My journey from tyrosine phosphorylation inhibitors to targeted immune therapy as strategies to combat cancer.

Since the 1980s there has been a drive toward personalized targeted therapy for cancer. "Targeted cancer therapy" originally focused on inhibiting essential tumor survival factors, primarily protein tyrosine kinases. The complexity and rapid mutability of tumors, however, enable them to develop resistance to tyrosine kinase inhibitors (TKIs), even when these are multitargeted or applied in combination. This has led to the development of targeted cancer immunotherapy, to enhance immune surveillance against the tumor. In this paper, we provide a personal view of the development of targeted therapy, from TKIs to targeted immunotherapy.

PSMA-targeted polyinosine/polycytosine vector induces prostate tumor regression and invokes an antitumor immune response in mice.

There is an urgent need for an effective treatment for metastatic prostate cancer (PC). Prostate tumors invariably overexpress prostate surface membrane antigen (PSMA). We designed a nonviral vector, PEI-PEG-DUPA (PPD), comprising polyethylenimine-polyethyleneglycol (PEI-PEG) tethered to the PSMA ligand, 2-[3-(1, 3-dicarboxy propyl)ureido] pentanedioic acid (DUPA), to treat PC. The purpose of PEI is to bind polyinosinic/polycytosinic acid (polyIC) and allow endosomal release, while DUPA targets PC cells. PolyIC activates multiple pathways that lead to tumor cell death and to the activation of bystander effects that harness the immune system against the tumor, attacking nontargeted neighboring tumor cells and reducing the probability of acquired resistance and disease recurrence. Targeting polyIC directly to tumor cells avoids the toxicity associated with systemic delivery. PPD selectively delivered polyIC into PSMA-overexpressing PC cells, inducing apoptosis, cytokine secretion, and the recruitment of human peripheral blood mononuclear cells (PBMCs). PSMA-overexpressing tumors in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with partially reconstituted immune systems were significantly shrunken following PPD/polyIC treatment, in all cases. Half of the tumors showed complete regression. PPD/polyIC invokes antitumor immunity, but unlike many immunotherapies does not need to be personalized for each patient. The potent antitumor effects of PPD/polyIC should spur its development for clinical use.

S101, an Inhibitor of Proliferating T Cells, Rescues Mice From Superantigen-Induced Shock.

Superantigens (SAgs) are extremely potent bacterial toxins, which evoke a virulent immune response, inducing nonspecific T-cell proliferation, rapid cytokine release, and lethal toxic shock, for which there is no effective treatment. We previously developed a small molecule, S101, which potently inhibits proliferating T cells. In a severe mouse model of toxic shock, a single injection of S101 given together with superantigen challenge rescued 100% of the mice. Even when given 2 hours after challenge, S101 rescued 40% of the mice. S101 targets the T-cell receptor, inflammatory response, and actin cytoskeleton pathways. S101 inhibits the aryl hydrocarbon receptor, a ligand-activated transcription factor that is involved in the differentiation of T-helper cells, especially Th17, and regulatory T cells. Our results provide the rationale for developing S101 to treat superantigen-induced toxic shock and other pathologies characterized by T-cell activation and proliferation.

Tyrosine kinase inhibitors: views of selectivity, sensitivity, and clinical performance.

With the manufacture of imatinib, researchers introduced tyrosine kinase inhibitors (TKIs) into the clinical setting in 2000 to treat cancers; approximately fifteen other TKIs soon followed. Imatinib remains the most successful agent, whereas all the others have had modest effects on the cancers that they target. The current challenge is to identify the agents that need to be combined with TKIs to maximize their efficacy. One of the most promising approaches is to combine immune therapy with TKI treatment. In this review, the therapeutic potential of TKIs for treatment is discussed.

On a fundamental structure of gene networks in living cells.

Computers are organized into hardware and software. Using a theoretical approach to identify patterns in gene expression in a variety of species, organs, and cell types, we found that biological systems similarly are comprised of a relatively unchanging hardware-like gene pattern. Orthogonal patterns of software-like transcripts vary greatly, even among tumors of the same type from different individuals. Two distinguishable classes could be identified within the hardware-like component: those transcripts that are highly expressed and stable and an adaptable subset with lower expression that respond to external stimuli. Importantly, we demonstrate that this structure is conserved across organisms. Deletions of transcripts from the highly stable core are predicted to result in cell mortality. The approach provides a conceptual thermodynamic-like framework for the analysis of gene-expression levels and networks and their variations in diseased cells.

EGF receptor family: twisting targets for improved cancer therapies.

