High affinity anti-TIM-3 and anti-KIR monoclonal antibodies cloned from healthy human individuals.

We report here the cloning of native high affinity anti-TIM-3 and anti-KIR IgG monoclonal antibodies (mAbs) from peripheral blood mononuclear cells (PBMC) of healthy human donors. The cells that express these mAbs are rare, present at a frequency of less than one per 105 memory B-cells. Using our proprietary multiplexed screening and cloning technology CellSpot™ we assessed the presence of memory B-cells reactive to foreign and endogenous disease-associated antigens within the same individual. When comparing the frequencies of antigen-specific memory B-cells analyzed in over 20 screening campaigns, we found a strong correlation of the presence of anti-TIM-3 memory B-cells with memory B-cells expressing mAbs against three disease-associated antigens: (i) bacterial DNABII proteins that are a marker for Gram negative and Gram positive bacterial infections, (ii) hemagglutinin (HA) of influenza virus and (iii) the extracellular domain of anaplastic lymphoma kinase (ALK). One of the native anti-KIR mAbs has similar characteristics as lirilumab, an anti-KIR mAb derived from immunization of humanized transgenic mice that is in ongoing clinical trials. It is interesting to speculate that these native anti-TIM-3 and anti-KIR antibodies may function as natural regulatory antibodies, analogous to the pharmacological use in cancer treatment of engineered antibodies against the same targets. Further characterization studies are needed to define the mechanisms through which these native antibodies may function in healthy and disease conditions.

Frequent alterations in the expression of serine/threonine kinases in human cancers.

Protein kinases constitute a large family of regulatory enzymes involved in the homeostasis of virtually every cellular process. Subversion of protein kinases has been frequently implicated in malignant transformation. Within the family, serine/threonine kinases (STK) have received comparatively lesser attention, vis-a-vis tyrosine kinases, in terms of their involvement in human cancers. Here, we report a large-scale screening of 125 STK, selected to represent all major subgroups within the subfamily, on nine different types of tumors ( approximately 200 patients), by using in situ hybridization on tissue microarrays. Twenty-one STK displayed altered levels of transcripts in tumors, frequently with a clear tumor type-specific dimension. We identified three patterns of alterations in tumors: (a) overexpression in the absence of expression in the normal tissues (10 kinases), (b) overexpression in the presence of expression by normal tissues (8 kinases), and (c) underexpression (3 kinases). Selected members of the three classes were subjected to in-depth analysis on larger case collections and showed significant correlations between their altered expression and biological and/or clinical variables. Our findings suggest that alteration in the expression of STK is a relatively frequent occurrence in human tumors. Among the overexpressed kinases, 10 were undetectable in normal controls and are therefore ideal candidates for further validation as potential targets of molecular cancer therapy.

PAK4 mediates morphological changes through the regulation of GEF-H1.

Precise spatial and temporal regulation of Rho GTPases is required in controlling F-actin-based changes in cell morphology. The molecular mechanisms through which microtubules (MTs) modulate the activity of RhoGTPases and regulate the actin cytoskeleton are unclear. Here we show that p21-activated-kinase 4 (PAK4) mediates morphological changes through its association with the Rho-family guanine nucleotide exchange factor (GEF), GEF-H1. We show that this association is dependent upon a novel GEF-H1 interaction domain (GID) within PAK4. Further, we show that PAK4-mediated phosphorylation of Ser810 acts as a switch to block GEF-H1-dependent stress fiber formation while promoting the formation of lamellipodia in NIH-3T3 cells. We found that the endogenous PAK4-GEF-H1 complex associates with MTs and that PAK4 phosphorylation of MT-bound GEF-H1 releases it into the cytoplasm of NIH-3T3 cells, which coincides with the dissolution of stress fibers. Our observations propose a novel role for PAK4 in GEF-H1-dependent crosstalk between MTs and the actin cytoskeleton.

Acquired expression of periostin by human breast cancers promotes tumor angiogenesis through up-regulation of vascular endothelial growth factor receptor 2 expression.

The late stages of human breast cancer development are poorly understood complex processes associated with the expression of genes by cancers that promote specific tumorigenic activities, such as angiogenesis. Here, we describe the identification of periostin as a mesenchyme-specific gene whose acquired expression by human breast cancers leads to a significant enhancement in tumor progression and angiogenesis. Undetectable in normal human breast tissues, periostin was found to be overexpressed by the vast majority of human primary breast cancers examined. Tumor cell lines engineered to overexpress periostin showed a phenotype of accelerated growth and angiogenesis as xenografts in immunocompromised animals. The underlying mechanism of periostin-mediated induction of angiogenesis was found to derive in part from the up-regulation of the vascular endothelial growth factor receptor Flk-1/KDR by endothelial cells through an integrin alpha(v)beta(3)-focal adhesion kinase-mediated signaling pathway. These findings demonstrate the presence of a novel mechanism by which tumor angiogenesis is acquired with the expression of a mesenchyme-specific gene as a crucial step in late stages of tumorigenesis.

