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PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Here, we identify PARP7 as a negative regulator of nucleic acid sensing in tumor cells. Inhibition of PARP7 restores type I interferon (IFN) signaling responses to nucleic acids in tumor models. Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression. Oral dosing of the PARP7 small-molecule inhibitor, RBN-2397, results in complete tumor regression in a lung cancer xenograft and induces tumor-specific adaptive immune memory in an immunocompetent mouse cancer model, dependent on inducing type I IFN signaling in tumor cells. PARP7 is a therapeutic target whose inhibition induces both cancer cell-autonomous and immune stimulatory effects via enhanced IFN signaling. These data support the targeting of a monoPARP in cancer and introduce a potent and selective PARP7 inhibitor to enter clinical development.

Summary PARP14 has been implicated by genetic knockout studies to promote protumor macrophage polarization and suppress the antitumor inflammatory response due to its role in modulating interleukin-4 (IL-4) and interferon-γ signaling pathways. Here, we describe structure-based design efforts leading to the discovery of a potent and highly selective PARP14 chemical probe. RBN012759 inhibits PARP14 with a biochemical half-maximal inhibitory concentration of 0.003 μM, exhibits >300-fold selectivity over all PARP family members, and its profile enables further study of PARP14 biology and disease association both in vitro and in vivo. Inhibition of PARP14 with RBN012759 reverses IL-4-driven protumor gene expression in macrophages and induces an inflammatory mRNA signature similar to that induced by immune checkpoint inhibitor therapy in primary human tumor explants. These data support an immune suppressive role of PARP14 in tumors and suggest potential utility of PARP14 inhibitors in the treatment of cancer.

Acquired and intrinsic resistance to receptor tyrosine kinase inhibitors (RTKi) represents a major hurdle in improving the management of clear cell renal cell carcinoma (ccRCC). Recent reports suggest that drug resistance is driven by tumor adaptation via epigenetic mechanisms that activate alternative survival pathways. The histone methyl transferase EZH2 is frequently altered in many cancers, including ccRCC. To evaluate its role in ccRCC resistance to RTKi, we established and characterized a spontaneously metastatic, patient-derived xenograft model that is intrinsically resistant to the RTKi sunitinib, but not to the VEGF therapeutic antibody bevacizumab. Sunitinib maintained its antiangiogenic and antimetastatic activity but lost its direct antitumor effects due to kinome reprogramming, which resulted in suppression of proapoptotic and cell-cycle–regulatory target genes. Modulating EZH2 expression or activity suppressed phosphorylation of certain RTKs, restoring the antitumor effects of sunitinib in models of acquired or intrinsically resistant ccRCC. Overall, our results highlight EZH2 as a rational target for therapeutic intervention in sunitinib-resistant ccRCC as well as a predictive marker for RTKi response in this disease. Cancer Res; 77(23); 6651–66. ©2017 AACR.

The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2. Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR.

Poly-ADP-ribose polymerases (PARPs) are a family of enzymes responsible for transferring individual or chains of ADP-ribose subunits to substrate targets as a type of post-translational modification. PARPs regulate a wide variety of important cellular processes, ranging from DNA damage repair to antiviral response. However, most research to date has focused primarily on the polyPARPs, which catalyze the formation of ADP-ribose polymer chains, while the monoPARPs, which transfer individual ADP-ribose monomers, have not been studied as thoroughly. This is partially due to the lack of robust assays to measure mono-ADP-ribosylation in the cell. In this study, the recently developed MAR/PAR antibody has been shown to detect mono-ADP-ribosylation in cells, enabling the field to investigate the function and therapeutic potential of monoPARPs. In this study, the antibody was used in conjunction with engineered cell lines that overexpress various PARPs to establish a panel of assays to evaluate the potencies of literature-reported PARP inhibitors. These assays should be generally applicable to other PARP family members for future compound screening efforts. A convenient and generalizable workflow to identify and validate PARP substrates has been established. As an initial demonstration, aryl hydrocarbon receptor was verified as a direct PARP7 substrate and other novel substrates for this enzyme were also identified and validated. This workflow takes advantage of commercially available detection reagents and conventional mass spectrometry instrumentation and methods. Ultimately, these assays and methods will help drive research in the PARP field and benefit future therapeutics development.

