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NASA NTRS1999

The Effect of Skeletal Unloading on Bone Formation: Role of IGF-I

The best documented change in bone during space flight is the near cessation of bone formation. Space flight leads to a decrease in osteoblast number and activity, likely the result of altered differentiation of osteoblast precursors. The net result of these space flight induced changes is weaker bone. To understand the mechanism for these changes poses a challenge. Space flight studies must overcome enormous technical problems, and are necessarily limited in size and frequency. Therefore, ground based models have been developed to evaluate the effects of skeletal unloading. The hindlimb elevation (tail suspension) model simulates space flight better than other models because it reproduces the fluid shifts seen in space travel, is reversible, and is well tolerated by the animals with minimal evidence of stress as indicated by continued weight gain and normal levels and circadian rhythms of corticosterone. This is the model we have used for our experiments. Skeletal unloading by the hindlimb elevation method simulates a number of features of space flight in that bone formation, mineralization, and maturation are inhibited, osteoblast number is decreased, serum and skeletal osteocalcin levels fall, the ash content of bone decreases, and bone strength diminishes. We and others have shown that when osteoblasts or osteoprogenitor cells from the bones of the unloaded limbs are cultured in vitro they proliferate and differentiate more slowly, suggesting that skeletal unloading causes a persistent change in cell function which can be assessed in vitro. In contrast to the unweighted bones of the hindlimbs, no significant change in bone mass or bone formation is observed in the humeri, mandible, and cervical vertebrae during hindlimb elevation. The lack of effect of hindlimb elevation on bones like the humeri, mandible, and cervical vertebrae which are not unloaded by this procedure suggests that local factors rather than systemic effects dominate the response of bone to skeletal unloading. We have focussed on the role of IGF- 1 as the local factor mediating the effects of skeletal unloading on bone formation. IGF-I is produced by bone cells and chondrocytes; these cells have receptors for IGF-I, and respond to IGF-I with an increase in proliferation and function (e.g. collagen, and glycosaminoglycan production, respectively). IGF-I production by bone is under hormonal control, principally by GH and PTH, and IGF-I is thought to mediate some if not all of the effects of GH and PTH on bone growth. Thus, systemic changes in hormones such as GH and PTH may still have effects which vary from bone to bone depending on the loading history.

Bikle, D. D.↗

DOE OSTI2022

Ablating Lgr5-expressing prostatic stromal cells activates the ERK-mediated mechanosensory signaling and disrupts prostate tissue homeostasis

The heterogeneity of the prostate stromal cells is widely appreciated but the functional implication remains incompletely understood. Using genetic lineage tracing and light-sheet imaging, we show that some fibroblast cells near the junction of the mouse proximal prostatic ducts and prostatic urethra highly express Lgr5. Genetic ablation of these anatomically restricted stromal cells, but not nonselective ablation of prostatic stromal cells, rapidly induces prostate epithelial turnover and dedifferentiation that are reversed following spontaneous restoration of the Lgr5 + stromal cells. RNA-Seq analysis implies that ablating the Lgr5 + stromal cells activates a mechanosensory response. We show that ablating the Lgr5 + stromal cells impairs the control of prostatic ductal outlet, increases prostate tissue stiffness, and activates the mitogen-activated protein kinase (MAPK). Suppressing MAPK overrides the increased epithelial proliferation. This study demonstrates that the Lgr5 + stromal cells regulate tissue homeostasis in a long-distance manner by maintaining anatomic integrity and implies that the cells near the transitional regions between organs likely control organ homeostasis by sustaining a balanced mechanoforce.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Peripheral blood hematology, plasma biochemistry, and the optimization of an in vitro immune-based assay in the brown watersnake ( Nerodia taxispilota )

