Defining the conformational states that enable transglutaminase 2 to promote cancer cell survival versus cell death.

Transglutaminase 2 (TG2) is a GTP-binding/protein-crosslinking enzyme that has been investigated as a therapeutic target for Celiac disease, neurological disorders, and aggressive cancers. TG2 has been suggested to adopt two conformational states that regulate its functions: a GTP-bound, closed conformation, and a calcium-bound, crosslinking-active open conformation. TG2 mutants that constitutively adopt an open conformation are cytotoxic to cancer cells. Thus, small molecules that maintain the open conformation of TG2 could offer a new therapeutic strategy. Here, we investigate TG2, using static and time-resolved small-angle X-ray scattering (SAXS) and single-particle cryoelectron microscopy (cryo-EM), to determine the conformational states responsible for conferring its biological effects. We also describe a newly developed TG2 inhibitor, LM11, that potently kills glioblastoma cells and use SAXS to investigate how LM11 affects the conformational states of TG2. Using SAXS and cryo-EM, we show that guanine nucleotide-bound TG2 adopts a monomeric closed conformation while calcium-bound TG2 assumes an open conformational state that can form higher order oligomers. SAXS analysis also suggests how a TG2 mutant that constitutively adopts the open state binds nucleotides through an alternative mechanism to wildtype TG2. Furthermore, we use time-resolved SAXS to show that LM11 increases the ability of calcium to drive TG2 to an open conformation, which is not reversible by guanine nucleotides and is cytotoxic to cancer cells. Taken together, our findings demonstrate that the conformational dynamics of TG2 are more complex than previously suggested and highlight how conformational stabilization of TG2 by LM11 maintains TG2 in a cytotoxic conformational state.

Confirmation of canine acanthomatous ameloblastoma using RAS Q61R immunohistochemical staining of formalin-fixed paraffin-embedded tissues.

Differentiating canine acanthomatous ameloblastoma (CAA) from oral squamous cell carcinoma (OSCC) based on routine histopathology can be challenging. We have previously shown that more than 95% of CAAs harbor an HRAS p.Q61R somatic mutation, while OSCCs carry either wild-type alleles or other MAPK pathway activating mutations (e.g., HRAS p.Q61L, BRAF p.V595E). Given that HRAS p.Q61R mutations are highly prevalent in CAA, we hypothesized that a RAS Q61R-specific rabbit monoclonal antibody may be a useful tool for confirmation of CAA by immunohistochemical (IHC) staining. In the present study, we assessed IHC staining of archived formalin-fixed and paraffin-embedded biopsy samples with a diagnosis of CAA (n = 23), using a RAS Q61R-specific rabbit monoclonal antibody (SP174) and an automated IHC stainer. Negative control samples consisted of HRAS p.Q61R mutation-negative OSCC tumors with either a known HRAS p.Q61L mutation (n = 1), BRAF p.V595E mutation (n = 4), or wild-type corresponding alleles (n = 3). We found that all 23 CAAs showed diffuse and strong membranous RAS Q61R immunoreactivity (100% sensitivity), while none of the 8 OSCCs showed immunoreactivity (100% specificity). The data supports the use of RAS Q61R-specific rabbit monoclonal antibody for diagnostic IHC confirmation of CAA and ruling out OSCC in dogs.

Inhibiting Glutamine Metabolism Blocks Coronavirus Replication in Mammalian Cells.