The dynamic structure of histones and DNA, also known as chromatin, is regulated by two classes of enzymes: those that mediate covalent modifications on either histone proteins or DNA and those that use the energy generated by ATP hydrolysis to mechanically alter chromatic structure. Both classes of enzymes are often found in large protein complexes. In this review, we describe two such complexes: polycomb repressive complex 2 (PRC2), with the protein methyltransferase EZH2 as its catalytic subunit, and the ATP-dependent chromatin remodeler switch/sucrose non-fermentable (SWI/SNF). EZH2 catalyzes the methylation of lysine 27 on histone H3, a covalent chromatin modification that is associated with repressed heterochromatin. The catalytic activity of SWI/SNF, in contrast, leads to a state of open chromatin associated with active transcription. In this review, we discuss the biochemical properties of both complexes, outline the principles of their regulation, and describe their opposing roles in normal development, which can be perturbed in disease settings such as cancer.

Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure-activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency.

Summary Background Activating enhancer of zeste homolog 2 (EZH2) mutations or aberrations of the switch/sucrose non-fermentable (SWI/SNF) complex (eg, mutations or deletions of the subunits INI1 or SMARCA4) can lead to aberrant histone methylation, oncogenic transformation, and a proliferative dependency on EZH2 activity. In this first-in-human study, we aimed to investigate the safety, clinical activity, pharmacokinetics, and pharmacodynamics of tazemetostat, a first-in-class selective inhibitor of EZH2. Methods We did an open-label, multicentre, dose-escalation, phase 1 study using a 3 + 3 design with planned cohort expansion at the two highest doses below the maximally tolerated dose. The study was done at two centres in France: Institut Gustave Roussy (Villejuif, Val de Marne) and Institut Bergonie (Bordeaux, Gironde). Eligible patients had relapsed or refractory B-cell non-Hodgkin lymphoma or an advanced solid tumour and were older than 18 years, with Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Tazemetostat was administered orally from 100 mg twice daily to 1600 mg twice daily in 28-day cycles. The primary endpoint was to establish the maximum tolerated dose or recommended phase 2 dose of tazemetostat, as determined by dose-limiting toxicities, laboratory values, and other safety or pharmacokinetic measures in cycle one according to local investigator assessment. Safety was assessed in patients who received at least one dose of tazemetostat; antitumour activity was assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01897571. The phase 1 part of the study is complete, and phase 2 is ongoing. Findings Between June 13, 2013, and Sept 21, 2016, 64 patients (21 with B-cell non-Hodgkin lymphoma, and 43 with advanced solid tumours) received doses of tazemetostat. The most common treatment-related adverse events, regardless of attribution, were asthenia (21 [33%] of 64 treatment-related events), anaemia (nine [14%]), anorexia (four [6%]), muscle spasms (nine [14%]), nausea (13 [20%]), and vomiting (six [9%]), usually grade 1 or 2 in severity. A single dose-limiting toxicity of grade 4 thrombocytopenia was identified at the highest dose of 1600 mg twice daily. No treatment-related deaths occurred; seven (11%) patients had non-treatment-related deaths (one at 200 mg twice daily, four at 400 mg twice daily, and two at 1600 mg twice daily). The recommended phase 2 dose was determined to be 800 mg twice daily. Durable objective responses, including complete responses, were observed in eight (38%) of 21 patients with B-cell non-Hodgkin lymphoma and two (5%) of 43 patients with solid tumours. Interpretation Tazemetostat showed a favourable safety profile and antitumour activity in patients with refractory B-cell non-Hodgkin lymphoma and advanced solid tumours, including epithelioid sarcoma. Further clinical investigation of tazemetostat monotherapy is ongoing in phase 2 studies in adults and a phase 1 study for children, which are currently enrolling patients who have B-cell non-Hodgkin lymphoma and INI1-negative or SMARCA4-negative tumours. Funding Epizyme and Eisai.

Cell proliferation assays are an important component of small molecule inhibitor screens for cancer therapies. An important but often overlooked variable involves the timing and timeframe of inhibitor treatment. Whereas many traditional chemotherapeutics kill or inhibit proliferation on the timeframe of hours or in a few days of treatment, more targeted therapies that affect other cancer-relevant pathways, including differentiation or cell stress responses, can take longer, often several days to weeks to impact cellular growth and survival. Many poly(ADP-ribose) polymerases (PARPs) are involved in cellular stress pathways; therefore, phenotypic effects of PARP inhibition are often only observed with long-term inhibitor treatment. Here we summarize several assays for analyzing long-term proliferation of both adherent and suspension cells, relying either on growth in two-dimensional tissue culture or on systems than enable growth in 3D.

