Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer

Even though SYK and ZAP70 kinases share high sequence homology and serve analogous functions, their expression in B and T cells is strictly segregated throughout evolution. Here, we identified aberrant ZAP70 expression as a common feature in a broad range of B cell malignancies. We validated SYK as the kinase that sets the thresholds for negative selection of autoreactive and premalignant clones. When aberrantly expressed in B cells, ZAP70 competes with SYK at the BCR signalosome and redirects SYK from negative selection to tonic PI3K signaling, thereby promoting B cell survival. In genetic mouse models for B-ALL and B-CLL, conditional expression of Zap70 accelerated disease onset, while genetic deletion impaired malignant transformation. Inducible activation of Zap70 during B cell development compromised negative selection of autoreactive B cells, resulting in pervasive autoantibody production. Strict segregation of the two kinases is critical for normal B cell selection and represents a central safeguard against the development of autoimmune disease and B cell malignancies.acceptedVersionPeer reviewe

Investigation of the role and mechanism of beta-catenin activation in acute myeloid leukaemia.

Aberrant activation of β-catenin is a common event in Acute Myeloid Leukaemia (AML), and accumulating evidence indicates this pathway plays a critical role in the establishment and maintenance of myeloid neoplasms. In AML, increased β-catenin signalling has been associated with activating mutations in the FLT3 receptor, and the oncogenic AML1-ETO and PML-RARα translocation products. In the absence of these lesions, however, it remains unclear which mechanisms may activate β-catenin in AML more broadly. Here we have explored a potential role for the multipotent haematopoietic cytokine, interleukin-3 (IL-3) in the regulation of β-catenin signalling in myeloid and leukaemic cells. We show that IL-3 can induce the dose dependent stabilisation of β-catenin in a myeloid model of Hox oncogenesis, and that β-catenin is required for IL-3 driven colony formation and growth. Enforced expression of β-catenin in this system allows cell survival at sub-optimal concentrations of IL-3 which may contribute to leukaemic transformation by providing a survival advantage to blast cells in the haematopoietic niche. We also demonstrate that IL-3 can promote β-catenin activation in the IL-3 dependent human erythroleukaemia cell line, TF-1.8, and in primary AML cells. Furthermore, Affymetrix gene expression analysis of bone marrow cells from four AML patients treated ± IL-3 revealed a strong correlation between the IL-3 induced signature and Wnt/β-catenin gene networks. Interestingly, the IL-3 receptor alpha subunit (IL-3Rα) has been previously shown to be overexpressed in AML leukaemic stem cells and progenitors compared to normal counterparts, and elevated levels of IL-3Rα are associated with poor prognosis and overall survival. Consistent with the regulation of β-catenin by IL-3, we show that a neutralising monoclonal antibody (7G3) which targets IL-3Rα, inhibits IL-3 mediated activation of β-catenin in TF-1.8 and primary AML cells. Modified versions of 7G3 are currently undergoing clinical trials for patients with AML, and our data indicates that this therapy may be more effective for patients with elevated levels of oncogenic β-catenin. As previous studies have demonstrated that cytokines can induce the inhibitory phosphorylation of GSK3β via activation of the PI3K/AKT pathway, we have also made use of pharmacological PI3K and AKT small molecule inhibitors to determine the importance of this axis in the IL-3 mediated regulation of β-catenin. On the whole, the work in this thesis reveals a novel mechanism which may contribute to β-catenin activation in AML, and provides further insight into the amplitude of IL-3 signalling in normal and malignant haematopoiesis.Thesis (Ph.D.) -- University of Adelaide, School of Molecular & Biomedical Science, 201

PON2 subverts metabolic gatekeeper functions in B cells to promote leukemogenesis