Developing therapeutic strategies against COVID-19 has gained widespread interest given the likelihood that new viral variants will continue to emerge. Here we describe one potential therapeutic strategy which involves targeting members of the glutaminase family of mitochondrial metabolic enzymes (GLS and GLS2), which catalyze the first step in glutamine metabolism, the hydrolysis of glutamine to glutamate. We show three examples where GLS expression increases during coronavirus infection of host cells, and another in which GLS2 is upregulated. The viruses hijack the metabolic machinery responsible for glutamine metabolism to generate the building blocks for biosynthetic processes and satisfy the bioenergetic requirements demanded by the 'glutamine addiction' of virus-infected host cells. We demonstrate how genetic silencing of glutaminase enzymes reduces coronavirus infection and that newer members of two classes of small molecule allosteric inhibitors targeting these enzymes, designated as SU1, a pan-GLS/GLS2 inhibitor, and UP4, which is specific for GLS, block viral replication in mammalian epithelial cells. Overall, these findings highlight the importance of glutamine metabolism for coronavirus replication in human cells and show that glutaminase inhibitors can block coronavirus infection and thereby may represent a novel class of anti-viral drug candidates. Teaser: Inhibitors targeting glutaminase enzymes block coronavirus replication and may represent a new class of anti-viral drugs.

Alone and together: current approaches to targeting glutaminase enzymes as part of anti-cancer therapies.

Metabolic reprogramming is a major hallmark of malignant transformation in cancer, and part of the so-called Warburg effect, in which the upregulation of glutamine catabolism plays a major role. The glutaminase enzymes convert glutamine to glutamate, which initiates this pathway. Inhibition of different forms of glutaminase (KGA, GAC, or LGA) demonstrated potential as an emerging anti-cancer therapeutic strategy. The regulation of these enzymes, and the molecular basis for their inhibition, have been the focus of much recent research. This review will explore the recent progress in understanding the molecular basis for activation and inhibition of different forms of glutaminase, as well as the recent focus on combination therapies of glutaminase inhibitors with other anti-cancer drugs.

Many strategies exist to inhibit cancer progression, from chemotherapy to more targeted therapies that exploit differences between tumors and healthy tissue. One such targeted strategy involves inhibition of the enzyme glutaminase, which converts glutamine obtained from the bloodstream into nutrients that fuel tumor growth. Research into glutaminase is ongoing, with regulation of the enzyme, and novel molecular approaches to inhibit its activity, being key focus areas. Here, we review recent progress on targeting glutaminase enzymes for anti-cancer therapy, including several approaches in which glutaminase inhibitors are combined with inhibitors of other cancer-relevant targets, to increase the overall effectiveness of the treatment.

Highly recurrent BRAF p.V595E mutation in canine papillary oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common oral epithelial malignancy in dogs. It exhibits locally aggressive biological behaviour with the potential to metastasize, and a reported 1-year survival rate of 0% when left untreated. Expression studies suggest that aberrant MAPK signalling plays a key role in canine OSCC tumorigenesis, which is consistent with BRAF and HRAS MAPK-activating mutations reported in some tumours. Several morphological subtypes of canine OSCC have been described, with papillary, conventional, and basaloid as the most common patterns. We hypothesized that mutational differences may underlie these phenotypic variations. In this study, targeted Sanger sequencing and restriction fragment length polymorphism assays demonstrate that up to 85.7% of canine papillary OSCC (n = 14) harbour a BRAF p.V595E mutation. Assessment of neoplastic epithelial cell proliferation using Ki67 immunolabelling (n = 10) confirmed a relatively high proliferation activity, consistent with their known aggressive clinical behaviour. These findings underscore a consistent genetic feature of canine papillary OSCC and provide a basis for the development of novel diagnostic and targeted therapeutic approaches that can improve the quality of veterinary care.

Exploring the Role of Transglutaminase in Patients with Glioblastoma: Current Perspectives.

Tissue transglutaminase (tTG) is a rather unique GTP-binding/protein crosslinking enzyme that has been shown to play important roles in a number of cellular processes that impact both normal physiology and disease states. This is especially the case in the context of aggressive brain tumors, such as glioblastoma. The diverse roles played by tTG in cancer survival and progression have led to significant interest in recent years in using tTG as a therapeutic target. In this review, we provide a brief overview of the transglutaminase family, and then discuss the primary biochemical activities exhibited by tTG with an emphasis on the role it plays in glioblastoma progression. Finally, we consider current approaches to target tTG which might eventually have clinical impact.

