A colorimetric chemical tongue detects and distinguishes between multiple analytes.

The rate-limiting step for diagnostics development is the discovery and validation of biomarker analytes. We describe a new analyte-agnostic and label-free approach based on colorimetric reactions involving type I polymerization photoinitiators. We demonstrate that a chemically diverse array of hydrogels embedded with cleaved type I photoinitiators could act as microreactors, undergoing colorimetric reactions with bound analytes. The colorimetric signatures produced were visually distinctive and readable with a flatbed document scanner. Signatures of a broad range of sample types were accurately differentiated by unsupervised clustering without knowledge of any analytes bound to the array. The principles described have the potential to enable scalable and cost-effective analysis of complex samples.

Glycogen synthase kinase 3 in MLL leukaemia maintenance and targeted therapy.

Glycogen synthase kinase 3 (GSK3) is a multifunctional serine/threonine kinase that participates in numerous signalling pathways involved in diverse physiological processes. Several of these pathways are implicated in disease pathogenesis, which has prompted efforts to develop GSK3-specific inhibitors for therapeutic applications. However, before now, there has been no strong rationale for targeting GSK3 in malignancies. Here we report pharmacological, physiological and genetic studies that demonstrate an oncogenic requirement for GSK3 in the maintenance of a specific subtype of poor prognosis human leukaemia, genetically defined by mutations of the MLL proto-oncogene. In contrast to its previously characterized roles in suppression of neoplasia-associated signalling pathways, GSK3 paradoxically supports MLL leukaemia cell proliferation and transformation by a mechanism that ultimately involves destabilization of the cyclin-dependent kinase inhibitor p27(Kip1). Inhibition of GSK3 in a preclinical murine model of MLL leukaemia provides promising evidence of efficacy and earmarks GSK3 as a candidate cancer drug target.

IMC-EB10, an anti-FLT3 monoclonal antibody, prolongs survival and reduces nonobese diabetic/severe combined immunodeficient engraftment of some acute lymphoblastic leukemia cell lines and primary leukemic samples.

The class III receptor tyrosine kinase FLT3 is expressed on the blasts of >90% of patients with B-lineage acute lymphoblastic leukemias (ALL). In addition, it is expressed at extremely high levels in ALL patients with mixed lineage leukemia rearrangements or hyperdiploidy and is sometimes mutated in these same patients. In this report, we investigate the effects of treating ALL cell lines and primary samples with human anti-FLT3 monoclonal antibodies (mAb) capable of preventing binding of FLT3 ligand. In vitro studies, examining the ability of two anti-FLT3 mAbs (IMC-EB10 and IMC-NC7) to affect FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. FLT3 phosphorylation was consistently inhibited by IMC-NC7 treatment, but in some cell lines, IMC-EB10 actually stimulated FLT3 activation, possibly as a result of antibody-mediated receptor dimerization. Through antibody-dependent, cell-mediated cytotoxicity, such an antibody could still prove efficacious against leukemia cells in vivo. In fact, IMC-EB10 treatment significantly prolonged survival and/or reduced engraftment of several ALL cell lines and primary ALL samples in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This occurred even when IMC-EB10 treatment resulted in FLT3 activation in vitro. Moreover, fluorescence-activated cell sorting and PCR analysis of IMC-EB10-treated NOD/SCID mice surviving 150 days post-leukemic cell injection revealed that FLT3 immunotherapy reduced leukemic engraftment below the level of detection in these assays (<0.001%). Furthermore, in vivo IMC-EB10 treatment did not select for resistant cells, because cells surviving IMC-EB10 treatment remain sensitive to IMC-EB10 cytotoxicity upon retransplantation. In vivo studies involving either partial depletion or activation of natural killer (NK) cells show that most of the cytotoxic effect of IMC-EB10 is mediated through NK cells. Therefore, such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

Inhibitory anti-FLT3 antibodies are capable of mediating antibody-dependent cell-mediated cytotoxicity and reducing engraftment of acute myelogenous leukemia blasts in nonobese diabetic/severe combined immunodeficient mice.

Aberrant FLT3 expression and/or mutation plays a significant role in leukemogenesis. This has prompted the development of selective small molecule tyrosine kinase inhibitors against FLT3. However, like most tyrosine kinase inhibitors, those against FLT3 are not completely specific and at the doses required to completely inhibit target, significant toxicities may occur. In addition, tyrosine kinase inhibitors for other kinases have been shown to select for cells that become resistant. To overcome some of these limitations we developed two fully human phage display monoclonal antibodies against FLT3 (IMC-EB10 and IMC-NC7). These antibodies inhibited ligand-mediated activation of wild-type FLT3 and constitutively activated mutant FLT3 and in most cell types affected downstream STAT5, AKT, and mitogen-activated protein kinase activation. In addition to interfering with FLT3 signaling, IMC-EB10 and, to a significantly lesser extent, IMC-NC7 initiated antibody-dependent cell-mediated cytotoxicity on FLT3-expressing cells. When IMC-EB10 was used in vivo to treat nonobese diabetic/severe combined immunodeficient mice given injections of primary FLT3/ITD acute myelogenous leukemia samples or myeloid cell lines with FLT3 expression, it significantly decreased engraftment of leukemic cells and increased survival, respectively. In contrast, IMC-EB10 treatment did not reduce engraftment of normal human CD34+ cord blood cells nor did it show any significant inhibition of normal murine hematopoiesis. Thus, these types of antibodies have the potential to be safe and effective new therapeutic agents for acute myelogenous leukemia and possibly other FLT3-expressing malignancies.

