Metabolic modulation of tumours with engineered bacteria for immunotherapy.

The availability of L-arginine in tumours is a key determinant of an efficient anti-tumour T cell response1-4. Consequently, increases of typically low L-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies5. However, currently no means are available to locally increase intratumoural L-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours6, to L-arginine. Colonization of tumours with these bacteria increased intratumoural L-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies.

Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity.

Synthetic biology is a powerful tool to create therapeutics which can be rationally designed to enable unique and combinatorial functionalities. Here we utilize non-pathogenic E coli Nissle as a versatile platform for the development of a living biotherapeutic for the treatment of cancer. The engineered bacterial strain, referred to as SYNB1891, targets STING-activation to phagocytic antigen-presenting cells (APCs) in the tumor and activates complementary innate immune pathways. SYNB1891 treatment results in efficacious antitumor immunity with the formation of immunological memory in murine tumor models and robust activation of human APCs. SYNB1891 is designed to meet manufacturability and regulatory requirements with built in biocontainment features which do not compromise its efficacy. This work provides a roadmap for the development of future therapeutics and demonstrates the transformative potential of synthetic biology for the treatment of human disease when drug development criteria are incorporated into the design process for a living medicine.

An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans.

The intestine is a major source of systemic ammonia (NH3); thus, capturing part of gut NH3 may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH3 to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH3 in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase-deficient spfash mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 1012 colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma 15N-nitrate (highest dose versus placebo, P = 0.0015), and urinary 15N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.

Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria.

Phenylketonuria (PKU) is a genetic disease that is characterized by an inability to metabolize phenylalanine (Phe), which can result in neurotoxicity. To provide a potential alternative to a protein-restricted diet, we engineered Escherichia coli Nissle to express genes encoding Phe-metabolizing enzymes in response to anoxic conditions in the mammalian gut. Administration of our synthetic strain, SYNB1618, to the Pahenu2/enu2 PKU mouse model reduced blood Phe concentration by 38% compared with the control, independent of dietary protein intake. In healthy Cynomolgus monkeys, we found that SYNB1618 inhibited increases in serum Phe after an oral Phe dietary challenge. In mice and primates, Phe was converted to trans-cinnamate by SYNB1618, quantitatively metabolized by the host to hippurate and excreted in the urine, acting as a predictive biomarker for strain activity. SYNB1618 was detectable in murine or primate feces after a single oral dose, permitting the evaluation of pharmacodynamic properties. Our results define a strategy for translation of live bacterial therapeutics to treat metabolic disorders.

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.

Systems biology driving drug development: from design to the clinical testing of the anti-ErbB3 antibody seribantumab (MM-121).

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR/ErbB1), human EGFR 2 (HER2/ErbB2), ErbB3/HER3, and ErbB4/HER4. The first two members of this family, EGFR and HER2, have been implicated in tumorigenesis and cancer progression for several decades, and numerous drugs have now been approved that target these two proteins. Less attention, however, has been paid to the role of this family in mediating cancer cell survival and drug tolerance. To better understand the complex signal transduction network triggered by the ErbB receptor family, we built a computational model that quantitatively captures the dynamics of ErbB signaling. Sensitivity analysis identified ErbB3 as the most critical activator of phosphoinositide 3-kinase (PI3K) and Akt signaling, a key pro-survival pathway in cancer cells. Based on this insight, we designed a fully human monoclonal antibody, seribantumab (MM-121), that binds to ErbB3 and blocks signaling induced by the extracellular growth factors heregulin (HRG) and betacellulin (BTC). In this article, we present some of the key preclinical simulations and experimental data that formed the scientific foundation for three Phase 2 clinical trials in metastatic cancer. These trials were designed to determine if patients with advanced malignancies would derive benefit from the addition of seribantumab to standard-of-care drugs in platinum-resistant/refractory ovarian cancer, hormone receptor-positive HER2-negative breast cancer, and EGFR wild-type non-small cell lung cancer (NSCLC). From preclinical studies we learned that basal levels of ErbB3 phosphorylation correlate with response to seribantumab monotherapy in mouse xenograft models. As ErbB3 is rapidly dephosphorylated and hence difficult to measure clinically, we used the computational model to identify a set of five surrogate biomarkers that most directly affect the levels of p-ErbB3: HRG, BTC, EGFR, HER2, and ErbB3. Preclinically, the combined information from these five markers was sufficient to accurately predict which xenograft models would respond to seribantumab, and the single-most accurate predictor was HRG. When tested clinically in ovarian, breast and lung cancer, HRG mRNA expression was found to be both potentially prognostic of insensitivity to standard therapy and potentially predictive of benefit from the addition of seribantumab to standard of care therapy in all three indications. In addition, it was found that seribantumab was most active in cancers with low levels of HER2, consistent with preclinical predictions. Overall, our clinical studies and studies of others suggest that HRG expression defines a drug-tolerant cancer cell phenotype that persists in most solid tumor indications and may contribute to rapid clinical progression. To our knowledge, this is the first example of a drug designed and clinically tested using the principles of Systems Biology.

