RIPK3 deficiency blocks R-2-hydroxyglutarate-induced necroptosis in IDH-mutated AML cells.

Mutant isocitrate dehydrogenases (IDHs) produce R-2-hydroxyglutarate (R-2HG), which inhibits the growth of most acute myeloid leukemia (AML) cells. Here, we showed that necroptosis, a form of programmed cell death, contributed to the antileukemia activity of R-2HG. Mechanistically, R-2HG competitively inhibited the activity of lysine demethylase 2B (KDM2B), an α-ketoglutarate-dependent dioxygenase. KDM2B inhibition increased histone 3 lysine 4 trimethylation levels and promoted the expression of receptor-interacting protein kinase 1 (RIPK1), which consequently caused necroptosis in AML cells. The expression of RIPK3 was silenced because of DNA methylation in IDH-mutant (mIDH) AML cells, resulting in R-2HG resistance. Decitabine up-regulated RIPK3 expression and repaired endogenous R-2HG-induced necroptosis pathway in mIDH AML cells. Together, R-2HG induced RIPK1-dependent necroptosis via KDM2B inhibition in AML cells. The loss of RIPK3 protected mIDH AML cells from necroptosis. Restoring RIPK3 expression to exert R-2HG's intrinsic antileukemia effect will be a potential therapeutic strategy in patients with AML.

All-trans retinoic acid enhances the cytotoxic effect of decitabine on myelodysplastic syndromes and acute myeloid leukaemia by activating the RARα-Nrf2 complex.

BACKGROUND: Decitabine (DAC) is used as the first-line therapy in patients with higher-risk myelodysplastic syndromes (HR-MDS) and elderly acute myeloid leukaemia (AML) patients unsuitable for intensive chemotherapy. However, the clinical outcomes of patients treated with DAC as a monotherapy are far from satisfactory. Adding all-trans retinoic acid (ATRA) to DAC reportedly benefitted MDS and elderly AML patients. However, the underlying mechanisms remain unclear and need further explorations from laboratory experiments. METHODS: We used MDS and AML cell lines and primary cells to evaluate the combined effects of DAC and ATRA as well as the underlying mechanisms. We used the MOLM-13-luciferase murine xenograft model to verify the enhanced cytotoxic effect of the drug combination. RESULTS: The combination treatment reduced the viability of MDS/AML cells in vitro, delayed leukaemia progress, and extended survival in murine xenograft models compared to non- and mono-drug treated models. DAC application as a single agent induced Nrf2 activation and downstream antioxidative response, and restrained reactive oxygen species (ROS) generation, thus leading to DAC resistance. The addition of ATRA blocked Nrf2 activation by activating the RARα-Nrf2 complex, leading to ROS accumulation and ROS-dependent cytotoxicity. CONCLUSIONS: These results demonstrate that combining DAC and ATRA has potential for the clinical treatment of HR-MDS/AML and merits further exploration.

15-days duration of venetoclax combined with azacitidine in the treatment of relapsed/refractory high-risk myelodysplastic syndromes: A retrospective single-center study.

The treatment of patients with refractory and/or relapsed (R/R) high-risk myelodysplastic syndrome (HR-MDS) remains a daunting clinical challenge. Venetoclax is a selective BCL-2 inhibitor, which combined with hypomethylating agents (HMAs), increased responses and prolonged survival in unfit and previously untreated acute myeloid leukemia. We performed a retrospective study of patients with R/R HR-MDS receiving combination azacytidine (AZA) plus 15-days duration of venetoclax (VEN-15d) in order to determine their efficacy and toxicity in this context. We showed that the overall response rate was 57.2% (20/35) and the median over survival was 14 months in R/R MDS. The most common treatment-emergent adverse events were peripheral blood cytopenias and infectious complications. Our retrospective study showed that the real-world experience of treating R/R MDS with AZA plus VEN-15d highlights an encouraging response rate with myelosuppression being the major toxicity. Of note, VEN-15d with AZA may salvage patients failing to respond optimally to HMAs and reduce the disease-burden for subsequent allogeneic stem cell transplantation in our analysis. These data of combination AZA plus VEN-15d in R/R MDS warrant further prospective evaluation in clinical trials.

Predictive values of mutational variant allele frequency in overall survival and leukemic progression of myelodysplastic syndromes.