Beta-Hemolytic Bacteria Selectively Trigger Liposome Lysis, Enabling Rapid and Accurate Pathogen Detection.

For more than a century, blood agar plates have been the only test for beta-hemolysis. Although blood agar cultures are highly predictive for bacterial pathogens, they are too slow to yield actionable information. Here, we show that beta-hemolytic pathogens are able to lyse and release fluorophores encapsulated in sterically stabilized liposomes whereas alpha and gamma-hemolytic bacteria have no effect. By analyzing fluorescence kinetics, beta-hemolytic colonies cultured on agar could be distinguished in real time with 100% accuracy within 6 h. Additionally, end point analysis based on fluorescence intensity and machine-extracted textural features could discriminate between beta-hemolytic and cocultured control colonies with 99% accuracy. In broth cultures, beta-hemolytic bacteria were detectable in under an hour while control bacteria remained negative even the next day. This strategy, called beta-hemolysis triggered-release assay (BETA) has the potential to enable the same-day detection of beta-hemolysis with single-cell sensitivity and high accuracy.

Next-generation NAMPT inhibitors identified by sequential high-throughput phenotypic chemical and functional genomic screens.

Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.

Knock-in of an internal tandem duplication mutation into murine FLT3 confers myeloproliferative disease in a mouse model.

Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) mutations is one of the most common molecular alterations known in acute myeloid leukemia (AML). To investigate the role FLT3/ITD mutations play in the development of leukemia, we generated a FLT3/ITD knock-in mouse model by inserting an ITD mutation into the juxtamembrane domain of murine Flt3. FLT3wt/ITD mice developed myeloproliferative disease, characterized by splenomegaly, leukocytosis, and myeloid hypercellularity, which progressed to mortality by 6 to 20 months. Bone marrow (BM) and spleen from FLT3wt/ITD mice had an increased fraction of granulocytes/monocytes and dendritic cells, and a decreased fraction of B-lymphocytes. No sign of acute leukemia was observed over the lifetime of these mice. BM from FLT3wt/ITD mice showed enhanced potential to generate myeloid colonies in vitro. BM from FLT3wt/ITD mice also produced more spleen colonies in the in vivo colony-forming unit (CFU)-spleen assay. In the long-term competitive repopulation assay, BM cells from FLT3wt/ITD mice outgrew the wild-type competitor cells and showed increased myeloid and reduced lymphoid expansion activity. In summary, our data indicate that expression of FLT3/ITD mutations alone is capable of conferring normal hematopoietic stem/progenitor cells (HSPCs) with enhanced myeloid expansion. It also appears to suppress B lymphoid maturation. Additional cooperative events appear to be required to progress to acute leukemia.

Prolonged exposure to FLT3 inhibitors leads to resistance via activation of parallel signaling pathways.

Continuous treatment of malignancies with tyrosine kinase inhibitors (TKIs) may select for resistant clones (ie, imatinib mesylate). To study resistance to TKIs targeting FLT3, a receptor tyrosine kinase that is frequently mutated in acute myelogenous leukemia (AML), we developed resistant human cell lines through prolonged coculture with FLT3 TKIs. FLT3 TKI-resistant cell lines and primary samples still exhibit inhibition of FLT3 phosphorylation on FLT3 TKI treatment. However, FLT3 TKI-resistant cell lines and primary samples often show continued activation of downstream PI3K/Akt and/or Ras/MEK/MAPK signaling pathways as well as continued expression of genes involved in FLT3-mediated cellular transformation. Inhibition of these signaling pathways restores partial sensitivity to FLT3 TKIs. Mutational screening of FLT3 TKI-resistant cell lines revealed activating N-Ras mutations in 2 cell lines that were not present in the parental FLT3 TKI-sensitive cell line. Taken together, these data indicate that FLT3 TKI-resistant cells most frequently become FLT3 independent because of activation of parallel signaling pathways that provide compensatory survival/proliferation signals when FLT3 is inhibited. Anti-FLT3 mAb treatment was still cytotoxic to FLT3 TKI-resistant clones. An approach combining FLT3 TKIs with anti-FLT3 antibodies and/or inhibitors of important pathways downstream of FLT3 may reduce the chances of developing resistance.

Constitutive Fms-like tyrosine kinase 3 activation results in specific changes in gene expression in myeloid leukaemic cells.

