Forward Genetic Screens Identify Mechanisms of Resistance to Small-Molecule Lactate Dehydrogenase Inhibitors.

Altered metabolism is a hallmark of cancer; however, it has been difficult to specifically target metabolism in cancer for therapeutic benefit. Cancers with genetically defined defects in metabolic enzymes constitute a subset of cancers where targeting metabolism is potentially accessible. Hürthle cell carcinoma of the thyroid (HTC) tumors frequently harbor deleterious mitochondrial DNA (mtDNA) mutations in subunits of complex I of the mitochondrial electron transport chain (ETC). Previous work has shown that HTC models with deleterious mtDNA mutations exhibit mitochondrial ETC defects that expose lactate dehydrogenase (LDH) as a therapeutic vulnerability. Here, we performed forward genetic screens to identify mechanisms of resistance to small-molecule LDH inhibitors. We identified two distinct mechanisms of resistance: upregulation of an LDH isoform and a compound-specific resistance mutation. Using these tools, we demonstrate that the anticancer activity of LDH inhibitors in cell line and xenograft models of complex I mutant HTC is through on-target LDH inhibition.

Forward genetic screens identify mechanisms of resistance to small molecule lactate dehydrogenase inhibitors.

Altered metabolism is a hallmark of cancer; however, it has been difficult to specifically target metabolism in cancer for therapeutic benefit. Cancers with genetically defined defects in metabolic enzymes constitute a subset of cancers where targeting metabolism is potentially accessible. Hürthle cell carcinoma of the thyroid (HTC) tumors frequently harbor deleterious mitochondrial DNA (mtDNA) mutations in subunits of complex I of the mitochondrial electron transport chain (ETC). Previous work has shown that HTC models with deleterious mtDNA mutations exhibit mitochondrial ETC defects that expose lactate dehydrogenase (LDH) as a therapeutic vulnerability. Here, we performed forward genetic screens to identify mechanisms of resistance to small molecule LDH inhibitors. We identified two distinct mechanisms of resistance: upregulation of an LDH isoform and a compound-specific resistance mutation. Using these tools, we demonstrate that the anti-cancer activity of LDH inhibitors in cell line and xenograft models of complex I-mutant HTC is through on-target LDH inhibition.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Role of Pellino-1 in Inflammation and Cardioprotection following Severe Sepsis: A Novel Mechanism in a Murine Severe Sepsis Model †.

OBJECTIVES: Intra-abdominal sepsis is commonly diagnosed in the surgical population and remains the second most common cause of sepsis overall. Sepsis-related mortality remains a significant burden in the intensive care unit despite advances in critical care. Nearly a quarter of the deaths in people with heart failure are caused by sepsis. We have observed that overexpression of mammalian Pellino-1 (Peli1), an E3 ubiquitin ligase, causes inhibition of apoptosis, oxidative stress, and preservation of cardiac function in a myocardial infarction model. Given these manifold applications, we investigated the role of Peli1 in sepsis using transgenic and knockout mouse models specific to this protein. Therefore, we aimed to explore further the myocardial dysfunction seen in sepsis through its relation to the Peli 1 protein by using the loss of function and gain-of-function strategy. METHODS: A series of genetic animals were created to understand the role of Peli1 in sepsis and the preservation of heart function. Wild-type, global Peli1 knock out (Peli1-/-), cardiomyocyte-specific Peli1 deletion (CP1KO), and cardiomyocyte-specific Peli1 overexpressing (alpha MHC (αMHC) Peli1; AMPEL1Tg/+) animals were divided into sham and cecal ligation and puncture (CLP) surgical procedure groups. Cardiac function was determined by two-dimensional echocardiography pre-surgery and at 6- and 24-h post-surgery. Serum IL-6 and TNF-alpha levels (ELISA) (6 h), cardiac apoptosis (TUNEL assay), and Bax expression (24 h) post-surgery were measured. Results are expressed as mean ± S.E.M. RESULTS: AMPEL1Tg/+ prevents sepsis-induced cardiac dysfunction assessed by echocardiographic analysis, whereas global and cardiomyocyte-specific deletion of Peli1 shows significant deterioration of cardiac functions. Cardiac function was similar across the sham groups in all three genetically modified mice. ELISA assay displayed how Peli 1 overexpression decreased cardo-suppressive circulating inflammatory cytokines (TNF-alpha, IL-6) compared to both the knockout groups. The proportion of TUNEL-positive cells varied according to Peli1 expression, with overexpression (AMPEL1Tg/+) leading to a significant reduction and Peli1 gene knockout (Peli1-/- and CP1KO) leading to a significant increase in their presence. A similar trend was also observed with Bax protein expression. The improved cellular survival associated with Peli1 overexpression was again shown with the reduction of oxidative stress marker 4-Hydroxy-2-Nonenal (4-HNE). CONCLUSION: Our results indicate that overexpression of Peli1 is a novel approach that not only preserved cardiac function but reduced inflammatory markers and apoptosis following severe sepsis in a murine genetic model.