New insights into the molecular mechanisms of glutaminase C inhibitors in cancer cells using serial room temperature crystallography.

Cancer cells frequently exhibit uncoupling of the glycolytic pathway from the TCA cycle (i.e., the "Warburg effect") and as a result, often become dependent on their ability to increase glutamine catabolism. The mitochondrial enzyme Glutaminase C (GAC) helps to satisfy this 'glutamine addiction' of cancer cells by catalyzing the hydrolysis of glutamine to glutamate, which is then converted to the TCA-cycle intermediate α-ketoglutarate. This makes GAC an intriguing drug target and spurred the molecules derived from bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (the so-called BPTES class of allosteric GAC inhibitors), including CB-839, which is currently in clinical trials. However, none of the drugs targeting GAC are yet approved for cancer treatment and their mechanism of action is not well understood. Here, we shed new light on the underlying basis for the differential potencies exhibited by members of the BPTES/CB-839 family of compounds, which could not previously be explained with standard cryo-cooled X-ray crystal structures of GAC bound to CB-839 or its analogs. Using an emerging technique known as serial room temperature crystallography, we were able to observe clear differences between the binding conformations of inhibitors with significantly different potencies. We also developed a computational model to further elucidate the molecular basis of differential inhibitor potency. We then corroborated the results from our modeling efforts using recently established fluorescence assays that directly read out inhibitor binding to GAC. Together, these findings should aid in future design of more potent GAC inhibitors with better clinical outlook.

Comparative transcriptional profiling of canine acanthomatous ameloblastoma and homology with human ameloblastoma.

Ameloblastomas are odontogenic tumors that are rare in people but have a relatively high prevalence in dogs. Because canine acanthomatous ameloblastomas (CAA) have clinicopathologic and molecular features in common with human ameloblastomas (AM), spontaneous CAA can serve as a useful translational model of disease. However, the molecular basis of CAA and how it compares to AM are incompletely understood. In this study, we compared the global genomic expression profile of CAA with AM and evaluated its dental origin by using a bulk RNA-seq approach. For these studies, healthy gingiva and canine oral squamous cell carcinoma served as controls. We found that aberrant RAS signaling, and activation of the epithelial-to-mesenchymal transition cellular program are involved in the pathogenesis of CAA, and that CAA is enriched with genes known to be upregulated in AM including those expressed during the early stages of tooth development, suggesting a high level of molecular homology. These results support the model that domestic dogs with spontaneous CAA have potential for pre-clinical assessment of targeted therapeutic modalities against AM.

Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer.

BACKGROUND: Glutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients. METHODS: A panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity. RESULTS: The anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness. CONCLUSION: This study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

Patents targeting the Warburg effect for cancer therapy: an interview with William P Katt.

Dr William Katt is a multidisciplinary scientist with particular focus in computational, synthetic and biological chemistry. He obtained his undergraduate degree at Rensselaer Polytechnic Institute and performed his graduate studies at Yale University, where he focused on designing small-molecule inhibitors of the Rho/Rho GEF interaction. Following those studies, Dr Katt accepted a fellowship from the American Cancer Society which funded his work at Cornell University, where he investigated small-molecule inhibitors of the enzyme glutaminase, a key player in cancer metabolism. Today, Dr Katt is a research associate at Cornell and maintains a number of collaborations with researchers across the nation examining glutaminase, cancer stem cells, nano-therapeutics and more, with the goal of developing therapeutic approaches that will eventually help patients in the clinic.

Liver-Type Glutaminase GLS2 Is a Druggable Metabolic Node in Luminal-Subtype Breast Cancer.

