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BackgroundGenome-wide association studies have found many genetic risk variants associated with Alzheimer’s disease (AD). However, how these risk variants affect deeper phenotypes such as disease progression and immune response remains elusive. Also, our understanding of cellular and molecular mechanisms from disease risk variants to various phenotypes is still limited. To address these problems, we performed an integrative multi-omics analysis of genotype, transcriptomics, and epigenomics for revealing gene regulatory mechanisms from disease variants to AD phenotypes.MethodFirst, given the population gene expression data of a cohort, we construct and cluster its gene co-expression network to identify gene co-expression modules for various AD phenotypes. Next, we predict transcription factors (TFs) regulating co-expressed genes and AD risk SNPs that interrupt TF binding sites on regulatory elements. Finally, we construct a gene regulatory network (GRN) linking SNPs, interrupted TFs, and regulatory elements to target genes and gene modules for each phenotype in the cohort. This network thus provides systematic insights into gene regulatory mechanisms from risk variants to AD phenotypes.ResultsOur analysis predicted GRNs in three major AD-relevant regions: Hippocampus, Dorsolateral Prefrontal Cortex (DLPFC), Lateral Temporal Lobe (LTL). Comparative analyses revealed cross-region-conserved and region-specific GRNs, in which many immunological genes are present. For instance, SNPs rs13404184 and rs61068452 disrupt SPI1 binding and regulation of AD gene INPP5D in the Hippocampus and LTL. However, SNP rs117863556 interrupts bindings of REST to regulate GAB2 in DLPFC only. Driven by emerging neuroinflammation in AD, we used Covid-19 as a proxy to identify possible regulatory mechanisms for neuroimmunology in AD. To this end, we looked at the GRN subnetworks relating to genes from shared AD-Covid pathways. From those subnetworks, our machine learning analysis prioritized the AD-Covid genes for predicting Covid-19 severity. Decision Curve Analysis also validated our AD-Covid genes outperform known Covid-19 genes for classifying severe Covid-19 patients. This suggests AD-Covid genes along with linked SNPs can be potential novel biomarkers for neuroimmunology in AD. Finally, our results are open-source available as a comprehensive functional genomic map for AD, providing a deeper mechanistic understanding of the interplay among multi-omics, brain regions, gene functions like neuroimmunology, and phenotypes.

A SHIP1/INPP5D Target Enabling Component focused on Chemical Probes

A Target Enabling Package Centered around INPP5D (SHIP1) as it relates to Alzheimer's disease

BackgroundGenome-wide association studies for atopic dermatitis (AD, eczema) have identified 25 reproducible loci associated in populations of European descent. We attempt to prioritise candidate causal genes at these loci using a multifaceted bioinformatic approach and extensive molecular resources compiled into a novel pipeline: ADGAPP (Atopic Dermatitis GWAS Annotation & Prioritisation Pipeline).MethodsWe identified a comprehensive list of 103 accessible molecular resources for AD aetiology, including expression, protein and DNA methylation QTL datasets in skin or immune-relevant tissues. These were used to test for overlap with GWAS signals (including colocalisation testing where possible). This was combined with functional annotation based on regulatory variant prediction, and independent genomic features such as chromatin accessibility, promoter-enhancer interactions, splicing sites, non-coding RNA regions, differential expression studies involving eczema patients and fine-mapping of causal variants. For each gene at each locus, we condensed the evidence into a prioritisation score.ResultsAcross the 25 AD loci investigated, we detected significant enrichment of genes with adaptive immune regulatory function and epidermal barrier formation among the top prioritised genes. At 8 loci, we were able to prioritise a single candidate gene (IL6R, ADO, PRR5L, IL7R, ETS1, INPP5D, MDM1, TRAF3). At a further 2 loci, 2 candidate genes emerge (IL18R1/IL18RAP, LRRC32/EMSY). For the majority of these, the prioritised gene has been previously proposed as a plausible candidate, but the evidence we combine here, strengthens the case for many of these. In addition, at 6 of the 25 loci, our ADGAPP analysis prioritises novel alternative candidates (SLC22A5, IL2RA, MDM1, DEXI, ADO, STMN3), highlighting the importance of this comprehensive approach.ConclusionsOur ADGAPP analysis provides additional support for previously implicated genes at several AD GWAS loci, as well as evidence for plausible novel candidates at others. We highlight several genes with good/converging evidence of involvement in AD that represent potential new targets for drug discovery.

