Adenylate kinase 1 overexpression increases locomotor activity in medaka fish

Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.

Virtual Screening of Adenylate Kinase 3 Inhibitors Employing Pharmacophoric Model, Molecular Docking, and Molecular Dynamics Simulations as Potential Therapeutic Target in Chronic Lymphocytic Leukemia

Adenylate kinase 3 (AK3) is an enzyme located in the mitochondrial matrix involved in purine homeostasis. This protein has been considered a potential therapeutic target in chronic lymphocytic leukemia (CLL), because the silencing of the AK3 gene has inhibited cell growth in CLL in vitro models. This study aimed to design potential AK3 inhibitors by applying molecular modeling techniques. Through the mapping of AK3 binding sites, essential interaction fields for pharmacophore design were identified. Online libraries were virtually screened by using a pharmacophore model, and 6891 compounds exhibited the functional groups for interaction with the target. These compounds underwent molecular docking simulations through Surflex and GOLD programs. After visual inspection, we selected 13 compounds for pharmacokinetic properties toxicology prediction via admetSAR and Protox web servers. Finally, six compounds were chosen for further analysis.

An Adenylate Kinase OsAK3 Involves Brassinosteroid Signaling and Grain Length in Rice (Oryza sativa L.)

Background Grain size is one of the major determinants of cereal crop yield. As a class of plant polyhydroxysteroids, brassinosteroids (BRs) play essential roles in the regulation of grain size and plant architecture in rice. In a previous research, we cloned qGL3/OsPPKL1 encoding a protein phosphatase with Kelch-like repeat domains, which negatively regulates BR signaling and grain length in rice. Results Here, we screened qGL3-interacting proteins (GIPs) via yeast two-hybrid assay and analyzed the phenotypes of the T-DNA insertion mutants of GIPs. Among these mutants, mutant osak3 presents shorter grain length and dwarfing phenotype. OsAK3 encodes an adenylate kinase, which regulates grain size by controlling cell expansion of rice spikelet glume. Overexpression of OsAK3 resulted in longer grain length. OsAK3 interacts with qGL3 in vivo and in vitro. Lamina inclination, coleoptile elongation and root inhibition experiments showed that the osak3 mutant was less sensitive to exogenous brassinolide (BL) treatment. The transcriptional level of OsAK3 was up-regulated under BL induction. In addition, RNA-Seq data indicate that OsAK3 is involved in a variety of biological processes that regulate BR signaling and grain development in rice. Conclusions Our study reveals a novel BR signaling component OsAK3 in the regulation of grain length, and provides novel clues for uncovering the potential functions of OsAK3 in rice growth and development.

Reticular Dysgenesis: A Rare Immunodeficiency in a Neonate With Cytopenias and Bacterial Sepsis

Severe combined immunodeficiency (SCID) consists of a group of disorders defined by abnormal B and T cell development that typically results in death within the first year of life if undiagnosed or untreated. Reticular dysgenesis (RD) is a rare but especially severe form of SCID that is caused by adenylate kinase 2 deficiency and is characterized not only by lymphopenia but also by profound neutropenia. RD predisposes patients to viral and fungal infections typical of SCID as well as serious bacterial infections atypical in the neonatal period in other SCID types. RD is also associated with sensorineural hearing loss not typically seen in other forms of SCID. Without rapid diagnosis and curative hematopoietic stem cell transplantation, RD is fatal within days to months due to overwhelming bacterial infection. The inclusion of the T cell receptor excision circle assay nationally in 2017 on the newborn screen has facilitated diagnosis of SCID in the neonatal period. This case reports on a male infant with RD who presented after preterm birth with severe cytopenias and a gastrointestinal anomaly and ultimately developed severe bacterial sepsis. Postmortem bone marrow evaluation and panel-based gene sequencing identifying 2 novel variants in the adenylate kinase 2 gene provided confirmation for a diagnosis of RD. This case emphasizes the importance of thorough diagnostic evaluation, including the newborn screen, in neonates and infants with persistent and unexplained cytopenias. Prompt hematology and/or immunology referral is advised for disease management and to facilitate hematopoietic stem cell transplantation to optimize long-term survival.

