Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1
ABSTRACT: Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GAB2 as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transduced Gab2-/- bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors PI3K or SHP2. GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph+ hematologic neoplasms. RNA-Seq expression profiling of 6 mouse bone marrow samples: 3 GAB2 WT (+/+) and 3 GAB2 NULL (-/-)
Project description:BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) have revolutionized treatment of Philadelphia chromosome-positive (Ph+) hematologic neoplasms. Nevertheless, acquired TKI resistance remains a major problem in chronic myeloid leukemia (CML), and TKIs are less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). GAB2, a scaffolding adaptor that binds and activates SHP2, is essential for leukemogenesis by BCR-ABL1, and a GAB2 mutant lacking SHP2 binding cannot mediate leukemogenesis. Using a genetic loss-of-function approach and bone marrow transplantation (BMT) models for CML and BCR-ABL1+ B-ALL, we show that SHP2 is required for BCR-ABL1-evoked myeloid and lymphoid neoplasia. Ptpn11 deletion impairs initiation and maintenance of CML-like myeloproliferative neoplasm, and compromises induction of BCR-ABL1+ B-ALL. SHP2, and specifically, its SH2 domains, PTP activity and C-terminal tyrosines, is essential for BCR-ABL1+, but not WT, pre-B cell proliferation. The MEK/ERK pathway is regulated by SHP2 in WT and BCR-ABL1+ pre-B cells, but is only required for the proliferation of BCR-ABL1+ cells. SHP2 is required for SRC family kinase (SFK) activation only in BCR-ABL1+ pre-B cells. RNAseq reveals distinct SHP2-dependent transcriptional programs in BCR-ABL1+ and WT pre-B cells. Our results suggest that SHP2, via SFKs and ERK, represses MXD3/4 to facilitate a MYC-dependent proliferation program in BCR-ABL1-transformed pre-B cells. Overall design: RNA-Seq expression profiling of 16 mouse pre-B cell samples: 4 BCR-ABL/SHP2+/+ 4 BCR-ABL/SHP2-/+ 4 BCR-ABL/SHP2+/- 4 BCR-ABL/SHP2-/-
Project description:The Philadelphia chromosome (Ph) encodes the oncogenic BCR-ABL1 tyrosine kinase, which is present in almost every patient with chronic myeloid leukemia. In this study, the tyrosine kinase inhibitor Imatinib was used for pharmacological inhibition of BCR-ABL1. Gene expression profiles of CML cell lines were analyzed in response to Imatinib treatment. Overall design: Three CML cell lines (KU-812, KCL-22, JURL-MK1) were either treated with 10 µM STI571 (Imatinib) for 16 hours or cultured in absence of STI571.
Project description:Chronic myeloid leukaemia (CML) is characterised by the presence of a fusion driver oncogene, BCR-ABL1, which is a constitutive tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are the central treatment strategy for CML patients and have significantly improved survival rates, but the T315I mutation in the kinase domain of BCR-ABL1 confers resistance to all clinically approved TKIs, except ponatinib. However, compound mutations can mediate resistance even to ponatinib and remain a clinical challenge in CML therapy. Here, we investigated a ponatinib-resistant CML patient through whole genome sequencing (WGS) to identify the cause of resistance and to find alternative therapeutic targets.
Project description:In chronic myeloid leukemia (CML) neoplastic stem cells (NCS) represent a critical target of therapy. However, little is known about markers and targets expressed in CML NSC. We examined the phenotype and function of CD34+/CD38─/Lin─ CML LSC by a multi-parameter screen approach employing antibody-phenotyping, mRNA expression profiling, and functional studies, followed by marker-validation using diverse control-cohorts and follow-up samples of CML patients treated with imatinib. We here show that in contrast to normal stem cells, CD34+/CD38− CML NSC express IL2RA (CD25), and that STAT5 induces expression of CD25 in Lin−/Sca-1+/Kit+ NSC (LSK) in C57/Bl6 mice. Correspondingly, expression of CD25 decreased in the human BCR/ABL1+ stem cell line KU812 upon shRNA-induced STAT5-depletion. The BCR/ABL1-inhibitors nilotinib and ponatinib were also found to decrease STAT5 activity and CD25-expression in KU812 cells and primary CML NSC. A CD25-targeting shRNA augmented the proliferation of KU812 cells in vitro and in vivo in NOD/SCID-IL2R-/- mice. In consecutive experiments the PI3K/mTOR-blocker BEZ235 was found to promote STAT5- and CD25 expression and to produce synergistic anti-neoplastic effects with nilotinib and ponatinib in CML cells. Together, CD25 is a novel STAT5-dependent marker and target in CML NSC. Overall design: To define differences in mRNA expression patterns in KU812 cells transduced with a random control shRNA or a shRNA against CD25, gene array analyses were performed.
