Hepatocellular Carcinoma: Old and Emerging Therapeutic Targets.

Liver cancer, predominantly hepatocellular carcinoma (HCC), globally ranks sixth in incidence and third in cancer-related deaths. HCC risk factors include non-viral hepatitis, alcohol abuse, environmental exposures, and genetic factors. No specific genetic alterations are unequivocally linked to HCC tumorigenesis. Current standard therapies include surgical options, systemic chemotherapy, and kinase inhibitors, like sorafenib and regorafenib. Immunotherapy, targeting immune checkpoints, represents a promising avenue. FDA-approved checkpoint inhibitors, such as atezolizumab and pembrolizumab, show efficacy, and combination therapies enhance clinical responses. Despite this, the treatment of hepatocellular carcinoma (HCC) remains a challenge, as the complex tumor ecosystem and the immunosuppressive microenvironment associated with it hamper the efficacy of the available therapeutic approaches. This review explores current and advanced approaches to treat HCC, considering both known and new potential targets, especially derived from proteomic analysis, which is today considered as the most promising approach. Exploring novel strategies, this review discusses antibody drug conjugates (ADCs), chimeric antigen receptor T-cell therapy (CAR-T), and engineered antibodies. It then reports a systematic analysis of the main ligand/receptor pairs and molecular pathways reported to be overexpressed in tumor cells, highlighting their potential and limitations. Finally, it discusses TGFß, one of the most promising targets of the HCC microenvironment.

SerpinB3 Upregulates Low-Density Lipoprotein Receptor-Related Protein (LRP) Family Members, Leading to Wnt Signaling Activation and Increased Cell Survival and Invasiveness.

Abnormal activation of the Wnt-ß-catenin signaling cascade is involved in tumor growth and dissemination. SerpinB3 has been shown to induce ß-catenin, and both molecules are overexpressed in tumors, particularly in those with poor prognoses. The aim of this study was to evaluate the ability of SerpinB3 to modulate the Wnt pathway in liver cancer and in monocytic cells, the main type of inflammatory cells in the tumor microenvironment. The Wnt cascade, Wnt co-receptors, and low-density lipoprotein receptor-related protein (LRP) members were analyzed in different cell lines and human monocytes in the presence or absence of SerpinB3. The Wnt-ß-catenin axis was also evaluated in liver tumors induced in mice with different extents of SeprinB3 expression. In monocytic cells, SerpinB3 induced a significant upregulation of Wnt-1/7, nuclear ß-catenin, and c-Myc, which are associated with increased cell lifespan and proliferation. In liver tumors in mice, the expression of ß-catenin was significantly correlated with the presence of SerpinB3. In hepatoma cells, Wnt co-receptors LRP-5/6 and LRP-1, implicated in cell survival and invasiveness, were upregulated by SerpinB3. The LRP pan-inhibitor RAP not only induced a decrease in LRP expression, but also a dose-dependent reduction in SerpinB3-induced invasiveness. In conclusion, SerpinB3 determines the activation of the Wnt canonical pathway and cell invasiveness through the upregulation of LRP family members.

Asparagine and Glutamine Deprivation Alters Ionizing Radiation Response, Migration and Adhesion of a p53null Colorectal Cancer Cell Line.

Colorectal cancer (CRC) is the most prominent form of colon cancer for both incidence (38.7 per 100,000 people) and mortality (13.9 per 100,000 people). CRC's poor response to standard therapies is linked to its high heterogeneity and complex genetic background. Dysregulation or depletion of the tumor suppressor p53 is involved in CRC transformation and its capability to escape therapy, with p53null cancer subtypes known, in fact, to have a poor prognosis. In such a context, new therapeutic approaches aimed at reducing CRC proliferation must be investigated. In clinical practice, CRC chemotherapy is often combined with radiation therapy with the aim of blocking the expansion of the tumor mass or removing residual cancer cells, though contemporary targeting of amino acid metabolism has not yet been explored. In the present study, we used the p53null Caco-2 model cell line to evaluate the effect of a possible combination of radiation and L-Asparaginase (L-ASNase), a protein drug that blocks cancer proliferation by impairing asparagine and glutamine extracellular supply. When L-ASNase was administered immediately after IR, we observed a reduced proliferative capability, a delay in DNA-damage response and a reduced capability to adhere and migrate. Our data suggest that a correctly timed combination of X-rays and L-ASNase treatment could represent an advantage in CRC therapy.

