Akinori Yoda

Down syndrome acute lymphoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group.

We report gene expression and other analyses to elucidate the molecular characteristics of acute lymphoblastic leukemia (ALL) in children with Down syndrome (DS). We find that by gene expression DS-ALL is a highly heterogeneous disease not definable as a unique entity. Nevertheless, 62% (33/53) of the DS-ALL samples analyzed were characterized by high expression of the type I cytokine receptor CRLF2 caused by either immunoglobulin heavy locus (IgH@) translocations or by interstitial deletions creating chimeric transcripts P2RY8-CRLF2. In 3 of these 33 patients, a novel activating somatic mutation, F232C in CRLF2, was identified. Consistent with our previous research, mutations in R683 of JAK2 were identified in 10 specimens (19% of the patients) and, interestingly, all 10 had high CRLF2 expression. Cytokine receptor-like factor 2 (CRLF2) and mutated Janus kinase 2 (Jak2) cooperated in conferring cytokine-independent growth to BaF3 pro-B cells. Intriguingly, the gene expression signature of DS-ALL is enriched with DNA damage and BCL6 responsive genes, suggesting the possibility of B-cell lymphocytic genomic instability. Thus, DS confers increased risk for genetically highly diverse ALLs with frequent overexpression of CRLF2, associated with activating mutations in the receptor itself or in JAK2. Our data also suggest that the majority of DS children with ALL may benefit from therapy blocking the CRLF2/JAK2 pathways.

Functional screening identifies CRLF2 in precursor B-cell acute lymphoblastic leukemia

The prognosis for adults with precursor B-cell acute lymphoblastic leukemia (B-ALL) remains poor, in part from a lack of therapeutic targets. We identified the type I cytokine receptor subunit CRLF2 in a functional screen for B-ALL–derived mRNA transcripts that can substitute for IL3 signaling. We demonstrate that CRLF2 is overexpressed in approximately 15% of adult and high-risk pediatric B-ALL that lack MLL, TCF3, TEL, and BCR/ABL rearrangements, but not in B-ALL with these rearrangements or other lymphoid malignancies. CRLF2 overexpression can result from translocation with the IGH locus or intrachromosomal deletion and is associated with poor outcome. CRLF2 overexpressing B-ALLs share a transcriptional signature that significantly overlaps with a BCR/ABL signature, and is enriched for genes involved in cytokine receptor and JAK-STAT signaling. In a subset of cases, CRLF2 harbors a Phe232Cys gain-of-function mutation that promotes constitutive dimerization and cytokine independent growth. A mutually exclusive subset harbors activating mutations in JAK2. In fact, all 22 B-ALLs with mutant JAK2 that we analyzed overexpress CRLF2, distinguishing CRLF2 as the key scaffold for mutant JAK2 signaling in B-ALL. Expression of WT CRLF2 with mutant JAK2 also promotes cytokine independent growth that, unlike CRLF2 Phe232Cys or ligand-induced signaling by WT CRLF2, is accompanied by JAK2 phosphorylation. Finally, cells dependent on CRLF2 signaling are sensitive to small molecule inhibitors of either JAKs or protein kinase C family kinases. Together, these findings implicate CRLF2 as an important factor in B-ALL with diagnostic, prognostic, and therapeutic implications.

Genetic resistance to JAK2 enzymatic inhibitors is overcome by HSP90 inhibition

Hsp90 inhibition in B cell acute lymphoblastic leukemia overcomes resistance to JAK2 inhibitors.

Molecular Ontogeny of Donor-Derived Follicular Lymphomas Occurring after Hematopoietic Cell Transplantation

UNLABELLED The relative timing of genetic alterations that contribute to follicular lymphoma remains unknown. We analyzed a donor-recipient pair who both developed grade 2/3A follicular lymphoma 7 years after allogeneic transplantation and donor lymphocyte infusions. Both patients harbored identical BCL2/IGH rearrangements also present in 1 in 2,000 cells in the donor lymphocyte infusion, and the same V(D)J rearrangement, which underwent somatic hypermutation both before and after clonal divergence. Exome sequencing of both follicular lymphomas identified 15 shared mutations, of which 14 (including alterations in EP300 and KLHL6) were recovered from the donor lymphocyte infusion by ultra-deep sequencing (average read coverage, 361,723), indicating acquisition at least 7 years before clinical presentation. Six additional mutations were present in only one follicular lymphoma and not the donor lymphocyte infusion, including an ARID1A premature stop, indicating later acquisition during clonal divergence. Thus, ultrasensitive sequencing can map clonal evolution within rare subpopulations during human lymphomagenesis in vivo. SIGNIFICANCE For the first time, we define the molecular ontogeny of follicular lymphoma during clonal evolution in vivo. By using ultrasensitive mutation detection, we mapped the time-course of somatic alterations after passage of a malignant ancestor by hematopoietic cell transplantation.

TSLP Signaling Network Revealed by SILAC-Based Phosphoproteomics

Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets for TSLP/TSLPR-associated diseases in humans.