The Ste20 kinase MST4 plays a role in prostate cancer progression.

MST4, a member of the Sterile 20 serine/threonine kinase family, was found to be expressed in prostate carcinoma tumor samples and cell lines. In addition, expression levels appeared to correlate with tumorigenicity and androgen receptor status of the cells. Ectopic expression of wild-type and kinase-inactive MST4 was used to alter cellular MST4 activity levels in three widely studied human prostate tumor cell lines: LNCaP, DU 145, and PC-3. Overexpression of wild-type MST4 induced anchorage-independent growth of the LNCaP cell line, and increased both in vitro proliferation and in vivo tumorigenesis of the DU 145 cell line. On the other hand, expression of a kinase-inactive form reverted the anchorage-independent growth phenotype and highly tumorigenic behavior of the PC-3 cell line. MST4 kinase activity was stimulated significantly by epidermal growth factor receptor ligands, which are known to promote growth of prostate cancer cells. Together, our studies suggest a potential role for MST4 in the signal transduction pathways involved in prostate cancer progression.

Stk10, a new member of the polo-like kinase kinase family highly expressed in hematopoietic tissue.

The Ste20 family of serine/threonine kinases plays an important role in numerous cellular functions such as growth, apoptosis, and morphogenesis. We have identified a previously cloned but uncharacterized family member termed Stk10, which is a human homolog of murine Lok, a serine/threonine kinase highly expressed in lymphocytes. Northern analysis demonstrated that the Stk10 transcript is present in many tissues, although highest expression levels are seen in hematopoietic cells. Due to close sequence homology to human Slk and Xenopus laevis xPlkk1, two polo-like kinase kinases, we investigated whether Stk10 might also play a role as a Plk1 activator. Plk1 has been shown to be overexpressed in multiple tumor types, thus attracting high interest to its potential upstream regulators. We show here that Stk10 can associate with Plk1 in cells and furthermore can phosphorylate Plk1 in vitro. Engineered NIH-3T3 cell lines that overexpress a dominant negative version of Stk10 display an altered cell cycle phenotype characterized by increased DNA content, raising the possibility that expression of a dominant negative Stk10 may impinge upon Plk1 function in vivo; it has previously been shown that unregulated expression of Plk1 can result in a variety of nuclear defects. We suggest, therefore, that Stk10 is a novel polo-like kinase kinase that cooperates with hSlk to regulate Plk1 function in human cells.

The lymphoid protein tyrosine phosphatase Lyp interacts with the adaptor molecule Grb2 and functions as a negative regulator of T-cell activation.

OBJECTIVE: Following activation of T cells, phosphorylation of tyrosine residues occurs through a complex signaling process involving protein tyrosine kinases, phosphatases, and a variety of adapter molecules including Grb2. We have attempted to identify new signaling molecules that are important for the activation response. METHODS: Using a protein interaction screening protocol based on phage display, T-cell signaling components that associate with the adapter molecule, Grb2, the lymphoid-specific tyrosine phosphatase Lyp was identified. Using transcriptional reporter assays, the role of Lyp in T-cell activation was studied by overexpression of wild-type or catalytically inactive mutants of Lyp. RESULTS: A GST fusion containing the C-terminal SH3 domain of Grb2 bound to the nucleotide exchange factor Sos or Grb2-associated binder 2 (Gab2). In contrast, the N-terminal SH3-containing fusion bound to the protein tyrosine phosphatase Lyp. Grb2 was co-immunoprecipitated with Lyp in 293T cells overexpressing both proteins. Using Northern blot analysis, Lyp was found to be expressed predominantly in hematopoietic tissue, including spleen, lymph node, thymus, peripheral blood leukocytes, bone marrow, and fetal liver. Two human T-cell lines, Jurkat and HuT78, expressed both Lyp mRNA and protein. Overexpression of wild-type Lyp or a catalytically inactive, substrate-trapping mutant (D195A) in Jurkat cells inhibited transcriptional activity initiated by anti-CD3 and anti-CD28 antibodies. In contrast, two other catalytically inactive mutants (R233M or C227S) had no effect. CONCLUSION: These data demonstrate a novel interaction between the phosphatase Lyp and the adaptor Grb2 and are consistent with a negative regulatory role for Lyp in T-cell signaling.