Novel Class Of Therapeutic Targets Research Articles

Targeting IGF2BP3 enhances antileukemic effects of menin-MLL inhibition in MLL-AF4 leukemia

AbstractRNA-binding proteins (RBPs) are emerging as a novel class of therapeutic targets in cancer, including in leukemia, given their important role in posttranscriptional gene regulation, and have the unexplored potential to be combined with existing therapies. The RBP insulin-like growth factor 2 messenger RNA–binding protein 3 (IGF2BP3) has been found to be a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Here, we study the combined effects of targeting IGF2BP3 and menin-MLL interaction in MLL-AF4–driven leukemia in vitro and in vivo, using genetic inhibition with CRISPR-Cas9–mediated deletion of Igf2bp3 and pharmacologic inhibition of the menin-MLL interaction with multiple commercially available inhibitors. Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the effects of menin-MLL inhibition on cell growth and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and menin-MLL inhibition led to increased differentiation in vitro and in vivo, seen in functional readouts and by gene expression analyses. IGF2BP3 knockdown had a greater effect on increasing survival and attenuating disease than pharmacologic menin-MLL inhibition with small molecule MI-503 alone and showed enhanced antileukemic effects in combination. Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4–mediated leukemogenesis and a potent therapeutic target, providing a paradigm for targeting leukemia at both the transcriptional and posttranscriptional level.

Immune Checkpoints, a Novel Class of Therapeutic Targets for Autoimmune Diseases.

Autoimmune diseases, such as multiple sclerosis and type-1 diabetes, are the outcomes of a failure of immune tolerance. Immune tolerance is sustained through interplays between two inter-dependent clusters of immune activities: immune stimulation and immune regulation. The mechanisms of immune regulation are exploited as therapeutic targets for the treatment of autoimmune diseases. One of these mechanisms is immune checkpoints (ICPs). The roles of ICPs in maintaining immune tolerance and hence suppressing autoimmunity were revealed in animal models and validated by the clinical successes of ICP-targeted therapeutics for autoimmune diseases. Recently, these roles were highlighted by the clinical discovery that the blockade of ICPs causes autoimmune disorders. Given the crucial roles of ICPs in immune tolerance, it is plausible to leverage ICPs as a group of therapeutic targets to restore immune tolerance and treat autoimmune diseases. In this review, we first summarize working mechanisms of ICPs, particularly those that have been utilized for therapeutic development. Then, we recount the agents and approaches that were developed to target ICPs and treat autoimmune disorders. These agents take forms of fusion proteins, antibodies, nucleic acids, and cells. We also review and discuss safety information for these therapeutics. We wrap up this review by providing prospects for the development of ICP-targeting therapeutics. In summary, the ever-increasing studies and results of ICP-targeting of therapeutics underscore their tremendous potential to become a powerful class of medicine for autoimmune diseases.

Epigenetic silencing of KLF2 by long non-coding RNA SNHG1 inhibits periodontal ligament stem cell osteogenesis differentiation

BackgroundExploring the effects of lncRNA SNHG1 in the process of osteogenic differentiation of periodontal ligament stem cells (PDLSCs) would provide novel therapeutic strategies for tissue regeneration.MethodsLoss-of-function and gain-of-function assays were induced by lentivirus. The osteogenic differentiation of PDLSCs were assessed by ALP staining and Alizarin Red staining as well as the mRNA and protein levels of osteogenic marker genes osterix, osteocalcin, and alkaline phosphatase through qRT-PCR and western blot. RNA immunoprecipitation assay and chromatin immunoprecipitation assays were performed to uncover the interaction between SNHG1 and EZH2.ResultsOur analysis revealed that SNHG1 was downregulated and KLF2 was upregulated during the osteogenic induction differentiation of PDLSCs. SNHG1 inhibited while KLF2 promoted osteogenic differentiation of PDLSCs. SNHG1 directly interact with the histone methyltransferase enhancer of the zeste homolog 2 (EZH2) and modulate the histone methylation of promoter of Kruppel-like factor 2 (KLF2) and altered the progress osteogenic differentiation of PDLSCs.ConclusionsTaken together, SNHG1 inhibited the osteogenic differentiation of PDLSCs through EZH2-mediated H3K27me3 methylation of KLF2 promotor and provided a novel class of therapeutic targets for regenerate dental tissues.