Project description:Philadelphia (Ph) chromosome-positive leukemia is characterized by the BCR/ABL1 fusion protein that affects a wide range of signal transduction pathways. The knowledge about its downstream target genes is, however, still quite limited. To identify novel BCR/ABL1-regulated genes we used global gene expression profiling of several Ph-positive and Ph-negative cell lines treated with imatinib. Following imatinib treatment, the Ph-positive cells showed decreased growth, viability, and reduced phosphorylation of BCR/ABL1 and STAT5. In total, 142 genes were identified as being dependent on BCR/ABL1-mediated signaling, mainly including genes involved in signal transduction, e.g. the JAK/STAT, MAPK, TGFB and insulin signaling pathways, and in regulation of metabolism. Interestingly, BCR/ABL1 was found to activate several genes involved in negative feedback regulation (CISH, SOCS2, SOCS3, PIM1, DUSP6, and TNFAIP3), which may act to indirectly suppress the tumor promoting effects exerted by BCR/ABL1. In addition, several genes identified as deregulated upon BCR/ABL1 expression could be assigned to the TGFB and NFkB signaling pathways, as well as to reflect the metabolic adjustments needed for rapidly growing cells. Apart from providing important pathogenetic insights into BCR/ABL1-mediated leukemogenesis, the present study also provides a number of pathways/individual genes that may provide attractive targets for future development of targeted therapies. Keywords: global gene expression profiling, Ph chromosome, BCR/ABL1, imatinib mesylate Overall design: Five Philadelphia-positive (Ph+) and five Philadelphia-negative (Ph-) cell lines were cultured in the absence or presence of 1 microM imatinib mesylate for 3 and 12 hours. Total RNA was extracted from untreated and treated cell cultures at both time points, resulting in 4 samples per cell line. RNA extraction, labeling, hybridization, washing, scanning, and feature analysis were performed as described (Håkansson et al., 2008, Genes Chromosomes Cancer). Dye-swap labeling was performed in 32 out of 40 samples. In total, 72 slides were hybridized and scanned. The transcriptional response was studied using the mean value of the 3 and 12 hour microarray measurements.
Project description:To identify differences in the gene regulation between BCL6+/+ and BCL6-/- CML cells a gene expression analysis has been performed. We investigated the gene expression pattern in BCL6+/+ cells in the presence or absence of Imatinib and a combination of Imatinib and RI-BPI (a novel retro-inverso BCL6 peptide inhibitor). In BCL6-/- CML cells, we investigated the gene expression pattern in the presence or absence of Imatinib. BCR-ABL1 transformed myeloid cells from BCL6+/+ mice were cultured in the presence or absence of 10µM Imatinib or 10µM Imatinib and 20µM RI-BPI for 16 hours. BCR-ABL1 transformed myeloid cells from BCL6-/- mice were cultured in the presence or absence of 10µM Imatinib. Two samples for each condition were processed.
Project description:To identify differences in the gene regulation between BCL6+/+ and BCL6-/- CML cells a gene expression analysis has been performed. We investigated the gene expression pattern in BCL6+/+ cells in the presence or absence of Imatinib and a combination of Imatinib and RI-BPI (a novel retro-inverso BCL6 peptide inhibitor). In BCL6-/- CML cells, we investigated the gene expression pattern in the presence or absence of Imatinib. Overall design: BCR-ABL1 transformed myeloid cells from BCL6+/+ mice were cultured in the presence or absence of 10µM Imatinib or 10µM Imatinib and 20µM RI-BPI for 16 hours. BCR-ABL1 transformed myeloid cells from BCL6-/- mice were cultured in the presence or absence of 10µM Imatinib. Two samples for each condition were processed.
