Christina L. Boulton

Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412

Constitutively activating FLT3 receptor mutations have been found in 35% of patients with acute myeloblastic leukemia (AML). Here we report the identification of a small molecule FLT3 tyrosine kinase inhibitor PKC412, which selectively induced G1 arrest and apoptosis of Ba/F3 cell lines expressing mutant FLT3 (IC(50) < 10 nM) by directly inhibiting the tyrosine kinase. Ba/F3-FLT3 cell lines made resistant to PKC412 demonstrated overexpression of mutant FLT3, confirming that FLT3 is the target of this drug. Finally, progressive leukemia was prevented in PKC412-treated Balb/c mice transplanted with marrow transduced with a FLT3-ITD-expressing retrovirus. PKC412 is a potent inhibitor of mutant FLT3 and is a candidate for testing as an antileukemia agent in AML patients with mutant FLT3 receptors.

CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML)

Up to 30% of acute myelogenous leukemia (AML) patients harbor an activating internal tandem duplication (ITD) within the juxtamembrane domain of the FLT3 receptor, suggesting that it may be a target for kinase inhibitor therapy. For this purpose we have developed CT53518, a potent antagonist that inhibits FLT3, platelet-derived growth factor receptor (PDGFR), and c-Kit (IC(50) approximately 200 nM), while other tyrosine or serine/threonine kinases were not significantly inhibited. In Ba/F3 cells expressing different FLT3-ITD mutants, CT53518 inhibited IL-3-independent cell growth and FLT3-ITD autophosphorylation with an IC(50) of 10-100 nM. In human FLT3-ITD-positive AML cell lines, CT53518 induced apoptosis and inhibited FLT3-ITD phosphorylation, cellular proliferation, and signaling through the MAP kinase and PI3 kinase pathways. Therapeutic efficacy of CT53518 was demonstrated both in a nude mouse model and in a murine bone marrow transplant model of FLT3-ITD-induced disease.

PML/RARα and FLT3-ITD induce an APL-like disease in a mouse model

Acute promyelocytic leukemia (APL) cells invariably express aberrant fusion proteins involving the retinoic acid receptor α (RARα). The most common fusion partner is promyelocytic leukemia protein (PML), which is fused to RARα in the balanced reciprocal chromosomal translocation, t(15;17)(q22:q11). Expression of PML/RARα from the cathepsin G promoter in transgenic mice causes a nonfatal myeloproliferative syndrome in all mice; about 15% go on to develop APL after a long latent period, suggesting that additional mutations are required for the development of APL. A candidate target gene for a second mutation is FLT3, because it is mutated in approximately 40% of human APL cases. Activating mutations in FLT3, including internal tandem duplication (ITD) in the juxtamembrane domain, transform hematopoietic cell lines to factor independent growth. FLT3-ITDs also induce a myeloproliferative disease in a murine bone marrow transplant model, but are not sufficient to cause AML. Here, we test the hypothesis that PML/RARα can cooperate with FLT3-ITD to induce an APL-like disease in the mouse. Retroviral transduction of FLT3-ITD into bone marrow cells obtained from PML/RARα transgenic mice results in a short latency APL-like disease with complete penetrance. This disease resembles the APL-like disease that occurs with long latency in the PML/RARα transgenics, suggesting that activating mutations in FLT3 can functionally substitute for the additional mutations that occur during mouse APL progression. The leukemia is transplantable to secondary recipients and is ATRA responsive. These observations document cooperation between PML/RARα and FLT3-ITD in development of the murine APL phenotype.

PKC412 overcomes resistance to imatinib in a murine model of FIP1L1-PDGFRα-induced myeloproliferative disease.