Loss of heterozygosity of essential genes represents a widespread class of potential cancer vulnerabilities

Alterations in non-driver genes represent an emerging class of potential therapeutic targets in cancer. Hundreds to thousands of non-driver genes undergo loss of heterozygosity (LOH) events per tumor, generating discrete differences between tumor and normal cells. Here we interrogate LOH of polymorphisms in essential genes as a novel class of therapeutic targets. We hypothesized that monoallelic inactivation of the allele retained in tumors can selectively kill cancer cells but not somatic cells, which retain both alleles. We identified 5664 variants in 1278 essential genes that undergo LOH in cancer and evaluated the potential for each to be targeted using allele-specific gene-editing, RNAi, or small-molecule approaches. We further show that allele-specific inactivation of either of two essential genes (PRIM1 and EXOSC8) reduces growth of cells harboring that allele, while cells harboring the non-targeted allele remain intact. We conclude that LOH of essential genes represents a rich class of non-driver cancer vulnerabilities.

Long Non-coding RNAs: At the Heart of Cardiac Dysfunction?

During the past decade numerous studies highlighted the importance of long non-coding RNAs (lncRNAs) in orchestrating cardiovascular cell signaling. Classified only by a transcript size of more than 200 nucleotides and their inability to code for proteins, lncRNAs constitute a heterogeneous group of RNA molecules with versatile functions and interaction partners, thus interfering with numerous endogenous signaling pathways. Intrinsic transcriptional regulation of lncRNAs is not only specific for different cell types or developmental stages, but may also change in response to stress factors or under pathological conditions. Regarding the heart, an increasing number of studies described the critical regulation of lncRNAs in multiple cardiac disorders, underlining their key role in the development and progression of cardiac diseases. In this review article, we will summarize functional cardiac lncRNAs with a detailed view on their molecular mode of action in pathological cardiac remodeling and myocardial infarction. In addition, we will discuss the use of circulating lncRNAs as biomarkers for prognostic and diagnostic purposes and highlight the potential of lncRNAs as a novel class of therapeutic targets for therapeutic purpose in heart diseases.

Tau-induced nuclear envelope invagination causes a toxic accumulation of mRNA in Drosophila.

The nucleus is a spherical dual‐membrane bound organelle that encapsulates genomic DNA. In eukaryotes, messenger RNAs (mRNA) are transcribed in the nucleus and transported through nuclear pores into the cytoplasm for translation into protein. In certain cell types and pathological conditions, nuclei harbor tubular invaginations of the nuclear envelope known as the “nucleoplasmic reticulum.” Nucleoplasmic reticulum expansion has recently been established as a mediator of neurodegeneration in tauopathies, including Alzheimer's disease. While the presence of pore‐lined, cytoplasm‐filled, nuclear envelope invaginations has been proposed to facilitate the rapid export of RNAs from the nucleus to the cytoplasm, the functional significance of nuclear envelope invaginations in regard to RNA export in any disorder is currently unknown. Here, we report that polyadenylated RNAs accumulate within and adjacent to tau‐induced nuclear envelope invaginations in a Drosophila model of tauopathy. Genetic or pharmacologic inhibition of RNA export machinery reduces accumulation of polyadenylated RNA within and adjacent to nuclear envelope invaginations and reduces tau‐induced neuronal death. These data are the first to point toward a possible role for RNA export through nuclear envelope invaginations in the pathogenesis of a neurodegenerative disorder and suggest that nucleocytoplasmic transport machinery may serve as a possible novel class of therapeutic targets for the treatment of tauopathies.

Cyclic nucleotide phosphodiesterases: potential therapeutic targets for alcohol use disorder.

Alcohol use disorder (AUD), which combines the criteria of both alcohol abuse and dependence, contributes as an important causal factor to multiple health and social problems. Given the limitation of current treatments, novel medications for AUD are needed to better control alcohol consumption and maintain abstinence. It has been well established that the intracellular signal transduction mediated by the second messengers cyclic AMP (cAMP) and cyclic GMP (cGMP) crucially underlies the genetic predisposition, rewarding properties, relapsing features, and systemic toxicity of compulsive alcohol consumption. On this basis, the upstream modulators phosphodiesterases (PDEs), which critically control intracellular levels of cyclic nucleotides by catalyzing their degradation, are proposed to play a role in modulating alcohol abuse and dependent process. Here, we highlight existing evidence that correlates cAMP and cGMP signal cascades with the regulation of alcohol-drinking behavior and discuss the possibility that PDEs may become a novel class of therapeutic targets for AUD.

10. Epigenetic Enzymes as Novel Therapeutic Targets in Alcohol Addiction

Gene expression in the mPFC is dysregulated in alcohol dependence. We have discovered epigenetic enzymes that take part in dependence-induced reprogramming of the mPFC transcriptome, and may offer a novel class of therapeutic targets.

