of plasma cells origin. However, MM with/without t(11;14) exists a huge therapeutic venetoclax response gap. To explore the clinical and genetic characteristics of MM patients, we divided the patients into two groups according to the t(11;14) translocation status. A total of 110 patients were enrolled in this analysis and 28 of them had t(11;14) translocation, of which 24 cases were MM patients and 4 cases were smoldering myeloma (SMM) patients. Compared to those t(11;14) counterparts, patients without t(11;14) has a higher ratio of early/later relapse, double/triple hit risk, higher mutation count as well as more hyper-karyotypes and other chromosome alterations (Figure 1A). Circos diagram analysis indicated the correlation between t(11;14) and most of cytogenetic alterations except del IGH, del 13q, t(6;14) and t(4;14),. To be specific, the strong connection between t(11;14) and Cyclin D was also in consistent with other reports (Figure 1B). With the normalized data of area under the curve (AUC), we further analyzed the drug sensitivity of patients with/without t(11;14) and 6 out of 76 drugs were identified to be significantly different. Compared to non-t(11;14), t(11;14) patients were significantly more sensitive to venetoclax as the lower AUC suggests. However, no significant difference in AUC of other potential myeloma drugs was found between the two groups (Figure 1C). In order to identify the critical genes associated with venetoclax sensitivity, we screened out the differentially expressed genes (DEGs) and applied weighted gene co-expression network analysis as previously described (Figure 1D and E). We combined clinical phenotype with the significant modules, and the key genes were screened based on the thresholds with cor. gene module membership > 0.5 and cor. gene significance > 0.3. Five modules were identified and of them brown and yellow modules showed significant negative correlations with AUC, with correlations being -0.4 and -0.31(p=0.01 and 0.07), respectively (Figure 1F). Furthermore, we analyzed these two modules in detail and finally screened 29 key genes associated with venetoclax sensitivity (Figure 1G), which were enriched in regulation of immune effector process pathway by Metascape, suggesting the immune dysfunction in these patients (Figure 1H). To identify the critical molecules affected by venetoclax, we analyzed the DEGs between the two groups divided by the median AUC value . In total, 143 genes were significantly upregulated and 38 genes downregulated in the high AUC group (i.e. low sensitivity to venetoclax) group . Next, we systematically analyzed the DEGs in brown module, AUC of venetoclax and of t(11;14). Notably, only NRG2 was identified to be involved in all these three aspects. NRG2, which presented a high sensitivity of venetoclax, was also validated to be more expressed in non-t(11;14) patients. Moreover, we could observe a trend of the expression of NRG2 increasing with the progression of myeloma, especially the significantly higher expression in disease stage of MM (P=0.0245) compared to sMM. In addition, NRG2 expression in myeloma is rather high compared to most hematological malignancies, while the extremely low expression was observed in AML which might correspond to its better reactivity to venetoclax. The expression of NRG2 in non-t(11;14) MM cells such as NCIH929, U266B1 and MM.1S was much higher than that in KMS12. BCL-2 expression was an important biomarker of venetoclax sensitivity. We found that NRG2 and BCL-2 had a strong positive correlation in non-t(11;14) MM patients (R 2=0.5016, P Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION HQP1351 (olverembatinib) is an orally active third-generation BCR-ABL TKI designed for treatment of the patients with CML, harboring T315I mutation, which confers resistance against all first- and second-generation TKIs. METHODS The TKI resistant CML patients harboring T315I mutations either in chronic-phase (CP), or in accelerated-phase (AP), were enrolled into two single-arm, multicenter, open-label pivotal studies: HQP1351-CC201 in and HQP1351-CC202, respectively. The HQP1351 was administered at 40 mg once every other day (QOD) for 28 consecutive days per cycle over 24 