Age and Comorbidities Predict COVID-19 Outcome, Regardless of Innate Immune Response Severity: A Single Institutional Cohort Study.

The COVID-19 pandemic has claimed over eight hundred thousand lives in the United States alone, with older individuals and those with comorbidities being at higher risk of severe disease and death. Although severe acute respiratory syndrome coronavirus 2-induced hyperinflammation is one of the mechanisms underlying the high mortality, the association between age and innate immune responses in COVID-19 mortality remains unclear. DESIGN: Flow cytometry of fresh blood and multiplexed inflammatory chemokine measurements of sera were performed on samples collected longitudinally from our cohort. Aggregate impact of comorbid conditions was calculated with the Charlson Comorbidity Index, and association between patient factors and outcomes was calculated via Cox proportional hazard analysis and repeated measures analysis of variance. SETTING: A cohort of severely ill COVID-19 patients requiring ICU admission was followed prospectively. PATIENTS: In total, 67 patients (46 male, age 59 ± 14 yr) were included in the study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Mortality in our cohort was 41.8%. We identified older age (hazard ratio [HR] 1.09 [95% CI 1.07-1.11]; p = 0.001), higher comorbidity index (HR 1.24 [95% CI 1.14-1.35]; p = 0.039), and hyponatremia (HR 0.90 [95% CI 0.82-0.99]; p = 0.026) to each independently increase risk for death in COVID-19. We also found that neutrophilia (R = 0.2; p = 0.017), chemokine C-C motif ligand (CCL) 2 (R = 0.3; p = 0.043), and C-X-C motif chemokine ligand 9 (CXCL9) (R = 0.3; p = 0.050) were weakly but significantly correlated with mortality. Older age was associated with lower monocyte (R = -0.2; p = 0.006) and cluster of differentiation (CD) 16+ cell counts (R = -0.2; p = 0.002) and increased CCL11 concentration (R = 0.3; p = 0.050). Similarly, younger patients (< 65 yr) demonstrated a rise in CD4 (b-coefficient = 0.02; p = 0.036) and CD8 (0.01; p = 0.001) counts, as well as CCL20 (b-coefficient = 6.8; p = 0.036) during their ICU stay. This CD8 count rise was also associated with survival (b-coefficient = 0.01; p = 0.023). CONCLUSIONS: Age, comorbidities, and hyponatremia independently predict mortality in severe COVID-19. Neutrophilia and higher CCL2 and CXCL9 levels are also associated with higher mortality, while independent of age.

Mixed-surface polyamidoamine polymer variants retain nucleic acid-scavenger ability with reduced toxicity.

Nucleic acid-binding polymers can have anti-inflammatory properties and beneficial effects in animal models of infection, trauma, cancer, and autoimmunity. PAMAM G3, a polyamidoamine dendrimer, is fully cationic bearing 32 protonable surface amines. However, while PAMAM G3 treatment leads to improved outcomes for mice infected with influenza, at risk of cancer metastasis, or genetically prone to lupus, its administration can lead to serosal inflammation and elevation of biomarkers of liver and kidney damage. Variants with reduced density of cationic charge through the interspersal of hydroxyl groups were evaluated as potentially better-tolerated alternatives. Notably, the variant PAMAM G3 50:50, similar in size as PAMAM G3 but with half the charge, was not toxic in cell culture, less associated with weight loss or serosal inflammation after parenteral administration, and remained effective in reducing glomerulonephritis in lupus-prone mice. Identification of such modified scavengers should facilitate their development as safe and effective anti-inflammatory agents.

Suppression of Fibrinolysis and Hypercoagulability, Severity of Hypoxemia, and Mortality in COVID-19 Patients: A Retrospective Cohort Study.