The epidermal growth factor receptor (EGFR) undergoes a conformational change in response to ligand binding. The ligand-induced changes in cell surface aggregation and mobility have a profound effect on the function of all the family members. Ligand also activates the EGFR intracellular kinase, stimulating proliferation and cell survival. The EGFR family are often activated, overexpressed or mutated in cancer cells and therapeutic drugs (including antibodies) can slow the progress of some cancers. This article provides a brief, annotated summary of the presentations and discussion which occurred at the Epidermal Growth Factor Receptor - Future Directions Conference held in Jerusalem in November 2013.

Optimization of liganded polyethylenimine polyethylene glycol vector for nucleic acid delivery.

The delivery of nucleic acids into cells is an attractive approach for cancer therapy. Polyethylenimine (PEI) is among the most efficient nonviral carriers. Recent studies have demonstrated that PEI can be conjugated to targeting ligands, such as epidermal growth factor (EGF) and transferrin (Schaffert et al., 2011; Abourbeh et al., 2012; Ogris et al., 1999). Herein we present a simplified protocol for producing homogeneous preparations of PEGylated linear PEI: LPEI-PEG2k. We generated two well-characterized copolymers, with ratios of LPEI to PEG of 1:1 and 1:3. These copolymers were further conjugated through disulfide bonds to a Her-2 targeting moiety, Her-2 affibody. This reaction yielded two triconjugates that target Her-2 overexpressing tumors. Polyplexes were formed by complexing plasmid DNA with the triconjugates. We characterized the biophysical properties of the conjugates, and found that the triconjugate 1:3 polyplex had lower ζ potential, larger particle size, and more heterogeneous shape than the triconjugate 1:1 polyplex. Triconjugate 1:1 and triconjugate 1:3 polyplexes were highly selective toward cells that overexpress Her-2 receptors, but triconjugate 1:1 polyplex was more efficient at gene delivery. Our studies show that the biophysical and biological properties of the conjugates can be profoundly affected by the ratio of LPEI:PEG2k:ligand. The procedure described here can be adapted to generate a variety of triconjugates, simply by changing the targeting moiety.

Degrasyn-like symmetrical compounds: possible therapeutic agents for multiple myeloma (MM-I).

A series of degrasyn-like symmetrical compounds have been designed, synthesized, and screened against B cell malignancy (multiple myeloma, mantle cell lymphoma) cell lines. The lead compounds T5165804 and CP2005 showed higher nanomolar potency against these tumor cells in comparison to degrasyn and inhibited Usp9x activity in vitro and in intact cells. These observations suggest that this new class of compounds holds promise as cancer therapeutic agents.

Killing time for cancer cells.

As the signalling pathways that control cellular proliferation and death are unravelled, a range of targets have emerged as candidates for molecular cancer therapy. For their survival, cancer cells depend on a few highly activated signalling pathways; inhibition of these pathways has a strong apoptotic effect and can lead to tumour regression. But drugs that exploit this weakness, such as imatinib, have not cured patients: withdrawal of the drug leads to disease recurrence, and sustained treatment leads to the emergence of drug-resistant clones. Can cancer be cured, or will it have to be controlled as a chronic disease?

Information-theoretic analysis of phenotype changes in early stages of carcinogenesis.

Cancer is a multistep process characterized by altered signal transduction, cell growth, and metabolism. To identify such processes in early carcinogenesis we use an information theoretic approach to characterize gene expression quantified as mRNA levels in primary keratinocytes (K) and human papillomavirus 16 (HPV16)-transformed keratinocytes (HF1 cells) from early (E) and late (L) passages and from benzo(a)pyrene-treated (BP) L cells. Our starting point is that biological signaling processes are subjected to the same quantitative laws as inanimate, nonequilibrium chemical systems. Environmental and genomic constraints thereby limit the maximal thermodynamic entropy that the biological system can reach. The procedure uncovers the changes in gene expression patterns in different networks and defines the significance of each altered network in the establishment of a particular phenotype. The development of transformed HF1 cells is shown to be represented by one major transcription pattern that is important at all times. Two minor transcription patterns are also identified, one that contributes at early times and a distinguishably different pattern that contributes at later times. All three transcription patterns defined by our analysis were validated by gene expression values and biochemical means. The major transcription pattern includes reduced transcripts participating in the apoptotic network and enhanced transcripts participating in cell cycle, glycolysis, and oxidative phosphorylation. The two minor patterns identify genes that are mainly involved in lipid or carbohydrate metabolism.

Up-regulation of AMP-activated protein kinase in cancer cell lines is mediated through c-Src activation.

We report that the activation level of AMP-dependent protein kinase AMPK is elevated in cancer cell lines as a hallmark of their transformed state. In OVCAR3 and A431 cells, c-Src signals through protein kinase Cα, phospholipase Cγ, and LKB1 to AMPK. AMPK controls internal ribosome entry site (IRES) dependent translation in these cells. We suggest that AMPK activation via PKC might be a general mechanism to regulate IRES-dependent translation in cancer cells.

