Plasma IgG aggregates as biomarkers for multiple sclerosis.

We recently reported that multiple sclerosis (MS) plasma contains IgG aggregates and induces complement-dependent neuronal cytotoxicity (Zhou et al., 2023). Using ELISA, we report herein that plasma IgG levels in the aggregates can be used as biomarkers for MS. We enriched the IgG aggregates from samples of two cohorts (190 MS and 160 controls) by collecting flow-through after plasma binding to Protein A followed by detection of IgG subclass. We show that there are significantly higher levels of IgG1, IgG3, and total IgG antibodies in MS IgG aggregates, with an AUC >90%; higher levels of IgG1 distinguish secondary progressive MS from relapsing-remitting MS (AUC = 91%). Significantly, we provided the biological rationale for MS plasma IgG biomarkers by demonstrating the strong correlation between IgG antibodies and IgG aggregate-induced neuronal cytotoxicity. These non-invasive, simple IgG-based blood ELISA assays can be adapted into clinical practice for diagnosing MS and SPMS and monitoring treatment responses.

Innate Signaling in the CNS Prevents Demyelination in a Focal EAE Model.

The pathological hallmark of multiple sclerosis (MS) is the formation of multifocal demyelinating lesions in the central nervous system (CNS). Stimulation of innate receptors has been shown to suppress experimental autoimmune encephalomyelitis (EAE), an MS-like disease in mice. Specifically, targeting Toll-like receptor 9 (TLR9) and NOD-like receptor 2 (NOD2) significantly reduced disease severity. In the present work we have developed a novel focal EAE model to further study the effect of innate signaling on demyelinating pathology. Focal lesions were induced by stereotactic needle insertion into the corpus callosum (CC) of mice previously immunized for EAE. This resulted in focal pathology characterized by infiltration and demyelination in the CC. We find that intrathecal delivery of MIS416, a TLR9 and NOD2 bispecific innate ligand, into the cerebrospinal fluid reduced focal lesions in the CC. This was associated with upregulation of type I and II interferons, interleukin-10, arginase-1, CCL-2 and CXCL-10. Analysis of draining cervical lymph nodes showed upregulation of type II interferons and interleukin 10. Moreover, intrathecal MIS416 altered the composition of early CNS infiltrates, increasing proportions of myeloid and NK cells and reducing T cells at the lesion site. This study contributes to an increased understanding of how innate immune responses can play a protective role, which in turn may lead to additional therapeutic strategies for the prevention and treatment of demyelinating pathologies.

Protective roles for myeloid cells in neuroinflammation.

Myeloid cells represent the major cellular component of innate immune responses. Myeloid cells include monocytes and macrophages, granulocytes (neutrophils, basophils and eosinophils) and dendritic cells (DC). The role of myeloid cells has been broadly described both in physiological and in pathological conditions. All tissues or organs are equipped with resident myeloid cells, such as parenchymal microglia in the brain, which contribute to maintaining homeostasis. Moreover, in case of infection or tissue damage, other myeloid cells such as monocytes or granulocytes (especially neutrophils) can be recruited from the circulation, at first to promote inflammation and later to participate in repair and regeneration. This review aims to address the regulatory roles of myeloid cells in inflammatory diseases of the central nervous system (CNS), with a particular focus on recent work showing induction of suppressive function via stimulation of innate signalling in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE).

First-in-human phase I clinical trial of the NY-ESO-1 protein cancer vaccine with NOD2 and TLR9 stimulants in patients with NY-ESO-1-expressing refractory solid tumors.