Structural Aspects of E. coli Type II Asparaginase in Complex with Its Secondary Product L-Glutamate.

Bacterial L-asparaginases are amidohydrolases (EC 3.5.1.1) capable of deaminating L-asparagine and, with reduced efficiency, L-glutamine. Interest in the study of L-asparaginases is driven by their use as biodrugs for the treatment of acute lymphoblastic leukemia. Here, we report for the first time the description of the molecular structure of type II asparaginase from Escherichia coli in complex with its secondary product, L-glutamate. To obtain high-quality crystals, we took advantage of the N24S variant, which has structural and functional features similar to the wild-type enzyme, but improved stability, and which yields more ordered crystals. Analysis of the structure of the N24S-L-glutamate complex (N24S-GLU) and comparison with its apo and L-aspartate-bound form confirmed that the enzyme-reduced catalytic efficiency in the presence of L-glutamine is due to L-glutamine misfitting into the enzyme-binding pocket, which causes a local change in the catalytic center geometry. Moreover, a tight interaction between the two protomers that form the enzyme active site limits the capability of L-glutamine to fit into (and to exit from) the binding pocket of E. coli L-asparaginase, explaining why the enzyme has lower glutaminolytic activity compared to other enzymes of the same family, in particular the Erwinia chrysanthemi one.

A Targeted Catalytic Nanobody (T-CAN) with Asparaginolytic Activity.

E. coli L-asparaginase is an amidohydrolase (EC 3.5.1.1) which has been successfully used for the treatment of Acute Lymphoblastic Leukemia for over 50 years. Despite its efficacy, its side effects, and especially its intrinsic immunogenicity, hamper its usage in a significant subset of cases, thus limiting therapeutic options. Innovative solutions to improve on these drawbacks have been attempted, but none of them have been truly successful so far. In this work, we fully replaced the enzyme scaffold, generating an active, miniaturized form of L-asparaginase by protein engineering of a camel single domain antibody, a class of antibodies known to have a limited immunogenicity in humans. We then targeted it onto tumor cells by an antibody scFv fragment directed onto the CD19 B-cell surface receptor expressed on ALL cells. We named this new type of nanobody-based antibody-drug conjugate "Targeted Catalytic Nanobody" (T-CAN). The new molecule retains the catalytic activity and the binding capability of the original modules and successfully targets CD19 expressing cells in vitro. Thanks to its theoretically reduced immunogenic potential compared to the original molecule, the T-CAN can represent a novel approach to tackle current limitations in L-asparaginase usage.

Revealing Escherichia coli type II L-asparaginase active site flexible loop in its open, ligand-free conformation.

Since 1993, when the structure of Escherichia coli type II L-asparaginase (EcAII) in complex with L-aspartate was firstly reported, many structures of the wild type and mutated enzyme have been deposited in the Protein Data Bank. None of them report the full structure of the monomer in its ligand-free, open conformation, mainly because of the high dynamic and flexibility of the active site flexible loop. Here we report for the first time the structure of EcAII wild type in its open conformation comprising, for at least one protomer, clear electron density for the active site flexible loop (PDB ID: 6YZI). The structural element is highly mobile and it is transposed onto the rigid part of the active site upon substrate binding to allow completion of the enzyme catalytic center, thanks to key residues that serve as hinges and anchoring points. In the substrate binding pocket, several highly conserved water molecules are coordinated by residues involved in substrate binding, comprising two water molecules very likely involved in the enzyme catalytic process. We also describe, by molecular dynamics simulations, how the transposition of the loop, besides providing the proximity of residues needed for catalysis, causes a general stabilization of the protein.

Anti-Cancer Auto-Antibodies: Roles, Applications and Open Issues.