FIP1L1-PDGFRalpha causes hypereosinophilic syndrome (HES) and is inhibited by the tyrosine kinase inhibitor imatinib (Gleevec). Imatinib is a potent inhibitor of ABL, ARG, PDGFRalpha, PDGFRbeta, and KIT and induces durable hematologic responses in HES patients. However, we observed relapse with resistance to imatinib as consequence of a T674I mutation in FIP1L1-PDGFRalpha, analogous to the imatinib-resistant T315I mutation in BCR-ABL. We developed a murine bone marrow transplant model of FIP1L1-PDGFRalpha-induced myeloproliferative disease to evaluate the efficacy of PKC412, an alternative inhibitor of PDGFRalpha, for the treatment of HES. PKC412 is effective for treatment of FIP1L1-PDGFRalpha-induced disease and of imatinib-induced resistance due to the T674I mutation. Our data establish PKC412 as molecularly targeted therapy for HES and other diseases expressing activated PDGFRalpha and demonstrate the potential of alternative kinase inhibitors to overcome resistance in target tyrosine kinases.

FLT3 internal tandem duplication mutations induce myeloproliferative or lymphoid disease in a transgenic mouse model.

Activating FMS-like tyrosine kinase 3 (FLT3) mutations have been identified in ∼30% of patients with acute myelogenous leukemia (AML), and recently in a smaller subset of patients with acute lymphoblastic leukemia (ALL). To explore the in vivo consequences of an activating FLT3 internal tandem duplication mutation (FLT3-ITD), we created a transgenic mouse model in which FLT3-ITD was expressed under the control of the vav hematopoietic promoter. Five independent lines of vav-FLT3-ITD transgenic mice developed a myeloproliferative disease with high penetrance and a disease latency of 6–12 months. The phenotype was characterized by splenomegaly, megakaryocytic hyperplasia, and marked thrombocythemia, but without leukocytosis, polycythemia, or marrow fibrosis, displaying features reminiscent of the human disease essential thrombocythemia (ET). Clonal immature B- or T-lymphoid disease was observed in two additional founder mice, respectively, that could be secondarily transplanted to recipient mice that rapidly developed lymphoid disease. Treatment of these mice with the FLT3 tyrosine kinase inhibitor, PKC412, resulted in suppression of disease and a statistically significant prolongation of survival. These results demonstrate that FLT3-ITD is capable of inducing myeloproliferative as well as lymphoid disease, and indicate that small-molecule tyrosine kinase inhibitors may be an effective treatment for lymphoid malignancies in humans that are associated with activating mutations in FLT3.

Predictive factors of distal femoral fracture nonunion after lateral locked plating: a retrospective multicenter case-control study of 283 fractures.

INTRODUCTION Reported initial success rates after lateral locked plating (LLP) of distal femur fractures have led to more concerning outcomes with reported nonunion rates now ranging from 0 to 21%. Reported factors associated with nonunion include comorbidities such as obesity, age and diabetes. In this study, our goal was to identify patient comorbidities, injury and construct characteristics that are independent predictors of nonunion risk in LLP of distal femur fractures; and to develop a predictive algorithm of nonunion risk, irrespective of institutional criteria for clinical intervention variability. PATIENTS AND METHODS A retrospective review of 283 distal femoral fractures in 278 consecutive patients treated with LLP at three Level1 academic trauma centers. Nonunion was liberally defined as need for secondary procedure to manage poor healing based on unrestricted surgeon criteria. Patient demographics (age, gender), comorbidities (obesity, smoking, diabetes, chronic steroid use, dialysis), injury characteristics (AO type, periprosthetic fracture, open fracture, infection), and management factors (institution, reason for intervention, time to intervention, plate length, screw density, and plate material) were obtained for all participants. Multivariable analysis was performed using logistic regression to control for confounding in order to identify independent risk factors for nonunion. RESULTS 28 of the 283 fractures were treated for nonunion, 13 were referred to us from other institutions. Obesity (BMI>30), open fracture, occurrence of infection, and use of stainless steel plate were significant independent risk factors (P<0.01). A predictive algorithm demonstrates that when none of these variables are present (titanium instead of stainless steel) the risk of nonunion requiring intervention is 4%, but increases to 96% with all factors present. When a stainless plate is used, obesity alone carries a risk of 44% while infection alone a risk of 66%. While Chi-square testing suggested no institutional differences in nonunion rates, the time to intervention for nonunion varied inversely with nonunion rates between institutions, indicating varying trends in management approach. DISCUSSION Obesity, open fracture, occurrence of infection, and the use of stainless steel are prognostic risk factors of nonunion in distal femoral fractures treated with LLP independent of differing trends in how surgeons intervene in the management of nonunion.