Reptiles represent a phylogenetic lineage that provides a unique link between ectothermic anamniotes and endothermic amniotes. Compared to mammalian and avian species, our understanding of the reptilian immune system is greatly lacking. This gap in knowledge is largely due to an absence of established immune-based assays or specific reagents for these species. In the present study, brown watersnakes (Nerodia taxispilota) were live-captured in the wild, sexed, weighed, measured, bled via the caudal vein and released. At 24 hr post-collection, peripheral blood leukocytes were enriched and evaluated with an established mammalian in-vitro lymphocyte proliferation assay. Snake peripheral blood leukocyte enrichment yielded > 90% lymphocytes with viabilities averaging 81.5%. Baseline physiologic data for N. taxispilota, including hematology and total solids, leukocyte differentials, cell recovery and plasma biochemistry, were also collected. Cells cultured with Concanavalin A exhibited significantly increased proliferation at both 72 and 96 hr. Furthermore, these preliminary results show that enriched peripheral blood from wild-caught N. taxispilota provides a sufficient yield of leukocytes that can be cultured and functionally evaluated using a standard mammalian in-vitro immune-based assay.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

CRL4A DTL degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation

Genomic instability induced by DNA damage and improper DNA damage repair is one of the main causes of malignant transformation and tumorigenesis. DNA double strand breaks (DSBs) are the most detrimental form of DNA damage, and nonhomologous end-joining (NHEJ) mechanisms play dominant and priority roles in initiating DSB repair. A well-studied oncogene, the ubiquitin ligase Cullin 4A (CUL4A), is reported to be recruited to DSB sites in genomic DNA, but whether it regulates NHEJ mechanisms of DSB repair is unclear. Here, we discovered that the CUL4A-DTL ligase complex targeted the DNA-PKcs protein in the NHEJ repair pathway for nuclear degradation. Overexpression of either CUL4A or DTL reduced NHEJ repair efficiency and subsequently increased the accumulation of DSBs. Moreover, we demonstrated that overexpression of either CUL4A or DTL in normal cells led to genomic instability and malignant proliferation. Consistent with the in vitro findings, in human precancerous lesions, CUL4A expression gradually increased with increasing malignant tendency and was negatively correlated with DNA-PKcs and positively correlated with γ-H2AX expression. Collectively, this study provided strong evidence that the CUL4A-DTL axis increases genomic instability and enhances the subsequent malignant transformation of normal cells by inhibiting NHEJ repair. These results also suggested that CUL4A may be a prognostic marker of precancerous lesions and a potential therapeutic target in cancer.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1998

Effects of 2-deoxy-D-glucose administration on immune parameters in mice

Physical exercise and diet alterations have been shown to affect immune parameters. Similar effects are also induced by the administration of the non-metabolizable glucose analog, 2-deoxy-D-glucose (2-DG). The current study was designed to characterize the effects of glucoprivation induced by 2-DG administration on leukocyte subset distribution and function. BDF1 mice (n = 8 per group) were injected intraperitoneally one or three times with 0, 500, 750, 1000 or 1500 mg/kg of 2-DG. Two hours after the last injection of 2-DG, immunological parameters were analyzed. A dose-dependent increase in plasma glucose concentrations of mice injected once with up to 1500 mg/kg of 2-DG was observed (p < 0.001). After either one or three injections of up to 1500 mg/kg of 2-DG, corticosterone levels, leukocyte counts in the spleen, and CD3+ cells in the thymus increased. In vitro proliferation of partially purified lymphocytes from the spleen in the presence of both concanavalin-A and lipopolysaccharide decreased in a dose dependent manner (p < 0.05). In addition, after three injections, the proportion of both thymocytes and splenocytes bearing alphabeta-TCR increased as the concentration of 2-DG increased (p < 0.01). These results demonstrate that 2-DG administration induced dose-dependent changes in both thymus and spleen cell distribution and function.

