Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells.

Background: The mitotic kinesin, KIF18A, is required for proliferation of cancer cells that exhibit chromosome instability (CIN), implicating it as a promising target for treatment of a subset of aggressive tumor types. Determining regions of the KIF18A protein to target for inhibition will be important for the design and optimization of effective small molecule inhibitors. Methods: In this study, we used cultured cell models to investigate the effects of mutating S284 within the alpha-4 helix of KIF18A, which was previously identified as a phosphorylated residue. Results: Mutations in S284 cause relocalization of KIF18A from the plus-ends of spindle microtubules to the spindle poles. Furthermore, KIF18A S284 mutants display loss of KIF18A function and fail to support proliferation in CIN tumor cells. Interestingly, similar effects on KIF18A localization and function were seen after treatment of CIN cells with KIF18A inhibitory compounds that are predicted to interact with residues within the alpha-4 helix. Conclusion: These data implicate the KIF18A alpha-4 helix as an effective target for inhibition and demonstrate that small molecules targeting KIF18A selectively limit CIN tumor cell proliferation and result in phenotypically similar effects on mitosis at the single cell level compared to genetic perturbations.

Identification of the KIF18A alpha-4 helix as a therapeutic target for chromosomally unstable tumor cells.

The mitotic kinesin, KIF18A, is required for proliferation of cancer cells that exhibit chromosome instability (CIN), implicating it as a promising target for treatment of a subset of aggressive tumor types. Determining regions of the KIF18A protein to target for inhibition will be important for the design and optimization of effective small molecule inhibitors. In this study, we investigated the effects of mutating S284 within the alpha-4 helix of KIF18A, which was previously identified as a phosphorylated residue. Mutations in S284 cause relocalization of KIF18A from the plus-ends of spindle microtubules to the spindle poles. Furthermore, KIF18A S284 mutants display loss of KIF18A function and fail to support proliferation in CIN tumor cells. Interestingly, similar effects on KIF18A localization and function were seen after treatment of CIN cells with KIF18A inhibitory compounds that are predicted to interact with residues within the alpha-4 helix. These data implicate the KIF18A alpha-4 helix as an effective target for inhibition and demonstrate that small molecules targeting KIF18A selectively limit CIN tumor cell proliferation and result in phenotypically similar effects on mitosis at the single cell level compared to genetic perturbations.

TGF-ß1 potentiates Vγ9Vδ2 T cell adoptive immunotherapy of cancer.

Despite its role in cancer surveillance, adoptive immunotherapy using γδ T cells has achieved limited efficacy. To enhance trafficking to bone marrow, circulating Vγ9Vδ2 T cells are expanded in serum-free medium containing TGF-ß1 and IL-2 (γδ[T2] cells) or medium containing IL-2 alone (γδ[2] cells, as the control). Unexpectedly, the yield and viability of γδ[T2] cells are also increased by TGF-ß1, when compared to γδ[2] controls. γδ[T2] cells are less differentiated and yet display increased cytolytic activity, cytokine release, and antitumor activity in several leukemic and solid tumor models. Efficacy is further enhanced by cancer cell sensitization using aminobisphosphonates or Ara-C. A number of contributory effects of TGF-ß are described, including prostaglandin E2 receptor downmodulation, TGF-ß insensitivity, and upregulated integrin activity. Biological relevance is supported by the identification of a favorable γδ[T2] signature in acute myeloid leukemia (AML). Given their enhanced therapeutic activity and compatibility with allogeneic use, γδ[T2] cells warrant evaluation in cancer immunotherapy.

Predicting CD4 T-cell epitopes based on antigen cleavage, MHCII presentation, and TCR recognition.

Accurate predictions of T-cell epitopes would be useful for designing vaccines, immunotherapies for cancer and autoimmune diseases, and improved protein therapies. The humoral immune response involves uptake of antigens by antigen presenting cells (APCs), APC processing and presentation of peptides on MHC class II (pMHCII), and T-cell receptor (TCR) recognition of pMHCII complexes. Most in silico methods predict only peptide-MHCII binding, resulting in significant over-prediction of CD4 T-cell epitopes. We present a method, ITCell, for prediction of T-cell epitopes within an input protein antigen sequence for given MHCII and TCR sequences. The method integrates information about three stages of the immune response pathway: antigen cleavage, MHCII presentation, and TCR recognition. First, antigen cleavage sites are predicted based on the cleavage profiles of cathepsins S, B, and H. Second, for each 12-mer peptide in the antigen sequence we predict whether it will bind to a given MHCII, based on the scores of modeled peptide-MHCII complexes. Third, we predict whether or not any of the top scoring peptide-MHCII complexes can bind to a given TCR, based on the scores of modeled ternary peptide-MHCII-TCR complexes and the distribution of predicted cleavage sites. Our benchmarks consist of epitope predictions generated by this algorithm, checked against 20 peptide-MHCII-TCR crystal structures, as well as epitope predictions for four peptide-MHCII-TCR complexes with known epitopes and TCR sequences but without crystal structures. ITCell successfully identified the correct epitopes as one of the 20 top scoring peptides for 22 of 24 benchmark cases. To validate the method using a clinically relevant application, we utilized five factor VIII-specific TCR sequences from hemophilia A subjects who developed an immune response to factor VIII replacement therapy. The known HLA-DR1-restricted factor VIII epitope was among the six top-scoring factor VIII peptides predicted by ITCall to bind HLA-DR1 and all five TCRs. Our integrative approach is more accurate than current single-stage epitope prediction algorithms applied to the same benchmarks. It is freely available as a web server (http://salilab.org/itcell).