Low frequency clonal mutations recoverable by deep sequencing in patients with aplastic anemia

Low frequency clonal mutations recoverable by deep sequencing in patients with aplastic anemia

TSLP signaling pathway map: a platform for analysis of TSLP-mediated signaling

Thymic stromal lymphopoietin (TSLP) is a four-helix bundle cytokine that plays a critical role in the regulation of immune responses and in the differentiation of hematopoietic cells. TSLP signals through a heterodimeric receptor complex consisting of an interleukin-7 receptor α chain and a unique TSLP receptor (TSLPR) [also known as cytokine receptor-like factor 2 (CRLF2)]. Cellular targets of TSLP include dendritic cells, B cells, mast cells, regulatory T (Treg) cells and CD4+ and CD8+ T cells. The TSLP/TSLPR axis can activate multiple signaling transduction pathways including the JAK/STAT pathway and the PI-3 kinase pathway. Aberrant TSLP/TSLPR signaling has been associated with a variety of human diseases including asthma, atopic dermatitis, nasal polyposis, inflammatory bowel disease, eosinophilic eosophagitis and, most recently, acute lymphoblastic leukemia. A centralized resource of the TSLP signaling pathway cataloging signaling events is not yet available. In this study, we present a literature-annotated resource of reactions in the TSLP signaling pathway. This pathway map is publicly available through NetPath (http://www.netpath.org/), an open access signal transduction pathway resource developed previously by our group. This map includes 236 molecules and 252 reactions that are involved in TSLP/TSLPR signaling pathway. We expect that the TSLP signaling pathway map will provide a rich resource to study the biology of this important cytokine as well as to identify novel therapeutic targets for diseases associated with dysregulated TSLP/TSLPR signaling. Database URL: http://www.netpath.org/pathways?path_id=NetPath_24

Mutations in G protein β subunits promote transformation and kinase inhibitor resistance

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4–5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

BCL2 Suppresses PARP1 Function and Nonapoptotic Cell Death

BCL2 suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating outer mitochondrial membrane permeabilization. Many tumor types, including B-cell lymphomas and chronic lymphocytic leukemia, are dependent on BCL2 for survival but become resistant to apoptosis after treatment. Here, we identified a direct interaction between the antiapoptotic protein BCL2 and the enzyme PARP1, which suppresses PARP1 enzymatic activity and inhibits PARP1-dependent DNA repair in diffuse large B-cell lymphoma cells. The BH3 mimetic ABT-737 displaced PARP1 from BCL2 in a dose-dependent manner, reestablishing PARP1 activity and DNA repair and promoting nonapoptotic cell death. This form of cell death was unaffected by resistance to single-agent ABT-737 that results from upregulation of antiapoptotic BCL2 family members. On the basis of the ability of BCL2 to suppress PARP1 function, we hypothesized that ectopic BCL2 expression would kill PARP inhibitor-sensitive cells. Strikingly, BCL2 expression reduced the survival of PARP inhibitor-sensitive breast cancer and lung cancer cells by 90% to 100%, and these effects were reversed by ABT-737. Taken together, our findings show that a novel interaction between BCL2 and PARP1 blocks PARP1 enzymatic activity and suppresses PARP1-dependent repair. Targeted disruption of the BCL2-PARP1 interaction therefore may represent a potential therapeutic approach for BCL2-expressing tumors resistant to apoptosis.

Differences in signaling through the B-cell leukemia oncoprotein CRLF2 in response to TSLP and through mutant JAK2

Approximately 10% of B-cell acute lymphoblastic leukemias (B-ALLs) overexpress the cytokine receptor subunit CRLF2, which may confer a poor prognosis. CRLF2 binds its ligand thymic stromal lymphopoietin (TSLP) as a heterodimer with IL7R. Subsets of CRLF2-overexpressing B-ALLs also have a gain-of-function CRLF2 F232C mutation or activating mutations in JAK2. Whether these mutant alleles confer differences in signaling has not been addressed. Through a domain mutation analysis, we demonstrate a distinct dependence on the CRLF2 intracellular tyrosine Y368 in signaling by CRLF2 F232C, but not signaling induced by TSLP or through CRLF2/mutant JAK2. In contrast, CRLF2 signaling in each context is strictly dependent on both the CRLF2 box1 domain and the intracellular tryptophan W286. Using a global quantitative analysis of tyrosine phosphorylation induced by TSLP, we previously identified TSLP-induced phosphorylation of multiple kinases implicated in B-cell receptor signaling, including Lyn, Btk, Hck, Syk, MAPK8, MAPK9, and MAPK10. We now demonstrate that cells dependent on CRLF2/mutant JAK2 have reduced phosphorylation at these targets, suggesting that the kinases promote TSLP-mediated proliferation but serve as negative regulators of CRLF2/mutant JAK2 signaling. Thus, targetable nodes downstream of CRLF2 differ based on the presence or absence of additional mutations in CRLF2 signaling components.