Serine phosphorylation by SYK is critical for nuclear localization and transcription factor function of Ikaros.

Ikaros is a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis through transcriptional regulation of the earliest stages of lymphocyte ontogeny and differentiation. Functional deficiency of Ikaros has been implicated in the pathogenesis of acute lymphoblastic leukemia, the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros activity is considered of paramount importance, but the operative molecular mechanisms responsible for its regulation remain largely unknown. Here we provide multifaceted genetic and biochemical evidence for a previously unknown function of spleen tyrosine kinase (SYK) as a partner and posttranslational regulator of Ikaros. We demonstrate that SYK phoshorylates Ikaros at unique C-terminal serine phosphorylation sites S358 and S361, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Mechanistically, we establish that SYK-induced Ikaros activation is essential for its nuclear localization and optimal transcription factor function.

STAT3 is a substrate of SYK tyrosine kinase in B-lineage leukemia/lymphoma cells exposed to oxidative stress.

We provide unprecedented genetic and biochemical evidence that the antiapoptotic transcription factor STAT3 serves as a substrate for SYK tyrosine kinase both in vitro and in vivo. Induction of SYK in an ecdysone-inducible mammalian expression system results in STAT3 activation, as documented by tyrosine phosphorylation and nuclear translocation of STAT3, as well as amplified expression of several STAT3 target genes. STAT3 activation after oxidative stress (OS) is strongly diminished in DT40 chicken B-lineage lymphoma cells rendered SYK-deficient by targeted disruption of the syk gene. Introduction of a wild-type, C-terminal or N-terminal SH2 domain-mutated, but not a kinase domain-mutated, syk gene into SYK-deficient DT40 cells restores OS-induced enhancement of STAT-3 activity. Thus, SYK plays an important and indispensable role in OS-induced STAT3 activation and its catalytic SH1 domain is critical for this previously unknown regulatory function. These results provide evidence for the existence of a novel mode of cytokine-independent cross-talk that operates between SYK and STAT3 pathways and regulates apoptosis during OS. We further provide experimental evidence that SYK is capable of associating with and phosphorylating STAT3 in human B-lineage leukemia/lymphoma cells challenged with OS. In agreement with a prerequisite role of SYK in OS-induced STAT3 activation, OS does not induce tyrosine phosphorylation of STAT3 in SYK-deficient human proB leukemia cells. Notably, inhibition of SYK with a small molecule drug candidate prevents OS-induced activation of STAT3 and overcomes the resistance of human B-lineage leukemia/lymphoma cells to OS-induced apoptosis.

CD22 Exon 12 deletion is a characteristic genetic defect of therapy-refractory clones in paediatric acute lymphoblastic leukaemia.

Gene expression profiling (GEP) of primary leukaemic cells (PLC) from 157 paediatric B-lineage acute lymphoblastic leukaemia (ALL) patients, including a direct comparison of matched pair initial diagnosis versus first relapse leukaemic specimens, provided previously unknown evidence that relapse clones are characterized by significantly higher expression levels of a CD22 exon 12 deletion (CD22ΔE12)-associated signature transcriptome than the PLC from newly diagnosed patients. In agreement with and validating these GEP results, reverse transcription polymerase chain reaction and Western blot analysis of PLC from 19 of 19 paediatric ALL patients in first bone marrow relapse occurring within 12 months of the completion of primary therapy confirmed them to be CD22ΔE12(+). Likewise, PLC in diagnostic initial bone marrow specimens from seven of seven therapy-refractory newly diagnosed paediatric B-lineage ALL patients with <7 months event-free survival (EFS), including four patients with induction failures and three patients with early relapses, were CD22ΔE12(+), whereas PLC from only one of five newly diagnosed paediatric B-lineage ALL patients with >18 months EFS was CD22ΔE12(+). CD22ΔE12(+) could be detected in PLC of therapy-refractory patients both at the time of initial diagnosis as well as at the time of documented treatment failure. Our study implicates the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory paediatric B-lineage ALL.

Recombinant human CD19-ligand protein as a potent anti-leukaemic agent.