Complex Structures of Human MDM2 with Interacting Proteins Reveal the Regulation Mechanism of p53

Human MDM2 (murine double minute 2), amplified and overexpressed frequently in most of tumors and cancers, directly interacts with and inhibits the tumor suppressor protein p53. Understanding the regulation mechanism of MDM2 has recently been highlighted because of its roles in the regulation of p53 in cancer biology. MDM2 contains three domains. The N–terminal region interacts with p53 and downregulates its activity. The RING finger domain has ubiquitin ligase activity that can mediate p53 ubiquitination and degradation. Many studies showed that the central region of MDM2 is critical for p53 activation and tumor suppression. To date, more than twenty proteins have been shown to bind to this central region and further to be involved in the regulations of p53. However, how these interacting proteins precisely interact with MDM2 remains to be elucidated. We solved the first structure of the human MDM2–RPL11 complex. Both the acidic domain and C4 zinc finger of MDM2 are essential for RPL11 binding. Moreover, another unexpectedly zinc finger that forms inter-molecularly within the complex. A small molecule imidazole was found to be chelated in this zinc finger, which might be a promising start of drug design. These structural and functional information combining with earlier studies of MDM2 with other interacting proteins establish MDM2 as a novel class of therapeutic targets in human diseases such as cancers, yield insights into the MDM2–p53 surveillance pathway and could also yield useful anticancer strategies.

Targeting Phosphodiesterases in Pharmacotherapy for Substance Dependence.

Substance dependence is a chronic relapsing brain disorder associated with adaptational changes in synaptic plasticity and neuronal functions. The high levels of substance consumption and relapse rate suggest more reliable medications are in need to better address the underlying causes of this disease. It has been well established that the intracellular second messengers cyclic AMP (cAMP) and cyclic GMP (cGMP) and their signaling systems play an important role in the molecular mechanisms of substance taking behaviors. On this basis, the phosphodiesterase (PDE) superfamily, which crucially controls cyclic nucleotide levels by catalyzing their hydrolysis, has been proposed as a novel class of therapeutic targets for substance use disorders. This chapter reviews the expression patterns of PDEs in the brain with regard to neural structures underlying the dependent process and highlights available evidence for a modulatory role of PDEs in substance dependence.

Feedback Regulation of STAT5 Is Critical to Balance MYC and BCL6-Dependent Transcriptional Programs That Regulate Cell Size and Glucose Metabolism

