DNA damage to bone marrow stromal cells by antileukemia drugs induces chemoresistance in acute myeloid leukemia via paracrine FGF10-FGFR2 signaling.

Chemoresistance remains a major challenge in the current treatment of acute myeloid leukemia (AML). The bone marrow microenvironment (BMM) plays a complex role in protecting leukemia cells from chemotherapeutics, and the mechanisms involved are not fully understood. Antileukemia drugs kill AML cells directly but also damage the BMM. Here, we determined antileukemia drugs induce DNA damage in bone marrow stromal cells (BMSCs), resulting in resistance of AML cell lines to adriamycin and idarubicin killing. Damaged BMSCs induced an inflammatory microenvironment through NF-κB; suppressing NF-κB with small molecule inhibitor Bay11-7082 attenuated the prosurvival effects of BMSCs on AML cell lines. Furthermore, we used an ex vivo functional screen of 507 chemokines and cytokines to identify 44 proteins secreted from damaged BMSCs. Fibroblast growth factor-10 (FGF10) was most strongly associated with chemoresistance in AML cell lines. Additionally, expression of FGF10 and its receptors, FGFR1 and FGFR2, was increased in AML patients after chemotherapy. FGFR1 and FGFR2 were also widely expressed by AML cell lines. FGF10-induced FGFR2 activation in AML cell lines operates by increasing P38 MAPK, AKT, ERK1/2, and STAT3 phosphorylation. FGFR2 inhibition with small molecules or gene silencing of FGFR2 inhibited proliferation and reverses drug resistance of AML cells by inhibiting P38 MAPK, AKT, and ERK1/2 signaling pathways. Finally, release of FGF10 was mediated by ß-catenin signaling in damaged BMSCs. Our data indicate FGF10-FGFR2 signaling acts as an effector of damaged BMSC-mediated chemoresistance in AML cells, and FGFR2 inhibition can reverse stromal protection and AML cell chemoresistance in the BMM.

When is the best time and grade to start ruxolitinib in corticosteroid-refractory acute graft-versus-host-disease: A multi-center research.

OBJECTIVE: Ruxolitinib was recently approved to treat corticosteroid-resistant acute graft-versus-host disease (GvHD). However, it is unknown as to whether starting ruxolitinib at a lower versus higher acute GvHD grade or earlier versus later affected outcomes. This study identified the impact of starting acute GvHD grade and start time after declaring corticosteroid resistance and the effect on complete and overall response rates to ruxolitinib therapy. METHODS: Retrospective, observational multi-center study. We divided cohorts into starting ruxolitinib ≤ 7 days (N = 45) versus at > 7 days after declaring corticosteroid resistance (N = 24). RESULTS: In ≤ 7 days cohort complete response (CR) rates at day 28 were 69% (54, 81%) versus 25% (11, 47%; p = .001) in > 7 days cohort, and overall response (OR) rates were 91% (78, 96%) versus 80% (48, 92%; p = .25). CONCLUSIONS: Our data suggest that starting ruxolitinib in ≤ 7 days of declaring corticosteroid failure regardless of G vHD grade improves complete response rate but not OR rates. Starting ruxolitinib at grade I and within 7 days may get a more significant response.

Impact of the International Consensus Classification of myelodysplastic syndromes.

The impact of the 2022 International Consensus Classification (ICC) of myelodysplastic syndromes (MDS) needs study. We analysed data from 989 MDS subjects classified using the 2016 World Health Organization (WHO) criteria to determine the impact of the new proposal. Our analyses suggested the ICC criteria of MDS-SF3B1 identifies a more homogenous disease entity than the WHO 2016 criteria of myelodysplastic syndromes with ring sideroblasts (MDS-RS). MDS, not otherwise specified with single lineage dysplasia (MDS, NOS-SLD) patients had a better prognosis than MDS, NOS with multilineage dysplasia (MDS, NOS-MLD) patients. MDS with mutated TP53 and MDS/acute myeloid leukaemia with mutated TP53 patients had the briefest survivals. These data support the ICC of MDS, which allows more accurate diagnoses and risk stratification.

A randomized phase 2 trial of pomalidomide in subjects failing prior therapy for chronic graft-versus-host disease.