Cholesteryl pullulan (CHP) is a novel antigen delivery system. CHP and New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen complexes (CHP-NY-ESO-1) present multiple epitope peptides to the MHC class I and II pathways. Adjuvants are essential for cancer vaccines. MIS416 is a non-toxic microparticle that activates immunity via the nucleotide-binding oligomerization domain 2 (NOD2) and TLR9 pathways. However, no reports have explored MIS416 as a cancer vaccine adjuvant. We conducted a first-in-human clinical trial of CHP-NY-ESO-1 with MIS416 in patients with NY-ESO-1-expressing refractory solid tumors. CHP-NY-ESO-1/MIS416 (µg/µg) was administered at 100/200, 200/200, 200/400 or 200/600 (cohorts 1, 2, 3 and 4, respectively) every 2 weeks for a total of 6 doses (treatment phase) followed by one vaccination every 4 weeks until disease progression or unacceptable toxicity (maintenance phase). The primary endpoints were safety and tolerability, and the secondary endpoint was the immune response. In total, 26 patients were enrolled. Seven patients (38%) continued vaccination in the maintenance phase. Grade 3 drug-related adverse events (AEs) were observed in six patients (23%): anorexia and hypertension were observed in one and five patients, respectively. No grade 4-5 drug-related AEs were observed. Eight patients (31%) had stable disease (SD). Neither augmentation of the NY-ESO-1-specific IFN-γ-secreting CD8+ T cell response nor an increase in the level of anti-NY-ESO-1 IgG1 was observed as the dose of MIS416 was increased. In a preclinical study, adding anti-PD-1 monoclonal antibody to CHP-NY-ESO-1 and MIS416 induced significant tumor suppression. This combination therapy is a promising next step.

Innate signaling within the central nervous system recruits protective neutrophils.

There is great interest in understanding how the central nervous system (CNS) communicates with the immune system for recruitment of protective responses. Infiltrating phagocytic monocytes and granulocytes are implicated in neuroinflammation in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). To investigate how CNS endogenous signals can be harnessed to promote anti-inflammatory programs, we have used a particulate Toll-like receptor 9 and nucleotide-oligomerization domain 2 bispecific innate ligand (MIS416), to address whether its phagocytosis within the CNS recruits protective myeloid cells. We find that MIS416 injected intrathecally into the cerebrospinal fluid via the cisterna magna induced a local chemokine response that recruited blood-derived monocytes and neutrophils to the CNS. These cells phagocytosed MIS416. The increase in EAE severity normally seen from time of onset did not occur in mice receiving MIS416. This suppression of disease symptoms was dependent on expression of the type I interferon receptor (IFNAR). Transfer of intrathecal MIS416-induced neutrophils suppressed EAE in recipient mice, while monocytes did not transfer protection. MIS416-induced neutrophils showed increased IL-10 expression that was IFNAR1-driven. In contrast to intrathecal administration, intravenous administration of MIS416 led to monocyte but not neutrophil infiltration to the CNS. We thus identify a CNS-intrinsic and -specific phagocytosis-induced recruitment of anti-inflammatory neutrophils that contribute to CNS homeostasis and may have therapeutic potential.

Oncolytic Maraba virus armed with tumor antigen boosts vaccine priming and reveals diverse therapeutic response patterns when combined with checkpoint blockade in ovarian cancer.

BACKGROUND: Cancer immunotherapies are emerging as promising treatment strategies for ovarian cancer patients that experience disease relapse following first line therapy. As such, identifying strategies to bolster anti-tumor immunity and limit immune suppression, while recognizing diverse patterns of tumor response to immunotherapy is critical to selecting treatment combinations that lead to durable therapeutic benefit. METHODS: Using a pre-clinical mouse model, we evaluated a heterologous prime/boost vaccine in combination with checkpoint blockade to treat metastatic intraperitoneal ovarian cancer. Vaccine-elicited CD8+ T cell responses and changes in the tumor microenvironment following treatment were analyzed and compared to treatment outcome. Kinetics of intraperitoneal tumor growth were assessed using non-invasive magnetic resonance imaging (MRI). RESULTS: Vaccine priming followed by antigen-armed oncolytic Maraba virus boosting elicited robust tumor-specific CD8+ T cell responses that improved tumor control and led to unique immunological changes in the tumor, including a signature that correlated with improved clinical outcome of ovarian cancer patients. However, this treatment was not curative and T cells in the tumor microenvironment (TME) were functionally suppressed. Combination PD-1 blockade partially overcame the adaptive resistance in the tumor observed in response to prime/boost vaccination, restoring CD8+ T cell function in the TME and enhancing the therapeutic response. Non-invasive MRI of tumors during the course of combination treatment revealed heterogeneous radiologic response patterns following treatment, including pseudo-progression, which was associated with improved tumor control prior to relapse. CONCLUSIONS: Our findings point to a key hierarchical role for PD-1 signaling and adaptive immune resistance in the ovarian TME in determining the functional fate of tumor-specific CD8+ T cells, even in the context of robust therapy mediated anti-tumor immunity, as well as the ability of multiple unique patterns of therapeutic response to result in durable tumor control.