Target Enablement Resource for INPP5D/SHIP1 specific to bioinformatics

Target Enablement Resource specific to INPP5D/SHIP1 Protein Structures

Target Enablement Resource for INPP5D/SHIP1 specific to screening

The present study investigated the association of SNPs involved in the regulation of immune response, cellular degenerative and neuroinflammatory pathways with the susceptibility and progression of idiopathic Parkinson’s Disease (PD). In particular, 342 PD patients were subjected to a genotyping analysis of a panel of 120 SNPs by Open Array Technology. As control group, 503 samples representative of the European general population were utilized. The genetic analysis identified 26 SNPs associated with PD susceptibility. Of them, 12 SNPs were described as significant expression Quantitative Loci (eQTL) variants in different brain regions associated with motor and non-motor PD phenomenology. Moreover, the study highlighted 11 novel susceptibility genes for PD, which may alter multiple signaling pathways critically involved in peripheral immune response, neuroinflammation, neurodegeneration and dopaminergic neurons wiring. The study of miRNA-target genes highlighted a possible role of miR-499a, miR-196a2, and miR-29a in the modulation of multiple neuroinflammatory and neurodegenerative mechanisms underlying PD physiopathology. The study described a network of interconnected genes (APOE, CLU, IL6, IL7R, IL12B, INPP5D, MAPK1, MEF2C, MIF, and TNFSF14), which may act as upstream regulators in the modulation of biological pathways relevant to PD. Intriguingly, IL6 stands out as a master gene regulator since it may indirectly regulate the network of interconnected genes. The study highlighted different genes and miRNAs interactions potentially involved in PD physiopathology, which are worth to be further explored to improve the knowledge of disease and the research of novel treatments strategies.

Deeply phenotyped cohort data can elucidate differences associated with genetic risk for common complex diseases across an age spectrum. Previous work has identified genetic variants associated with Alzheimer’s disease (AD) risk from large-scale genome-wide association study meta-analyses. To explore effects of known AD-risk variants, we performed a phenome-wide association study on ~2000 clinical, proteomic, and metabolic blood-based analytes obtained from 2,831 cognitively normal adult clients of a consumer-based scientific wellness company. Results uncovered statistically significant SNP-analyte associations for five genetic variants after correction for multiple testing (for SNPs in or nearNYAP1, ABCA7, INPP5D, andAPOE). These effects were detectable from early adulthood. Sex modified the effects of four genetic variants, with multiple interrelated immune-modulating effects associated with thePICALMvariant. Sex-stratified GWAS results from an independent AD case-control meta-analysis supported sexspecific disease effects of thePICALMvariant, highlighting the importance of sex as a biological variable. These analyses support evidence from previous functional genomics studies in the identification of a causal variant within thePILRAgene. Taken together, this study highlights clues to the earliest effects of AD genetic risk variants in individuals where disease symptoms have not (yet) arisen.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, robust microgliosis, neuroinflammation, and neuronal loss. Genome-wide association studies recently highlighted a prominent role for microglia in late-onset AD (LOAD). Specifically, inositol polyphosphate-5-phosphatase (INPP5D), also known as SHIP1, is selectively expressed in brain microglia and has been reported to be associated with LOAD. Although INPP5D is likely a crucial player in AD pathophysiology, its role in disease onset and progression remains unclear. We performed differential gene expression analysis to investigate INPP5D expression in AD and its association with plaque density and microglial markers using transcriptomic (RNA-Seq) data from the Accelerating Medicines Partnership for Alzheimer's Disease (AMP-AD) cohort. We also performed quantitative real-time PCR, immunoblotting, and immunofluorescence assays to assess INPP5D expression in the 5xFAD amyloid mouse model. Differential gene expression analysis found that INPP5D expression was upregulated in LOAD and positively correlated with amyloid plaque density. In addition, in 5xFAD mice, Inpp5d expression increased as the disease progressed, and selectively in plaque-associated microglia. Increased Inpp5d expression levels in 5xFAD mice were abolished entirely by depleting microglia with the colony-stimulating factor receptor-1 antagonist PLX5622. Our findings show that INPP5D expression increases as AD progresses, predominantly in plaque-associated microglia. Importantly, we provide the first evidence that increased INPP5D expression might be a risk factor in AD, highlighting INPP5D as a potential therapeutic target. Moreover, we have shown that the 5xFAD mouse model is appropriate for studying INPP5D in AD.

Funder: ZHANGJIANG LAB, Tianqiao and Chrissy Chen Institute, and the State Key Laboratory of Neurobiology and Frontiers Center for Brain Science of Ministry of Education, Fudan University, Background: Genome-wide association studies have identified more than 30 Alzheimer’s disease (AD) risk genes, although the detailed mechanism through which all these genes are associated with AD pathogenesis remains unknown. We comprehensively evaluate the roles of the variants in top 30 non-APOE AD risk genes, based on whether these variants were associated with altered mRNA transcript levels, as well as brain amyloidosis, tauopathy, and neurodegeneration. Methods: Human brain gene expression data were obtained from the UK Brain Expression Consortium (UKBEC), while other data used in our study were obtained from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) cohort. We examined the association of AD risk allele carrier status with the levels of gene expression in blood and brain regions and tested the association with brain amyloidosis, tauopathy, and neurodegeneration at baseline, using a multivariable linear regression model. Next, we analyzed the longitudinal effects of these variants on the change rates of pathology using a mixed effect model. Results: Altogether, 27 variants were detected to be associated with the altered expression of 21 nearby genes in blood and brain regions. Eleven variants (especially novel variants in ADAM10, IGHV1-68, and SLC24A4/RIN3) were associated with brain amyloidosis, 7 variants (especially in INPP5D, PTK2B) with brain tauopathy, and 8 variants (especially in ECHDC3, HS3ST1) with brain neurodegeneration. Variants in ADAMTS1, BZRAP1-AS1, CELF1, CD2AP, and SLC24A4/RIN3 participated in more than one cerebral pathological process. Conclusions: Genetic variants might play functional roles and suggest potential mechanisms in AD pathogenesis, which opens doors to uncover novel targets for AD treatment.

BackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, robust microgliosis, neuroinflammation, and neuronal loss. Genome-wide association studies recently highlighted a prominent role for microglia in late-onset AD (LOAD). Specifically, inositol polyphosphate-5-phosphatase (INPP5D), also known as SHIP1, is selectively expressed in brain microglia and has been reported to be associated with LOAD. Although INPP5D is likely a crucial player in AD pathophysiology, its role in disease onset and progression remains unclear.MethodsWe performed differential gene expression analysis to investigate INPP5D expression in LOAD and its association with plaque density and microglial markers using transcriptomic (RNA-Seq) data from the Accelerating Medicines Partnership for Alzheimer’s Disease (AMP-AD) cohort. We also performed quantitative real-time PCR, immunoblotting, and immunofluorescence assays to assess INPP5D expression in the 5xFAD amyloid mouse model.ResultsDifferential gene expression analysis found that INPP5D expression was upregulated in LOAD and positively correlated with amyloid plaque density. In addition, in 5xFAD mice, Inpp5d expression increased as the disease progressed, and selectively in plaque-associated microglia. Increased Inpp5d expression levels in 5xFAD mice were abolished entirely by depleting microglia with the colony-stimulating factor receptor-1 antagonist PLX5622.ConclusionsOur findings show that INPP5D expression increases as AD progresses, predominantly in plaque-associated microglia. Importantly, we provide the first evidence that increased INPP5D expression might be a risk factor in AD, highlighting INPP5D as a potential therapeutic target. Moreover, we have shown that the 5xFAD mouse model is appropriate for studying INPP5D in AD.

Liu et al. compare epigenetic and expression profiles in isogenic ESC-derived human microglia-like cells (hMGLs) comprising Alzheimer’s disease (AD)–associated mutations. Using this model, convergent dysregulation at the APOE locus is observed with AD-associated mutations in SORL1 and TREM2., Variations in many genes linked to sporadic Alzheimer’s disease (AD) show abundant expression in microglia, but relationships among these genes remain largely elusive. Here, we establish isogenic human ESC–derived microglia-like cell lines (hMGLs) harboring AD variants in CD33, INPP5D, SORL1, and TREM2 loci and curate a comprehensive atlas comprising ATAC-seq, ChIP-seq, RNA-seq, and proteomics datasets. AD-like expression signatures are observed in AD mutant SORL1 and TREM2 hMGLs, while integrative multi-omic analysis of combined epigenetic and expression datasets indicates up-regulation of APOE as a convergent pathogenic node. We also observe cross-regulatory relationships between SORL1 and TREM2, in which SORL1R744X hMGLs induce TREM2 expression to enhance APOE expression. AD-associated SORL1 and TREM2 mutations also impaired hMGL Aβ uptake in an APOE-dependent manner in vitro and attenuated Aβ uptake/clearance in mouse AD brain xenotransplants. Using this modeling and analysis platform for human microglia, we provide new insight into epistatic interactions in AD genes and demonstrate convergence of microglial AD genes at the APOE locus., Graphical Abstract

More than 20 genetic loci have been associated with risk for Alzheimer’s disease (AD), but reported genome-wide significant loci do not account for all the estimated heritability and provide little information about underlying biological mechanisms. Genetic studies using intermediate quantitative traits such as biomarkers, or endophenotypes, benefit from increased statistical power to identify variants that may not pass the stringent multiple test correction in case–control studies. Endophenotypes also contain additional information helpful for identifying variants and genes associated with other aspects of disease, such as rate of progression or onset, and provide context to interpret the results from genome-wide association studies (GWAS). We conducted GWAS of amyloid beta (Aβ42), tau, and phosphorylated tau (ptau181) levels in cerebrospinal fluid (CSF) from 3146 participants across nine studies to identify novel variants associated with AD. Five genome-wide significant loci (two novel) were associated with ptau181, including loci that have also been associated with AD risk or brain-related phenotypes. Two novel loci associated with Aβ42 near GLIS1 on 1p32.3 (β = −0.059, P = 2.08 × 10−8) and within SERPINB1 on 6p25 (β = −0.025, P = 1.72 × 10−8) were also associated with AD risk (GLIS1: OR = 1.105, P = 3.43 × 10−2), disease progression (GLIS1: β = 0.277, P = 1.92 × 10−2), and age at onset (SERPINB1: β = 0.043, P = 4.62 × 10−3). Bioinformatics indicate that the intronic SERPINB1 variant (rs316341) affects expression of SERPINB1 in various tissues, including the hippocampus, suggesting that SERPINB1 influences AD through an Aβ-associated mechanism. Analyses of known AD risk loci suggest CLU and FERMT2 may influence CSF Aβ42 (P = 0.001 and P = 0.009, respectively) and the INPP5D locus may affect ptau181 levels (P = 0.009); larger studies are necessary to verify these results. Together the findings from this study can be used to inform future AD studies.