Mitochondrial-Encoded Complex I Impairment Induces a Targetable Dependency on Aerobic Fermentation in Hürthle Cell Carcinoma of the Thyroid.

A metabolic hallmark of cancer identified by Warburg is the increased consumption of glucose and secretion of lactate, even in the presence of oxygen. Although many tumors exhibit increased glycolytic activity, most forms of cancer rely on mitochondrial respiration for tumor growth. We report here that Hürthle cell carcinoma of the thyroid (HTC) models harboring mitochondrial DNA-encoded defects in complex I of the mitochondrial electron transport chain exhibit impaired respiration and alterations in glucose metabolism. CRISPR-Cas9 pooled screening identified glycolytic enzymes as selectively essential in complex I-mutant HTC cells. We demonstrate in cultured cells and a patient-derived xenograft model that small-molecule inhibitors of lactate dehydrogenase selectively induce an ATP crisis and cell death in HTC. This work demonstrates that complex I loss exposes fermentation as a therapeutic target in HTC and has implications for other tumors bearing mutations that irreversibly damage mitochondrial respiration. SIGNIFICANCE: HTC is enriched in somatic mtDNA mutations predicted to affect complex I of the electron transport chain (ETC). We demonstrate that these mutations impair respiration and induce a therapeutically tractable reliance on aerobic fermentation for cell survival. This work provides a rationale for targeting fermentation in cancers harboring irreversible genetically encoded ETC defects. See related article by Gopal et al., p. 1904. This article is highlighted in the In This Issue feature, p. 1749.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Differential requirements for mitochondrial electron transport chain components in the adult murine liver.

Mitochondrial electron transport chain (ETC) dysfunction due to mutations in the nuclear or mitochondrial genome is a common cause of metabolic disease in humans and displays striking tissue specificity depending on the affected gene. The mechanisms underlying tissue-specific phenotypes are not understood. Complex I (cI) is classically considered the entry point for electrons into the ETC, and in vitro experiments indicate that cI is required for basal respiration and maintenance of the NAD+/NADH ratio, an indicator of cellular redox status. This finding has largely not been tested in vivo. Here, we report that mitochondrial complex I is dispensable for homeostasis of the adult mouse liver; animals with hepatocyte-specific loss of cI function display no overt phenotypes or signs of liver damage, and maintain liver function, redox and oxygen status. Further analysis of cI-deficient livers did not reveal significant proteomic or metabolic changes, indicating little to no compensation is required in the setting of complex I loss. In contrast, complex IV (cIV) dysfunction in adult hepatocytes results in decreased liver function, impaired oxygen handling, steatosis, and liver damage, accompanied by significant metabolomic and proteomic perturbations. Our results support a model whereby complex I loss is tolerated in the mouse liver because hepatocytes use alternative electron donors to fuel the mitochondrial ETC.

Mitochondria are specialised structures inside cells that help to convert nutrients into energy. They take electrons from nutrients and use them to power biochemical reactions that supply chemical fuel. Previous studies of cells grown in the laboratory have found that electrons enter this process via a large assembly of proteins in mitochondria called complex I. Understanding the mechanism of energy production is important, as issues with mitochondria can lead to a variety of metabolic diseases. However, it is still unclear how complex I acts in living animals. Lesner et al. addressed this knowledge gap by genetically removing a key protein from complex I in the liver of mice. Surprisingly, the animals did not develop any detectable symptoms and maintained healthy liver function. Mice did not seem to compensate by making energy in a different way, suggesting that complex I is not normally used by the mouse liver for this process. This research suggests that biologists should reconsider the mechanism that mitochondria use to power cells in animals. While the role of Complex I in electron transfer is well established in laboratory-grown cells and some organs, like the brain, it cannot be assumed this applies to the whole body. Understanding energy production in specific organs could help researchers to develop nutrient-based therapies for metabolic diseases.