NASA Discipline Regulatory Physiology↗

DOE OSTI2021

Structural analysis of the catalytic domain of Artemis endonuclease/SNM1C reveals distinct structural features

The endonuclease Artemis is responsible for opening DNA hairpins during V(D)J recombination and for processing a subset of pathological DNA double-strand breaks. Artemis is an attractive target for the development of therapeutics to manage various B cell and T cell tumors, because failure to open DNA hairpins and accumulation of chromosomal breaks may reduce the proliferation and viability of pre-T and pre-B cell derivatives. However, structure-based drug discovery of specific Artemis inhibitors has been hampered by a lack of crystal structures. Here, we report the structure of the catalytic domain of recombinant human Artemis. The catalytic domain displayed a polypeptide fold similar overall to those of other members in the DNA cross-link repair gene SNM1 family and in mRNA 3'-end-processing endonuclease CPSF-73, containing metallo-β-lactamase and β-CASP domains and a cluster of conserved histidine and aspartate residues capable of binding two metal atoms in the catalytic site. As in SNM1A, only one zinc ion was located in the Artemis active site. However, Artemis displayed several unique features. Unlike in other members of this enzyme class, a second zinc ion was present in the β-CASP domain that leads to structural reorientation of the putative DNA-binding surface and extends the substrate-binding pocket to a new pocket, pocket III. Moreover, the substrate-binding surface exhibited a dominant and extensive positive charge distribution compared with that in the structures of SNM1A and SNM1B, presumably because of the structurally distinct DNA substrate of Artemis. The structural features identified here may provide opportunities for designing selective Artemis inhibitors.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRSDate not supplied

A Novel Bone Marrow Single Cell Atlas of Mechanotransduction in Microgravity, Normal Gravity, Exercise, and Hindlimb Unloading for WT and CDKN1A-Null Regenerative Mice

Mechanical loading of adult stem cell progenitors is a key factor in modulating their proliferation, differentiation, and tissue regenerative potential, with loading generally promoting tissue formation and unloading mediating tissue loss. To address the role of mechanotransduction in maintaining stem cell-based tissue regenerative health, we generated a single cell transcriptomic atlas mapping responses of femur bone marrow mesenchymal and hematopoietic lineages to a range of altered mechanical loading conditions, both in WT B1629SF2/J mice as well as the p21/CDKN1A-null regenerative mice. We selected the femur marrow compartment as a model because of the diversity and high numbers of stem cell progenitor stages present, and because it undergoes static and cyclic hydrostatic pressure loading associated with weight-bearing and ambulation. Our study included normally loaded mice at 1g, unloading in microgravity during spaceflight as well as tail suspension hindlimb unloading, and voluntary running wheel exercise. Basal, one-year natural aging, and habitat controls were also conducted. Overall, the atlas encompasses 18 different experimental conditions with N=3 mice per condition and includes more than 500,000 single cell expressomes. Key specific findings include: increased mature reticulocyte populations in aging and unloading mice compared to active mice; greater hematopoietic and mesenchymal differentiating progenitors cluster identification in CDKN1A-null samples, especially the exercise model; distinct pseudotime cell trajectory shifts in the hematopoietic lineage for spaceflight and unloaded mice; and shifts in monocytic cell populations toward osteoclastic bone degenerative lineages in unloading and spaceflight samples. Overall mechanical loading shows increased marrow progenitor population differentiation while unloading is associated with increased CDKN1A expression and maintenance of marrow population stemness. Deletion of CDKN1A appears to remove a negative check on progenitor lineages commitment to differentiation. Finally, the Bone Marrow Mechanotransduction Single Cell Atlas will serve as a reference tool for studying marrow regenerative responses across a range of mechanical environments as they relate to CDKN1A status.

single-cell↗

DOE OSTI2021

Competing endogenous RNA network associated with oxygen-induced retinopathy: Expression of the network and identification of the MALAT1/miR-124–3p/EGR1 regulatory axis

