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.

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.

HER2-Targeted Polyinosine/Polycytosine Therapy Inhibits Tumor Growth and Modulates the Tumor Immune Microenvironment.

The development of targeted therapies that affect multiple signaling pathways and stimulate antitumor immunity is greatly needed. About 20% of patients with breast cancer overexpress HER2. Small molecules and antibodies targeting HER2 convey some survival benefits; however, patients with advanced disease succumb to the disease under these treatment regimens, possibly because HER2 is not completely necessary for the survival of the targeted cancer cells. In the present study, we show that a polyinosine/polycytosine (pIC) HER2-homing chemical vector induced the demise of HER2-overexpressing breast cancer cells, including trastuzumab-resistant cells. Targeting pIC to the tumor evoked a number of cell-killing mechanisms, as well as strong bystander effects. These bystander mechanisms included type I IFN induction, immune cell recruitment, and activation. The HER2-targeted pIC strongly inhibited the growth of HER2-overexpressing tumors in immunocompetent mice. The data presented here could open additional avenues in the treatment of HER2-positive breast cancer. Cancer Immunol Res; 4(8); 688-97. ©2016 AACR.

Intrinsically active variants of Erk oncogenically transform cells and disclose unexpected autophosphorylation capability that is independent of TEY phosphorylation.

The receptor-tyrosine kinase (RTK)/Ras/Raf pathway is an essential cascade for mediating growth factor signaling. It is abnormally overactive in almost all human cancers. The downstream targets of the pathway are members of the extracellular regulated kinases (Erk1/2) family, suggesting that this family is a mediator of the oncogenic capability of the cascade. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Here we used spontaneously active Erk variants to check whether Erk's activity per se is sufficient for oncogenic transformation. We show that Erk1(R84S) is an oncoprotein, as NIH3T3 cells that express it form foci in tissue culture plates, colonies in soft agar, and tumors in nude mice. We further show that Erk1(R84S) and Erk2(R65S) are intrinsically active due to an unusual autophosphorylation activity they acquire. They autophosphorylate the activatory TEY motif and also other residues, including the critical residue Thr-207 (in Erk1)/Thr-188 (in Erk2). Strikingly, Erk2(R65S) efficiently autophosphorylates its Thr-188 even when dually mutated in the TEY motif. Thus this study shows that Erk1 can be considered a proto-oncogene and that Erk molecules possess unusual autoregulatory properties, some of them independent of TEY phosphorylation.

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.

Therapeutic destruction of insulin receptor substrates for cancer treatment.

Insulin receptor substrates 1 and 2 (IRS1/2) mediate mitogenic and antiapoptotic signaling from insulin-like growth factor 1 receptor (IGF-IR), insulin receptor (IR), and other oncoproteins. IRS1 plays a central role in cancer cell proliferation, its expression is increased in many human malignancies, and its upregulation mediates resistance to anticancer drugs. IRS2 is associated with cancer cell motility and metastasis. Currently, there are no anticancer agents that target IRS1/2. We present new IGF-IR/IRS-targeted agents (NT compounds) that promote inhibitory Ser-phosphorylation and degradation of IRS1 and IRS2. Elimination of IRS1/2 results in long-term inhibition of IRS1/2-mediated signaling. The therapeutic significance of this inhibition in cancer cells was shown while unraveling a novel mechanism of resistance to B-RAF(V600E/K) inhibitors. We found that IRS1 is upregulated in PLX4032-resistant melanoma cells and in cell lines derived from patients whose tumors developed PLX4032 resistance. In both settings, NT compounds led to the elimination of IRS proteins and evoked cell death. Treatment with NT compounds in vivo significantly inhibited the growth of PLX4032-resistant tumors and displayed potent antitumor effects in ovarian and prostate cancers. Our findings offer preclinical proof-of-concept for IRS1/2 inhibitors as cancer therapeutics including PLX4032-resistant melanoma. By the elimination of IRS proteins, such agents should prevent acquisition of resistance to mutated-B-RAF inhibitors and possibly restore drug sensitivity in resistant tumors.

Targeted cancer immunotherapy.

The understanding that the immune system plays a dual role in cancer progression has led to the recent development of targeted immunotherapies. These treatments, which aim to harness the immune system against cancer, include monoclonal antibodies, immune adjuvants, cell-based therapy and vaccines. Although numerous immune-targeted treatment modalities have entered the clinic, most have shown limited efficacy. The intrinsic heterogeneity and genomic instability of the tumor, coupled with immune suppression induced by both the tumor and its microenvironment, remain the main obstacles to the success of these therapies. We believe that the primary objective of the new generation of therapies must be to reinstate immune surveillance against primary and metastatic tumor cells, while inhibiting the immune suppressive microenvironment. Most probably this will be achieved by combining several treatment modalities. This paper will briefly review current immunotherapies and their promise, as well as the obstacles associated with them.

