Genome-wide association studies implicate the PTPN2 gene locus (18p11.21) in risk for several autoimmune diseases, including inflammatory bowel disease. Through genetic fine mapping, we identified the single-nucleotide polymorphism rs80262450 in the PTPN2 gene as the putative causal variant. Analysis of GTEx tissue samples and genetically engineered myeloid cell lines carrying risk and nonrisk alleles of rs80262450 demonstrated increased expression of the PTPN2 splice isoform 4 (PTPN2.4), suggesting that the rs80262450 enhances disease susceptibility by favoring production of PTPN2.4. Furthermore, we found that PTPN2.4 contains a nuclear export sequence (NES) that leads to its retention in the cytoplasm. Differential localization of PTPN2.4 isoform results in a distinct protein binding profile revealed by mass-spectrometry analysis, and its overexpression increased TNF-α. PTPN2.4 knockdown reduced pro-inflammatory cytokines in human macrophages. Mutations within the NES motif abolished the unique localization and function of PTPN2.4. Lastly, increased expression of PTPN2.4 was found in Crohn's disease tissues, demonstrating its involvement in the disease. Together, we identified the pathogenic isoform PTPN2.4 as a novel driver of intestinal inflammation and a potential target to attenuate inflammation in IBD.

Effects on cultured (human and mouse) immune cells

Figure S1. A schematic representation for the method of transforming the log2 linear RPPA values to a 5 point categorical scale suitable for BMA modeling. Figure S2. Differential AML signaling responses to single agent PIM inhibition with AZD1208 and combination with FLT3 inhibitor AC220 Figure S3. Synergic combinations of drugs are sought to increase sensitivity. Figure S4. Susceptibility identified in AZD1208 Resistant Cells. Figure S5. Generation of a Boolean model to predict phosphorylation events, responses to single treatment and synergistic combinations of treatments. Figure S6. Generation of an AND/OR model to predict phosphorylation events, responses to single treatment and synergistic combinations of treatments. Table S1. Categorized RPPA. RPPA results for MOLM16, MV411, KG1A and EOL1 cells, categorized to 5 point scale from 0-4 Table S2. PhosphoScan MassSpec. LC-MS/MS phosphorylation proteomic in MOLM16 cells treated with 2 uM AZD1208 for 3 hours Table S3. QMN RPPA. Quadrant median normalized (QMN) calculated for RPPA protein levels for MOLM16, MV411, KG1A and EOL1 cells. Table S4. RPPA Stats. Calling statistically significant total protein and phosphorylation changes (determined by log2 QMN differences greater than or equal to 0.5 and Wilcoxon Rank Sum Tests p-values less than or equal to 0.1) Table S5. Exome. Whole exome DNA sequencing

<p>The following tables are supplied in the excel file due to their sizes: Table S6. Phosphosite Overlap. PhosphoScan mass-spectrometry in MOLM16 cells after 3 hour treatment with AZD1208 Table S7. Literature Entries. Full literature entries. Table S8. Model Target Functions. Target functions for AML general model and cell specific calibrated AML model. Table S9. AML genotype state. Nodes settings for model initialization for each cell-specific state. Table S10. Immediate downstream effect of modelled AML genotypes. Direct modelled effect of each genotype. Table S11. Cell-specific AML network model replicates response to treatments reported in external publications. Summary of experimental conclusions compared to model predictions. Table S12. Combination strategy. showing predicted activity for combinations of PI3K inhibitor, AKT inhibitor, MEK inhibitor, and AKT inhibitor with the PIM inhibitor AZD1208 across the 4 AML cell lines. Table S13. MOLM16_Res_DNAseq. Whole exome DNA-seq for PIM inhibition resistant populations of MOLM16 cell line. Table S14. BIOGRID Interactions. BIOGRID interactions between putative resistant deriving genes to the RAS/PI3K and/or the AKT/MTOR signaling pathways.</p>