Retinopathy of prematurity (ROP) is a severe retinal dysfunction in prematurely born babies. The relationship between non-coding RNAs and retinopathy of prematurity (ROP) remain unclear. Microarray analysis of lncRNAs, miRNAs, and mRNAs was conducted in a mouse model of ROP. A competing endogenous RNA (ceRNA) network was constructed. The relationship among MALAT1, miR-124–3p, and Early growth response protein 1 (EGR1) was assessed in hypoxia-induced primary human umbilical vein endothelial cells (HUVECs) and ROP mouse model. In the study, we found 2252 lncRNAs, 1239 mRNAs, and 36 miRNAs were differentially regulated. ceRNA network consisting of 21 lncRNAs, 10 miRNAs, and 19 mRNAs was established. Of the most down-regulated miRNAs, miR-124–3p was selected for additional study. miR-124–3p ceased the migration and proliferation of primary HUVECs in hypoxic conditions, and directly suppressed EGR1. Additionally, MALAT1 directly sponged miR-124–3p. Knockdown of MALAT1 decreased EGR1 expression and inhibited the migration and proliferation of primary HUVECs in hypoxia. Furthermore, these changes were rescued by depletion of miR-124–3p. In vivo, intravitreal injection of miR-124–3p, shMALAT1 decreased EGR1 expression and markedly suppressed retinal neovascularization in OIR models. Intravitreal injection of shMALAT1 and miR-124–3p antagomir at the same time can promote retinal neovascularization, which reversed the suppression of retinal neovascularization functioned by shMALAT1. In conclusion, the expression profiles of lncRNAs and miRNAs and the ceRNA network in a mouse model of ROP may be indicative of the underlying mechanisms of retinal angiogenesis and neural activity. The MALAT1/miR-124–3p/EGR1 regulatory axis is partly responsible for retinal neovascularization, which may provide a novel theoretical basis for the pathogenesis of ROP.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2018

CDKN1a/p21 Plays a Critical Role in Suppressing Stem Cell Regenerative Potential During Aging

Unloading during spaceflight is known to adversely affect mammalian physiology. Mechanical stimulation is required for repair and regeneration by stem cell lineages to maintain tissue health and mass. CDKN1a/p21 functions as a potent cell cycle arrest molecule and we previously found that CDKN1a/p21 was overexpressed in mouse bone during 15-days of spaceflight on STS-131 and localized to osteoprecursor cells in the femur. Therefore, we hypothesized that altered expression of CDKN1a/p21 leads to an arrest of bone formation during spaceflight in response to altered load. To study CDKN1a/p21 and its role in stem cell-based tissue regeneration, we use a CDKN1a/p21 knockout (KO) mouse to investigate the impact on bone structure, osteoprogenitor proliferation, and mineralized nodule formation. We have shown that bone marrow stem cells isolated from juvenile (11-week-old) and skeletally mature (18-week-old) KO mice have an increased bone formation potential as evidenced by increased proliferation and mineralization rates. In addition, we have shown that juvenile KO mice display significantly increased bone volume fraction (BV/TV) relative to wildtype (WT) mice, but not in skeletally mature KO mice, indicating increased resorption and bone turnover in adult mice. To more closely examine age differences in the KO mouse, we will study a wider spectrum of mice ranging from 4 weeks to 12 months in age. To do this, we will analyze differences in bone morphometric parameters using MicroCT and osteoblastogenesis assays. The pelvis, femur, and tibia are key in distributing weight and we expect to see altered remodeling and stem cell potential with age. In combination with histomorphometry, these results will help elucidate the complex mechanisms underlying bone tissue maintenance and stem cell regeneration.

stem cells↗

NASA NTRS1989

The effects of simulated hypogravity on murine bone marrow cells

Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

Lawless, Desales↗

NASA NTRS1993

The identification of a naturally occurring cell surface growth inhibitor related to a previously described bovine sialoglycopeptide