Unlike other cell types, developing B cells undergo multiple rounds of somatic recombination and hypermutation to evolve high-affinity antibodies. Reflecting the high frequency of DNA double-strand breaks, adaptive immune protection by B cells comes with an increased risk of malignant transformation. B lymphoid transcription factors (e.g., IKZF1 and PAX5) serve as metabolic gatekeepers by limiting glucose to levels insufficient to fuel transformation. We here identified aberrant expression of the lactonase PON2 in B cell acute lymphoblastic leukemia (B-ALL) as a mechanism to bypass metabolic gatekeeper functions. Compared to normal pre-B cells, PON2 expression was elevated in patient-derived B-ALL samples and correlated with poor clinical outcomes in pediatric and adult cohorts. Genetic deletion of Pon2 had no measurable impact on normal B cell development. However, in mouse models for BCR-ABL1 and NRASG12D-driven B-ALL, deletion of Pon2 compromised proliferation, colony formation, and leukemia initiation in transplant recipient mice. Compromised leukemogenesis resulted from defective glucose uptake and adenosine triphosphate (ATP) production in PON2-deficient murine and human B-ALL cells. Mechanistically, PON2 enabled glucose uptake by releasing the glucose-transporter GLUT1 from its inhibitor stomatin (STOM) and genetic deletion of STOM largely rescued PON2 deficiency. While not required for glucose transport, the PON2 lactonase moiety hydrolyzes the lactone-prodrug 3OC12 to form a cytotoxic intermediate. Mirroring PON2 expression levels in B-ALL, 3OC12 selectively killed patient-derived B-ALL cells but was well tolerated in transplant recipient mice. Hence, while B-ALL cells critically depend on aberrant PON2 expression to evade metabolic gatekeeper functions, PON2 lactonase activity can be leveraged as synthetic lethality to overcome drug resistance in refractory B-ALL

The GM-CSF receptor utilizes β-catenin and Tcf4 to specify macrophage lineage differentiation

Granulocyte–macrophage colony stimulating factor (GM-CSF) promotes the growth, survival, differentiation and activation of normal myeloid cells and is essential for fully functional macrophage differentiation in vivo. To better understand the mechanisms by which growth factors control the balance between proliferation and self-renewal versus growth-suppression and differentiation we have used the bi-potent FDB1 myeloid cell line, which proliferates in IL-3 and differentiates to granulocytes and macrophages in response to GM-CSF. This provides a manipulable model in which to dissect the switch between growth and differentiation. We show that, in the context of signaling from an activating mutant of the GM-CSF receptor β subunit, a single intracellular tyrosine residue (Y577) mediates the granulocyte fate decision. Loss of granulocyte differentiation in a Y577F second-site mutant is accompanied by enhanced macrophage differentiation and accumulation of β-catenin together with activation of Tcf4 and other Wnt target genes. These include the known macrophage lineage inducer, Egr1. We show that forced expression of Tcf4 or a stabilised β-catenin mutant is sufficient to promote macrophage differentiation in response to GM-CSF and that GM-CSF can regulate β-catenin stability, most likely via GSK3β. Consistent with this pathway being active in primary cells we show that inhibition of GSK3β activity promotes the formation of macrophage colonies at the expense of granulocyte colonies in response to GM-CSF. This study therefore identifies a novel pathway through which growth factor receptor signaling can interact with transcriptional regulators to influence lineage choice during myeloid differentiation.

B-Cell-Specific Diversion of Glucose Carbon Utilization Reveals a Unique Vulnerability in B Cell Malignancies

B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD

Comparative performance of δ13C, δ18O and δ15N for phenotyping durum wheat adaptation to a dryland environment

International audienceGrain yield and the natural abundance of the stable isotope compositions of carbon (delta C-13), oxygen (delta O-18) and nitrogen (delta N-15) of mature kernels were measured during 3 consecutive years in 10 durum wheat genotypes (five landraces and five modern cultivars) subjected to different water and N availabilities in a Mediterranean location and encompassing a total of 12 trials. Water limitation was the main environmental factor affecting yield, delta C-13 and delta O-18, whereas N fertilisation had a major effect on delta N-15. The genotypic effect was significant for yield, yield components, delta C-13, delta O-18 and delta N-15. Landraces exhibited a higher delta C-13 and delta N-15 than cultivars. Phenotypic correlations of delta C-13 and delta O-18 with grain yield were negative, suggesting that genotypes able to sustain a higher water use and stomatal conductance were the most productive and best adapted; delta N-15 was also negatively correlated with grain yield regardless of the growing conditions. delta C-13 was the best isotopic trait in terms of genetic correlation with yield and heritability, whereas delta O-18 was the worst of the three isotopic abundances. The physiological basis for the different performance of the three isotopes explaining the genotypic variability in yield is discussed