The Role of c-Abl in Jak2 Activation in IL-3-Dependent Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1672-1672
Author(s):  
Wenjing Tao ◽  
Xiaohong Leng ◽  
Ralph B. Arlinghaus
Keyword(s):  
Cell Survival ◽  
Kinase Activity ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Chromosomal Translocation ◽  
Kinase Domain ◽  
Bimolecular Fluorescence Complementation ◽  
Abl Kinase ◽  
A Minor

Abstract Abstract 1672 The reciprocal chromosomal translocation of Abl and Bcr locus [t(9: 22)] is present in 95% of chronic myeloid leukemia (CML) patients. The resulting Bcr-Abl oncoprotein contains a persistently activated tyrosine kinase activity that activates Jak2/Stat5 signaling pathways. Little is known about the molecular mechanism of Jak2 activation in Bcr-Abl positive CML cells, except that the IL-3 receptor is required (Tao et al., Oncogene 2008). We found that the Jak2 activity (measured by pY1007/1008) in 32D mouse hematopoietic cells steadily diminished immediately upon IL-3 withdrawal. However, expression of kinase-inactive form of Bcr-Abl (p210K1172R) in 32D cells maintained Jak2 activity for up to 8 hrs after IL-3 withdrawal. Our previous studies have shown that the C-terminal region (CT-4) of c-Abl binds to Jak2 as does the kinase domain of c-Abl (Xie et al. Oncogene, 2001). We found that Jak2 activation depends on its binding to the CT-4 region of c-Abl using Bimolecular fluorescence complementation assays. In order to examine the role of c-Abl in Jak2 activation, we expressed c-Abl in both 32D cells (32D-Abl) and 32D cells expressing p210K1172R (32D-p210K1172R+Abl). We found that unlike 32D-Abl cells which remained cytokine-dependent, a minor population (∼7%) of 32D- p210K1172R+Abl cells gained growth independency of IL-3. Compared to 32D-Abl cells in which the level of Jak2 activity was barely detected by pY1007/1008 antibody, 32D-p210K1172R+Abl cells showed a dramatic elevation of Jak2 activation, indicating that c-Abl alone is unable to induce Jak2 activation in hematopoietic cells. Phosphorylation on p210Y177 in 32D-p210K1172R+Abl cells was also strongly increased, indicative of activated Jak2 activity (Samanta et al., Leukemia 2011). We found that 32D-p210K1172R+Abl cells were sensitive to Imatinib Mesylate (IM), as 80% of 32D-p210K1172R+Abl cells were apoptotic after treatment with 5μM IM for 24hrs, indicating that the cell survival depends on the activated c-Abl kinase. The apoptosis induced by IM in 32D-p210K1172R+Abl cells could be effectively rescued by addition of IL-3, indicating the importance of Jak2 activation through IL-3 pathway in maintaining cell survival. The above results suggest that a higher level of c-Abl enables cells expressing a Bcr-Abl kinase defective protein to acquire cytokine-independent growth. The elevation of Jak2 activity in 32D-p210K1172R+ABL cells correlated with the increased c-Abl kinase activity. We propose that the c-Abl kinase plays two crucial roles in these Bcr-Abl kinase mutant cells: 1) making cells cytokine-independent for growth, and 2) promoting persistent Jak2 activation. These results lead us to propose that the Abl kinase domain within Bcr-Abl promotes Jak2 activation by binding to the Jak2 kinase. As our recent findings indicate that Jak2 is a dominant player in CML (Samanta et al., Leukemia 2011) and particularly in later stages of Bcr-Abl positive CML, we propose that the inhibition of both Jak2 and Bcr-Abl kinase activities will result in a near complete elimination of leukemia cells including CD34+CML progenitor cells. Disclosures: No relevant conflicts of interest to declare.