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.

Nucleic acid-based therapeutics for glioblastoma.

Nucleic acid based therapeutics offer the possibility of tailor-made treatment of malignant diseases. For recurrent glioblastoma multiforme (GBM), the most aggressive type of brain tumor, no accepted treatment exists, making therapeutically active nucleic acids a viable option. In this review, current preclinical and clinical studies harnessing the potential of antitumoral nucleic acids for GBM treatment will be considered. These include gene therapy to over-express antitumoral gene products, RNA interference to knock down components that promote tumor progression, and the tumor-targeted delivery of antitumoral double stranded RNA. Vectors applied in GBM for the delivery of nucleic acids will be discussed. These include non-replicating and replicating (oncolytic) viruses, as well as non-viral delivery vectors based on polycations or cationic lipids.

EGFR-homing dsRNA activates cancer-targeted immune response and eliminates disseminated EGFR-overexpressing tumors in mice.

PURPOSE: The cause of most cancer deaths is incurable dissemination of cancer cells into vital organs. Current systemic therapies for disseminated cancers provide limited efficacy and are often accompanied by toxic side effects. We have recently shown that local application of epidermal growth factor receptor (EGFR)-targeted polyinosine-cytosine (polyIC) eradicates preestablished EGFR-overexpressing tumors. Here we show for the first time the high efficiency of systemic application of polyIC/melittin-polyethyleneimine-polyethyleneglycol-EGF (polyIC/MPPE) in combination with human immune cells. EXPERIMENTAL DESIGN: Cancer-targeted activation of immune cells was examined in vitro and in vivo following transfection with polyIC/MPPE. The therapeutic efficiency of the strategy was then examined on disseminated EGFR-overexpressing tumors grown in severe combined immunodeficient (SCID) mice. RESULTS: Intravenous delivery of polyIC/MPPE followed by intraperitoneal injection of peripheral blood mononuclear cells induced the complete cure of SCID mice with preestablished disseminated EGFR-overexpressing tumors, with no adverse toxic effects. The immune cells and the cytokines they produce are localized to the tumor site of the treated animal and contribute decisively to the demise of the tumor cells. The immune system homes to the tumors, due to the chemokines produced by the internalized polyIC. CONCLUSION: The EGFR-homing vector loaded with polyIC can be used to treat and possibly cure patients with disseminated EGFR-overexpressing tumors. The possibility of adopting this strategy to treat other tumors that express a protein capable of ligand induced internalization is discussed.

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.

EGF receptor-targeted synthetic double-stranded RNA eliminates glioblastoma, breast cancer, and adenocarcinoma tumors in mice.

BACKGROUND: Glioblastoma multiforme (GBM) is the most lethal form of brain cancer. With the available treatments, survival does not exceed 12-14 mo from the time of diagnosis. We describe a novel strategy to selectively induce the death of glioblastoma cells and other cancer cells that over-express the EGF receptor. Using a non-viral delivery vector that homes to the EGF receptor, we target synthetic anti-proliferative dsRNA (polyinosine-cytosine [poly IC]), a strong activator of apoptosis, selectively to cancer cells. METHODS AND FINDINGS: Poly IC was delivered by means of a non-viral vector: 25kDa polyethylenimine-polyethyleneglycol-EGF (PEI25-PEG-EGF). EGFR-targeted poly IC induced rapid apoptosis in the target cells in vitro and in vivo. Expression of several cytokines and "bystander killing" of untransfected tumor cells was detected in vitro and in vivo. Intra-tumoral delivery of the EGFR-targeted poly IC induced the complete regression of pre-established intracranial tumors in nude mice, with no obvious adverse toxic effects on normal brain tissue. A year after treatment completion the treated mice remain cancer-free and healthy. Similarly, non-viral delivery of poly IC completely eliminated pre-established breast cancer and adenocarcinoma xenografts derived from EGFR over-expressing cancer cell lines, suggesting that the strategy is applicable to other EGFR-over-expressing tumors. CONCLUSION: The strategy described has yielded an effective treatment of EGFR over-expressing GBM in an animal model. If this strategy is translated successfully to the clinical setting, it may actually offer help to GBM patients. Moreover the elimination of two additional EGFR over-expressing cancers in vivo suggests that in principle this strategy can be applied to treat other tumors that over-express EGFR.

