Leukemogenic Tyrosine Kinases Research Articles

The Effects of Tiger Milk Mushroom Lignosus rhinocerus TM02® (Agaricomycetes) on Leukemogenicity Tyrosine Kinase Cell Lines.

Leukemia can be a result of genetic changes associated with protein tyrosine kinase activity such as in MPL W515L and BCR/ABL genes. However, the current conventional treatment of leukemia produces severe side effects that urge the approach to use natural products. A medicinal mushroom, Lignosus rhinocerus shows potential as an anti-cancer treatment. To investigate the efficacy and mechanism of action of the L. rhinocerus cultivar (TM02®) extract on leukemogenic tyrosine kinase cell lines, a cold-water extract (CWE) was produced by using TM02® sclerotia powder at 4°C. The carbohydrate and protein contents were found to be 77.24% and 1.75% respectively. In comparison to the normal Ba/F3 cell, the CWE TM02® shows significant effects on exhibiting proliferation of Ba/F3 expressed MPL W515L and BCR/ABL, possibly due to the presence of phenolic compounds and antioxidant properties of TM02®, which contribute to act on various signaling pathways, and the reported apoptotic activity of CWE TM02®. In contrast, CWE TM02® significantly exhibited high scavenging activity of both Ba/F3 expressed MPL W515L and BCR/ABL. At concentrations of 125 μg/mL and 500 μg/mL of CWE TM02® decreased 49.5% and 67.5% of cell migration activity of Ba/F3 expressed MPL W515L and BCR/ABL respectively. Therefore, we postulate that CWE TM02® has the capability to mediate the migration route of the leukemogenic tyrosine kinase cell lines.

Chronic myeloid leukemia: the concepts of resistance and persistence and the relationship with the BCR-ABL1 transcript type.

Chronic myeloid leukemia is driven by a hybrid gene, BCR-ABL1, that codes for a leukemogenic tyrosine kinase (TK) protein of 210 KDa (p210BCR-ABL1). Resistance to TK inhibitor (TKI) therapy occurs in relatively few patients, no more than 10%, while persistence of minimal residual disease during TKI therapy occurs in the great majority of patients. Resistance is a cause of death, persistence is compatible with a fairly normal length and quality of life, but may require lifelong treatment. The causes of resistance are heterogeneous, including the development of other genomic abnormalities or the altered expression of other genes, requiring different treatments. The causes of persistence may not be the same as those of resistance. We hypothesize that the variability in breakpoint position within the Major-breakpoint cluster region (M-bcr), resulting in two different messenger RNAs that may or may not include exon 14 of BCR (e13a2 and e14a2, respectively), and, as a consequence, in two p210BCR-ABL1 proteins that differ by 25 amino acids, may be a cause of persistence. The hypothesis is based on a critical review of the relationships between the BCR-ABL1 transcript types, the response to TKIs, the outcome of treatment, and the immune response, suggesting that the e14a2 transcript is associated with more and deeper molecular responses, hence with a higher probability of achieving treatment-free remission (TFR). Investigating this putative cause of persistence may help bringing more patients into stable TFR.

A Specific PTPRC/CD45 Phosphorylation Event Governed by Stem Cell Chemokine CXCL12 Regulates Primitive Hematopoietic Cell Motility

CXCL12 governs cellular motility, a process deregulated by hematopoietic stem cell oncogenes such as p210-BCR-ABL. A phosphoproteomics approach to the analysis of a hematopoietic progenitor cell line treated with CXCL12 and the Rac 1 and 2 inhibitor NSC23766 has been employed to objectively discover novel mechanisms for regulation of stem cells in normal and malignant hematopoiesis. The proteomic data sets identified new aspects of CXCL12-mediated signaling and novel features of stem cell regulation. We also identified a novel phosphorylation event in hematopoietic progenitor cells that correlated with motile response and governed by the chemotactic factor CXCL12. The novel phosphorylation site on PTPRC/CD45; a protein tyrosine phosphatase, was validated by raising an antibody to the site and also using a mass spectrometry absolute quantification strategy. Site directed mutagenesis and inhibitor studies demonstrated that this single phosphorylation site governs hematopoietic progenitor cell and lymphoid cell motility, lies downstream from Rac proteins and potentiates Src signaling. We have also demonstrated that PTPRC/CD45 is down-regulated in leukemogenic tyrosine kinase expressing cells. The use of discovery proteomics has enabled further understanding of the regulation of PTPRC/CD45 and its important role in cellular motility in progenitor cells.

