RNAi-Mediated β-Catenin Inhibition Promotes T Cell Infiltration and Antitumor Activity in Combination with Immune Checkpoint Blockade.

Wnt/β-catenin signaling mediates cancer immune evasion and resistance to immune checkpoint therapy, in part by blocking cytokines that trigger immune cell recruitment. Inhibition of β-catenin may be an effective strategy for increasing the low response rate to these effective medicines in numerous cancer populations. DCR-BCAT is a nanoparticle drug product containing a chemically optimized RNAi trigger targeting CTNNB1, the gene that encodes β-catenin.

β-Catenin mRNA Silencing and MEK Inhibition Display Synergistic Efficacy in Preclinical Tumor Models.

Colorectal carcinomas harbor well-defined genetic abnormalities, including aberrant activation of Wnt/β-catenin and MAPK pathways, often simultaneously. Although the MAPK pathway can be targeted using potent small-molecule drugs, including BRAF and MEK inhibitors, β-catenin inhibition has been historically challenging. RNAi approaches have advanced to the stage of clinical viability and are especially well suited for transcriptional modulators, such as β-catenin.

Direct Pharmacological Inhibition of β-Catenin by RNA Interference in Tumors of Diverse Origin.

The Wnt/β-catenin pathway is among the most frequently altered signaling networks in human cancers. Despite decades of preclinical and clinical research, efficient therapeutic targeting of Wnt/β-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets.

A single dose of EGLN1 siRNA yields increased erythropoiesis in nonhuman primates.

Decreased production of erythropoietin (EPO) causes anemia in patients with chronic kidney disease, and recombinant human EPO is used to treat renal failure associated anemia. The liver, the main EPO-producing organ in utero, maintains the capacity to produce EPO in the adult but in insufficient quantities to restore hemoglobin levels to normal in patients with impaired renal function. Inhibition of prolyl-4-hydroxylase domain (PHD) proteins is known to cause an increase in EPO production through its effects on hypoxia inducible factor.

Development of a liver-targeted siRNA delivery platform with a broad therapeutic window utilizing biodegradable polypeptide-based polymer conjugates.

The greatest challenge standing in the way of effective in vivo siRNA delivery is creating a delivery vehicle that mediates a high degree of efficacy with a broad therapeutic window. Key structure-activity relationships of a poly(amide) polymer conjugate siRNA delivery platform were explored to discover the optimized polymer parameters that yield the highest activity of mRNA knockdown in the liver. At the same time, the poly(amide) backbone of the polymers allowed for the metabolism and clearance of the polymer from the body very quickly, which was established using radiolabeled polymers to demonstrate the time course of biodistribution and excretion from the body.

An in vivo evaluation of amphiphilic, biodegradable peptide copolymers as siRNA delivery agents.

A series of amphiphilic, biodegradable polypeptide copolymers were prepared for the delivery of siRNA (short interfering ribonucleic acid). The molecular weight (or polymer chain length) of the linear polymer was controlled by reaction stoichiometry for the 11.5, 17.

Quantitative evaluation of siRNA delivery in vivo.

Effective small interfering RNA (siRNA)-mediated therapeutics require the siRNA to be delivered into the cellular RNA-induced silencing complex (RISC). Quantitative information of this essential delivery step is currently inferred from the efficacy of gene silencing and siRNA uptake in the tissue. Here we report an approach to directly quantify siRNA in the RISC in rodents and monkey.

Evaluation of efficacy, biodistribution, and inflammation for a potent siRNA nanoparticle: effect of dexamethasone co-treatment.

Despite recent progress, systemic delivery remains the major hurdle for development of safe and effective small inhibitory RNA (siRNA)-based therapeutics. Encapsulation of siRNA into liposomes is a promising option to overcome obstacles such as low stability in serum and inefficient internalization by target cells. However, a major liability of liposomes is the potential to induce an acute inflammatory response, thereby increasing the risk of numerous adverse effects.

Development of thioquinazolinones, allosteric Chk1 kinase inhibitors.

A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability.

Kinesin spindle protein (KSP) inhibitors. 9. Discovery of (2S)-4-(2,5-difluorophenyl)-n-[(3R,4S)-3-fluoro-1-methylpiperidin-4-yl]-2-(hydroxymethyl)-N-methyl-2-phenyl-2,5-dihydro-1H-pyrrole-1-carboxamide (MK-0731) for the treatment of taxane-refractory cancer.

Inhibition of kinesin spindle protein (KSP) is a novel mechanism for treatment of cancer with the potential to overcome limitations associated with currently employed cytotoxic agents. Herein, we describe a C2-hydroxymethyl dihydropyrrole KSP inhibitor ( 11) that circumvents hERG channel binding and poor in vivo potency, issues that limited earlier compounds from our program. However, introduction of the C2-hydroxymethyl group caused 11 to be a substrate for cellular efflux by P-glycoprotein (Pgp).

Synthesis and evaluation of substituted benzoisoquinolinones as potent inhibitors of Chk1 kinase.

