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CXCR4 antagonists sensitize FLT3/ITD

Background The hypothesis of the Developmental Origins of Health and Disease states that environmental factors during fetal and infantile life are risk factors for some chronic diseases in adulthood. Few studies, however, have confirmed this hypothesis early in childhood. Therefore, we assessed how premature birth and low‐birthweight (LBW) affect the renal function of Japanese children. Methods This retrospective study surveyed 168 patients who were born before 35 weeks of gestation and were cared for at the present neonatal intensive care unit. Follow‐up duration was >2 years. Serum creatinine (sCr) and estimated glomerular filtration rate (eGFR) recorded in medical records were reviewed. Results The eGFR at 2 years of age was significantly correlated with birthweight and gestational age (P

Laryngeal web is a rare congenital or acquired disease that results in airway stenosis. Depending on the severity of atresia, patients with laryngeal web show a wide variety of symptoms ranging from asymptomatic to life-threatening respiratory dysfunction that may require emergency tracheostomy immediately after birth. We report a neonatal case of laryngeal web with 22q11.2 deletion syndrome. Post-delivery, the infant showed dysphonia and had a ventricular septal defect with characteristic craniofacial features. The infant underwent an endoscopic incision of the web and cardiac surgery. Among patients with laryngeal web, 30% have 22q11.2 deletion syndrome. 22q11.2 deletion syndrome is the most common chromosomal microdeletion syndrome and the second most common chromosomal abnormality associated with congenital heart disease. Therefore, if an infant has laryngeal web with comorbidities such as congenital heart disease, 22q11.2 deletion syndrome should be considered in differential diagnosis.

Background Urinary tract infections caused by extended-spectrum beta-lactamase-producing bacteria are increasing worldwide. At our hospital, the number of pediatric patients hospitalized because of an upper urinary tract infection has dramatically increased since 2016. In total, 60.5% of urinary tract infections are caused by extended-spectrum beta-lactamase-producing Escherichia coli. Such a high prevalence of extended-spectrum beta-lactamase-producing E. coli has not been detected previously in Japan. Therefore, we evaluated the clinical and bacteriologic characteristics and efficacy of antibiotics against upper urinary tract infections caused by E. coli in children. Methods This retrospective study surveyed 152 patients who were hospitalized in the pediatric department of Shimane Prefectural Central Hospital because of upper urinary tract infections caused by E. coli. Medical records were reviewed to examine patient characteristics. O antigens, antibiotic susceptibility, gene typing, and pulse-field gel electrophoresis were studied at the Shimane Prefectural Institute of Public Health and Environmental Science. Results Urine sample analyses showed extended-spectrum beta-lactamase types such as CTX-M-9 and plural virulence genes. We changed the primary antibiotic treatment to flomoxef or cefmetazole to treat upper urinary tract infections caused by Gram-negative bacilli. After changing treatment, the time to fever alleviation was significantly shortened. Conclusion Extended-spectrum beta-lactamase-producing E. coli should be suspected in community-acquired upper urinary tract infections. Therefore, when treating patients, it is necessary to focus on antibiotic susceptibility and the prevalence of extended-spectrum beta-lactamase-producing bacteria found in each area. Flomoxef and cefmetazole are useful primary treatments for upper urinary tract infections caused by extended-spectrum beta-lactamase-producing E. coli.

Background Invasive pneumococcal disease (IPD) is defined by the detection of Streptococcus pneumoniae on culture from samples obtained from a normally sterile site. Pneumococcal conjugate vaccines (PCV) have been developed for the prevention of IPD that is caused by highly virulent serotypes. Despite the effective reduction of IPD caused by vaccine serotypes after the introduction of PCV, there has been a rapid increase in the incidence of IPD caused by non-vaccine serotypes, and serotype replacement has become a global issue. Case presentation We report a previously healthy 4-month-old girl presenting with a large subcutaneous abscess caused by S. pneumoniae, identified as non-vaccine serotype 28F. The patient had received routine vaccination, including PCV vaccination. After the incision and drainage of the subcutaneous abscess, the patient was treated with antibiotics. She was discharged on Day 7 of hospitalization without any residual sequelae. Conclusions Subcutaneous abscess is a common pediatric skin and soft tissue infection, whereas pneumococcal subcutaneous abscesses are quite rare. As the pneumococcal serotype 28F caused a subcutaneous abscess, this serotype possibly has a high virulence. The incidence of IPD caused by non-vaccine serotypes, such as 28F, is expected to increase in the future. The consolidation of international data on pneumococcal serotypes is important for the development of novel PCV.

