Tofacitinib enhances delivery of antibody-based therapeutics to tumor cells through modulation of inflammatory cells.

The routes by which antibody-based therapeutics reach malignant cells are poorly defined. Tofacitinib, an FDA-approved JAK inhibitor, reduced tumor-associated inflammatory cells and allowed increased delivery of antibody-based agents to malignant cells. Alone, tofacitinib exhibited no antitumor activity, but combinations with immunotoxins or an antibody-drug conjugate resulted in increased antitumor responses. Quantification using flow cytometry revealed that antibody-based agents accumulated in malignant cells at higher percentages following tofacitinib treatment. Profiling of tofacitinib-treated tumor-bearing mice indicated that cytokine transcripts and various proteins involved in chemotaxis were reduced compared with vehicle-treated mice. Histological analysis revealed significant changes to the composition of the tumor microenvironment, with reductions in monocytes, macrophages, and neutrophils. Tumor-associated inflammatory cells contributed to non-target uptake of antibody-based therapeutics, with mice treated with tofacitinib showing decreased accumulation of therapeutics in intratumoral inflammatory cells and increased delivery to malignant cells. The present findings serve as a rationale for conducting trials where short-term treatments with tofacitinib could be administered in combination with antibody-based therapies.

Chemical Screens Identify Drugs that Enhance or Mitigate Cellular Responses to Antibody-Toxin Fusion Proteins.

The intersection of small molecular weight drugs and antibody-based therapeutics is rarely studied in large scale. Both types of agents are currently part of the cancer armamentarium. However, very little is known about how to combine them in optimal ways. Immunotoxins are antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. For fusion proteins derived from Pseudomonas exotoxin (PE), potency relies on the enzymatic domain of the toxin which catalyzes the ADP-ribosylation of EF2 causing inhibition of protein synthesis leading to cell death. Candidate immunotoxins have demonstrated clear value in clinical trials but generally have not been curative as single agents. Therefore we undertook three screens to discover effective combinations that could act synergistically. From the MIPE-3 library of compounds we identified various enhancers of immunotoxin action and at least one major class of inhibitor. Follow-up experiments confirmed the screening data and suggested that immunotoxins when administered with everolimus or nilotinib exhibit favorable combinatory activity and would be candidates for preclinical development. Mechanistic studies revealed that everolimus-immunotoxin combinations acted synergistically on elements of the protein synthetic machinery, including S61 kinase and 4E-BP1 of the mTORC1 pathway. Conversely, PARP inhibitors antagonized immunotoxins and also blocked the toxicity due to native ADP-ribosylating toxins. Thus, our goal of investigating a chemical library was justified based on the identification of several approved compounds that could be developed preclinically as 'enhancers' and at least one class of mitigator to be avoided.

Immunotoxin Therapies for the Treatment of Epidermal Growth Factor Receptor-Dependent Cancers.

Many epithelial cancers rely on enhanced expression of the epidermal growth factor receptor (EGFR) to drive proliferation and survival pathways. Development of therapeutics to target EGFR signaling has been of high importance, and multiple examples have been approved for human use. However, many of the current small molecule or antibody-based therapeutics are of limited effectiveness due to the inevitable development of resistance and toxicity to normal tissues. Recombinant immunotoxins are therapeutic molecules consisting of an antibody or receptor ligand joined to a protein cytotoxin, combining the specific targeting of a cancer-expressed receptor with the potent cell killing of cytotoxic enzymes. Over the decades, many bacterial- or plant-based immunotoxins have been developed with the goal of targeting the broad range of cancers reliant upon EGFR overexpression. Many examples demonstrate excellent anti-cancer properties in preclinical development, and several EGFR-targeted immunotoxins have progressed to human trials. This review summarizes much of the past and current work in the development of immunotoxins for targeting EGFR-driven cancers.

Targeting a Cancer-Specific Epitope of the Epidermal Growth Factor Receptor in Triple-Negative Breast Cancer.