Syngeneic mouse tumor efficacy, STAT3, and gene expression changes

Gating and antibodies for flow cytometry and CyTOF

Supplementary Materials and Methods: Cell line treatment Gene expression microarray Whole exome DNA sequencing Theranostics health reverse phase protein array Protein array data transformation for executable network model construction PhosphoScan phosphorylation proteomics Qualitative networks (QN) Construction of a cell-specific AML model

Patient tumor gene expression data

Cytokine, T, and NK cell changes in CT26 tumors

Patient STAT reductions and reproducibility of replicates

AbstractPurpose:Danvatirsen is a therapeutic antisense oligonucleotide (ASO) that selectively targets STAT3 and has shown clinical activity in two phase I clinical studies. We interrogated the clinical mechanism of action using danvatirsen-treated patient samples and conducted back-translational studies to further elucidate its immunomodulatory mechanism of action.Experimental Design:Paired biopsies and blood samples from danvatirsen-treated patients were evaluated using immunohistochemistry and gene-expression analysis. To gain mechanistic insight, we used mass cytometry, flow cytometry, and immunofluorescence analysis of CT26 tumors treated with a mouse surrogate STAT3 ASO, and human immune cells were treated in vitro with danvatirsen.Results:Within the tumors of treated patients, danvatirsen uptake was observed mainly in cells of the tumor microenvironment (TME). Gene expression analysis comparing baseline and on-treatment tumor samples showed increased expression of proinflammatory genes. In mouse models, STAT3 ASO demonstrated partial tumor growth inhibition and enhanced the antitumor activity when combined with anti–PD-L1. Immune profiling revealed reduced STAT3 protein in immune and stromal cells, and decreased suppressive cytokines correlating with increased proinflammatory macrophages and cytokine production. These changes led to enhanced T-cell abundance and function in combination with anti–PD-L1.Conclusions:STAT3 ASO treatment reverses a suppressive TME and promotes proinflammatory gene expression changes in patients' tumors and mouse models. Preclinical data provide evidence that ASO-mediated inhibition of STAT3 in the immune compartment is sufficient to remodel the TME and enhance the activity of checkpoint blockade without direct STAT3 inhibition in tumor cells. Collectively, these data provide a rationale for testing this combination in the clinic.

Background Alzheimer’s disease (AD) is a chronic progressive neurodegenerative disease impacting an estimated 44 million adults worldwide. The causal pathology of AD (accumulation of amyloid-beta and tau), precedes hallmark symptoms of dementia by more than a decade, necessitating development of early diagnostic markers of disease onset, particularly for new drugs that aim to modify disease processes. To evaluate differentially methylated positions (DMPs) as novel blood-based biomarkers of AD, we used a subset of 653 individuals with peripheral blood (PB) samples in the Alzheimer’s disease Neuroimaging Initiative (ADNI) consortium. The selected cohort of AD, mild cognitive impairment (MCI), and age-matched healthy controls (CN) all had imaging, genetics, transcriptomics, cerebrospinal protein markers, and comprehensive clinical records, providing a rich resource of concurrent multi-omics and phenotypic information on a well-phenotyped subset of ADNI participants. Results In this manuscript, we report cross-diagnosis differential peripheral DNA methylation in a cohort of AD, MCI, and age-matched CN individuals with longitudinal DNA methylation measurements. Epigenome-wide association studies (EWAS) were performed using a mixed model with repeated measures over time with a P value cutoff of 1 × 10 −5 to test contrasts of pairwise differential peripheral methylation in AD vs CN, AD vs MCI, and MCI vs CN. The most highly significant differentially methylated loci also tracked with Mini Mental State Examination (MMSE) scores. Differentially methylated loci were enriched near brain and neurodegeneration-related genes (e.g., BDNF, BIN1, APOC1 ) validated using the genotype tissue expression project portal (GTex). Conclusions Our work shows that peripheral differential methylation between age-matched subjects with AD relative to healthy controls will provide opportunities to further investigate and validate differential methylation as a surrogate of disease. Given the inaccessibility of brain tissue, the PB-associated methylation marks may help identify the stage of disease and progression phenotype, information that would be central to bringing forward successful drugs for AD.