Novel Oxindole Sulfonamides and Sulfamides: EPZ031686, the First Orally Bioavailable Small Molecule SMYD3 Inhibitor.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies.

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.

HSP90 inhibition enhances antimitotic drug-induced mitotic arrest and cell death in preclinical models of non-small cell lung cancer.

HSP90 inhibitors are currently undergoing clinical evaluation in combination with antimitotic drugs in non-small cell lung cancer (NSCLC), but little is known about the cellular effects of this novel drug combination. Therefore, we investigated the molecular mechanism of action of IPI-504 (retaspimycin HCl), a potent and selective inhibitor of HSP90, in combination with the microtubule targeting agent (MTA) docetaxel, in preclinical models of NSCLC. We identified a subset of NSCLC cell lines in which these drugs act in synergy to enhance cell death. Xenograft models of NSCLC demonstrated tumor growth inhibition, and in some cases, regression in response to combination treatment. Treatment with IPI-504 enhanced the antimitotic effects of docetaxel leading to the hypothesis that the mitotic checkpoint is required for the response to drug combination. Supporting this hypothesis, overriding the checkpoint with an Aurora kinase inhibitor diminished the cell death synergy of IPI-504 and docetaxel. To investigate the molecular basis of synergy, an unbiased stable isotope labeling by amino acids in cell culture (SILAC) proteomic approach was employed. Several mitotic regulators, including components of the ubiquitin ligase, anaphase promoting complex (APC/C), were specifically down-regulated in response to combination treatment. Loss of APC/C by RNAi sensitized cells to docetaxel and enhanced its antimitotic effects. Treatment with a PLK1 inhibitor (BI2536) also sensitized cells to IPI-504, indicating that combination effects may be broadly applicable to other classes of mitotic inhibitors. Our data provide a preclinical rationale for testing the combination of IPI-504 and docetaxel in NSCLC.

Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

Hedgehog-Gli pathway activation during kidney fibrosis.

The Hedgehog (Hh) signaling pathway regulates tissue patterning during development, including patterning and growth of limbs and face, but whether Hh signaling plays a role in adult kidney remains undefined. In this study, using a panel of hedgehog-reporter mice, we show that the two Hh ligands (Indian hedgehog and sonic hedgehog ligands) are expressed in tubular epithelial cells. We report that the Hh effectors (Gli1 and Gli2) are expressed exclusively in adjacent platelet-derived growth factor receptor-ß-positive interstitial pericytes and perivascular fibroblasts, suggesting a paracrine signaling loop. In two models of renal fibrosis, Indian Hh ligand was upregulated with a dramatic activation of downstream Gli effector expression. Hh-responsive Gli1-positive interstitial cells underwent 11-fold proliferative expansion during fibrosis, and both Gli1- and Gli2-positive cells differentiated into α-smooth muscle actin-positive myofibroblasts. In the pericyte-like cell line 10T1/2, hedgehog ligand triggered cell proliferation, suggesting a possible role for this pathway in the regulation of cell cycle progression of myofibroblast progenitors during the development of renal fibrosis. The hedgehog antagonist IPI-926 abolished Gli1 induction in vivo but did not decrease kidney fibrosis. However, the transcriptional induction of Gli2 was unaffected by IPI-926, suggesting the existence of smoothened-independent Gli activation in this model. This study is the first detailed description of paracrine hedgehog signaling in adult kidney, which indicates a possible role for hedgehog-Gli signaling in fibrotic chronic kidney disease.

Antitumor activity of the Hsp90 inhibitor IPI-504 in HER2-positive trastuzumab-resistant breast cancer.

Hsp90 facilitates the maturation and stability of numerous oncoproteins, including HER2. The aim of this study was to assess the antitumor activity of the Hsp90 inhibitor IPI-504 in trastuzumab-resistant, HER2-overexpressing breast cancer cells. Therapy with trastuzumab, IPI-504, and the combination of trastuzumab and IPI-504 was evaluated in trastuzumab-sensitive and trastuzumab-resistant cells. Inhibition of protein targets, cell proliferation, and tumor growth was assessed in vitro and in xenograft models. IPI-504 inhibited proliferation of both trastuzumab-sensitive and trastuzumab-resistant cells. Administration of IPI-504 markedly reduced total levels of HER2 and Akt, as well as phosphorylated Akt and mitogen-activated protein kinase (MAPK), to an equal extent in trastuzumab-sensitive and trastuzumab-resistant cells. IPI-504, used as single agent or in combination with trastuzumab, also inhibited in vivo the growth of both trastuzumab-sensitive and -resistant tumor xenografts. As a mechanism for the observed antitumor activity, IPI-504 resulted in a marked decrease in the levels of HER2, Akt, p-Akt, and p-MAPK in trastuzumab-resistant xenografts as early as 12 hours after a single dose of IPI-504. IPI-504-mediated Hsp90 inhibition may represent a novel therapeutic approach in trastuzumab refractory HER2-positive breast cancer.

