RNAi mediated silencing of STAT3/PD-L1 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors.

Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical rodent tumor models, chemically stabilized acylated small interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types, reduced STAT3 protein levels, and increased cytotoxic T cell infiltration. In a murine model of CPI-resistant pancreatic cancer, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced in combination with CPIs. To further exemplify the utility of RNAi for cancer immunotherapy, this technology was used to silence Cd274, the gene encoding the immune checkpoint protein programmed death-ligand 1 (PD-L1). Interestingly, silencing of Cd274 was effective in tumor models that are resistant to PD-L1 antibody therapy. These data represent the first demonstration of systemic delivery of RNAi agents to the TME and suggest applying this technology for immuno-oncology applications.

Loss of hepatocyte ß-catenin protects mice from experimental porphyria-associated liver injury.

BACKGROUND & AIMS: Porphyrias result from anomalies of heme biosynthetic enzymes and can lead to cirrhosis and hepatocellular cancer. In mice, these diseases can be modeled by administration of a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes accumulation of porphyrin intermediates, resulting in hepatobiliary injury. Wnt/ß-catenin signaling has been shown to be a modulatable target in models of biliary injury; thus, we investigated its role in DDC-driven injury. METHODS: ß-Catenin (Ctnnb1) knockout (KO) mice, Wnt co-receptor KO mice, and littermate controls were fed a DDC diet for 2 weeks. ß-Catenin was exogenously inhibited in hepatocytes by administering ß-catenin dicer-substrate RNA (DsiRNA), conjugated to a lipid nanoparticle, to mice after DDC diet and then weekly for 4 weeks. In all experiments, serum and livers were collected; livers were analyzed by histology, western blotting, and real-time PCR. Porphyrin was measured by fluorescence, quantification of polarized light images, and liquid chromatography-mass spectrometry. RESULTS: DDC-fed mice lacking ß-catenin or Wnt signaling had decreased liver injury compared to controls. Exogenous mice that underwent ß-catenin suppression by DsiRNA during DDC feeding also showed less injury compared to control mice receiving lipid nanoparticles. Control livers contained extensive porphyrin deposits which were largely absent in mice lacking ß-catenin signaling. Notably, we identified a network of key heme biosynthesis enzymes that are suppressed in the absence of ß-catenin, preventing accumulation of toxic protoporphyrins. Additionally, mice lacking ß-catenin exhibited fewer protein aggregates, improved proteasomal activity, and reduced induction of autophagy, all contributing to protection from injury. CONCLUSIONS: ß-Catenin inhibition, through its pleiotropic effects on metabolism, cell stress, and autophagy, represents a novel therapeutic approach for patients with porphyria. LAY SUMMARY: Porphyrias are disorders resulting from abnormalities in the steps that lead to heme production, which cause build-up of toxic by-products called porphyrins. Liver is commonly either a source or a target of excess porphyrins, and complications can range from minor abnormalities to liver failure. In this report, we inhibited Wnt/ß-catenin signaling in an experimental model of porphyria, which resulted in decreased liver injury. Targeting ß-catenin affected multiple components of the heme biosynthesis pathway, thus preventing build-up of porphyrin intermediates. Our study suggests that drugs inhibiting ß-catenin activity could reduce the amount of porphyrin accumulation and help alleviate symptoms in patients with porphyria.

Hepatocyte-Specific ß-Catenin Deletion During Severe Liver Injury Provokes Cholangiocytes to Differentiate Into Hepatocytes.