BACKGROUND: The implication of mutational variant allelic frequency (VAF) has been increasingly considered in the prognostic interpretation of molecular data in myeloid malignancies. However, the impact of VAF on outcomes of myelodysplastic syndromes (MDS) has not been extensively explored. METHODS: Targeted next-generation sequencing was performed in 350 newly diagnosed MDS cases. The associations of mutational VAF of each gene with overall survival (OS) and leukemia-free survival (LFS) were examined by multivariate Cox regression after univariate analysis. RESULTS: Shorter OS was independently associated with DNMT3A VAF (HR 1.020 per 1% VAF increase; 95% CI 1.005-1.035; p = 0.011) and TP53 VAF (HR 1.014 per 1% VAF increase; 95% CI 1.006-1.022; p = 0.001). LFS analyses revealed that TET2 VAF (HR 1.013 per 1% VAF increase; 95% CI 1.005-1.022; p = 0.003) and TP53 VAF (HR 1.012 per 1% VAF increase; 95% CI 1.004-1.021; p = 0.005) were independently associated with faster leukemic transformation. Furthermore, we established nomograms to predict OS and LFS, respectively, by integrating independent mutational predictors into the revised International Prognostic Scoring System. CONCLUSION: Our study highlights that VAF of certain genes should be incorporated into routine clinical prognostication of survival and leukemic transformation of MDS.

Clinical significance of cytogenetic and molecular genetic abnormalities in 634 Chinese patients with myelodysplastic syndromes.

PURPOSE: To explore the relevance of cytogenetic or molecular genetic abnormalities to clinical variables, including clinical and laboratory characteristics and prognosis in Chinese patients with myelodysplastic syndromes (MDS). METHODS: A total of 634 consecutive patients diagnosed with MDS at The First Affiliated Hospital, Zhejiang University School of Medicine from June 2008 to May 2018 were retrospectively included in this study. All patients had evaluable cytogenetic analysis, and 425 patients had MDS-related mutations sequencing. RESULTS: 38.6% of patients displayed abnormal karyotypes. The most common cytogenetic abnormality was +8 (31%). Sole +8 was related to female (p = 0.002), hemoglobin >10 g/dL (p = 0.03), and <60 years old (p = 0.046). TP53 mutations were associated with complex karyotype (CK) (p < 0.001). DNMT3A mutations correlated with -Y (p = 0.01) whereas NRAS mutations correlated with 20q- (p = 0.04). The overall survival (OS) was significantly inferior in patients with +8 compared with those with normal karyotype (NK) (p = 0.003). However, the OS of sole +8 and +8 with one additional karyotypic abnormality was not different from NK (p = 0.16), but +8 with two or more abnormalities had a significantly shorter OS than +8 and +8 with one additional karyotypic abnormality (p = 0.02). In multivariable analysis, ≥60 years old, marrow blasts ≥5% and TP53 mutations were independent predictors for poor OS (p < 0.05), whereas SF3B1 mutations indicated better prognosis. Male IDH1 and IDH2 mutations and marrow blasts ≥5% were independent risk factors for worse leukemia free survival (LFS) (p < 0.05). CONCLUSION: In this population of Chinese patients, trisomy 8 is the most common karyotypic abnormality. Patients with +8 showed a poorer OS compared with patients with NK. Sole +8 and +8 with one additional karyotypic abnormality had similar OS with NK, whereas +8 with two or more abnormalities had a significantly shorter OS. DNMT3A mutations correlated with -Y and NRAS mutations correlated with 20q-. TP53 mutations were associated with CK and had a poor OS. SF3B1 mutations indicated a favorable OS. IDH1 and IDH2 mutations independently indicated inferior LFS.

Real-world data of chronic myelomonocytic leukemia: A chinese single-center retrospective study.