Background: The BCR-ABL1 tyrosine kinase in Ph+ ALL and other ABL1-kinase fusions and JAK-kinase lesions that are found in the emerging subset of Ph-like ALL cause malignant transformation through constitutive phosphorylation of STAT5 at Y694. In addition, genetic lesions of genes encoding IL7R, CRLF2, EPOR are frequently found in Ph-like ALL and cause constitutive STAT5 Y694-phosphorylation. Ph+ ALL represents the most frequent subtype of human ALL in adults and accounts for ~30% of adult B-lineage ALL cases. Ph-like ALL is frequent among children (~10%) and young adults (~25%). Both Ph+ and Ph-like ALL represent highly aggressive diseases and define the group of patients with the worst clinical outcome.STAT5 induces its own negative feedback through transcriptional activation of CISH and SOCS proteins. The cytokine inducible SH2-containing protein (CISH) binds to tyrosine kinase with its SH2 domain and induces its degradation through poly-ubiquitinylation and recruitment of cullins and other ubiquitin-ligases to its SOCS-box. Suppressor of cytokine signaling (SOCS) proteins can interfere with oncogenic tyrosine kinase signaling directly through its kinase inhibitory region (KIR). Interestingly, SOCS2, SOCS3 and CISH are highly expressed in Ph+ ALL and represent the focus of this study. Results: Gene expression analyses showed that mRNA levels of SOCS2 and CISH were 10-fold (p<0.0001) and 3-fold (p<0.0001) upregulated in Ph+ ALL cells (n=15) compared to normal pre-B cells (n=8) respectively. As measured by qRT-PCR and Western blot, tyrosine kinase inhibitor (TKI)-treatment rapidly decreased the expression of SOCS2, SOCS3 and CISH in Ph+ ALL cells, indicating that high expression levels of SOCS2, SOCS3 and CISH are induced by tyrosine kinase activity.Importantly, we found that high expression levels of SOCS and CISH feedback inhibitors are predictive of poor outcome in the COG trial (P9906; n=207) for children with high-risk ALL: Patients with early minimal residual disease (MRD) findings on day 29 had much higher mRNA levels of SOCS2, SOCS3 or CISH (n=207; p<0.04), and patients with higher than median expression of SOCS2, SOCS3 or CISH had decreased overall survival rate (p<0.05) and relapse free survival rate (p<0.01) compared to patients with lower than median expression of SOCS2, SOCS3 or CISH. These results collectively indicate that inhibitory signaling from SOCS2, SOCS3 and CISH molecules may contribute to disease progression of pre-B ALL.To study the function of Socs2, Socs3 and Cish in genetic experiments, we transformed pre-B cells from mutant mice. Deletion of Socs2, Socs3 or Cish resulted in increased STAT5-activation, increased ROS (reactive oxygen species), accumulation of Arf, p53 and p21, cellular senescence and subsequent cell death of leukemia cells. Studying BCR-ABL1 transformed Cishfl/fl ALL cells in vivo, we found that genetic deletion of Cish resulted in rapid leukemia regression and prolonged survival of recipient NOD-SCID mice (13 days vs 30 days; n=7; p<0.001).To study the mechanism of Cish-dependency of pre-B ALL cells, we studied the consequences of inducible expression of a constitutively tyrosine-phosphorylated form (pY) or inactive form that cannot be tyrosine-phosphorylated (YF) of Stat5 in BCR-ABL1 ALL cells. While both pY and YF forms of Stat5 were highly toxic, the phenotypic outcome was dramatically different. While Stat5-YF caused rapid cell shrinkage, Stat5-pY increased cell size by nearly 2-fold. Stat5-YF upregulated Bcl6 expression, decreased Myc expression, glucose consumption and ATP production. In contrast, Stat5-pY increased Myc expression, glucose consumption and ATP production. Metabolomics and RNA-seq results indicated that Stat5-pY increased glycolytic and TCA metabolism through mTOR activation. Inhibition of mTOR by rapamycin or knocking down Myc largely rescued leukemia cells that were transduced with Stat5-pY, while overexpression of Myc rescued Stat5-YF transduced cells.Conclusion: Our results suggest that STAT5 feedback control is required to keep a balance between p-STAT5 (pY) and non-p-STAT5 (YF), which promote divergent, MYC (pY) and BCL6 (YF)-dependent transcriptional programs. Even minor imbalances between these two programs are toxic to ALL cells. These findings also identify SOCS proteins as members of a fundamentally novel class of therapeutic targets for ALL. DisclosuresAlexander:Childrens Cancer Institute Australia for Medical Research: Membership on an entity's Board of Directors or advisory committees; National Health and Medical Research Council, Australia: Membership on an entity's Board of Directors or advisory committees, Research Funding; Murigen Therapeutics: Equity Ownership; The walter and Eliza Hall Institute of Medical Reserach: Employment.

MicroRNA regulation of airway smooth muscle function.

Airway smooth muscle (ASM) controls airway narrowing and plays a pivotal role in the pathogenesis of asthma. MicroRNAs are small yet powerful gene tuners that regulate diverse cellular processes. Recent studies have demonstrated the versatile role of microRNAs in regulating multiple ASM phenotypes that are critically involved in asthma pathogenesis. These ASM phenotypes include proliferation, cell size, chemokine secretion, and contractility. Here we review microRNA-mediated regulation of ASM functions and discuss the potential of microRNAs as a novel class of therapeutic targets to improve ASM function for asthma therapy.

Erk and Stat5 Feedback Control Enables Pre-B Cell Transformation and Represents a Therapeutic Target in Acute Lymphoblastic Leukemia