A 66-kDa sialoglycoprotein has been identified as the parental membrane molecule of an earlier described sialoglycopeptide (SGP), an 18-kDa molecule released by protease treatment of intact bovine cerebral cortex cells that was shown to be a potent inhibitor of cellular proliferation. The 66-kDa parental sialoglycoprotein (p-SGP) was purified approximately 2,400-fold, to apparent homogeneity, from bovine cerebral cortex cell membranes by its release during incubation with 3 M NaCl, preparative isoelectric focusing and lectin affinity chromatography. Although a membrane-associated molecule, the p-SGP appeared to be tightly bound to the cell membrane, since it was not released during incubations in the absence of 3 M NaCl. Incubation of the membrane preparations with 3 M urea proved to be too harsh, and the antigenicity required to follow the purification of the p-SGP was abolished. Analyses by SDS-PAGE, under reducing and nonreducing conditions, suggested that the p-SGP membrane component was a single polypeptide without subunit structure. The p-SGP was shown to be structurally related to the SGP fragment by immunoblots with IgG raised to the SGP inhibitor, and functionally related to the SGP by its ability to inhibit Swiss 3T3 proliferation at concentrations strikingly similar to that previous measured with the SGP fragment.

Non-NASA Center↗

DOE OSTI2020

Beyond regulations at DNA levels: A review of epigenetic therapeutics targeting cancer stem cells

Abstract In the past few years, the paramount role of cancer stem cells (CSCs), in terms of cancer initiation, proliferation, metastasis, invasion and chemoresistance, has been revealed by accumulating studies. However, this level of cellular plasticity cannot be entirely explained by genetic mutations. Research on epigenetic modifications as a complementary explanation for the properties of CSCs has been increasing over the past several years. Notably, therapeutic strategies are currently being developed in an effort to reverse aberrant epigenetic alterations using specific chemical inhibitors. In this review, we summarize the current understanding of CSCs and their role in cancer progression, and provide an overview of epigenetic alterations seen in CSCs. Importantly, we focus on primary cancer therapies that target the epigenetic modification of CSCs by the use of specific chemical inhibitors, such as histone deacetylase (HDAC) inhibitors, DNA methyltransferase (DNMT) inhibitors and microRNA‐based (miRNA‐based) therapeutics.

Zhang, Shunhao↗

DOE OSTI2024

Sustainable production of extracellular polymeric substances and iron or copper complex from glutinous rice processing wastewater

Essential trace minerals play vital roles in maintaining human and animal health. However, an overdose of the existing inorganic trace minerals is prone to induce detrimental effects that outweigh positive benefits. In this study, an extracellular polymeric substances (EPS)-producing bacterium, identified as Bacillus licheniformis CCTCC M2020298, was isolated from marine using glutinous rice processing wastewater as enrichment medium. The EPS yield of Bacillus licheniformis CCTCC M2020298 could reach 8.62 g/L by using glutinous rice-processing wastewater containing medium. Furthermore, the potential of the EPS as a carrier for synthesizing EPS-iron (Fe) and EPS-copper (Cu) complex was explored. The results showed that the optimum condition for the synthesis EPS-Fe were the reaction temperature 70°C, pH 8.5–9.0 and mass ratio of EPS to trisodium citrate 2:1. The iron content of EPS-Fe reached 77.4 mg/g. Under the same condition, the copper content of EPS-Cu reached 90.7 mg/g. The elemental composition, functional groups and valence state of the mineral elements of EPS-Fe and EPS-Cu were well characterized. The EPS-Fe and EPS-Cu exhibited antioxidant activity in scavenging ·OH, DPPH and ·O 2− free radicals, thereby leading to reduced oxidative stress and apoptosis levels in human colonic epithelial cells in vitro . They also inhibited the proliferation of mouse hepatocellular carcinoma H22 and the growth of intestinal pathogens in vitro . This study provided an effective avenue for EPS production from glutinous rice processing wastewater and proved the potential of EPS-Fe and EPS-Cu complexes as a new-type comprehensive essential trace mineral supplement.