TK216 targets microtubules in Ewing sarcoma cells.

Ewing sarcoma (EWS) is a pediatric malignancy driven by the EWSR1-FLI1 fusion protein formed by the chromosomal translocation t(11; 22). The small molecule TK216 was developed as a first-in-class direct EWSR1-FLI1 inhibitor and is in phase II clinical trials in combination with vincristine for patients with EWS. However, TK216 exhibits anti-cancer activity against cancer cell lines and xenografts that do not express EWSR1-FLI1, and the mechanism underlying cytotoxicity remains unresolved. We apply a forward-genetics screening platform utilizing engineered hypermutation in EWS cell lines and identify recurrent mutations in TUBA1B, encoding ⍺-tubulin, that prove sufficient to drive resistance to TK216. Using reconstituted microtubule (MT) polymerization in vitro and cell-based chemical probe competition assays, we demonstrate that TK216 acts as an MT destabilizing agent. This work defines the mechanism of cytotoxicity of TK216, explains the synergy observed with vincristine, and calls for a reexamination of ongoing clinical trials with TK216.

Scinderin promotes fusion of electron transport chain dysfunctional muscle stem cells with myofibers.

Muscle stem cells (MuSCs) experience age-associated declines in number and function, accompanied by mitochondrial electron transport chain (ETC) dysfunction and increased reactive oxygen species (ROS). The source of these changes, and how MuSCs respond to mitochondrial dysfunction, is unknown. We report here that in response to mitochondrial ROS, murine MuSCs directly fuse with neighboring myofibers; this phenomenon removes ETC-dysfunctional MuSCs from the stem cell compartment. MuSC-myofiber fusion is dependent on the induction of Scinderin, which promotes formation of actin-dependent protrusions required for membrane fusion. During aging, we find that the declining MuSC population accumulates mutations in the mitochondrial genome, but selects against dysfunctional variants. In the absence of clearance by Scinderin, the decline in MuSC numbers during aging is repressed; however, ETC-dysfunctional MuSCs are retained and can regenerate dysfunctional myofibers. We propose a model in which ETC-dysfunctional MuSCs are removed from the stem cell compartment by fusing with differentiated tissue.

Quality control of image sensors using gaseous tritium light sources.

We propose a practical method for radiometrically calibrating cameras using widely available gaseous tritium light sources (betalights). Along with the gain (conversion factor) and read noise level, the predictable photon flux of the source allows us to gauge the quantum efficiency. The design is easily reproducible with a 3D printer (three-dimensional printer) and three inexpensive parts. Suitable for common image sensors, we believe that the method has the potential to be a useful tool in microscopy facilities and optical laboratories alike. This article is part of the theme issue 'Super-resolution structured illumination microscopy (part 2)'.

Symptoms COVID 19 Positive Vapers Compared to COVID 19 Positive Non-vapers.

OBJECTIVES: The purpose of the present study was to assess and describe the severity of symptoms reported by Covid-19 positive patients who vaped (smoked e-cigarettes) when compared to those who did not vape or smoke at the time of the diagnosis of Covid-19. METHODS: Patients from this study are from a well-characterized patient cohort collected at Mayo Clinic between March 1, 2020 and February 28, 2021; with confirmed COVID-19 diagnosis defined as a positive result on reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays from nasopharyngeal swab specimens. Among the 1734 eligible patients, 289 patients reported current vaping. The cohort of vapers (N = 289) was age and gender matched to 1445 covid-19 positive patients who did not vape. The data analyzed included: date of birth, gender, ethnicity, race, marital status, as well as lifestyle history such as vaping and smoking and reported covid-19 symptoms experienced. RESULTS: A logistic regression analysis was performed separately for each symptom using generalized estimating equations (GEE) with robust variance estimates in order to account for the 1:5 age, sex, and race matched set study design. Patients who vaped and developed Covid-19 infection were more likely to have chest pain or tightness (16% vs 10%, vapers vs non vapers, P = .005), chills (25% vs 19%, vapers vs non vapers, P = .0016), myalgia (39% vs 32%, vapers vs non vapers, P = .004), headaches (49% vs 41% vapers vs non vapers, P = .026), anosmia/dysgeusia (37% vs 30%, vapers vs non vapers, P = .009), nausea/vomiting/abdominal pain (16% vs 10%, vapers vs non vapers, P = .003), diarrhea (16% vs 10%, vapers vs non vapers, P = .004), and non-severe light-headedness (16% vs 9%, vapers vs non vapers, P < .001). CONCLUSION: Vapers experience higher frequency of covid-19 related symptoms when compared with age and gender matched non-vapers. Further work should examine the impact vaping has on post-covid symptom experience.