▪Background and Hypothesis: Targeted therapy of cancer typically focuses on the development of agents that will inactivate a transforming oncogene. In this study, we tested the concept that besides the oncogene itself, factors that enable permissiveness of a normal cell to oncogenic signaling represent a novel class of therapeutic targets. This hypothesis was based on three findings. First, acute activation of oncogenes in normal pre-B cells typically caused immediate cell death, unless pre-B cells were capable of adapting quickly enough to a high level of signaling output. Second, few surviving pre-B cell clones achieved permissiveness to oncogenic signaling by strong activation of negative feedback control of Erk and Stat5. Third, robust feedback control of Erk and Stat5 distinguishes normal pre-B cells from fully transformed pre-B acute lymphoblastic leukemia (ALL) cells.Results: To test the significance of strong feedback control of Erk and Stat5 signaling in pre-B ALL cells, we developed genetic loss-of function models for six central molecules in Erk (DUSP6, SPRY2, ETV5) and Stat5 (Cish, SOCS2, SOCS3) feedback control. Genetic deletion of the sprouty family Ras inhibitor Spry2, the Erk dual specificity phosphatase Dusp6 and their transcriptional activator Etv5, decreased robustness of Erk feedback control and compromised oncogenic transformation in mouse models for pre-B ALL. Likewise, ablation of Stat5 feedback control through deletion of the suppressors of cytokine signaling (SOCS) family molecules Cish, Socs2 and Socs3 reversed permissiveness of pre-B cells. Studying deletion of Spry2 (Erk) and Cish (Stat5) in an in vivo transplant model using inducible, Cre-mediated deletion of Spry2 and Cish in pre-B ALL cells confirmed that Erk and Stat5 feedback control are essential for malignant transformation and development of lethal leukemia. Genetic deletion of Erk (Dusp6, Spry2, Etv5) and Stat5 (Cish, Socs2, Socs3) feedback control impairs leukemic transformation of pre-B cells.Searching for factors that restrict permissiveness to oncogene signaling, we identified the pre-B cell tumor suppressor IKZF1, which is deleted in a large fraction of pre-B ALL cases. IKZF1 directly bound to and transcriptionally repressed multiple promoters of Erk and Stat5 feedback control and IKZF1 deletion raised the limit of maximum allowable oncogene signaling strength in pre-B ALL cells. We propose that the pre-B cell tumor suppressor IKZF1 functions as transcriptional repressor of Erk and Stat5 feedback control and thereby retains pre-B cells in a Non-permissive state.Clinical relevance: To assess potential usefulness of this finding for the development of future treatment strategies, we tested the effect of a specific small molecule inhibitor of DUSP6, E-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI), which was designed as an allosteric inhibitor of the interaction between DUSP6 and phospho-ERK1/2. Interestingly, BCI acutely subverted Erk feedback control and selectively induced cell death in pre-B ALL cells. Small molecule inhibition of DUSP6 was sufficient to overcome conventional mechanisms of drug-resistance in pre-B ALL and selectively killed patient-derived pre-B ALL cells in a leukemia transplant model. BCI treatment, similar to Dusp6-deletion in our leukemia mouse model, led to the accumulation of P53 and ARF in patient-derived pre-B ALL cells. In addition, small molecule inhibition of DUSP6 had strong selective activity on drug-resistant patient-derived pre-B ALL cells that were injected into NOD/SCID transplant recipient mice. These findings identify permissive negative feedback control of oncogenic signaling as a previously unrecognized vulnerability of pre-B ALL cells and a new class of potential therapeutic targets.Conclusion: Targeting negative feedback regulation of both Erk and Stat5 signaling for the treatment of pre-B ALL seems counter-intuitive because it represents effectively the opposite of current efforts of targeted inhibition of oncogenic signaling. Our results, however, demonstrate that a robust negative feedback regulation is required for the leukemic transformation and development of fatal leukemia in pre-B ALL. We demonstrate that feedback control of Erk and Stat5 signaling represents a previously unrecognized vulnerability and, potentially, a novel class of therapeutic targets in human pre-B ALL. DisclosuresNo relevant conflicts of interest to declare.

Harnessing Negative B Cell Selection to Overcome Drug-Resistance in Acute Lymphoblastic Leukemia