Toll-like receptor 9-dependent interferon production prevents group 2 innate lymphoid cell-driven airway hyperreactivity.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) are important mediators of allergic asthma. Bacterial components, such as unmethylated CpG DNA, a Toll-like receptor (TLR) 9 agonist, are known to possess beneficial immunomodulatory effects in patients with T cell-mediated chronic asthma. However, their roles in regulating ILC2s remain unclear. OBJECTIVE: We sought to determine the role of TLR9 activation in regulating ILC2 function and to evaluate the therapeutic utility of an immunomodulatory microparticle containing natural TLR9 ligand (MIS416). METHODS: We evaluated the immunomodulatory effects of CpG A in IL-33-induced airway hyperreactivity (AHR) and airway inflammation. The roles of interferons were examined in vivo and in vitro by using signal transducer and activator of transcription 1 (Stat1)-/- mice and neutralizing antibodies against IFN-γ and IFN-α/ß receptor subunit 1, and their cellular sources were identified. The therapeutic utility of MIS416 was investigated in the Alternaria alternata model of allergic asthma and in humanized NSG mice. RESULTS: We show that TLR9 activation by CpG A suppresses IL-33-mediated AHR and airway inflammation through inhibition of ILC2s. Activation of TLR9 leads to production of IFN-α, which drives IFN-γ production by natural killer cells. Importantly, IFN-γ is essential for TLR9-driven suppression, and IFN-α cannot compensate for impaired IFN-γ signaling. We further show that IFN-γ directly inhibits ILC2 function through a STAT1-dependent mechanism. Finally, we demonstrate the therapeutic potential of MIS416 in A alternata-induced airway inflammation and validated these findings in human subjects. CONCLUSION: TLR9 activation alleviates ILC2-driven AHR and airway inflammation through direct suppression of cell function. Microparticle-based delivery of TLR9 ligands might serve as a therapeutic strategy for asthma treatment.

MIS416 Enhances Therapeutic Functions of Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Against Experimental Colitis by Modulating Systemic Immune Milieu.

Human adult stem cells, including umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), have recently been considered a promising alternative treatment for inflammatory bowel disease (IBD) due to their unique immunomodulatory properties and ability to promote tissue regeneration. However, despite many years of research and pre-clinical studies, results from clinical trials using these cells have been diverse and conflicting. This discrepancy is caused by several factors, such as poor engraftment, low survival rate, and donor-dependent variation of the cells. Enhancement of consistency and efficacy of MSCs remains a challenge for the feasibility of cell-based therapy. In this study, we investigated whether administration of MIS416, a novel microparticle that activates NOD2 and TLR9 signaling, could enhance the therapeutic efficacy of hUCB-MSCs against Crohn's disease, using dextran sulfate sodium (DSS)-induced colitis model. Colitis was experimentally induced in mice by using 3% DSS, and mice were administered a retro-orbital injection of MIS416 and subsequent intraperitoneal injection of hUCB-MSCs. Mice were examined grossly, and blood, spleen, and colon tissues were subsequently collected for further ex vivo analyses. To explore the effects of MIS416 on the therapeutic process, hUCB-MSCs and primary isolated immune cells were cultured with MIS416, and in vitro assays were performed. Compared to the single administration of hUCB-MSCs, co-administration with MIS416 improved the therapeutic efficiency of the stem cells by significantly alleviating the symptoms of IBD. Interestingly, MIS416 did not exert any direct effect on the immunomodulatory capacity of hUCB-MSCs. Instead, systemically injected MIS416 altered the immune milieu in the colon which caused hUCB-MSCs to be more readily recruited toward the lesion site and to suppress inflammation more efficiently. In addition, considerable numbers of regulatory immune cells were stimulated as a result of the cooperation of MIS416 and hUCB-MSCs. These findings indicate that co-administration with MIS416 enhances the therapeutic potential of hUCB-MSCs by systemically regulating the immune response, which might be an effective strategy for overcoming the current obstacles to stem cell therapy in clinical practice.