A Novel BCR-ABL Mutation Predicts Resistance to Tyrosine Kinase Inhibitor (TKI) Therapy by a Unique Mechanism in Patients (pts) with Philadelphia-Positive (Ph+) Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4773-4773
Author(s):  
Alfonso Quintas-Cardama ◽  
Jorge Cortes ◽  
Hagop M. Kantarjian ◽  
Moshe Talpaz ◽  
Ji Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase ◽  
High Level ◽  
A Minor ◽  
Unique Mechanism

Abstract Albeit most pts with chronic myeloid leukemia (CML) treated with imatinib (IM) have a favorable outcome, some will acquire resistance, mainly due to the development of Abl kinase domain mutations, which confer varying levels of TKI resistance. We describe a novel V304D mutation in pts with Ph+ leukemia who failed TKI therapy. Expression of V304D mutation in BCR-ABL failed to induce cytokine-independence in Ba/F3 cells. Studies in Cos-7 cells demonstrated that this mutant did not induce autophosphorylation and was deficient in kinase activity. We detected V304D mutation in 13 (18%) of 70 IM-resistant pts screened (12 CML, 1 Ph+ acute lymphoblastic leukemia [ALL]), and it was present in a median of 37% (range, 20% to 80%) resistant clones. Median age was 60 years (range, 30 to 81) and median time from diagnosis to IM therapy was 39 months (range, 1 to 91). Eleven (92%) of 12 pts with CML were in chronic phase (CP) at IM start and 1 was in blast phase (BP). Pts received IM for a median of 35 months (range, 2 to 66). Nine pts with CML had failed interferon and 2 (1 CML, 1 Ph+ALL) allogeneic stem cell transplantation prior to IM. Ten (83%) of 12 pts started IM at 400 mg/d but all eventually received ≥600 mg/d. Six pts with CML achieved a complete hematologic response (CHR), 1 BP returned to chronic phase (RCP), and 6 (5 CML, 1 Ph+ALL) had primary hematologic resistance (HR). No cytogenetic (CG) responses were observed and 7 pts with CML CP progressed (4 to AP and 3 to BP) after IM discontinuation. Four pts with CML (1 CP, 2 AP, 1 BP) received nilotinib after IM failure for a median of 2 months (range, 1 to 3.5). Two pts (1 CP, 1 AP) showed primary HR, 1 AP progressed to BP, and 1 BP (on 600 mg twice daily) had a transient (6 weeks) CHR before showing secondary HR. Twelve pts (11 CML, 1 Ph+ALL) received dasatinib: 7 at 70 mg twice daily, 1 at 90 mg daily, 1 at 140 mg daily, 1 at 180 mg daily, 1 at 90 mg twice daily, and 1 at 120 mg twice daily. Dasatinib was administered for a median of 8 months (range, 1 to 23). Two pts achieved CHR and a minor CG response in 1 analysis (75% and 65% Ph+ cells, respectively), 1 RCP, 1 no evidence of leukemia, and 8 (67%) primary HR. One of 4 pts who started dasatinib in CP progressed to AP. Responders to dasatinib had V304D mutation in 20%, 20%, and 25% of clones, respectively. Four pts exhibited concomitant Abl kinase mutations developed prior to dasatinib therapy: 3 with F317L and 1 with G250E. One pt had a 6 base pair in-frame insertion in the TK domain. T315I mutation evolved in 1 pt after dasatinib discontinuation. Eight pts discontinued dasatinib due to disease progression (7 died), 2 were lost to follow-up, and 2 remain on CHR after 17+ and 23+ months on dasatinib. In vitro studies of cells from one pt in CP with V304D mutation (50% of clones) failed to detect CrkL phosphorylation despite detectable expression of the Bcr-Abl protein. In summary, the V304D mutation in the Abl kinase domain results in kinase inactivation and is associated with high-level resistance to TKI therapy, transformation to AP/BP in CML and a particularly poor prognosis. Loss of kinase activity by mutation represents a very unique mechanism of kinase inhibitor resistance and predicts acquisition of other transforming events that support CML cell survival.

scholarly journals Regulation of Jak2 Function by Phosphorylation of Tyr317 and Tyr637 during Cytokine Signaling

2009 ◽  
Vol 29 (12) ◽  
pp. 3367-3378 ◽  
Author(s):  
Scott A. Robertson ◽  
Rositsa I. Koleva ◽  
Lawrence S. Argetsinger ◽  
Christin Carter-Su ◽  
Jarrod A. Marto ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Feedback Inhibition ◽  
Dominant Role ◽  
Kinase Domain ◽  
Cytokine Signaling ◽  
Tyrosine Kinase Domain ◽  
Phosphorylation Sites ◽  
Cytokine Stimulation