Auto-antibodies are classically associated with autoimmune diseases, where they are an integral part of diagnostic panels. However, recent evidence is accumulating on the presence of auto-antibodies against single or selected panels of auto-antigens in many types of cancer. Auto-antibodies might initially represent an epiphenomenon derived from the inflammatory environment induced by the tumor. However, their effect on tumor evolution can be crucial, as is discussed in this paper. It has been demonstrated that some of these auto-antibodies can be used for early detection and cancer staging, as well as for monitoring of cancer regression during treatment and follow up. Interestingly, certain auto-antibodies were found to promote cancer progression and metastasis, while others contribute to the body's defense against it. Moreover, auto-antibodies are of a polyclonal nature, which means that often several antibodies are involved in the response to a single tumor antigen. Dissection of these antibody specificities is now possible, allowing their identification at the genetic, structural, and epitope levels. In this review, we report the evidence available on the presence of auto-antibodies in the main cancer types and discuss some of the open issues that still need to be addressed by the research community.

High resolution structure of human apolipoprotein (a) kringle IV type 2: beyond the lysine binding site.

Lipoprotein (a) [Lp(a)] is characterized by an LDL-like composition in terms of lipids and apoB100, and by one copy of a unique glycoprotein, apo(a). The apo(a) structure is mainly based on the repetition of tandem kringle domains with high homology to plasminogen kringles 4 and 5. Among them, kringle IV type 2 (KIV-2) is present in a highly variable number of genetically encoded repeats, whose length is inversely related to Lp(a) plasma concentration and cardiovascular risk. Despite it being the major component of apo(a), the actual function of KIV-2 is still unclear. Here, we describe the first high-resolution crystallographic structure of this domain. It shows a general fold very similar to other KIV domains with high and intermediate affinity for the lysine analog, ε-aminocaproic acid. Interestingly, KIV-2 presents a lysine binding site (LBS) with a unique shape and charge distribution. KIV-2 affinity for predicted small molecule binders was found to be negligible in surface plasmon resonance experiments; and with the LBS being nonfunctional, we propose to rename it "pseudo-LBS". Further investigation of the protein by computational small-molecule docking allowed us to identify a possible heparin-binding site away from the LBS, which was confirmed by specific reverse charge mutations abolishing heparin binding. This study opens new possibilities to define the pathogenesis of Lp(a)-related diseases and to facilitate the design of specific therapeutic drugs.

Enzymes in Metabolic Anticancer Therapy.

Cancer treatment has greatly improved over the last 50 years, but it remains challenging in several cases. Useful therapeutic targets are normally unique peculiarities of cancer cells that distinguish them from normal cells and that can be tackled with appropriate drugs. It is now known that cell metabolism is rewired during tumorigenesis and metastasis as a consequence of oncogene activation and oncosuppressors inactivation, leading to a new cellular homeostasis typically directed towards anabolism. Because of these modifications, cells can become strongly or absolutely dependent on specific substrates, like sugars, lipids or amino acids. Cancer addictions are a relevant target for therapy, as removal of an essential substrate can lead to their selective cell-cycle arrest or even to cell death, leaving normal cells untouched. Enzymes can act as powerful agents in this respect, as demonstrated by asparaginase, which has been included in the treatment of Acute Lymphoblastic Leukemia for half a century. In this review, a short outline of cancer addictions will be provided, focusing on the main cancer amino acid dependencies described so far. Therapeutic enzymes which have been already experimented at the clinical level will be discussed, along with novel potential candidates that we propose as new promising molecules. The intrinsic limitations of their present molecular forms, along with molecular engineering solutions to explore, will also be presented.

Helicobacter pylori L-asparaginase: A Novel Bacterial Antigen that May Contribute to Infection Detection.

Helicobacter pylori is responsible for gastric inflammation and for an increased risk of cancer development in humans. Several bacterial antigens contribute to stimulate the immune system, but their relative role has not yet been defined. H. pylori (strain CCUG) type II L-asparaginase (L-ASNase) induces an immune response in mice. To verify if an immune response could also be detected in humans, sera positive (n=11) or negative (n=11), respectively, to H. pylori according to a commercial test were assayed for their reactivity towards purified H. pylori L-ASNase. Among positive samples, 8/11 (72%) were positive to L-ASNase. We conclude that H. pylori L-ASNase is immunogenic in humans and contributes to the generation of the antibody response induced by the bacterium.