BACKGROUND: COVID-19 causes hypercoagulability, but the association between coagulopathy and hypoxemia in critically ill patients has not been thoroughly explored. This study hypothesized that severity of coagulopathy would be associated with acute respiratory distress syndrome severity, major thrombotic events, and mortality in patients requiring intensive care unit-level care. METHODS: Viscoelastic testing by rotational thromboelastometry and coagulation factor biomarker analyses were performed in this prospective observational cohort study of critically ill COVID-19 patients from April 2020 to October 2020. Statistical analyses were performed to identify significant coagulopathic biomarkers such as fibrinolysis-inhibiting plasminogen activator inhibitor 1 and their associations with clinical outcomes such as mortality, extracorporeal membrane oxygenation requirement, occurrence of major thrombotic events, and severity of hypoxemia (arterial partial pressure of oxygen/fraction of inspired oxygen categorized into mild, moderate, and severe per the Berlin criteria). RESULTS: In total, 53 of 55 (96%) of the cohort required mechanical ventilation and 9 of 55 (16%) required extracorporeal membrane oxygenation. Extracorporeal membrane oxygenation-naïve patients demonstrated lysis indices at 30 min indicative of fibrinolytic suppression on rotational thromboelastometry. Survivors demonstrated fewer procoagulate acute phase reactants, such as microparticle-bound tissue factor levels (odds ratio, 0.14 [0.02, 0.99]; P = 0.049). Those who did not experience significant bleeding events had smaller changes in ADAMTS13 levels compared to those who did (odds ratio, 0.05 [0, 0.7]; P = 0.026). Elevations in plasminogen activator inhibitor 1 (odds ratio, 1.95 [1.21, 3.14]; P = 0.006), d-dimer (odds ratio, 3.52 [0.99, 12.48]; P = 0.05), and factor VIII (no clot, 1.15 ± 0.28 vs. clot, 1.42 ± 0.31; P = 0.003) were also demonstrated in extracorporeal membrane oxygenation-naïve patients who experienced major thrombotic events. Plasminogen activator inhibitor 1 levels were significantly elevated during periods of severe compared to mild and moderate acute respiratory distress syndrome (severe, 44.2 ± 14.9 ng/ml vs. mild, 31.8 ± 14.7 ng/ml and moderate, 33.1 ± 15.9 ng/ml; P = 0.029 and 0.039, respectively). CONCLUSIONS: Increased inflammatory and procoagulant markers such as plasminogen activator inhibitor 1, microparticle-bound tissue factor, and von Willebrand factor levels are associated with severe hypoxemia and major thrombotic events, implicating fibrinolytic suppression in the microcirculatory system and subsequent micro- and macrovascular thrombosis in severe COVID-19.

Targeting DAMPs with nucleic acid scavengers to treat lupus.

Systemic lupus erythematosus (SLE) is a chronic and often progressive autoimmune disorder marked clinically by a variable constellation of symptoms including fatigue, rash, joint pains, and kidney damage. The lungs, heart, gastrointestinal system, and brain can also be impacted, and individuals with lupus are at higher risk for atherosclerosis, thrombosis, thyroid disease, and other disorders associated with chronic inflammation . Autoimmune diseases are marked by erroneous immune responses in which the target of the immune response is a "self"-antigen, or autoantigen, driven by the development of antigen-specific B or T cells that have overcome the normal systems of self-tolerance built into the development of B and T cells. SLE is specifically characterized by the production of autoantibodies against nucleic acids and their binding proteins, including anti-double stranded DNA, anti-Smith (an RNA binding protein), and many others . These antibodies bind their nuclear-derived antigens to form immune complexes that cause injury and scarring through direct deposition in tissues and activation of innate immune cells . In over 50% of SLE patients, immune complex aggregation in the kidneys drives intrarenal inflammation and injury and leads to lupus nephritis, a progressive destruction of the glomeruli that is one of the most common causes of lupus-related death . To counter this pathology increasing attention has turned to developing approaches to reduce the development and continued generation of such autoantibodies. In particular, the molecular and cellular events that lead to long term, continuous activation of such autoimmune responses have become the focus of new therapeutic strategies to limit renal and other pathologies in lupus patients. The focus of this review is to consider how the innate immune system is involved in the development and progression of lupus nephritis and how a novel approach to inhibit innate immune activation by neutralizing the activators of this response, called Damage Associated Molecular Patterns, may represent a promising approach to treat this and other autoimmune disorders.

DAMPs/PAMPs induce monocytic TLR activation and tolerance in COVID-19 patients; nucleic acid binding scavengers can counteract such TLR agonists.

