Introduction Bronchiolitis obliterans syndrome (BOS) is the major form of chronic lung dysfunction that occurs in allogeneic hematopoietic cell transplant recipients (HCT), reported in approximately 5% of transplants, and associated with poor quality of life and long-term survival. In 2020, a NIH Consensus Development Project formalized guidelines for pulmonary function test (PFT) performance, with PFT’s recommended pre-HCT, then q.3 months over the initial year post-HCT (PMID 33839317). Full PFT (spirometry, lung volumes, and diffusion capacity) are recommended pre-HCT, D100 and 1-year post-HCT, with spirometry at 6 and 9 months post-HCT. A single center, retrospective analysis of adherence to NIH guidelines, with clinical outcomes in those that did (or did not) adhere to these guidelines for PFT monitoring now reported. Methods Case records from 418 patients (8 – 75 years, median 58 years) that underwent allogeneic HCT from 2021 – 2024 were reviewed. Patient demographics, clinical outcomes and PFT performance were analyzed. Diagnostic criteria for BOS were based upon 2014 NIH Consensus Criteria (PMID 25529383). PFT non-compliance was defined as failure to meet the NIH criteria (i.e. baseline + q.3 month PFT) within the initial year post-HCT. Patients were censored at the time of relapse or death. Patients who relapsed or died within the initial 100 days post-HCT were excluded from the post-HCT analysis. Results Pre-HCT spirometry was performed in 416 of 418 (99.5%) patients, with full PFT (spirometry, plethysmography, diffusion capacity) performed in 15.0% of patients pre-HCT. PFT performance was low at all time points, including 3 months (39.6%), 6 months (40.9%), 9 months (32.7%) and 1-year (55.8%) post-HCT in patients evaluable at that time point. Eleven of 267 (4.1%) patients who survived 1-year post-HCT (without relapse) were compliant with NIH guidelines for PFT performance. Overall, 24 patients (5.7%) met the diagnostic criteria for BOS, with a 2.4% incidence of BOS in patients who missed 0-1 PFT versus an 8.0% incidence in those missing 2 (or more) PFT in the first year post-HCT. The mean FEV1 at the time of diagnostic recognition of BOS was 60% in patients missing 0-1 PFT, 58% in patients missing 2 PFT and 48% in patients missing 3-4 PFT within the first year post-HCT. Likewise, the mean time to diagnostic onset of BOS was shorter in patients missing 0-1 versus 2-4 PFT in the first year post-HCT, 344 days (± 60 days) vs 645 days (± 62 days). Conclusion Adherence to NIH Consensus Guidelines for monitoring lung function is very low (<10%) in allogeneic HCT recipients. Significant delays in the diagnostic recognition of BOS occur in PFT non-compliant patients. Given the potential morbidity and mortality associated with BOS, our transplant community must recognize the need to perform PFT routinely in the first year post-HCT.