ABSTRACT Jak2, the cognate tyrosine kinase for numerous cytokine receptors, undergoes multisite phosphorylation during cytokine stimulation. To understand the role of phosphorylation in Jak2 regulation, we used mass spectrometry to identify numerous Jak2 phosphorylation sites and characterize their significance for Jak2 function. Two sites outside of the tyrosine kinase domain, Tyr317 in the FERM domain and Tyr637 in the JH2 domain, exhibited strong regulation of Jak2 activity. Mutation of Tyr317 promotes increased Jak2 activity, and the phosphorylation of Tyr317 during cytokine signaling requires prior activation loop phosphorylation, which is consistent with a role for Tyr317 in the feedback inhibition of Jak2 kinase activity after receptor stimulation. Comparison to several previously identified regulatory phosphorylation sites on Jak2 revealed a dominant role for Tyr317 in the attenuation of Jak2 signaling. In contrast, mutation of Tyr637 decreased Jak2 signaling and activity and partially suppressed the activating JH2 V617F mutation, suggesting a role for Tyr637 phosphorylation in the release of JH2 domain-mediated suppression of Jak2 kinase activity during cytokine stimulation. The phosphorylation of Tyr317 and Tyr637 act in concert with other regulatory events to maintain appropriate control of Jak2 activity and cytokine signaling.

scholarly journals The Functional Interplay Between the t(9;22)-Associated Fusion Proteins BCR/ABL and ABL/BCR in Philadelphia Chromosome Positive Acute Lymphatic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2402-2402
Author(s):  
Afsar Mian ◽  
Anahita Rafiei ◽  
Claudia Oancea ◽  
Oliver G. Ottmann ◽  
Martin Ruthardt
Keyword(s):  
Stem Cell ◽  
Fusion Proteins ◽  
Conflicts Of Interest ◽  
Philadelphia Chromosome ◽  
Acute Lymphatic Leukemia ◽  
Abl Kinase ◽  
Lymphatic Leukemia ◽  
Syngeneic Mouse Model ◽  
Philadelphia Chromosome Positive

Abstract The successful targeting of BCR/ABL by selective ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib alone is unable to eradicate the leukemic clone in Philadelphia chromosome positive (Ph+ ) leukemia. The t(9;22)(q34;q11) is a balanced translocation. Der22 involves the BCR (breakpoint cluster region) gene locus with two principal breaks: the M-bcr, encoding for the p210BCR/ABL and the m-bcr, encoding for the 185BCR/ABL fusion proteins, respectively. The constitutively activated BCR/ABL kinase is responsible for the leukemic transformation through an aberrant activation of multiple signaling pathways, such as Stat, Pi3K and Ras/Erk. The der9 encodes for the reciprocal ABL/BCR fusion proteins the p40ABL/BCR, present in 65% of patients with chronic myeloid leukemia (CML) and the p96ABL/BCR, detectable in 100% of patients with Ph+ acute lymphatic leukemia (ALL). ABL/BCRs are oncogenes able to influence the lineage commitment of hematopoietic progenitors. Aim of this study was to further disclose the role of p96ABL/BCR for the pathogenesis of Ph+ ALL. We co-expressed p96ABL/BCRand p185BCR/ ABL from a p2A peptide-linked multi-cistronic retroviral vector, which allows the expression of multiple proteins from a single open reading frame (ORF) to identical levels. The co- expression of p96ABL/BCR enhanced the kinase activity and, as a consequence, the transformation potential of p185BCR/ABL in factor dependent progenitor cells and untransformed fibroblasts. Targeting p96ABL/BCR by RNAi inhibited growth of Ph+ ALL cell lines and primary Ph+ ALL patient-derived long-term cultures (PD-LTCs). Furthermore p96ABL/BCR negatively influenced the response to AKI in these models as shown by an increased response to AKI when p96ABL/BCR was down-regulated. Our in vitro and in vivo stem cell studies on murine fetal liver cells and adult HSCs revealed a functional hierarchy between p96ABL/BCR and p185BCR/ABL. In fact, p96ABL/BCR strongly increased stem cell capacity in replating efficiency and colony forming unit-spleen day 12 (CFU-S12) assays, whereas p185BCR/ABL showed no effect. In contrast co-expression of p96ABL/BCR and p185BCR/ABL increased significantly both serial replating potential and CFU-S12 colony formation as compared to p96ABL/BCR alone. In a syngeneic mouse model co-expression of p96ABL/BCR abolished the capacity of p185BCR/ABL to induce a CML-like disease and led to the induction of ALL. Taken together our here presented data reveal an important role of p96ABL/BCR for the pathogenesis of Ph + ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Death-domain dimerization-mediated activation of RIPK1 controls necroptosis and RIPK1-dependent apoptosis