PolyIC GE11 polyplex inhibits EGFR-overexpressing tumors.

Phage display has identified the dodecapeptide YHWYGYTPQNVI (GE11) as a ligand that binds to the epidermal growth factor receptor (EGFR) but does not activate the receptor. Here, we compare the EGFR binding affinities of GE11, EGF, and their polyethyleneimine-polyethyleneglycol (PEI-PEG) conjugates. We found that although GE11 by itself does not exhibit measurable affinity to the EGFR, tethering it to PEI-PEG increases its affinity markedly, and complex formation with polyinosine/cytosine (polyIC) further enhances the affinity to the submicromolar range. PolyIC/PPGE11 has a similar strong antitumor effect against EGFR overexpressing tumors in vitro and in vivo, as polyIC/polyethyleneimine-polyetheleneglycol-EGF (polyIC/PP-EGF). Absence of EGFR activation, as previously shown by us and easier production of GE11 and GE11 conjugates, confer polyIC/PPGE11 a significant advantage over similar EGF-based polyplexes as a potential therapy of EGFR overexpressing tumors.

Studying protein-peptide interactions using benzophenone units: a case study of protein kinase B/Akt and its inhibitor PTR6154.

Protein-protein interactions (PPIs) govern nearly all processes in living cells. Peptides play an important role in studying PPIs. Peptides carrying photoaffinity labels that covalently bind the interacting protein can be used to obtain more accurate information regarding PPIs. Benzophenone (BP) is a useful photoaffinity label that is widely used to study PPIs. We developed a one-pot two-step synthesis for the preparation of novel BP units. To map the binding site more thoroughly, linkers of various lengths were attached to the BP moiety. These units can be incorporated into peptide sequences using well-established solid phase peptide synthesis (SPPS) protocols. As a proof of concept, we studied the interaction between protein kinase B (PKB/Akt) and its synthetic peptide inhibitor, PTR6154. The methodology is general and can be implemented to study PPIs in a variety of biological systems.

Microwave-assisted solid-phase aza-peptide synthesis: aza scan of a PKB/Akt inhibitor using aza-arginine and aza-proline precursors.

Aza-peptides are peptidomimetics in which one or more of the α-carbons, bearing the side-chain residues, has been replaced by a nitrogen. These peptidomimetics have been shown to be promising for the generation of drug leads and for structure-activity relationship studies. Aza-scan is the systematic replacement of amino acid residues in a given peptide with their aza counterparts. We report here an aza-scan of a potent, peptide-based PKB/Akt inhibitor, PTR6154. Procedures for microwave-assisted, Fmoc/t-Bu chemistry, solid-phase aza-peptide synthesis were developed which significantly reduce standard reaction time and are suitable for automation. Novel substituted hydrazines have been prepared for the straightforward incorporation of aza-arginine and aza-proline residues. This work will enable aza-scan to become a more common and standard method for structure-activity relationship studies of peptides.

Poly(I:C)-mediated tumor growth suppression in EGF-receptor overexpressing tumors using EGF-polyethylene glycol-linear polyethylenimine as carrier.

PURPOSE: To develop a novel polyethylenimine (PEI)-based polymeric carrier for tumor-targeted delivery of cytotoxic double-stranded RNA polyinosinic:polycytidylic acid, poly(I:C). The novel carrier should be chemically less complex but at least as effective as a previously developed tetra-conjugate containing epidermal growth factor (EGF) as targeting ligand, polyethylene glycol (PEG) as shielding spacer, 25 kDa branched PEI as RNA binding and endosomal buffering agent, and melittin as endosomal escape agent. METHODS: Novel conjugates were designed employing a simplified synthetic strategy based on 22 kDa linear polyethylenimine (LPEI), PEG spacers, and recombinant EGF. The efficacy of various conjugates (different PEG spacers, with and without targeting EGF) in poly(I:C)-mediated cell killing was evaluated in vitro using two human U87MG glioma cell lines. The most effective polyplex was tested for in vivo activity in A431 tumor xenografts. RESULTS: Targeting conjugate LPEI-PEG2 kDa-EGF was found as most effective in poly(I:C)-triggered killing of tumor cells in vitro. The efficacy correlated with glioma cell EGFR levels. Repeated intravenous administration of poly(I:C) polypexes strongly retarded growth of A431 human tumor xenograft in mice. CONCLUSIONS: The optimized LPEI-PEG2 kDa-EGF conjugate displays reduced chemical complexity and efficient poly(I:C)-mediated killing of EGFR overexpressing tumors in vitro and in vivo.

Tyrphostin-like compounds with ubiquitin modulatory activity as possible therapeutic agents for multiple myeloma.