IntroductionFanconi anemia (FA) is an inherited bone marrow failure syndrome caused by defects in DNA repair. Patients often require hematopoietic stem cell transplantation (HSCT) to cure their severe bone marrow failure. Owing to the defects in DNA repair, patients with Fanconi anemia have significant morbidity with myeloablative conditioning prior to HSCT. Previous studies have reported good outcomes with reduced intensity conditioning (RIC) prior to HSCT for patients with FA. There are limited studies that compare outcomes between matched related donors and alternative donors.ObjectiveIn this analysis, we describe our single center experience of 22 patients with FA who received HSCT with RIC from various graft sources, including haploidentical donors.MethodsA retrospective review was performed for 22 patients with FA who received HSCT for bone marrow failure from matched related donor (4), matched unrelated donor (5), mismatched unrelated donor (4), haploidentical donor (7), and unrelated cord blood unit (1) between 2000-2022. Median age at time of last follow up was 14 years (range: 7 to 28). Patients received fludarabine-based RIC with 16/22 patients receiving TBI (200-450 cGy) and 13/22 receiving alemtuzumab. Patients who received HSCT for MDS/AML were excluded. Overall survival (OS), event free survival (EFS), and cumulative incidence of acute (aGVHD) and chronic (cGVHD) graft-vs-host disease were analyzed using Kaplan-Meier estimates. Events were defined as graft failure or death.ResultsThe 5-year OS for the entire cohort was 95.2% (95% CI 70.7-100%) and the 5-year EFS was 85.7% (95% CI 62.0-95.1%) with a median follow-up of 7 years, (range: 0.5-21). No difference in OS was observed in patients who received HSCT from matched donors compared with those who had alternative donors (100% vs. 91.7% respectively, P=0.69); however, there was a trend toward improved EFS for matched donors compared to alternative donors (100% vs. 75%, P=0.09). The difference in EFS was driven by higher rates of graft failure in patients receiving haploidentical transplants. Primary and secondary graft failure developed in 9.1% (2) and 13.6% (3) of patients, respectively. Cumulative incidence of aGVHD grade 2-4 and 3-4 was 19.0% and 5.3%, respectively. Cumulative incidence of cGVHD was 23.8% with two patients being extensive.ConclusionPatients with FA undergoing HSCT have excellent OS and EFS using fludarabine/low-dose TBI based RIC regimens. Haploidentical transplants may have higher rates of graft failure, resulting in lower EFS. The use of alemtuzumab results in low rates of aGVHD and cGVHD, with especially low rates of severe GVHD. Further studies are needed to seek optimal conditioning regimens that improve transplant outcomes, particularly for patients lacking well matched donors.

PURPOSE Time to antibiotic administration (TTA) in <60 minutes for children with neutropenic fever presenting to an emergency room is associated with reduced incidence of sepsis and intensive care admission. As such, TTA is used as a national quality metric for pediatric oncology patients. At our center, in 2020, 19% of the hospitalized patients with a new fever encounter were receiving antibiotics in <60 minutes, prompting a multidisciplinary approach to reach a goal of >90% in all pediatric patients with cancer with a new fever. METHODS A multidisciplinary team completed four Plan-Do-Study-Act cycles between March 2021 and September 2023. We implemented education initiatives, an updated handoff smartphrase guiding the on-call team, an antibiotic champion (AC) nursing role, a revised fever plan for handoff, a rapid-response team to address new fevers, and an algorithm for blood culture collection. Data were collected, analyzed, and reported biweekly with feedback sought for delays in TTA. RESULTS There was a total of 639 new fevers in 329 unique oncology patients. As of September 4, 2023, average TTA decreased from 89 minutes at baseline to 46.4 minutes for more than 12 months. The percentage of patients receiving first dose of antibiotic in <60 minutes also increased from 19% to 93.7%, which was sustained as well. The most effective interventions were creation of the AC role and streamlining the blood culture collection process. CONCLUSION This project demonstrates the importance of multidisciplinary involvement for providing optimal care. Specific implementation of targeted education, an AC role, and development of an algorithm streamlining the processes led to meaningful targeted improvements. Further analyses will explore the impact of these interventions on patient outcomes.

