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.

Antagonism of Ang-Tie2 and Dll4-Notch signaling has opposing effects on tumor endothelial cell proliferation, evidenced by a new flow cytometry method.

Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietin-Tie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.

Adductor-related groin pain in recreational athletes: role of the adductor enthesis, magnetic resonance imaging, and entheseal pubic cleft injections.

BACKGROUND: Adductor dysfunction can cause groin pain in athletes and may emanate from the adductor enthesis. Adductor enthesopathy may be visualized with magnetic resonance imaging and may be treated with entheseal pubic cleft injections. We have previously reported that pubic cleft injections can provide predictable pain relief at one year in competitive athletes who have no evidence of enthesopathy on magnetic resonance imaging and immediate relief only in patients with findings of enthesopathy on magnetic resonance imaging. In this follow-up study, we attempted to determine if the same holds true for recreational athletes. METHODS: We reviewed a consecutive case series of twenty-eight recreational athletes who had presented to our sports medicine clinic with groin pain secondary to adductor longus dysfunction. A period of conservative treatment had failed for all of these athletes. The adductor longus origin was assessed with magnetic resonance imaging for the presence or absence of enthesopathy. All patients were treated with a single pubic cleft injection of a local anesthetic and corticosteroid into the adductor enthesis. The patients were assessed for recurrence of symptoms at one year after treatment. RESULTS: On clinical reassessment five minutes after the injection, all twenty-eight athletes reported resolution of the groin pain. Fifteen patients (Group 1) had no evidence of enthesopathy on magnetic resonance imaging, and thirteen patients (Group 2) had findings of enthesopathy on magnetic resonance imaging. At one year after the injection, five of the fifteen patients in Group 1 had experienced a recurrence; these recurrences were noted at a mean of fourteen weeks (range, seven to twenty weeks) after the injection. Four of the thirteen patients in Group 2 had experienced a recurrence of the symptoms at one year, and these recurrences were noted at a mean of eight weeks (range, two to nineteen weeks) after the injection. Overall, nineteen (68%) of the twenty-eight athletes had a good result following the injection. Of the remaining nine athletes, two were treated successfully with repeat injection; therefore, overall, twenty-one (75%) of the twenty-eight athletes had a good result after entheseal pubic cleft injection. CONCLUSIONS: Most recreational athletes with adductor enthesopathy have pain relief at one year after entheseal pubic cleft injection, regardless of the findings on magnetic resonance imaging. There were similarities between this group of recreational athletes and the competitive athletes in our previous study, in that the adductor enthesis was the source of pain and entheseal pubic cleft injection was a valuable treatment option. The main difference was that, in this group of recreational athletes, magnetic resonance imaging evidence of adductor enthesopathy did not correlate with the outcome of the injection.

Adductor-related groin pain in competitive athletes. Role of adductor enthesis, magnetic resonance imaging, and entheseal pubic cleft injections.

BACKGROUND: Adductor dysfunction is a condition that can cause groin pain in competitive athletes, but the source of the pain has not been established and no specific interventions have been evaluated. We previously defined a magnetic resonance imaging protocol to visualize adductor enthesopathy. The aim of this study was to elucidate, in the context of adductor-related groin pain in the competitive athlete, the role of the adductor enthesis (origin), the relevance of adductor enthesopathy diagnosed with magnetic resonance imaging, and the efficacy of entheseal pubic cleft injections of local anesthetic and steroids. METHODS: We reviewed the findings in a consecutive series of twenty-four competitive athletes who had presented to our sports medicine clinic with groin pain secondary to adductor longus dysfunction. Magnetic resonance imaging was performed to assess the adductor longus origin for the presence or absence of enthesopathy. Seven patients (Group 1) had no evidence of enthesopathy on magnetic resonance imaging, and seventeen patients (Group 2) had enthesopathy confirmed on magnetic resonance imaging. All patients were treated with a single pubic cleft injection of local anesthetic and steroid into the adductor enthesis. At one year after this treatment, the patients were assessed for recurrence of symptoms. RESULTS: On clinical reassessment five minutes after the injection, all twenty-four athletes reported resolution of the groin pain. At one year, none of the seven patients in Group 1 had experienced a recurrence. Sixteen of the seventeen patients in Group 2 had a recurrence of the symptoms (p < 0.001) at a mean of five weeks (range, one to sixteen weeks) after the injection. CONCLUSIONS: A single entheseal pubic cleft injection can be expected to afford at least one year of relief of adductor-related groin pain in a competitive athlete with normal findings on a magnetic resonance imaging scan; however, it should be employed only as a diagnostic test or short-term treatment for a competitive athlete with evidence of enthesopathy on magnetic resonance imaging.

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.

Ultrasound in Arthritis and Inflammation.

High resolution ultrasound (US) provides a versatile and sensitive method of demonstrating both soft-tissue and osseous changes in early arthritis and other inflammatory conditions. A brief outline is provided regarding the potential usage of US in the management of patients with musculoskeletal inflammatory disease.