months. The primary objective was to evaluate efficacy by major cytogenetic response (MCyR) in patients with CML-CP and major hematologic response (MaHR) in CML-AP. Secondary objectives included safety, tolerability, and pharmacokinetics (PK). RESULTS Baseline characteristics Study CC201 (CML-CP) As of the study cut-off date of March 23, 2020, total 41 patients were enrolled, of whom 38 (92.7%) completed ≥ 6 cycles and 21 (51.2%) were male. Median (range) follow-up was 7.9 (3.1-11.1) months, median age was 47 (22-70) years old. Median (range) interval from CML diagnosis to first HQP1351 treatment was 5.31 (0.6-23.2) years, and 32 (78.1%) patients received ≥ 2 prior lines of TKI. Three patients withdrew from the study-due to progressive disease (PD), intolerance, or consent withdrawal. Study CC202 (CML-AP) As of the cut-off date of February 11, 2020, total 23 patients were enrolled, of whom 18 (78.3%) completed ≥ 6 cycles and 18 (78.3%) were male. Follow-up duration was 8.2 (1.4-9.6) months, median age was 41 (21-74) years old. Median interval from CML diagnosis to first dose of HQP1351 was 4.96 (0.4-10.2) years, and 18 (78.3%) patients received ≥ 2 prior TKIs. Five patients withdrew because of PD or intolerance before Cycle 6. Efficacy Study CC201 (CML-CP) Across a median follow-up of 7.9 months, the mean (95% CI) 3-month progression-free survival (PFS) was 100% (100-100%) and 6-month PFS 96.7% (78.6-99.5%) because of 1 PD. In 31 evaluable patients who did not have a complete hematologic response (CHR) at baseline, 30 (96.8%) achieved CHR. In 41 evaluable patients who did not have a complete CyR (CCyR) at baseline, 31 (75.6%) achieved MCyR, including 27 (65.9%) CCyR and 4 (9.8%) partial CyR (PCyR). Total 20 out of 41 (48.8%) evaluable patients achieved major molecular response (MMR; Figure 1). Study CC202 (CML-AP) Across a median follow-up of 8.2 months, the 3-month PFS was 100% (100-100%) and the 6- month PFS 95.5% (71.9-99.3%) because of 2 PDs. A total of 18 (78.3%) of 23 evaluable patients without MaHR at baseline achieved MaHR, including 14 (60.9%) with CHR. In the 23 evaluable patients without MCyR at baseline, 12 (52.2%) patients achieved MCyR, including 9 (39.1%) CCyR and 3 (13.1%) PCyR. A total of 6 out of 23 (26.1%) evaluable patients achieved MMR (Figure 1). HQP1351 was highly and durably efficacious in the CML patients with T315I mutation; the probability and depth of clinical response may increase with prolonged treatment period. Safety/tolerability Study CC201 Frequent treatment-related adverse events (TRAEs; all grades; G3-4) included thrombocytopenia (65.9%; 48.8%), followed by anemia (48.8%; 24.4%), leukopenia (46.3%; 12.2%), and neutropenia (36.6%; 19.5%). Common non-hematologic TRAEs of any grade included skin pigmentation (53.7%) as well as elevations in creatine kinase (48.8%), ALT (31.7%) and AST (26.8% which most were grade 1 - 2. No death occurred. Study CC202 Common TRAEs (all grades; G3-4; serious AE [SAE]) included thrombocytopenia (73.9%; 52.2%; 17.4%), anemia (65.2%; 39.1%; 13.0%), leukopenia (56.5%; 30.4%; 0%), and neutropenia (26.1%; 21.7%; 0%;). Common non-hematologic AEs included skin pigmentation (69.6%), hypocalcemia (52.2%), proteinuria (52.2%), hypertriglyceridemia (47.8%), hyperphosphatemia (43.5%), arthralgia (34.8%), and fatigue (26.1%), of which most were grade 1- 2. CONCLUSIONS HQP1351 has been shown highly efficacious in heavily TKI-pretreated patients with T315I-mutated CML-CP or CML-AP and was well tolerated. Registration: Clinicaltrials.gov identifier NCT03883087 (Study HQP1351-CC201) NCT03883100 (Study HQP1351-CC202). Disclosures Chen: Ascentage Pharma Group: Current Employment. Niu:Ascentage Pharma Group: Current Employment. Zeng:Ascentage Pharma Group: Current Employment. Men:Ascentage Pharma Group: Current Employment. Lu:Ascentage Pharma Group: Current Employment, Current equity holder in publicly-traded company. Wang:Ascentage Pharma Group: Current Employment. JI:Ascentage Pharma Group: Current Employment. Yue:Ascentage Pharma Group: Current Employment. Yang:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests. Zhai:Ascentage Pharma (SuZhou) Co., Ltd: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests.