Patients with severe hypophosphatasia (HPP) develop osteogenic impairment with extremely low alkaline phosphatase (ALP) activity, resulting in a fatal course during infancy. Mesenchymal stem cells (MSCs) differentiate into various mesenchymal lineages, including bone and cartilage. The efficacy of allogeneic hematopoietic stem cell transplantation for congenital skeletal and storage disorders is limited, and therefore we focused on MSCs for the treatment of HPP. To determine the effect of MSCs on osteogenesis, we performed multiple infusions of ex vivo expanded allogeneic MSCs for two patients with severe HPP who had undergone bone marrow transplantation (BMT) from asymptomatic relatives harboring the heterozygous mutation. There were improvements in not only bone mineralization but also muscle mass, respiratory function, and mental development, resulting in the patients being alive at the age of 3. After the infusion of MSCs, chimerism analysis of the mesenchymal cell fraction isolated from bone marrow in the patients demonstrated that donor-derived DNA sequences existed. Adverse events of BMT were tolerated, whereas those of MSC infusion did not occur. However, restoration of ALP activity was limited, and normal bony architecture could not be achieved. Our data suggest that multiple MSC infusions, following BMT, were effective and brought about clinical benefits for patients with lethal HPP. Allogeneic MSC-based therapy would be useful for patients with other congenital bone diseases and tissue disorders if the curative strategy to restore clinically normal features, including bony architecture, can be established.

Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression.

Internal tandem duplication in the FLT3 gene (FLT3/ITD), which is found in patients with acute myeloid leukemia (AML), causes resistance to FLT3 inhibitors. We found that RUNX1, a transcription factor that regulates normal hematopoiesis, is up-regulated in patients with FLT3/ITD(+) AML. While RUNX1 can function as a tumor suppressor, recent data have shown that RUNX1 is required for AML cell survival. In the present study, we investigated the functional role of RUNX1 in FLT3/ITD signaling. FLT3/ITD induced growth factor-independent proliferation and impaired G-CSF mediated myeloid differentiation in 32D hematopoietic cells, coincident with up-regulation of RUNX1 expression. Silencing of RUNX1 expression significantly decreased proliferation and secondary colony formation, and partially abrogated the impaired myeloid differentiation of FLT3/ITD(+) 32D cells. Although the number of FLT3/ITD(+) 32D cells declined after incubation with the FLT3/ITD inhibitor AC220, the cells became refractory to AC220, concomitant with up-regulation of RUNX1. Silencing of RUNX1 abrogated the emergence and proliferation of AC220-resistant FLT3/ITD(+) 32D cells in the presence of AC220. Our data indicate that FLT3/ITD deregulates cell proliferation and differentiation and confers resistance to AC220 by up-regulating RUNX1 expression. These findings suggest an oncogenic role for RUNX1 in FLT3/ITD(+) cells and that inhibition of RUNX1 function represents a potential therapeutic strategy in patients with refractory FLT3/ITD(+) AML.

Traumatic asphyxia is a crush injury of the chest characterized by facial edema, cyanosis, conjunctival hemorrhage, and petechiae on the face and chest. The prognosis depends on the duration of chest compression and early cardiopulmonary resuscitation after cardiopulmonary arrest. Here we report a case of full recovery from cardiopulmonary arrest caused by traumatic asphyxia. The chest of a 56-year-old man was compressed by a machine while working. Immediately, his colleague started cardiopulmonary resuscitation, which was successful. When he was admitted to our hospital, his consciousness level was E1V2M2(Glasgow coma scale). Our treatment included therapeutic hypothermia, the duration of which was 24 hours at 34 °C. Rewarming his body to 36 °C took place over 48 hours. Thereafter, he recovered completely and was discharged on the 12th hospital day without neurologic sequela. Therapeutic hypothermia was possibly effective in this case.