MIS416 as a siRNA Delivery System with the Ability to Target Antigen-Presenting Cells.

MIS416 is a microparticulate formulation derived from propionibacterium acnes cell wall skeletons with intrinsic adjuvant activity. Conjugates of MIS416-SS-peptide containing a disulfide linkage facilitate the cytoplasmic delivery and release of peptides in antigen-presenting cells (APCs). We hypothesized that MIS416-siRNA (small interfering RNA) conjugates, containing a disulfide linkage between MIS416 and the siRNA, would allow cytoplasmic release of siRNA in APCs. MIS416-SS-siStat3 conjugates added to cell culture medium of monolayers of DCs in culture flasks successfully targeted Stat3 mRNA in DCs in vitro without transfection, downregulating Stat3 mRNA and protein levels. These results suggest that MIS416-SS-siRNA conjugates can be used as a novel siRNA delivery system for the knockdown of mRNA levels in APCs.

Innate IFN-γ ameliorates experimental autoimmune encephalomyelitis and promotes myeloid expansion and PDL-1 expression.

The innate immune system plays a central role in the immune-mediated pathology of multiple sclerosis, and is a therapeutic target for progressive disease. Recently, it has been demonstrated that MIS416, a novel immunomodulatory microparticle that activates NOD-2 and TLR-9-signaling, has disease-modifying activity in multiple sclerosis models. This activity is dependent on innate IFN-γ; however, the precise immune regulatory mechanisms amplified by MIS416 have not previously been determined. Using the experimental autoimmune encephalomyelitis model, MIS416 treatment was associated with IFN-γ-dependant expansion of Treg number and increased suppressive function; however, these cells did not account for disease reduction. Additionally, MIS416 treatment stimulated increased nitric oxide production that was IFN-γ-dependant but dispensable for protection. Finally, MIS416-mediated protection was shown to correlate with IFN-γ-dependant expansion of PDL-1-expressing peripheral myeloid cells, a subset of which was found to be selectively recruited to the brain. This central nervous system trafficking was independent of neuro-inflammatory signals as it occurred in MIS416-treated healthy mice. Together, these findings provide insight into regulatory myeloid cell activities amplified by MIS416-mediated NOD-2 and TLR-9 signalling and highlight the potential importance of these cells in accessing the brain where they may act locally and contribute to the control of neuroinflammation.

Evaluation of neurological changes in secondary progressive multiple sclerosis patients treated with immune modulator MIS416: results from a feasibility study.