Liver regeneration after injury is normally mediated by proliferation of hepatocytes, although recent studies have suggested biliary epithelial cells (BECs) can differentiate into hepatocytes during severe liver injury when hepatocyte proliferation is impaired. We investigated the effect of hepatocyte-specific ß-catenin deletion in recovery from severe liver injury and BEC-to-hepatocyte differentiation. To induce liver injury, we administered choline-deficient, ethionine-supplemented (CDE) diet to three different mouse models, the first being mice with deletion of ß-catenin in both BECs and hepatocytes (Albumin-Cre; Ctnnb1flox/flox mice). In our second model, we performed hepatocyte lineage tracing by injecting Ctnnb1flox/flox ; Rosa-stopflox/flox -EYFP mice with the adeno-associated virus serotype 8 encoding Cre recombinase under the control of the thyroid binding globulin promoter, a virus that infects only hepatocytes. Finally, we performed BEC lineage tracing via Krt19-CreERT ; Rosa-stopflox/flox -tdTomato mice. To observe BEC-to-hepatocyte differentiation, mice were allowed to recover on normal diet following CDE diet-induced liver injury. Livers were collected from all mice and analyzed by quantitative real-time polymerase chain reaction, western blotting, immunohistochemistry, and immunofluorescence. We show that mice with lack of ß-catenin in hepatocytes placed on the CDE diet develop severe liver injury with impaired hepatocyte proliferation, creating a stimulus for BECs to differentiate into hepatocytes. In particular, we use both hepatocyte and BEC lineage tracing to show that BECs differentiate into hepatocytes, which go on to repopulate the liver during long-term recovery. Conclusion: ß-catenin is important for liver regeneration after CDE diet-induced liver injury, and BEC-derived hepatocytes can permanently incorporate into the liver parenchyma to mediate liver regeneration.

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

Wnt/ß-catenin signaling mediates cancer immune evasion and resistance to immune checkpoint therapy, in part by blocking cytokines that trigger immune cell recruitment. Inhibition of ß-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 ß-catenin. In syngeneic mouse tumor models, ß-catenin inhibition with DCR-BCAT significantly increased T cell infiltration and potentiated the sensitivity of the tumors to checkpoint inhibition. The combination of DCR-BCAT and immunotherapy yielded significantly greater tumor growth inhibition (TGI) compared to monotherapy in B16F10 melanoma, 4T1 mammary carcinoma, Neuro2A neuroblastoma, and Renca renal adenocarcinoma. Response to the RNAi-containing combination therapy was not dependent on Wnt activation status of the tumor. Importantly, this drug combination was associated with elevated levels of biomarkers of T cell-mediated cytotoxicity. Finally, when CTLA-4 and PD-1 antibodies were combined with DCR-BCAT in MMTV-Wnt1 transgenic mice, a genetic model of spontaneous Wnt-driven tumors, complete regressions were achieved in the majority of treated subjects. These data support RNAi-mediated ß-catenin inhibition as an effective strategy to increase response rates to cancer immunotherapy.

Inhibition of Glycogen Synthase II with RNAi Prevents Liver Injury in Mouse Models of Glycogen Storage Diseases.

Glycogen storage diseases (GSDs) of the liver are devastating disorders presenting with fasting hypoglycemia as well as hepatic glycogen and lipid accumulation, which could lead to long-term liver damage. Diet control is frequently utilized to manage the potentially dangerous hypoglycemia, but there is currently no effective pharmacological treatment for preventing hepatomegaly and concurrent liver metabolic abnormalities, which could lead to fibrosis, cirrhosis, and hepatocellular adenoma or carcinoma. In this study, we demonstrate that inhibition of glycogen synthesis using an RNAi approach to silence hepatic Gys2 expression effectively prevents glycogen synthesis, glycogen accumulation, hepatomegaly, fibrosis, and nodule development in a mouse model of GSD III. Mechanistically, reduction of accumulated abnormally structured glycogen prevents proliferation of hepatocytes and activation of myofibroblasts as well as infiltration of mononuclear cells. Additionally, we show that silencing Gys2 expression reduces hepatic steatosis in a mouse model of GSD type Ia, where we hypothesize that the reduction of glycogen also reduces the production of excess glucose-6-phosphate and its subsequent diversion to lipid synthesis. Our results support therapeutic silencing of GYS2 expression to prevent glycogen and lipid accumulation, which mediate initial signals that subsequently trigger cascades of long-term liver injury in GSDs.

Recent preclinical and clinical advances in oligonucleotide conjugates.