Constitutively activating internal tandem duplication (ITD) mutations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) play an important role in leukaemogenesis. We have examined, by cDNA microarray analysis, the changes in gene expression induced by FLT3/ITD or constitutively activated wild type FLT3 signalling. A limited set of genes was consistently affected by FLT3 inhibition. In confirmation of their FLT3 dependence, these genes returned toward pretreatment levels of expression after reversal of FLT3 inhibition. Several of the most significantly affected genes are involved in the RAS/mitogen-activated protein kinase, Janus kinase/signal transducer and activator of transcription and phosphatidylinositol 3 kinase (PI3K)/AKT pathways. These data suggest that constitutively activated FLT3 works through multiple signal transduction pathways. PIM1, MYC and CCND3 were chosen from this gene set to explore their biological roles. Knock-down of these genes by small interfering RNA showed that these genes play important roles in constitutively activated FLT3 expressing cells. The alterations of the gene expression profiles in these cells help to further elucidate the mechanisms of FLT3-mediated leukaemogenesis.

FLT3/ITD expression increases expansion, survival and entry into cell cycle of human haematopoietic stem/progenitor cells.

Activating mutation of FLT3 by internal tandem duplications (ITDs) in the juxtamembrane region is the most common molecular aberration found in acute myeloid leukaemia (AML). In this study, a lentiviral vector containing two promoters achieved consistent and efficient co-expression of FLT3/ITD and GFP in transduced human CD34(+) haematopoietic stem/progenitor cells (HSPCs). When cultured in medium containing stem cell factor, thrombopoietin and FLT3 ligand (FL), FLT3/ITD-transduced cells demonstrated enhanced self-renewal and survival potential, unaffected by the withdrawal of FL. These cells retained a CD34(+)CD38(-/dim) immunophenotype, typical of HSPCs. Compared to cells transduced with a vector expressing GFP alone, FLT3/ITD-transduced HSPCs had a higher fraction of cells in active cell cycle. FLT3/ITD-transduced HSPCs were more sensitive to the induction of cytotoxicity by CEP-701, a selective FLT3 inhibitor, indicating a rapid 'addiction' to signalling through this oncogenic pathway. The FLT3/ITD-transduced HSPCs showed increased expression of Pim-1, c-Myc and Cyclin D3 (CCND3), each of which may contribute to the altered genetic programme instituted by FLT3/ITD signalling. Taken together, these results indicate that FLT3/ITD mutations may contribute to leukaemic transformation of normal HSPCs by prolonging survival, promoting proliferation and partially blocking differentiation. CEP-701 may act as a potent therapeutic agent for AML stem cells harbouring FLT3/ITD mutations.

FLT3 ligand causes autocrine signaling in acute myeloid leukemia cells.

The FLT3 receptor tyrosine kinase is highly expressed in most acute leukemias and frequently mutated in acute myeloid leukemia (AML). The mutated form of the receptor is constitutively activated and known to play an important role in AML, but the activation state of the overexpressed wild-type (wt) receptor is, at present, unknown. In this study, we examined the activation state of the wild-type receptor in AML. We found that the wild-type receptor was constitutively phosphorylated/activated in 8 of 12 primary AML samples and 4 of 13 leukemia cell lines. To explain why wtFLT3 is often activated, we investigated the expression of its ligand, FL, by these same cells. Coexpression of FL with FLT3 was a universal finding in both primary AML samples and leukemic-derived cell lines. To further prove that autocrine signaling was accounting for the activation, we showed that conditioned media but not fresh media was able to activate FLT3. In addition, an antibody that blocks binding of ligand to the receptor blocks FLT3 activation. Finally, depletion of FL from conditioned media is able to block the activation of FLT3. Taken together, these findings represent strong evidence that wtFLT3 is often constitutively activated in AML and thus, like its mutated form, might contribute to the altered signaling that characterizes leukemogenesis.

Suppression of leukemia expressing wild-type or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing antibody.

FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, is expressed at high levels in the blasts of approximately 90% of patients with acute myelogenous leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and point mutations in the kinase domain of FLT3 are found in approximately 37% of AML patients and are associated with a poor prognosis. We report here the development and characterization of a fully human anti-FLT3 neutralizing antibody (IMC-EB10) isolated from a human Fab phage display library. IMCEB10 (immunoglobulin G1 [IgG1], kappa) binds with high affinity (KD=158 pM) to soluble FLT3 in enzyme-linked immunosorbent assay (ELISA) and to FLT3 receptor expressed on the surfaces of human leukemia cell lines. IMC-EB10 blocks the binding of FLT3 ligand (FL) to soluble FLT3 in ELISA and competes with FL for binding to cell-surface FLT3 receptor. IMC-EB10 treatment inhibits FL-induced phosphorylation of FLT3 in EOL-1 and EM3 leukemia cells and FL-independent constitutive activation of ITD-mutant FLT3 in BaF3-ITD and MV4;11 cells. Activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and AKT is also inhibited in these cell lines by antibody treatment. The antibody inhibits FL-stimulated proliferation of EOL-1 cells and ligand-independent proliferation of BaF3-ITD cells. In both EOL-1 xenograft and BaF3-ITD leukemia models, treatment with IMC-EB10 significantly prolongs the survival of leukemia-bearing mice. No overt toxicity is observed with IMC-EB10 treatment. Taken together, these data demonstrate that IMC-EB10 is a specific and potent inhibitor of wild-type and ITD-mutant FLT3 and that it deserves further study for targeted therapy of human AML.