BACKGROUND: While disease progression can be readily monitored in early stage relapsing multiple sclerosis (MS), it is more challenging for secondary progressive multiple sclerosis (SPMS). This advanced stage of disease has distinct pathophysiology due to compartmentalization of neuroinflammatory activity within the central nervous system, resulting in increased incidence and severity of cognitive dysfunction. The shift in the dominant disease pathways is underscored by the failure of relapsing therapies to benefit SPMS patients, highlighting the need for novel treatment strategies and clinical trial endpoints that are well-aligned with potential benefits. The Expanded Disability Status Scale (EDSS) is widely used but is weighted towards ambulatory ability, lacking sensitivity to other aspects of neurological impairment experienced in more severely disabled SPMS patients, so may not effectively capture their clinical status.To investigate the feasibility of an alternative clinical trial endpoint model for a phase 2B trial of an immune modulator for SPMS, the potential for treatment efficacy-based patient-centered outcomes was assessed within the context of a before and after, 12-week clinical trial of safety and tolerability. METHODS: Patients treated with MIS416 for 12 weeks were evaluated for clinical status at baseline and end of dosing, using the established Multiple Sclerosis Functional Composite, Short Form Health Survey, and Expanded Disability Status Scale. Responder status was determined for eight outcome measures based on minimally important change, defined using published studies. To evaluate the patients' immune response to MIS416, blood plasma samples collected at baseline and pre- and 24-h post doses 1-4 were analyzed using multiplex cytokine quantification assays. RESULTS: Using a combination of patient-centered outcomes, MIS416 treatment was associated with improved clinical status for 10/11 patients: eight patients showed improvement on two to five outcome measures, five of which also showed improvement by EDSS. Multi-dimensional scaling analysis of MIS416-induced factors quantified in individual patients, revealed immune response patterns which had a strong concordance with the extent of the patients' clinical response. CONCLUSIONS: The data support the feasibility of using patient-centered outcomes as additional clinical trial endpoints, for determining the efficacy of disease-modifying therapies, in secondary progressive multiple sclerosis patients. TRIAL REGISTRATION: ClinicalTrial.gov, NCT01191996.

Bridging Small Molecules to Modified Bacterial Microparticles Using a Disulphide Linkage: MIS416 as a Cargo Delivery System.

MIS416 is an intact minimal cell wall skeleton derived from Proprionibacterium acnes that is phagocytosed by antigen presenting cells, including dendritic cells (DCs). This property allows MIS416 to be exploited as a vehicle for the delivery of peptide antigens or other molecules (for example, nucleic acids) to DCs. We previously showed that covalent (non-cleavable) conjugation of OVA, a model antigen derived from ovalbumin, to MIS416 enhanced immune responses in DCs in vivo, compared to unconjugated MIS416 and OVA. Intracellular trafficking promotes the lysosomal degradation of MIS416, leading to the destruction of MIS416 plus the associated cargos conjugated to MIS416. However, lysosomal degradation of cargo may not be desired for some MIS416 conjugates. Here we have investigated whether a cleavable linkage could facilitate release of the cargo in the cytoplasm of DCs to avoid lysosomal degradation. DCs were treated in vitro with disulfide-containing conjugates, and as hypothesised faster release of SIINFEKL peptide in the cytoplasm of DCs was observed with the inclusion of a disulfide bond between MIS416 and cargo. The inclusion of a cleavable disulfide bond in the conjugates did not significantly alter the amount of SIINFEKL antigens presented on MHC I molecules on DCs as compared with conjugates without a disulfide bond. However, the conjugates containing disulfide-linkages performed either slightly better (p<0.05) than, or the same as conjugates without a disulfide bond with respect to in vitro OT-1 T-cell proliferation induced by the presentation of SIINFEKL antigens on DCs, or DC activation studies, respectively. However, disulfide-containing conjugates were less effective than conjugates without a disulfide bond in in vivo cytotoxicity assays. In conclusion, inclusion of a disulfide bond in MIS416-peptide conjugates was associated with efficient release of peptides in the cytoplasm of DCs, an important consideration for MIS416-mediated delivery of degradation-sensitive cargoes. However, treatment of DCs with disulfide-containing conjugates did not significantly alter the presentation of peptide antigens on MHC class I molecules to T-cells, or greatly enhance antigen-associated T-cell proliferation in vitro.