IntroductionOutcomes in hematopoietic stem cell transplantation (HSCT) for severe aplastic anemia (SAA) have improved greatly over time; however, primary (PGF) and secondary graft failure (SGF) remain a significant concern. Patients experiencing graft failure require additional HSCT for cure. We report our single center experience, including long term follow-up, of 111 pediatric patients with SAA who underwent HSCT.MethodsWe performed a retrospective review of 111 patients with idiopathic SAA who received HSCT at the Center for Cell and Gene Therapy at Baylor College of Medicine/Texas Children's Hospital between 1997-2023. Overall survival (OS), event free survival (EFS) and cumulative incidence of acute (aGVHD) and chronic (cGVHD) graft vs. host disease were calculated using Kaplan-Meier estimates. Events included PGF, SGF and death. Comparison of total nucleated cell (TNC) doses between patients that developed SGF to those that did not was performed using the Mann-Whitney test.HSCT was performed using a matched sibling donor (MSD) in 46 patients, matched unrelated donor (MUD) in 41, mismatched unrelated donor (MMUD) in 14 and haploidentical donor in 10. All patients received reduced intensity conditioning regimens based on a cyclophosphamide backbone; a majority of MSD HSCTs also received ATG (43/46), MUD/MMUD HSCTs received low dose TBI and alemtuzumab (51/55) and haploidentical HSCTs received low dose TBI and post-transplant cyclophosphamide (6/10).ResultsThe 5-year OS for the entire cohort was 92.8% (95% CI 89.1-100%) and the 5-year EFS was 70.0% (95% CI 61.2-81.3%) with a median follow-up of 4 years (range 3 months-23 years). While patients who received HSCT from MSD had excellent OS (97.7%, 95% CI 84.9-100%), it did not differ significantly (P = 0.35) from those who received HSCT from other donors (fig. 1). EFS for patients receiving HSCT from MSD was 64.5% (95% CI 49.0-86.7%), comparable to those receiving HSCT from other donors (P = 0.49). The lower EFS was largely driven by high rates of SGF (cumulative incidence 25.9%). The median TNC dose for the entire cohort was 4.6 × 108 TNC/kg; the median TNC for those that developed SGF was 3.3 × 108 TNC/kg compared to 5.0 × 108 TNC/kg (P = 0.02) for those that did not have graft failure (fig. 2). The cumulative incidence of grade 2-4 acute GVHD was 7.7%, of grade 3-4 acute GVHD was 2.3% and of chronic GVHD was 21.0%; however, no severe cGHVD was observed (fig. 3).ConclusionsExcellent long-term OS was comparable among all donor types. EFS remains a challenge but no difference was seen among all donor types. Patients that received HSCT with higher TNC had a lower risk for SGF. Rates of aGVHD and cGVHD are very low, likely owing to the predominance of matched donors in this cohort and the use of alemtuzumab in patients with unrelated donors. Further studies are needed to determine whether more immunosuppressive conditioning regimens are needed to prevent SGF.

Background HLH is a syndrome characterized by an immune hyperactivation due either to an underlying genetic or inflammatory trigger. Relapsed/refractory HLH remains associated with dismal outcomes, prompting the need for novel therapies including hematopoietic stem cell transplant (HSCT). Novel agents for T cell depletion and cytokine blockade, such as alemtuzumab and emapalumab have been increasingly prevalent, but appropriate dosing and correlations with survival after HSCT are limited. Thus, we summarize our center's outcomes with treating relapsed/refractory HLH in the setting of alemtuzumab with the goal of proposing an optimized transplant regimen for these patients. Methods We reviewed 39 pediatric patients treated at Texas Children's Hospital from June 2008-January 2023 who had an underlying diagnosis of HLH. We performed outcome analysis of overall survival, and causes of death and correlate them with the dosing and use of cumulative alemtuzumab (both pre- and post-HSCT), as well as treatment with emapalumab. Results Thirty-nine patients with a diagnosis of HLH who underwent HSCT (median age 3.4 years; range 0.6-19.8). Twenty-nine received a matched unrelated donor transplant, three were matched related, eight were mismatched related. Nineteen patients received a reduced intensity conditioning with fludarabine, melphalan, and alemtuzumab while 16 patients received the same backbone but with thiotepa added. One patient received a myeloablative regimen with busulfan, fludarabine, melphalan, and alemtuzumab. 22 patients received alemtuzumab IV (dose range<15kg: 3 mg x 4 doses; 15-30 kg: 5 mg x 4 doses; >30 kg: 10 mg x 4 doses) compared to 18 subcutaneous (SQ) (dose: 1 mg/kg). Emapalumab was given to six patients prior to HSCT. 5-year overall survival (OS) for all patients was 59% with 18 patients dying at a median time of 118 days after transplant; (range: 9-894). Causes of death included: infections (9), organ failure (5), disease progression (2), and GVHD (1). Seven out of nine patients who died of infection had a cumulative alemtuzumab of 0.9 mg/kg or greater. Patients who died had a significantly higher median alemtuzumab dose/kg (M=1.49, SD=0.59) compared to survivors (M=1.15, SD=0.40), p=.02. Additionally, pre-conditioning alemtuzumab salvage therapy was associated with worse outcome with 5/8 patients dying following HSCT. When comparing the route of administration of alemtuzumab, patients receiving SQ dosing trended toward improved survival compared to IV treatment (66.6% vs. 50%; p=0.27). Conclusions HSCT is required for cure for patients with primary HLH. Optimizing conditioning therapy by adjusting overall dosing of alemtuzumab lead to improved long-term survival in patients with HLH.