Millions of COVID-19 patients have succumbed to respiratory and systemic inflammation. Hyperstimulation of toll-like receptor (TLR) signaling is a key driver of immunopathology following infection by viruses. We found that severely ill COVID-19 patients in the Intensive Care Unit (ICU) display hallmarks of such hyper-stimulation with abundant agonists of nucleic acid-sensing TLRs present in their blood and lungs. These nucleic acid-containing Damage and Pathogen Associated Molecular Patterns (DAMPs/PAMPs) can be depleted using nucleic acid-binding microfibers to limit the patient samples' ability to hyperactivate such innate immune receptors. Single-cell RNA-sequencing revealed that CD16+ monocytes from deceased but not recovered ICU patients exhibit a TLR-tolerant phenotype and a deficient anti-viral response after ex vivo TLR stimulation. Plasma proteomics confirmed such myeloid hyperactivation and revealed DAMP/PAMP carrier consumption in deceased patients. Treatment of these COVID-19 patient samples with MnO nanoparticles effectively neutralizes TLR activation by the abundant nucleic acid-containing DAMPs/PAMPs present in their lungs and blood. Finally, MnO nanoscavenger treatment limits the ability of DAMPs/PAMPs to induce TLR tolerance in monocytes. Thus, treatment with microfiber- or nanoparticle-based DAMP/PAMP scavengers may prove useful for limiting SARS-CoV-2 induced hyperinflammation, preventing monocytic TLR tolerance, and improving outcomes in severely ill COVID-19 patients.

Generation of an anticoagulant aptamer that targets factor V/Va and disrupts the FVa-membrane interaction in normal and COVID-19 patient samples.

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.

PEG-Like Brush Polymer Conjugate of RNA Aptamer That Shows Reversible Anticoagulant Activity and Minimal Immune Response.

Ribonucleic acid (RNA) therapeutics are an emerging class of drugs. RNA aptamers are of significant therapeutic and clinical interest because their activity can be easily reversed in vivo-a useful feature that is difficult to achieve using other therapeutic modalities. Despite their therapeutic promise, RNA aptamers are limited by their poor blood circulation. The attachment of polyethylene glycol (PEG) to RNA aptamers addresses this limitation. However, an RNA aptamer-PEG conjugate that is a reversible anticoagulant fails in a clinical trial due to the reactivity of the conjugate with pre-existing PEG antibodies and has cast a pall over PEGylation of aptamers and other biologics, despite its long history of utility in drug delivery. Here, PEG antibody-reactivity of this RNA aptamer is eliminated by conjugating it to a next-generation PEG-like brush polymer-poly[(oligoethylene glycol) methyl ether methacrylate)] (POEGMA). The conjugate retained the drug's therapeutic action and the ability to be easily reversed. Importantly, this conjugate does not bind pre-existing PEG antibodies that are prevalent in humans and does not induce a humoral immune response against the polymer itself in mice. These findings suggest a path to rescuing the PEGylation of RNA therapeutics and vaccines from the deleterious side-effects of PEG.

Combining Heparin and a FX/Xa Aptamer to Reduce Thrombin Generation in Cardiopulmonary Bypass and COVID-19.

Known limitations of unfractionated heparin (UFH) have encouraged the evaluation of anticoagulant aptamers as alternatives to UFH in highly procoagulant settings such as cardiopulmonary bypass (CPB). Despite progress, these efforts have not been totally successful. We take a different approach and explore whether properties of an anticoagulant aptamer can complement UFH, rather than replace it, to address shortcomings with UFH use. Combining RNA aptamer 11F7t, which targets factor X/Xa, with UFH (or low molecular weight heparin) yields a significantly enhanced anticoagulant cocktail effective in normal and COVID-19 patient blood. This aptamer-UFH combination (1) supports continuous circulation of human blood through an ex vivo membrane oxygenation circuit, as is required for patients undergoing CPB and COVID-19 patients requiring extracorporeal membrane oxygenation, (2) allows for a reduced level of UFH to be employed, (3) more effectively limits thrombin generation compared to UFH alone, and (4) is rapidly reversed by the administration of protamine sulfate, the standard treatment for reversing UFH clinically following CPB. Thus, the combination of factor X/Xa aptamer and UFH has significantly improved anticoagulant properties compared to UFH alone and underscores the potential of RNA aptamers to improve medical management of acute care patients requiring potent yet rapidly reversible anticoagulation.

ß-Cyclodextrin-containing polymer treatment of cutaneous lupus and influenza improves outcomes.