Steroid-refractory chronic graft-versus-host disease (cGVHD) is a therapeutic challenge. Sclerotic skin manifestations are especially difficult to treat. We conducted a randomized phase 2 clinical trial (#NCT01688466) to determine the safety, efficacy, and preferred dose of pomalidomide in persons with moderate to severe cGVHD unresponsive to corticosteroids and/or subsequent lines of therapy. Thirty-four subjects were randomized to receive pomalidomide 0.5 mg per day orally (n = 17; low-dose cohort) or 2 mg per day at a starting dose of 0.5 mg per day increasing to 2 mg per day over 6 weeks (n = 17; high-dose cohort). The primary endpoint was overall response rate (ORR) at 6 months according to the 2005 National Institutes of Health cGVHD Response Criteria. Thirty-two patients had severe sclerotic skin and received a median of 5 (range, 2-10) previous systemic therapies. ORR was 47% (95% confidence interval, 30-65) in the intention-to-treat analyses. All were partial responses, with no difference in ORR between the cohorts. ORR was 67% (45%-84%) in the 24 evaluable subjects at 6 months. Nine had improvement in National Institutes of Health joint/fascia scores (P = .018). Median change from the baseline in body surface area involvement of skin cGVHD was -7.5% (-10% to 35%; P = .002). The most frequent adverse events were lymphopenia, infection, and fatigue. Eight subjects in the high-dose cohort had dose decreases because of adverse events. There was 1 death in the low-dose cohort from bacterial pneumonia. Our data indicate antifibrotic effects of pomalidomide and possible association with increases in concentrations of blood regulatory T-cell and interleukin-2. Pomalidomide 0.5 mg per day is a safe and effective therapy for advanced corticosteroid-refractory cGVHD.

The mutational landscape in chronic myelomonocytic leukemia and its impact on allogeneic hematopoietic cell transplantation outcomes: a Center for Blood and Marrow Transplantation Research (CIBMTR) analysis.

Somatic mutations are recognized as an important prognostic factor in chronic myelomonocytic leukemia (CMML). However, limited data are available regarding their impact on outcomes after allogeneic hematopoietic cell transplantation (HCT). In this registry analysis conducted in collaboration with the Center for International Blood and Marrow Transplantation Registry database/sample repository, we identified 313 adult patients with CMML (median age: 64 years, range, 28- 77) who underwent allogeneic HCT during 2001-2017 and had an available biospecimen in the form of a peripheral blood sample obtained prior to the start of conditioning. In multivariate analysis, a CMML-specific prognostic scoring system (CPSS) score of intermediate-2 (HR=1.46, P=0.049) or high (HR=3.22, P=0.0004) correlated significantly with overall survival. When the molecularly informed CPSS-Mol prognostic model was applied, a high CPSS-Mol score (HR=2 P=0.0079) correlated significantly with overall survival. The most common somatic mutations were in ASXL1 (62%), TET2 (35%), KRAS/NRAS (33% combined), and SRSF2 (31%). DNMT3A and TP53 mutations were associated with decreased overall survival (HR=1.70 [95% CI: 1.11-2.60], P=0.0147 and HR=2.72 [95% CI: 1.37-5.39], P=0.0042, respectively) while DNMT3A, JAK2, and TP53 mutations were associated with decreased disease-free survival (HR=1.66 [95% CI: 1.11-2.49], P=0.0138, HR=1.79 [95% CI: 1.06-3.03], P=0.0293, and HR=2.94 [95% CI: 1.50-5.79], P=0.0018, respectively). The only mutation associated with increased relapse was TP53 (HR=2.94, P=0.0201). Nonetheless, the impact of TP53 mutations specifically should be interpreted cautiously given their rarity in CMML. We calculated the goodness of fit measured by Harrell's C-index for both the CPSS and CPSS-Mol, which were very similar. In summary, via registry data we have determined the mutational landscape in patients with CMML who underwent allogeneic HCT, and demonstrated an association between CPSS-Mol and transplant outcomes although without major improvement in the risk prediction beyond that provided by the CPSS.

Association between the choice of the conditioning regimen and outcomes of allogeneic hematopoietic cell transplantation for myelofibrosis.

Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only curative treatment for myelofibrosis. However, the optimal conditioning regimen either with reduced-intensity conditioning (RIC) or myeloablative conditioning (MAC) is not well known. Using the Center for International Blood and Marrow Transplant Research database, we identified adults aged ≥18 years with myelofibrosis undergoing allo-HCT between 2008-2019 and analyzed the outcomes separately in the RIC and MAC cohorts based on the conditioning regimens used. Among 872 eligible patients, 493 underwent allo-HCT using RIC (fludarabine/ busulfan n=166, fludarabine/melphalan n=327) and 379 using MAC (fludarabine/busulfan n=247, busulfan/cyclophosphamide n=132). In multivariable analysis with RIC, fludarabine/melphalan was associated with inferior overall survival (hazard ratio [HR]=1.80; 95% confidenec interval [CI]: 1.15-2.81; P=0.009), higher early non-relapse mortality (HR=1.81; 95% CI: 1.12-2.91; P=0.01) and higher acute graft-versus-host disease (GvHD) (grade 2-4 HR=1.45; 95% CI: 1.03-2.03; P=0.03; grade 3-4 HR=2.21; 95%CI: 1.28-3.83; P=0.004) compared to fludarabine/busulfan. In the MAC setting, busulfan/cyclophosphamide was associated with a higher acute GvHD (grade 2-4 HR=2.33; 95% CI: 1.67-3.25; P<0.001; grade 3-4 HR=2.31; 95% CI: 1.52-3.52; P<0.001) and inferior GvHD-free relapse-free survival (GRFS) (HR=1.94; 95% CI: 1.49-2.53; P<0.001) as compared to fludarabine/busulfan. Hence, our study suggests that fludarabine/busulfan is associated with better outcomes in RIC (better overall survival, lower early non-relapse mortality, lower acute GvHD) and MAC (lower acute GvHD and better GRFS) in myelofibrosis.

Impacts of early therapy response, interval to therapy interruption, and cumulative therapy interruption duration on outcome of ibrutinib therapy in relapsed/refractory chronic lymphocytic leukemia.

To investigate the impact of early response and treatment interruption on the survival of patients with relapsed/refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (r/r CLL/SLL) treated with ibrutinib. This post hoc analysis used data of patients received ibrutinib treatment from an open-label, multicenter phase 3 study comparing ibrutinib with rituximab in patients with r/r CLL/SLL. The association of complete or partial response at 6 months, interruption within the first 6 months, cumulative interruption durations during the ibrutinib-treated period with progression-free survival (PFS) and overall survival (OS) were evaluated using the adjusted Cox hazard proportional model. The study included 87 patients treated with ibrutinib, of which 74 patients had at least 6 months of ibrutinib treatment and were analyzed. The response at 6 months did not affect PFS (HR = 0.58, 95%CI: 0.22-1.49) or OS (HR = 0.86, 95%CI: 0.22-3.31). The onset of interruption before or after 6 months was not associated with PFS (HR = 0.88, 95%CI: 0.34-2.30) or OS (HR = 0.75, 95%CI: 0.23-2.52). However, a cumulative interruption of more than 35 days was independently associated with worse PFS (HR = 2.4, 95%CI: 0.99-5.74) and OS (HR = 2.6, 95%CI: 0.88-7.44). Continuous interruption for more than 14 days was associated with a numerically lower 3-year PFS rate (> 14 vs. ≤ 14 days: 42% vs. 73%) and 3-year OS rate (> 14 vs. ≤ 14 days: 58% vs. 84%, both P > 0.05). Response status at 6 months or early therapy interruptions did not affect survival in patients with r/r CLL/SLL treated with ibrutinib. However, a cumulative temporary interruption of more than 35 days could potentially impact patient outcomes.

Clonal Megakaryocyte Dysplasia with Isolated Thrombocytosis Is a Distinct Myeloproliferative Neoplasm Phenotype.

INTRODUCTION: About 15% of people with a myeloproliferative neoplasm (MPN) are identified as MPN, unclassifiable using the 2016 WHO classification. METHODS: We tested whether persons with platelet concentration ≥450 × 10E+9/L, bone marrow megakaryocyte morphology typical of prefibrotic/early myelofibrosis (pre-MF), and no minor criteria of pre-MF should be classified as a distinct MPN subtype, clonal megakaryocyte dysplasia with isolated thrombocytosis (CMD-IT). RESULTS: 139 subjects meet these criteria who we compared with primary myelofibrosis (PMF) including 402 with pre-MF and 521 with overt myelofibrosis. CMD-IT subjects were more likely female and younger. They had lower frequencies of JAK2V617F compared with persons with PMF (55% vs. 70%; p < 0.001) and higher frequencies of CALR mutations (37% vs. 17%; p < 0.001). They also had lower frequency of variations associated with JAK2V617F susceptibility, JAK2 46/1 (35% vs. 47%; p = 0.021), and VEGFA rs3025039 (12% vs. 17%; p = 0.030). Subjects with CMD-IT had lower incidences of thrombotic events compared with those with pre-MF (9.7% vs. 26%; p < 0.001) and longer survival (median, not reached vs. 23 years; HR = 0.34 (0.10, 0.30); p < 0.001). CONCLUSION: Our data indicate CMD-IT is a distinct MPN subtype and should be included in the classification of myeloid neoplasms.