Internal tandem duplication mutations in the Flt3 gene (ITD-FLT3) enhance cell migration toward the chemokine Cxcl12, which is highly expressed in the therapy-protective bone marrow niche, providing a potential mechanism underlying the poor prognosis of ITD-FLT3(+) acute myeloid leukemia. We aimed to investigate the mechanisms linking ITD-FLT3 to increased cell migration toward Cxcl12. Classification of the expression of Cxcl12-regulated genes in ITD-FLT3(+) cells demonstrated that the enhanced migration of ITD-FLT3(+) cells toward Cxcl12 was associated with the differential expression of genes downstream of Cxcl12/Cxcr4, which are functionally distinct from those expressed in ITD-FLT3(-) cells but are independent of the Cxcr4 expression levels. Among these differentially regulated genes, the expression of Rock1 in the ITD-FLT3(+) cells that migrated toward Cxcl12 was significantly higher than in ITD-FLT3(-) cells that migrated toward Cxcl12. In ITD-FLT3(-) cells, Rock1 expression and Mypt1 phosphorylation were transiently up-regulated but were subsequently down-regulated by Cxcl12. In contrast, the presence of ITD-FLT3 blocked the Cxcl12-induced down-regulation of Rock1 and early Mypt1 dephosphorylation. Likewise, the FLT3 ligand counteracted the Cxcl12-induced down-regulation of Rock1 in ITD-FLT3(-) cells, which coincided with enhanced cell migration toward Cxcl12. Rock1 antagonists or Rock1 shRNA abolished the enhanced migration of ITD-FLT3(+) cells toward Cxcl12. Our findings demonstrate that ITD-FLT3 increases cell migration toward Cxcl12 by antagonizing the down-regulation of Rock1 expression. These findings suggest that the aberrant modulation of Rock1 expression and activity induced by ITD-FLT3 may enhance acute myeloid leukemia cell chemotaxis to the therapy-protective bone marrow niche, where Cxcl12 is abundantly expressed.

We describe not only fleeting assembly of photoisomerizable azobenzene-based palladium complexes into microstructured crystalline architectures but also their light-responsive functions. A transformation in the crystalline morphology from two-dimensional (2D) parallelogram-like sheets to three-dimensional (3D) cuboid- or rhombus-like structures was achieved by changing the solvent from tetrahydrofuran (THF) to acetone and N,N-dimethylformamide (DMF). The sizes of the structures, ranging from a few hundred nanometers to several hundred micrometers, were also modified by varying the complex concentration. In stark contrast to the very stable 2D sheets in the THF–H2O suspensions, exposure of 3D structures in polar DMF–H2O suspensions to ultraviolet (UV) light led to fast disassembly of the structures into isolated metal complexes and further dissociation of free azobenzene ligands from the complexes. In acetone–H2O suspensions, interestingly, disassembly of 3D cuboid-like structures into isolated complex components occurred upon exposure to UV light without further dissociation of azobenzene ligands from the palladium complexes. Considering the photoisomerization ability of the azobenzene-based palladium complex in common organic solvents, the π-stacking interactions that support 3D structures are likely to be sufficiently weak that they might be broken by the UV-induced trans-to-cis isomerization in more polar solvent mixtures. As a consequence, disassembly proceeded under UV light irradiation. Moreover, the effect of solvent polarity on the UV-assisted dissociation (in DMF–H2O) may be associated with the coordination ability of solvent molecules with the metal center.

The facile formation of anisotropic two-dimensional sheets with different sizes, ranging from nanometre-scale to micrometre-scale, was achieved by the assembly of rod-shaped palladium complexes.