Background: B cells are selected for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: attenuation below minimum (e.g. non-functional BCR) or hyperactivation above maximum (e.g. autoreactive BCR) thresholds of signaling strength causes negative selection and cell death. About 25% of B cell acute lymphoblastic leukemia (ALL) is driven by oncogenic tyrosine kinases (e.g. BCR-ABL1 in Ph+ ALL), which mimics constitutively active pre-BCR signaling and defines the ALL subgroup with the worst clinical outcome. Currently more potent tyrosine kinase inhibitors (TKI) are developed for Ph+ ALL to suppress oncogenic signaling below a minimum threshold for survival. However Ph+ ALL cells invariably develop resistance against TKI. Here, we tested the hypothesis that targeted hyperactivation of oncogenic signaling above a maximum threshold will trigger B cell-inherent mechanisms of negative selection and selectively kill Ph+ALL cells.Results: The Ph+ ALL cells don not express a functional pre-BCR and BCR-ABL1 oncogene mimics a constitutively active pre-BCR by phosphorylating SYK, LYN, BTK and PLCg2. An incremental increase of pre-BCR downstream signaling (ITAM or SYK overexpression) was indeed sufficient to induce cell death in Ph+ ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ALL cells in this experimental setting.Ph+ ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1 (CD31), CD300A and LAIR1. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials. In the COG trial (P9906; n=207) for children high-risk ALL, mRNA levels of PECAM1, CD300A and LAIR1at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p<0.0005), and negatively correlated with overall survival (OS) rate (p<0.02) or relapse free survival (RFS) rate (p<0.05). In the ECOG trial (E2993; n=215) for adults ALL, PECAM1 mRNA level negatively correlated with OS rate (p=0.0285).Genetic studies revealed that Pecam1, Cd300a and Lair1 receptors are critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases Ptpn6 (SHP1) and Inpp5d (SHIP1). Deletion of Pecam1, Cd300a or Lair1 in Ph+ ALL cells caused increased ROS levels, G0/G1cell cycle arrest, decreased colony formation capacity and cellular senescence. Phosphorylation of pre-BCR downstream molecules (SYK, LYN, BTK and PLCg2) was increased after Lair1 deletion and this hyper-signaling could not be tolerated by Ph+ ALL cells. Lair1 deletion resulted in rapid leukemia regression and prolonged survival of recipient mice in a transplant experiment. Leukemia cell death caused by Lair1-deletion could be rescued by overexpression of the active inhibitory phosphatase Ptpn6 (CD8-SHP1) or Inpp5d (CD8-SHIP1). Genetic deletion of Ptpn6 and Inpp5d caused increased pre-BCR signaling and cell death in BCR-ABL1 ALL cells but not myeloid cells (normal and BCR-ABL1-transformed), which -unlike B cells- are not subject to negative selection of auto-reactive clones. Decreasing pre-BCR signaling by SYK inhibition rescued cell death after Ptpn6- or Inpp5d- deletion.Blocking inhibitory receptors by using chimeric PECAM1, CD300A and LAIR1 receptor decoys inhibited proliferation and caused cell death in Ph+ ALL xenograft cells. More potently, a novel small molecule inhibitor of INPP5D (SHIP1) selectively killed Ph+ ALL xenograft cells through inducing hyper pre-BCR signaling, regardless of TKI resistance. We demonstrate that inhibitory phosphatase signaling represents a potential novel class of therapeutic targets for Ph+ALL.Conclusions: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified ITIM-receptors and phosphatases as members of a potential novel class of therapeutic targets. The concept of pharmacological perturbance of oncogenic signaling equilibrium in leukemia cells by inhibition (e.g. TKI-treatment) or exaggeration of signaling strength (e.g. blockade of ITIM-receptors) may lead to the discovery of multiple additional therapeutic targets and broaden our repertoire of currently available therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Ribosomal proteins as unrevealed caretakers for cellular stress and genomic instability

Ribosomal proteins (RPs) have gained much attention for their extraribosomal functions particularly with respect to p53 regulation. To date, about fourteen RPs have shown to bind to MDM2 and regulate p53. Upon binding to MDM2, the RPs suppress MDM2 E3 ubiquitin ligase activity resulting in the stabilization and activation of p53. Of the RPs that bind to MDM2, RPL5 and RPL11 are the most studied and RPL11 appears to have the most significant role in p53 regulation. Considering that more than 17% of RP species have been shown to interact with MDM2, one of the questions remains unresolved is why so many RPs bind MDM2 and modulate p53. Genes encoding RPs are widely dispersed on different chromosomes in both mice and humans. As components of ribosome, RP expression is tightly regulated to meet the appropriate stoichiometric ratio between RPs and rRNAs. Once genomic instability (e.g. aneuploidy) occurs, transcriptional and translational changes due to change of DNA copy number can result in an imbalance in the expression of RPs including those that bind to MDM2. Such an imbalance in RP expression could lead to failure to assemble functional ribosomes resulting in ribosomal stress. We propose that RPs have evolved ability to regulate MDM2 in response to genomic instability as an additional layer of p53 regulation. Full understanding of the biological roles of RPs could potentially establish RPs as a novel class of therapeutic targets in human diseases such as cancer.