The phosphoglycerate kinase 1 variants found in carcinoma cells display different catalytic activity and conformational stability compared to the native enzyme.

Cancer cells are able to survive in difficult conditions, reprogramming their metabolism according to their requirements. Under hypoxic conditions they shift from oxidative phosphorylation to aerobic glycolysis, a behavior known as Warburg effect. In the last years, glycolytic enzymes have been identified as potential targets for alternative anticancer therapies. Recently, phosphoglycerate kinase 1 (PGK1), an ubiquitous enzyme expressed in all somatic cells that catalyzes the seventh step of glycolysis which consists of the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to ADP, has been discovered to be overexpressed in many cancer types. Moreover, several somatic variants of PGK1 have been identified in tumors. In this study we analyzed the effect of the single nucleotide variants found in cancer tissues on the PGK1 structure and function. Our results clearly show that the variants display a decreased catalytic efficiency and/or thermodynamic stability and an altered local tertiary structure, as shown by the solved X-ray structures. The changes in the catalytic properties and in the stability of the PGK1 variants, mainly due to the local changes evidenced by the X-ray structures, suggest also changes in the functional role of PGK to support the biosynthetic need of the growing and proliferating tumour cells.

A protease-resistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro.

L-Asparaginases (ASNases) have been used as first line drugs for paediatric Acute Lymphoblastic Leukaemia (ALL) treatment for more than 40 years. Both the Escherichia coli (EcAII) and Erwinia chrysanthemi (ErAII) type II ASNases currently used in the clinics are characterized by high in vivo instability, short half-life and the requirement of several administrations to obtain a pharmacologically active concentration. Moreover, they are sensitive to proteases (cathepsin B and asparagine endopeptidase) that are over-expressed by resistant leukaemia lymphoblasts, thereby impairing drug activity and pharmacokinetics. Herein, we present the biochemical, structural and in vitro antiproliferative characterization of a new EcAII variant, N24S. The mutant shows completely preserved asparaginase and glutaminase activities, long-term storage stability, improved thermal parameters, and outstanding resistance to proteases derived from leukaemia cells. Structural analysis demonstrates a modification in the hydrogen bond network related to residue 24, while Normal Mode-based geometric Simulation and Molecular Dynamics predict a general rigidification of the monomer as compared to wild-type. These improved features render N24S a potential alternative treatment to reduce the number of drug administrations in vivo and to successfully address one of the major current challenges of ALL treatment: spontaneous, protease-dependent and immunological inactivation of ASNase.

Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies.

Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).

Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes.

Glutaminase activity determines cytotoxicity of L-asparaginases on most leukemia cell lines.

L-Asparaginase (ASNase) is a front-line chemotherapy for acute lymphoblastic leukemia (ALL), which acts by deaminating asparagine and glutamine. To evaluate the importance of glutaminase activity, we exploited a recently developed mutant of Helicobacter pylori ASNase (dm HpA), with amino acid substitutions M121C/T169M. The mutant form has the same asparaginase activity as wild-type but lacks glutaminase activity. Wild-type and dm HpA were compared with the clinically used ASNases from Escherichia coli (l-ASP) and Erwinia chrysanthemi (ERWase). Asparaginase activity was similar for all isoforms, while glutaminase activity followed the rank order: ERWase>l-ASP>wild-type HpA>dm HpA. Cytotoxic efficacy of ASNases was tested on 11 human leukemia cell lines and two patient-derived ALL samples. Two cell lines which we had previously shown to be asparagine-dependent were equally sensitive to the asparaginase isoforms. The other nine lines and the two patient-derived samples were more sensitive to isoforms with higher glutaminase activities. ERWase was overall the most effective ASNase on all cell lines tested whereas dm HpA, having the lowest glutaminase activity, was the least effective. These data demonstrate that asparaginase activity alone may not be sufficient for ASNase cytotoxicity, and that glutaminase activity may be required for full anti-leukemic efficacy.

Tackling Critical Catalytic Residues in Helicobacter pylori L-Asparaginase.

