Transient exposure to quizartinib mediates sustained inhibition of FLT3 signaling while specifically inducing apoptosis in FLT3-activated leukemia cells.

Fms-like tyrosine kinase 3 (FLT3) is implicated in the pathogenesis of acute myeloid leukemia (AML). FLT3-activating internal tandem duplication (ITD) mutations are found in approximately 30% of patients with AML and are associated with poor outcome in this patient population. Quizartinib (AC220) has previously been shown to be a potent and selective FLT3 inhibitor. In the current study, we expand on previous observations by showing that quizartinib potently inhibits the phosphorylation of FLT3 and downstream signaling molecules independent of FLT3 genotype, yet induces loss of viability only in cells expressing constitutively activated FLT3. We further show that transient exposure to quizartinib, whether in vitro or in vivo, leads to prolonged inhibition of FLT3 signaling, induction of apoptosis, and drastic reductions in tumor volume and pharmacodynamic endpoints. In vitro experiments suggest that these prolonged effects are mediated by slow binding kinetics that provide for durable inhibition of the kinase following drug removal/clearance. Together these data suggest quizartinib, with its unique combination of selectivity and potent/sustained inhibition of FLT3, may provide a safe and effective treatment against FLT3-driven leukemia.

CEP-32496: a novel orally active BRAF(V600E) inhibitor with selective cellular and in vivo antitumor activity.

Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60% to 70% of melanomas, 29% to 83% of papillary thyroid carcinomas, 4% to 16% colorectal cancers, and a lesser extent in serous ovarian and non-small cell lung cancers. The V600E mutation is found in the vast majority of cases and is an activating mutation, conferring transforming and immortalization potential to cells. CEP-32496 is a potent BRAF inhibitor in an in vitro binding assay for mutated BRAF(V600E) (K(d) BRAF(V600E) = 14 nmol/L) and in a mitogen-activated protein (MAP)/extracellular signal-regulated (ER) kinase (MEK) phosphorylation (pMEK) inhibition assay in human melanoma (A375) and colorectal cancer (Colo-205) cell lines (IC(50) = 78 and 60 nmol/L). In vitro, CEP-32496 has multikinase binding activity at other cancer targets of interest; however, it exhibits selective cellular cytotoxicity for BRAF(V600E) versus wild-type cells. CEP-32496 is orally bioavailable in multiple preclinical species (>95% in rats, dogs, and monkeys) and has single oral dose pharmacodynamic inhibition (10-55 mg/kg) of both pMEK and pERK in BRAF(V600E) colon carcinoma xenografts in nude mice. Sustained tumor stasis and regressions are observed with oral administration (30-100 mg/kg twice daily) against BRAF(V600E) melanoma and colon carcinoma xenografts, with no adverse effects. Little or no epithelial hyperplasia was observed in rodents and primates with prolonged oral administration and sustained exposure. CEP-32496 benchmarks favorably with respect to other kinase inhibitors, including RAF-265 (phase I), sorafenib, (approved), and vemurafenib (PLX4032/RG7204, approved). CEP-32496 represents a novel and pharmacologically active BRAF inhibitor with a favorable side effect profile currently in clinical development.

Identification of 1-(3-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-(5-(1,1,1-trifluoro-2-methylpropan-2-yl)isoxazol-3-yl)urea hydrochloride (CEP-32496), a highly potent and orally efficacious inhibitor of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF) V600E.

The Ras/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) signaling pathway plays a central role in the regulation of cell growth, differentiation, and survival. Expression of mutant BRAF(V600E) results in constitutive activation of the MAPK pathway, which can lead to uncontrolled cellular growth. Herein, we describe an SAR optimization campaign around a series of quinazoline derived BRAF(V600E) inhibitors. In particular, the bioisosteric replacement of a metabolically sensitive tert-butyl group with fluorinated alkyl moieties is described. This effort led directly to the identification of a clinical candidate, compound 40 (CEP-32496). Compound 40 exhibits high potency against several BRAF(V600E)-dependent cell lines and selective cytotoxicity for tumor cell lines expressing mutant BRAF(V600E) versus those containing wild-type BRAF. Compound 40 also exhibits an excellent PK profile across multiple preclinical species. In addition, significant oral efficacy was observed in a 14-day BRAF(V600E)-dependent human Colo-205 tumor xenograft mouse model, upon dosing at 30 and 100 mg/kg BID.

