The Role Of Bca2 In Regulation Of Warburg-Like Glucose And Lactate Metabolism In Breast Cancer Cell Lines

Modified metabolism is a hallmark of cancer cell biology known as “The Warburg Effect,” characterized by increased glucose-consumption and lactate-production, providing the metabolites and energy necessary for rapid proliferation. Breast-cancer-associated-gene-2 (BCA2) is an E3-Ub-ligase known to modulate AMPK, the “master-regulator of energy-homeostasis.” BCA2 is an oncogene associated with poor patient-survival and breast cancer invasiveness. Phospho-proteomic analysis of siBCA2-MDA-MB-231 knockdown-(KD) revealed enrichment of Nicotinamide-Nucleotide-Adenylyl-transferase-1 (NMNAT1), an essential enzyme in nuclear NAD-synthesis and a tumor-suppressor. Initial experiments demonstrated that glucose concentration was positively correlated with BCA2 and inversely with NMNAT1 protein expression. shBCA2-KD in MDA-MB-468/-231 reproduced the inverse correlation between BCA2 and NMNAT1. shBCA2 -KD resulted in a decrease in BCA2 protein (MDA-MB-468: 54% and 49%; MDA-MB-231: 31% and 19%), qPCR-transcript (MDA-MB-468: 45%; MDA-MB-231: 31%) expression. shBCA2-KD also resulted in an inverse increase In NMNAT1 qPCR-transcript (MDA-MB-468: 146%; MDA-MB-231: 150%) and promoter levels (MDA-MB-468: 409% and 459%; MDA-MB-231: 175% and 169%). shBCA2-KD was also associated with a decrease in the ratio of key enzyme associated with Warburg metabolism (LDHA/LDHB); high LDHA/LDHB is associated with Warburg-like metabolism, and low LDHA/LDHB is associated with decreased Warburg-like metabolism. Consistent with these findings, shBCA2 -KD resulted in decreased glucose consumption (MDA-MB-468: 70% and 76%; MDA-MB-231: 75% and 77%), decreased lactate production (MDA-MB-468: 69% and 72%; MDA-MB-231: 68% and 64%), and decrease cellular proliferation (MDA-MB-468: 54% and 49%; MDA-MB-231: 31% and 19%). All of these data are consistent with a role for BCA2 in maintaining Warburg-like metabolism in breast cancer cell lines, and that inhibition of BCA2 decreases Warburg-like metabolism and tumor cell proliferation. In order to investigate the reciprocal role of NMNAT1 in triple negative breast cancer cells NMNAT1 was knocked down. The shNMNAT1-KD induced BCA2 protein, indicating a potential reciprocal feedback loop. shNMNAT1 -KD resulted in a decrease in NMNAT1 protein (MDA-MB-468: 7070%, 6286 and 8427%; MDA-MB-231: 212%, 202% and 19%), qPCR-transcript (MDA-MB-468: 45%; MDA-MB-231: 31%) expression. shBCA2-KD also resulted in an inverse increase in NMNAT1 qPCR-transcript (MDA-MB-468: 146%; MDA-MB-231: 150%) and promoter levels (MDA-MB-468: 409% and 459%; MDA-MB-231: 175% and 169%). shBCA2-KD was also associated with a decrease in the ratio of key enzyme associated with Warburg metabolism (LDHA/LDHB); high LDHA/LDHB is associated with Warburg-like metabolism, and low LDHA/LDHB is associated with decreased Warburg-like metabolism. Consistent with these findings, shBCA2 -KD resulted in decreased glucose consumption (MDA-MB-468: 70% and 76%; MDA-MB-231: 75% and 77%), decreased lactate production (MDA-MB-468: 69% and 72%; MDA-MB-231: 68% and 64%), and decrease cellular proliferation (MDA-MB-468: 54% and 49%; MDA-MB-231: 31% and 19%). All of these data are consistent with the role of BCA2 in maintaining Warburg-like metabolism in breast cancer cell lines, and that inhibition of BCA2 decreases Warburg-like metabolism and tumor cell proliferation. In addition, shBCA2/NMNAT1-KD differentially regulates the expression of LDHA/LDHB. The ratio of LDHA/LDHB modulates lactate/pyruvate equilibria and is a recognized marker of the relative level of Warburg metabolism. High LDHA/LDHB is positively correlated with enhanced Warburg activity. NMNAT1, differentially regulates glucose-consumption and lactate-production. shBCA2-KD diminished LDHA/LDHB-ratio, lactate-production, and glucose-consumption. shNMNAT1-KD increased the LDHA/LDHB-ratio and lactate-production significantly, with and on glucose-consumption. shBCA2/NMNAT1-double-KD reversed the effects of shBCA2-KD, resulting in significant increases in lactate production with an increase of LDHA and LDHB relative to shNT and shBCA2, and increased LDHA/LDHB ratios relative to shBCA2-KD, as well as rescuing the effects of shBCA2 on glucose consumption relative to shNT and shBCA2. Collectively, these data suggest that BCA2 and NMNAT1 modulate breast-cancer-cell-metabolism. The role of energy status indicated by the role of glucose in the regulation of BCA2/NMNAT1 could provide clues to the significance of these findings and thereby direct future investigations

