Biosynthesis of Polyhydroxyalkanoate Terpolymer from Methanol via the Reverse β-Oxidation Pathway in the Presence of Lanthanide

Methylorubrum extorquens AM1 is the attractive platform for the production of value-added products from methanol. We previously demonstrated that M. extorquens equipped with PHA synthase with broad substrate specificity synthesized polyhydroxyalkanoates (PHAs) composed of (R)-3-hydroxybutyrate and small fraction of (R)-3-hydroxyvalerate (3HV) and (R)-3-hydroxyhexanoate (3HHx) units on methanol. This study further engineered M. extorquens for biosynthesis of PHAs with higher 3HV and 3HHx composition focusing on the EMC pathway involved in C1 assimilation. The introduction of ethylmalonyl-CoA decarboxylase, catalyzing a backward reaction in the EMC pathway, aiming to increase intracellular propionyl/butyryl-CoA precursors did not affect PHA composition. Reverse b-oxidation pathway and subsequent (R)-specific hydration of 2-enoyl-CoA were then enhanced by heterologous expression of four genes derived from Ralstonia eutropha for the conversion of propionyl/butyryl-CoAs to the corresponding (R)-3-hydroxyacyl-CoA monomers. The resulting strains produced PHAs with higher 3HV and 3HHx compositions, while the methylotrophic growth was severely impaired. This growth impairment was interestingly restored by the addition of La3+ without a negative impact on PHA biosynthesis, suggesting the activation of the EMC pathway by La3+. The engineered M. extorquens synthesized PHA terpolymer composed of 5.4 mol% 3HV and 0.9% of 3HHx with 41% content from methanol as a sole carbon source in the presence of La3+.

CD38 Is a Key Regulator of Tumor Growth By Modulating the Metabolic Signature of Malignant Plasma Cells

Introduction: Multiple myeloma (MM) is a disease of malignant plasma cells, characterized by high CD38 expression. Although the CD38-targeting monoclonal antibodies are highly effective, resistance invariably arises. Tumor CD38 levels decrease after anti-CD38 therapy, but the expression is rarely permanently silenced. This suggests that CD38 expression may offer a tumor cell survival advantage, but the direct impact of CD38 loss on tumor dynamics has not been extensively characterized. Methods: CD38 knockout (KO) cell lines were generated by CRISPR-Cas9. Immunocompetent Balb/c and immunodeficient NSG mice were injected subcutaneously with either non-targeting (NT) or CD38 KO J558 cells. Stromal adhesion was compared using labeled NT and KO cells, with OP-9 murine stroma cells. Cellular NAD content was quantified using the Promega Glo Assay. Mitochondria were isolated with the Mitochondria Isolation Kit (Thermo Scientific). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were quantified using the Seahorse Assay. Response to hypoxia was evaluated using a modular hypoxic chamber. Cell cycle was quantified using propidium iodine staining. Results: To examine the role of CD38 in murine models, we utilized the CD38-expressing, murine plasmacytoma cell line J558. Strikingly, CD38 KO cells injected into Balb/c mice demonstrated significantly decreased tumor volume compared to NT (113 mm 3 (KO) vs. 1293 mm 3 (NT) at day 25, p <0.001). In contrast, in vitro cell proliferation and colony formation between KO and NT J558 cells were nearly identical, suggesting that the effects of CD38-loss were highly context dependent. Since tumoral CD38 expression may negatively modulate the immune response, we next compared CD38 KO and NT cells injected into immunodeficient NSG mice. CD38 KOs demonstrated an approximately 2.2-fold decreased tumor volume compared to the NT (708 mm 3 (KO) vs. 1592 mm 3 (NT), p=0.07). Further examination of the role of CD38 on the immune microenvironment are ongoing. Considering that some tumor growth impairment was maintained in immunodeficient mice, we next interrogated the effect of CD38 loss on other aspects of cell proliferation using J558 as well as human MM cell lines RPMI-8226 and NCI-H929. Daratumumab induced CD38 internalization has been shown to reduce stromal adhesion of MM cells. Similarly, CD38 KO cells demonstrated reduced stromal adhesion (2.5-fold decrease for J558, p<0.005 and 2-fold decrease for H929, p<0.005). Although stroma is a known promoter of cell survival and proliferation, we further questioned whether the NAD-metabolizing activity of CD38 modulates tumor growth. CD38 overexpression can drive down intracellular NAD and impair mitochondrial biogenesis. Accordingly, we found significantly higher NAD levels in the KO J558 tumor cells compared to NT (2-fold change, p <0.05). Additionally, CD38 KO cells demonstrated significantly higher levels of mitochondrial protein compared with the NTs (5-fold in J558 and 2-fold in H929). CD38 KO cell lines also showed markedly increased metabolic activity, with nearly 2-fold increase in basal OCR and ECAR, as well as in spare respiratory and glycolytic capacity. Given the contrast between in vivo and in vitro growth capacity, we questioned whether changes in mitochondrial content and metabolic function could confer an advantage for CD38-expressing cells under conditions of hypoxia, which is an important characteristic of the tumor microenvironment. Strikingly, under hypoxia, but not normoxia, CD38 KO MM cells demonstrated significantly more cell cycle arrest, defined by G0/G1 blockage (p=0.003 for H929 and p=0.004 for RPMI). Conclusion: We have shown that CD38 KO cells demonstrate decreased tumor growth in vivo but not in vitro. While the immune modulatory potential of CD38 is recognized, some of the growth impairment we observed may be explained by non-immune mediated mechanisms such as reduced stroma adherence as well as changes in cell metabolism. Loss of CD38 was associated with increased mitochondrial respiration, but also elevated ECAR and glycolytic rate. Higher reliance on mitochondrial respiration could explain impaired CD38 KO proliferation rates under hypoxia, possibly as a result of increased generation of reactive oxygen species. Disclosures Ghiaur: Menarini Richerche: Research Funding; Syros Pharmaceuticals: Consultancy.