Chronic myelomonocytic leukemia (CMML) is a rare disease of elderly people characterized by the presence of sustained peripheral blood monocytosis, overlapping features of myeloproliferation, and myelodysplasia. We present a large retrospective study of 156 CMML patients in China. Mean age at diagnosis was 68 years old (range 23-91). According to the CMML-specific prognostic scoring system (CPSS), 10 patients (8.3%) were low risk, 27 patients (22.5%) were intermediate-1 risk, 72 patients (60%) were intermediate-2 risk, and 11 patients (9.2%) were high risk. A total of 90 patients (57.7%) received hypomethylating agents (HMAs) treatment, 19 patients (12.2%) received chemotherapy and 47 patients (30.1%) received the best supportive care. Seventeen patients (10.9%) underwent allogeneic hematopoietic stem cell transplantation (allo-SCT) after HMAs treatment or chemotherapy. With a median follow-up of 35.3 months, overall response rate (ORR) was 69.5% in the HMAs ± chemotherapy group, 79.5% in the HMAs monotherapy group, 60.0% in the HMAs + chemotherapy group, and 37.5% in the chemotherapy group. HMAs monotherapy group had prolonged OS compared with the chemotherapy group (23.57 months vs. 11.73 months; p = 0.035). Patients who achieved ORR had prolonged OS (25.83 months vs. 8.00 months; p < 0.001) and LFS (20.53 months vs. 6.80 months; p < 0.001) compared with those not achieved ORR in the HMA ± chemotherapy group. By univariate analysis, only higher hemoglobulin (≥80 g/L) and lower serum LDH levels (<300 U/L) predicted for better OS and LFS. By multivariate analysis, only Hb ≥ 80 g/L predicted for prolonged OS, Hb ≥ 80 g/L, and monocytes < 3 × 109/L predicted for prolonged LFS. In summary, our study highlights the benefit of HMAs therapy in CMML, but we still need to develop novel therapeutics to achieve better outcomes.

TP53 mutations are associated with very complex karyotype and suggest poor prognosis in newly diagnosed myelodysplastic syndrome patients with monosomal karyotype.

AIM: The aim of this study was to evaluate the clinical and molecular characteristics of myelodysplastic syndrome (MDS) patients with monosomal karyotype (MK). METHODS: Eighty MDS patients with MK diagnosed between January 2010 and December 2018 were included in the retrospective study. Seventy-three had complex karyotype (CK) and 46 had very CK (vCK, ≥ 5 abnormalities). Clinical information was collected, and a panel of 37 genes, on which mutations have been previously reported to be associated with MDS patients, was analyzed by next-generation sequencing. Genetic and biological features and their association with survival were evaluated. RESULTS: Monosomy 5, 7, and 17 were the most frequent and mainly occurred in patients with vCK. While median overall survival (OS) for all patients was 12.8 months with 95% CI 9.1-16.5, patients with vCK had shorter OS (8.4 months with 95% CI 3.9-12.8) than those with non-vCK (16.1 months with 95% CI 11.5-20.8) (P = .02). At least one gene mutation was detected in 76 patients (95%), TP53 mutations were detected in 57 patients, and their median OS was significantly shorter than those without TP53 mutations (9.5 months with 95% CI 7.5-11.5 vs 26.1 months with 95% CI 8.0-44.2, P < .01). In 34 patients who received treatment with decitabine, 25 with TP53 mutations had higher overall response rate than those with wild-type TP53 (60% vs 22.2%, P = .03). However, OS was still significantly shorter in those with TP53 mutations (10.1 vs 26.1 months, P = .03). Multivariate analysis confirmed that TP53 mutations was an independent poor prognostic factor on OS. CONCLUSIONS: CK and vCK overlap in most of the MDS patients with MK. TP53 mutations occur more frequently in MDS patients with vCK, and both TP53 mutations and vCK are adverse prognostic factors.

Impact of mutational variant allele frequency on prognosis in myelodysplastic syndromes.

The clinical relevance of variant allele frequency (VAF) of recurrent mutations in myelodysplastic syndromes (MDS) has been increasingly reported. However, the prognostic value of mutational VAF across the genetic spectrum of MDS has not been extensively evaluated. In this study, we profiled the mutational spectrum of 382 newly diagnosed MDS patients using targeted next-generation sequencing. Exploratory analysis found that mutational VAF of some genes including TET2, TP53, and SF3B1 had significant associations with patient survival. Specifically, TET2 VAF ≥ 32% (HR 1.69, P = 0.025) and TP53 VAF ≥ 27% (HR 3.58, P < 0.001) were independently associated with shorter overall survival (OS). In contrast, SF3B1 VAF ≥ 15% had an independent association with better prognosis (HR 0.52, P = 0.048). In addition, high TET2 VAF was associated with an increased response to hypomethylating agents relative to low TET2 VAF (P = 0.009). Patients with high TP53 VAF more often possessed complex karyotypes than those with low VAF (P = 0.034). And patients with high SF3B1 VAF were more frequently classified as MDS with ring sideroblasts (MDS-RS) category than those with low VAF (P = 0.012). Meanwhile, we found that for some other genes like EZH2 and NRAS, once their mutations appeared, it meant poor survival regardless of mutational VAF. These findings suggest that mutational VAF of certain genes should be considered into the routine prognostic prediction and risk stratification of MDS patients.