Adenylate Kinase 2 Is a Selective Multiple Myeloma Cell Dependency That Is Preferentially Essential in NSD2-Overexpressing Cells

Background: Multiple myeloma (MM) is the second most common hematologic malignancy and remains incurable. Advances in MM therapy have come about due to therapies that target vulnerabilities of the plasma cell such as high protein load (proteasome inhibitors; PIs), dependence on specific transcription factors such as IKZF1 and IKZF3 which are degraded by immunomodulatory drugs (IMiDs), the susceptibility of B cells to glucocorticoids and the presence of specific B cell markers that can serve as targets for monoclonal antibodies and CAR-T cells. Gene editing screens offer a way to identify novel MM therapeutic targets. Objectives: The molecular heterogeneity of MM imposes challenges to discovering generalized therapeutic targets. Therefore, identification of selective dependencies associated with a particular recurrent genetic lesion is a promising strategy to personalize therapy. Here, we aim to identify vulnerabilities linked to the chromosomal translocation t(4;14), a recurrent rearrangement in MM characterized by overexpression of the histone methyltransferase NSD2. Methods: Genome-wide CRISPR-based loss-of-function screens were performed in NSD2-high and low isogenic cells derived from the t(4;14) MM cell line KMS11 to define selective dependencies associated with NSD2 overexpression. High-confidence hits were corroborated by in vitro competitive growth assays where individual candidates are genetically knocked out or suppressed or chemically inhibited. Detailed investigation was performed for selected candidates using various molecular and biochemical assays to elucidate mechanisms by which these genes contribute to MM cell fitness. Results: A fitness screen in NSD2-high and low isogenic MM cells identified 1118 essential genes which are common between the cell pair. We further revealed 282 genes whose loss is more detrimental to cells overexpressing NSD2 and 139 genes that are preferentially essential when NSD2 levels are low. Pathway analysis of NSD2-high selectively essential genes indicated that these cells are more dependent on mitochondrial processes including oxidative phosphorylation. Although proteasomal degradation is essential for all MM cells, our screens indicated that NSD2-high cells are more dependent on the proteasome, which was validated by increased sensitivity to the PI bortezomib. One of the high-confidence selective NSD2-high hits was the mitochondrial adenine nucleotide regulator adenylate kinase 2 (AK2). Analyzing the dependence of hundreds of human cell lines on AK2 using the cancer dependency map portal (depmap.org/portal/), we found that AK2 is not a common essential gene. The top enriched linages with AK2 dependency included MM with notable representation of t(4;14)-positive cell lines. Analysis of the multiple myeloma research foundation (MMRF)-CoMMPass data demonstrated that MM patients with high NSD2 expression, despite poor prognosis, display enhanced overall survival when AK2 levels are low. In vitro competitive growth assays in NSD2-high and low MM cells confirmed the increased dependence of NSD2-overexpressing cells on AK2. In addition, NSD2-high MM cells displayed elevated sensitivity to AK2 inhibitors. Moreover, AK2 knockdown in t(4;14) MM cell lines increased sensitivity to the PI bortezomib. Mechanistically, we showed that AK2 disruption activates apoptotic unfolded protein response (UPR) signaling in MM cells. Metabolomic profiling in NSD2-high and low MM cells revealed accumulation of purine metabolites and reduction of pyrimidine metabolites upon NSD2 overexpression. Intriguingly, purine supplementation rescued MM cell depletion due to AK2 loss. These observations suggested that MM cells, especially those with NSD2 overexpression, are addicted to elevated purine levels and that lethality of MM cells upon AK2 loss is due to perturbed purine metabolism. How impaired purine metabolism activates UPR signaling is currently under investigation. Conclusions: Our work indicated that NSD2 overexpression resulting from chromosomal translocation t(4;14), despite its oncogenic role, generates metabolic dependencies in MM cells. Our findings further suggest that inhibition of AK2, a mitochondrial enzyme involved in purine metabolism, can induce UPR-mediated apoptosis in MM cells and could be used in combination with PI therapy to treat MM patients with t(4;14) translocations. Disclosures Licht: Epizyme: Research Funding.

Reticular Dysgenesis-Associated Adenylate Kinase 2 Deficiency Impairs Purine Metabolism and Ribosomal Biogenesis during Myelopoiesis