2018 ◽  
Vol 115 (9) ◽  
pp. E2001-E2009 ◽  
Author(s):  
Huyan Meng ◽  
Zhen Liu ◽  
Xingyan Li ◽  
Huibing Wang ◽  
Taijie Jin ◽  
...  
Keyword(s):  
Cell Death ◽  
Proinflammatory Cytokine ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Death Domain ◽  
Degenerative Diseases ◽  
Intermediate Domain ◽  
A Charge ◽  
Mutant Cells

RIPK1 is a critical mediator of cell death and inflammation downstream of TNFR1 upon stimulation by TNFα, a potent proinflammatory cytokine involved in a multitude of human inflammatory and degenerative diseases. RIPK1 contains an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (DD). The kinase activity of RIPK1 promotes cell death and inflammation. Here, we investigated the involvement of RIPK1-DD in the regulation of RIPK1 kinase activity. We show that a charge-conserved mutation of a lysine located on the surface of DD (K599R in human RIPK1 or K584R in murine RIPK1) blocks RIPK1 activation in necroptosis and RIPK1-dependent apoptosis and the formation of complex II. Ripk1K584R/K584R knockin mutant cells are resistant to RIPK1 kinase-dependent apoptosis and necroptosis. The resistance of K584R cells, however, can be overcome by forced dimerization of RIPK1. Finally, we show that the K584R RIPK1 knockin mutation protects mice against TNFα-induced systematic inflammatory response syndrome. Our study demonstrates the role of RIPK1-DD in mediating RIPK1 dimerization and activation of its kinase activity during necroptosis and RIPK1-dependent apoptosis.

Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 438-438 ◽  
Author(s):  
Xiaoyan Jiang ◽  
Kyi Min Saw ◽  
Allen Eaves ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

Kinase Activity of RTKs Is Dispensable for Factor Independent Growth and Transformation of a Myeloid Cell Line 32D in the Presence of Cbl Mutants.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3970-3970
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Cell Line ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Myeloid Cell ◽  
Dominant Negative ◽  
Class Iii ◽  
Primary Resistance

Abstract Abstract 3970 Poster Board III-906 Purpose The Cbl proto-oncogene products have emerged as important components of the signal transduction cascades downstream of both non-receptor and receptor tyrosine kinases (RTKs). By regulation of receptor trafficking and degradation, they have been shown to tightly regulate the intensity and amplitude of RTK activation. c-Kit belongs to the family of the class-III RTKs and plays an important role in the pathogenesis of acute myeloid leukemia (AML). So far, very little is known about the role of c-Cbl mutants in the role of c-Kit signaling. Results We analyzed the interaction of c-Cbl with c-Kit and the functional relevance of this interaction in the IL-3-dependent murine myeloid progenitor cell line 32Dcl3. We recently identified the first c-Cbl mutation in human disease in an AML patient, named Cbl-R420Q. Co-expression of two different dominant negative mutants of c-Cbl (Cbl-R420Q or Cbl-70Z) with Kit induced cytokine-independent proliferation, survival and clonogenic growth. Importantly, transformation was observed also with kinase-dead forms of Kit and Flt3 in the presence of Cbl-70Z, but not in the absence of Kit or Flt3, suggesting a mechanism dependent on RTKs, but independent of their kinase activity. Instead, transformation appeared to depend on Src family kinases (SFKs), as c-Cbl co-immunoprecipitated with SFKs and SFK inhibition abolished transformation. Conclusion Our results indicate that c-Cbl has an important role in c-Kit signal mitigation. They demonstrate that disturbed mechanisms of c-Kit internalization have important implications for its transforming potential, possibly in the development of AML. Furthermore, these findings may explain primary resistance to tyrosine kinase inhibitors targeted at RTKs. Disclosures: No relevant conflicts of interest to declare.