Rapamycin inhibits proliferation of Epstein-Barr virus-positive B-cell lymphomas through modulation of cell-cycle protein expression.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV+ B cells. METHODS: SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies. RESULTS: We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA. CONCLUSION: The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways of EBV+ B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV+ lymphomas.

Enhancement of gene transfer to human myeloid cells by adenovirus-fiber complexes.

Adenoviral (Ad) vectors are currently being developed for immunotherapy and vaccine delivery, particularly for cancer and antiviral treatment (e.g., human immunodeficiency virus vaccine). However, the inefficient transduction of antigen-presenting cells of myeloid lineage such as dendritic cells (DCs) by conventional Ad5-based vectors limits these applications. DCs lack the coxsackie and adenovirus receptor but express CD46, a member of the family of complement regulatory proteins, as well as sialic acid glycoconjugates, a situation that allows infection by Ad type 37 as well as most subgroup B Ads. In this study, we generated recombinant Ad fiber knobs (FKs) to probe Ad37 receptor usage on immune cells. Remarkably, treatment of human myeloid cells with Ad37 FK (37FK) enhanced, rather than inhibited transduction by Ad5 or Ad37 pseudotyped virions. The enhanced gene transfer was dose dependent, involved association with alpha(2,6)-linked sialic acid residues, and was limited to blood cells of myeloid lineage, including immature and mature DCs. We also provide evidence that 37FK binds directly to Ad5 virus particles, likely acting as a bridge to facilitate greater virus-cell interaction.

CD46-utilizing adenoviruses inhibit C/EBPbeta-dependent expression of proinflammatory cytokines.

The majority of adenovirus serotypes utilize the coxsackievirus-adenovirus receptor (CAR) for virus-host cell attachment, but subgroup B and subgroup D (adenovirus type 37 [Ad37]) viruses recognize CD46. CD46 is a ubiquitously expressed receptor that serves as a cofactor for the inactivation of the complement components C3b and C4b, and it also serves as a receptor for diverse microbial pathogens. A reported consequence of CD46 engagement is a reduced capability of human immune cells to express interleukin-12 (IL-12), a cytokine involved in both the innate and adaptive immune responses. Studies were thus undertaken to determine whether CD46-utilizing Ads alter the expression of proinflammatory cytokines. Subgroup B (Ad16 and -35) and Ad37, but not Ad2 or -5, significantly reduced IL-12 production by human peripheral blood mononuclear cells stimulated with gamma interferon (IFN-gamma) and lipopolysaccharide. IL-12 mRNA (p35 and p40 subunits) levels as well as other cytokine mRNA levels (IL-1alpha and -beta, IL-1Ra, and IL-6) were decreased upon interaction with CD46-utilizing Ads. Analysis of transcription factor activity required for cytokine expression indicated that CD46-utilizing Ads preferentially inhibited IFN-gamma-induced C/EBPbeta protein expression, consequently reducing its ability to form DNA complexes. Interference with IFN-gamma signaling events by CD46-utilizing Ads, but not CAR-utilizing Ads, reveals a potentially critical difference in the host immune response against distinct Ad vectors, a situation that has implications for gene delivery and vaccine development.

CryoEM structure at 9A resolution of an adenovirus vector targeted to hematopoietic cells.