Nucleic acid (NA)-containing damage- and pathogen-associated molecular patterns (DAMPs and PAMPs, respectively) are implicated in numerous pathological conditions from infectious diseases to autoimmune disorders. Nucleic acid-binding polymers, including polyamidoamine (PAMAM) dendrimers, have demonstrated anti-inflammatory properties when administered to neutralize DAMPs/PAMPs. The PAMAM G3 variant has been shown to have beneficial effects in a cutaneous lupus erythematosus (CLE) murine model and improve survival of mice challenged with influenza. Unfortunately, the narrow therapeutic window of cationic PAMAM dendrimers makes their clinical development challenging. An alternative nucleic acid-binding polymer that has been evaluated in humans is a linear ß-cyclodextrin-containing polymer (CDP). CDP's characteristics prompted us to evaluate its anti-inflammatory potential in CLE autoimmune and influenza infectious disease mouse models. We report that CDP effectively inhibits NA-containing DAMP-mediated activation of Toll-like receptors (TLRs) in cell culture, improves healing in lupus mice, and does not immunocompromise treated animals upon influenza infection but improves survival even when administered 3 days after infection. Finally, as anticipated, we observe limited toxicity in animals treated with CDP compared with PAMAM G3. Thus, CDP is a new anti-inflammatory agent that may be readily translated to the clinic to combat diseases associated with pathological NA-containing DAMPs/PAMPs.

Rapid test to assess the escape of SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are concerning in the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here, we developed a rapid test, termed CoVariant-SCAN, that detects neutralizing antibodies (nAbs) capable of blocking interactions between the angiotensin-converting enzyme 2 receptor and the spike protein of wild-type (WT) SARS-CoV-2 and three other variants: B.1.1.7, B.1.351, and P.1. Using CoVariant-SCAN, we assessed neutralization/blocking of monoclonal antibodies and plasma from COVID-19­positive and vaccinated individuals. For several monoclonal antibodies and most plasma samples, neutralization against B.1.351 and P.1 variants is diminished relative to WT, while B.1.1.7 is largely cross-neutralized. We also showed that we can rapidly adapt the platform to detect nAbs against an additional variant­B.1.617.2 (Delta)­without reengineering or reoptimizing the assay. Results using CoVariant-SCAN are consistent with live virus neutralization assays and demonstrate that this easy-to-deploy test could be used to rapidly assess nAb response against multiple SARS-CoV-2 variants.

COVID-19: Thrombosis, thromboinflammation, and anticoagulation considerations.

Vascular endothelial injury is a hallmark of acute infection at both the microvascular and macrovascular levels. The hallmark of SARS-CoV-2 infection is the current COVID-19 clinical sequelae of the pathophysiologic responses of hypercoagulability and thromboinflammation associated with acute infection. The acute lung injury that initially occurs in COVID-19 results from vascular and endothelial damage from viral injury and pathophysiologic responses that produce the COVID-19-associated coagulopathy. Clinicians should continue to focus on the vascular endothelial injury that occurs and evaluate potential therapeutic interventions that may benefit those with new infections during the current pandemic as they may also be of benefit for future pathogens that generate similar thromboinflammatory responses. The current Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) studies are important projects that will further define our management strategies. At the time of writing this report, two mRNA vaccines are now being distributed and will hopefully have a major impact on slowing the global spread and subsequent thromboinflammatory injury we see clinically in critically ill patients.

Multiplexed, quantitative serological profiling of COVID-19 from blood by a point-of-care test.

Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage coronavirus disease 2019 (COVID-19). Here, we report on a microfluidic POC test that can profile the antibody response against multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens-spike S1 (S1), nucleocapsid (N), and the receptor binding domain (RBD)-simultaneously from 60 µl of blood, plasma, or serum. We assessed the levels of antibodies in plasma samples from 31 individuals (with longitudinal sampling) with severe COVID-19, 41 healthy individuals, and 18 individuals with seasonal coronavirus infections. This POC assay achieved high sensitivity and specificity, tracked seroconversion, and showed good concordance with a live virus microneutralization assay. We can also detect a prognostic biomarker of severity, IP-10 (interferon-γ-induced protein 10), on the same chip. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed to combat COVID-19.

Key Pathogenic Factors in Coronavirus Disease 2019-Associated Coagulopathy and Acute Lung Injury Highlighted in a Patient With Copresentation of Acute Myelocytic Leukemia: A Case Report.