Enhanced SH3:Linker Interaction Suppresses Activating Mutations of the c-Abl Protein-Tyrosine Kinase.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1208-1208
Author(s):  
Shoghag Panjarian ◽  
Shugui Chen ◽  
John Engen ◽  
Thomas Smithgall
Keyword(s):  
Tyrosine Kinase ◽  
Myristic Acid ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Binding Pocket ◽  
Sh3 Domain ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Defective Mutant ◽  
T315i Mutation

Abstract Abstract 1208 Bcr-Abl, the chimeric protein-tyrosine kinase expressed as a result of the Philadelphia chromosome translocation, plays a pivotal role in the initiation and maintenance of chronic myelogenous leukemia (CML). Imatinib (Gleevec) is an ATP-competitive Bcr-Abl inhibitor that selectively kills Bcr-Abl+ CML cells. Despite its clinical success, imatinib is less effective in the advanced stages of CML due to the emergence of drug resistance caused by point mutations in the Abl kinase domain. Second generation Bcr-Abl inhibitors such as dasatinib and nilotinib are active against most imatinib-resistant forms of Bcr-Abl, with the exception of the T315I “gatekeeper” mutant. The Abl gatekeeper residue (Thr315) is located between the ATP-binding site and an adjacent hydrophobic pocket, and forms a key hydrogen bond with imatinib. Additionally, the T315I mutation produces a strong activating effect on the downregulated c-Abl “core,” consisting of the myristoylated N-terminal Ncap, tandem SH3 and SH2 regulatory domains, the SH2-kinase linker, which forms a polyproline type II helix for internal SH3 docking, and the tyrosine kinase domain. Using hydrogen-exchange mass spectrometry, we recently found that the T315I mutation not only induced conformational changes in the Abl kinase domain as expected, but also at a distance in the RT-loop of the SH3 domain. Such changes may allosterically contribute to kinase domain activation by disturbing the negative regulatory influence of SH3:linker interaction. Recently, a new class of allosteric Bcr-Abl inhibitors has been reported that targets the myristate-binding pocket of Abl, which localizes to C-lobe of the kinase domain and away from the active site. Together with our finding that the T315I mutation perturbs SH3:linker interaction, these inhibitors support the existence of an extensive network of allosteric interactions that work together to regulate Abl kinase activity. In this project, we investigated whether enhanced SH3:linker interaction can allosterically reverse the activating effects of the T315I imatinib resistance mutation as well as mutations of the N-terminal myristoylation site and myristic acid binding pocket. We created modified versions of Abl [High Affinity Linker proteins (HALs)] by mutating multiple residues within the SH2-kinase linker to proline, thereby enhancing the SH3 domain binding affinity. Using mammalian cell-based expression assays and immunoblotting with phosphospecific antibodies, we identified five of eleven Abl-HAL proteins that did not exhibit changes in basal kinase activity. The Abl-HAL protein with the greatest enhancement of SH3:linker interaction was then combined with the T315I mutation, a myristoylation-defective mutant, and a myristic acid binding pocket mutation. Remarkably, this HAL substitution completely reversed the activating effect of the myristic acid binding pocket mutation, while substantially suppressing the activity of Abl T315I and the myristoylation-defective mutant. These results indicate that stabilization of SH3:linker interaction allosterically represses Abl activation by a wide variety of mechanisms, and suggests a new approach to allosteric control of Bcr-Abl kinase activity. Disclosures: No relevant conflicts of interest to declare.