Inhibitory Receptors and Phosphatases Enable Oncogenic Tyrosine Kinase Signaling In B Cell Lineage Leukemia

BackgroundB cells are selected at multiple developmental checkpoints for an intermediate level of (pre-) B cell receptor (BCR) signaling strength: either insufficient or hyperactive signaling (e.g. from an autoreactive BCR) results in cell death. Acute lymphoblastic leukemia (ALL) is the most frequent type of cancer in children and typically arises from pre-B cells, a large fraction of which are autoreactive. In ∼25% of patients, ALL is driven by an oncogenic tyrosine kinase (e.g. BCR-ABL1 in Ph+ ALL) and defines the ALL subgroup with the worst clinical outcome. Ph+ ALL cells invariably develop resistance against tyrosine kinase inhibitors (TKI). Here we tested the hypothesis that inherent mechanisms of negative selection to eliminate autoreactive clones with hyperactive pre-BCR signaling are still active in transformed pre-B cells and identified a potential therapeutic target for ALL patients. ResultsThe BCR-ABL1 oncogene mimics a constitutively active pre-BCR and an incremental increase of pre-BCR downstream signaling (ITAM overexpression) was indeed sufficient to induce cell death in Ph+ ALL, but not in normal pre-B cells with low baseline signaling strength. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting.Patient-derived Ph+ ALL cells differ from normal pre-B cells by expression of high levels of ITIM containing inhibitory receptors including PECAM1, CD300A and LAIR1. However, ITAM containing activation receptors like CD79B was absent on the cell surface, and there was point or frame-shift mutation for both CD79A and CD79B. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials. In the COG trial (P9906; n=207) for children high-risk ALL, mRNA levels of PECAM1, CD300A and LAIR1 at diagnosis positively correlated with early minimal residual disease (MRD) findings on day 29 (p<0.0005), and negatively correlated with overall survival (OS) rate (p<0.02) or relapse free survival (RFS) rate (p<0.05). In the ECOG trial (E2993; n=215) for adults ALL, PECAM1 mRNA level negatively correlated with OS rate (p=0.0285).Genetic studies revealed that Pecam1, Cd300a and Lair1 receptors are critical to calibrate oncogenic signaling strength and prevent excessive tyrosine kinase activity through recruitment of the inhibitory phosphatases Ptpn6 (SHP1) and Inpp5d (SHIP1). Deletion of Pecam1, Cd300a or Lair1 in Ph+ ALL cells caused increased ROS levels, accumulation of p53, p21 and p27 protein, G0/G1cell cycle arrest and cellular senescence. P-STAT5 and p-ERK were increased after Lair1 deletion. Transplant experiments indicated that Lair1 deletion resulted in rapid leukemia regression and prolonged survival of recipient mice. Leukemia cell death caused by Lair1 deletion could be rescued by overexpression of the inhibitory phosphatase Ptpn6 (SHP1) or Inpp5d (SHIP1). Genetic deletion of Ptpn6 and Inpp5d caused cell death in BCR-ABL1 ALL cells but neither in normal pre-B cells with weak baseline signaling, nor myeloid cells (normal and BCR-ABL1-transformed), which -unlike B cells- are not subject to negative selection of auto-reactive clones. To test if these findings are also relevant to other subtypes of ALL, we used a model for NRASG12D-driven ALL. BCR-ABL1 (∼25%) and NRAS lesions (∼30%) account for more than half cases of ALL. Consistently, NRASG12D Pecam1-/-, Cd300a-/- leukemia cells were prone to G0/G1 cell cycle arrest and cellular senescence (p<0.01).Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of INPP5D (SHIP1), we demonstrate that inhibitory phosphatase signaling represents a potential novel class of therapeutic targets for tyrosine kinase-driven ALL. ConclusionThese results indicated that inhibitory receptors and downstream phosphatases are critical regulators of oncogenic signaling strength in tyrosine kinase-driven ALL, and identified ITIM-receptors and phosphatases as members of a potential novel class of therapeutic targets. The concept of pharmacological perturbance of oncogenic signaling equilibrium in leukemia cells by inhibition (e.g. TKI-treatment) or exaggeration of signaling strength (e.g. blockade of ITIM-receptors) may lead to the discovery of multiple additional therapeutic targets and broaden our repertoire of currently available therapeutic intervention. Disclosures:No relevant conflicts of interest to declares.

MicroRNAs in Gastric Cancer: From Benchtop to Bedside

Gastric carcinogenesis represents a stepwise progression from chronic inflammation to invasive adenocarcinomas and distant metastasis. It has been widely accepted that these pathologic changes are contributed by aberrant activation or inactivation of protein-coding proto-oncogenes and tumor suppressor genes. However, recent discoveries in microRNA research have reshaped our understanding of the role of non-protein-coding genes in carcinogenesis. MicroRNAs (miRNAs) are a family of 18-25-nucleotide small RNAs that negatively regulate gene expression at the post-transcriptional level during various crucial cell processes such as apoptosis, differentiation and development. Changes in miRNA expression profiles have been observed in a variety of human tumors, including gastric cancer. Further studies demonstrated that miRNAs may function as tumor suppressors and oncogenes. These findings have shown great potential of miRNAs as a novel class of therapeutic targets. In addition, it was found that some miRNAs were directly involved in patients with gastric cancer, including prognosis prediction, treatment selection, and in the search for unknown primary sites. MiRNAs have also been proved to be detectable in serum and plasma. In this review, we summarize the function of miRNAs in gastric cancer. Furthermore, we describe the pathophysiological roles of these miRNAs and their clinical potential as diagnostic biomarkers and therapeutic targets.