Transcriptional regulation of immediate-early gene response by THOC5, a member of mRNA export complex, contributes to the M-CSF-induced macrophage differentiation

Hematopoiesis and commitment to a restricted lineage are guided by a timely expressed set of cytokine receptors and their downstream transcription factors. A member of the mRNA export complex, THOC5 (suppressors of the transcriptional defects of hpr1 delta by overexpression complex 5) is a substrate for several tyrosine kinases such as macrophage colony-stimulating factor (M-CSF) receptor and various leukemogenic tyrosine kinases, such as Bcr-Abl, or NPM-ALK. THOC5 tyrosine phosphorylation is elevated in stem cells from patients with chronic myeloid leukemia, suggesting that THOC5 may be involved in leukemia development. THOC5 is also an essential element in the maintenance of hematopoiesis in adult mice. In this report, we show that THOC5 is located in the nuclear speckles, and that it is translocated from the nucleus to cytoplasm during M-CSF-induced bone marrow-derived macrophage differentiation. Furthermore, we have identified THOC5 target genes by trancriptome analysis, using tamoxifen-inducible THOC5 knockout macrophages. Although only 99 genes were downregulated in THOC5-depleted macrophages, half of the genes are involved in differentiation and/or migration. These include well-known regulators of myeloid differentiation inhibitor of DNA binding (Id)1, Id3, Smad family member 6 (Smad6) and Homeobox (Hox)A1. In addition, a subset of M-CSF-inducible genes, such as Ets family mRNAs are THOC5 target mRNAs. Upon depletion of THOC5, unspliced v-ets erythroblastosis virus E26 oncogene homolog (Ets1) mRNA was accumulated in the nucleus. Furthermore, THOC5 was recruited to chromatin where Ets1 was transcribed and bound to unspliced and spliced Ets1 transcripts, indicating that THOC5 has a role in processing/export of M-CSF-inducible genes. In conclusion, regulation of immediate-early gene response by THOC5, a member of mRNA export complex contributes to the M-CSF-induced macrophage differentiation.

Correction: Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

Correction: Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases.

Compensatory Signaling From ROR1 and the Pre-B Cell Receptor Promote Survival of t(1;19) Acute Lymphoblastic Leukemia

Abstract▪2466▪This icon denotes a clinically relevant abstract BACKGROUND:Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the t(1;19) ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against t(1;19) ALL cells due to its capacity to inhibit tyrosine kinases necessary for transduction of pre-B cell receptor (preBCR) signaling. Finally, we show that ROR1 and the preBCR activate mutually compensatory signaling pathways, suggesting that optimal therapeutic regimens would include agents targeting both pathways. METHODS:To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients with siRNAs and determined cell viability using an MTS assay. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cells lines and primary ALL cells by treating samples with a library of small-molecule inhibitors and cell viability was assessed by MTS. Signaling pathways disrupted by inhibitor treatment or ROR1 knockdown were interrogated by phospho-protein arrays and confirmed by immunoblot analysis. RESULTS:The RAPID assay identified a unique sensitivity to the cell surface receptor ROR1 in a subject identified with t(1;19) pediatric ALL. Similar sensitivity was not observed in ALL patients of alternative cytogenetic subtypes. Examination of additional ALL patient samples revealed conserved overexpression of the ROR1 transcript in t(1;19)-positive specimens with absence of ROR1 expression in t(1;19)-negative samples. Cell lines and early passage xenograft cells confirmed overexpression and functional dependence of t(1;19)-positive cells on ROR1. A subsequent kinase inhibitor screen of t(1;19) ALL cell lines and patient samples revealed universal sensitivity to the FDA-approved drug dasatinib. Further examination revealed the dasatinib targets, BTK and LYN, which signal downstream of the preBCR as the viability dependent targets of dasatinib in t(1;19) ALL. Inhibition of the preBCR results in transient loss of AKT activity and, surprisingly, upregulation of ROR1. Analysis of signaling pathways after silencing of ROR1 or dasatinib treatment revealed compensatory signaling pathways emanating from ROR1 and the preBCR that both serve to regulate AKT activity. Consequently, combination of ROR1 knockdown and dasatinib treatment resulted in additive impairment of cell viability compared with either treatment alone. CONCLUSION:The cell surface receptor ROR1 is consistently overexpressed in t(1;19) ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Similarly, t(1;19) cells are sensitive to the kinase inhibitor dasatinib due to activity against the preBCR. Combined targeting of ROR1 and the preBCR with dasatinib yields an additive viability effect due to compensatory signaling pathways aimed at regulating AKT. These results demonstrate a novel mechanism of AKT regulation in t(1;19) ALL as well as a therapeutic strategy for treatment of t(1;19) ALL. Disclosures:Druker:MolecularMD: Equity Ownership, OHSU and Dr. Druker have a financial interest in MolecularMD. Technology used in this research has been licensed to MolecularMD. This potential conflict of interest has been reviewed and managed by the OHSU Conflict of Interest in Research Committee and t; Ariad Pharmaceuticals: Consultancy; OHSU patent #843: Mutated ABL Kinase Domains: Patents & Royalties; Bristol-Myers Squibb: OHSU has clinical trial contracts with Bristol-Myers-Squibb to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Novartis: OHSU has clinical trial contracts with Novartis to pay for patient costs, nurse and data manager salaries, and institutional overhead. Dr. Druker does not derive salary, nor does his lab receive funds from these contracts.; Cylene: Consultancy.