Can Immune Therapy Cure Acute Myeloid Leukemia?

OPINION STATEMENT: Although safe and effective immune therapies have been developed in several cancers, this has not been so in acute myeloid leukaemia (AML). Studies of antibodies to CD33, CD123 and CLL-1 report with unconvincing efficacy and substantial adverse events. Lacking AML-specific target antigens, these approaches using non-specific antigen targets often cause unacceptable bone marrow toxicity and off-target adverse events. Studies of AML incidence in persons with immune deficiency indicate little if any immune surveillance against AML. In contrast, data studies of recipients of haematopoietic cell transplants support an effective allogeneic anti-AML effect associated with graft-versus-host disease (GvHD) and possibly a specific graft-versus-leukaemia (GvL) effect. A special problem in the immune therapy of AML is few neo-antigens compared with solid cancers because of a relatively low mutation frequency. Studies of CAR-T-, CAR-NK-adaptor CAR-T- and allogeneic NK-cells are progressing as are approaches using synthetic biology. Presently, there are no convincing data of efficacy of immune therapy in AML.

Survival prediction optimization of acute myeloid leukaemia based on T-cell function-related genes and plasma proteins.

Interactions between acute myeloid leukaemia (AML) cells and immune cells are postulated to corelate with outcomes of AML patients. However, data on T-cell function-related signature are not included in current AML survival prognosis models. We examined data of RNA matrices from 1611 persons with AML extracted from public databases arrayed in a training and three validation cohorts. We developed an eight-gene T-cell function-related signature using the random survival forest variable hunting algorithm. Accuracy of gene identification was tested in a real-world cohort by quantifying cognate plasma protein concentrations. The model had robust prognostic accuracy in the training and validation cohorts with five-year areas under receiver-operator characteristic curve (AUROC) of 0.67-0.76. The signature was divided into high- and low-risk scores using an optimum cut-off value. Five-year survival in the high-risk groups was 6%-23% compared with 42%-58% in the low-risk groups in all the cohorts (all p values <0.001). In multivariable analyses, a high-risk score independently predicted briefer survival with hazard ratios of death in the range 1.28-2.59. Gene set enrichment analyses indicated significant enrichment for genes involved in immune suppression pathways in the high-risk groups. Accuracy of the gene signature was validated in a real-world cohort with 88 pretherapy plasma samples. In scRNA-seq analyses most genes in the signature were transcribed in leukaemia cells. Combining the gene expression signature with the 2017 European LeukemiaNet classification significantly increased survival prediction accuracy with a five-year AUROC of 0.82 compared with 0.76 (p < 0.001). Our T-cell function-related risk score complements current AML prognosis models.

Ruxolitinib combined with prednisone, thalidomide and danazol in patients with myelofibrosis: Results of a pilot study.

Ruxolitinib is a safe and effective therapy of myeloproliferative neoplasm-associated (MPN) myelofibrosis. However, often there are dose reductions and/or therapy interruptions because of therapy-related adverse events (AEs), especially anemia and thrombocytopenia. We previously reported combined therapy with prednisone, thalidomide and danazol (PTD) reversed anemia and thrombocytopenia in people with MPN-associated myelofibrosis. We wondered whether adding PTD to ruxolitinib might mitigate the hematologic AEs and thereby avoid the dose reduction of ruxolitinib and improve the efficacy. To test this hypothesis, we conducted a baseline hemoglobin and platelet concentration assignment prospective observational study in 72 patients comparing 3-month dose adjustment and efficacy of ruxolitinib with (N = 53, the study group) or without (N = 19, the control group) PTD. According to the platelet counts, the median daily ruxolitinib doses in the study group increased from 30 to 40 mg by week 12, whereas in the control group it remained at 30 mg (p = 0.019). In the study group 35 patients had a hemoglobin increase ≥10 g/L compared with no patient receiving ruxolitinib only (p < 0.001). Platelet increases >100 × 10E+9/L were seen in 56.6% and 5.3% of patients in the two groups, respectively (p < 0.001). In patients with anemia and thrombocytopenia, 18 patients in the study group had an anemia response at week 12 and 12 had a platelet increase of ≥50 × 10E+9/L. No patient in the control group achieved either response (p < 0.001 and p = 0.078). The study group had a more spleen response than the control group (p = 0.046). Peripheral edema and transaminase elevation were the main nonhematologic AEs of PTD. These AEs can be alleviated by adjusting the danazol dose. In conclusion, adding PTD to ruxolitinib improved ruxolitinib-associated anemia and thrombocytopenia, and resulted in a higher ruxolitinib dose.