We report a sub-nanometer resolution cryo-electron microscopy (cryoEM) structural analysis of an adenoviral vector, Ad35F, comprised of an adenovirus type 5 (Ad5) capsid pseudo-typed with an Ad35 fiber. This vector transduces human hematopoietic cells via association of its fiber protein with CD46, a member of the complement regulatory protein family. Major advances in data acquisition and image processing allowed a significant improvement in resolution compared to earlier structures. Analysis of the cryoEM density was enhanced by docking the crystal structures of both the hexon and penton base capsid proteins. CryoEM density was observed for hexon residues missing from the crystal structure that include hypervariable regions and the epitope of a neutralizing monoclonal antibody. Within the penton base, density was observed for the integrin-binding RGD loop missing from the crystal structure and for the flexible beta ribbon of the variable loop on the side of the penton base. The Ad35 fiber is flexible, consistent with the sequence insert in the third beta-spiral repeat. On the inner capsid surface density is revealed at the base of the hexons and below the penton base. A revised model is presented for protein IX within the virion. Well-defined density was assigned to a conserved domain in the N terminus of protein IX required for incorporation into the virion. For the C-terminal domain of protein IX two alternate conformations are proposed, either binding on the capsid surface or extending away from the capsid. This model is consistent with the tolerance of the C terminus for inserted ligands and its potential use in vector retargeting. This structural study increases our knowledge of Ad capsid assembly, antibody neutralization mechanisms, and may aid further improvements in gene delivery to important human cell types.

Identification, cloning, and characterization of a novel rat natural killer receptor, RNKP30: a molecule expressed in liver allografts.

BACKGROUND: As a component of the innate immune system, natural killer (NK) cells may play a significant role in the early events after solid-organ transplantation. Activated NK cells have been shown to infiltrate allografts in transplant models. To better understand NK cells and the role of NK cell receptors in transplantation, we have cloned and begun characterizing a novel rat molecule, rNKp30. METHODS: RNKp30 cDNA was cloned by 5' rapid amplification of cDNA ends polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR from mononuclear cells infiltrating a rejecting liver allograft. Southern blot analysis was used to determine the rNKp30 gene copy number. RT-PCR and Northern blotting were used to examine rNKp30 RNA expression in NK cells, multiple tissues, and liver grafts. Immunocytochemistry, immunoprecipitation, and Western blot analysis with two anti-rNKp30 polyclonal antibodies, CA680 and CA1071, were performed. Tunicamycin and endoglycosidase treatments determined the extent of rNKp30 glycosylation. RESULTS: RNKp30 is homologous to human and macaque NKp30. It is a single copy gene with five identified single-nucleotide polymorphisms. RNKp30 is expressed by NK cells and is detectable as a single transcript by Northern blot in normal spleen, lymph node, and lung tissues. RNKp30 is a variably N-glycosylated cell surface molecule with a protein backbone of approximately 21 kDa. Elevated transcript expression of rNKp30 is detected in both rejected and spontaneously accepted liver allografts, but not in syngeneic or cyclosporine A-treated allografts. CONCLUSIONS: RNKp30 is a glycosylated surface NK cell receptor with limited polymorphism. This putative activation receptor is expressed in liver allografts and may participate in the innate immune response after transplantation.

Rapamycin inhibits the interleukin 10 signal transduction pathway and the growth of Epstein Barr virus B-cell lymphomas.

EBV-infected B-cell lymphomas are a potentially life-threatening complication in bone marrow and solid organ transplant recipients. Immunosuppressive drugs required to prevent allograft rejection also impair anti-EBV T-cell immunity, thereby increasing the risk of EBV-associated disease. Here we demonstrate that the immunosuppressant rapamycin (RAPA) has a strong antiproliferative effect in vitro on B-cell lines derived from organ transplant recipients with EBV-associated posttransplant lymphoproliferative disorder (PTLD). Furthermore, RAPA significantly inhibits or delays the growth of solid tumors established from EBV-infected B-cell lines in a xenogeneic mouse model of PTLD. RAPA acts via cell cycle arrest, induction of apoptosis, and, most importantly, inhibition of interleukin 10 secretion, a necessary autocrine growth factor. The reduced interleukin 10 production is accompanied by corresponding decreases in the constitutive activation of the growth-promoting transcription factors signal transducer and activator of transcription 1 and 3. Thus, RAPA can limit B-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection in patients who are otherwise at risk for EBV-associated PTLD. Moreover, these findings may have application to other EBV-associated malignancies.