Blinatumomab, a bispecific T cell engager that binds CD19 in leukemic cells and CD3 in cytotoxic T cells and leads to leukemic blast lysis, is often used in pediatric patients with relapsed/refractory (R/R) B cell acute lymphoblastic leukemia (B-ALL) prior to allogeneic hematopoietic cell transplantation (allo-HCT). Concerns about the potential risk of blinatumomab-related immune-mediated toxicities after allo-HCT have not been adequately addressed. These include graft-versus-host disease (GVHD), delayed engraftment, and graft failure or rejection. Pediatric-specific data reporting post-HCT outcomes of patients treated with blinatumomab are scarce and limited to small cohorts. We sought to investigate the clinical outcomes of pediatric patients with R/R B-ALL who received blinatumomab therapy pre-HCT, focusing on overall survival (OS), leukemia-free survival (LFS), cumulative incidence of relapse (CIR), and nonrelapse mortality (NRM), as well as the incidence of immune-mediated post-HCT complications including GVHD, delayed neutrophil or platelet engraftment, graft failure, and graft rejection. We also investigated blinatumomab's effects on B cell reconstitution based on achievement of i.v. immunoglobulin (IVIG) independence post-HCT. This single-center, retrospective study included patients with B-ALL receiving blinatumomab therapy before undergoing allo-HCT, with transplantation performed between 2016 and 2021 at our institution. Patients receiving blinatumomab for relapse after allo-HCT were excluded. Patients receiving chemotherapy alone before allo-HCT during the same period composed the control group. Seventy-two patients were included, 31 of whom received blinatumomab before allo-HCT. Survival estimates were obtained using the Kaplan-Meier method, and the log-rank test was used to analyze differences between groups. Categorical variables were compared between groups using the chi-square test or Fisher exact test, and continuous variables were compared using the Wilcoxon rank-sum test. Cumulative incidences were estimated using the competing risks method, and Gray's test was used to analyze differences between groups. A Cox proportional hazards regression model was used for univariate and multivariable analyses for OS. Landmark analysis was performed at the set time points of 30 days and 100 days post-allo-HCT. Most patients in the study cohort had high-risk relapsed B-ALL. Blinatumomab therapy induced minimal residual disease (MRD)-negative remissions in all patients, whereas 5 patients (12.2%) receiving chemotherapy alone had persistent MRD pre-allo-HCT. Time from the start of therapy to the date of allo-HCT was shorter for patients who received blinatumomab compared with those who received chemotherapy (P < .0001). Blinatumomab therapy was associated with greater LFS compared to chemotherapy alone (P = .049), but when limited to 1 year, LFS was not significantly different from control (P = .066). There appeared to be higher OS, lower CIR, and lower NRM in patients receiving blinatumomab compared to the control group; however, the differences were not significant. None of the variables assessed in multivariable analysis was associated with differences in OS. When compared to the controls, blinatumomab therapy did not result in a higher incidence of acute or chronic GVHD, delayed neutrophil or platelet engraftment, or graft failure or rejection. The time to IVIG infusion independence post-allo-HCT was similar in the 2 groups. This study supports the use of blinatumomab salvage therapy for R/R B-ALL before allo-HCT given its efficacy in inducing MRD-negative remissions and optimizing LFS, as well as its lack of association with an increased incidence of post-allo-HCT adverse immune-mediated toxicities. Larger, prospective studies are needed to confirm these findings and to investigate blinatumomab's effects in long-term post-allo-HCT events.