Background: Myelodysplastic syndrome (MDS) was characterized as ineffective hematopoiesis, increased transformation to acute myeloid leukemia (AML), and accompanied by immune system dysfunction. However, the immune signature of MDS remains elusive. Methods: The clinical data (age, sex, international prognostic score system (IPSS), hemoglobin, blast, RBC transfusion dependence, and corresponding subject-level survival) as well as expression profiles of MDS (CD34+ cells) were obtained from Gene Expression Omnibus (GEO: GSE 58831; GSE 114922). A robust prognosis model of immune genes was constructed by the least absolute shrinkage and selection operator (LASSO) regression analysis. Survival analysis for prognostic model was carried out through the Kaplan-Meier curve and Log-rank test. The receiver operating characteristic (ROC) curves and area under the curve (AUC) were used to assess the accuracy of prognostic models. Immune score for different subtype were calculated further by single sample gene set enrichment analysis (ssGSEA). Result: A novel robust immune gene prognostic model indicate that subtype with lower risk score were longer overall survival (OS) than subtype with higher risk score in training cohort (Figure1 A, C). The model was further verified by the validation cohort (Figure1 B, D). The multivariate Cox regression analysis demonstrated the model was an independent prognostic factor for OS prediction with hazard ratios of 56.694 (95% CIs: 9.038−355.648), 3.009 (95% CIs: 1.042−8.692) both in train cohort and external validation cohort respectively (Figure1 G, H). The AUC of 5- year were 0.92 (95% CIs: 0.86 - 0.97) and 0.7 (95% CIs: 0.51 - 0.89) for OS respectively in training cohort and validation cohort (Figure1 E, F). Furthermore, ssGSEA showed higher risk score subtype was significantly associated with higher immune score of check point, human leukocyte antigen (HLA), T cell co-inhibition and type I interferon (IFN) response (Figure1 K-N), which indicating that the poor outcome might be caused by tumor-associated immune response dysfunction partly. Conclusion: We constructed a robust immune gene prognostic model, which have a potential prognostic value for MDS patients and may provide evidence for personalized immunotherapy. Figure Disclosures No relevant conflicts of interest to declare.

Significance DNMT3A is a critical epigenetic modifier and tumor suppressor in the hematopoietic system. This gene is frequently mutated in hematopoietic malignancies, including acute myeloid leukemia (AML), with Dnmt3a R878H being the most common mutant. By using a conditional knockin approach, this study shows that Dnmt3a R878H is sufficient to initiate AML and recapitulate human leukemic features in mice. The leukemia-initiating cells are enriched in hematopoietic stem/progenitor cells. Through gene expression profiling, DNA methylation and histone modification analysis, and functional tests on important regulators for cell proliferation and differentiation in an animal model, this study has not only discovered mTOR pathway activation as a key player in the disease mechanism but also revealed the potential therapeutic effects of mTOR inhibition on DNMT3A mutation-related leukemia.

An error appeared in our paper entitled “Enhanced Biosynthesis of Furoic Acid via the Effective Pretreatment of Corncob into Furfural in the Biphasic Media” published in Catalysis Letters. Unfortunately, there are some mistakes in the values and equation in the Sect. 2.3 Conversion of Corncob to FAL and in Table 2.