Centrosomes function as the main microtubuleorganizing centers (MTOCs) and play an essential role during cellular mitosis. As such, centrosome number is tightly controlled, with centrosomes duplicated only once per cell cycle [1]. Unsurprisingly, centrosome abnormality, in particular centrosome amplification, has been frequently observed in human malignancies and is linked with aneuploidy and tumorigenesis. Recently the causative role of centrosome amplification in cancer has been demonstrated in a mouse model [2]. The resulting extra centrosomes from amplification pose a challenge for the proper execution of mitosis since multipolar mitosis is often detrimental to dividing cells and can lead to cell death [3]. Cancer cells, however, have developed tactics to avoid deleterious spindle multipolarity during mitosis through mechanisms such as centrosome clustering, a process that involves assembling multiple centrosomes into pseudo-bipolar spindles and thereby promotes tumor survival [4]. In recent years, a therapeutic strategy to pharmacologically impair centrosome clustering has emerged and demonstrated early promise in the preclinical setting: the goal is to exploit the vulnerability of cancer cells in handling spindle multipolarity by inducing centrosome de-clustering and achieve selective killing of cancer cells while sparing normal tissues. We recently reported the involvement of monoADP-ribosyltransferase PARP6 in maintaining centrosome integrity during mitosis by demonstrating that pharmacological inhibition of PARP6 induced multipolar spindle formation and elicited therapeutic effects in breast cancer [5]. This study was largely enabled by the AstraZeneca collection of PARP inhibitors consisting of mostly phthalazinone and quinazolinone based NAD+ mimetic cores that display differential selectivity towards polyand mono-ADP-ribosyltranferases [6]. Previous siRNA screens implicated PARP activity in centrosome clustering and bipolar spindle formation as a specific mitotic susceptibility in cancer cells [7]. We leveraged this knowledge and conducted parallel biochemical screens against multiple PARP enzymes together with cell-based phenotypic profiling in cancer cells to find that only PARP6 inhibition could drive multipolar spindle formation, with a strong correlation (R2 = 0.76) observed between PARP6 enzymatic inhibition and multipolar spindle induction potency. We recapitulated the mitotic phenotype with siRNA-based PARP6 knockdown. Together, these results link the suppression of PARP6 enzymatic activity to the induced multipolar spindle centrosome phenotype. We further showed that targeting PARP6 with the optimized small molecule inhibitor AZ0108 effectively induced spindle multipolarity and demonstrated promising anti-tumor activity in various breast cancer models in vitro and in vivo. To gain mechanistic understanding of the mitotic phenotype associated with PARP6 inhibition, we sought to identify PARP6 enzyme substrates using a powerful high-content ProtoArray screen consisting of 9,000+ full length human proteins. Centrosome related proteins were enriched as screening hits, including Chk1, which was further confirmed as a specific PARP6 substrate by in vitro enzymatic assays and mass spectrometric analysis in cells. Beyond its role in mediating DNA damage responses, Chk1 is also involved in regulating cellcycle checkpoints such as mitosis. During undisturbed cell cycle, Chk1 was reported to localize to interphase centrosomes and modulate cyclinB-Cdk1 activity, thereby impacting centrosomic mitotic events [8]. Our experiments demonstrated that intracellular ADP-ribosylation of Chk1 accumulated upon mitotic arrest and was abolished by treatment with PARP6 inhibitor AZ0108. Intriguingly, suppressing PARP6 using AZ0108 resulted in marked elevation of Chk1 phosphorylation at S345 and a dosedependent increase in centrosome-associated Chk1 expression. These events were accompanied by diminished activation of mitotic proteins such as FoxM1, histone H3 and Aurora kinases. Together, these observations support a novel mechanism for PARP6 in regulating Chk1 activation in mitosis via ADP-ribosylation and provide a mechanistic basis for multipolar spindle induction through PARP6 inhibition. Editorial