Bacterial asparaginases (amidohydrolases, EC 3.5.1.1) are important enzymes in cancer therapy, especially for Acute Lymphoblastic Leukemia. They are tetrameric enzymes able to catalyze the deamination of L-ASN and, to a variable extent, of L-GLN, on which leukemia cells are dependent for survival. In contrast to other known L-asparaginases, Helicobacter pylori CCUG 17874 type II enzyme (HpASNase) is cooperative and has a low affinity towards L-GLN. In this study, some critical amino acids forming the active site of HpASNase (T16, T95 and E289) have been tackled by rational engineering in the attempt to better define their role in catalysis and to achieve a deeper understanding of the peculiar cooperative behavior of this enzyme. Mutations T16E, T95D and T95H led to a complete loss of enzymatic activity. Mutation E289A dramatically reduced the catalytic activity of the enzyme, but increased its thermostability. Interestingly, E289 belongs to a loop that is very variable in L-asparaginases from the structure, sequence and length point of view, and which could be a main determinant of their different catalytic features.

Clinical Severity of PGK1 Deficiency Due To a Novel p.E120K Substitution Is Exacerbated by Co-inheritance of a Subclinical Translocation t(3;14)(q26.33;q12), Disrupting NUBPL Gene.

Carriers of cytogenetically similar, apparently balanced familial chromosome translocations not always exhibit the putative translocation-associated disease phenotype. Additional genetic defects, such as genomic imbalance at breakpoint regions or elsewhere in the genome, have been reported as the most plausible explanation.By means of comprehensive molecular and functional analyses, additional to careful dissection of the t(3;14)(q26.33;q12) breakpoints, we unveil a novel X-linked PGK1 mutation and examine the contribution of these to the extremely severe clinical phenotype characterized by hemolytic anemia and neuromyopathy.The 3q26.33 breakpoint is 40 kb from the 5' region of tetratricopeptide repeat domain 14 gene (TTC14), whereas the 14q12 breakpoint is within IVS6 of nucleotide-binding protein-like gene (NUBPL) that encodes a mitochondrial complex I assembly factor. Disruption of NUBPL in translocation carriers leads to a decrease in the corresponding mRNA accompanied by a decrease in protein level. Exclusion of pathogenic genomic imbalance and reassessment of familial clinical history indicate the existence of an additional causal genetic defect. Consequently, by WES a novel mutation, c.358G>A, p.E120K, in the X-linked phosphoglycerate kinase 1 (PGK1) was identified that segregates with the phenotype. Specific activity, kinetic properties, and thermal stability of this enzyme variant were severely affected. The novel PGK1 mutation is the primary genetic alteration underlying the reported phenotype as the translocation per se only results in a subclinical phenotype. Nevertheless, its co-inheritance presumably exacerbates PGK1-deficient phenotype, most likely due to a synergistic interaction of the affected genes both involved in cell energy supply.

Engineering of Helicobacter pylori L-asparaginase: characterization of two functionally distinct groups of mutants.

Bacterial L-asparaginases have been used as anti-cancer drugs for over 4 decades though presenting, along with their therapeutic efficacy, several side effects due to their bacterial origin and, seemingly, to their secondary glutaminase activity. Helicobacter pylori type II L-asparaginase possesses interesting features, among which a reduced catalytic efficiency for L-GLN, compared to the drugs presently used in therapy. In the present study, we describe some enzyme variants with catalytic and in vitro cytotoxic activities different from the wild type enzyme. Particularly, replacements on catalytic threonines (T16D and T95E) deplete the enzyme of both its catalytic activities, once more underlining the essential role of such residues. One serendipitous mutant, M121C/T169M, had a preserved efficiency vs L-asparagine but was completely unable to carry out L-glutamine hydrolysis. Interestingly, this variant did not exert any cytotoxic effect on HL-60 cells. The M121C and T169M single mutants had reduced catalytic activities (nearly 2.5- to 4-fold vs wild type enzyme, respectively). Mutant Q63E, endowed with a similar catalytic efficiency versus asparagine and halved glutaminase efficiency with respect to the wild type enzyme, was able to exert a cytotoxic effect comparable to, or higher than, the one of the wild type enzyme when similar asparaginase units were used. These findings may be relevant to determine the role of glutaminase activity of L-asparaginase in the anti-proliferative effect of the drug and to shed light on how to engineer the best asparaginase/glutaminase combination for an ever improved, patients-tailored therapy.