From HTS lead 1, a novel benzoisoquinolinone class of ATP-competitive Chk1 inhibitors was devised and synthesized via a photochemical route. Using X-ray crystallography as a guide, potency was rapidly enhanced through the installation of a tethered basic amine designed to interact with an acidic residue (Glu91) in the enzyme pocket. Further SAR was explored at the solvent front and near to the H1 pocket and resulted in the discovery of low MW, sub-nanomolar inhibitors of Chk1.

Kinesin spindle protein (KSP) inhibitors. Part V: discovery of 2-propylamino-2,4-diaryl-2,5-dihydropyrroles as potent, water-soluble KSP inhibitors, and modulation of their basicity by beta-fluorination to overcome cellular efflux by P-glycoprotein.

Installation of a C2-aminopropyl side chain to the 2,4-diaryl-2,5-dihydropyrrole series of kinesin spindle protein (KSP) inhibitors results in potent, water soluble compounds, but the aminopropyl group induces susceptibility to cellular efflux by P-glycoprotein (Pgp). We show that by carefully modulating the basicity of the amino group by beta-fluorination, this series of inhibitors maintains potency against KSP and has greatly improved efficacy in a Pgp-overexpressing cell line. The discovery that cellular efflux by Pgp can be overcome by carefully modulating the basicity of an amine may be of general use to medicinal chemists attempting to transform leading compounds into cancer cell- or CNS-penetrant drugs.

EGFR-dependent downregulation of Bim in epithelial cells requires MAPK and PKC-delta activities.

Activation of the epidermal growth factor receptor (EGFR) provides a measure of protection to immortalized epidermal keratinocytes (HaCaT cells) against apoptosis induced by diverse cellular stressors. This effect is due, in part, to sustained MAPK-dependent Bcl-xL expression. Here, we report a second EGFR/MAPK-dependent signaling event that protects HaCaT cells against apoptosis incurred during forced suspension culture (anoikis).

RNAi-mediated silencing of insulin receptor substrate 1 (IRS-1) enhances tamoxifen-induced cell death in MCF-7 breast cancer cells.

Insulin receptor substrate 1 (IRS-1) is a major downstream signaling protein for insulin and insulin-like growth factor I (IGF-I) receptors, conveying signals to PI-3K/Akt and ERK1/2 pathways. In breast cancer, IRS-1 overexpression has been associated with tumor development, hormone-independence and antiestrogen-resistance. In part, these effects are related to potentiation of IRS-1/PI-3K/Akt signaling.

Evaluation of glucocorticoid sensitivity in 697 pre-B acute lymphoblastic leukemia cells after overexpression or silencing of MAP kinase phosphatase-1.

Purpose: To determine the effect of modulating MAP kinase phosphatase-1 (MKP-1) expression levels on cell death induced by glucocorticoid (GC) or hydroxyurea (HU) treatment in the human pre-B acute lymphoblastic leukemia cell line 697.

Methods: Stable MKP-1 overexpressing transformants of the 697 pre-B acute lymphoblastic leukemia cell line were created and tested for sensitivity to the GC triamcinolone acetonide (TA) and HU, and compared to a control 697 cell line containing normal MKP-1 expression levels. Small interfering RNAs (siRNAs) were designed to inhibit MKP-1 expression and evaluated for their effect on GC-mediated cell death.

Inhibition of glucocorticoid-induced apoptosis by targeting the major splice variants of BIM mRNA with small interfering RNA and short hairpin RNA.

Glucocorticoids (GCs) induce apoptosis in lymphocytes and are effective agents for the treatment of leukemia. The activated glucocorticoid receptor initiates a transcriptional program leading to caspase activation and cell death, but the critical signaling intermediates in GC-induced apoptosis remain largely undefined. We have observed that GC induction of the three major protein products of the Bcl-2 relative Bim (BimEL, BimS, and BimL) correlates with GC sensitivity in a panel of human precursor B-cell (pre-B) acute lymphoblastic leukemia (ALL) cell lines.

Macrocyclic piperazinones as potent dual inhibitors of farnesyltransferase and geranylgeranyltransferase-I.

A series of macrocyclic piperazinone compounds with dual farnesyltransferase/geranylgeranyltransferase-I inhibitory activity was prepared. These compounds were found to be potent inhibitors of protein prenylation in cell culture. A hypothesis for the binding mode of compound 3o in FPTase is proposed.

Role of apical caspases and glucocorticoid-regulated genes in glucocorticoid-induced apoptosis of pre-B leukemic cells.

Glucocorticoid (GC) sensitivity in hematopoietic cells requires the activation and nuclear translocation of the glucocorticoid receptor (GR) and the subsequent activation of caspases. To gain insight into the caspase cascade responsible for the execution phase of GC-induced apoptosis, 697 pre-B leukemic cells were stably transfected with dominant negative forms of caspase-8, caspase-9, or caspase-10 and the caspase-8 inhibitor CrmA. We observed that inhibition of caspase-9 or caspase-10 activity, but not caspase-8, caused partial resistance of 697 cells to GC-induced apoptosis.

The synthesis and biological evaluation of a series of potent dual inhibitors of farnesyl and geranyl-Geranyl protein transferases.

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.