Tigecycline, a first representative of the new class of glycylcycline antibacterial drugs, has been approved by FDA for using in the treatment of complicated skin and skin structure infections and community-acquired pneumonia in adults in 2005. It has been widely used in hospitals due to its broad-spectrum activity against pathogens. Pre-clinical studies showed that tigecycline has a significant anti-bacterial effect with low and mild adverse drug reactions (ADRs), including nausea, vomit or some other ADRs. Accompany with the huge increase of tigecycline usage in China, more and more side effects began to arouse people's attention. Here, we found that tigecycline can cause coagulation abnormalities in cancer patients, especially with hematological malignance, through clinical observation and retrospective analysis. To analyze the effect and risk factors of tigecycline on coagulation index in severely infected patients with hematologic cancer, we screened the cases admitted to department of hematology from three hospitals treated with tigecycline from Nov, 2015 to Oct, 2019. Then we compared the differences among the prothrombin time (PT), prothrombin time activity (PT %), the international standardization ratio (INR), part of the activation of prothrombin time (APTT), fibrinogen (FBG), thrombin time (TT), D-Dimer, fibrin degradation products (FDP), platelet (PLT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CRE), and c-reactive protein (CRP) before and after the tigecycline treatment. Meanwhile, we tend to establish the relationship between coagulation function and the time of tigecycline usage. In addition, for exploring the potential cause of abnormal coagulation induced by tigecycline, we evaluated the underlying biological mechanisms via Chinese hamster ovary (CHO) cells treated by tigecycline in vitro and subsequent RNA-sequencing analysis. A total of 129 qualified cases were included for analysis, including 71 males (55%) and 58 females (45%), with average age of 49.5±15.9 years old. After the treatment with tigecycline, PT, APTT and TT were significantly prolonged (P < 0.05). The INR, D-Dimer and FDP levels also increased (P < 0.05), but the FBG concentration was significantly reduced (P < 0.05) which indicated the possibility of hypofibrinogenaemia. Moreover, the coagulation parameters were tremendously changed in the early stage after intravenous infusion of tigecycline for anti-infection. There was no significant change in PLT count, but the levels of ALT, AST, CRE and CRP in the patients were statistically significant declined (P < 0.05). Most of all, the liver and kidney function of the patients was recovered after the infection was controlled by tigecycline treatment. In addition, the adhesion and stretching function of CHO cells were significantly suppressed after tigecycline treatmentin vitro. Lastly, bulk RNA-sequencing assay showed significant abnormalities in gene sets associated with platelet-activation and coagulation, such as PKFP, PLA2g7, PDGFRA, PDGFRB, PAFAH1b3, PDGFA, PECAM1, PDGFC and MPIG6b. Furthermore, adhesion regulating molecules RPN13, CHL1, ICAM5, TLCN were also downregulated by tigecycline treatment. What's more, these key genes which showed above need to be further validated in blood samples of AML patients when treated with tigecycline. Taken together, tigecycline can cause coagulation disorders in blood cancer patients. As we known, blood cancer could disturb the normal hematopoietic reconstitution in bone marrow, which resulted in abnormal numbers and functions of the peripheral blood lineages. Hence, these ADRs can accelerate the disease progression, cause severe bleeding and increase the risk of mortality in these patients. The biological mechanism study of this abnormality showed that tigecycline could inhibit platelet activation and the expression of adhesion regulators, which may increase the risk of bleeding and threaten the safety of patients. Therefore, the monitoring of coagulation index during tigecycline treatment should be adopted. Disclosures No relevant conflicts of interest to declare.