Hsp90 (heat shock protein 90) inhibitor occupancy is a direct determinant of client protein degradation and tumor growth arrest in vivo.

Several Hsp90 (heat shock protein 90) inhibitors are currently under clinical evaluation as anticancer agents. However, the correlation between the duration and magnitude of Hsp90 inhibition and the downstream effects on client protein degradation and cancer cell growth inhibition has not been thoroughly investigated. To investigate the relationship between Hsp90 inhibition and cellular effects, we developed a method that measures drug occupancy on Hsp90 after treatment with the Hsp90 inhibitor IPI-504 in living cells and in tumor xenografts. In cells, we find the level of Hsp90 occupancy to be directly correlated with cell growth inhibition. At the molecular level, the relationship between Hsp90 occupancy and Hsp90 client protein degradation was examined for different client proteins. For sensitive Hsp90 clients (e.g. HER2 (human epidermal growth factor receptor 2), client protein levels directly mirror Hsp90 occupancy at all time points after IPI-504 administration. For insensitive client proteins, we find that protein abundance matches Hsp90 occupancy only after prolonged incubation with drug. Additionally, we investigate the correlation between plasma pharmacokinetics (PK), tumor PK, pharmacodynamics (PD) (client protein degradation), tumor growth inhibition, and Hsp90 occupancy in a xenograft model of human cancer. Our results indicate Hsp90 occupancy to be a better predictor of PD than either plasma PK or tumor PK. In the nonsmall cell lung cancer xenograft model studied, a linear correlation between Hsp90 occupancy and tumor growth inhibition was found. This novel binding assay was evaluated both in vitro and in vivo and could be used as a pharmacodynamic readout in the clinic.

The antiproliferative activity of the heat shock protein 90 inhibitor IPI-504 is not dependent on NAD(P)H:quinone oxidoreductase 1 activity in vivo.

IPI-504, a water-soluble ansamycin analogue currently being investigated in clinical trials, is a potent inhibitor of the protein chaperone heat shock protein 90 (Hsp90). Inhibition of Hsp90 by IPI-504 triggers the degradation of important oncogenic client proteins. In cells, the free base of IPI-504 hydroquinone exists in a dynamic redox equilibrium with its corresponding quinone (17-AAG); the hydroquinone form binding 50 times more tightly to Hsp90. It has been proposed recently that the NAD(P)H:quinone oxidoreductase NQO1 can produce the active hydroquinone and could be essential for the activity of IPI-504. Here, we have devised a method to directly measure the intracellular ratio of hydroquinone to quinone (HQ/Q) and have applied this measurement to correlate NQO1 enzyme abundance with HQ/Q ratio and cellular activity of IPI-504 in 30 cancer cell lines. Interestingly, the intracellular HQ/Q ratio was correlated with NQO1 levels only in a subset of cell lines and overall was poorly correlated with the growth inhibitory activity of IPI-504. Although artificial overexpression of NQO1 is able to increase the level of hydroquinone and cell sensitivity to IPI-504, it has little effect on the activity of 17-amino-17-demethoxy-geldanamycin, the major active metabolite of IPI-504. This finding could provide an explanation for the biological activity of IPI-504 in xenograft models of cell lines that are not sensitive to IPI-504 in vitro. Our results suggest that NQO1 activity is not a determinant of IPI-504 activity in vivo and, therefore, unlikely to become an important resistance mechanism to IPI-504 in the clinic.

Ansamycin inhibitors of Hsp90: nature's prototype for anti-chaperone therapy.

The ansamycin class of natural products is well known for its anti-tumor effects and has been extensively studied by cancer researchers for nearly four decades. The first description of geldanamycin in the scientific literature appeared in 1970 and nearly thirty years later the semi-synthetic derivative 17-AAG, or tanespimycin, entered Phase 1 clinical trials. In the subsequent years, three additional geldanamycin derivatives have entered clinical evaluation. Kosan Biosciences developed 17-DMAG or alvespimycin hydrochloride for clinical evaluation as both an intravenous and oral product. Infinity Pharmaceuticals is developing IPI-504 or retaspimycin hydrochloride as an intravenous product, which is in several Phase 2 clinical trials; IPI-504 is the hydroquinone hydrochloride salt of 17-AAG. More recently, Infinity Pharmaceuticals initiated a Phase 1 clinical trial with an oral formulation of 17-AG (IPI-493), the major metabolite of 17-AAG and IPI-504. Since a vast amount of scientific literature exists regarding the ansamycin field and Hsp90 inhibition, this review will survey key milestones in the development of the natural product class as anti-cancer drugs including discovery of the compounds and their anti-tumor effects, identification of Hsp90 as their biological target, the structure-activity relationships that have been identified in this interesting class of compounds, and development of clinical candidates for the treatment of cancer patients. A brief overview of important pre-clinical development data from each clinical lead is also provided.