Project description:BCR-ABL1 lymphoblastic leukaemia is characterized by the deletion of Ikaros. The Philadelphia chromosome, encoding BCR-ABL1, is the defining lesion of chronic myelogenous leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL) cases. To define oncogenic lesions that cooperate with BCR-ABL1 to induce ALL, we performed genome-wide analysis of leukemic samples from 23 CML cases and 304 ALL cases, including 43 BCR-ABL1 B-ALL cases. IKZF1 (encoding the transcription factor Ikaros) was deleted in 83.7% of BCR-ABL1 B-ALL cases, but not in chronic phase CML. Deletion of IKZF1 was also identified as an acquired lesion in lymphoid blast crisis of CML. The IKZF1 deletions resulted in haploinsufficiency, expression of a dominant negative Ikaros isoform or the complete loss of Ikaros expression. Sequencing of IKZF1 deletion breakpoints suggested that aberrant V(D)J recombination is responsible for the deletions. These findings suggest that genetic lesions resulting in the loss of Ikaros function are a key event in the development of BCR-ABL1 ALL. *** Due to privacy concerns, the primary SNP array data is no longer available with unrestricted access. Individuals wishing to obtain this data for research purposes may request access using the Web links below. *** This SuperSeries is composed of the SubSeries listed below. Overall design: Affymetrix SNP arrays were performed according to the maufacturers directions on DNA extracted from cryopreserved diagnostic bone marrow or peripheral blood samples. Copy number analysis of Affymetrix 100K and 500K SNP arrays was performed for 304 pediatric and adult ALL samples, 23 chronic myeloid leukemia samples, and 36 ALL cell lines. There are also 50 samples from leukemia in remission, which were used as references for copy number inference.
Project description:KCL-22 is a chronic myeloid leukemia (CML) cell line derived from a patient in blast crisis phase and harbors the BCR-ABL translocation. The catalytic (ATP-competitive) BCR-ABL inhibitors imatinib and nilotinib have dramatically improved CML patient outcome, but the development of resistance remains a clinical challenge. The recent identification of allosteric BCR-ABL inhibitors, such as GNF-2, which target the enzyme by binding to the myristoyl pocket rather than catalytic site of ABL1, may provide a strategy to broadly overcome resistance to the class of ABL1 ATP competitive inhibitors. We therefore wanted to use the ClonTracer barcoding system to compare the clonal responses of KCL-22 to imatinib, nilotinib and GNF-2. RNA-seq was employed to characterize genetic alterations and gene expression signatures in the pooled cell populations resistant to BCR-ABL inhibitors as well as single clones showing differential response to the three inhibitors. mRNA profiling of the subpopulations and single clones of human CML cell line KCL-22 that contribute to BCR-ABL inhibitor resistance
Project description:The BCR-ABL1 translocation product is the cause of Chronic Myeloid Leukemia (CML) and of a significant fraction of adult-onset B-Acute Lymphoblastic Leukemia (B-ALL) cases. Here we identify an essential role for gamma-catenin (junction plakoglobin) in B lineage restricted cells for the progression of B-ALL in a mouse model. The array analysis of preleukemic B cells aimed at identifying genes that explain the deficient B-ALL progression in the absence gamma-catenin. Total bone marrow cells from chimeric mice expressing or lacking gamma-catenin in the hematopoietic compartment were transduced with BCR-ABL1+ IRES GFP retrovirus before transplantation into lethally irradiated wild type recipients. Three weeks later, GFP+ (BCR-ABL1+) B220+ BP-1+ B cells were flow sorted from the bone marrow of preleukemic recipient mice. The analysis includes 3 replicates for gamma-catenin WT and 3 replicates for gamma-catenin KO BCR-ABL1+ BP1+ B cells.