FMS-like tyrosine kinase 3 (FLT3) is a membrane type tyrosine kinase and has important roles for the proliferation and differentiation of the hematopoietic cells. The Internal Tandem Duplications of FLT3 (FLT3/ITD) is detected in approximately 30 % of patients with acute myeloid leukemia (AML) and the prognoses of FLT3/ITD+ AML are very poor. While a number of inhibitors targeting FLT3 tyrosine kinase have been developed, few drugs are effective for the FLT3/ITD+ AML because of emergence of resistant cells against the drugs. Recently, AC220 (Quizartinib), a second generation class III tyrosine kinase inhibitor (TKI) for FLT3/ITD+ AML was developed and used in clinical trials. Although AC220 is a more potent and specific inhibitor for FLT3/ITD compared to the other TKIs, report demonstrates that prolonged exposure to AC220 can generate resistant clones to AC220 in FLT3/ITD+ cells (Smith et al. Nature 2012). These findings underscore the need to develop additional therapeutic strategies to overcome the resistance of FLT3/ITD+ AML to TKIs. However, the mechanism responsible for drug resistance of FLT3/ITD+ AML cells remains to be investigated. We previously reported that mRNA expression of RUNX1, a core-binding transcription factor that regulates the differentiation and proliferation of hematopoietic stem cells, is significantly higher in FLT3/ITD+AML cells compared to FLT3/ITD-AML cells (Hirade et al. ASH 2013). Although loss of RUNX1 function (i.e. RUNX1/ETO fusion gene) contributes to the development of AML, RUNX1 also promotes survival of AML cells (Goyama et al. JCI 2013) and can function as an oncogene in cancer cells (Kilbey et al. Cancer Research 2010). These findings lead us hypothesize that RUNX1 may confer resistance of AML cells to TKIs. In the present study, we investigated if Runx1 is involved in the refractory phenotype of Flt3/ITD+cells to AC220. Transduction of Flt3/ITD into IL3-dependent mouse 32D cells allowed the cells proliferate in a growth factor independent fashion, concomitant with up-regulation of Runx1 mRNA level, similar to the patients’ samples with FLT3/ITD+AML. Silencing Runx1 expression using shRNA resulted in 70% reduction of Flt3/ITD+32D cells that proliferated in the absence of growth factors. Similarly, incubating the Flt3/ITD+32D cells with 0.5nM AC220 inhibited their factor independent proliferation by 95%, which was further accentuated up to 99% by the combination with shRNA mediated silencing of Runx1. Although the number of factor independent Flt3/ITD+32D cells cultured in the presence of 2nM AC220 rapidly declined within 96 hours, the residual cells subsequently re-proliferate within 14 days and became no longer sensitive to AC220. Surprisingly, the expression of Runx1 mRNA in the resistant cells to AC220 was 5.0±0.2 fold higher (P Our data indicates that knocking down Runx1 expression enhances the cytotoxic effect of AC220 on Flt3/ITD+32D cells and that Runx1 expression is significantly up-regulated by the AC220 resistance cells. Moreover, Runx1 knockdown recovered the cytotoxicity of AC220 in the refractory Flt3/ITD+32D cells, demonstrating that Flt3/ITD confers resistance to AC220 by up-regulating the expression of Runx1. These findings demonstrate that antagonizing RUNX1 may represent potential therapeutic strategy in the patients with FLT3/ITD+ AML that become refractory to AC220. Disclosures No relevant conflicts of interest to declare.