Im Rahmen dieser Arbeit sollte die molekulare Basis des erstaunlich benignen Verhaltens der SPN angesichts des aktivierten Wnt-Signalweges und der Cyclin D1-Überexpression, welche normalerweise beide mit aggressiven Tumoren assoziiert sind, beleuchtet werden. Hierzu wurde die RNA-Expression der Gene FLI1 sowie DKK1 und INPP5D (SHIP1) als durch FLI1 regulierte Gene und BCL9 als Bestandteil des Wnt/β-Catenin Signalweges untersucht. Neben der SPN wurde zusätzlich eine mögliche Bedeutung dieser Gene im aggressiven PDAC sowie dessen Vorläuferläsion der IPMN geprüft. Die anfangs aufgestellte Hypothese einer Beteiligung der Gene DKK1 und BCL9 sowohl an der SPN- als auch an der PDAC- und IPMN-Tumorgenese musste aufgrund der erhobenen Daten jedoch vorerst verworfen werden. Jedoch scheint FLI1 eine entscheidende Rolle in der SPN-Tumorgenese zu spielen. Zudem sind sowohl FLI1 als auch INPP5D in der IPMN signifikant überexprimiert, allerdings unabhängig voneinander. Die Frage, über welchen intrazellulären Signalweg FLI1 und INPP5D diese Rolle ausüben, konnte noch nicht geklärt werden. Auch der molekulargenetische Grund für das niedrig maligne Verhalten der SPN bzw. intermediär-maligne Verhalten der IPMN bleibt weiterhin ein nur zum Teil gelöstes Rätsel und wirft noch immer eine Vielzahl von Fragen auf.

The vascular hypothesis of Alzheimer's disease (AD) has proposed the involvement of brain hypoperfusion in AD pathogenesis, where cognitive decline and dysfunction result from dwindling cerebral blood flow (CBF). Based on the vascular hypothesis of Alzheimer's disease, we focused on exploring how genetic factors influence AD pathogenesis via the cerebrovascular system. To investigate the role of CBF endophenotypes in AD pathogenesis, we performed a targeted genetic analysis of 258 subjects from the Alzheimer's Disease Neuroimaging Initiative cohort to examine associations between 4033 single-nucleotide polymorphisms of 24 AD genes and CBF measures in 4 brain regions. A novel association with CBF measure in the left angular gyrus was identified in an INPP5D single-nucleotide polymorphism (i.e., rs61068452; p = 1.48E-7; corrected p = 2.39E-3). The gene-based analysis discovered both INPP5D and CD2AP associated with the left angular gyrus CBF. Further analyses on nonoverlapping samples revealed that rs61068452-G was associated with lower CSF t-tau/Aβ1–42 ratio. Our findings suggest a protective role of rs61068452-G in an AD-relevant cerebrovascular endophenotype, which has the potential to provide novel insights for better mechanistic understanding of AD.

Purpose: Myelodysplastic syndromes (MDS) with deletion of chromosome 7q/7 [-7/(del)7q MDS] is associated with worse outcomes and needs novel insights into pathogenesis. Reduced expression of signaling protein dedicator of cytokinesis 4 (DOCK4) in patients with -7/(del)7q MDS leads to a block in hematopoietic stem cell (HSC) differentiation. Identification of targetable signaling networks downstream of DOCK4 will provide means to restore hematopoietic differentiation in MDS. Experimental Design: We utilized phosphoproteomics approaches to identify signaling proteins perturbed as a result of reduced expression of DOCK4 in human HSCs and tested their functional significance in primary model systems. Results: We demonstrate that reduced levels of DOCK4 lead to increased global tyrosine phosphorylation of proteins in primary human HSCs. LYN kinase and phosphatases INPP5D (SHIP1) and PTPN6 (SHP1) displayed greatest levels of tyrosine phosphorylation when DOCK4 expression levels were reduced using DOCK4-specific siRNA. Our data also found that increased phosphorylation of SHIP1 and SHP1 phosphatases were due to LYN kinase targeting these phosphatases as substrates. Increased migration and impediment of HSC differentiation were consequences of these signaling alterations. Pharmacologic inhibition of SHP1 reversed these functional aberrations in HSCs expressing low DOCK4 levels. In addition, differentiation block seen in DOCK4 haplo-insufficient [-7/(del)7q] MDS was rescued by inhibition of SHP1 phosphatase. Conclusions: LYN kinase and phosphatases SHP1 and SHIP1 are perturbed when DOCK4 expression levels are low. Inhibition of SHP1 promotes erythroid differentiation in healthy HSCs and in -7/(del)7q MDS samples with low DOCK4 expression. Inhibitors of LYN, SHP1 and SHIP1 also abrogated increased migratory properties in HSCs expressing reduced levels of DOCK4.