Men with type 2 diabetes are often characterized by abnormal plasma testosterone levels. This study was aimed at investigating whether testosterone treatment has an impact on cardiovascular risk factors in patients with type 2 diabetes and late-onset hypogonadism (LOH), chronically treated with hypoglycemic agents.This study included 51 men with type 2 diabetes, 26 of whom had already been treated with metformin and 25 with glimepiride for at least 6 months. On the basis of patient preference, 15 men receiving metformin and 12 receiving glimepiride were treated with intramuscular testosterone enanthate (100 mg weekly) for 12 weeks. Plasma lipids, glucose homeostasis markers, as well as plasma levels of androgens, uric acid, high-sensitivity C-reactive protein (hsCRP), homocysteine and fibrinogen were determined before and at the end of the study.With the exception of insulin sensitivity, plasma hsCRP and homocysteine, there were no differences between patients treated with metformin and glimepiride. Testosterone enanthate administered to both groups of patients increased plasma testosterone, reduced plasma hsCRP and improved insulin sensitivity. Testosterone-metformin combination therapy reduced also circulating levels of uric acid, homocysteine and fibrinogen. These effects, stronger in patients treated with metformin than glimepiride, correlated with the impact of testosterone on insulin sensitivity.Our results suggest that testosterone may bring more clinical benefits to metformin- than sulfonylurea-treated men with diabetes and LOH.

Chimeric antigen receptor (CAR) T cell (CAR-T) therapy represents a revolutionary treatment for patients with relapsed/refractory hematologic malignancies. However, its use can result in significant toxicities, including cytokine release syndrome (CRS), a potentially life-threatening clinical syndrome resulting from the release of proinflammatory cytokines upon T cell activation. In addition, patients who develop CRS often experience prolonged cytopenias, and those with the most severe CRS also have the longest delays in full marrow recovery. Although an association between CRS and delayed bone marrow recovery has been established, the precise mechanism underlying this phenomenon remains unknown. This study was conducted to test our hypothesis that delayed bone marrow recovery following CAR-T therapy is caused by elevation of proinflammatory cytokines, leading to apoptosis and depletion of hematopoietic stem and progenitor cells (HSPCs). SCID-beige mice bearing intraperitoneal CD19+ Raji cell tumors were treated with injection of human CD19.28z CAR T cells. Bone marrow was then harvested for analysis by flow cytometry, and HSPCs were isolated for whole-transcriptome analysis by RNA sequencing. Complete blood counts and serum cytokine levels were measured as well. A second model was developed in which SCID-beige mice were treated with murine IFN-γ (mIFN-γ), murine IL-6 (mIL-6), or both. Bone marrow was harvested, and flow cytometry assays were conducted to evaluate the degree of apoptosis and proliferation on specific HSPC populations. SCID-beige mice bearing intraperitoneal Raji cell tumors that were treated with CAR-T therapy developed CRS, with elevations of several proinflammatory cytokines, including profound elevation of human IFN-γ. Gene set enrichment analysis of RNA sequencing data revealed that genes associated with apoptosis were significantly upregulated in HSPCs from mice that developed CRS. Endothelial protein C receptor (EPCR)-negative HSCs, a subset of HSCs that is poised for terminal differentiation, was found to be specifically decreased in mice that were treated with CAR T cells. Furthermore, HSPCs were found to have increased levels of apoptosis upon treatment with mIFN-γ and mIL-6, whereas short-term HSCs and multipotent progenitors exhibited increases in proliferation with mIFN-γ treatment alone. The results from this study provide evidence that the elevation of proinflammatory cytokines following CAR-T therapy impacts the bone marrow through a combined mechanism: pluripotent HSCs that are exposed to elevated levels of IFN-γ and IL-6 undergo increased cell death, while more committed progenitor cells become more proliferative in response to elevated IFN-γ. These combined effects lead to depleted stores of repopulating HSCs and ultimately cytopenias. © 2023 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Wnt/beta-catenin signaling is active in small subpopulations of Ewing sarcoma cells, and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these beta-catenin-activated Ewing sarcoma cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell-autonomous mechanisms, Wnt/beta-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from beta-catenin-active versus-nonactive tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of beta-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that beta-catenin-activated Ewing cells secreted factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of proangiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-beta. Mechanistically, Wnt/beta-catenin signaling antagonizes EWS-FLI1-dependent repression of TGF-beta receptor type 2, thereby sensitizing tumor cells to TGF-beta ligands. Together, these findings suggest that Wnt/beta-catenin-active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.