Perspective: Pivotal translational hematology and therapeutic insights in chronic myeloid hematopoietic stem cell malignancies.

Despite much of the past 2 years being engulfed by the devastating consequences of the SAR-CoV-2 pandemic, significant progress, even breathtaking, occurred in the field of chronic myeloid malignancies. Some of this was show-cased at the 15th Post-American Society of Hematology (ASH) and the 25th John Goldman workshops on myeloproliferative neoplasms (MPN) held on 9th-10th December 2020 and 7th-10th October 2021, respectively. The inaugural Post-ASH MPN workshop was set out in 2006 by John Goldman (deceased) and Tariq Mughal to answer emerging translational hematology and therapeutics of patients with these malignancies. Rather than present a resume of the discussions, this perspective focuses on some of the pivotal translational hematology and therapeutic insights in these diseases.

Safety and efficacy of a humanized CD19 chimeric antigen receptor T cells for relapsed/refractory acute lymphoblastic leukemia.

CD19-targeted chimeric antigen receptor T (CAR-T) cells using murine single-chain variable fragment (scFv) has shown substantial clinical efficacy in treating relapsed/refractory acute lymphoblastic leukemia (R/R ALL). However, potential immunogenicity of the murine scFv domain may limit the persistence of CAR-T cells. In this study, we treated 52 consecutive subjects with R/R ALL with humanized CD19-specific CAR-T cells (hCART19s). Forty-six subjects achieved complete remission (CR) (N = 43) or CR with incomplete count recovery (CRi) (N = 3) within 1 month post infusion. During the follow-up with a median time of 20 months, the 1-year cumulative incidence of relapse was 25% (95% confidence interval [CI] 13-46), and 1-year event-free survival was 45% (95% CI 29-60). To the cutoff date, 20 patients presented CD19+ relapse and 2 had CD19- relapse. Among the 22 relapsed patients, 14 had treatment-mediated and treatment-boosted antidrug antibodies (ADA) as detected in a sensitive and specific cell-based assay. ADA positivity was correlated with the disease relapse risk. ADA-positive patients had a significantly lower CAR copy number than ADA-negative patients at the time of recurrence (p < .001). In conclusion, hCART19s therapy is safe and highly active in R/R ALL patients, and the hCART19s treatment could induce the emergence of ADA, which is related to the recurrence of the primary disease.

Exposure to 17α-Ethinylestradiol Results in Differential Susceptibility of Largemouth Bass (Micropterus salmoides) to Bacterial Infection.

Disease outbreaks, skin lesions, mortality events, and reproductive abnormalities have been observed in wild populations of centrarchids. The presence of estrogenic endocrine disrupting compounds (EEDCs) has been implicated as a potential causal factor for these effects. The effects of prior EEDC exposure on immune response were examined in juvenile largemouth bass (Micropterus salmoides) exposed to a potent synthetic estrogen (17α-ethinylestradiol, EE2) at a low (EE2Low, 0.87 ng/L) or high (EE2High, 9.08 ng/L) dose for 4 weeks, followed by transfer to clean water and injection with an LD40 dose of the Gram-negative bacteria Edwardsiella piscicida. Unexpectedly, this prior exposure to EE2High significantly increased survivorship at 10 d post-infection compared to solvent control or EE2Low-exposed, infected fish. Both prior exposure and infection with E. piscicida led to significantly reduced hepatic glycogen levels, indicating a stress response resulting in depletion of energy stores. Additionally, pathway analysis for liver and spleen indicated differentially expressed genes associated with immunometabolic processes in the mock-injected EE2High treatment that could underlie the observed protective effect and metabolic shift in EE2High-infected fish. Our results demonstrate that exposure to a model EEDC alters metabolism and immune function in a fish species that is ecologically and economically important in North America.