The role of concurrent illness in coronavirus disease 2019 (COVID-19) is unknown. Patients with leukemia may display altered thromboinflammatory responses. We report a 53-year-old man presenting with acute leukemia and COVID-19 who developed thrombotic complications and acute respiratory distress syndrome. Multiple analyses, including rotational thromboelastometry and flow cytometry on blood and bronchoalveolar lavage, are reported to characterize coagulation and immune profiles. The patient developed chemotherapy-induced neutropenia that may have protected his lungs from granulocyte-driven hyperinflammatory acute lung injury. However, neutropenia also alters viral clearing, potentially enabling ongoing viral propagation. This case depicts a precarious equilibrium between leukemia and COVID-19.

Multiplexed, quantitative serological profiling of COVID-19 from a drop of blood by a point-of-care test.

Highly sensitive, specific, and point-of-care (POC) serological assays are an essential tool to manage the COVID-19 pandemic. Here, we report on a microfluidic, multiplexed POC test that can profile the antibody response against multiple SARS-CoV-2 antigens - Spike S1 (S1), Nucleocapsid (N), and the receptor binding domain (RBD) - simultaneously from a 60 microliter drop of blood, plasma, or serum. We assessed the levels of anti-SARS-CoV-2 antibodies in plasma samples from 19 individuals (at multiple time points) with COVID-19 that required admission to the intensive care unit and from 10 healthy individuals. This POC assay shows good concordance with a live virus microneutralization assay, achieved high sensitivity (100%) and specificity (100%), and successfully tracked the longitudinal evolution of the antibody response in infected individuals. We also demonstrated that we can detect a chemokine, IP-10, on the same chip, which may provide prognostic insight into patient outcomes. Because our test requires minimal user intervention and is read by a handheld detector, it can be globally deployed in the fight against COVID-19 by democratizing access to laboratory quality tests.

Streptococcus salivarius Prosthetic Joint Infection following Dental Cleaning despite Antibiotic Prophylaxis.

We present the case of a 92-year-old man with septic arthritis of a prosthetic hip joint due to Streptococcus salivarius one week following a high-risk dental procedure despite preprocedure amoxicillin. S. salivarius is a commensal bacterium of the human oral mucosa that is an uncommon cause of bacteremia. S. salivarius has previously been described as a causative agent of infective endocarditis and spontaneous bacterial peritonitis but was only recently recognized as a cause of prosthetic joint infection. This case highlights the potential pathogenicity of a common commensal bacteria and the questionable utility of prophylactic antibiotics before dental procedures to prevent periprosthetic joint infections.

The effects of living in an outdoor enclosure on hippocampal plasticity and anxiety-like behavior in response to nematode infection.

The hippocampus of rodents undergoes structural remodeling throughout adulthood, including the addition of new neurons. Adult neurogenesis is sensitive to environmental enrichment and stress. Microglia, the brain's resident immune cells, are involved in adult neurogenesis by engulfing dying new neurons. While previous studies using laboratory environmental enrichment have investigated alterations in brain structure and function, they do not provide an adequate reflection of living in the wild, in which stress and environmental instability are common. Here, we compared mice living in standard laboratory settings to mice living in outdoor enclosures to assess the complex interactions among environment, gut infection, and hippocampal plasticity. We infected mice with parasitic worms and studied their effects on adult neurogenesis, microglia, and functions associated with the hippocampus, including cognition and anxiety regulation. We found an increase in immature neuron numbers of mice living in outdoor enclosures regardless of infection. While outdoor living prevented increases in microglial reactivity induced by infection in both the dorsal and ventral hippocampus, outdoor mice with infection had fewer microglia and microglial processes in the ventral hippocampus. We observed no differences in cognitive performance on the hippocampus-dependent object location task between infected and uninfected mice living in either setting. However, we found that infection caused an increase in anxiety-like behavior in the open field test but only in outdoor mice. These findings suggest that living conditions, as well as gut infection, interact to produce complex effects on brain structure and function.

Microglia Play an Active Role in Obesity-Associated Cognitive Decline.