Local and metastatic progression of solid tumors depends on crosstalk between tumor cells and the tumor microenvironment (TME), including both stromal cells and the extracellular matrix (ECM). We recently showed that high Wnt/beta-catenin activity in Ewing sarcoma correlates with diminished patient survival and that canonical Wnt signaling alters the tumor secretome, influencing ECM protein composition. In light of this, we investigated the hypothesis that Wnt/beta-catenin supports tumor progression by modulating tumor: TME crosstalk. Our results reveal that, in discrete tumor cell sub-populations, beta-catenin activation sensitizes cells to TGF-beta ligands through derepression of the TGF-beta receptor, TGFBR2, resulting in canonical Wnt-induced, TGF-beta-dependent upregulation of TGF-beta targets. Significantly, these Wnt/TGF-beta responsive targets include multiple AngioMatrix genes that are known to alter the TME to promote angiogenesis, including tenascin-C and collagens. Studies of Ewing sarcoma models, in vitro and in vivo, as well as in two independent patient cohorts, confirm that a direct relationship exists between beta-catenin and TGF-beta activation in tumor cells and angiogenesis in the local TME. Mechanistically, this is due, in part, to tenascin C-mediated promotion of endothelial cell proliferation. Thus, functional cooperation between canonical Wnt and TGF-beta signaling in Ewing cells induces secretion of pro-angiogenic factors. This study reveals a novel link between Wnt and TGF-beta signaling in Ewing sarcoma and illustrates the critical contribution of tumor cell heterogeneity and tumor: TME crosstalk to sarcoma progression.Citation Format: Allegra G. Hawkins, Elisabeth A. Pedersen, Wei Jiang, Sydney Treichel, Colin Sperring, Jay Read, Brian Magnuson, Rajiv M. Patel, Dafydd Thomas, Rashmi Chugh, Elizabeth R. Lawlor. Cooperation between canonical Wnt and TGF-beta pathways promotes sarcoma angiogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3645.

We report three new cases of a germline heterozygous gain-of-function missense (p.(Met1141Lys)) mutation in the C2 domain of phospholipase C gamma 2 (PLCG2) associated with symptoms consistent with previously described auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome and pediatric common variable immunodeficiency (CVID). Functional evaluation showed platelet hyper-reactivity, increased B cell receptor-triggered calcium influx and ERK phosphorylation. Expression of the altered p.(Met1141Lys) variant in a PLCγ2-knockout DT40 cell line showed clearly enhanced BCR-triggered influx of external calcium when compared to control-transfected cells. Our results further expand the molecular basis of pediatric CVID and phenotypic spectrum of PLCγ2-related defects.