AbstractPARP proteins represent a class of post-translational modification enzymes with diverse cellular functions. Targeting PARPs has proven to be efficacious clinically, but exploration of the therapeutic potential of PARP inhibition has been limited to targeting poly(ADP-ribose) generating PARP, including PARP1/2/3 and tankyrases. The cancer-related functions of mono(ADP-ribose) generating PARP, including PARP6, remain largely uncharacterized. Here, we report a novel therapeutic strategy targeting PARP6 using the first reported PARP6 inhibitors. By screening a collection of PARP compounds for their ability to induce mitotic defects, we uncovered a robust correlation between PARP6 inhibition and induction of multipolar spindle (MPS) formation, which was phenocopied by PARP6 knockdown. Treatment with AZ0108, a PARP6 inhibitor with a favorable pharmacokinetic profile, potently induced the MPS phenotype, leading to apoptosis in a subset of breast cancer cells in vitro and antitumor effects in vivo. In addition, Chk1 was identified as a specific substrate of PARP6 and was further confirmed by enzymatic assays and by mass spectrometry. Furthermore, when modification of Chk1 was inhibited with AZ0108 in breast cancer cells, we observed marked upregulation of p-S345 Chk1 accompanied by defects in mitotic signaling. Together, these results establish proof-of-concept antitumor efficacy through PARP6 inhibition and highlight a novel function of PARP6 in maintaining centrosome integrity via direct ADP-ribosylation of Chk1 and modulation of its activity.Significance:These findings describe a new inhibitor of PARP6 and identify a novel function of PARP6 in regulating activation of Chk1 in breast cancer cells.

BACKGROUND:T-cell checkpoint blockade and MEK inhibitor combinations are under clinical investigation. Despite progress elucidating the immuno-modulatory effects of MEK inhibitors as standalone therapies, the impact of MEK inhibition on the activity of T-cell checkpoint inhibitors remains incompletely understood. Here we sought to characterize the combined effects of MEK inhibition and anti-CTLA-4 mAb (anti-CTLA-4) therapy, examining effects on both T-cells and tumor microenvironment (TME).METHODS:In mice, the effects of MEK inhibition, via selumetinib, and anti-CTLA-4 on immune responses to keyhole limpet haemocyanin (KLH) immunization were monitored using ex vivo functional assays with splenocytes. In a KRAS-mutant CT26 mouse colorectal cancer model, the impact on the tumor microenvironment (TME) and the spleen were evaluated by flow cytometry. The TME was further examined by gene expression and immunohistochemical analyses. The combination and sequencing of selumetinib and anti-CTLA-4 were also evaluated in efficacy studies using the CT26 mouse syngeneic model.RESULTS:Anti-CTLA-4 enhanced the generation of KLH specific immunity following KLH immunization in vivo; selumetinib was found to reduce, but did not prevent, this enhancement of immune response by anti-CTLA-4 in vivo. In the CT26 mouse model, anti-CTLA-4 treatment led to higher expression levels of the immunosuppressive mediators, Cox-2 and Arg1 in the TME. Combination of anti-CTLA-4 with selumetinib negated this up-regulation of Cox-2 and Arg1, reduced the frequency of CD11+ Ly6G+ myeloid cells, and led to the accumulation of differentiating monocytes at the Ly6C+ MHC+ intermediate state in the tumor. We also report that MEK inhibition had limited impact on anti-CTLA-4-mediated increases in T-cell infiltration and T-cell activation in CT26 tumors. Finally, we show that pre-treatment, but not concurrent treatment, with selumetinib enhanced the anti-tumor activity of anti-CTLA-4 in the CT26 model.CONCLUSION:These data provide evidence that MEK inhibition can lead to changes in myeloid cells and immunosuppressive factors in the tumor, thus potentially conditioning the TME to facilitate improved response to anti-CTLA-4 treatment. In summary, the use of MEK inhibitors to alter the TME as an approach to enhance the activities of immune checkpoint inhibitors warrants further investigation in clinical trials.