Therapeutically targeting ErbB3: a key node in ligand-induced activation of the ErbB receptor-PI3K axis.

The signaling network downstream of the ErbB family of receptors has been extensively targeted by cancer therapeutics; however, understanding the relative importance of the different components of the ErbB network is nontrivial. To explore the optimal way to therapeutically inhibit combinatorial, ligand-induced activation of the ErbB-phosphatidylinositol 3-kinase (PI3K) axis, we built a computational model of the ErbB signaling network that describes the most effective ErbB ligands, as well as known and previously unidentified ErbB inhibitors. Sensitivity analysis identified ErbB3 as the key node in response to ligands that can bind either ErbB3 or EGFR (epidermal growth factor receptor). We describe MM-121, a human monoclonal antibody that halts the growth of tumor xenografts in mice and, consistent with model-simulated inhibitor data, potently inhibits ErbB3 phosphorylation in a manner distinct from that of other ErbB-targeted therapies. MM-121, a previously unidentified anticancer therapeutic designed using a systems approach, promises to benefit patients with combinatorial, ligand-induced activation of the ErbB signaling network that are not effectively treated by current therapies targeting overexpressed or mutated oncogenes.

Inhibition of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway but not the MEK/ERK pathway attenuates laminin-mediated small cell lung cancer cellular survival and resistance to imatinib mesylate or chemotherapy.

The fact that small cell lung cancer (SCLC) is commonly incurable despite being initially responsive to chemotherapy, combined with disappointing results from a recent SCLC clinical trial with imatinib, has intensified efforts to identify mechanisms of SCLC resistance. Adhesion to extracellular matrix (ECM) is one mechanism that can increase therapeutic resistance in SCLC cells. To address whether adhesion to ECM increases resistance through modulation of signaling pathways, a series of SCLC cell lines were plated on various ECM components, and activation of two signaling pathways that promote cellular survival, the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway and the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) pathway, was assessed. Although differential activation was observed, adhesion to laminin increased Akt activation, increased cellular survival after serum starvation, and caused the cells to assume a flattened, epithelial morphology. Inhibitors of the PI3K/Akt/mTOR pathway (LY294002, rapamycin) but not the MEK/ERK pathway (U0126) abrogated laminin-mediated survival. SCLC cells plated on laminin were not only resistant to serum starvation-induced apoptosis but were also resistant to apoptosis caused by imatinib. Combining imatinib with LY294002 or rapamycin but not U0126 caused greater than additive increases in apoptosis compared with apoptosis caused by the inhibitor or imatinib alone. Similar results were observed when adenoviruses expressing mutant Akt were combined with imatinib, or when LY294002 was combined with cisplatin or etoposide. These studies identify laminin-mediated activation of the PI3K/Akt/mTOR pathway as a mechanism of cellular survival and therapeutic resistance in SCLC cells and suggest that inhibition of the PI3K/Akt/mTOR pathway is one strategy to overcome SCLC resistance mediated by ECM.

Preferential inhibition of Akt and killing of Akt-dependent cancer cells by rationally designed phosphatidylinositol ether lipid analogues.

Activation of the PI3k/Akt pathway controls key cellular processes and contributes to tumorigenesis in vivo, but investigation of the PI3k/Akt pathway has been limited by the lack of specific inhibitors directed against Akt. To develop Akt inhibitors, we used molecular modeling of the pleckstrin homology (PH) domain of Akt to guide synthesis of structurally modified phosphatidylinositol ether lipid analogues (PIAs). Here, we characterize the biochemical and cellular effects of PIAs. Of 24 compounds tested, five PIAs with modifications at two sites on the inositol ring inhibited Akt with IC(50)s < 5 micro M. Molecular modeling identified putative interactions of PIAs with the phosphoinositide-binding site in the PH domain of Akt, and growth factor-induced translocation of Akt to the plasma membrane was inhibited by PIA administration. Inhibition of Akt occurred rapidly and was maintained for hours. PIAs decreased phosphorylation of many downstream targets of Akt without affecting upstream kinases, such as PI3k or phosphoinositide-dependent kinase-1, or members of other kinase pathways such as extracellular signal-regulated kinase. Importantly, PIAs increased apoptosis 20-30-fold in cancer cell lines with high levels of endogenous Akt activity but only 4-5-fold in cancer cell lines with low levels of Akt activity. These studies identify PIAs as effective Akt inhibitors, and provide proof of principle for targeting the PH domain of Akt.