INTRODUCTION: Oligonucleotide therapeutics have the potential to change the way disease is treated due to their ability to modulate gene expression of any therapeutic target in a highly specific and potent manner. Unfortunately, this drug class is plagued with inherently poor pharmacological characteristics, which need to be overcome. The development of a chemical modification library for oligonucleotides has addressed many of the initial challenges, but delivery of these payloads across plasma membranes remains difficult. The latest technological advances in oligonucleotide therapeutics utilizes direct conjugation to targeting ligands, which has improved bioavailability and target tissue exposure many-fold. The success of this approach has resulted in numerous clinical programs over the past 5 years. AREAS COVERED: We review the literature on oligonucleotide conjugate strategies which have proven effective preclinically and clinically. We summarize the chemical modifications which allow parenteral administration as well as evaluate the efficacy of a multitude of conjugate approaches including lipids, peptides, carbohydrates, and antibodies. EXPERT OPINION: The success of future conjugate strategies will likely rely on the effective combination of characteristics from earlier technologies. High-affinity ligand-receptor interactions can be critical to achieving meaningful accumulation in target tissues, but pharmacokinetic modulators which increase the circulating half-life may also be necessary. Synthesis of these approaches has the potential to bring the next breakthrough in oligonucleotide therapeutics.

ß-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/ß-catenin and MAPK pathways, often simultaneously. Although the MAPK pathway can be targeted using potent small-molecule drugs, including BRAF and MEK inhibitors, ß-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 ß-catenin. In this study, we report therapeutic effects of combined targeting of these pathways with pharmacologic agents. Using a recently described tumor-selective nanoparticle containing a ß-catenin-targeting RNAi trigger, in combination with the FDA-approved MEK inhibitor (MEKi) trametinib, we demonstrate synergistic tumor growth inhibition in in vivo models of colorectal cancer, melanoma, and hepatocellular carcinoma. At dose levels that were insufficient to significantly impact tumor growth as monotherapies, combination regimens resulted in synergistic efficacy and complete tumor growth inhibition. Importantly, dual MEKi/RNAi therapy dramatically improved survival of mice bearing colorectal cancer liver metastases. In addition, pharmacologic silencing of ß-catenin mRNA was effective against tumors that are inherently resistant or that acquire drug-induced resistance to trametinib. These results provide a strong rationale for clinical evaluation of this dual-targeting approach for cancers harboring Wnt/ß-catenin and MAPK pathway mutations. Mol Cancer Ther; 17(2); 544-53. ©2017 AACR.

Targeting ß-catenin in hepatocellular cancers induced by coexpression of mutant ß-catenin and K-Ras in mice.

Recently, we have shown that coexpression of hMet and mutant-ß-catenin using sleeping beauty transposon/transposase leads to hepatocellular carcinoma (HCC) in mice that corresponds to around 10% of human HCC. In the current study, we investigate whether Ras activation, which can occur downstream of Met signaling, is sufficient to cause HCC in association with mutant-ß-catenin. We also tested therapeutic efficacy of targeting ß-catenin in an HCC model. We show that mutant-K-Ras (G12D), which leads to Ras activation, cooperates with ß-catenin mutants (S33Y, S45Y) to yield HCC in mice. Affymetrix microarray showed > 90% similarity in gene expression in mutant-K-Ras-ß-catenin and Met-ß-catenin HCC. K-Ras-ß-catenin tumors showed up-regulation of ß-catenin targets like glutamine synthetase (GS), leukocyte cell-derived chemotaxin 2, Regucalcin, and Cyclin-D1 and of K-Ras effectors, including phosphorylated extracellular signal-regulated kinase, phosphorylated protein kinase B, phosphorylated mammalian target of rapamycin, phosphorylated eukaryotic translation initiation factor 4E, phosphorylated 4E-binding protein 1, and p-S6 ribosomal protein. Inclusion of dominant-negative transcription factor 4 at the time of K-Ras-ß-catenin injection prevented HCC and downstream ß-catenin and Ras signaling. To address whether targeting ß-catenin has any benefit postestablishment of HCC, we administered K-Ras-ß-catenin mice with EnCore lipid nanoparticles (LNP) loaded with a Dicer substrate small interfering RNA targeting catenin beta 1 (CTNNB1; CTNNB1-LNP), scrambled sequence (Scr-LNP), or phosphate-buffered saline for multiple cycles. A significant decrease in tumor burden was evident in the CTNNB1-LNP group versus all controls, which was associated with dramatic decreases in ß-catenin targets and some K-Ras effectors, leading to reduced tumor cell proliferation and viability. Intriguingly, in relatively few mice, non-GS-positive tumors, which were evident as a small subset of overall tumor burden, were not affected by ß-catenin suppression. CONCLUSION: Ras activation downstream of c-Met is sufficient to induce clinically relevant HCC in cooperation with mutant ß-catenin. ß-catenin suppression by a clinically relevant modality is effective in treatment of ß-catenin-positive, GS-positive HCCs. (Hepatology 2017;65:1581-1599).