BACKGROUND: Triple-negative breast cancers (TNBCs) are typically more aggressive and result in poorer outcomes than other breast cancers because treatment options are limited due to lack of hormone receptors or amplified human epidermal growth factor receptor 2 (HER2). Many TNBCs overexpress the epidermal growth factor receptor (EGFR) or manifest amplification of theEGFRgene, supporting EGFR as a therapeutic target. While EGFR-directed small molecule inhibitors have shown limited effectiveness in clinical settings, use of EGFR as a mechanism of delivering enzymatic cytotoxins to TNBC has not been demonstrated. METHODS: Using the single-chain variable fragment (scFv) of the 806 antibody that binds only cells with overexpressed, misfolded, or mutant variants of the EGFR, a recombinant immunotoxin was engineered through gene fusion withPseudomonas aeruginosaExotoxin A (806-PE38). The potency of 806-PE38 on reducing TNBC cell growth in vitro and in xenograft models (n ≥ 6) was examined for six TNBC cell lines. All statistical tests were two-sided. RESULTS: 806-PE38 statistically significantly reduced the viability of all tested TNBC lines, with IC50values below 10 ng/mL for three of six cell lines, while not affecting cells with wild-type EGFR (IC50>300 ng/mL). Systemic treatments with 806-PE38 vs vehicle resulted in statistically significantly reduced tumor burdens (806-PE38 mean = 128 mm(3)[SD = 46 mm(3)] vs vehicle mean = 749 mm(3)[SD = 395 mm(3)], P = .001) and increased median survival (806-PE38 median = 82 days vs vehicle median = 50 days,P= .01) in a MDA-MB-468 TNBC mouse xenograft. Deletion of the catalytic residue eliminated both cytotoxic activity in vitro and the reduction in tumor burden and survival (P= .52). CONCLUSIONS: These data support the further development of the 806-PE38 immunotoxin as a therapeutic agent for the treatment of patients with EGFR-positive TNBC. Follow-up experiments with combination therapies will be attempted to achieve full remissions.

Novel bacterial ADP-ribosylating toxins: structure and function.

Bacterial ADP-ribosyltransferase toxins (bARTTs) transfer ADP-ribose to eukaryotic proteins to promote bacterial pathogenesis. In this Review, we use prototype bARTTs, such as diphtheria toxin and pertussis toxin, as references for the characterization of several new bARTTs from human, insect and plant pathogens, which were recently identified by bioinformatic analyses. Several of these toxins, including cholix toxin (ChxA) from Vibrio cholerae, SpyA from Streptococcus pyogenes, HopU1 from Pseudomonas syringae and the Tcc toxins from Photorhabdus luminescens, ADP-ribosylate novel substrates and have unique organizations, which distinguish them from the reference toxins. The characterization of these toxins increases our appreciation of the range of structural and functional properties that are possessed by bARTTs and their roles in bacterial pathogenesis.

Bacillus cereus Certhrax ADP-ribosylates vinculin to disrupt focal adhesion complexes and cell adhesion.

Bacillus cereus is often associated with mild to moderate gastroenteritis; however, some recent isolates cause inhalational anthrax-like diseases and death. These potential emerging human pathogens express multiple virulence factors. B. cereus strain G9241 expresses anthrax toxin, several polysaccharide capsules, and the novel ADP-ribosyltransferase, Certhrax. In this study, we show that Certhrax ADP-ribosylates Arg-433 of vinculin, a protein that coordinates actin cytoskeleton and extracellular matrix interactions. ADP-ribosylation of vinculin disrupted focal adhesion complexes and redistributed vinculin to the cytoplasm. Exogenous vinculin rescued these phenotypes. This provides a mechanism for strain G9241 to breach host barrier defenses and promote bacterial growth and spread. Certhrax is the first bacterial toxin to add a post-translational modification to vinculin to disrupt the actin cytoskeleton.

The chaperone protein SmgGDS interacts with small GTPases entering the prenylation pathway by recognizing the last amino acid in the CAAX motif.

Ras family small GTPases localize at the plasma membrane, where they can activate oncogenic signaling pathways. Understanding the mechanisms that promote membrane localization of GTPases will aid development of new therapies to inhibit oncogenic signaling. We previously reported that SmgGDS splice variants promote prenylation and trafficking of GTPases containing a C-terminal polybasic region and demonstrated that SmgGDS-607 interacts with nonprenylated GTPases, whereas SmgGDS-558 interacts with prenylated GTPases in cells. The mechanism that SmgGDS-607 and SmgGDS-558 use to differentiate between prenylated and nonprenylated GTPases has not been characterized. Here, we provide evidence that SmgGDS-607 associates with GTPases through recognition of the last amino acid in the CAAX motif. We show that SmgGDS-607 forms more stable complexes in cells with nonprenylated GTPases that will become geranylgeranylated than with nonprenylated GTPases that will become farnesylated. These binding relationships similarly occur with nonprenylated SAAX mutants. Intriguingly, farnesyltransferase inhibitors increase the binding of WT K-Ras to SmgGDS-607, indicating that the pharmacological shunting of K-Ras into the geranylgeranylation pathway promotes K-Ras association with SmgGDS-607. Using recombinant proteins and prenylated peptides corresponding to the C-terminal sequences of K-Ras and Rap1B, we found that both SmgGDS-607 and SmgGDS-558 directly bind the GTPase C-terminal region, but the specificity of the SmgGDS splice variants for prenylated versus nonprenylated GTPases is diminished in vitro. Finally, we present structural homology models and data from functional prediction software to define both similar and unique features of SmgGDS-607 when compared with SmgGDS-558.

