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XLS file 3927K, STK33 enzymatic activity and cellular activities for molecules a-l from Figure 6E

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XLS file - 175K, Effects of 515 small molecules on STK33 enzymatic activity vs. phosphorylation of RPS6 in cell lines

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Objective Antibodies to human granulocyte colony-stimulating factor receptor (HuG-CSFR) are widely available and have been used in numerous studies to evaluate the expression of this protein on normal and malignant cells of hematopoietic and nonhematopoietic origin. Spurred by recent studies that demonstrated that two commonly used antibodies against the erythropoietin and thrombopoietin receptors can in fact bind to completely unrelated and more broadly expressed proteins, we screened 27 commercially available monoclonal and polyclonal antibodies with claimed specificity to HuG-CSFR to determine if they are specific to this receptor. Materials and Methods Antibodies were evaluated by Western blotting, flow cytometry, and immunohistochemistry using 293T cells engineered to overexpress HuG-CSFR protein, immortalized human hematopoietic cell lines expressing endogenous G-CSFR, and purified human neutrophils. Results Only two monoclonal antibodies and one polyclonal antibody could be employed using defined Western blotting or flow cytometry protocols to detect G-CSFR protein in cell lysates or on the surface of cells that express G-CSFR messenger RNA with no binding to cells that did not express the gene. None of the antibodies were suitable for immunohistochemistry. Competitive inhibition with soluble G-CSFR extracellular domain and small inhibitory RNA-mediated knock-down of G-CSFR messenger RNA further demonstrated the limited specificity of these antibodies for HuG-CSFR expressed on the cell surface. Conclusions Most commercially available anti−HuG-CSFR antibodies do not bind specifically to this protein. These studies highlight the need for investigators to validate antibodies in their own systems to avoid the inadvertent use of nonspecifically binding antibodies that could lead, as exemplified in this case with a hematopoietic growth factor receptor, to erroneous conclusions about protein expression.

Yeast and human Eme1 protein, in complex with Mus81, constitute an endonuclease that cleaves branched DNA structures, especially those arising during stalled DNA replication. We identified mouse Eme1, and show that it interacts with Mus81 to form a complex that preferentially cleaves 3′‐flap structures and replication forks rather than Holliday junctions in vitro . We demonstrate that Eme1 −/− embryonic stem (ES) cells are hypersensitive to the DNA cross‐linking agents mitomycin C and cisplatin, but only mildly sensitive to ionizing radiation, UV radiation and hydroxyurea treatment. Mammalian Eme1 is not required for the resolution of DNA intermediates that arise during homologous recombination processes such as gene targeting, gene conversion and sister chromatid exchange (SCE). Unlike Blm‐deficient ES cells, increased SCE was seen only following induced DNA damage in Eme1‐deficient cells. Most importantly, Eme1 deficiency led to spontaneous genomic instability. These results reveal that mammalian Eme1 plays a key role in DNA repair and the maintenance of genome integrity.

The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS null allele ( WAS − / − ). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44 − CD25 + to the CD44 − CD25 − stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-ζ, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH 2 -terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS − / − cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS − / − lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS − / − neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.

Mutation in the gene encoding the recently isolated WASP protein has now been identified as the genetic defect responsible for the X-linked Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency disease associated with extensive phenotypic variability. To elucidate the range of WASP mutations responsible for WAS, we used PCR-SSCP analysis to screen for WASP gene mutation in 19 unrelated boys with the diagnosis of classical or attenuated WAS or isolated thrombocytopenia. All 19 patients had WASP mutations, each of which localized to the initial three or terminal three exons of the gene, and the majority of which were unique in each case. However, a missense mutation which results in substitution of the arginine at WAS codon 86 was identified in three boys with severe WAS as well as in one boy presenting with thrombocytopenia alone. While the three mutations found in the isolated thrombocytopenia patients leave the reading frame intact, about one-half of the gene alterations detected in both severe and attenuated WAS patients result in frameshifted transcript and premature translation termination. These findings therefore confirm the association of WAS with WASP mutation and identify WASP mutation as a cause for isolated congenital thrombocytopenia in males. While the WASP gene defects responsible for isolated thrombocytopenia and other mild presentations of WAS do not appear distinct from those resulting in severe WAS, these data indicate that analysis of WASP gene mutation provides a valuable tool for distinguishing the spectrum of WAS patients and the subset of males with isolated thrombocytopenia who represent mild cases of WAS.