We report the cloning and characterization of a novel 54-kDa high-mobility group (HMG)-box protein as the ligand for the human pan-B cell co-receptor CD19 (CD19-L), which interacts with the extracellular domain of CD19 in trans. CD19-L is the first CD19-specific recombinant human protein with potent anti-leukaemic activity against B-lineage acute lymphoblastic leukaemia (ALL), the most common form of childhood cancer and the second most common form of acute leukaemia in adults. Soluble recombinant CD19-L protein exhibited exquisite specificity for the extracellular domain of CD19 and strong binding to the surface of B-lineage leukaemia/lymphoma cells. Engagement of CD19 co-receptor on B-lineage ALL cells with CD19-L perturbed the CD19-associated signalling network, altering the expression levels of multiple genes directly involved in regulation of apoptosis, and triggered rapid apoptotic cell death in a CD19-specific manner. The identification of human CD19-L may lead to therapeutic innovation for B-lineage ALL and other B-lineage lymphoid malignancies as well as B-cell lymphoproliferative states and systemic autoimmunity.

Inducing apoptosis in chemotherapy-resistant B-lineage acute lymphoblastic leukaemia cells by targeting HSPA5, a master regulator of the anti-apoptotic unfolded protein response signalling network.

We present previously unknown evidence that the immunoglobulin heavy chain binding protein BIP/HSPA5, also known as glucose regulated protein (GRP)78, serving as a pivotal component of the pro-survival axis of the unfolded protein response (UPR) signalling network, is abundantly expressed in relapsed B-lineage acute lymphoblastic leukaemia (ALL) and contributes to chemotherapy resistance of leukaemic B-cell precursors. The resistance of B-lineage ALL cells to the standard anti-leukaemic drug vincristine was overcome by the HSPA5 inhibitor epigallocatechin gallate, which inhibits the anti-apoptotic function of HSPA5 by targeting its ATP-binding domain. Notably, chemotherapy-resistant B-lineage ALL cells underwent apoptosis within 48 h of exposure to a doxorubicin-conjugated cell-penetrating cyclic anti-HSPA5 peptide targeting surface-expressed HSPA5 molecules on leukaemia cells. The identification of the HSPA5 as a chemoresistance biomarker and molecular target for B-lineage ALL may lead to new anti-leukaemic treatment strategies that are much needed.

Polo-like-kinase 1 (PLK1) as a molecular target to overcome SYK-mediated resistance of B-lineage acute lymphoblastic leukaemia cells to oxidative stress.

SYK tyrosine kinase has emerged as a master regulator of cellular resistance to oxidative stress (OS) by mediating the activation of the anti-apoptotic nuclear factor kappaB and phosphatidylinositol-3 kinase/AKT pathways after OS exposure. Here, we present unprecedented experimental evidence that polo-like kinase 1 (PLK1) is the upstream regulator of SYK in B-lineage acute lymphoblastic leukaemia (ALL) cells. Selective inhibition of PLK-1 with the leflunomide metabolite analogue alpha-cyano-beta-hydroxy-beta-methyl-N-[4-(trifluoromethoxy) phenyl]-propenamide/LFM-A12 abolished the resistance of B-lineage ALL cells to OS by preventing the activation of the anti-apoptotic SYK signal transduction pathway. Notably, LFM-A12 treatments at non-cytotoxic concentrations resulted in marked augmentation of clonogenic death in resistant human B-lineage ALL cell lines challenged with OS. Further, LFM-A12 augmented OS-induced apoptosis of chemotherapy-resistant primary leukaemic cells from relapsed B-lineage ALL patients in vitro and markedly potentiated the in vivo anti-leukaemic activity of total body irradiation (TBI) against leukaemia-initiating cells in severe combined immunodeficient mouse xenograft models of B-lineage ALL. This study is the first to identify PLK1 as a regulator of SYK tyrosine kinase and a molecular target to overcome SYK-mediated resistance of B-lineage ALL cells to OS.

One-Step, Rapid, 18F-19F Isotopic Exchange Radiolabeling of Difluoro-dioxaborinins: Substituent Effect on Stability and In Vivo Applications.

The ß-diketone moiety is commonly present in many anticancer drugs, antibiotics, and natural products. We describe a general method for radiolabeling ß-diketone-bearing molecules with fluoride-18. Radiolabeling was carried out via 18F-19F isotopic exchange on nonradioactive difluoro-dioxaborinins, which were generated by minimally modifying the ß-diketone as a difluoroborate. Radiochemistry was one-step, rapid (<10 min), and high-yielding (>80%) and proceeded at room temperature to accommodate the half-life of F-18 (t1/2 = 110 min). High molar activities (7.4 Ci/µmol) were achieved with relatively low starting activities (16.4 mCi). It was found that substituents affected both the solvolytic stability and fluorescence properties of difluoro-dioxaborinins. An F-18 radiolabeled difluoro-dioxaborinin probe that was simultaneously fluorescent showed sufficient stability for in vivo positron emission tomography (PET)/fluorescence imaging in mice, rabbits, and patients. These findings will guide the design of probes with specific PET/fluorescence properties; the development of new PET/fluorescence dual-modality reporters; and accurate in vivo tracking of ß-diketone molecules.