Introduction: Clinical features and genetic backgrounds of acute myeloid Leukemia (AML) are heterogenous and the prognosis is poor. Cooperating alterations of several genes including oncogenes or tumor suppressor genes lead to development of Leukemia. AML leukemogenesis is thought to require at least two different types of genetic change: class I mutations, which confer a proliferative or survival advantage; and class II mutations, which inhibit myeloid differentiation and apoptotic activity. In hematological malignancies with 11p15 translocations, the NUP98 gene is reportedly fused to various partner genes, often including homeobox genes, such as HOXA9, A11, A13, C11, C13, D11, D13 and PMX. We previously revealed that high frequencies of FLT3 internal tandem duplication (FLT3-ITD), NRAS mutations (NRASMT), and WT1 mutations (WT1MT) in myeloid malignancies with NUP98-HOX fusions were found and their concurrent mutations with NUP98-HOX fusions were associated with clinical prognosis of the patients. However, biological outcome of the combination of NUP98-HOX fusions with FLT3-ITD, NRASMT or WT1MT remains to be investigated. To elucidate the mechanism resposible for the leukemogenesis, we examined the function of NUP98-HOX fusions and the concomitant somatic mutations in vitro. Methods: Full length of NUP98-HOXA9 (NHA9), FLT3-ITD (51 bp-duplication), NRASMT (G13V), and WT1MT (Arg250trp) was cloned into a plasmid that has a mammalian replication initiation region and a nuclear matrix attachment region, which possesses great ability to persist gene amplification for long periods. Each plasmid or the combination of the two plasmids (NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT) were transfected into IL3-dependent mouse myeloid 32D cell line. Cell proliferation was determined in medium without IL3. Cellular differentiation was examined in the medium containing G-CSF using May-Giemsa staining and fluorescence-activated cell sorting (FACS). Apoptosis was quantitated using FACS. Colony forming assay was performed in methyl cellulose medium with stem cell factor and IL3. Moreover, autophagy induced by tamoxifen was checked. Results: Compared to the empty vector, IL3-independent cellular growth was significantly increased in the cells transfected with NHA9 and FLT3-ITD alone, but not in NRASMT and WT1MT alone. In contrast, co-transfection of NRASMT or WT1MT with NHA9 significantly increased the IL3 independent proliferation of 32D cells. Similarly, cells transfected with NHA9/FLT3-ITD showed the enhanced IL3-independent proliferation. CD11b and Gr1, of which represent myeloid differentiation marker, decreased in all the transfected cells except for NRASMT. While transfection with FLT3-ITD, NRASMT and WT1MT increased the morphological nuclear segmentation of the cells, NHA9/FLT3-ITD, NHA9/NRASMT, and NHA9/WT1MT abrogated the nuclear segmentation. Both Annexin V positive/propidium iodide negative cells and active caspase 3-positive cells declined in all transfected cells. Transfection of NHA9/FLT3-ITD, NHA9/NRASMT, NHA9/WT1MT tended to increase the number of colony forming unit compared to the single gene-transfected cells. After treatment with tamoxifen, monodansylcadaverine, which was used to reveal autophagic vacuoles, was incorporated into empty vector-transfected cells, not into all transfected cells. Conclusions: NHA9 acquired proliferation capacity, blockage of differentiation, and anti-apoptosis activity, FLT3-ITD did proliferation capacity and anti-apoptosis activity, NRASMT and WT1MT did anti-apoptosis activity. By contrast, NHA9 with FLT3-ITD, NRASMT, or WT1MT conferred growth advantage, inhibited differentiation and apoptosis, augmented self-renewality. In vivo study demonstrated that NHA9 or each genetic mutation (FLT3-ITD and NRASMT) alone was insufficient to achieve myeloid leukemia. These suggested that leukemogenesis of NUP98-HOX fusion genes with simultaneous gene mutations might be induced by not only harmonization between class I and class II mutations but also self-renewality and anti-autophagic cell death. Disclosures No relevant conflicts of interest to declare.