BACKGROUND: Acute myeloid leukemia (AML) is a type of cancer that consists of a group of hematological malignancies with high heterogeneity. DNA methyltransferase 3A (DNMT3A)-mutated AML patients have a poor prognosis. Some long non-coding RNAs (lncRNAs) have been reported to enhance therapeutic sensitivity, and so could affect the overall survival rate of elderly cytogenetically normal acute myeloid leukemia (CN-AML) patients; however, studies on the lncRNA signature in DNMT3A-mutated AML are rare. METHOD: The DNMT3A R878H conditional knock-in mouse model was constructed to explore the lncRNAs of DNMT3A mutation by using the Cuffcomparison method. Cis and trans regulation networks were used to predict candidate genes. The expression levels in leukemic cell lines and the prognostic index of these candidate genes were analyzed with the Broad Institute Cancer Cell Line Encyclopedia (CCLE) and OncoLnc databases. The data for each sample were statistically analyzed using GraphPad Prism. RESULTS: In this study, we applied the DNMT3A R878H conditional knock-in mouse model to explore the lncRNA epigenetic landscape of DNMT3A mutation by using the Cuffcomparison method. Twenty-three differentially expressed lncRNAs were identified in Dnmt3a(R878H/WT)Mx1-Cre(+) mice. We next predicted the downstream targetable genes regulated by these lncRNAs through cis and trans regulation networks and found 124 candidate genes are related to these lncRNAs. In further analysis of 124 genes, we found that increased mRNA expression levels of interleukin 1 receptor type 2 (IL1R2), Krüppel-like factor 13 (KLF13), ATPase H+ transporting V1 subunit A (ATP6V1A), proteasome 26S Subunit, non-ATPase 3 (PSMD3), and pyrroline-5-carboxylate reductase 2 (PYCR2) were associated with poor prognosis in AML. Functional analysis of these genes demonstrated that the pathways involved in autophagy, cell cycle, and hematopoietic stem cell differentiation were more enriched in Dnmt3a(R878H/WT)Mx1-Cre(+) mice. CONCLUSION: Our study was the first to use DNMT3A R878H conditional knock-in mouse model to predict the specific lncRNAs regulated by the DNMT3A mutation in AML. Six candidate genes were found to be associated with DNMT3A mutation with poor prognosis. Our results provided a possible treatment strategy for this disease.

Background: The present prognosis for AML mainly rely on the cyto- and molecular genetic features. However, the bone marrow microenvironment also plays a critical role in the development and progression of the disease, and the immune signature within the bone marrow microenvironment of AML remains unknown, and should be clarified. Patients and methods: Here, we applied the robust algorithm CIBERSORT method to generate the distribution and frequency of 22 immune subsets in the bone marrow of AML from public datasets. The associations between the overall survival and proportions of putative marrow immune cell types were analyzed to generate an immune risk score in a public training cohort according to LASSO cox regression model and validated in other validation cohorts. Gene Set Enrichment Analysis was adopted to analyze the immune risk score associated biological phenotypes. Results: Data from GSE37642 (n = 531) were used for immune model estimation, and samples from GSE425 (n =81) and TCGA (n =111) were served as external cohorts for validation. We first derived the 22 immune subsets through CIBERSORT, and found that monocytes, resting mast cells, eosinophils, CD8+T cells, resting NK cells, resting dendritic cells and resting memory CD4+ T cells were the most represented cell fractions within AML bone marrow microenvironment. LASSO cox regression was then applied to identify a total of 10 immune subsets to generate the immune risk score that may predict the survival prognosis of AML patients. This model has an excellent reproducibility, with the area under curve of the immune risk score at 5- year OS of 0.65, 0.84 and 0.74 in the training, external 1 and external 2 cohort. Patients with low immune risk score had a significant survival advantage than those with high immune risk score (5-year OS: 39.2 vs 19.6%, P < 0.001 for the training cohort; 77.4 vs 20.1%, P = 0.004 for the external cohort 1; 27.2 vs 0.0%, P = 0.006 for the external cohort 2). After adjusting the confounding variables of the classic ELN risk category and other clinical factors, the multivariable analysis revealed that immune risk score remained an independent prognostic factor for OS in all the three cohorts (HR: 1.34, 5.42 and 2.16 for the training, external cohort1 and 2 cohorts; all P < 0.05). Next, we explored the related biological phenotypes to immune risk score and found that the immune risk score was significantly associated with the markers of immune checkpoint (HMGB1, CEACAM1, LAG3 and CTLA-4), cell proliferation (NOTCH1, LYN, TSC2, SETBP1, TGFBR2, MAP3K14, TP53, NFKB2 and FOXO1), DNA repairment (RAD50 and BRCA1), epigenetic regulation and transcriptional regulation (SRSF6, DNMT3, TLE1). Furthermore, GSEA analysis indicated a significant enrichment of active immune-related pathways in low immune risk group. Finally, we noted that the immune risk score consistently increased follow the increased cytogenetic risk level. Conclusions: The study identified a novel immune prognostic category of AML, which may be used as another prognostic tool that complements the existing genetic risk classification. Figure Disclosures No relevant conflicts of interest to declare.

