A replicable CD271+ mesenchymal stromal cell density score: bringing the dysfunctional myelodysplastic syndrome niche to the diagnostic laboratory.
Leukemia & lymphoma
E2A-PBX1 remodels oncogenic signaling networks in B-cell precursor acute lymphoid leukemia.
Mesenchymal stromal cell density is increased in higher grade myelodysplastic syndromes and independently predicts survival.
American journal of clinical pathology
2014; 142 (6): 795-802
There is limited understanding of how signaling pathways are altered by oncogenic fusion transcription factors that drive leukemogenesis. To address this, we interrogated activated signaling pathways in a comparative analysis of mouse and human leukemias expressing the fusion protein E2A-PBX1, which is present in 5%-7% of pediatric and 50% of pre-B-cell receptor (preBCR(+)) acute lymphocytic leukemia (ALL). In this study, we describe remodeling of signaling networks by E2A-PBX1 in pre-B-ALL, which results in hyperactivation of the key oncogenic effector enzyme PLCγ2. Depletion of PLCγ2 reduced proliferation of mouse and human ALLs, including E2A-PBX1 leukemias, and increased disease-free survival after secondary transplantation. Mechanistically, E2A-PBX1 bound promoter regulatory regions and activated the transcription of its key target genes ZAP70, SYK, and LCK, which encode kinases upstream of PLCγ2. Depletion of the respective upstream kinases decreased cell proliferation and phosphorylated levels of PLCγ2 (pPLCγ2). Pairwise silencing of ZAP70, SYK, or LCK showed additive effects on cell growth inhibition, providing a rationale for combination therapy with inhibitors of these kinases. Accordingly, inhibitors such as the SRC family kinase (SFK) inhibitor dasatinib reduced pPLCγ2 and inhibited proliferation of human and mouse preBCR(+)/E2A-PBX1(+) leukemias in vitro and in vivo Furthermore, combining small-molecule inhibition of SYK, LCK, and SFK showed synergistic interactions and preclinical efficacy in the same setting. Our results show how the oncogenic fusion protein E2A-PBX1 perturbs signaling pathways upstream of PLCγ2 and renders leukemias amenable to targeted therapeutic inhibition. Cancer Res; 76(23); 6937-49. ©2016 AACR.
View details for PubMedID 27758892
SH2B1 (SH2-B) and JAK2: a multifunctional adaptor protein and kinase made for each other
TRENDS IN ENDOCRINOLOGY AND METABOLISM
2007; 18 (1): 38-45
We retrospectively tested the prognostic and diagnostic significance of CD271+ mesenchymal stromal cell (MSC) density in cytopenic patients who underwent bone marrow biopsy to evaluate for myelodysplastic syndromes (MDS).CD271+ MSC density was quantitated by automated image analysis of tissue microarray cores in 125 cytopenic patients: 40 lower grade MDS (<5% marrow blasts), 24 higher grade MDS, and 61 benign.CD271+ MSC density was increased in higher grade MDS compared with benign (P = .006) and lower grade MDS (P = .02). CD271+ MSC density was predictive of survival among patients with MDS independent of Revised International Prognostic Scoring System (IPSS-R), history of transfusion, therapy-related MDS, and fibrosis (hazard ratio, 3.4; P < .001). Among low or intermediate IPSS-R patients, median survival was significantly shorter in the high CD271+ MSC density group (47 vs 18 months, P < .02).High CD271+ MSC density is characteristic of higher grade MDS and is associated with poor risk independent of known prognostic factors.
View details for DOI 10.1309/AJCP71OPHKOTLSUG
View details for PubMedID 25389333
Binding of SH2-B family members within a potential negative regulatory region maintains JAK2 in an active state
MOLECULAR AND CELLULAR BIOLOGY
2006; 26 (17): 6381-6394
Src homology 2 (SH2) B adaptor protein 1 (SH2B1; originally named SH2-B) is a member of a family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase (JAK) and receptor tyrosine kinases. Although SH2B1 performs classical adaptor functions, such as recruitment of specific proteins to activated receptors, it also demonstrates a unique ability to enhance the kinase activity of the cytokine receptor-associated tyrosine kinase JAK2, as well as that of several receptor tyrosine kinases. SH2B1 is also among a small number of adaptor proteins shown to undergo nucleocytoplasmic shuttling, although its exact role within the nucleus is not yet clear. Deletion of the SH2B1 gene results in severe obesity and both leptin and insulin resistance, as well as infertility, which might be a consequence of resistance to insulin-like growth factor I. Thus, knockout mice support a role for SH2B1 as a positive regulator of JAK2 signaling pathways initiated by leptin, as well as of pathways initiated by insulin and, potentially, by insulin-like growth factor I.