Identification of Epstein-Barr virus-specific CD8+ T lymphocytes in the circulation of pediatric transplant recipients.

BACKGROUND: Pediatric transplant recipients are at increased risk for Epstein Barr virus (EBV)-related B cell lymphomas. In healthy individuals, the expansion of EBV-infected B cells is controlled by CD8+ cytotoxic T cells. However, immunosuppressive therapy may compromise antiviral immunity. We identified and determined the frequency of EBV-specific T cells in the peripheral blood of pediatric transplant recipients. METHODS: HLA-B*0801 and HLA-A*0201 tetramers folded with immunodominant EBV peptides were used to detect EBV-specific CD8+ T cells by flow cytometry in peripheral blood mononuclear cells from 24 pediatric liver and kidney transplant recipients. The expression of CD38 and CD45RO on EBV-specific, tetramer-binding cells was also examined in a subset of patients by immunofluorescent staining and flow cytometry. RESULTS: Tetramer-binding CD8+ T cells were identified in 21 of 24 transplant recipients. EBV-specific CD8+ T cells were detected as early as 4 weeks after transplant in EBV seronegative patients receiving an organ from an EBV seropositive donor. The frequencies (expressed as a percentage of the CD8+ T cells) of the tetramer-binding cells were HLA-B8-RAKFKQLL (BZLF1 lytic antigen peptide) tetramer, range=0.96 to 3.94%; HLA-B8-FLRGRAYGL (EBNA3A latent antigen peptide) tetramer, range=0.03 to 0.59%; and HLA-A2-GLCTLVAML (BMLF1 lytic antigen peptide) tetramer, range=0.06 to 0.76%. The majority of tetramer reactive cells displayed an activated/memory phenotype. CONCLUSIONS: Pediatric transplant recipients receiving immunosuppression can generate EBV-specific CD8+ T cells. Phenotypic and functional analysis of tetramer cells may prove useful in defining and monitoring EBV infection in the posttransplant patient.

Constitutive activation of Jak/STAT proteins in Epstein-Barr virus-infected B-cell lines from patients with posttransplant lymphoproliferative disorder.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a major complication after bone marrow and solid organ transplantation. The disease encompasses a spectrum of abnormal, Epstein-Barr virus (EBV)-associated B-cell proliferations. We have previously shown that EBV-infected, spontaneous lymphoblastoid cell lines (SLCL) derived from PTLD patients require autocrine interleukin (IL)-10 to proliferate. To determine if cytokine signal transduction is involved in the autonomous growth of the SLCL, the activation states of the Jak/STAT signaling pathway proteins were analyzed in three different SLCL, termed JB7, MF4, and VB4. METHODS: The tyrosine phosphorylation (P-tyr) states of the Janus kinases (Jaks) and signal transducers and activators of transcription (STAT) proteins were examined by immunoprecipitation and immunoblot. Activated STAT dimer formation was determined by electromobility shift assays. RESULTS: All three SLCL, but not the Daudi Burkitt's lymphoma B-cell line, expressed the four known Jak kinases constitutively tyrosine phosphorylated, with particularly high levels of P-tyr Jak1 in the JB7 line. STAT1 and STAT3, but not STAT2 or STAT5, are also constitutively activated in all SLCL. The ability of the activated STAT proteins to form DNA-binding dimers was confirmed by electromobility shift assay. The SLCL, but not the Daudi line, express activated STAT complexes composed of STAT1 and STAT3. Another EBV-infected B-cell line, isolated from a lymph node biopsy after kidney transplantation, is phenotypically similar to the other SLCL in both surface antigen and activated STAT1 and STAT3 expression. CONCLUSION: These data support the presence of a constitutively active autocrine signaling pathway consistent with IL-10 in the SLCL.