EPID-15. AGE AND DOSIMETRIC IMPACTS ON RADIOTHERAPY ASSOCIATED GROWTH IMPAIRMENT FOR PATIENTS WITH INTRACRANIAL GERM CELL TUMORS (GCTs)

PURPOSE Intracranial germ cell tumors (GCTs) are rare pediatric central nervous system (CNS) tumors. Growth impairment induced by radiation treatments was rarely evaluated. We aimed to study the impacts of radiotherapy on height development and identify the dosimetric constraints. MATERIALS AND METHODS A total of 148 pediatric patients diagnosed with GCTs were retrospectively analyzed. Sex, age at irradiation, physical doses and biologically effective dose (BED), height, and endocrine status were obtained from patient records. The cumulative change in height was assessed using age-matched normalized height (ANH). Variables were assessed for correlations and statistical significance. RESULTS Cumulative physical doses and BEDs for the whole brain and pituitary were derived via DVHs and BEDVHs. In contrast to patients >11.5 yr, linear correlations between ANH and cumulative physical doses as well as BEDs to the whole brain and pituitary were identified in patients≤11.5 yr. Dosimetric constrains to the pituitary was 36 Gy for physical dose (AUC=0.70 [95% CI, 0.54-0.86], P< 0.05) and 63 Gy2 BED (AUC=0.69 [95% CI, 0.53-0.86], P< 0.05). Intriguingly, no significant differences in ANH were found among different CSI dose groups in patients ≤11.5 yr. (all P>0.05). Impaired hormone secretions in terms of GH and TSH were identified following cranial irradiation (both P< 0.001), particularly for those with tumors at the suprasellar region (GH: P< 0.01, TSH: P< 0.001). CONCLUSION Our study revealed the impacts of age, dosimetrics and tumor locations for growth impairment. This study will contribute to the optimization of radiation treatment planning and facilitate more individualized therapeutic strategies in pediatric patient with GCTs.

Growth Outcome and Metabolic Profile of PWS Patients Treated With GH and Differences Between AGA and SGA Group

Objective: Prader-Willi syndrome (PWS) is a complex genetic disease associated with growth impairment, severe obesity and metabolic dysfunctions. High proportion of PWS patients are born small for gestational age (SGA), which also increase the risk of growth impairment and metabolic dysfunction. The aim of this study was to describe growth outcome and and metabolic profiles in GH treated PWS patients. We also investigated the differences in clinical outcomes between AGA and SGA group Methods: Data of 55 children and adults with genetically verified PWS aged more than 2 years old (32 male and 23 female, age 2-18.8 years) from single center were studied. Only patients who were treated with GH were included. The clinical characteristics and laboratory findings were reviewed retrospectively. Results: Among 55 subjects, 39 had 15q11-13 deletion and 16 had uniparental disomy (UPD). Twenty (36.3%) were born SGA. All patients received GH treatment, and 11 (20%) discontinued GH treatemnt. Mean age at GH treatment initiation was 2.5 (range 0.3-12.4) years, and mean duration of treatment was 6.3 (range 1.0-11.3) years. Current height-SDS (-0.36 vs -0.16) and BMI-SDS (1.44 vs 1.33) did not differ between AGA and SGA group. Two patients in SGA group, but none in AGA group had diabetes mellitus. Mean glucose level was also higher in SGA group (100.1 vs 114.4 mg/dL) Conclusions: Our report gives an overview of growth profile and metabolic dysfunctions recorded in GH treated PWS patients. Growth profile did not differ between AGA and SGA group. Glucose level was higher in SGA group, so more careful monitoring and prevention for DM will be required in SGA group.

Influence of maternal use of tenofovir disoproxil fumarate or zidovudine in Vietnamese pregnant women with HIV on infant growth, renal function, and bone health

Tenofovir disoproxil fumarate (TDF) is still widely prescribed for human immunodeficiency virus (HIV)-infected pregnant women, despite its renal and bone toxicity. Although TDF-exposed infants often show transient growth impairment, it is not clear whether maternal TDF causes infantile rickets via maternal/fetal renal dysfunction in Asian populations. This prospective observational study was conducted in Vietnam and involved pregnant HIV-infected women treated with TDF-based regimen (TDF group) or zidovudine-based regimen (AZT-group). At birth, 3, 12, and 18 months of age, and included body length, weight, head circumference, serum alkaline phosphatase (ALP), creatinine, calcium, phosphorus, urine-β2-microglobulin (U-BMG), percentage of tubular reabsorption of phosphate (%TRP), and radiographic wrist score for rickets. Age-adjusted multivariate linear regression analysis evaluated the association of TDF/AZT use during pregnancy with fetal renal function and bone health. The study included 63 mother-infant pairs (TDF group = 53, AZT group = 10). In the mothers, detectable U-BMG (>252 μg/L) was observed more frequently in the TDF- than AZT group (89 vs 50%, p<0.001), but other renal/bone parameters were similar. In infants, maternal TDF use was not associated with growth impairment, renal dysfunction, or abnormal bone findings, but with a slightly higher ALP levels (p = 0.019). However, shorter length was associated with maternal AZT (p = 0.021), and worse radiographic scores were associated with LPV/r (p = 0.024). In Vietnamese population, TDF usage during pregnancy was not associated with infant transient rickets, growth impairment, or renal dysfunction, despite mild maternal tubular impairment. Maternal AZT and LPV/r influenced infant growth and bone health, though further studies are needed to confirm this finding.