View details for DOI 10.1016/j.tem.2006.11.007
View details for Web of Science ID 000243706900006
View details for PubMedID 17140804
Capillary electrophoresis and fluorescence anisotropy for quantitative analysis of peptide-protein interactions using JAK2 and SH2-B beta as a model system
2005; 77 (8): 2482-2489
The tyrosine kinase Janus kinase 2 (JAK2) transduces signaling for the majority of known cytokine receptor family members and is constitutively activated in some cancers. Here we examine the mechanisms by which the adapter proteins SH2-Bbeta and APS regulate the activity of JAK2. We show that like SH2-Bbeta, APS binds JAK2 at multiple sites and that binding to phosphotyrosine 813 is essential for APS to increase active JAK2 and to be phosphorylated by JAK2. Binding of APS to a phosphotyrosine 813-independent site inhibits JAK2. Both APS and SH2-Bbeta increase JAK2 activity independent of their N-terminal dimerization domains. SH2-Bbeta-induced increases in JAK2 dimerization require only the SH2 domain and only one SH2-Bbeta to be bound to a JAK2 dimer. JAK2 mutations and truncations revealed that amino acids 809 to 811 in JAK2 are a critical component of a larger regulatory region within JAK2, most likely including amino acids within the JAK homology 1 (JH1) and JH2 domains and possibly the FERM domain. Together, our data suggest that SH2-Bbeta and APS do not activate JAK2 as a consequence of their own dimerization, recruitment of an activator of JAK2, or direct competition with a JAK2 inhibitor for binding to JAK2. Rather, they most likely induce or stabilize an active conformation of JAK2.
View details for DOI 10.1128/MCB.00570-06
View details for Web of Science ID 000239848800006
View details for PubMedID 16914724
Tyrosine 813 is a site of JAK2 autophosphorylation critical for activation of JAK2 by SH2-B beta
MOLECULAR AND CELLULAR BIOLOGY
2004; 24 (10): 4557-4570
Fluorescence anisotropy capillary electrophoresis (FACE) and affinity probe capillary electrophoresis (APCE) with laser-induced fluorescence detection were evaluated for analysis of peptide-protein interactions with rapid binding kinetics. The Src homology 2 domain of protein SH2-Bbeta (SH2-Bbeta (525-670)) and a tyrosine-phosphorylated peptide corresponding to the binding sequence of JAK2 were used as a model system. For peptide labeled with fluorescein, the K(d) = 82 +/- 7 nM as measured by fluorescence anisotropy (FA). APCE assays had a limit of detection (LOD) of 100 nM or 12 amol injected for SH2-Bbeta (525-670). The separation time of 4 s, achieved using an electric field of 2860 V/cm on 7-cm-long capillaries, was on the same time scale as complex dissociation allowing K(d) (101 +/- 12 nM in good agreement with FA measurements) and dissociation rate (k(off) = 0.95 +/- 0.02 s(-)(1) corresponding to a half-life of 0.73 s) to be determined. This measurement represents a 30-fold higher rate of complex dissociation than what had previously been measurable by nonequilibrium CE analysis of equilibrium mixtures. Using FACE, the protein was detected with an LOD of 300 nM or 7.5 fmol injected. FACE was not used for determining K(d) or k(off); however, this method provided better separation resolution for multiple forms of the protein than APCE. Both methods were found suitable for analysis of cell lysate. These results demonstrate that FACE and APCE may be useful complements to existing techniques for exploring binding interactions with rapid kinetics.
View details for DOI 10.1021/ac048307u
View details for Web of Science ID 000228605100027
View details for PubMedID 15828784
The tyrosine kinase Janus kinase 2 (JAK2) binds to the majority of the known members of the cytokine family of receptors. Ligand-receptor binding leads to activation of the associated JAK2 molecules, resulting in rapid autophosphorylation of multiple tyrosines within JAK2. Phosphotyrosines can then serve as docking sites for downstream JAK2 signaling molecules. Despite the importance of these phosphotyrosines in JAK2 function, only a few sites and binding partners have been identified. Using two-dimensional phosphopeptide mapping and a phosphospecific antibody, we identified tyrosine 813 as a site of JAK2 autophosphorylation of overexpressed JAK2 and endogenous JAK2 activated by growth hormone. Tyrosine 813 is contained within a YXXL sequence motif associated with several other identified JAK2 phosphorylation sites. We show that phosphorylation of tyrosine 813 is required for the SH2 domain-containing adapter protein SH2-B beta to bind JAK2 and to enhance the activity of JAK2 and STAT5B. The homologous tyrosine in JAK3, tyrosine 785, is autophosphorylated in response to interleukin-2 stimulation and is required for SH2-B beta to bind JAK3. Taken together these data strongly suggest that tyrosine 813 is a site of autophosphorylation in JAK2 and is the SH2-B beta-binding site within JAK2 that is required for SH2-B beta to enhance activation of JAK2.
View details for DOI 10.1128/MCB.24.10.4557-4570.2004
View details for Web of Science ID 000221440900042
View details for PubMedID 15121872