// Hua Jing 1,* , Jun-Xia Zhu 1,* , Hui-Fu Wang 1 , Wei Zhang 2 , Zhan-Jie Zheng 3 , Ling-Li Kong 3 , Chen-Chen Tan 1 , Zi-Xuan Wang 1 , Lin Tan 4 and Lan Tan 1,4 1 Department of Neurology, Qingdao Municipal Hospital, School of Medicine, Qingdao University, Qingdao, PR China 2 Department of Emergency, Qingdao Municipal Hospital, School of Medicine, Qingdao University, Qingdao, PR China 3 Department of Geriatric, Qingdao Mental Health Center, Qingdao, PR China 4 College of Medicine and Pharmaceutics, Ocean University of China, Qingdao, PR China * The first two authors should be regarded as co-first authors Correspondence to: Lan Tan, email: // Keywords : Alzheimer’s disease; INPP5D; rs35349669; association study; meta-analysis; Gerotarget Received : May 05, 2016 Accepted : October 02, 2016 Published : October 13, 2016 Abstract Inositol polyphosphate-5-phosphatase ( INPP5D ) was reported to be associated with Alzheimer’s disease (AD) through modulating the inflammatory process and immune response. A recent genome-wide association study discovered a new locus single nucleotide polymorphism (SNP, rs35349669) of INPP5D which was significantly associated with susceptibility to late-onset Alzheimer’s disease (LOAD) in Caucasians. In this study, we investigated the relations between the INPP5D polymorphism rs35349669 and LOAD in Han Chinese population comprising 984 LOAD cases and 1352 healthy controls being matched for age and gender. Our results showed no obvious differences in the genotypic or allelic distributions of rs35349669 polymorphism between LOAD cases and healthy controls (genotype: p = 0.167; allele: p = 0.094). Additionally, when these data were stratified by APOE e4 status, there are still no evident differences in the genotypic or allelic distributions in APOEe4 carriers ( p > 0.05). Furthermore, meta-analysis of 81964 individuals confirmed that rs35349669 was significantly associated with the risk for LOAD (OR=1.08, 95%CI=1.06-1.11), but the results remained negative in Chinese subgroup (OR=0.77, 95%CI=0.53-1.13). Overall, the current evidence did not indicate that INPP5D rs35349669 polymorphism play a role in the genetic predisposition to LOAD in Chinese population.

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch., Author summary Understanding the molecular mechanisms regulating EBV latency and entry into the lytic replication cycle is important in developing therapies for viral disease. We demonstrate here that a subset of EBV miRNAs target host factors within the BCR signaling pathway and consequently, negatively regulate cellular responses to BCR cross-linking. Disrupting activity of individual EBV miRNAs, specifically miR-BHRF-1-2-5p and miR-BART2-5p, enhanced lytic gene expression following BCR engagement, suggesting a key role for these miRNAs in attenuating viral reactivation. Our experiments establish a link between EBV miRNAs and signaling through the BCR, providing new insight into miRNA-mediated aspects of latency and reactivation.

Alzheimer's disease (AD) is a complex age-related neurodegenerative disorder of the central nervous system. Since the first description of AD in 1907, many hypotheses have been established to explain its causes. The inflammation theory is one of them. Pathological and biochemical studies of brains from AD individuals have provided solid evidence of the activation of inflammatory pathways. Furthermore, people with long-term medication of anti-inflammatory drugs have shown a reduced risk to develop the disease. After three decades of genetic study in AD, dozens of loci harboring genetic variants influencing inflammatory pathways in AD patients has been identified through genome-wide association studies (GWAS). The most well-known GWAS risk factor that is responsible for immune response and inflammation in AD development should be APOE ε4 allele. However, a growing number of other GWAS risk AD candidate genes in inflammation have recently been discovered. In the present study, we try to review the inflammation in AD and immunity-associated GWAS risk genes like HLA-DRB5/DRB1, INPP5D, MEF2C, CR1, CLU and TREM2.

B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In ∼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.

Here, we review the genetic risk factors for late onset Alzheimer's disease (AD) and their role in AD pathogenesis. Recent advances in our understanding of the human genome, namely technological advances in methods to analyze millions of polymorphisms in thousands of subjects, have revealed new genes associated with AD risk: ABCA7, BIN1, CASS4, CD33, CD2AP, CELF1, CLU, CR1, DSG2, EPHA1, FERMT2, HLA-DRB5-DBR1, INPP5D, MS4A, MEF2C, NME8, PICALM, PTK2B, SLC24H4 RIN3, SORL1, ZCWPW1. Emerging technologies to analyze the entire genome in large datasets have also revealed coding variants that increase AD risk: PLD3 and TREM2. We review the relationship between these AD risk genes and the cellular and neuropathological features of AD. Together, understanding the mechanisms underlying the association of these genes with risk for disease will provide the most meaningful targets for therapeutic development to date.

Alzheimer’s disease (AD) is a highly heritable complex disease with no current effective prevention or treatment. The majority of drugs developed for AD focus on the amyloid cascade hypothesis, which implicates Aß plaques as a causal factor in the disease. However, it is possible that other underexplored disease-associated pathways may be more fruitful targets for drug development. Findings from gene network analyses implicate immune networks as being enriched in AD; many of the genes in these networks fall within genomic regions that contain common and rare variants that are associated with increased risk of developing AD. Of these genes, several (including CR1, SPI1, the MS4As, TREM2, ABCA7, CD33, and INPP5D) are expressed by microglia, the resident immune cells of the brain. We summarize the gene network and genetics findings that implicate that these microglial genes are involved in AD, as well as several studies that have looked at the expression and function of these genes in microglia and in the context of AD. We propose that these genes are contributing to AD in a non-Aß-dependent fashion.