The role of BRCA1 in breast and ovarian tumor suppression has been primarily ascribed to the maintenance of genome integrity. BRCA1 interacts with components of the non-homologous end-joining pathway previously shown to play a role in telomere maintenance in yeast. Here, we provide evidence that links Brca1 with telomere integrity. Brca1(-/-) T-cells display telomere dysfunction in both loss of telomere repeats as well as defective telomere capping. Loss of Brca1 synergizes with p53 deficiency in the onset and frequency of tumorigenesis. Karyotyping of tBrca1(-/-)p53(-/-) thymic lymphomas revealed the presence of telomere dysfunction accompanied by clonal chromosomal translocations. The telomere dysfunction phenotype in Brca1-deficient cells suggests that loss of telomere integrity might contribute to chromosome end dysfunction and permit the formation of potentially oncogenic translocations.

The Drosophila melanogaster warts/lats tumour suppressor has two mammalian counterparts LATS1/Warts-1 and LATS2/Kpm. Here, we show that mammalian Lats orthologues exhibit distinct expression profiles according to germ cell layer origin. Lats2(-/-) embryos show overgrowth in restricted tissues of mesodermal lineage; however, lethality ultimately ensues on or before embryonic day 12.5 preceded by defective proliferation. Lats2(-/-) mouse embryonic fibroblasts (MEFs) acquire growth advantages and display a profound defect in contact inhibition of growth, yet exhibit defective cytokinesis. Lats2(-/-) embryos and MEFs display centrosome amplification and genomic instability. Lats2 localizes to centrosomes and overexpression of Lats2 suppresses centrosome overduplication induced in wild-type MEFs and reverses centrosome amplification inherent in Lats2(-/-) MEFs. These findings indicate an essential role of Lats2 in the integrity of processes that govern centrosome duplication, maintenance of mitotic fidelity and genomic stability.

Defects in death receptor-mediated apoptosis have been linked to cancer and autoimmune disease in humans. The in vivo role of caspase 8, a component of this pathway, has eluded analysis in postnatal tissues because of the lack of an appropriate animal model. Targeted disruption ofcaspase 8is lethal in utero. We generated mice with a targetedcaspase 8mutation that is restricted to the T-cell lineage. Despite normal thymocyte development in the absence of caspase 8, we observed a marked decrease in the number of peripheral T-cells and impaired T-cell response ex vivo to activation stimuli.caspase 8ablation protected thymocytes and activated T-cells from CD95 ligand but not anti-CD3-induced apoptosis, or apoptosis activated by agents that are known to act through the mitochondria.caspase 8mutant mice were unable to mount an immune response to viral infection, indicating that caspase 8 deletion in T-cells leads to immunodeficiency. These findings identify an essential, cell-stage-specific role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. This is consistent with the recent identification of caspase 8 mutations in human immunodeficiency.

Brca1 (breast cancer1, early onset) deficiency results in early embryonic lethality. As Brca1 is highly expressed in the T cell lineage, a T cell–specific disruption of Brca1 was generated to assess the role of Brca1 in relation to T lymphocyte development. We found that thymocyte development in Brca1−/− mice was impaired not as a result of V(D)J T cell receptor (TCR) recombination but because thymocytes had increased expression of tumor protein p53. Chromosomal damage accumulation and abnormal cell death were observed in mutant cells. We found that cell death inhibitor Bcl-2 overexpression, or p53−/− backgrounds, completely restored survival and development of Brca1−/− thymocytes; peripheral T cell numbers were not totally restored in Brca1−/− p53−/− mice; and that a mutant background for p21 (cyclin-dependent kinase inhibitor 1A) did not restore Brca1−/− thymocyte development, but partially restored peripheral T cell development. Thus, the outcome of Brca1 deficiency was dependent on cellular context, with the major defects being increased apoptosis in thymocytes, and defective proliferation in peripheral T cells.