Internal-Tandem-Duplication mutations in the FLT3 (FLT3/ITD) gene are detected in 30% of patients with acute myeloid leukemia (AML) and are associated with extremely poor prognoses. The lack of significant efficacy of FLT3/ITD inhibitors underscores the need to identify FLT3/ITD-specific signaling pathways that are distinct from those that occur in normal hematopoietic cells to develop novel therapeutic approaches. FLT3/ITD is classified as a “class I mutation” that drives the proliferation of leukemia cells. In addition to mutation of FLT3/ITD, a “class II mutation” that blocks differentiation of the pre-leukemic clone is generally required for the development of AML. For instance, dominant negative mutations of RUNX1 are occasionally found in patients with AML. These mutations of RUNX1 cause AML by blocking the differentiation of leukemia cells in combination with the mutation of FLT3/ITD. RUNX1 is a core-binding transcription factor and plays an important role in hematopoietic homeostasis, particularly differentiation and proliferation. Loss of RUNX1 blocks hematopoietic differentiation and is associated with the emergence of a primitive hematopoietic compartment, suggesting that RUNX1 generally induces differentiation of hematopoietic cells. However, the functional role of RUNX1 as a down-stream effector of FLT3/ITD has not been characterized. Herein, we investigated the role of Runx1 in aberrant proliferation and differentiation of hematopoietic cells induced by Flt3 /ITD. A comparison of RUNX1 expression levels in AML patients for whom information has been deposited in the public gene expression profile database (GSE1159) revealed that RUNX1 mRNA expression was significantly higher in FLT3/ITD+AML cells (N=78) than in FLT3/ITD-AML cells (N=190, P Our results suggest that Flt3/ITD blocks myeloid differentiation of Flt3/ITD+cells by up-regulating Runx1 expression. The blocking of differentiation mediated by Runx1 in Flt3/ITD+cells is in contrast to the cell differentiation-inducing role of Runx1 in normal hematopoiesis, suggesting that the function of Runx1 in Flt3/ITD+cells may be distinct from that in normal cells. The reduction of secondary CFU colonies by Runx1-shRNA suggests that Runx1 may mediate self-renewal of Flt3/ITD+hematopoietic progenitor cells. These findings suggest that antagonizing RUNX1 may represent a novel therapeutic strategy to induce terminal differentiation of FLT3/ITD+AML cells in AML patients, in addition to inhibiting their aberrant proliferation. Disclosures: No relevant conflicts of interest to declare.

We synthesized an amphiphilic fluorescent compound (PyAzEG) having a pyrene fluorophore directly attached to twisted ortho-alkylated azobenzene (i) to suppress pyrene excimer formation and (ii) to facilitate pyrene monomer fluorescence switching. PyAzEG molecules in a dilute solution underwent azobenzene-based trans ↔ cis photoisomerization under UV and visible light irradiation. When such amphiphilic molecules dispersed in an aqueous solution, they could assemble into spheres through a delicate balance between π–π stacking among aromatic rings and hydrophilic interactions among tetra(ethylene glycol) chains. For a PyAzEG suspension containing micrometer-sized spheres, typical pyrene monomer fluorescence was observed in the range of 370–460 nm, whereas excimer fluorescence did not emerge from the fluorescence spectrum. Monomer fluorescence switching as well as structural transformation from spheres to less packed vesicle-like structures were achieved by changing the wavelength of light. Furthermore, PyAzEG spheres exhibited enhanced fluorescence relative to the monomeric solution, which is probably due to moderate intermolecular interactions among chromophores in a head-to-tail fashion.

We describe the important determinants of stabilizing/destabilizing azobenzene-based palladium complexes capable of undergoing repeated light-triggered conformation changes. Inspired by highly warped palladium complexes with distorted azobenzene ligands, three different types of azobenzene ligands (1–3) were chosen to modify both the molecular structure (from a ubiquitous planar to a twisted trans-azobenzene) and the solubility of the azobenzenes in organic solvents. Single-crystal X-ray structure analyses were carried out for ortho-alkylated azobenzene (ortho-Az: 1b), meta-alkylated azobenzene (meta-Az: 2b) and PdCl2(ortho-Az)2 (1-Pd). Whereas both 2b and 3 lacking the meta and ortho substituents had planar structures, ortho-alkylated 1b was highly distorted by 68.06° from planarity. In stark contrast to PdCl2(meta-Az)2 (2-Pd) and PdCl2(Az)2 (3-Pd), which quickly dissociated in organic solvents, 1-Pd was very stable, especially in nonpolar solvents, so that it was possible to successfully purify and characterize them. The results suggest that unusually distorted trans-azobenzene is hardly influenced by the complexation reaction which requires considerable distortion of the azobenzene unit, thus stabilizing mononuclear palladium complexes. In nonpolar solvents, 1-Pd underwent repeated conformation changes under alternating UV and visible light irradiation. However, in polar solvents, the UV-triggered conformation change was accompanied by facile light-assisted breaking of the N:→Pd bond. Even dark incubation in polar organic solvents caused the dissociation of azobenzene ligands from the complexes. The breaking rate of the N:→Pd bond increased in the order of benzene ≈ dichloromethane < acetone < DMF, with more polar solvents inducing faster dissociation. The solvent polarity effect on the stability of azobenzene-based complexes can be interpreted in terms of the degree of polarization of the metal–ligand bond formed as a consequence of interactions between the palladium ion as a soft acid and azobenzene nitrogen as a hard base.