Late-onset Alzheimer’s disease (LOAD) is a devastating neurodegenerative disease with no effective treatment or cure. In addition to APOE, recent large genome-wide association studies have identified variation in over 20 loci that contribute to disease risk: CR1, BIN1, INPP5D, MEF2C, TREM2, CD2AP, HLA-DRB1/HLA-DRB5, EPHA1, NME8, ZCWPW1, CLU, PTK2B, PICALM, SORL1, CELF1, MS4A4/MS4A6E, SLC24A4/RIN3,FERMT2, CD33, ABCA7, CASS4. In addition, rare variants associated with LOAD have also been identified in APP, TREM2 and PLD3 genes. Previous research has identified inflammatory response, lipid metabolism and homeostasis, and endocytosis as the likely modes through which these gene products participate in Alzheimer’s disease. Despite the clustering of these genes across a few common pathways, many of their roles in disease pathogenesis have yet to be determined. In this review, we examine both general and postulated disease functions of these genes and consider a comprehensive view of their potential roles in LOAD risk.

Background Solid pseudopapillary neoplasms of the pancreas (SPN) are rare tumors affecting mainly women. They show an activating mutation in CTNNB1, the gene for β-catenin, and consequently an overactivation of the Wnt/β-catenin pathway. This signaling pathway is implied in the pathogenesis of various aggressive tumors, including pancreatic adenocarcinomas (PDAC). Despite this, SPN are characterized by an unusually benign clinical course. Attempts to explain this lack of malignancy have led to the discovery of an aberrant expression of the transcription factor FLI1 in SPN. Methods In 42 primary pancreatic tumors the RNA-expression of the FLI1 targets DKK1, INPP5D, IGFBP3 and additionally two members of the Wnt/β-catenin pathway, namely BCL9 and BCL9L, was investigated using quantitative real time PCR. Expression of these genes was evaluated in SPN (n = 18), PDAC (n = 12) and the less aggressive intraductal papillary mucinous neoplasm IPMN (n = 12) and compared to normal pancreatic tissue. Potential differences between the tumor entities were evaluated using students t-test. Results The results demonstrated a differential RNA-expression of BCL9L with a lack of expression in SPN (p

Acute myeloid leukemia (AML) is a malignant disease characterized by deregulated proliferation of immature myeloid cells. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is frequently detected in approximately 50-70% of AML patients. The gene INPP5D encodes the SH2-containing inositol 5-phosphatase 1 (SHIP1), which is a negative regulator of PI3K/AKT signaling. After lentiviral-mediated gene transfer of INPP5D into CD34(+) cells derived from AML patients (n=12) the granulocyte macrophage-colony stimulating factor (GM-CSF)-dependent proliferation was reduced in all samples analyzed (average 86%; range 72-93%). An enzymatically inactive form of SHIP1 (D672A) had no effect. In addition, SHIP1 reduced the autonomous proliferation of CD34(+) cells from a patient with a secondary AML who had a very high peripheral blast count (300 x 10(9) l(-1)). These data show that SHIP1 can effectively block GM-CSF-dependent and autonomous proliferation of AML cells.

The action of lipid phosphatases on inositol phosphates is thought to be one method that the cell uses to attenuate growth factor- or cytokine-induced mobilization of calcium. We have cloned a cDNA from a mouse spleen library that is virtually identical to the recently described inositol tetraphosphate and phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase, SHIP. Chromosomal localization studies in human and mouse by FISH mapping and interspecies backcrossing in mice have demonstrated that human SHIP is localized to 2q36-q37.1 and that mouse SHIP is localized to 1C5.

Recent genome-wide association studies (GWAS) have successfully identified several gene loci associated with multiple sclerosis (MS) susceptibility, severity or interferon-beta (IFN-ß) response. However, due to the nature of these studies, the functional relevance of these loci is not yet fully understood. We have utilized a systems biology based approach to explore the genetic interactomes of these MS related traits. We hypothesised that genes and pathways associated with the 3 MS related phenotypes might interact collectively to influence the heterogeneity and unpredictable clinical outcomes observed. Individual genetic interactomes for each trait were constructed and compared, followed by prioritization of common interactors based on their frequencies. Pathway enrichment analyses were performed to highlight shared functional pathways. Biologically relevant genes ABL1, GRB2, INPP5D, KIF1B, PIK3R1, PLCG1, PRKCD, SRC, TUBA1A and TUBA4A were identified as common to all 3 MS phenotypes. We observed that the highest number of first degree interactors were shared between MS susceptibility and MS severity (p = 1.34×10−79) with UBC as the most prominent first degree interactor for this phenotype pair from the prioritisation analysis. As expected, pairwise comparisons showed that MS susceptibility and severity interactomes shared the highest number of pathways. Pathways from signalling molecules and interaction, and signal transduction categories were found to be highest shared pathways between 3 phenotypes. Finally, FYN was the most common first degree interactor in the MS drugs-gene network. By applying the systems biology based approach, additional significant information can be extracted from GWAS. Results of our interactome analyses are complementary to what is already known in the literature and also highlight some novel interactions which await further experimental validation. Overall, this study illustrates the potential of using a systems biology based approach in an attempt to unravel the biological significance of gene loci identified in large GWAS. Refereed/Peer-reviewed

