Synthetic Antibodies Detect Distinct Cellular States of Chromosome Passenger Complex Proteins.

High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An Immunoprecipitation/Mass Spectrometry analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.

Direct Phosphorylation and Stabilization of MYC by Aurora B Kinase Promote T-cell Leukemogenesis.

Deregulation of MYC plays an essential role in T cell acute lymphoblastic leukemia (T-ALL), yet the mechanisms underlying its deregulation remain elusive. Herein, we identify a molecular mechanism responsible for reciprocal activation between Aurora B kinase (AURKB) and MYC. AURKB directly phosphorylates MYC at serine 67, counteracting GSK3ß-directed threonine 58 phosphorylation and subsequent FBXW7-mediated proteasomal degradation. Stabilized MYC, in concert with T cell acute lymphoblastic leukemia 1 (TAL1), directly activates AURKB transcription, constituting a positive feedforward loop that reinforces MYC-regulated oncogenic programs. Therefore, inhibitors of AURKB induce prominent MYC degradation concomitant with robust leukemia cell death. These findings reveal an AURKB-MYC regulatory circuit that underlies T cell leukemogenesis, and provide a rationale for therapeutic targeting of oncogenic MYC via AURKB inhibition.

Salivary glands require Aurora Kinase B for regeneration after transient innate immune-mediated injury.

Severe, irreversible salivary gland disease and oral dryness is experienced by sufferers of Sjögren's syndrome and those treated with irradiation for head and neck cancer. Therefore, major efforts have been made in the last decade to unravel key molecular signals that can drive salivary gland (SG) regeneration and functional restoration. However, the earliest molecular determinants that accompany SG regeneration remain incompletely defined. The present study examined the initial mitogenic events marking the regenerative response of the murine submandibular gland (SMG), following innate immune-mediated injury. Local intraductal administration of the synthetic double stranded (ds) RNA polyinosinic-polycytidylic acid (poly (I:C)) widely, but transiently, depleted the acinar and progenitor cells, 24 hours post poly (I:C) introduction. While the progenitor and duct cells started to proliferate and expand at 72 hours, the Mist1-positve acinar cells did not re-appear until 96 hours post poly (I:C) injury. The cellular replenishment during regeneration involved significant upregulation of the cell cycle promoter Aurora kinase B (AURKB). AURKB, which is expressed in healthy proliferating and cancerous cells, is a serine/threonine protein kinase, well known to orchestrate key events in cell division and cytokinesis. However, the expression and role of AURKB in regeneration of post mitotic salivary gland cells has not been previously explored. In vivo inhibition of AURKB using the selective inhibitor Barasertib (AZD1152-HQPA) interfered with SMG recovery from the transient, but severe poly (I:C)-mediated injury and cellular depletion. AURKB deficiency during regeneration of the injured tissues: disrupted cell cycle progression, repressed renewal of Mist1-positive acinar cells and prevented recovery of salivary secretion. The knowledge gained in this study may be utilized in the development of therapeutic targets for irreversible salivary gland disease.

Suppression of cell division-associated genes by Helicobacter pylori attenuates proliferation of RAW264.7 monocytic macrophage cells.

Helicobacter pylori at multiplicity of infection (MOI ≥ 50) have been shown to cause apoptosis in RAW264.7 monocytic macrophage cells. Because chronic gastric infection by H. pylori results in the persistence of macrophages in the host's gut, it is likely that H. pylori is present at low to moderate, rather than high numbers in the infected host. At present, the effect of low-MOI H. pylori infection on macrophage has not been fully elucidated. In this study, we investigated the genome-wide transcriptional regulation of H. pylori-infected RAW264.7 cells at MOI 1, 5 and 10 in the absence of cellular apoptosis. Microarray data revealed up- and down-regulation of 1341 and 1591 genes, respectively. The expression of genes encoding for DNA replication and cell cycle-associated molecules, including Aurora-B kinase (AurkB) were down-regulated. Immunoblot analysis verified the decreased expression of AurkB and downstream phosphorylation of Cdk1 caused by H. pylori infection. Consistently, we observed that H. pylori infection inhibited cell proliferation and progression through the G1/S and G2/M checkpoints. In summary, we suggest that H. pylori disrupts expression of cell cycle-associated genes, thereby impeding proliferation of RAW264.7 cells, and such disruption may be an immunoevasive strategy utilized by H. pylori.

Specific immune responses against epitopes derived from Aurora kinase A and B in acute myeloid leukemia.

Aurora kinases are serine/threonine kinases which play an important role in the process of mitosis and cell cycle regulation. Aurora kinase inhibitors are described to sensitize malignant cells to cytosine arabinoside and specific antibodies by mediating apoptosis. Aurora kinases are overexpressed in most acute leukemias but also in solid tumors. In this study we investigated whether epitopes derived from Aurora kinase A and B are able to elicit cellular immune responses in patients with acute myeloid leukemia (AML) to investigate their role as potential targets for specific immunotherapy. Samples of eight patients with AML were analyzed in enzyme-linked immunosorbent spot (ELISpot) assays and compared with immune responses of nine healthy volunteers (HVs). Specific CD8 + T cell responses were detected against the epitopes Aura A1, A2, B1, B2, B3, B4 and B5. Immune responses for epitopes derived from Aura B were induced more frequently compared to Aura A. The antigens with the most frequent cytotoxic T-lymphocyte (CTL) responses were Aura B3, B4 and B5, although the number of patients tested for these antigens was low. Aura B5 did not elicit specific CTL responses in HVs. For epitope Aura B6 no immune response was detected in HVs or patients. Taken together, with the combination of Aurora kinase inhibitors and an immunotherapeutic approach, an effective blast and minimal residual disease elimination might be achieved.