Negative Effects of GM-CSF Signaling In t(8;21)-Induced Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3163-3163
Author(s):  
Shinobu Matsuura ◽  
Ming Yan ◽  
Eun-Young Ahn ◽  
Miao-Chia Lo ◽  
David Dangoor ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Sex Chromosome ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Receptor Activation ◽  
Pseudoautosomal Region ◽  
Wild Type ◽  
Gm Csf

Abstract Abstract 3163 The t(8;21)(q22;q22) translocation is one of the most common chromosomal translocations in de novo acute myeloid leukemia (AML). The 8;21 translocation is often associated with additional cytogenetic abnormalities. The loss of the sex chromosome (LOS) is by far the most frequent abnormality found in association with the t(8;21) leukemia, accounting for 32–59% of patients, in contrast to other types of AML in which the LOS occurs in less than 5% of patients. To evaluate the role of sex chromosome deletion in t(8;21)-related leukemogenesis, hematopoietic cells from a mouse line with only one sex chromosome were used in retrovirus-mediated t(8;21) (AML1-ETO) expression and transplantation assays. The absence of leukemia in those animals suggested that a gene present in the pseudoautosomal region of sex chromosomes in humans but not in mice may be the target gene in LOS. The granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα) gene is one such gene and is also known to be involved in myeloid cell survival, proliferation and differentiation. The GM-CSFRα gene is specifically down-regulated in AML patients with t(8;21), but not in other common translocations (Valk PJM et al, NEJM, 2004). The GM-CSFR complex is composed of α and βc subunits that assemble into a complex for receptor activation and signaling. To investigate the role of GM-CSFR signaling in t(8;21)-mediated leukemogenesis, GM-CSFR common β subunit knockout (GM-CSFRβc-/-) mice were used in our studies as a model for deficient GM-CSFR signaling. Transduction of AML1-ETO in hematopoietic cells from GM-CSFRβc-/- resulted in myeloid leukemia of a median survival time of 225 days, high percentage of blasts in peripheral blood and bone marrow, anemia, thrombocytopenia, hepatomegaly and splenomegaly. Comparison of wild-type and GM-CSFRβc-/- cells in the same transplantation resulted in development of AML1-ETO-induced leukemia at higher penetrance in GM-CSFRβc-/- cells (28.5% vs 100%). Moreover, the latency of leukemia was shorter in GM-CSFRβc-/- cells than in wild-type cells after transduction of AML1-ETO9a. Analysis of the hematopoietic compartment of healthy GM-CSFRβc-/- mice detected no significant abnormalities in the immature hematopoietic compartment (LSK, CMP, GMP, MEP), suggesting that AML1-ETO expression is required for leukemia to occur. In vitro, expression of AML1-ETO alone is sufficient for the immortalization of normal hematopoietic cells, as demonstrated by serial replating capacity of cells in methylcellulose colony assay. Addition of mGM-CSF to the basic cytokine cocktail (mIL-3, hIL-6, mSCF, hEPO) did not significantly affect number, type, size, and cell composition of colony cells. In contrast, the addition of mGM-CSF eliminated the replating capacity of AML1-ETO expressing cells, although they survived longer than control vector-infected cells. The results suggest that activation of GM-CSF signaling can specifically abrogate the self-renewal ability of potential leukemic stem cells in the early immortalization phase. These results support a possible tumor suppressor role of GM-CSF in leukemogeneis by AML1-ETO and may provide clues to understand how AML1-ETO corrupts normal GM-CSF signals to its own advantage for leukemogenic transformation. Disclosures: No relevant conflicts of interest to declare.

p27 Is Mislocalized to the Cytoplasm by BCR-ABL In a Kinase-Independent Manner and Contributes to Leukemogenesis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 512-512
Author(s):  
Anupriya Agarwal ◽  
Ryan J Meckenzie ◽  
Thomas O'Hare ◽  
Kavin B Vasudevan ◽  
Dorian H LaTocha ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Kinase Activity ◽  
Immunofluorescence Microscopy ◽  
Independent Effect ◽  
Histopathological Analysis ◽  
Imatinib Treatment ◽  
Wild Type ◽  
Abl Kinase