The Wiskott-Aldrich syndrome (WAS), an X-linked immunodeficiency disease caused by mutation in the recently isolated gene encoding WAS protein (WASP), is known to be associated with extensive clinical heterogeneity. Cumulative mutation data have revealed that WASP genotypes are also highly variable among WAS patients, but the relationship of phenotype with genotype in this disease remains unclear. To address this issue we characterized WASP mutations in 24 unrelated WAS patients, including 18 boys with severe classical WAS and 6 boys expressing mild forms of the disease, and then examined the degree of correlation of these as well as all previously published WASP mutations with disease severity. By analysis of these compiled mutation data, we demonstrated clustering of WASP mutations within the four most N-terminal exons of the gene and also identified several sites within this region as hotspots for WASP mutation. These characteristics were observed, however, in both severe and mild cases of the disease. Similarly, while the cumulative data revealed a predominance of missense mutations among the WASP gene lesions observed in boys with isolated thrombocytopenia, missense mutations were not exclusively associated with milder WAS phenotypes, but also comprised a substantial portion (38%) of the WASP gene defects found in patients with severe disease. These findings, as well as the detection of identical WASP mutations in patients with disparate phenotypes, reveal a lack of phenotype concordance with genotype in WAS and thus imply that phenotypic outcome in this disease cannot be reliably predicted solely on the basis of WASP genotypes.

Tumors harboring KRAS mutations remain an elusive target for oncology therapeutics. A recent publication (Scholl et al 2009) described a high throughput cellular RNAi screen which suggested a synthetic lethal relationship between STK33 and mutant KRAS. STK33 is a member of the calcium/calmodulin kinase family with a poorly characterized function. Although no genetic alterations in the STK33 gene have been reported in cancer, inhibiting STK33 kinase activity could offer a novel therapeutic approach to target mutant KRAS tumors. Thus, we sought to validate whether the survival of cancer cell lines with mutant KRAS is dependent on STK33, in parallel with a program to identify small molecule inhibitors of STK33 (Zhang Y et al AACR 2011). We focused on mutant KRAS leukemia cells because of their reported dependence on STK33. Using a panel of siRNA, 50-80% knockdown of STK33 at the RNA and protein level was achieved in NOMO-1KRASG13D and SKM-1KRASK117N cells. However we did not observe any alteration in cell viability. In addition, putative STK33 downstream signaling, (phosphorylation of p70 S6K thr389 and RPS6 ser235/236) was unaltered by modulation of STK33 expression. In contrast, knockdown of KRAS caused a significant reduction in both viability and signaling in these cell lines, confirming their dependence on KRAS. A panel of 27 cancer cell lines was screened with an siRNA library representing 1500 druggable genes. STK33 siRNA had no significant effect on viability regardless of KRAS mutational status. As expected, knockdown of KRAS significantly reduced cell viability, especially in mutant KRAS cells. A kinase dead mutant STK33 was used to test the dependence on STK33 kinase activity in mutant KRAS cell lines. Transient over-expression of kinase dead STK33 in PANC-1KRASG12D and DLD-1KRASG13D cells had no effect on viability, nor on the phosphorylation of p70 S6K thr389 and RPS6 ser235/236. In summary we have been unable to confirm a synthetic lethal relationship between STK33 and KRAS. Our data do not support inhibition of STK33 as a promising therapeutic approach for targeting mutant KRAS tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2011-252