Chen, Zhen↗

NASA NTRS1981

Effect of weightlessness on lymphocyte proliferation

An experiment to study the effect of weightlessness on lymphocyte proliferation to detect possible alteration of the cells responsible for the immune response during long-duration space flights is described. Human lymphocytes in culture medium will be delivered shortly before launch in an incubator which will be kept at 37C. Mitogen will be added to the culture. A control without mitogen will be run in parallel. After 70 hours of incubation, radioactive thymidine will be added. After two hours, cellular activity will be stopped by fixation and incubator power switched off. Later, the amount of incorporated thymidine will be determined and the cell morphology and the distribution of cell organelles will be investigated.

Cogoli, A.↗

DOE OSTI2021

Combination therapy with lenvatinib and radiation significantly inhibits thyroid cancer growth by uptake of tyrosine kinase inhibitor

Highlights: • Lenvatinib combined with radiation significantly inhibits thyroid cancer growth. • Combined therapy induced apoptosis and G2/M phase cell cycle arrest. • Irradiation increased cellular uptake of lenvatinib into cancer cells. • Combined therapy suggests a new therapeutic strategy for advanced thyroid cancer. Although surgical treatment cures >90% of differentiated thyroid cancer (DTC) patients, the remaining patients, including advanced DTC cases, have poor clinical outcomes. These patients with inoperable disease have only two choices of radioactive iodine therapy and tyrosine kinase inhibitors such as lenvatinib, which have a high incidence of treatment-related adverse events and can only prolong progression free survival by approximately 5–15 months. In this study, we investigated the antitumor effects of combination therapy with lenvatinib and radiation (CTLR) for DTC. CTLR synergistically inhibited cell replication and colony formation in vitro and tumor growth in nude mice without apparent toxicities and suppressed the expression of proliferation marker (Ki-67). CTLR also induced apoptosis and G2/M phase cell cycle arrest. Moreover, quantitative analysis of the intracellular uptake of lenvatinib using liquid chromatography and mass spectrometry demonstrated that intracellular uptake of lenvatinib was significantly increased 48 h following irradiation. These data suggest that increased membrane permeability caused by irradiation increases the intracellular concentration of levatinib, contributing to the synergistic effect. This mechanism-based potential of combination therapy suggests a powerful new therapeutic strategy for advanced thyroid cancer with fewer side effects and might be a milestone for developing a regimen in clinical practice.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

DOE OSTI2021

Extracellular IL-37 promotes osteogenic and odontogenic differentiation of human dental pulp stem cells via autophagy

The osteogenic and odontogenic differentiation of dental pulp stem cells (DPSCs) contribute to restoration and regeneration of dental tissue. Previous study indicated that interleukin-37 (IL-37) was an anti-inflammatory factor that affected other pro-inflammatory signals. The aim of this study was to explore the effects of IL-37 on the differentiation of DPSCs. DPSCs were cultured in growth medium with different concentrations of IL-37. We selected the optimal concentration for the following experiments by alkaline phosphatase (ALP) activity analysis, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot. Cell counting kit assay (CCK-8) and 5-Ethynyl-2-Deoxyuridine (EdU) assay were conducted to assess the effects of IL-37 on the proliferation of DPSCs. ALP activity assay and staining, alizarin red S (ARS) staining, qRT‐PCR, Western blot as well as immunofluorescence staining were conducted to assess differentiation ability of DPSCs. Western blot, immunofluorescence staining and transmission electron microscopy (TEM) were utilized to examine cell autophagy. Results showed that IL-37 enhanced the osteogenic and odontogenic differentiation ability of DPSCs with no significant influence on the proliferation of DPSCs. Autophagy in DPSCs was activated by IL-37. Activation of autophagy enhanced osteogenesis and odontogenesis of DPSCs, whereas inhibition of autophagy suppressed DPSCs osteogenic and odontogenic differentiation. In conclusion, IL-37 increased osteogenic and odontogenic differentiation via autophagy.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Blocking C-terminal processing of KRAS4b via a direct covalent attack on the CaaX-box cysteine