Background: Acute myeloid leukemia (AML) is a type of cancer that consists of a group of hematological malignancies with high heterogeneity. AML patients with DNMT3A mutations often have a poor prognosis. Metabolic alterations have long been recognized in cancer cells. Metabolic homeostasis is a fundamental property of cells that becomes dysregulated in cancer to meet the altered, often heightened, demand for metabolism for increased growth and proliferation. Oncogenic mutations can directly change the cellular metabolism, priming cells for malignancy. It is well known that 2-hydroxyglutarate (2-HG) is an oncometabolite resulting from mutations of the isocitrate dehydrogenase 1 and 2 (IDH1and IDH2) genes and isa strong prognostic predictor independent of other clinical and molecular features. Alpha-ketoglutarate-dependent dioxygenase encodedby the TET2 gene could catalyze 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), thereby regulating DNA methylation and inducing hematopoietic malignancies. Methods: Metabolomic profiles of all serum samples were achieved using the GC-TOF MS platform. CCK-8 assays were used to examine cell viability and cytotoxic activity of 2-ketoisocaproic acid (2-KA) to leukemia cells. Methyl-Cellulose colony-forming cell assays and flow cytometry were performed to assess cell proliferation and cell differentiation. The pIpC was administrated in the Dnmt3aR878H/WTMx1Cre+mice at the time of weaning (4 weeks old) to activate transgene expression in vivo. The expressions of genes/proteins were detected by RNA sequencing, quantitative RT-PCR and western blot. Results: We report a metabolomics study with a total of 171 newly diagnosed AML patients and identified a distinct leucine metabolic signature in AML patients with DNMT3A mutations. The prognostic value of 2-KA was demonstrated in cytogenetically normal AML patients, and a low 2-KA level predicted a poor overall patient survival. We further compared the gene expression patterns of the blast cells in bone marrow (BM) between AML groups with and without a DNMT3A mutation; these patterns correlated well with those of the leucine metabolic pathways and exhibited enhanced ACAA1 and decreased ALDH7A1 gene expression in DNMT3A-mutated leukemic cells. In vitro results demonstrated that a high 2-KA level could inhibit the proliferation of DNMT3A-mutated cells but did not affect the differentiation of these leukemic cells. Conclusions:Our data suggest that 2-KA isa unique feature of AML with a DNMT3A mutation and might be a metabolic marker of early-stage hematopoietic malignances.This study also suggests that ACAA1 and ALDH7A1 were aberrantly expressed in DNMT3A mutant leukemic cells and may be potential targetsfor AML therapy. We also hypothesized that a combination of the metabolic inhibitor and chemotherapeutic agent may be a possible treatment strategy for DNMT3A-mutated AML. Disclosures No relevant conflicts of interest to declare.