HCV core expression in hepatocytes protects against autoimmune liver injury and promotes liver regeneration in mice.

Hepatitis C virus (HCV) infection causes acute and chronic liver disease often leading to liver cirrhosis and hepatocellular carcinoma. Numerous studies have shown that despite induction of virus specific immunity, a curative response is often not attained; this has led to the hypothesis that HCV genes modulate immunity, thereby enabling chronic infections. This study examined the effects on immune-mediated liver injury in transgenic mice expressing core protein throughout the body and bone marrow chimeras expressing core protein in either the lymphoid compartment or liver parenchyma. Presence of core protein in the liver parenchyma but not in lymphoid cells protects from autoimmune hepatitis induced by mitogen concanavalin A (ConA). Consistent with this observation, core transgenic hepatocytes are relatively resistant to death induced by anti-Fas antibody and tumor necrosis factor alpha (TNFalpha). This protective effect is associated with preferential activation of signal transducer and activation of transcription factor 3 (STAT3) versus STAT1 in livers of ConA-injected animals. In agreement with this effect of core protein on the Janus kinase (JAK)-STAT signaling pathway, transgenic mice accelerate liver regeneration after partial hepatectomy but are not protected from hepatocyte death. In conclusion, HCV core inhibits STAT1 and stimulates STAT3 activation, which protects infected hepatocytes from attack by the cell-mediated immune system and promotes their proliferation.

P2X7 receptor expression levels determine lethal effects of a purine based danger signal in T lymphocytes.

Contact of T lymphocytes with nicotinamide adenine dinucleotide (NAD) or ATP causes cell death that requires expression of purinergic receptor P2X(7) (P2X(7)R). T cell subsets differ in their responses to NAD and ATP, which awaits a mechanistic explanation. Here, we show that sensitivity to ATP correlates with P2X(7)R expression levels in CD4 cells, CD8 cells and CD4(+)CD25(+) cells from both C57BL/6 and BALB/c mice. But P2X(7)R ligands do not only induce cell death but also shedding of CD62L. It is shown here that in CD62L(high) T cells, CD62L shedding correlates with low expression of P2X(7)Rs and lower cell death, whereas in CD62L(low) cells P2X(7)R expression and death are higher. The possibility is therefore investigated that P2X(7)Rs induce T cell activation. Experiments show that spontaneous T cell proliferation is somewhat higher in cells expressing P2X(7)Rs, but this effect we suggest is caused by P2X(7)R expression on accessory cells.

High sensitivity of CD4+CD25+ regulatory T cells to extracellular metabolites nicotinamide adenine dinucleotide and ATP: a role for P2X7 receptors.

Although regulatory lymphocytes play an important role in the immune system, the regulation of their functions is poorly understood and remains to be elucidated. In this study we demonstrate that micromolar concentrations of the common cell metabolite NAD induce death in murine forkhead/winged helix transcription factor gene-expressing CD4+CD25+ regulatory T cells with high efficiency and within minutes. Similar, but less dramatic, effects are demonstrable with ATP and its nonhydrolysable derivative, benzoylbenzoyl-ATP. Other T cell subsets are more resistant, with CD8 cells being the least sensitive and CD4 cells expressing intermediate sensitivity. The higher sensitivity of CD4+CD25+ cells is demonstrable in vivo. Injection of NAD or benzoylbenzoyl-ATP causes preferential induction of a cell death signal in CD4+CD25+ cells. Transmission of the death signal requires functional P2X7 receptors, pointing to a role for these receptors in regulation and homeostasis of CD4+CD25+ regulatory T cells. Consistent with this, P2X7R gene-deleted mice possess increased levels of forkhead/winged helix transcription factor gene-expressing CD4+CD25+ cells.

P2X7 receptor-dependent and -independent T cell death is induced by nicotinamide adenine dinucleotide.

Adding NAD to murine T lymphocytes inhibits their functions and induces annexin V binding. This report shows that NAD induces cell death in a subset of T cells within seconds whereas others do not die until many hours later. Low NAD concentrations (<10 microM) suffice to trigger rapid cell death, which is associated with annexin V binding and membrane pore formation, is not blocked by the caspase inhibitor Z-VADfmk, and requires functional P2X7 receptors. The slower induction of death requires higher NAD concentrations (>100 microM), is blocked by caspase inhibitor Z-VADfmk, is associated with DNA fragmentation, and does not require P2X7 receptors. T cells degrade NAD to ADP-ribose (ADPR), and adding ADPR to T cells leads to slow but not rapid cell death. NAD but not ADPR provides the substrate for ADP-ribosyltransferase (ART-2)-mediated attachment of ADP-ribosyl groups to cell surface proteins; expression of ART-2 is required for NAD to trigger rapid but not slow cell death. These results support the hypothesis that cell surface ART-2 uses NAD but not ADPR to attach ADP-ribosyl groups to the cell surface, and that these groups act as ligands for P2X7 receptors that then induce rapid cell death. Adding either NAD or ADPR also triggers a different set of mechanisms, not requiring ART-2 or P2X7 receptors that more slowly induce cell death.