Abstract 1319 Internal Tandem Duplication mutations of the Flt3 gene (ITD-Flt3) and the deregulation of CXCR4 expression in patients with AML are associated with a poor prognosis (Rombouts et al. Blood 2004). ITD-Flt3 induces myeloproliferative disease with extramedullary infiltration in transplantation models, thereby suggesting that ITD-Flt3 can regulate the dissemination of leukemia cells. We have reported that ITD-Flt3 mutations enhance the migration of mouse hematopoietic cells toward SDF1 and modulate their homing in vivo (Fukuda et al. Blood 2005, Exp Hematology 2006). However, the molecular mechanism responsible for the aberrant migration of ITD-Flt3+ cells toward SDF1 remains unknown. A recent report has demonstrated that Rho-Associated Kinase (Rock) regulates the proliferation of ITD-Flt3+ hematopoietic cells (Mali et al. Cancer Cell 2012). In this report, we investigate the functional role of Rock1 in the aberrant hematopoietic cell migration induced by ITD-Flt3. The migration of Ba/F3 cells containing 3 different ITD-Flt3 variants (N51, N73, N78: ITD-Flt3+ cells) toward SDF1 (0–500 ng/ml) was significantly increased compared with Ba/F3 cells lacking ITD-Flt3 (wild-type Flt3+ cells; P Our data demonstrates that SDF1 down-regulates Rock1 expression in the absence of Flt3 signaling, while ITD-Flt3 mutations augment cell migration toward SDF1 by blocking the down-regulation of Rock1 that is induced by SDF1 signaling. Manipulating the SDF1/Rock1 axis that is modulated by ITD-Flt3 may be clinically beneficial for antagonizing the aberrant dissemination of AML cells in patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 2340 Internal-Tandem-Duplication (ITD) Flt3 mutations are found in 30% of patients with AML; however, Flt3-inhibitors are not significantly efficacious in the therapy. We have reported that ITD-Flt3 down-regulates Pbx1 expression, a transcriptional factor that regulates the self-renewal of hematopoietic stem cells, in mouse bone marrow c-kit+-Sca-1+-Lin− (KSL) cells, and that Pbx1 expression levels are reduced in patients with ITD-Flt3+AML compared with those lacking ITD-Flt3 (ASH 2011). However, the function of Pbx1 in ITD-Flt3 signaling remains unknown. In this report, we investigate the function of Pbx1 in hematopoietic progenitor cells transformed by ITD-Flt3. ITD-Flt3 transduction in mouse marrow cells led to a decrease in Pbx1 mRNA expression levels in KSL cells (80±8% reduction compared with normal KSL cells, P Our data indicates that expression of Pbx1, which supports the growth-factor-independent proliferation of ITD-Flt3+ cells, was down-regulated by p21, which was elevated in the presence of ITD-Flt3. The enhanced proliferation of ITD-Flt3+ cells induced by p21 deletion was partially abrogated by knocking down Pbx1, thereby indicating that the down-regulation of Pbx1 is secondary to the up-regulation of p21 that is induced by ITD-Flt3, which resulted in the growth inhibition of ITD-Flt3+cells. The data demonstrates that ITD-Flt3 can generate growth inhibitory signals by reducing the Pbx1 expression levels via p21.This implies that Flt3-inhibitors may destroy the Pbx1/p21 growth inhibitory signaling that is generated by ITD-Flt3, thereby contributing to subsequent recurrence of ITD-Flt3+AML cells. Disclosures: No relevant conflicts of interest to declare.