EZH2 and EZH1 are protein methyltransferases (PMTs) responsible for histone H3, lysine 27 (H3K27) methylation. Trimethylation of H3K27 (H3K27me3) is a hallmark of many cancers, including non-Hodgkin lymphoma (NHL). Heterozygous EZH2 point mutations at Tyr641, Ala677, and Ala687 have been observed in NHL. The Tyr641 mutations enhance activity on H3K27me2 but have weak or no activity on unmethylated H3K27, whereas the Ala677 and Ala687 mutations use substrates of all methylation states effectively. It has been proposed that enzymatic coupling of the wild-type and mutant enzymes leads to the oncogenic H3K27me3 mark in mutant-bearing NHL. We show that coupling with the wild-type enzyme is needed to achieve H3K27me3 for several mutants, but that others are capable of achieving H3K27me3 on their own. All forms of PRC2 (wild-type and mutants) display kinetic signatures that are consistent with a distributive mechanism of catalysis.

The SWI/SNF complex is a major regulator of gene expression and is increasingly thought to play an important role in human cancer, as evidenced by the high frequency of subunit mutations across virtually all cancer types. We previously reported that in preclinical models, malignant rhabdoid tumors, which are deficient in the SWI/SNF core component INI1 (SMARCB1), are selectively killed by inhibitors of the H3K27 histone methyltransferase EZH2. Given the demonstrated antagonistic activities of the SWI/SNF complex and the EZH2-containing PRC2 complex, we investigated whether additional cancers with SWI/SNF mutations are sensitive to selective EZH2 inhibition. It has been recently reported that ovarian cancers with dual loss of the redundant SWI/SNF components SMARCA4 and SMARCA2 are characteristic of a rare rhabdoid-like subtype known as small-cell carcinoma of the ovary hypercalcemic type (SCCOHT). Here, we provide evidence that a subset of commonly used ovarian carcinoma cell lines were misdiagnosed and instead were derived from a SCCOHT tumor. We also demonstrate that tazemetostat, a potent and selective EZH2 inhibitor currently in phase II clinical trials, induces potent antiproliferative and antitumor effects in SCCOHT cell lines and xenografts deficient in both SMARCA2 and SMARCA4. These results exemplify an additional class of rhabdoid-like tumors that are dependent on EZH2 activity for survival.

Reply to "Uveal melanoma cells are resistant to EZH2 inhibition regardless of BAP1 status"

Tazemetostat (EPZ-6438) is a selective inhibitor of the histone methyltransferase EZH2 and currently in clinical development for non-Hodgkin lymphoma and genetically defined tumors.Tazemetostat was tested against the Pediatric Preclinical Testing Program (PPTP) solid tumor xenografts using a dose of 400 mg/kg administered twice daily by oral gavage for 28 days. H3K27me3:H3 ratios were determined in control and treated tumors.Tazemetostat induced significant differences in event-free survival (EFS) distribution compared with control in nine of 30 (30%) of the xenografts studied. Significant differences in EFS distribution were observed in five of seven (71%) rhabdoid tumor xenograft lines compared with four of 23 (17%) nonrhabdoid xenograft lines (chi-square [χTazemetostat demonstrated significant antitumor activity in rhabdoid tumor models but showed no consistent activity against any other histology. Tazemetostat reduced H3K27me3 levels irrespective of tumor response. Further preclinical testing to evaluate tazemetostat in combination with other anticancer agents is warranted.

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.

Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry-based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

Gene expression studies have been widely used in an effort to identify signatures that can predict clinical progression of cancer. In this study we focused instead on identifying gene expression differences between breast tumors and adjacent normal tissue, and between different subtypes of tumor classified by clinical marker status. We have collected a set of 20 breast cancer tissues, matched with the adjacent pathologically normal tissue from the same patient. The cancer samples representing each subtype of breast cancer identified by estrogen receptor ER(+/-) and Her2(+/-) status and divided into four subgroups (ER+/Her2+, ER+/Her2-, ER-/Her2+, and ER-/Her2-) were hybridized on Affymetrix HG-133 Plus 2.0 microarrays. By comparing cancer samples with their matched normal controls we have identified 3537 overall differentially expressed genes using data analysis methods from Bioconductor. When we looked at the genes in common of the four subgroups, we found 151 regulated genes, some of them encoding known targets for breast cancer treatment. Unique genes in the four subgroups instead suggested gene regulation dependent on the ER/Her2 markers selection. In conclusion, the results indicate that microarray studies using robust analysis of matched tumor and normal samples from the same patients can be used to identify genes differentially expressed in breast cancer tumor subtypes even when small numbers of samples are considered and can further elucidate molecular features of breast cancer.