P90RSK2 is essential for FLT3-ITD– but dispensable for BCR-ABL–induced myeloid leukemia

Abstractp90 ribosomal S6 kinase 2 (p90RSK2) is important in diverse cellular processes including gene expression, cell proliferation, and survival. We found that p90RSK2 is commonly activated in diverse leukemia cell lines expressing different leukemogenic tyrosine kinases, including BCR-ABL and FMS-like tyrosine kinase 3–internal tandem duplication (FLT3-ITD). Interestingly, in a murine BM transplantation (BMT) model, genetic deficiency of RSK2 did not affect the pathogenesis or disease progression of BCR-ABL–induced myeloproliferative neoplasm (PN). In contrast, FLT3-ITD induced a T-cell acute lymphoblastic leukemia in BMT mice receiving RSK2 knockout (KO) BM cells, phenotypically distinct from the myeloproliferative neoplasm induced by FLT3-ITD using wild-type BM cells. In consonance with these results, inhibition of RSK2 by an RSK inhibitor, fmk, did not effectively induce apoptosis in BCR-ABL–expressing murine Ba/F3 cells, human K562 cells or primary tissue samples from CML patients, whereas fmk treatment induced significant apoptotic cell death not only in FLT3-ITD–positive Ba/F3 cells, human Molm14 and Mv(4;11) leukemia cells, but also in primary tissue samples from AML patients. These results suggest that RSK2 is dispensable for BCR-ABL–induced myeloid leukemia, but may be required for pathogenesis and lineage determination in FLT3-ITD–induced hematopoietic transformation. RSK2 may thus represent an alternative therapeutic target in the treatment of FLT3-ITD–positive leukemia.

P90RSK2 Is Essential for FLT3-ITD-, but Dispensable for BCR-ABL-Induced Myeloid Leukemia

AbstractAbstract 1716p90 ribosomal S6 kinase 2 (p90RSK2) is a serine/threonine kinase that plays an active role in diverse cellular processes, including gene expression, cell proliferation, and survival. We previously demonstrated that RSK2 signaling plays a key role in the pathogenesis and disease progression of leukemogenic FGFR3 associated hematopoietic malignancies, including FGFR3-expressing t(4;14) multiple myeloma and TEL-FGFR3-expressing t(4;12)(p16;p13) peripheral T cell lymphoma. In this study, we found that p90RSK2 is commonly activated in diverse leukemia cell lines expressing different leukemogenic tyrosine kinases, including K562 (BCR-ABL), Molm14 and Mv4;11 (FLT3-ITD), HEL (JAK2 V617F), and EOL-1(FIP1L1-PDGFR alpha). We next examined the role of RSK2 in myeloid transformation induced by BCR-ABL and FLT3-ITD due to their high frequency of occurrence in CML and AML, respectively. Interestingly, although RSK2 is activated by BCR-ABL in both stably transduced Ba/F3 cells and K562 human leukemia cells, we found that genetic deficiency of RSK2 does not affect the pathogenesis or disease progression of myeloproliferative disease induced by BCR-ABL in a murine bone marrow transplant (BMT) model using wild type or RSK2-/- donor bone marrow cells. This finding suggests that RSK2 is dispensable for BCR-ABL induced myeloproliferative disease. Moreover, targeting RSK2 by treatment with potent and highly specific RSK inhibitor fmk did not effectively induce apoptosis in K562 human leukemia cells expressing BCR-ABL, or in primary leukemia cells from BCR-ABL positive CML patients. In contrast, we found that targeting RSK2 may represent an effective therapy to treat patients with FLT3-ITD positive AML. Treatment with fmk induced significant apoptotic cell death in Molm14 and Mv4;11 human leukemia cells expressing FLT3-ITD, as well as in primary leukemia cells from FLT3-ITD positive AML patients. In consonance with these results, FLT3-ITD induced T-cell lymphoma in a BMT assay using RSK2-/- donor bone marrow cells, phenotypically distinct from the myeloproliferative disease induced by FLT3-ITD using wild type donor bone marrow cells. These results suggest that RSK2 is required for FLT3-ITD induced hematopoietic transformation, likely playing a role in pathogenesis and lineage determination. Together these findings suggest that the role of RSK2 in hematopoietic transformation may depend on different upstream oncogenic signals mediated by different leukemogenic tyrosine kinases. Our data also demonstrate that RSK2 may represent an alternative therapeutic target in the treatment of FLT3-ITD positive leukemia. Disclosures:No relevant conflicts of interest to declare.