Anti-inflammatory activity profile of JANEX-1 in preclinical animal models.

We examined the biologic activity of the rationally designed JAK3 inhibitor, JANEX-1, in several cellular and animal models of inflammation. Notably, JANEX-1 exhibited potent anti-inflammatory activity in each of these preclinical models, including mouse models of peritonitis, colitis, cellulitis, and systemic inflammatory response syndrome. Therefore, JANEX-1 may prove useful as a broad-spectrum anti-inflammatory agent. The present study may provide the basis for new and effective treatment as well as prevention programs for inflammatory disorders.

Recombinant human CD19L-sTRAIL effectively targets B cell precursor acute lymphoblastic leukemia.

Patients with B cell precursor acute lymphoblastic leukemia (BPL) respond well to chemotherapy at initial diagnosis; however, therapeutic options are limited for individuals with BPL who relapse. Almost all BPL cells express CD19, and we recently cloned the gene encoding a natural ligand of the human CD19 receptor (CD19L). We hypothesized that fusion of CD19L to the soluble extracellular domain of proapoptotic TNF-related apoptosis-inducing ligand (sTRAIL) would markedly enhance the potency of sTRAIL and specifically induce BPL cell apoptosis due to membrane anchoring of sTRAIL and simultaneous activation of the CD19 and TRAIL receptor (TRAIL-R) apoptosis signaling pathways. Here, we demonstrate that recombinant human CD19L-sTRAIL was substantially more potent than sTRAIL and induced apoptosis in primary leukemia cells taken directly from BPL patients. CD19L-sTRAIL effectively targeted and eliminated in vivo clonogenic BPL xenograft cells, even at femtomolar-picomolar concentrations. In mice, CD19L-sTRAIL exhibited a more favorable pharmacokinetic (PK) profile than sTRAIL and was nontoxic at doses ranging from 32 fmol/kg to 3.2 pmol/kg. CD19L-sTRAIL showed potent in vivo antileukemic activity in NOD/SCID mouse xenograft models of relapsed and chemotherapy-resistant BPL at nontoxic fmol/kg dose levels. Together, these results suggest that recombinant human CD19L-sTRAIL has clinical potential as a biotherapeutic agent against BPL.

A PREVIOUSLY UNKNOWN UNIQUE CHALLENGE FOR INHIBITORS OF SYK ATP-BINDING SITE: ROLE OF SYK AS A CELL CYCLE CHECKPOINT REGULATOR.

The identification of SYK as a molecular target in B-lineage leukemia/lymphoma cells prompted the development of SYK inhibitors as a new class of anti-cancer drug candidates. Here we report that induction of the SYK gene expression in human cells causes a significant down-regulation of evolutionarily conserved genes associated with mitosis and cell cycle progression providing unprecedented evidence that SYK is a master regulator of cell cycle regulatory checkpoint genes in human cells. We further show that SYK regulates the G2 checkpoint by physically associating with and inhibiting the dual-specificity phosphatase CDC25C via phosporylation of its S216 residue. SYK depletion by RNA interference or treatment with the chemical SYK inhibitor prevented nocodazole-treated human cell lines from activating the G2 checkpoint via CDC25C S216-phoshorylation and resulted in polyploidy. Our study provides genetic and biochemical evidence that spleen tyrosine kinase (SYK) has a unique role in the activation of the G2 checkpoint in both nonlymphohematopoietic and B-lineage lymphoid cells. This previously unknown role of SYK as a cell cycle checkpoint regulator represents an unforeseen and significant challenge for inhibitors of SYK ATP binding site.

Regulatory phosphorylation of Ikaros by Bruton's tyrosine kinase.

Diminished Ikaros function has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of Ikaros is of paramount importance for normal lymphocyte ontogeny. Here we provide genetic and biochemical evidence for a previously unknown function of Bruton's tyrosine kinase (BTK) as a partner and posttranslational regulator of Ikaros, a zinc finger-containing DNA-binding protein that plays a pivotal role in immune homeostasis. We demonstrate that BTK phosphorylates Ikaros at unique phosphorylation sites S214 and S215 in the close vicinity of its zinc finger 4 (ZF4) within the DNA binding domain, thereby augmenting its nuclear localization and sequence-specific DNA binding activity. Our results further demonstrate that BTK-induced activating phosphorylation is critical for the optimal transcription factor function of Ikaros.