Hematopoietic stem cell transplant effectively rescues lymphocyte differentiation and function in DOCK8-deficient patients

Biallelic inactivating mutations in DOCK8 cause a combined immunodeficiency characterized by severe pathogen infections, eczema, allergies, malignancy, and impaired humoral responses. These clinical features result from functional defects in most lymphocyte lineages. Thus, DOCK8 plays a key role in immune cell function. Hematopoietic stem cell transplant (HSCT) is curative for DOCK8 deficiency. While previous reports have described clinical outcomes for DOCK8 deficiency following HSCT, the effect on lymphocyte reconstitution and function has not been investigated. Our study determined whether defects in lymphocyte differentiation and function in DOCK8-deficient patients were restored following HSCT. DOCK8-deficient T and B lymphocytes exhibited aberrant activation and effector function in vivo and in vitro. Frequencies of alpha beta T and MAIT cells were reduced, while gamma delta T cells were increased in DOCK8-deficient patients. HSCT improved abnormal lymphocyte function in DOCK8-deficient patients. Elevated total and allergen-specific IgE in DOCK8-deficient patients decreased over time following HSCT. Our results document the extensive catalog of cellular defects in DOCK8-deficient patients and the efficacy of HSCT in correcting these defects, concurrent with improvements in clinical phenotypes. Overall, our findings reveal mechanisms at a functional cellular level for improvements in clinical features of DOCK8 deficiency after HSCT, identify biomarkers that correlate with improved clinical outcomes, and inform the general dynamics of immune reconstitution in patients with monogenic immune disorders following HSCT

Initial presenting manifestations in 16,486 patients with inborn errors of immunity include infections and noninfectious manifestations

Background: Inborn errors of immunity (IEI) are rare diseases, which makes diagnosis a challenge. A better description of the initial presenting manifestations should improve awareness and avoid diagnostic delay. Although increased infection susceptibility is a well-known initial IEI manifestation, less is known about the frequency of other presenting manifestations. Objective: We sought to analyze age-related initial presenting manifestations of IEI including different IEI disease cohorts. Methods: We analyzed data on 16,486 patients of the European Society for Immunodeficiencies Registry. Patients with autoinflammatory diseases were excluded because of the limited number registered. Results: Overall, 68% of patients initially presented with infections only, 9% with immune dysregulation only, and 9% with a combination of both. Syndromic features were the presenting feature in 12%, 4% had laboratory abnormalities only, 1.5% were diagnosed because of family history only, and 0.8% presented with malignancy. Two-third of patients with IEI presented before the age of 6 years, but a quarter of patients developed initial symptoms only as adults. Immune dysregulation was most frequently recognized as an initial IEI manifestation between age 6 and 25 years, with male predominance until age 10 years, shifting to female predominance after age 40 years. Infections were most prevalent as a first manifestation in patients presenting after age 30 years. Conclusions: An exclusive focus on infection-centered warning signs would have missed around 25% of patients with IEI who initially present with other manifestations. (J Allergy Clin Immunol 2021;148:1332-41.