A Large-Scale Screen for Mutagen-Sensitive Loci in Drosophila

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212–mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314–mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215ZIII-2059, that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.

Intramedullary Cannulated Headless Screw Fixation of a Comminuted Subcapital Metacarpal Fracture: Case Report

This case report describes an alternative technique for the fixation of displaced comminuted subcapital fractures of the metacarpal with limited distal bone stock. Using a cannulated headless screw as an intramedullary device placed through the articular surface, we were able to secure proximal and distal bone purchase without excessive soft tissue stripping or disruption of the fracture hematoma. This technique allows early rehabilitation, and our patient went on to uneventful healing with excellent functional results.

Internal anterior fixators for pelvic ring injuries: Do monaxial pedicle screws provide more stiffness than polyaxial pedicle screws?

OBJECTIVES Little is known about the mechanical properties of internal anterior fixators (known as INFIX), which have been proposed as subcutaneous alternatives to traditional anterior external fixators for pelvic ring disruptions. We hypothesised that INFIX has superior biomechanical performance compared with traditional external fixators because the distance from the bar to the bone is reduced. METHODS Using a commercially available synthetic bone model, 15 unstable pelvic ring injuries were simulated by excising the pubic bone through the bilateral superior and inferior rami anteriorly and the sacrum through the bilateral sacral foramen posteriorly. Three test groups were established: (1) traditional supra-acetabular external fixation, (2) INFIX with polyaxial screws, (3) INFIX with monaxial screws. Load was applied, simulating lateral compression force. Outcome measure was construct stiffness. RESULTS The traditional external fixator constructs had an average stiffness of 6.21 N/mm ± 0.40 standard deviation (SD). INFIX with monaxial screws was 23% stiffer than the traditional external fixator (mean stiffness, 7.66 N/mm ± 0.86 SD; p = .01). INFIX with polyaxial screws was 26% less stiff than INFIX with monaxial screws (mean stiffness, 5.69 N/mm ± 1.24 SD; p = .05). No significant difference was noted between polyaxial INFIX and external fixators (mean stiffness, 6.21 N/mm ± 0.40 SD; p=.65). CONCLUSIONS The performance of INFIX depends on the type of screw used, with monaxial screws providing significantly more stiffness than polyaxial screws. Despite the mechanical advantage of being closer to the bone, polyaxial INFIX was not stiffer than traditional external fixation.

Do Locking Screws Work in Plates Bent at Holes

Objectives: To assess whether plate bending at a hole significantly changes the biomechanical properties of a locked screw. Methods: Coronal plane bends of 5-, 15-, or 45-degree angles were placed in 3.5-mm locking compression plates with the apex at a locking hole. An additional 45-degree angle test group was created in which a threaded screw head insert was placed before bending. Ten plates were tested in each group and compared with nonbent controls in a stepwise cyclic loading protocol. Results: Statistically significant differences in protocol survival were shown between the control group and the 15-degree angle (P = 0.006) and 45-degree angle (P = 0.0007) groups. An apparent decrease in protocol survival in the 5-degree angle group did not reach statistical significance (P = 0.17). The average number of cycles survived was significantly different between the control group and the 15-degree angle (P = 0.027) and 45-degree angle (P = 0.0002) groups. The mean cycles to failure for the 5-degree angle group was 16% lower than for controls but did not reach statistical significance (P = 0.37). The test group bent to an angle of 45 degrees after placement of a threaded screw head insert showed no difference in protocol survival or in mean number of cycles survived compared with the regular 45-degree angle group. Conclusion: Bending of a 3.5-mm locking compression plate by more than 5 degrees at a locking hole results in a statistically significant decrease in survival of the corresponding locked screw. This effect cannot be prevented by the placement of a threaded screw head insert before bending.