The SH2 domain-containing protein-tyrosine phosphatase PTPN11 (Shp2) is required for normal development and is an essential component of signaling pathways initiated by growth factors, cytokines, and extracellular matrix. In many of these pathways, Shp2 acts upstream of Ras. About 50% of patients with Noonan syndrome have germ-line PTPN11 gain of function mutations. Associations between Noonan syndrome and an increased risk of some malignancies, notably leukemia and neuroblastoma, have been reported, and recent data indicate that somatic PTPN11 mutations occur in children with sporadic juvenile myelomonocytic leukemia, myelodysplasic syndrome, B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia (AML). Juvenile myelomonocytic leukemia patients without PTPN11 mutations have either homozygotic NF-1 deletion or activating RAS mutations. Given the role of Shp2 in Ras activation and the frequent mutation of RAS in human tumors, these data raise the possibility that PTPN11 mutations play a broader role in cancer. We asked whether PTPN11 mutations occur in other malignancies in which activating RAS mutations occur at low but significant frequency. Sequencing of PTPN11 from 13 different human neoplasms including breast, lung, gastric, and neuroblastoma tumors and adult AML and acute lymphoblastic leukemia revealed 11 missense mutations. Five are known mutations predicted to result in an activated form of Shp2, whereas six are new mutations. Biochemical analysis confirmed that several of the new mutations result in increased Shp2 activity. Our data demonstrate that mutations in PTPN11 occur at low frequency in several human cancers, especially neuroblastoma and AML, and suggest that Shp2 may be a novel target for antineoplastic therapy.

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma-a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein-display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.

The tumor suppressors BAP1 and ASXL1 interact to form a polycomb deubiquitinase complex that removes monoubiquitin from histone H2A lysine 119 (H2AK119Ub). However, BAP1 and ASXL1 are mutated in distinct cancer types, consistent with independent roles in regulating epigenetic state and malignant transformation. Here we demonstrate that Bap1 loss in mice results in increased trimethylated histone H3 lysine 27 (H3K27me3), elevated enhancer of zeste 2 polycomb repressive complex 2 subunit (Ezh2) expression, and enhanced repression of polycomb repressive complex 2 (PRC2) targets. These findings contrast with the reduction in H3K27me3 levels seen with Asxl1 loss. Conditional deletion of Bap1 and Ezh2 in vivo abrogates the myeloid progenitor expansion induced by Bap1 loss alone. Loss of BAP1 results in a marked decrease in H4K20 monomethylation (H4K20me1). Consistent with a role for H4K20me1 in the transcriptional regulation of EZH2, expression of SETD8-the H4K20me1 methyltransferase-reduces EZH2 expression and abrogates the proliferation of BAP1-mutant cells. Furthermore, mesothelioma cells that lack BAP1 are sensitive to EZH2 pharmacologic inhibition, suggesting a novel therapeutic approach for BAP1-mutant malignancies.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers. Mol Cancer Ther; 13(4); 842–54. ©2014 AACR.

p120-catenin (p120(ctn)) interacts with the cytoplasmic tail of cadherins and is thought to regulate cadherin clustering during formation of adherens junctions. Several observations suggest that p120 can both positively and negatively regulate cadherin adhesiveness depending on signals that so far remain unidentified. Although p120 tyrosine phosphorylation is a leading candidate, the role of this modification in normal and Src-transformed cells remains unknown. Here, as a first step toward pinpointing this role, we have employed two-dimensional tryptic mapping to directly identify the major sites of Src-induced p120 phosphorylation. Eight sites were identified by direct mutation of candidate tyrosines to phenylalanine and elimination of the accompanying spots on the two-dimensional maps. Identical sites were observed in vitro and in vivo, strongly suggesting that the physiologically important sites have been correctly identified. Changing all of these sites to phenylalanine resulted in a p120 mutant, p120-8F, that could not be efficiently phosphorylated by Src and failed to interact with SHP-1, a tyrosine phosphatase shown previously to interact selectively with tyrosine-phosphorylated p120 in cells stimulated with epidermal growth factor. Using selected tyrosine to phenylalanine p120 mutants as dominant negative reagents, it may now be possible to selectively block events postulated to be dependent on p120 tyrosine phosphorylation.