Ibrutinib in Advanced Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma: Lower Risk of Hepatitis B Virus Reactivation.

INTRODUCTION: Therapy of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) with drugs such as ibrutinib and rituximab is often associated with immune suppression, opportunistic infections, and reactivation of virus infections such as hepatitis B virus (HBV). This risk is especially important in geographical regions like Asia where many potential therapy recipients have HBV infection. Also, whether safety and efficacy of ibrutinib in Asians and Europeans with advanced CLL/SLL are similar is unknown. We determined the safety and efficacy of ibrutinib compared with rituximab in advanced CLL/SLL including persons with HBV infection. We compared outcomes with data published from trials in persons of European descent. METHODS: This is a post hoc analysis of a multicenter, phase-3 trial (NCT01973387). Subjects with advanced CLL/SLL were randomized 2:1 to receive ibrutinib, 420 mg/day, or rituximab, 500 mg/mE + 2, for 6 cycles. Subjects with resolved HBV infection were included. Endpoints were progression-free survival (PFS), overall response rate (ORR), survival, and adverse events including resolved HBV reactivation. RESULTS: 131 subjects received ibrutinib (N = 87) or rituximab (N = 44) including 53 with resolved HBV infection. Median follow-up was 31 months (95% confidence interval: 28, 32 months). ORR was 61% (50, 71%) versus 7% (2, 18%; p < 0.001). Median PFS was not reached in the ibrutinib cohort but must be >40 months versus 8 months (7, 9 months; p < 0.0001) in the rituximab cohort. Median survival was not reached but must be >40 months versus 27 months (17 months, NE; p = 0.0006). In multivariable analyses, receiving ibrutinib increased PFS (hazard rate [HR] for failure = 0.12 [0.06, 0.23]; p < 0.001) and decreased risk of death (HR = 0.31 [0.15, 0.63]; p < 0.001). Median duration of exposure to ibrutinib was significantly longer than exposure to rituximab (28 vs. 5 months). The safety profile of ibrutinib was consistent with that observed in previous studies with no new safety signal. No subject receiving ibrutinib had HBV reactivation versus 2 receiving rituximab, despite much greater use of drugs to prevent HBV reactivation in the rituximab cohort. Outcomes were like those reported in persons of European descent, except ORR which, was unreliably correlated with PFS in Asians. CONCLUSION: Ibrutinib is safe and effective in persons with advanced CLL/SLL and better than rituximab in all therapy outcomes including risk of HBV reactivation. Outcomes with ibrutinib in Chinese were like those reported in persons of predominately European descent.

Clonal Megakaryocyte Dysplasia with Normal Blood Values Is a Distinct Myeloproliferative Neoplasm.

INTRODUCTION: In 1991, we reported 18 persons with a clinical-pathologic entity and termed atypical myeloproliferative disorder because they did not meet the contemporary diagnostic criteria for a myeloproliferative neoplasm. We sought to gain further knowledge on this disease entity. METHODS: This retrospective cohort study included consecutive subjects registered in the database of the Center for the Study of Myelofibrosis in Pavia, Italy, from 1998 to 2020 (June), and diagnosed with atypical myeloproliferative disorder according to our adjudicated criteria. We studied clinical, histological, cytogenetic, and molecular covariates and risks of thrombosis, disease progression, and death. Data were compared with those of concurrent subjects with prefibrotic myelofibrosis. RESULTS: Fifteen new subjects with atypical myeloproliferative disorder were identified. Seven were male. Median age was 50 years (IQR, 41-54 years). Thirteen were diagnosed with a synchronous symptomatic or incidentally detected thrombotic event. The bone marrow showed megakaryocyte hyperplasia with dysplasia. JAK2V617F was present in 10 subjects and CALR mutation in one. No other somatic mutations were identified in next generation sequencing. After a median follow-up of 101 months (IQR, 40-160 months), no subject had disease progression or blast transformation. Incidence of post-diagnosis or recurrent thrombosis was 3.9 events (95% confidence interval, 3.5-4.0) and 5.0 events (4.6-5.6) per 100 person-years. Features of subjects with atypical myeloproliferative disorder differed markedly from those of 546 subjects with prefibrotic myelofibrosis. CONCLUSION: Our data indicate that these 15 persons have a distinct myeloproliferative neoplasm. We propose naming this new disorder clonal megakaryocyte dysplasia with normal blood values.