Background: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the B cell receptor (BCR): If B-cells fail to express a functional BCR, signaling output is too weak, resulting in “death by neglect”. If the BCR binds to ubiquitous self-antigen, BCR signals are exceedingly strong. Both attenuation below minimum (non-functional BCR; death by neglect) and hyperactivation above maximum (autoreactive BCR) thresholds of signaling strength trigger negative selection and cell death. Rationale: Unlike any other types of cancer, we recently discovered that pre-B acute lymphoblastic leukemia (ALL) cells are bound by the same rules that also govern normal B cell selection. Oncogenic tyrosine kinases in pediatric Ph-like ALL mimic active pre-BCR signaling. The Ph-like group defines the ALL subset with particularly poor clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph-like ALL cells through a mechanism that is functionally equivalent to removal of self-reactive B cells. Results: Ph-like ALL typically originates from pre-B cells that critically depend on survival signals emanating from a functional pre-BCR. Despite their pre-B cell origin, ALL cells lack expression of pre-BCR signaling chains Igα and Igβ, indicating lack of a functional pre-BCR. Reconstitution of Igα and Igβ was indeed sufficient to induce cell death in Ph-like ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph-like ALL cells in this experimental setting. These findings suggest that Ph-like ALL cells are selectively sensitive to even incremental increases of pre-BCR signaling. Consistent with this concept, patient-derived Ph-like ALL cells express high levels of inhibitory surface receptors PECAM1, CD300A and LAIR1 that recruit and activate phosphatases SHP1 and SHIP1, which terminate pre-BCR signaling. Importantly, high expression levels of these surface receptors were strong predictors of poor outcome of patients with ALL in two clinical trials, including Ph-like ALL patients (COG; P9906). Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases SHP1 and SHIP1. Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 transformed ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of an autoreactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in Ph-like ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of SHIP1, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in pediatric Ph-like ALL. Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph-like ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. This abstract is also presented as Poster B06. Citation Format: Zhengshan Chen, Huimin Geng, Clifford A. Lowell, Stephen P. Hunger, Markus Müschen. Targeted engagement of B cell autoimmunity checkpoints to overcome drug resistance in pediatric Ph-like ALL. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR11.

Helminth infection leads to the local proliferation and accumulation of macrophages in tissues. However, the function of macrophages during helminth infection remains unclear. SH2-containing inositol 5'-phosphatase 1 (Ship1, Inpp5d) is a lipid phosphatase that has been shown to play a critical role in macrophage function. Here, we identify a critical role for Ship1 in the negative regulation of interleukin (IL)-12/23p40 production by macrophages during infection with the intestinal helminth parasite Trichuris muris. Mice with myeloid cell-specific deletion of Ship1 (Ship1(ΔLysM) mice) develop a non-protective T-helper type 1 cell response and fail to expel parasites. Ship1-deficient macrophages produce heightened levels of IL-12/23p40 in vitro and in vivo and antibody blockade of IL-12/23p40 renders Ship1(ΔLysM) mice resistant to Trichuris infection. Our results identify a critical role for the negative regulation of IL-12/23p40 production by macrophages in the development of a protective T(H)2 cell response.

Ikaros family transcription factors have a key role in lymphoid development, and their aberrant function contributes to a multitude of lymphoid malignancies. Ikaros and Helios bind to similar DNA sequences, and Helios associates with Ikaros‐containing chromatin remodeling complexes. Previously, we have shown that loss of Ikaros leads to diminished BCR‐signaling strength. In this study, we describe a Helios‐deficient chicken DT40 B‐cell line with a BCR signaling phenotype that is the opposite to that of Ikaros‐deficient cells. In contrast to Ikaros‐deficient cells, Helios−/− B cells exhibit increased calcium release to the cytoplasm after BCR crosslinking, but diminished BCR‐induced phosphorylation of signaling molecules. The inositol 5‐phosphatase SHIP, an important regulator in several signaling pathways, is differentially expressed in Ikaros‐ and Helios‐deficient cells. In the absence of Ikaros, SHIP is upregulated, whereas Helios deficiency leads to the downregulation of SHIP expression. We also show with ChIP that Ikaros binds to the promoter of the INPP5D gene‐encoding SHIP. Considering the critical role of SHIP in the BCR signaling pathway, our findings provide insight into the mechanism of how both Helios and Ikaros are involved in the regulation of BCR signaling.