Reticular Dysgenesis (RD) is a particularly grave form of severe combined immunodeficiency (SCID), characterized by maturation arrest of both myeloid and lymphoid lineages paired with sensorineural hearing loss. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). AK2 catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. Using a CRISPR/Cas9 AK2 biallelic knock out model in human hematopoietic stem and progenitor cells (HSPCs), we have shown that AK2 -/- HSPCs mimic the neutrophil maturation defect in RD patients. Mitochondrial respiration is compromised in AK2 -/- HSPCs, which leads to a decreased NAD +/NADH ratio resulting in reductive stress. Metabolomics analysis by LC-MS/MS showed a significant accumulation of AMP, along with increased AMP/ADP and AMP/ATP ratios in AK2 -/- HSPCs, suggesting that purine metabolism is compromised by AK2 deficiency. Purine metabolism defects, such as deficiencies in adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP), have long been recognized as a cause of SCID. Furthermore, pharmacological interference with purine metabolism is a highly effective antiproliferative strategy in cancer therapy. In this study, we sought to investigate whether impaired purine metabolism contributes to the myelopoietic defect caused by AK2 deficiency. Results We explored how purine metabolism affects myelopoiesis by differentiating HSPCs in media containing no nucleosides (nucleoside-), mixed nucleosides (nucleoside+) or adenosine only (adenosine+). We observed no difference in proliferation or neutrophil maturation between nucleosides- and nucleoside+ media for both control and AK2 -/- HSPCs, suggesting that AK2 -/- HSPCs do not rely on exogenous nucleosides. Interestingly, control HSPCs cultured in adenosine+ media showed severe proliferation and neutrophil maturation defects that mimic AK2 deficiency, suggesting that purine imbalance is detrimental to myelopoiesis. Previous metabolomics analysis showed a significant accumulation of inosine monophosphate (IMP) in AK2 -/- HSPCs. Since IMP can be produced through AMP deamination by AMPD, we asked whether the IMP accumulation in AK2 -/- HSPCs is caused by converting excess AMP to IMP. An LC-MS/MS analysis showed that AMPD inhibitor (AMPDi) treatment significantly lowered IMP levels and increased AMP levels in AK2 -/- HSPCs, indicating that AMP deamination is a major source of IMP accumulation in AK2 -/- HSPCs. Furthermore, AMPDi treatment did not improve, but rather slightly aggravated neutrophil differentiation in AK2 -/- HSPCs, suggesting that the AK2 -/- neutrophil maturation defect is not caused by IMP accumulation. This raises the possibility that AK2 -/- HSPCs employ AMP deamination as a mechanism to curtail the toxicity of excess AMP. Since purine is a building block of RNA, and ribosomal RNA (rRNA) constitutes >85% of cellular RNA content, we asked whether rRNA synthesis is compromised by AK2 deficiency. Pyronin Y staining showed a significant decrease in rRNA content in AK2 -/- HSPCs. Nascent peptide synthesis rate was also decreased in AK2 -/- HSPCs, as quantified by OP-puromycin uptake. These findings are corroborated by RNA-seq analysis of AK2 -/- and control HSPCs, which showed that ribosomal subunits, ribosomal biogenesis and ribonucleoprotein complex assembly are among the top down-regulated pathways. The data suggest that defective purine metabolism in AK2 -/- HSPCs impairs ribosomal biogenesis and protein synthesis. Conclusion Our studies showed that purine imbalance in HSPCs impairs myeloid proliferation and neutrophil maturation. AK2 depletion in HSPCs leads to AMP accumulation and defective ribosomal biogenesis. AK2 -/- HSPCs convert excess AMP to IMP, possibly as a means to mitigate AMP toxicity. However, AMP deamination activities alone are not sufficient to lower AMP levels to those of control HSPCs. We are currently testing whether boosting 5'-nucleotidase activities (cNIA, cN1B and cNII) in AK2 -/- HSPCs can decrease AMP levels and rescue the neutrophil maturation defect. As purine metabolic defects are associated with diverse immune and non-immune abnormalities, further understanding of how purine metabolism governs differentiation of human HSPCs will enable us to develop novel therapeutic strategies for RD and other purine disorders. Disclosures Porteus: CRISPR Therapeutics: Current equity holder in publicly-traded company; Allogene Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Versant Ventures: Consultancy; Ziopharm: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Morrison: Garuda Therapeutics: Other: founder and SAB member ; Kojin Therapeutics: Other: SAB member ; Frequency Therapeutics: Other: SAB member ; Ona Terapeutics: Other: SAB member ; Protein Fluidics: Other: SAB member .

Adenylate Kinase 4—A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.

Adenylate kinase 1 overexpression increases locomotor activity in medaka fish

Maintenance of the energy balance is indispensable for cell survival and function. Adenylate kinase (Ak) is a ubiquitous enzyme highly conserved among many organisms. Ak plays an essential role in energy regulation by maintaining adenine nucleotide homeostasis in cells. However, its role at the whole organism level, especially in animal behavior, remains unclear. Here, we established a model using medaka fish (Oryzias latipes) to examine the function of Ak in environmental adaptation. Medaka overexpressing the major Ak isoform Ak1 exhibited increased locomotor activity compared to that of the wild type. Interestingly, this increase was temperature dependent. Our findings suggest that cellular energy balance can modulate locomotor activity.