Declines in Reproductive Condition of Male Largemouth Bass (Micropterus salmoides) Following Seasonal Exposure to Estrogenic Endocrine-Disrupting Compounds.

Reproductive abnormalities, that could lead to possible effects at the population level, have been observed in wild fish throughout the United States, with high prevalence in largemouth bass (LMB; Micropterus salmoides) and smallmouth bass (Micropterus dolomieu). Estrone (E1) and atrazine (ATR) are common environmental contaminants often associated with agricultural land use. 17alpha-ethinylestradiol (EE2) is a contaminant associated with wastewater treatment effluent, and a representative, well-studied estrogen commonly used for fish toxicity testing. Our objective was to assess whether early gonad recrudescence in adult fish was a period of sensitivity for alterations in reproductive condition and function. Adult male LMB were exposed from post-spawning to early gonad recrudescence to either a mixture of E1 (47.9 ng/L) + ATR (5.4 µg/L), or EE2 (2.4 ng/L) in outdoor experimental ponds. Gonad samples were collected from fish just prior to the start of exposure (July), at the end of the exposure period (December), the following spring just prior to spawning (April), and post spawning (May). Gonadosomatic index (GSI) was significantly reduced in E1 + ATR-exposed and EE2-exposed males compared to control at every post-exposure time point. Reduced sperm count and sperm motility were observed in the mixture treatment (E1 + ATR) compared to the control. Sperm motility was also reduced in the EE2 treatment. These data together indicate that estrogenic endocrine-disrupting compounds can lessen the reproductive condition of adult male LMB, and that effects of exposure during early gonad recrudescence can persist at least through the subsequent spawning cycle.

Crystallographic analysis of TarI and TarJ, a cytidylyltransferase and reductase pair for CDP-ribitol synthesis in Staphylococcus aureus wall teichoic acid biogenesis.

The cell wall of many pathogenic Gram-positive bacteria contains ribitol-phosphate wall teichoic acid (WTA), a polymer that is linked to virulence and regulation of essential physiological processes including cell division. CDP-ribitol, the activated precursor for ribitol-phosphate polymerization, is synthesized by a cytidylyltransferase and reductase pair known as TarI and TarJ, respectively. In this study, we present crystal structures of Staphylococcus aureus TarI and TarJ in their apo forms and in complex with substrates and products. The TarI structures illustrate the mechanism of CDP-ribitol synthesis from CTP and ribitol-phosphate and reveal structural changes required for substrate binding and catalysis. Insights into the upstream step of ribulose-phosphate reduction to ribitol-phosphate is provided by the structures of TarJ. Furthermore, we propose a general topology of the enzymes in a heterotetrameric form built using restraints from crosslinking mass spectrometry analysis. Together, our data present molecular details of CDP-ribitol production that may aid in the design of inhibitors against WTA biosynthesis.

Crystallographic analysis of Staphylococcus aureus LcpA, the primary wall teichoic acid ligase.

Gram-positive bacteria, including major clinical pathogens such as Staphylococcus aureus, are becoming increasingly drug-resistant. Their cell walls are composed of a thick layer of peptidoglycan (PG) modified by the attachment of wall teichoic acid (WTA), an anionic glycopolymer that is linked to pathogenicity and regulation of cell division and PG synthesis. The transfer of WTA from lipid carriers to PG, catalyzed by the LytR-CpsA-Psr (LCP) enzyme family, offers a unique extracellular target for the development of new anti-infective agents. Inhibitors of LCP enzymes have the potential to manage a wide range of bacterial infections because the target enzymes are implicated in the assembly of many other bacterial cell wall polymers, including capsular polysaccharide of streptococcal species and arabinogalactan of mycobacterial species. In this study, we present the first crystal structure of S. aureus LcpA with bound substrate at 1.9 Å resolution and those of Bacillus subtilis LCP enzymes, TagT, TagU, and TagV, in the apo form at 1.6-2.8 Å resolution. The structures of these WTA transferases provide new insight into the binding of lipid-linked WTA and enable assignment of the catalytic roles of conserved active-site residues. Furthermore, we identified potential subsites for binding the saccharide core of PG using computational docking experiments, and multiangle light-scattering experiments disclosed novel oligomeric states of the LCP enzymes. The crystal structures and modeled substrate-bound complexes of the LCP enzymes reported here provide insights into key features linked to substrate binding and catalysis and may aid the structure-guided design of specific LCP inhibitors.