RNA molecules as anti-cancer agents.

The inhibition of cancer growth and progression is one of the major challenges facing modern medicine. Despite significant progress in the development of therapies against cancer, only in a few cases are these therapies effective. Because cancer is a complex disease, agents that target a single oncogenic pathway have low efficacy, in addition to allowing the emergence of drug resistance. There is a need for specific therapy, which can affect a broad range of cancers, with minimal side effects. Here we summarize several novel anti-cancer strategies that answer the above criteria. These strategies utilize the multiple anti-proliferative effects of double stranded RNA (dsRNA): (1) the classical antisense RNA hybridizes with its target mRNA, leading to reduced levels of a specific oncoprotein; (2) short dsRNA of specific sequence, known as small inhibitory RNA (siRNA), can selectively and efficiently inhibit expression of specific oncogenes, expressed in cancer cells but not in normal cells. Shutting down the expression of cancer-promoting genes by siRNA has proven to be an effective approach against several cancers; (3) long dsRNA, frequently expressed in cells infected with viruses, activates mechanisms that efficiently kill the infected cells, thereby preventing spread of the virus. The dsRNA killing strategy (DKS), involving the in situ generation of dsRNA of sufficient length to induce antiviral defenses specifically in cancer cells, is a novel strategy developed in our laboratory. DKS has the potential to be applicable to a wide range of cancers. Thus dsRNA-based anti-cancer strategies could be powerful tools for cancer treatment.

Tumor specific activation of PKR as a non-toxic modality of cancer treatment.

Over the past decade progress has been made in the development of therapies against cancer. Small molecules, mainly tyrosine kinase inhibitors (tyrphostins) like Gleevec, Iressa targeting CML and EGFR overexpressing tumors have entered the clinic, where a large number of other tyrphostins are at various stages of clinical development. In parallel a few antibodies like Herceptin targeting breast cancer overexpressing Her-2 and Rituxan targeting B cell malignancies are utilized in the clinic. In all these cases success is moderate and restricted to a narrow population of patients, except for Gleevec which is effective for a long duration for chronic CML. The cancer community agrees that this is actually a unique exception that proves the rule. Over the past few years a few modalities of cancer gene therapies have emerged. In this short review we shall summarize our efforts to develop methods to activate PKR selectively in cancer cells.

Inhibition of glioma growth by tumor-specific activation of double-stranded RNA-dependent protein kinase PKR.

Activated double-stranded RNA (dsRNA-dependent protein kinase PKR is a potent growth inhibitory protein that is primarily activated in virally infected cells, inducing cell death. Here we investigate whether selective activation of PKR can be used to kill cancer cells that express mutated genes containing deletions or chromosomal translocations. We show that antisense (AS) RNA complementary to fragments flanking the deletion or translocation can produce a dsRNA molecule of sufficient length to activate PKR and induce cell death following hybridization with mutated but not wild-type mRNA. Using the U87MG Delta EGFR cell line, which expresses a truncated form of epidermal growth factor receptor (EGFR), Delta(2-7) EGFR, we found that expression of a 39-nucleotide (nt) AS RNA complementary to the unique exon 1 to 8 junction caused selective death of cells harboring the truncated EGFR both in vitro and in vivo but did not affect cells expressing wild-type EGFR. A lentiviral vector expressing the 39-nt AS sequence strongly inhibited glioblastoma growth in mouse brain when injected after tumor cell implantation. This PKR-mediated killing strategy may be useful in treating many cancers that express a unique RNA species.

Human interferon-gamma mRNA autoregulates its translation through a pseudoknot that activates the interferon-inducible protein kinase PKR.

PKR, an interferon (IFN)-inducible protein kinase activated by double-stranded RNA, inhibits translation by phosphorylating the initiation factor eIF2alpha chain. We show that human IFN-gamma mRNA uses local activation of PKR in the cell to control its own translation yield. IFN-gamma mRNA activates PKR through a pseudoknot in its 5' untranslated region. Mutations that impair pseudoknot stability reduce the ability to activate PKR and strongly increase the translation efficiency of IFN-gamma mRNA. Nonphosphorylatable mutant eIF2alpha, knockout of PKR and PKR inhibitors 2-aminopurine, transdominant-negative PKR, or vaccinia E3L correspondingly enhances translation of IFN-gamma mRNA. The potential to form the pseudoknot is phylogenetically conserved. We propose that the RNA pseudoknot acts to adjust translation of IFN-gamma mRNA to the PKR level expressed in the cell.