Male “viable motheaten” (mev) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of mev mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH2-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

A prominent tyrosine-phosphorylated protein of approximately 100 kDa (designated pp100) in epidermal growth factor (EGF)-stimulated A431 cells was found to be a main interaction partner of the protein-tyrosine phosphatase SHP-1 in pull-down experiments with a glutathione S-transferase-SHP-1 fusion protein. Binding was largely mediated by the N-terminal SH2 domain of SHP-1 and apparently direct and independent from the previously described association of SHP-1 with the activated EGF receptor. pp100 was partially purified and identified by mass spectrometric analysis of tryptic fragments, partial amino acid sequencing, and use of authentic antibodies as the 3A isoform of the Armadillo repeat protein superfamily member p120 catenin (p120(ctn)). Different p120(ctn) isoforms expressed in human embryonal kidney 293 cells, exhibited differential binding to SHP-1 that correlated partly with the extent of EGF-dependent p120(ctn) tyrosine phosphorylation. Despite strong phosphorylation, p120(ctn) isoforms 3B and 3AB bound, however, less readily to SHP-1. SHP-1 associated transiently with p120(ctn) in EGF-stimulated A431 cells stably transfected with a tetracycline-responsive SHP-1 expression construct, and p120(ctn) exhibited elevated phosphorylation upon a tetracycline-mediated decrease in the SHP-1 level. Functions of p120(ctn), which are regulated by tyrosine phosphorylation, may be modulated by the described SHP-1-p120(ctn) interaction.

The protein-tyrosine phosphatase SHP-1 binds to and dephosphorylates the epidermal growth factor receptor (EGFR), and both SH2 domains of SHP-1 are important for this interaction (Tenev, T., Keilhack, H., Tomic, S., Stoyanov, B., Stein-Gerlach, M., Lammers, R., Krivtsov, A. V., Ullrich, A., and Böhmer, F. D. (1997) J. Biol. Chem. 272, 5966-5973). We mapped the EGFR phosphotyrosine 1173 as the major binding site for SHP-1 by a combination of phosphopeptide activation, phosphopeptide competition, and receptor YF mutant analysis. Mutational conversion of the EGFR sequence 1171-1176 AEYLRV into the high affinity SHP-1 binding sequence LEYLYL of the erythropoietin receptor (EpoR) led to a highly elevated SHP-1 binding to the mutant EGFR (EGFR1171-1176EpoR) and in turn to an enhanced dephosphorylation of the receptor. SHP-1 expression interfered with EGF-dependent mitogen-activated protein kinase stimulation, and this effect was more pronounced in case of EGFR1171-1176EpoR. Reduced SHP-1 binding to the EGFR Y1173F mutant resulted in a reduced receptor dephosphorylation by coexpressed SHP-1 and less interference with EGF-dependent mitogen-activated protein kinase stimulation. The effects of receptor mutations on SHP-1 binding were, however, stronger than those on receptor dephosphorylation by SHP-1. Therefore, receptor dephosphorylation may be the result of the combined activity of receptor-bound SHP-1 and SHP-1 bound to an auxiliary docking protein.

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1 -deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4′-(morpholinomethyl)-[1,1′-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.

Heterozygous point mutations at Y641 and A677 in the EZH2 SET domain are prevalent in about 10–24% of Non-Hodgkin lymphomas (NHL). Previous studies indicate that these are gain-of-function mutations leading to the hypertrimethylation of H3K27. These EZH2 mutations may drive the proliferation of lymphoma and make EZH2 a molecular target for patients harboring these mutations. Here, another EZH2 SET domain point mutation, A687V, occurring in about 1–2% of lymphoma patients, is also shown to be a gain-of-function mutation that greatly enhances its ability to perform dimethylation relative to wild-type EZH2 and is equally proficient at catalyzing trimethylation. We propose that A687V EZH2 also leads to hypertrimethylation of H3K27 and may thus be a driver mutation in NHL.

