Zahide Ozer

Bruton’s Tyrosine Kinase as an Inhibitor of the Fas/CD95 Death-inducing Signaling Complex

Bruton’s tyrosine kinase (BTK) is a member of the Src-related Tec family of protein tyrosine kinases. Mutations in the btk gene have been linked to severe developmental blocks in human B-cell ontogeny leading to X-linked agammaglobulinemia. Here, we provide unique biochemical and genetic evidence that BTK is an inhibitor of the Fas/APO-1 death-inducing signaling complex in B-lineage lymphoid cells. The Src homology 2, pleckstrin homology (PH), and kinase domains of BTK are all individually important and apparently indispensable, but not sufficient, for its function as a negative regulator of Fas-mediated apoptosis. BTK associates with Fas via its kinase and PH domains and prevents the FAS-FADD interaction, which is essential for the recruitment and activation of FLICE by Fas during the apoptotic signal. Fas-resistant DT-40 lymphoma B-cells rendered BTK-deficient through targeted disruption of the btk gene by homologous recombination knockout underwent apoptosis after Fas ligation, but wild-type DT-40 cells or BTK-deficient DT-40 cells reconstituted with wild-type human btk gene did not. Introduction of an Src homology 2 domain, a PH domain, or a kinase domain mutant humanbtk gene into BTK-deficient cells did not restore the resistance to Fas-mediated apoptosis. Introduction of wild-type BTK protein by electroporation rendered BTK-deficient DT-40 cells resistant to the apoptotic effects of Fas ligation. BTK-deficient RAMOS-1 human Burkitt’s leukemia cells underwent apoptosis after Fas ligation, whereas BTK-positive NALM-6-UM1 human B-cell precursor leukemia cells expressing similar levels of Fas did not. Treatment of the anti-Fas-resistant NALM-6-UM1 cells with the leflunomide metabolite analog α-cyano-β-methyl-β-hydroxy-N-(2, 5-dibromophenyl)propenamide, a potent inhibitor of BTK, abrogated the BTK-Fas association without affecting the expression levels of BTK or Fas and rendered them sensitive to Fas-mediated apoptosis. The ability of BTK to inhibit the pro-apoptotic effects of Fas ligation prompts the hypothesis that apoptosis of developing B-cell precursors during normal B-cell ontogeny may be reciprocally regulated by Fas and BTK.

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.

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.

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 κB 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 α‐cyano‐β‐hydroxy‐β‐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.

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.

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 phosphorylation 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-phosphorylation 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 non-lymphohematopoietic 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.

Abstract LB-365: Unfolded protein response regulator BIP/HSPA5 as a chemoresistance biomarker and molecular target in acute lymphoblastic leukemia

BIP/HSPA5, also known as glucose regulated protein (GRP) 78, is an endoplasmic reticulum (ER)-resident multifunctional molecular chaperone that represents a pivotal component of the pro-survival arm of the unfolded protein response (UPR) signaling pathway. We performed a comprehensive meta-analysis of 17 datasets and 668 leukemia/lymphoma cases using the Oncomine Clinical Research Database. 7 of 10 contrasts for high-grade B-lineage lymphoid malignancies exhibited signigficant up-regulation of BIP/HSPA5, including B-lineage acute lymphoblastic leukemia (ALL) (2-fold, P=5.9×10–10). Notably, the gene expression profiles of primary leukemia cells in matched-pair leukemic specimens obtained from 27 B-lineage ALL patients both at the time of initial diagnosis and at the time of relapse were extracted from the NCBI Gene Expression Omnibus (GSE18497) revealed a significant 1.4-fold up-regulation of the BIP8/HSPA5 gene in relapse clones (P=0.0016). Likewise, anti-BIP/HSPA5 Western blot analysis of whole cell lysates from primary leukemic cells from relapsed and newly diagnosed B-lineage ALL patients revealed higher levels of BIP/HSPA5 protein in relapse specimens. Confocal microscopy revealed abundant expression of BIP/HSPA5 protein in the plasma membrane of chemotherapy-resistant ALL cells. Pep42, a cyclic 13-mer cell-penetrating peptide that was identified by screening a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cells, was found to not only specifically bind to surface expressed BIP/HSPA5 but also enter BIP/HSPA5+ cells by receptor-mediated endocytosis. We examined the ability of a doxorubicin conjugate of Pep42 (PEP-DOX) containing a cathepsin B-cleavable linker to target and induce apoptosis in surface BIP/HSPA5+ chemotherapy-resistant B-lineage ALL cells. By using a quantitative flow cytometric apoptosis assay, we documented that within 48 hours of treatment with 0.1–1.0 μM PEP-DOX (but not unconjugated drug-free Pep42, which was included as a control) apoptosis is induced in ≥99.9% of surface BIP/HSPA5+ ALL cells. As PEP-DOX specifically targets surface expressed BIP/HSPA5, 100-fold molar excess of drug-free Pep42 was able to compete with PEP-DOX for surface BIP/HSPA5 binding sites and thereby prevent PEP-DOX-induced apoptosis. Excess recombinant EGF, that was included as a negative control, did not affect PEP-DOX- induced apoptosis. The identification of the BIP/HSPA5 as a chemoresistance biomarker and molecular target for B-lineage ALL may lead to new anti-leukemic treatment strategies for relapsed disease that are much needed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-365. doi:10.1158/1538-7445.AM2011-LB-365