Targeting myeloid cells in the tumor microenvironment enhances vaccine efficacy in murine epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is typically diagnosed at advanced stages, and is associated with a high relapse rate. Patients in remission are ideal candidates for immunotherapy aimed at cure or prolonging disease-free periods. However, immunosuppressive pathways in the tumor microenvironment are obstacles to durable anti-tumor immunity. In a metastatic syngeneic mouse model of EOC, immunosuppressive macrophages and myeloid-derived suppressor cells (MDSCs) accumulate in the local tumor environment. In addition, resident peritoneal macrophages from non-tumor-bearing mice were highly immunosuppressive, abrogating stimulated T cell proliferation in a cell contact-dependent manner. Immunization with microparticles containing TLR9 and NOD-2 ligands (MIS416) significantly prolonged survival in tumor-bearing mice. The strategy of MIS416 immunization followed by anti-CD11b administration further delayed tumor progression, thereby establishing the proof of principle that myeloid depletion can enhance vaccine efficacy. In patients with advanced EOC, ascites analysis showed substantial heterogeneity in the relative proportions of myeloid subsets and their immunosuppressive properties. Together, these findings point to immunosuppressive myeloid cells in the EOC microenvironment as targets to enhance vaccination. Further studies of myeloid cell accumulation and functional phenotypes in the EOC microenvironment may identify patients who are likely to benefit from vaccination combined with approaches that deplete tumor-associated myeloid cells.

Safety, tolerability and pharmacodynamics of a novel immunomodulator, MIS416, in patients with chronic progressive multiple sclerosis.

BACKGROUND: Preclinical studies have demonstrated that MIS416, a bacterially derived immune modulator, targets myeloid cells following systemic delivery. MIS416 stimulated myeloid cells have the capacity to regulate innate inflammation, a potential therapeutic target for progressive multiple sclerosis. OBJECTIVES: To determine the safety, tolerability, pharmacodynamics and maximum tolerated dose and/or recommended Phase 2 dose of MIS416. METHODS: An open-label, non-randomized, phase II, dose-escalation study, in patients with progressive multiple sclerosis: dose-escalation phase, with MIS416 administered once weekly for four weeks to determine maximum tolerated dose; and dose-confirmation phase, administered once weekly for up to 12 weeks. RESULTS: The safety profile indicates the majority of adverse events were mild or moderate, tolerable, self-limiting and consistent with the known bioactivity of MIS416 (acute flu-like symptoms). Maximum tolerated dose was not reached. A dose of 500 µg/week was recommended for the Phase 2 dose. CONCLUSION: MIS416 is well tolerated at a dose of 500 µg/week. The adverse event profile is consistent with the mechanism of action of MIS416, indicating bioactivity within the signal transduction pathways and supported by induction of a known MIS416 pharmacodynamic marker. It is recommended that safety and efficacy of MIS416 is investigated further in a larger randomized controlled trial. http://clinicaltrials.gov reference NCT01191996.

Targeting innate receptors with MIS416 reshapes Th responses and suppresses CNS disease in a mouse model of multiple sclerosis.

Modification of the innate immune cell environment has recently been recognized as a viable treatment strategy for reducing autoimmune disease pathology. MIS416 is a microparticulate immune response modifier that targets myeloid cells, activating cytosolic receptors NOD2 and TLR9, and has completed a phase 1b/2a trial for the treatment of secondary progressive multiple sclerosis. Using a mouse model of multiple sclerosis, we are investigating the pathways by which activation of TLR9 and NOD2 may modify the innate immune environment and the subsequent T cell-mediated autoimmune responses. We have found that MIS416 has profound effects on the Th subset balance by depressing antigen-specific Th1, Th17, and Th2 development. These effects coincided with an expansion of specific myeloid subpopulations and increased levels of MIS416-stimulated IFN-γ by splenocytes. Additionally, systemic IFN-γ serum levels were enhanced and correlated strongly with disease reduction, and the protective effect of MIS416 was abrogated in IFN-γ-deficient animals. Finally, treatment of secondary progressive MS patients with MIS416 similarly elevated the levels of IFN-γ and IFN-γ-associated proteins in the serum. Together, these studies demonstrate that administration of MIS416, which targets innate cells, reshapes autoimmune T cell responses and leads to a significant reduction in CNS inflammation and disease.