Mutation status and burden can improve prognostic prediction of patients with lower-risk myelodysplastic syndromes.

Patients with lower-risk myelodysplastic syndromes (LR-MDS) as defined by the International Prognostic Scoring System (IPSS) have more favorable prognosis in general, but significant inter-individual heterogeneity exists. In this study, we examined the molecular profile of 15 MDS-relevant genes in 159 patients with LR-MDS using next-generation sequencing. In univariate COX regression, shorter overall survival (OS) was associated with mutation status of ASXL1 (P = .001), RUNX1 (P = .031), EZH2 (P = .049), TP53 (P = .016), SRSF2 (P = .046), JAK2 (P = .040), and IDH2 (P = .035). We also found significantly shorter OS in patients with an adjusted TET2 variant allele frequency (VAF) ≥18% versus those with either an adjusted TET2 VAF <18% or without TET2 mutations (median: 20.4 vs 47.8 months; P = .020; HR = 2.183, 95%CI: 1.129-4.224). After adjustment for IPSS, shorter OS was associated with mutation status of ASXL1 (P < .001; HR = 4.306, 95% CI: 2.144-8.650), TP53 (P = .004; HR = 4.863, 95% CI: 1.662-14.230) and JAK2 (P = .002; HR = 5.466, 95%CI: 1.848-16.169), as well as adjusted TET2 VAF ≥18% (P = .008; HR = 2.492, 95% CI: 1.273-4.876). Also, OS was increasingly shorter as the number of mutational factors increased (P < .001). A novel prognostic scoring system incorporating the presence/absence of the four independent mutational factors into the IPSS further stratified LR-MDS patients into three prognostically different groups (P < .001). The newly developed scoring system redefined 10.1% (16/159) of patients as a higher-risk group, who could not be predicted by the currently prognostic models. In conclusion, integration of the IPSS with mutation status/burden of certain MDS-relevant genes may improve the prognostication of patients with LR-MDS and could help identify those with worse-than-expected prognosis for more aggressive treatment.

The relation of SF3B1 mutation and intracellular iron in myelodysplastic syndrome with less than 5% bone marrow blasts.

According to 2008 WHO classification RARS is regarded as less than 5% blasts and more than 15% ring sideroblasts in the bone marrow. In 2016 WHO classification MDS-RS is revised as more than 15% ring sideroblasts or more than 5% ring sideroblasts in the presence of the SF3B1 mutation. In our study, we classified intracellular iron in bone marrow into four types according to the size and quantity of iron granules. We found that there was a significant difference between SF3B1-mutant and SF3B1-wild-type MDS patients in intracellular iron III, intracellular iron IV and ring sideroblasts. We defined intracellular iron (III + IV + RS)%×100 as 'Iron score'. We suggest that the patients carrying SF3B1 mutation with Iron score ≥10 will extend the subtype of MDS-RS, in addition to the current WHO classification criteria. This study gives us a new insight into the relation of SF3B1 mutation and intracellular iron in lower-risk MDS.

RNA-Seq Analysis of the Antioxidant Status and Immune Response of Portunus trituberculatus Following Aerial Exposure.

Desiccation tolerance has been long considered as an important trait for the life survival under acute environmental stress. One of the biggest problems for modern commercial crab farming is desiccation during transportation; high mortality could occur following the aerial exposure. In this regard, here, we utilized RNA-seq-based transcriptome profiling to characterize the molecular responses of swimming crab in response to aerial exposure. In present study, following aerial exposure, the gill samples were sequenced at 0, 6, 12, and 18 h. And the sequenced reads were assembled into 274,594 contigs, with average length of 735.59 bp and N50 size of 1262 bp. After differential expression analysis, a total of 1572 genes were captured significantly differentially expressed, and were categorized into antioxidant/oxidative stress response, chaperones/heat shock proteins, immune alteration, cell proliferation/apoptosis, and cytoskeletal. Our analysis revealed the dramatic tissue oxidant stress and the alteration of the tissue epithelial integrity, especially many genes that have not been reported in crab species. With the limited functional information in crab, further studies are needed and underway in our lab to further characterize the key cellular actors governing the crab tolerance to aerial exposure. Taken together, our results provide molecular resources for further identification of key genes for desiccation tolerance, and to facilitate the molecular selection and breeding of desiccation tolerant strain and family.