Exoenzyme S ADP-ribosylates Rab5 effector sites to uncouple intracellular trafficking.

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylates multiple eukaryotic targets to promote cytopathology and bacterial colonization. ADP-ribosylation of the small GTPase Rab5 has previously been shown to block fluid-phase endocytosis and trafficking of plasma membrane receptors to the early endosomes as well as inhibit phagocytosis of the bacterium. In this study, ExoS is shown to be capable of ADP-ribosylating 6 candidate arginine residues that are located in the effector binding region or in the C terminus of Rab5. Two Rab5 derivatives were engineered, which contained Arg→Ala mutations at four Arg residues within the effector binding region (EF) or two Arg residues within the C-terminal tail (TL). Expression of Rab5(TL) does not affect the ability of ExoS to modify intracellular trafficking, while expression of Rab5(EF) rescued the ability of ExoS to inhibit intracellular trafficking. ADP-ribosylation of effector arginines likely uncouples Rab5 signaling to downstream effectors. This is a different mechanism for inhibition than observed for the ADP-ribosylation of Ras by ExoS, where ADP-ribosylated Ras loses the ability to bind guanine nucleotide exchange factor (GEF). Other experiments showed that expression of dominant negative Rab5(Ser34Asn) does not inhibit ExoS trafficking to the perinuclear region of intoxicated cells. This study provides insight into a mechanism for how ExoS ADP-ribosylation of Rab5 inhibits Rab5 function.

Host cell cytotoxicity and cytoskeleton disruption by CerADPr, an ADP-ribosyltransferase of Bacillus cereus G9241.

Bacillus cereus G9241 was isolated from a welder suffering from an anthrax-like inhalation illness. B. cereus G9241 encodes two megaplasmids, pBCXO1 and pBC210, which are analogous to the toxin- and capsule-encoding virulence plasmids of Bacillus anthracis. Protein modeling predicted that the pBC210 LF homologue contained an ADP-ribosyltransferase (ADPr) domain. This putative bacterial ADP-ribosyltransferase domain was denoted CerADPr. Iterative modeling showed that CerADPr possessed several conserved ADP-ribosyltransferase features, including an α-3 helix, an ADP-ribosyltransferase turn-turn loop, and a "Gln-XXX-Glu" motif. CerADPr ADP-ribosylated an ~120 kDa protein in HeLa cell lysates and intact cells. EGFP-CerADPr rounded HeLa cells, elicited cytoskeletal changes, and yielded a cytotoxic phenotype, indicating that CerADPr disrupts cytoskeletal signaling. CerADPr(E431D) did not possess ADP-ribosyltransferase or NAD glycohydrolase activities and did not elicit a phenotype in HeLa cells, implicating Glu431 as a catalytic residue. These experiments identify CerADPr as a cytotoxic ADP-ribosyltransferase that disrupts the host cytoskeleton.

Adenylate cyclase activity of Pseudomonas aeruginosa ExoY can mediate bleb-niche formation in epithelial cells and contributes to virulence.

We previously showed that ADP-ribosylation (ADP-r) activity of ExoS, a type III secreted toxin of Pseudomonas aeruginosa, enables bacterial replication in corneal and respiratory epithelial cells and correlates with bacterial trafficking to plasma membrane blebs (bleb-niche formation). Here, we explored another type III secreted toxin, ExoY, for its impact on intracellular trafficking and survival, and for virulence in vivo using a murine corneal infection model. Chromosomal or plasmid-mediated expression of exoY in invasive P. aeruginosa (strain PAO1) enabled bacteria to form and traffic to epithelial membrane blebs in the absence of other known effectors. In contrast, plasmid expression of any of four adenylate cyclase mutant forms of exoY did not enable bleb-niche formation, and bacteria localized to perinuclear vacuoles as for effector-null mutant controls. None of the plasmid-complemented bacteria used in this study showed ADP-r activity in the absence of ExoS and ExoT. In contrast to ADP-r activity of ExoS, bleb-niche formation induced by ExoY's adenylate cyclase activity was not accompanied by enhanced intracellular replication. In vivo results showed that ExoY-adenylate cyclase activity promoted P. aeruginosa corneal virulence in susceptible mice. Together the data show that adenylate cyclase activity of P. aeruginosa ExoY, similarly to the ADP-r activity of ExoS, can mediate bleb-niche formation in epithelial cells. While this activity did not promote intracellular replication in vitro, ExoY conferred increased virulence in vivo in susceptible mice. Mechanisms for bleb-niche formation and relationships to intracellular replication and virulence in vivo require further investigation for both ExoS and ExoY.