Efforts to target glutamine metabolism for cancer therapy have focused on the glutaminase isozyme GLS. The importance of the other isozyme, GLS2, in cancer has remained unclear, and it has been described as a tumor suppressor in some contexts. Here, we report that GLS2 is upregulated and essential in luminal-subtype breast tumors, which account for >70% of breast cancer incidence. We show that GLS2 expression is elevated by GATA3 in luminal-subtype cells but suppressed by promoter methylation in basal-subtype cells. Although luminal breast cancers resist GLS-selective inhibitors, we find that they can be targeted with a dual-GLS/GLS2 inhibitor. These results establish a critical role for GLS2 in mammary tumorigenesis and advance our understanding of how to target glutamine metabolism in cancer.

Starving the Devourer: Cutting Cancer Off from Its Favorite Foods.

Increased consumption of glucose and glutamine are metabolic hallmarks of tumorigenesis. In this issue of Cell Chemical Biology, Reckzeh et al. (2019) describe the discovery of Glutor, a potent inhibitor of cellular glucose uptake. Combining Glutor with the glutaminase inhibitor CB-839 creates a metabolic crisis in cancer cells, synergistically impeding proliferation.

Inhibition of cancer metabolism: a patent landscape.

Cancer metabolism is currently a hot topic. Since it was first realized that cancer cells rely upon an altered metabolic program to sustain their rapid proliferation, the enzymes that support those metabolic changes have appeared to be good targets for pharmacological intervention. Here, we discuss efforts pertaining to targets in cancer metabolism, focusing upon the tricarboxylic acid cycle and the mechanisms which feed nutrients into it. We describe a broad landscape of small-molecule inhibitors, targeting a dozen different proteins, each implicated in cancer progression. We hope that this will serve as a reference both to the areas being most highly examined today and, relatedly, the areas that are still ripe for novel intervention.

Amorphous Quantum Nanomaterials.

In quantum materials, macroscopic behavior is governed in nontrivial ways by quantum phenomena. This is usually achieved by exquisite control over atomic positions in crystalline solids. Here, it is demonstrated that the use of disordered glassy materials provides unique opportunities to tailor quantum material properties. By borrowing ideas from single-molecule spectroscopy, single delocalized π-electron dye systems are isolated in relatively rigid ultrasmall (<10 nm diameter) amorphous silica nanoparticles. It is demonstrated that chemically tuning the local amorphous silica environment around the dye over a range of compositions enables exquisite control over dye quantum behavior, leading to efficient probes for photodynamic therapy (PDT) and stochastic optical reconstruction microscopy (STORM). The results suggest that efficient fine-tuning of light-induced quantum behavior mediated via effects like spin-orbit coupling can be effectively achieved by systematically varying averaged local environments in glassy amorphous materials as opposed to tailoring well-defined neighboring atomic lattice positions in crystalline solids. The resulting nanoprobes exhibit features proven to enable clinical translation.

A small molecule regulator of tissue transglutaminase conformation inhibits the malignant phenotype of cancer cells.

The protein crosslinking enzyme tissue transglutaminase (tTG) is an acyltransferase which catalyzes transamidation reactions between two proteins, or between a protein and a polyamine. It is frequently overexpressed in several different types of human cancer cells, where it has been shown to contribute to their growth, survival, and invasiveness. tTG is capable of adopting two distinct conformational states: a protein crosslinking active ("open") state, and a GTP-bound, crosslinking inactive ("closed") state. We have previously shown that the ectopic expression of mutant forms of tTG, which constitutively adopt the open conformation, are toxic to cells. This raises the possibility that strategies directed toward causing tTG to maintain an open state could potentially provide a therapeutic benefit for cancers in which tTG is highly expressed. Here, we report the identification of a small molecule, TTGM 5826, which stabilizes the open conformation of tTG. Treatment of breast and brain cancer cell lines, as well as glioma stem cells, with this molecule broadly inhibits their transformed phenotypes. Thus, TTGM 5826 represents the lead compound for a new class of small molecules that promote the toxicity of cancer cells by stabilizing the open state of tTG.