Background Patients with DLBCL exhibit widely divergent outcomes despite harboring histologically identical tumors. Currently, GEP and IHC algorithms assign patients to 1 of 2 main subtypes: germinal center B cell-like (GCB), or activated B cell-like (ABC), the latter of which historically carries a less favorable prognosis. However, it remains controversial as to whether these prognostic groupings remain valid in the era of rituximab therapy. Materials and Methods A systematic literature review identified 24 articles from which meta-analyses were conducted, comparing survival outcomes for patients assigned to either GCB or ABC/non-GCB subtype using GEP and/or Hans, Choi, or Muris IHC algorithms. Results Patients designated as GCB DLBCL using GEP fared significantly better in terms of overall survival than those with ABC DLBCL (hazard ratio, 1.85; P < .0001). In contrast, the Hans and Choi algorithms failed to identify significant differences in overall survival (P = .07 and P = .76, respectively) between GCB and non-GCB groups. Conclusions Our study illustrates a lack of evidence supporting the use of the Hans and Choi algorithms for stratifying patients into distinct prognostic groups. Rather, GEP remains the preferred method for predicting the course of a patient's disease and informing decisions regarding treatment options.

In comparison with the large number of nonribosomal peptide synthetases (NRPSs) that release their peptide products by hydrolytic cleavage of the peptide carrier protein (PCP) bound thioester, there are relatively few NRPSs that have been shown to use a nicotinamide cofactor to reduce this PCP-peptidyl thioester to an aldehyde or imine moiety. This work describes the first example of a reductase domain within a NRPS scaffold shown to reduce a PCP-peptidyl thioester to the corresponding primary alcohol, via an aldehyde intermediate, using two equivalents of reduced nicotinamide adenine dinucleotide phosphate (NADPH). By employing a ketone mimic of the aldehyde intermediate, as well as a specifically deuterated NADPH, it was further demonstrated that the pro-S hydride of the cofactor is transferred to the re face of the carbonyl group.

The first positive evidence for the utilization of a direct C-6' ' oxidation/reduction mechanism by ADP-l-glycero-d-manno-heptose 6-epimerase is reported here. The epimerase (HldD or AGME, formerly RfaD) operates in the biosynthetic pathway of l-glycero-d-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. The stereochemical inversion catalyzed by the epimerase is interesting as it occurs at an "unactivated" stereocenter that lacks an acidic C-H bond, and therefore, a direct deprotonation/reprotonation mechanism cannot be employed. Instead, the epimerase employs a transient oxidation strategy involving a tightly bound NADP(+) cofactor. A recent study ruled out mechanisms involving transient oxidation at C-4' ' and C-7' ' and supported a mechanism that involves an initial oxidation directly at the C-6' ' position to generate a 6' '-keto intermediate (Read, J. A., Ahmed, R. A., Morrison, J. P., Coleman, W. G., Jr., Tanner, M. E. (2004) J. Am. Chem. Soc. 126, 8878-8879). A subsequent nonstereospecific reduction of the ketone intermediate can generate either epimer of the ADP-heptose. In this work, an intermediate analogue containing an aldehyde functionality at C-6' ', ADP-beta-d-manno-hexodialdose, is prepared in order to probe the ability of the enzyme to catalyze redox chemistry at this position. It is found that incubation of the aldehyde with a catalytic amount of the epimerase leads to a dismutation process in which one-half of the material is oxidized to ADP-beta-d-mannuronic acid and the other half is reduced to ADP-beta-d-mannose. Transient reduction of the enzyme-bound NADP(+) was monitored by UV spectroscopy and implicates the cofactor's involvement during catalysis.

[reaction: see text] A chemoenzymatic synthesis of ADP-D-glycero-beta-D-manno-heptose (ADP-D,D-Hep) is described in which D,D-Hep 7-phosphate is converted to ADP-D,D-Hep by two biosynthetic enzymes. This strategy allows access to the 6''-deuterated analogue, which upon incubation with the epimerase showed complete retention of the isotopic label at the 6''-position. This provides evidence for a direct oxidation mechanism in which the hydride initially transferred to the NADP+ cofactor is subsequently returned to the same carbon in a nonstereospecific manner.