Abstract AZD9150, a gen2.5 antisense oligonucleotide (ASO) targeting human STAT3, has improved drug-like properties compared to previous generation ASO therapeutics, including increased stability and resistance to nucleases, reduced proinflammatory effects, and enhanced potency. We have previously reported that in tumors, STAT3 ASOs are taken up preferentially in stromal and immune cells of the tumor microenvironment (TME). Since AZD9150 is selective for human STAT3, we used a surrogate ASO (muSTAT3 ASO) to explore the pharmacodynamics of ASO-mediated STAT3 inhibition in syngeneic and genetically engineered mouse (GEM) tumor models, focusing on effects in the TME. In mice bearing subcutaneous CT-26 tumors, treatment with muSTAT3 ASO at 50 mg/kg, s.c., on a qdx5/wk schedule decreased STAT3 levels in immune cell subsets in the tumor and in circulating leukocytes by 40 - 60%, similar to the decrease in STAT3 achievable in circulating leukocytes in human patients after AZD9150 treatment. In a Nanostring analysis (nCounter mouse immunology panel) of CT-26 tumors from muSTAT3 ASO treated mice, CD163 (M2 immune suppressive macrophage marker) was the gene most consistently and significantly downregulated, by an average of 84% in three independent experiments, and was confirmed by immunohistochemistry (IHC). Flow cytometry analysis of myeloid subpopulations - tumor associated macrophages (F4/80+ TAMs), monocytic myeloid derived suppressor cells, and granulocytic cells - showed a decrease in TAMs averaging 69% across three independent experiments. The analysis was extended to include IHC for arginase (Arg, a marker of functional immune suppression activity). Subpopulations of cells identified included Arg+, CD163+, and Arg+CD163+. Treatment with muSTAT3 ASO decreased these populations by 79%, 88% and 97% respectively, compared to control treatment. These populations were also analyzed in two GEM tumor models - the KPC pancreatic cancer model, and a PTEN -/- prostate cancer model - which have a TME more representative of that found in tumors in the clinic. While the specific changes varied across the models, likely reflecting differences in TME makeup, a reduction in immune suppressive cell populations was present in both GEM models, including a decrease in CD163+ cells of 79% (along with modest antitumor activity) in the PTEN -/- prostate model after muSTAT3 ASO treatment. These results indicate that selective STAT3 inhibition can reduce immune suppressive cell populations in the TME, and suggest that STAT3 inhibition has the potential to enhance the antitumor activity of T-cell targeted therapies, such as those targeting the PD1-PDL1 axis. In support of this hypothesis, we observed that addition of muSTAT3 ASO to anti-PD-L1 Ab treatment significantly enhanced the antitumor activity of PD-L1 Ab treatment in two subcutaneous syngeneic tumor models, CT-26 and A20. Citation Format: Rich Woessner, Vasu Sah, Patricia McCoon, Shaun Grosskurth, Nanhua Deng, Rachel DuPont, Deborah Lawson, Lourdes Pablo, Corinne Reimer, Marco A. De Velasco, Hirotsugu Uemura, Juliana Candido, Paul Lyne. Inhibition of STAT3 by antisense oligonucleotide treatment decreases the immune suppressive tumor microenvironment in syngeneic and GEM tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3684. doi:10.1158/1538-7445.AM2017-3684