Abstract Abstract 512 Background: BCR-ABL promotes cell cycle progression by interfering with the regulatory functions of p27, a cyclin dependent kinase (Cdk) inhibitor and tumor suppressor. We have previously shown that BCR-ABL kinase activity promotes degradation of nuclear p27 (Agarwal, A. et al. Blood 2008). Additionally, in primary CML cells, p27 is mislocalized to the cytoplasm, thereby relieving Cdks from p27 inhibition. Results from studies of solid tumors show that cytoplasmic p27 can actively contribute to oncogenesis, raising the question of whether cytoplasmic p27 in CML cells may actively promote leukemogenesis rather than merely compromise Cdk inhibition. We hypothesize that BCR-ABL disrupts p27 function in a dual manner by reducing nuclear p27, where p27 normally serves as a tumor suppressor, and by increasing cytoplasmic p27, where it might have oncogenic activity. Experimental Approach and Results: Immunoblotting of nuclear and cytoplasmic lysates of CD34+ cells from 11 CML patients revealed that p27 localization is predominantly cytoplasmic in the majority of patients (10/11; 91%) irrespective of disease phase, while p27 was mostly nuclear in normal controls. Similar results were obtained by immunofluorescence microscopy. Imatinib treatment increased nuclear p27 suggesting that nuclear p27 levels are regulated by BCR-ABL kinase activity. However, imatinib does not alter cytoplasmic p27 levels, suggesting that cytoplasmic mislocalization of p27 is a kinase-independent effect of BCR-ABL. Kinase-independent regulation of cytoplasmic p27 localization was also tested by immunofluorescence microscopy of p27−/− MEFs engineered to express active or kinase-dead BCR-ABL in combination with wild-type p27. In these cells cytoplasmic p27 abundance was increased both by kinase-active or kinase-dead BCR-ABL as compared to the vector control. To interrogate the role of p27 in vivo we retrovirally transduced p27+/+ or p27−/− bone marrow with BCR-ABL-GFP retrovirus and sorted Lin-/c-Kit+/Sca-I+ cells by FACS, allowing for injection of exactly matched numbers of BCR-ABL-expressing GFP+ cells (5000/animal). Median survival was significantly reduced for recipients of p27−/− marrow as compared to p27+/+ controls (34 days vs. 93 days p<0.0001). Recipients of p27−/− marrow also exhibited significantly increased white blood cell (4.5-fold) and platelet counts (3.9-fold) as well as spleen size (6-fold) and liver size (1.6-fold). Accordingly, there was more pronounced leukemic infiltration of myeloid precursors on histopathology as compared to controls. An in vivo competition experiment performed by injecting equal numbers of BCR-ABL-transduced p27−/− and p27+/+ marrow cells in congenic recipients resulted in leukemias in recipient mice (N=8) that were derived exclusively from p27−/− cells. In total, these results suggest that the net function of p27 in CML is tumor suppressive. To functionally dissect the role of nuclear and cytoplasmic p27, we used p27T187A transgenic mice (in which nuclear p27 degradation is reduced) and p27S10A mice (in which p27 export to the cytoplasm is reduced resulting in predominantly nuclear p27). Mice of matched genetic background were used as p27WT controls in CML retroviral transduction/transplantation experiments. In both cases, survival was prolonged compared to controls: 25 vs. 21 days for p27T187A (p=0.05) and 32 vs. 23 days for p27S10A (p=0.01). This suggests that stabilization of nuclear p27 (p27T187A) and more significantly lack of cytoplasmic p27 (p27S10A) attenuate BCR-ABL-mediated leukemogenesis. Consistent with this, autopsy and histopathological analysis revealed reduced hepatosplenomegaly (p27T187A mice) and improved cell differentiation with a relative increase of mature neutophils (p27S10A mice) as compared to wild-type controls. Conclusions: These results provide in vivo evidence that p27 has genetically separable dual roles in CML as both a nuclear tumor suppressor and cytoplasmic oncogene. A kinase-independent activity of BCR-ABL contributes to leukemogenesis through aberrant p27 localization to the cytoplasm. This oncogene activity is independent from the kinase-dependent degradation of nuclear p27. We speculate that the inability of tyrosine kinase inhibitors to reverse cytoplasmic p27 mislocalization may contribute to disease persistence despite effective inhibition of BCR-ABL kinase activity. Disclosures: Deininger: Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; genzyme: Research Funding.