Background: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the B cell receptor (BCR): If B-cells fail to express a functional BCR, signaling output is too weak, resulting in "death by neglect". If the BCR binds to ubiquitous self-antigen, BCR signals are exceedingly strong. Both attenuation below minimum (non-functional BCR; death by neglect) and hyperactivation above maximum (autoreactive BCR) thresholds of signaling strength trigger negative selection and cell death. Rationale: Unlike any other types of cancer, we recently discovered that pre-B acute lymphoblastic leukemia (ALL) cells are bound by the same rules that also govern normal B cell selection. The oncogenic BCR-ABL1 tyrosine kinase mimics active pre-BCR signaling in Ph+ acute lymphoblastic leukemia which defines the ALL subgroup with the worst clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph+ ALL cells through a mechanism that is functionally equivalent to removal of self-reactive B cells. Results: ALL typically originates from pre-B cells that critically depend on survival signals emanating from a functional pre-BCR. Despite their pre-B cell origin, Ph+ ALL cells lack expression of pre-BCR signaling chains Iga and Igb, indicating lack of a functional pre-BCR. Reconstitution of Iga and Igb was indeed sufficient to induce cell death in BCR-ABL1 ALL. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. These findings suggest that Ph+ ALL are uniquely sensitive to even incremental increases of pre-BCR signaling. Consistent with this concept, patient-derived Ph+ ALL cells express high levels of inhibitory surface receptors PECAM1, CD300A and LAIR1 that recruit and activate phosphatases SHP1 and SHIP1, which terminate pre-BCR signaling. Importantly, high expression levels of these surface receptors were strong predictors of poor outcome of patients with ALL in two clinical trials, including both pediatric and adult ALL patients. Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases SHP1 and SHIP1. Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 transformed ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of an autoreactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in Ph+ ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of SHIP1, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in human Ph+ ALL. Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Introduction: Unlike other cell types, B cells are selected for an intermediate level of signaling strength. Critical survival and proliferation signals emanate from the (pre-) B cell receptor (BCR): Both attenuation below minimum (e.g. non-functional pre-BCR) and hyperactivation above maximum (e.g. autoreactive pre-BCR) thresholds of signaling strength trigger negative selection and cell death. The oncogenic BCR-ABL1 tyrosine kinase mimics active pre-BCR signaling in Ph+ acute lymphoblastic leukemia (ALL) which defines the ALL subgroup with the worst clinical outcome. Current therapy approaches are largely focused on the development of more potent tyrosine kinase inhibitors (TKI) to suppress oncogenic signaling. However resistance to TKI is developed invariably. Here, we test the hypothesis that targeting hyperactivation above a maximum threshold will selectively kill Ph+ ALL cells, similar to removal of self-reactive B cells. Results: The Ph+ ALL cells don not express ITAM (immunoreceptor tyrosine-based activation motif) receptor Igα or Igβ on the cell surface, indicating defects for a functional pre-BCR. Reconstitution of ITAM receptor was sufficient to induce cell death through increasing pre-BCR signaling strength indicated by phosphorylation of SYK, SRC, BTK and PLCγ2. TKI-treatment, while designed to kill leukemia cells, seemingly paradoxically rescued Ph+ ALL cells in this experimental setting. Surprisingly, patient-derived Ph+ ALL cells express the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptors PECAM1, CD300A and LAIR1 at high levels compared to normal pre-B cells. Importantly, high expression levels of ITIM-receptors are predictive of poor outcome in two clinical trials, including both pediatric and adult ALL patients. Genetic studies revealed that Pecam1, Cd300a and Lair1 were critical to calibrate pre-BCR signaling strength through recruitment of the inhibitory phosphatases Ptpn6 (Shp1) and Inpp5d (Ship1). Genetic deletion of Lair1, Ptpn6 or Inpp5d in BCR-ABL1 ALL caused cell death in vitro and in vivo through hyperactivation of pre-BCR signaling. Testing various components of proximal pre-BCR signaling, we found that an incremental increase of SYK tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive SYK was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Using chimeric PECAM1, CD300A and LAIR1 receptor decoys and a novel small molecule inhibitor of INPP5D, we demonstrated that pharmacological hyperactivation of pre-BCR signaling and engagement of negative B cell selection represents a promising new strategy to overcome drug-resistance in human Ph+ ALL. Conclusion: These results indicated that inhibitory receptors and downstream phosphatases are critical regulators of pre-BCR signaling strength in Ph+ ALL, and identified targeting hyperactivation of pre-BCR signaling as a potential novel class of therapeutic strategy. Note: This abstract was not presented at the meeting. Citation Format: Zhengshan Chen, Seyedmehdi Shojaee, Maike Buchner, Huimin Geng, Jae Woong Lee, Lars Klemm, Eugene Park, Ying Xim Tan, Anne Satterthwaite, Elisabeth Paietta, Stephen P. Hunger, Mignon L. Loh, Jae U. Jung, John E. Coligan, Silvia Bolland, Tak W. Mak, Andre Limnander, Hassan Jumaa, Michael Reth, Arthur Weiss, Clifford A. Lowell, Markus Müschen. Signaling thresholds and negative B cell selection in acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2075. doi:10.1158/1538-7445.AM2015-2075

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 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. Disclosures No relevant conflicts of interest to declare.