MIS416, a non-toxic microparticle adjuvant derived from Propionibacterium acnes comprising immunostimulatory muramyl dipeptide and bacterial DNA promotes cross-priming and Th1 immunity.

Propionibacterium acnes was modified using biochemical extraction methods generating a suspension of microparticles (MIS416) comprising a minimal cell wall skeleton rich in immunostimulatory crosslinked muramyl dipeptide repeats and native bacterial DNA fragments, each which have known adjuvant activity. In vitro studies demonstrated that MIS416 was readily internalized by human myeloid and plasmacytoid DC inducing cytokine secretion and cell activation/maturation. Vaccination studies in mice using OVA as a model antigen demonstrated that MIS416 acts as a Th1 adjuvant, promoting cross-priming of cytotoxic CD8(+) T cell responses and enhanced anti-tumour immunity. Covalent attachment of OVA to MIS416 enabling simultaneous delivery of antigen and adjuvant to the antigen presentation system resulted in a dose-sparing vaccine formulation. Preclinical GLP toxicology studies demonstrated that MIS416 has a favorable safety profile in mouse and rabbit supporting its use in human vaccine formulations.

Potent subunit-specific effects on cell growth and drug sensitivity from optimised siRNA-mediated silencing of ribonucleotide reductase.

Ribonucleotide reductase (RR) has an essential role in DNA synthesis and repair and is a therapeutic target in a number of different cancers. Previous studies have shown that RNAi-mediated knockdown of either the RRM1 or RRM2 subunit sensitizes cells to the cytotoxic effects of the nucleoside analogs and more recently it has been shown that RRM2 knockdown itself has a growth inhibitory effect. Here we compare the effects of siRNA-mediated knockdown of both RRM1 and RRM2 subunits of RR in A549 and HCT-116 cells using an optimized transfection protocol. Growth of A549 cells was strongly inhibited by efficient siRNA-mediated silencing of either RRM1 or RRM2, and knockdown of each subunit led to long-term growth inhibition and cell-cycle arrest. Knockdown with sub growth inhibitory siRNA concentrations sensitized A549 and HCT-116 cells to gemcitabine when RRM1 was targeted, whereas RRM2 knockdown led to hydroxyurea sensitization. These results suggest that the inhibition of cell growth, rather than drug sensitization, is the major effect of RRM1 and RRM2 knockdown. In an A549 xenograft model, cells transfected with RRM1-specific siRNA failed to form tumors in 6 out of 8 CD1 nude mice, whereas those transfected with RRM2-specific siRNA grew but at a reduced rate. Taken together, these data demonstrate that siRNA-mediated knockdown of the RRM1 subunit is more effective than knockdown of RRM2 in inhibiting the growth of cancer cell lines and suggest that RRM1 is a potential target for nucleic acid-based cancer therapies, either alone or in combination with gemcitabine.

A novel form of the RelA nuclear factor kappaB subunit is induced by and forms a complex with the proto-oncogene c-Myc.

Members of both Myc and nuclear factor kappaB (NF-kappaB) families of transcription factors are found overexpressed or inappropriately activated in many forms of human cancer. Furthermore, NF-kappaB can induce c-Myc gene expression, suggesting that the activities of these factors are functionally linked. We have discovered that both c-Myc and v-Myc can induce a previously undescribed, truncated form of the RelA(p65) NF-kappaB subunit, RelA(p37). RelA(p37) encodes the N-terminal DNA binding and dimerization domain of RelA(p65) and would be expected to function as a trans-dominant negative inhibitor of NF-kappaB. Surprisingly, we found that RelA(p37) no longer binds to kappaB elements. This result is explained, however, by the observation that RelA(p37), but not RelA(p65), forms a high-molecular-mass complex with c-Myc. These results demonstrate a previously unknown functional and physical interaction between RelA and c-Myc with many significant implications for our understanding of the role that both proteins play in the molecular events underlying tumourigenesis.