Modeling CML Development and Drug Resistance Using iPSC Technology,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3767-3767
Author(s):  
Kran Suknuntha ◽  
Yuki Ishii ◽  
Kejin Hu ◽  
Jean YJ Wang ◽  
Igor Slukvin
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Stem Cell ◽  
Blood Cells ◽  
Kinase Activity ◽  
Hematopoietic Cells ◽  
Patient Specific ◽  
Hematopoietic Progenitors ◽  
Abl Kinase

Abstract Abstract 3767 Reprogramming of neoplastic cells to pluripotency provides a unique tool to personalize the exploration of tumor pathogenic mechanisms and drug resistance using iPSCs with patient-specific chromosomal abnormalities. We have developed a technology to generate transgene-free iPSCs from bone marrow and cord blood cells employing episomal vectors. Using this approach we created transgene-free iPSCs from a patient with CML in the chronic phase. CMLiPSCs showed a unique complex chromosomal translocation identified in the patinet's marrow sample while displaying typical embryonic stem cell phenotype and pluripotent differentiation potential. Importantly, these CMLiPSCs are devoid of genomic integration and expression of reprogramming factors, which are incompatible for modeling tumor development and drug response (Hu et al. Blood 117:e109). We have also shown that these CMLiPSCs contain the BCR-ABL oncogene without any detectable mutations in its kinase domain. By coculture with OP9, we generated APLNR+ mesodermal cells, MSCs, and lin-CD34+CD45+ hematopoietic progenitors from CMLiPSCs, and control BMiPSCs from a normal subject and analyzed the levels of BCR-ABL protein and tyrosine-phosphorylated (pTyr) cellular proteins in the different cell populations. The highest level of BCR-ABL protein expression was found in the in undifferentiated iPSCs, however, the overall cellular pTyr levels was lower than the control BMiPSCs, suggesting that BCR-ABL kinase activity was suppressed in the CMLiPScs. Consistent with these findings, imatinib does not inhibit the growth and survival of these CMLiPSCs. The levels of BCR-ABL protein decreased upon differentiation with a major reduction observed when cells became mesoderm. Following differentiation of CMLiPSC-derived mesoderm into the MSCs and lin-CD34+CD45+ hematopoietic progenitors, the levels of BCR-ABL protein did not change significantly, indicating that the major epigenetic regulation of BCR-ABL expression occurs during the transition to mesoderm. In spite of the decrease in BCR-ABL expression, the total pTyr levels significantly increased following transition of CMLiPSCs to mesoderm and blood cells, suggesting recovery of BCR-ABL kinase activity during differentiation. Interestingly, we found that imatinib had no effect on CFC potential of the most primitive lin-CD34+CD45+ hematopoietic progenitors derived from CMLiPSCs, while significant inhibition in hematopoietic CFC potential was observed when we used the patient's bone marrow cells. Following expansion of lin-CD34+CD45+ progenitors in serum-free medium with cytokines, we found that more differentiated hematopoietic cells became imatinib sensitive. The differential response of progenitors versus more differentiated cells to imatinib recapitulate the clinical observation that CML stem cells display innate resistance to imatinib but their differentiated progenies become sensitive to this BCR-ABL kinase inhibitor. The iPSC-based models provide several advantages for the study of CML pathogenesis. iPSCs can provide an unlimited supply of hematopoietic cells carrying patient-specific genetic abnormalities. Using well-defined temporal windows and surface markers, distinct cell subsets with tumor-initiating/tumor-propagating potential after transplantation in immunodeficient mice could be identified and used for drug screening. iPSC models make it possible to address CML stem-cell potential at various stages of differentiation for which it may be difficult to obtain samples from the patient, for example, at the hemangioblast stage. They also provide a unique opportunity to explore the interplays between epigenetics and oncogene function, as we have demonstrated using the CMLiPSCs. The major unsolved question is why CML stem cells are naturally resistant to imatinib, and this question can be addressed using the iPS system. Disclosures: Slukvin: CDI: Consultancy, Equity Ownership.

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