Low-density Lipoprotein Receptor-related Protein 1 (LRP1)-dependent Cell Signaling Promotes Axonal Regeneration

Low-density lipoprotein receptors (LRPs) are present extensively on cells outside of the nervous system and classically exert roles in lipoprotein metabolism. It has been reported recently that LRP1 activation could phosphorylate the neurotrophin receptor TrkA in PC12 cells and increase neurite outgrowth from developing cerebellar granule cells. These intriguing findings led us to explore the hypothesis that LRP1 activation would activate canonical neurotrophic factor signaling in adult neurons and promote axonal regeneration after spinal cord injury. We now find that treatment of adult rat dorsal root ganglion neurons in vitro with LRP1 agonists (the receptor binding domain of α-2-macroglobulin or the hemopexin domain of matrix metalloproteinase 9) induces TrkC, Akt, and ERK activation; significantly increases neurite outgrowth (p < 0.01); and overcomes myelin inhibition (p < 0.05). These effects require Src family kinase activation, a classic LRP1-mediated Trk transactivator. Moreover, intrathecal infusions of LRP1 agonists significantly enhance sensory axonal sprouting and regeneration after spinal cord injury in rats compared with control-infused animals (p < 0.05). A significant role is established for lipoprotein receptors in sprouting and regeneration after CNS injury, identifying a novel class of therapeutic targets to explore for traumatic neurological disorders.

Targeting Noncoding RNAs in Disease: Challenges and Opportunities

Noncoding RNAs make up the majority of transcribed RNA and have a wide range of functions in cellular and developmental processes. Consequently, they are also implicated in the development and pathophysiology of many diseases and represent potential targets for therapeutic intervention. microRNAs are one class of noncoding RNA that has been intensely studied. Effective inhibition (or silencing) of microRNAs in vivo has enabled scientists to make groundbreaking discoveries about the contribution of these short regulating RNAs to some of the major human diseases, such as cancer, heart disease, and diabetes. Long noncoding RNAs are less well studied, but have recently emerged as another novel class of therapeutic targets in a variety of diseases. Both long and short noncoding RNAs represent new avenues of investigation for drug discovery with several advantages over traditional protein-based targets; however, they come with their own unique set of challenges.

Abstract 5292: MicroRNA signatures predictive of response to chemotherapy in mCRC.

Abstract MicroRNAs (miRNAs) are post-transcriptional regulators involved in numerous biological and pathological processes including colorectal cancer (CRC). From a clinical perspective, several studies have identified groups of miRNAs with potential utility for early diagnosis or prognostic stratification of CRC patients. However, very few have evaluated the potential ability of miRNA to predict response to selected chemotherapy regimens. The aim of our study was to evaluate the ability of miRNA expression patterns to predict chemotherapy response in a cohort of 78 patients with metastatic CRC (mCRC). We examined tumor expression levels of 667 miRNAs in the training cohort by TaqMan Low Density Arrays (TLDA). In training cohort (N=39), we identified a miRNA molecular signature with significant association in relevant clinical endpoints (objective response, progression-free and overall survival). In validation cohort (N=39), we validated significantly higher expression levels of miR-107, and miR99a* according to objective response to fluoropyrimidine-based regimens. These results support a substantial role for miRNAs in determining drug response, and highlight their potential value as biomarkers for personalized treatment strategies and as a novel class of therapeutic targets. Citation Format: Sonia Molina-Pinelo, Amancio Carnero, Fernando Rivera, Purificacion Estevez-Garcia, Jose M. Bozada, Maria L. Limon, Javier Gomez, Maria D. Pastor, Rocio Suarez, Luis G. Paz-Ares, Fernando de la Portilla, Andres Carranza, Isabel Sevilla, Luis Vicioso, Rocio Garcia Carbonero. MicroRNA signatures predictive of response to chemotherapy in mCRC. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5292. doi:10.1158/1538-7445.AM2013-5292