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

The Wnt/ß-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/ß-catenin has been elusive. RNA interference (RNAi) technology silences genes at the mRNA level and therefore can be applied to previously undruggable targets. Lipid nanoparticles (LNP) represent an elegant solution for the delivery of RNAi-triggering oligonucleotides to disease-relevant tissues, but have been mostly restricted to applications in the liver. In this study, we systematically tuned the composition of a prototype LNP to enable tumor-selective delivery of a Dicer-substrate siRNA (DsiRNA) targeting CTNNB1, the gene encoding ß-catenin. This formulation, termed EnCore-R, demonstrated pharmacodynamic activity in subcutaneous human tumor xenografts, orthotopic patient-derived xenograft (PDX) tumors, disseminated hematopoietic tumors, genetically induced primary liver tumors, metastatic colorectal tumors, and murine metastatic melanoma. DsiRNA delivery was homogeneous in tumor sections, selective over normal liver and independent of apolipoprotein-E binding. Significant tumor growth inhibition was achieved in Wnt-dependent colorectal and hepatocellular carcinoma models, but not in Wnt-independent tumors. Finally, no evidence of accelerated blood clearance or sustained liver transaminase elevation was observed after repeated dosing in nonhuman primates. These data support further investigation to gain mechanistic insight, optimize dose regimens, and identify efficacious combinations with standard-of-care therapeutics. Mol Cancer Ther; 15(9); 2143-54. ©2016 AACR.

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, 17.2, and 24.6 kDa polypeptides, and the highest molecular weight polypeptide was prepared using a sequential addition method to obtain a polypeptide having a molecular weight of 38.6 kDa. These polymers were used to prepare polymer conjugate systems designed to target and deliver an apolipoprotein B (ApoB) siRNA to hepatocyte cells and to help delineate the effect of polymer molecular weight or polymer chain length on siRNA delivery in vivo. A clear trend in increasing potency was found with increasing molecular weight of the polymers examined (at a constant polymer:siRNA (w/w) ratio), with minimal toxicity found. Furthermore, the biodegradability of these polymer conjugates was examined and demonstrates the potential of these systems as siRNA delivery vectors.

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. The fast degradation and clearance of the polymers provided for very low toxicity at efficacious doses, and the therapeutic window of this poly(amide)-based siRNA delivery platform was shown to be much broader than a comparable polymer platform. The results of this work illustrate that the poly(amide) platform has a promising future in the development of a siRNA-based drug approved for human use.

Expression of asialoglycoprotein receptor 1 in human hepatocellular carcinoma.

Human hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. Currently, surgical resection is the only effective treatment for HCC if the tumor is resectable. Small molecule, biologics and siRNA anti-cancer drugs have been explored for the treatment of HCC. Selective targeting to tumor tissue rather than normal liver in HCC patients is still a challenge. Galactosamine-mediated targeting delivery of anti-cancer drugs in the liver has been tested because its receptor, asialoglycoprotein receptor 1 (ASGPR1), is expressed in the liver and not in other human tissues. We examined ASGPR1 expression levels by immunohistochemistry in HCC with different grades. Guidance for a targeting delivery strategy for anti-cancer drugs to HCC is suggested in this report.