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitive kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc., link_to_OA_fulltext

Mutations in the Src homology 2 (SH2)-containing protein-tyrosine phosphatase Shp2 (PTPN11) underlie half of the cases of the autosomal dominant genetic disorder Noonan syndrome, and somatic Shp2 mutations are found in several hematologic and solid malignancies. Earlier studies of small numbers of mutants suggested that disease-associated mutations cause constitutive (SH2 binding-independent) activation and that cancer-associated mutants are more active than those associated with Noonan syndrome. We have characterized a larger panel of Shp2 mutants and find that this "activity-centric" model cannot explain the behaviors of all pathogenic Shp2 mutations. Instead, enzymatic, structural, and mathematical modeling analyses show that these mutants can affect basal activation, SH2 domain-phosphopeptide affinity, and/or substrate specificity to varying degrees. Furthermore, there is no absolute correlation between the mutants' extents of basal activation and the diseases they induce. We propose that activated mutants of Shp2 modulate signaling from specific stimuli to a subset of effectors and provide a theoretical framework for understanding the complex relationship between Shp2 activation, intracellular signaling, and pathology.

Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in EZH2 mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone – a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.

Activation of the SH2 domain-possessing protein-tyrosine phosphatase SHP-1 by acidic phospholipids as phosphatidic acid (PA) has been described earlier and suggested to participate in regulation of SHP-1 activity toward cellular substrates. The mechanism of this activation is poorly understood. Direct binding of phosphatidic acid to recombinant SHP-1 could be demonstrated by measuring the extent of [(14)C]PA binding in a chromatographic assay, by measuring the extent of binding of SHP-1 to PA-coated ELISA plates or silica beads (TRANSIL), and by spectroscopic assays employing fluorescently labeled PA liposomes. In addition to PA, phosphatidylinositol 3,4, 5-trisphosphate (PIP3), dipalmitoylphosphatidylglycerol, phosphatidylinositol 4,5-bisphosphate, and phosphatidylserine (PS) were found to bind to SHP-1, albeit to a lesser extent. A high-affinity binding site for PA and PIP3 was mapped to the 41 C-terminal amino acids of SHP-1. This site was absent from the related protein-tyrosine phosphatase SHP-2 and conferred activation of SHP-1 by PA toward two different substrates at low lipid concentrations. A SHP-1 mutant missing this binding site could, however, still be activated toward phosphorylated myelin basic protein as a substrate at high PA concentrations. This activation is likely to be mediated by a second, low-affinity binding site for PA in the N-terminal part of SHP-1 within the SH2 domains. High-affinity phospholipid binding to the C-terminus of SHP-1 may present a specific mechanism of regulating activity and/or cellular localization.

The previously demonstrated functional and physical interaction of the SH2 domain protein-tyrosine phosphatase SHP-1 with the epidermal growth factor (EGF) receptor (Tomic, S., Greiser, U., Lammers, R., Kharitonenkov, A., Imyanitov, E., Ullrich, A., and Bohmer, F. D. (1995) J. Biol. Chem. 270, 21277-21284) was investigated with respect to the involved structural elements of SHP-1. Various mutants of SHP-1 were transiently expressed in 293 or COS-7 cells and analyzed for their capacity to associate with immobilized autophosphorylated EGF receptor in vitro and to dephosphorylate coexpressed EGF receptor in intact cells. Inactivating point mutation of the C-terminal SH2 domain reduced the association weakly, point mutation of the N-terminal SH2 domain reduced association strongly and the respective double mutation abolished association totally. The capacity of SHP-1 to dephosphorylate coexpressed EGF receptor was impaired by all point mutations. Truncation of the N-terminal or of both SH2 domains strongly reduced or abolished association, respectively, but the truncated SHP-1 derivatives still dephosphorylated coexpressed EGF receptor effectively. Various chimeric protein-tyrosine phosphatases constructed from SHP-1 and the closely homologous SHP-2 dephosphorylated the EGF receptor when they contained the catalytic domain of SHP-1. As native SHP-2, the chimera lacked activity toward the receptor when they contained the catalytic domain of SHP-2, despite their capacity to associate with the receptor and to dephosphorylate an artificial phosphopeptide. We conclude that the differential interaction of SHP-1 and SHP-2 with the EGF receptor is due to the specificity of the respective catalytic domains rather than to the specificity of the SH2 domains. Functional interaction of native SHP-1 with the EGF receptor requires association mediated by both SH2 domains.