Genetics, Diagnosis, and Management of Hürthle Cell Thyroid Neoplasms.

Hürthle cell lesions have been a diagnostic conundrum in pathology since they were first recognized over a century ago. Controversy as to the name of the cell, the origin of the cell, and even which cells in particular may be designated as such still challenge pathologists and confound those treating patients with a diagnosis of "Hürthle cell" anything within the diagnosis, especially if that anything is a sizable mass lesion. The diagnosis of Hürthle cell adenoma (HCA) or Hürthle cell carcinoma (HCC) has typically relied on a judgement call by pathologists as to the presence or absence of capsular and/or vascular invasion of the adjacent thyroid parenchyma, easy to note in widely invasive disease and a somewhat subjective diagnosis for minimally invasive or borderline invasive disease. Diagnostic specificity, which has incorporated a sharp increase in molecular genetic studies of thyroid tumor subtypes and the integration of molecular testing into preoperative management protocols, continues to be challenged by Hürthle cell neoplasia. Here, we provide the improving yet still murky state of what is known about Hürthle cell tumor genetics, clinical management, and based upon what we are learning about the genetics of other thyroid tumors, how to manage expectations, by pathologists, clinicians, and patients, for more actionable, precise classifications of Hürthle cell tumors of the thyroid.

Engineered resveratrol-loaded fibrous scaffolds promotes functional cardiac repair and regeneration through Thioredoxin-1 mediated VEGF pathway.

Despite recent advancements, mortality due to coronary heart disease (CHD) remains high. Recently, the use of tissue-engineered grafts and scaffolds has emerged as a candidate for supporting the myocardium after an ischemic event. Resveratrol is a naturally occurring plant-based non-flavonoid polyphenolic compound found in many natural foods, including grapes and red wine. We embedded resveratrol in a polycaprolactone (PCL) scaffold and evaluated the cardio-therapeutic effects in a murine model of myocardial infarction (MI), with animals being grouped into Sham (S), Myocardial Infarction (MI), MI + PCL, and MI + PCL-Resveratrol (MI + PCL-R). After 4 and 8 weeks, echocardiography was performed to assess ejection fraction (EF) and fractional shortening (FS), which was followed by immunohistochemistry and immunofluorescence analysis at 8 weeks. The MI + PCL-R group showed a significant improvement in EF and FS compared with the MI + PCL group at 4 and 8-weeks post-surgery. PCL-R scaffolds treated hearts revealed decreased inflammatory cell infiltration, improved collagen extracellular matrix (ECM) secretion and blood vessel network formation following MI. The immunofluorescence analysis revealed resveratrol-loaded scaffolds promote increased expression of cTnT, Cx-43, Trx-1, and VEGF proteins. This study reports resveratrol-mediated rescue of ischemic myocardium when delivered through a biodegradable polymeric scaffold system after MI.

A Multipronged Approach Establishes Covalent Modification of ß-Tubulin as the Mode of Action of Benzamide Anti-cancer Toxins.

A phenotypic high-throughput screen identified a benzamide small molecule with activity against small cell lung cancer cells. A "clickable" benzamide probe was designed that irreversibly bound a single 50 kDa cellular protein, identified by mass spectrometry as ß-tubulin. Moreover, the anti-cancer potency of a series of benzamide analogs strongly correlated with probe competition, indicating that ß-tubulin was the functional target. Additional evidence suggested that benzamides covalently modified Cys239 within the colchicine binding site. Consistent with this mechanism, benzamides impaired growth of microtubules formed with ß-tubulin harboring Cys239, but not ß3 tubulin encoding Ser239. We therefore designed an aldehyde-containing analog capable of trapping Ser239 in ß3 tubulin, presumably as a hemiacetal. Using a forward genetics strategy, we identified benzamide-resistant cell lines harboring a Thr238Ala mutation in ß-tubulin sufficient to induce compound resistance. The disclosed chemical probes are useful to identify other colchicine site binders, a frequent target of structurally diverse small molecules.