KU HAPLOINSUFFIENCY CAUSES A LYMPHOPROLIFERATIVE DISORDER OF IMMATURE T-CELL PRECURSORS DUE TO IKAROS MALFUNCTION.

Ikaros (IK) malfunction has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL), the most common form of childhood cancer. Therefore, a stringent regulation of IK activity is very important. Here we provide unique genetic and biochemical evidence that the Ku protein components Ku70 and Ku80 act as positive regulators of IK function via formation of IK-Ku70 and IK-Ku80 heterodimers with augmented sequence-specific DNA binding activity. siRNA-mediated depletion of Ku70 or Ku80 reduced the sequence-specific DNA binding activity of IK in EMSA as well as the RT-PCR measured IK target gene expression levels in human cells. The interaction of Ku components with IK likely contributes to the anti-leukemic effects of IK as a tumor suppressor, because Ku70 as well as Ku80 haploinsuffiency in mice caused development of a lymphoproliferative disorder (LPD) involving CD2+CD4+CD8+CD1+IL7R+ thymic T-cell precursors with functional IK deficiency.

Bruton's tyrosine kinase prevents activation of the anti-apoptotic transcription factor STAT3 and promotes apoptosis in neoplastic B-cells and B-cell precursors exposed to oxidative stress.

Bruton's tyrosine kinase (BTK) was previously demonstrated to be a mediator of oxidative stress-induced apoptosis in irradiated neoplastic B-cells and B-cell precursors. Defective BTK expression in leukaemic B-cell precursors from infants with t(4;11) acute lymphoblastic leukaemia has been associated with radiation resistance. The present study examined whether BTK mediates apoptosis during oxidative stress by interfering with the anti-apoptotic function of signal transducer and activator of transcription 3 (STAT3). BTK physically associated with and tyrosine phosphorylated STAT3; this association was promoted by pervanadate (PV)-induced oxidative stress. The BTK/STAT3 interaction appeared to prevent STAT3 response to oxidative stress, because PV-induced STAT3 activation was markedly enhanced in DT40 chicken lymphoma B-cells that were rendered BTK-deficient by targeted disruption of the btk gene as well as in BTK-deficient RAMOS-1 human lymphoma B-cells. These BTK-deficient cells were highly resistant to oxidative stress-induced apoptosis triggered by PV treatment. Reconstitution of BTK-deficient DT40 cells with wild-type human BTK gene eliminated the amplification of the STAT3 response and restored the PV-induced apoptotic signal. Similarly, while the BTK-positive NALM-6 human leukaemic B-cell precursor cell line showed no STAT3 activation after PV treatment and was exquisitely sensitive to PV-induced apoptosis, PV failed to induce apoptosis in BTK-deficient RAMOS-1 human lymphoma B-cells that showed a robust STAT3 response. These results provide unprecedented biochemical and genetic evidence for a unique mode of cross-talk that occurs between BTK and STAT3 pathways during oxidative stress, whereby BTK may trigger apoptosis via negative regulation of the anti-apoptotic STAT3 activity.

Structure-based design and engineering of a nontoxic recombinant pokeweed antiviral protein with potent anti-human immunodeficiency virus activity.

A molecular model of pokeweed antiviral protein (PAP)-RNA interactions was used to rationally engineer FLP-102((151)AA(152)) and FLP-105((191)AA(192)) as nontoxic PAPs with potent anti-human immunodeficiency virus (anti-HIV) activities. FLP-102 and FLP-105 have been produced in Escherichia coli and tested both in vitro and in vivo. These proteins depurinate HIV type 1 (HIV-1) RNA much better than rRNA and are more potent anti-HIV agents than native PAP or recombinant wild-type PAP. They are substantially less toxic than native PAP in BALB/c mice and exhibit potent in vivo activities against genotypically and phenotypically nucleoside reverse transcriptase inhibitor-resistant HIV-1 in a surrogate human peripheral blood lymphocyte (Hu-PBL) SCID mouse model of human AIDS. Rationally engineered nontoxic recombinant PAPs such as FLP-102 and FLP-105 may provide the basis for effective salvage therapies for patients harboring highly drug-resistant strains of HIV-1. The documented in vitro potencies of FLP-102 and FLP-105, their in vivo antiretroviral activities in the HIV-infected Hu-PBL SCID mouse model, and their favorable toxicity profiles in BALB/c mice warrant the further development of these promising new biotherapeutic agents.