RAS is the most frequently mutated oncogene in cancer. RAS proteins show high sequence similarities in their G-domains but are significantly different in their C-terminal hypervariable regions (HVR). These regions interact with the cell membrane via lipid anchors that result from posttranslational modifications (PTM) of cysteine residues. KRAS4b is unique as it has only one cysteine that undergoes PTM, C185. Small molecule covalent modification of C185 would block any form of prenylation and subsequently inhibit attachment of KRAS4b to the cell membrane, blocking its biological activity. We translated this concept to the discovery and development of disulfide tethering screen hits into irreversible covalent modifiers of C185. These compounds inhibited proliferation of KRAS4b-driven mouse embryonic fibroblasts, but not cells driven by N-myristoylated KRAS4b that harbor a C185S mutation and are not dependent on C185 prenylation. Top–down proteomics was used to confirm target engagement in cells. These compounds bind in a pocket formed when the HVR folds back between helix 3 and 4 in the G-domain (HVR-α3-α4). This interaction can happen in the absence of small molecules as predicted by molecular dynamics simulations and is stabilized in the presence of C185 binders as confirmed by small-angle X-ray scattering and solution NMR. NOESY-HSQC, an NMR approach that measures internuclear distances of 6 Å or less, and structure analysis identified the critical residues and interactions that define the HVR-α3-α4 pocket. Further development of compounds that bind to this pocket could be the basis of a new approach to targeting KRAS cancers.

C185↗

DOE OSTI2021

Disrupted NOS2 metabolism drives myoblast response to wasting-associated cytokines

Highlights: • Amino acid metabolism is impacted in myoblasts treated with cancer cell conditioned media. • Inflammatory cytokines induce nitric oxide synthase 2 in myoblasts. • Elevated nitric oxide synthase 2 activity impairs myoblast proliferation and differentiation. Skeletal muscle wasting drives negative clinical outcomes and is associated with a spectrum of pathologies including cancer. Cancer cachexia is a multi-factorial syndrome that encompasses skeletal muscle wasting and remains understudied, despite being a frequent and serious co-morbidity. Deviation from the homeostatic balance between breakdown and regeneration leads to muscle wasting disorders, such as cancer cachexia. Muscle stem cells (MuSCs) are the cellular compartment responsible for muscle regeneration, which makes MuSCs an intriguing target in the context of wasting muscle. Molecular studies investigating MuSCs and skeletal muscle wasting largely focus on transcriptional changes, but our group and others propose that metabolic changes are another layer of cellular regulation underlying MuSC dysfunction in cancer cachexia. In the present study, we combined gene expression and non-targeted metabolomic profiling of myoblasts exposed to wasting conditions (cancer cell conditioned media, CC-CM) to derive a more complete picture of the myoblast response to wasting factors. After mapping these features to annotated pathways, we found that more than half of the mapped pathways were amino acid-related, linking global amino acid metabolic disruption to conditioned media-induced myoblast defects. Notably, arginine metabolism was a highly enriched pathway in combined metabolomic and transcriptomic data. Arginine catabolism generates nitric oxide (NO), an important signaling molecule known to have negative effects on mature muscle. We hypothesize that tumor-derived disruptions in Nitric Oxide Synthase (NOS)2-regulated arginine catabolism impair differentiation of MuSCs. The work presented here further investigates the effect of NOS2 overactivity on myoblast proliferation and differentiation. We show that NOS2 inhibition is sufficient to rescue wasting phenotypes associated with inflammatory cytokines. Ultimately, this work provides new insights into MuSC biology and opens up potential therapeutic avenues for addressing disrupted MuSC dynamics in cancer cachexia.

60 APPLIED LIFE SCIENCES↗