Background Cytogenetic aberrations and gene mutations have long been regarded as independent prognostic markers in AML, both of which can lead to misexpression of some key genes related to hematopoiesis. It is believed that the expression level of the key genes is associated with the treatment outcome of AML. Methods In this study, we analyzed the clinical features and molecular aberrations of 560 newly diagnosed non-M3 AML patients, including mutational status of CEBPA, NPM1, FLT3, C-KIT, NRAS, WT1, DNMT3A, MLL-PTD and IDH1/2, as well as expression levels of MECOM, ERG, GATA2, WT1, BAALC, MEIS1 and SPI1. Results Certain gene expression levels were associated with the cytogenetic aberration of the disease, especially for MECOM, MEIS1 and BAALC. FLT3, C-KIT and NRAS mutations contained conversed expression profile regarding MEIS1, WT1, GATA2 and BAALC expression, respectively. FLT3, DNMT3A, NPM1 and biallelic CEBPA represented the mutations associated with the prognosis of AML in our group. Higher MECOM and MEIS1 gene expression levels showed a significant impact on complete remission (CR) rate, disease free survival (DFS) and overall survival (OS) both in univariate and multivariate analysis, respectively; and an additive effect could be observed. By systematically integrating gene mutational status results and gene expression profile, we could establish a more refined system to precisely subdivide AML patients into distinct prognostic groups. Conclusions Gene expression abnormalities contained important biological and clinical informations, and could be integrated into current AML stratification system. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1279-4) contains supplementary material, which is available to authorized users.

Genomic landscapes of 92 adult and 111 pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) were investigated using next-generation sequencing and copy number alteration analysis. Recurrent gene mutations and fusions were tested in an additional 87 adult and 93 pediatric patients. Among the 29 newly identified in-frame gene fusions, those involving MEF2D and ZNF384 were clinically relevant and were demonstrated to perturb B-cell differentiation, with EP300-ZNF384 inducing leukemia in mice. Eight gene expression subgroups associated with characteristic genetic abnormalities were identified, including leukemia with MEF2D and ZNF384 fusions in two distinct clusters. In subgroup G4 which was characterized by ERG deletion, DUX4-IGH fusion was detected in most cases. This comprehensive dataset allowed us to compare the features of molecular pathogenesis between adult and pediatric B-ALL and to identify signatures possibly related to the inferior outcome of adults to that of children. We found that, besides the known discrepancies in frequencies of prognostic markers, adult patients had more cooperative mutations and greater enrichment for alterations of epigenetic modifiers and genes linked to B-cell development, suggesting difference in the target cells of transformation between adult and pediatric patients and may explain in part the disparity in their responses to treatment., Highlights • The genomic landscapes of adult and pediatric B-ALL were defined by next-generation sequencing of patient samples. • MEF2D and ZNF384 fusions could perturb B-cell differentiation or induce leukemia in mice and exhibited clinical relevance. • Adult patients showed greater enrichment for alterations of genes linked to epigenetic modification and B-cell development. This study comprehensively addressed the genomic signatures of adult versus pediatric B-ALL. The identification of distinct MEF2D and ZNF384 fusions expands the existing knowledge about molecular subtypes of B-ALL in both age groups. RNA-seq data allowed most of the B-ALL cases to be clustered into 8 subgroups related to genetic abnormalities. Notably, adult patients have more cooperative sequence variation mutations than pediatric patients, especially in genes involved in epigenetic regulation and B-cell development. These findings may improve our understanding of leukemogenesis in B-ALL, leading to a more precise genetic classification and the further development of targeted therapy in this disease.

Pardosa pseudoannulata is one of the most common wandering spiders in agricultural fields and a potentially good bioindicator for heavy metal contamination. However, little is known about the mechanism by which spiders respond to heavy metals at the molecular level. In this study, high-throughput transcriptome sequencing was employed to characterize the de novo transcriptome of the spiders and to identify differentially expressed genes (DEGs) after cadmium exposure. We obtained 60,489 assembled unigenes, 18,773 of which were annotated in the public databases. Ultimately, 3450 cDNA simple sequence repeats were identified and validated as potential molecular markers in the unigenes. A total of 2939, 2491 and 3759 DEGs were detected among the three libraries of two Cd-treated groups and the control. Functional enrichment analysis revealed that metabolism processes and digestive system function were predominately enriched in response to Cd stress. At the cellular and molecular levels, significantly enriched pathways in lysosomes and phagosomes as well as replication, recombination and repair demonstrated that oxidative damage resulted from Cd exposure. Based on the selected DEGs, certain critical genes involved in defence and detoxification were analysed. These results may elucidate the molecular mechanism underlying spiders' responses to heavy metal stress.