Mitochondrial DNA Haplotypes as Genetic Modifiers of Cancer.

Mitochondria play an essential role in cellular metabolism, generation of reactive oxygen species (ROS), and the initiation of apoptosis. These properties enable mitochondria to be crucial integrators in the pathways of tumorigenesis. An open question is to what extent variation in the mitochondrial genome (mtDNA) contributes to the biological heterogeneity observed in human tumors. In this review, we summarize our current understanding of the role of mtDNA genetics in relation to human cancers.

Deletion of newly described pro-survival molecule Pellino-1 increases oxidative stress, downregulates cIAP2/NF-κB cell survival pathway, reduces angiogenic response, and thereby aggravates tissue function in mouse ischemic models.

INTRODUCTION: In the present study, we aimed to explore the functional role of Pellino-1 (Peli1) in inducing neovascularization after myocardial infarction (MI) and hindlimb ischemia (HLI) using Peli1 global knockout mice (Peli1-/-). Recently we have shown that Peli1, an E3 ubiquitin ligase, induce angiogenesis and improve survivability, with decreased necrosis of ischemic skin flaps. METHODS: Peli1fl/fl and Peli1-/- mice were subjected to either permanent ligation of the left anterior descending coronary artery (LAD) or sham surgery (S). Tissues from the left ventricular risk area were collected at different time points post-MI. In addition, Peli1fl/fl and Peli1-/- mice were also subjected to permanent ligation of the right femoral artery followed by motor function scores, Doppler analysis for blood perfusion and immunohistochemical analysis. RESULTS: Global Peli1 knockout exacerbated myocardial dysfunction, 30 and 60 days after MI compared to wild type (WT) mice as measured by echocardiogram. In addition, Peli1-/- mice also showed decreased motor function scores and perfusion ratios compared with Peli1fl/fl mice 28 days after the induction of HLI. The use of Peli1 in adenoviral gene therapy following HLI in CD1 mice improved the perfusion ratio at 28 days compared to Ad.LacZ-injected mice. CONCLUSION: These results suggest new insights into the protective role of Peli1 on ischemic tissues and its influence on survival signaling.

New Approaches to SCLC Therapy: From the Laboratory to the Clinic.

The outcomes of patients with SCLC have not yet been substantially impacted by the revolution in precision oncology, primarily owing to a paucity of genetic alterations in actionable driver oncogenes. Nevertheless, systemic therapies that include immunotherapy are beginning to show promise in the clinic. Although, these results are encouraging, many patients do not respond to, or rapidly recur after, current regimens, necessitating alternative or complementary therapeutic strategies. In this review, we discuss ongoing investigations into the pathobiology of this recalcitrant cancer and the therapeutic vulnerabilities that are exposed by the disease state. Included within this discussion, is a snapshot of the current biomarker and clinical trial landscapes for SCLC. Finally, we identify key knowledge gaps that should be addressed to advance the field in pursuit of reduced SCLC mortality. This review largely summarizes work presented at the Third Biennial International Association for the Study of Lung Cancer SCLC Meeting.

Engineering Forward Genetics into Cultured Cancer Cells for Chemical Target Identification.

Target identification for biologically active small molecules remains a major barrier for drug discovery. Cancer cells exhibiting defective DNA mismatch repair (dMMR) have been used as a forward genetics system to uncover compound targets. However, this approach has been limited by the dearth of cancer cell lines that harbor naturally arising dMMR. Here, we establish a platform for forward genetic screening using CRISPR/Cas9 to engineer dMMR into mammalian cells. We demonstrate the utility of this approach to identify mechanisms of drug action in mouse and human cancer cell lines using in vitro selections against three cellular toxins. In each screen, compound-resistant alleles emerged in drug-resistant clones, supporting the notion that engineered dMMR enables forward genetic screening in mammalian cells.