Obesity affects >600 million people worldwide, a staggering number that appears to be on the rise. One of the lesser known consequences of obesity is its deleterious effects on cognition, which have been well documented across many cognitive domains and age groups. To investigate the cellular mechanisms that underlie obesity-associated cognitive decline, we used diet-induced obesity in male mice and found memory impairments along with reductions in dendritic spines, sites of excitatory synapses, increases in the activation of microglia, the brain's resident immune cells, and increases in synaptic profiles within microglia, in the hippocampus, a brain region linked to cognition. We found that partial knockdown of the receptor for fractalkine, a chemokine that can serve as a "find me" cue for microglia, prevented microglial activation and cognitive decline induced by obesity. Furthermore, we found that pharmacological inhibition of microglial activation in obese mice was associated with prevention of both dendritic spine loss and cognitive degradation. Finally, we observed that pharmacological blockade of microglial phagocytosis lessened obesity-associated cognitive decline. These findings suggest that microglia play an active role in obesity-associated cognitive decline by phagocytosis of synapses that are important for optimal function.SIGNIFICANCE STATEMENT Obesity in humans correlates with reduced cognitive function. To investigate the cellular mechanisms underlying this, we used diet-induced obesity in mice and found impaired performance on cognitive tests of hippocampal function. These deficits were accompanied by reduced numbers of dendritic spines, increased microglial activation, and increased synaptic profiles within microglia. Inhibition of microglial activation by transgenic and pharmacological methods prevented cognitive decline and dendritic spine loss in obese mice. Moreover, pharmacological inhibition of the phagocytic activity of microglia was also sufficient to prevent cognitive degradation. This work suggests that microglia may be responsible for obesity-associated cognitive decline and dendritic spine loss.

The BCKDH Kinase and Phosphatase Integrate BCAA and Lipid Metabolism via Regulation of ATP-Citrate Lyase.

Branched-chain amino acids (BCAA) are strongly associated with dysregulated glucose and lipid metabolism, but the underlying mechanisms are poorly understood. We report that inhibition of the kinase (BDK) or overexpression of the phosphatase (PPM1K) that regulates branched-chain ketoacid dehydrogenase (BCKDH), the committed step of BCAA catabolism, lowers circulating BCAA, reduces hepatic steatosis, and improves glucose tolerance in the absence of weight loss in Zucker fatty rats. Phosphoproteomics analysis identified ATP-citrate lyase (ACL) as an alternate substrate of BDK and PPM1K. Hepatic overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis. BDK and PPM1K transcript levels were increased and repressed, respectively, in response to fructose feeding or expression of the ChREBP-ß transcription factor. These studies identify BDK and PPM1K as a ChREBP-regulated node that integrates BCAA and lipid metabolism. Moreover, manipulation of the BDK:PPM1K ratio relieves key metabolic disease phenotypes in a genetic model of severe obesity.

Inactivation of the Dlc1 gene cooperates with downregulation of p15INK4b and p16Ink4a, leading to neoplastic transformation and poor prognosis in human cancer.

The tumor suppressor gene deleted in liver cancer-1 (DLC1), which encodes a protein with strong RhoGAP (GTPase activating protein) activity and weak Cdc42GAP activity, is inactivated in various human malignancies. Following Dlc1 inactivation, mouse embryo fibroblasts (MEF) with a conditional Dlc1 knockout allele reproducibly underwent neoplastic transformation. In addition to inactivation of Dlc1 and increased activity of Rho and Cdc42, transformation depended on the subsequent decreased expression of the Cdk4/6 inhibitors p15(Ink4b) and p16(Ink4a) together with increased expression and activation of Cdk4/6. The level of expression of these cell-cycle regulatory genes was relevant to human tumors with low DLC1 expression. Analysis of publicly available annotated datasets of lung and colon cancer with gene expression microarray profiles indicated that, in pairwise comparisons, low DLC1 expression occurred frequently together (P < 0.01) with downregulation of p15(Ink4b) or p16(Ink4a) or upregulation of CDK4 or CDK6. In addition, an unfavorable prognosis (P < 0.05) was associated with low DLC1 and low p15(Ink4b) in lung cancer and colon cancer, low DLC1 and low p16(Ink4a) in lung cancer, low DLC1 and high CDK4 in lung cancer, and low DLC1 and high CDK6 in colon cancer. Thus, several genes and biochemical activities collaborate with the inactivation of DLC1 to give rise to cell transformation in MEFs, and the identified genes are relevant to human tumors with low DLC1 expression.