Significance Epigenetic modifications are required for the regulation of hematopoiesis. DNA methyltransferase 3A (DNMT3A), a critical epigenetic modifier responsible for de novo DNA methylation, was reported recently to be a frequently mutated gene in hematopoietic malignancies. However, the role of mutated DNMT3A in hematopoiesis remains largely unknown. Here we show that the Arg882 (R882) mutation of DNMT3A disrupts the normal function of this enzyme and results in chronic myelomonocytic leukemia (CMML) in mice. Meanwhile, the gene expression, DNA methylation, and protein–protein interaction assays suggest that DNMT3A R882 mutation drives CMML by disturbing the transcriptional expression/DNA methylation program and cell-cycle regulation of hematopoietic cells. This study may shed light on the function of DNMT3A mutant in myeloid leukemogenesis.

Introduction DNMT3A is a gene frequently mutated in human acute myeloid leukemia (AML), with DNMT3A R882H as the hot spot. It had been long postulated that DNMT3A mutation should play a key role in AML pathogenesis, so far the main animal models used were Dnmt3a-/- or transplantation of retrovirally transduced bone marrow cells expressing human DNMT3A R882H mutations (BMT). To recapitulate the features of human AML associated with DNMT3A mutation, this study generated a conditional knock-in mouse model to express Dnmt3a R878H mutation (homologous to human DNMT3A R882H) from the endogenous promoter/enhancer. We investigated epigenetic changes, including gene expression profiles, DNA methylation, and chromatin modification as affected by the mutation. We also explored the potential mechanisms that can explain the process by which DNMT3Amutation hierarchically induces abnormal hematopoiesis and the manner by which specific regulators of relevant pathways in murine and human settings can be targeted for potential therapeutic applications. Method We performed the single-cell RNA-seq (scRNA-seq) of LSKs and MEPs, RNA-seq and Methylated DNA immunoprecipitation sequencing (MeDIP-seq) of Gr-1 cells and whole exome sequence (WES) of BMs and tails in Dnmt3a R878H conditional knock-in mice. Result Approximately 4-6 months after birth with interferon induction, all Dnmt3aR878H/WTMx1-Cre+ knock-in mice developed AML of myelomonocytic subtype, characterized by massive expansion of immature cells and infiltration of bone marrow, spleen and lymph node, along with hyperleukocytosis, thrombocytosis, splenomegaly and lymphadenectasis. The leukemic mice also showed severe diffuse skin ulceration and alopecia. The transcriptome and DNA methylation profiling of bulk Gr-1 leukemic cells and the single-cell RNA-sequencing of LSKs/MEPs revealed significant changes in gene expression and epigenetic regulatory patterns that could cause differentiation arrest and growth advantage. Consistent with leukemic cell accumulation in G2/M phase, CDK1 was found overexpressed as a result of mTOR gene activation due to DNA hypomethylation in the gene body region. We then discovered that overexpressed CDK1 could compete with EZH2 in binding to DNMT3A, induce EZH2 phosphorylation and reduction, and result in abnormal histone methylation. Notably, we showed a very significant response from Dnmt3aR878H/WTto the therapeutic effect of the mTOR inhibitor rapamycin, particularly in terms of prolongation of lifespan in treatment group as compared to the control group (p Conclusions We established a novel mouse model for the expression of mutant Dnmt3a R878H from endogenous locus to investigate the role of Dnmt3a abnormality in leukemogenesis. Indeed, Dnmt3aR878H/WTMx1-Cre+ mice developed AML of myelomonocytic subtype with skin injury. We discovered unique gene expression and DNA methylation patterns in concordance with enhanced proliferation and suppressed differentiation in leukemic cells. The heterogeneity of gene expression in individual leukemic stem/progenitor cells implied the presence of clonal diversity, which could underlie disease evolution. The activation of mTOR and the resultant overexpression of CDK1 might contribute to malignant transformation. Evidence has been obtained in both murine and human settings to suggest DNMT3A mutation-related AML as a potential disease target for rapamycin. Disclosures No relevant conflicts of interest to declare.