Abstract Personalized therapy is a major goal of modern oncology, as patient responses vary greatly even within a histologically defined cancer subtype. This is especially true in acute myeloid leukemia (AML), which exhibits striking heterogeneity in molecular segmentation. When calibrated to cell-specific data, executable network models can reveal subtle differences in signaling that help explain differences in drug response. Furthermore, they can suggest drug combinations to increase efficacy and combat acquired resistance. Here, we experimentally tested dynamic proteomic changes and phenotypic responses in diverse AML cell lines treated with pan-PIM kinase inhibitor and fms-related tyrosine kinase 3 (FLT3) inhibitor as single agents and in combination. We constructed cell-specific executable models of the signaling axis, connecting genetic aberrations in FLT3, tyrosine kinase 2 (TYK2), platelet-derived growth factor receptor alpha (PDGFRA), and fibroblast growth factor receptor 1 (FGFR1) to cell proliferation and apoptosis via the PIM and PI3K kinases. The models capture key differences in signaling that later enabled them to accurately predict the unique proteomic changes and phenotypic responses of each cell line. Furthermore, using cell-specific models, we tailored combination therapies to individual cell lines and successfully validated their efficacy experimentally. Specifically, we showed that cells mildly responsive to PIM inhibition exhibited increased sensitivity in combination with PIK3CA inhibition. We also used the model to infer the origin of PIM resistance engineered through prolonged drug treatment of MOLM16 cell lines and successfully validated experimentally our prediction that this resistance can be overcome with AKT1/2 inhibition. Cancer Res; 77(4); 827–38. ©2016 AACR.

Agents that target components of the PI3K/AKT/mTOR pathway are under investigation for the treatment of diffuse large B cell lymphoma (DLBCL). Given the highly heterogeneous nature of DLBCL, it is not clear whether all subtypes of DLBCL will be susceptible to PI3K pathway inhibition, or which kinase within this pathway is the most favorable target. Pharmacological profiling of a panel of DLBCL cell lines revealed a subset of DLBCL that was resistant to AKT inhibition. Strikingly, sensitivity to AKT inhibitors correlated with the ability of these inhibitors to block phosphorylation of S6K1 and ribosomal protein S6. Cell lines resistant to AKT inhibition activated S6K1 independent of AKT either through upregulation of PIM2 or through activation by B cell receptor (BCR) signaling components. Finally, combined inhibition of AKT and BTK, PIM2, or S6K1 proved to be an effective strategy to overcome resistance to AKT inhibition in DLBCL.

Abstract Purpose: The emergence of EGFR inhibitors such as gefitinib, erlotinib, and osimertinib has provided novel treatment opportunities in EGFR-driven non–small cell lung cancer (NSCLC). However, most patients with EGFR-driven cancers treated with these inhibitors eventually relapse. Recent efforts have identified the canonical Wnt pathway as a mechanism of protection from EGFR inhibition and that inhibiting tankyrase, a key player in this pathway, is a potential therapeutic strategy for the treatment of EGFR-driven tumors. Experimental Design: We performed a preclinical evaluation of tankyrase inhibitor AZ1366 in combination with multiple EGFR-inhibitors across NSCLC lines, characterizing its antitumor activity, impingement on canonical Wnt signaling, and effects on gene expression. We performed pharmacokinetic and pharmacodynamic profiling of AZ1366 in mice and evaluated its therapeutic activity in an orthotopic NSCLC model. Results: In combination with EGFR inhibitors, AZ1366 synergistically suppressed proliferation of multiple NSCLC lines and amplified global transcriptional changes brought about by EGFR inhibition. Its ability to work synergistically with EGFR inhibition coincided with its ability to modulate the canonical Wnt pathway. Pharmacokinetic and pharmacodynamic profiling of AZ1366-treated orthotopic tumors demonstrated clinically relevant serum drug levels and intratumoral target inhibition. Finally, coadministration of an EGFR inhibitor and AZ1366 provided better tumor control and improved survival for Wnt-responsive lung cancers in an orthotopic mouse model. Conclusions: Tankyrase inhibition is a potent route of tumor control in EGFR-dependent NSCLC with confirmed dependence on canonical Wnt signaling. These data strongly support further evaluation of tankyrase inhibition as a cotreatment strategy with EGFR inhibition in an identifiable subset of EGFR-driven NSCLC. Clin Cancer Res; 23(6); 1531–41. ©2016 AACR.