With the goal of developing small molecules as novel regulators of signal transduction and apoptosis, a series of tyrphostin-like compounds were synthesized and screened for their activity against MM-1 (multiple myeloma) cells and other cell lines representing this malignancy. Synthesis was completed in solution-phase initially and then adopted to solid-phase for generating a more diverse set of compounds. A positive correlation was noted between compounds capable of inducing apoptosis and their modulation of protein ubiquitination. Further analysis suggested that ubiquitin modulation occurs through inhibition of cellular deubiquitinase activity. Bulky groups on the sidechain near the α,ß-unsaturated ketone caused a complete loss of activity, whereas cyclization on the opposite side was tolerated. Theoretical calculations at the B3LYP/LACV3P(∗∗) level were completed on each molecule, and the resulting molecular orbitals and Fukui reactivity values for C(ß) carbon were utilized in developing a model to explain the compound activity.

Amino acid starvation sensitizes cancer cells to proteasome inhibition.

We explored the crosstalk between protein degradation and synthesis in cancer cells. The tumorigenic cell line, MCF7, showed enhanced proteasome activity compared to the nontumorigenic line, MCF10A. Although there was no difference in the sensitivity of MCF7 and MCF10A cells to proteasome inhibition in complete growth medium, combining proteasome inhibition with amino acid deprivation led to reduced protein synthesis and survival of MCF7 cells, with a lesser effect on MCF10A cells. Additional cancer cell lines (including CAG and A431) could be strongly sensitized to proteasome inhibition by concomitant amino acid deprivation, whereas others were completely resistant to proteasome inhibition. We hypothesize that protein catabolism contributes to the pool of free amino acids available for protein synthesis, leading to a crucial role of the proteasome in cell survival during amino acid depletion, in some tumor cell lines.

Design, synthesis, and evaluation of quinazoline T cell proliferation inhibitors.

We report here on a class of quinazoline molecules that inhibit T cell proliferation. The most potent compound N-p-tolyl-2-(3,4,5-trimethoxyphenyl)quinazolin-4-amine (S101) and its close analogs were found to inhibit the proliferation of T cells from human peripheral blood mononuclear cells (PBMC) and Jurkat cells, with IC(50) in the sub-micromolar range. The inhibitor induced G2 cell cycle arrest but did not inhibit IL-2 secretion. The anti-proliferative effect correlated with inhibition of the tyrosine phosphorylation of SLP-76, a molecular element in the signaling pathway of the T cell receptor (TCR). The inhibitor restrained proliferation of lymphocytes with much higher potency than non-hematopoietic cells. This new class of specific T cell proliferation inhibitors may serve as lead molecules for the development of agents aimed at diseases in which T cell signaling plays a role and agents to induce tolerance to grafted tissues or organs.

Synthesis and structure-activity relationship studies of peptidomimetic PKB/Akt inhibitors: the significance of backbone interactions.

Elevated levels of activated Protein Kinase B (PKB/Akt) have been detected in many types of human cancer. In contrast to ATP site inhibitors, substrate-based inhibitors are more likely to be selective because of extensive interactions with the specific substrate binding site. Unfortunately, peptide-based inhibitors lack important pharmacological properties that are required of drug candidates. Chemical modifications of potent peptide inhibitors, such as peptoids and N(alpha)-methylated amino acids, may overcome these drawbacks, while maintaining potency. We present a structure-activity relationship study of a potent, peptide-based PKB/Akt inhibitor, PTR6154. The study was designed to evaluate backbone modifications on the inhibitory activity of PTR6154. Two peptidomimetic libraries, peptoid and N(alpha)-methylation, based on PTR6154, were synthesized and evaluated for in vitro PKB/Akt inhibition efficiency. All the peptoid analogs reduced potency significantly, as well as most of the members of the N-methyl library, suggesting that the backbone conformation and/or hydrogen bond interactions of PTR6154 derivatives are essential for inhibition activity. Two N-terminal members of the N-methyl library did not decrease potency and can be used as future drug leads.

Novel method for the synthesis of urea backbone cyclic peptides using new Alloc-protected glycine building units.

Cyclization of bioactive peptides, utilizing functional groups serving as natural pharmacophors, is often accompanied with loss of activity. The backbone cyclization approach was developed to overcome this limitation and enhance pharmacological properties. Backbone cyclic peptides are prepared by the incorporation of special building units, capable of forming amide, disulfide and coordinative bonds. Urea bridge is often used for the preparation of cyclic peptides by connecting two amine functionalized side chains. Here we present urea backbone cyclization as an additional method for the preparation of backbone cyclic peptide libraries. A straightforward method for the synthesis of crystalline Fmoc-N(alpha) [omega-amino(Alloc)-alkyl] glycine building units is presented. A set of urea backbone cyclic Glycogen Synthase Kinase 3 analogs was prepared and assessed for protein kinase B inhibition as anticancer leads.