Targeting Therapy Resistance: When Glutamine Catabolism Becomes Essential.

Identifying contexts in which cancer cells become addicted to specific nutrients is critical for developing targeted metabolic therapies. In this issue of Cancer Cell, Momcilovic et al. report that suppressed glycolysis following mTOR inhibition is countered by adaptive glutamine catabolism in lung squamous cell carcinoma, sensitizing tumors to glutaminase inhibition.

Characterization of the interactions of potent allosteric inhibitors with glutaminase C, a key enzyme in cancer cell glutamine metabolism.

Altered glycolytic flux in cancer cells (the "Warburg effect") causes their proliferation to rely upon elevated glutamine metabolism ("glutamine addiction"). This requirement is met by the overexpression of glutaminase C (GAC), which catalyzes the first step in glutamine metabolism and therefore represents a potential therapeutic target. The small molecule CB-839 was reported to be more potent than other allosteric GAC inhibitors, including the parent compound bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl (BPTES), and is in clinical trials. Recently, we described the synthesis of BPTES analogs having distinct saturated heterocyclic cores as a replacement for the flexible chain moiety, with improved microsomal stability relative to CB-839 and BPTES. Here, we show that one of these new compounds, UPGL00004, like CB-839, more potently inhibits the enzymatic activity of GAC, compared with BPTES. We also compare the abilities of UPGL00004, CB-839, and BPTES to directly bind to recombinant GAC and demonstrate that UPGL00004 has a similar binding affinity as CB-839 for GAC. We also show that UPGL00004 potently inhibits the growth of triple-negative breast cancer cells, as well as tumor growth when combined with the anti-vascular endothelial growth factor antibody bevacizumab. Finally, we compare the X-ray crystal structures for UPGL00004 and CB-839 bound to GAC, verifying that UPGL00004 occupies the same binding site as CB-839 or BPTES and that all three inhibitors regulate the enzymatic activity of GAC via a similar allosteric mechanism. These results provide insights regarding the potency of these inhibitors that will be useful in designing novel small-molecules that target a key enzyme in cancer cell metabolism.

The diamond anniversary of tissue transglutaminase: a protein of many talents.

Tissue transglutaminase (tTG) is capable of binding and hydrolyzing GTP, as well as catalyzing an enzymatic transamidation reaction that crosslinks primary amines to glutamine residues. tTG adopts two vastly different conformations, depending on whether it is functioning as a GTP-binding protein or a crosslinking enzyme. It has been shown to have important roles in several different aspects of cancer progression, making it an attractive target for therapeutic intervention. Here, we highlight many of the major findings involving tTG since its discovery 60 years ago, and describe recent drug discovery efforts that target specific activities or conformations of this unique protein.

Opening up about Tissue Transglutaminase: When Conformation Matters More than Enzymatic Activity.

Tissue transglutaminase (tTG), also referred to as type 2 transglutaminase or Gαh, can bind and hydrolyze GTP, as well as function as a protein crosslinking enzyme. tTG is widely expressed and can be detected both inside cells and in the extracellular space. In contrast to many enzymes, the active and inactive conformations of tTG are markedly different. The catalytically inactive form of tTG adopts a compact "closed-state" conformation, while the catalytically active form of the protein adopts an elongated "open-state" conformation. tTG has long been appreciated as an important player in numerous diseases, including celiac disease, neuronal degenerative diseases, and cancer, and its roles in these diseases often depend as much upon its conformation as its catalytic activity. While its ability to promote these diseases has been traditionally thought to be dependent on its protein crosslinking activity, more recent findings suggest that the conformational state tTG adopts is also important for mediating its effects. In particular, we and others have shown that the closed-state of tTG is important for promoting cell growth and survival, while maintaining tTG in the open-state is cytotoxic. In this review, we examine the two unique conformations of tTG and how they contribute to distinct biological processes. We will also describe how this information can be used to generate novel therapies to treat diseases, with a special focus on cancer.