Dual Targeting of Aurora Kinases with AMG 900 Exhibits Potent Preclinical Activity Against Acute Myeloid Leukemia with Distinct Post-Mitotic Outcomes.

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B-selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein-expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288-11 cells and mouse Jak2 V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic antimitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3'-deoxy-3'-18F-fluorothymidine [18F]FLT positron emission tomographic (PET)-CT imaging to measure the antiproliferative effects of AMG 900 in skeletal tissues in mice.

Involvement of alpha4 integrins in maintenance of cardiac sympathetic axons.

Sympathetic neurons extend and maintain axons that innervate the myocardium, and proper innervation is important for cardiac function. However, the molecular basis for axon outgrowth and maintenance is not well understood. We have shown previously that the integrin alpha4beta1 is expressed on developing axons, and the alpha4 function is important for the development of innervation in vivo [Wingerd, K.L., Goodman, N.L., Tresser, J.W., Smail, M.M., Leu, S.T., Rohan, S.J., Pring, J.L., Jackson, D.Y., and Clegg, D.O., 2002. Alpha 4 integrins and vascular cell adhesion molecule-1 play a role in sympathetic innervation of the heart. J. Neurosci. 22,10772-10780]. Here we examine the function of alpha4beta1 integrins in the maintenance of cardiac sympathetic innervation in vitro and in vivo, and investigate integrin expression and function after myocardial infarction and in hypertensive rats. On substrates of vascular cell adhesion molecule-1 (VCAM-1), alpha4beta1 was required for both initial outgrowth and maintenance of neurites in vitro. On fibronectin substrates, initial outgrowth requires only alpha4 integrins, but maintenance requires both alpha4 integrins and RGD-dependent integrins. In vivo, in adult Long Evans rats, inhibition of alpha4 integrins resulted in decreased maintenance of sympathetic fibers innervating the apex of the heart. However, alpha4 integrins were not detected on most sympathetic axons that sprout after myocardial infarction, and alpha4 function was not required for sprouting. Spontaneously hypertensive rats (SHR) have increased numbers of cardiac sympathetic fibers compared to the parental Wistar strain, but many of these lack alpha4 expression, and alpha4 function is not required for maintenance of these fibers in the heart. These results suggest that developing sympathetic axons and sprouting sympathetic axons use different mechanisms of outgrowth, and that maintenance of cardiac sympathetic innervation involves alpha4 integrins in some rat strains.

Integrin alpha4beta1 (VLA-4) expression and activity in retinal and peripheral neurons.

The integrin alpha4beta1 fulfills important roles in inflammation and hematopoesis, but its functions in neurons are not well understood. Here we show that the alpha4 subunit is expressed on mouse retinal ganglion cells (RGCs) and undifferentiated retinal neuroblasts during the period of axon extension and migration. To determine if alpha4 integrins expressed by retinal neurons were active, neurons were cultured on known alpha4 ligands in vitro. Recombinant soluble vascular cell adhesion molecule 1 (rsVCAM-1), fibronectin, and osteopontin (OPN) induced neurite outgrowth that was diminished by function blocking antibodies specific for alpha4. Neurite outgrowth on OPN was also blocked by antibodies to the integrin beta1 subunit, implicating the alpha4beta1 heterodimer as one integrin receptor mediating outgrowth on OPN. OPN immunoreactivity was detected in the RGC fiber layer and optic nerve, suggesting that it may act as an alpha4 ligand in vivo. Neurons from chick lumbar sympathetic ganglia, chick dorsal root ganglia, and mouse superior cervical ganglia also extended neurites on rsVCAM-1, suggesting that integrin alpha4beta1 may play a role in the development of multiple neuronal cell types.