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). Utilizing knowledge garnered from previous KSP inhibitors, we found that beta-fluorination modulated the p K a of the piperidine nitrogen and reduced Pgp efflux, but the resulting compound ( 14) generated a toxic metabolite in vivo. Incorporation of fluorine in a strategic, metabolically benign position by synthesis of an N-methyl-3-fluoro-4-(aminomethyl)piperidine urea led to compound 30 that has an optimal in vitro and metabolic profile. Compound 30 (MK-0731) was recently studied in a phase I clinical trial in patients with taxane-refractory solid tumors.

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.

Development of 6-substituted indolylquinolinones as potent Chek1 kinase inhibitors.

Through a comparison of X-ray co-crystallographic data for 1 and 2 in the Chek1 active site, it was hypothesized that the affinity of the indolylquinolinone series (2) for Chek1 kinase would be improved via C6 substitution into the hydrophobic region I (HI) pocket. An efficient route to 6-bromo-3-indolyl-quinolinone (9) was developed, and this series was rapidly optimized for potency by modification at C6. A general trend was observed among these low nanomolar Chek1 inhibitors that compounds with multiple basic amines, or elevated polar surface area (PSA) exhibited poor cell potency. Minimization of these parameters (basic amines, PSA) resulted in Chek1 inhibitors with improved cell potency, and preliminary pharmacokinetic data are presented for several of these compounds.

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). This pathway targets Bim, a pro-apoptotic BH3-only Bcl-2 family member. Bim expression was functionally relevant to HaCaT cell survival as demonstrated by partial protection against anoikis provided by siRNA-induced Bim downregulation. Growth factor starvation of attached and suspended cells was associated with enhanced Bim expression whereas EGFR activation reduced Bim expression by inducing Bim phosphorylation and proteasomal degradation. EGFR-dependent Bim phosphorylation required MAPK activation. Furthermore, PKC-delta activity contributed to both MEK/MAPK phosphorylation and Bim phosphorylation as demonstrated using both pharmacological inhibitors of PKC-delta and siRNA-mediated PKC-delta knockdown. In addition to HaCaT cells, EGFR activation supported survival and induced Bim phosphorylation in several squamous carcinoma cell lines in a strictly MAPK-dependent fashion. These results establish that EGFR activation attenuates susceptibility of immortalized and malignant keratinocytes to apoptosis by post-translational control of Bim-EL expression through a pathway requiring PKC-delta and MEK/MAPK activation.

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. In estrogen sensitive breast cancer cell lines, tamoxifen treatment reduces IRS-1 expression and function; consequently, inhibiting IRS-1/PI-3K signaling. We tested whether anti-IRS1 siRNA could inhibit growth and survival of estrogen-sensitive MCF-7 breast cancer cells, when used alone or in combination with TAM. Our results indicated: (a) out of four tested anti-IRS1 siRNAs, two siRNAs reduced IRS-1 protein by approximately three-fold in both growing and IGF-I-stimulated cells without affecting a closely related protein, IRS-2; (b) these effects paralleled IRS1 mRNA downregulation by approximately three-fold, measured by quantitative real time-polymerase chain reaction; (c) action of anti-IRS1 siRNAs induced the apoptotic response, observed by altered mitochondrial membrane potential coupled with downregulation of NF-kappaB target Bcl-xL and reduced cell viability; (d) anti-IRS1 siRNA treatment enhanced the cytotoxic effects of TAM by approximately 20%. In summary, anti-IRS1 RNAi strategy could become a potent tool to induce breast cancer cell death, especially if combined with standard TAM therapy.

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. RESULTS: MKP-1 overexpression caused a phenotype of partial resistance to HU-induced apoptosis but not to GC-induced apoptosis. Electroporation of siRNAs effectively silenced MKP-1 expression, and increased sensitivity to TA by 9.6+/-1.9%. CONCLUSIONS: Because MKP-1 protects certain tumor cells from chemotherapy-induced apoptosis, its inhibition is being considered as a possible strategy for combination cancer therapy. However, this study suggests that while MKP-1 inhibition may improve the efficacy of DNA damaging agents, it may have only limited utility in combination with glucocorticoids.