ROR1 as a Therapeutic Target In E2A-PBX1-Positive Acute Lymphoblastic Leukemia

AbstractAbstract 539 Background:Aberrant tyrosine kinase activity is commonly implicated in the pathogenesis of leukemia and other cancers. Identification of these leukemogenic tyrosine kinases has proven invaluable for diagnostic and prognostic stratification of patients as well as for the development of novel strategies for therapeutic intervention. We previously demonstrated that siRNA screening of mononuclear cells from leukemia patients can determine sensitivity to individual tyrosine kinases. With the goal of uncovering novel viability-dependent tyrosine kinases in leukemia patients, we have employed an RNAi-assisted protein target identification (RAPID) assay to screen cytogenetic subtypes of acute lymphoblastic leukemia (ALL). ALL is the most common pediatric cancer, accounting for one-quarter of all childhood malignancies. Childhood ALL has a primarily B cell precursor phenotype and is characterized by chromosomal abnormalities, primarily translocations and duplications. These lesions can result in aberrant tyrosine kinase expression and activity required for leukemogenesis. One of the most common recurring translocations associated with pediatric ALL, t(1;19)(q23;p13.3), generates the E2A-PBX1 fusion product. The role of E2A-PBX1 in the development of acute leukemia remains unclear. Here we show unique viability-dependent expression of a receptor tyrosine kinase, ROR1, in the E2A-PBX1 ALL background. In addition, we identify a kinase inhibitor, dasatinib, with significant activity against E2A-PBX1-positive ALL cells. Methods:To identify targets required for viability of leukemic cells, we screened cell lines as well as primary cells from ALL patients by electroporating siRNAs individually targeting each member of the tyrosine kinase gene family. Four days later, we determined the cell viability and tabulated sensitivity of the cells to any individual tyrosine kinase. ROR1 expression levels were determined by RT-PCR, immunoblot analysis and flow cytometry. Kinase inhibitor screening was performed on both cell lines and primary ALL cells by treating samples with a library of small-molecule inhibitors consisting of 90 cell-permeable inhibitor compounds. Inhibitors are plated at four serial dilutions to allow IC50 calculations. The effect of each drug on cell viability is determined at day three by an MTS cell viability assay. Results:The RAPID assay identified a unique sensitivity to the receptor tyrosine kinase-like orphan receptor 1 (ROR1) in a subject identified with E2A-PBX1-positive B cell precursor pediatric ALL. Similar sensitivity was not observed in patients of other leukemic backgrounds or ALL patients of alternative cytogenetic subtypes. Examination of mononuclear cells from this patient by reverse-transcriptase-PCR revealed overexpression of the ROR1 transcript compared with ALL patients lacking the E2A-PBX1 fusion product. Examination of 12 additional E2A-PBX1-positive ALL patient samples revealed universal overexpression of ROR1 within the E2A-PBX1 background. Analysis of E2A-PBX1-positive cell lines and early passage xenograft cells showed both overexpression of ROR1 and sensitivity to siRNA-mediated ROR1 silencing, confirming the ROR1 dependent survival observed in primary cells with the RAPID assay. Finally, since ROR1 is defined as a tyrosine kinase, we performed a kinase inhibitor screen and identified universal sensitivity of E2A-PBX1-positive cell lines and patient samples to the FDA-approved drug dasatinib. Hence, dasatinib is suggested as a potential therapeutic for E2A-PBX1-positive ALL patients. Conclusion:The cell surface receptor ROR1 is consistently overexpressed in E2A-PBX1-positive ALL. RNAi mediated downregulation of ROR1 impairs the viability of these cells. Finally, the kinase inhibitor dasatinib is suggested as a novel therapeutic tool for treatment of E2A-PBX1-positive ALL based on universal sensitivity of E2A-PBX1 samples to this kinase inhibitor. Disclosures:Druker:Molecular MD: Equity Ownership.

ABL fusion oncogene transformation and inhibitor sensitivity are mediated by the cellular regulator RIN1

ABL gene translocations create constitutively active tyrosine kinases that are causative in chronic myeloid leukemia, acute lymphocytic leukemia and other hematopoietic malignancies. Consistent retention of ABL SH3/SH2 autoinhibitory domains, however, suggests that these leukemogenic tyrosine kinase fusion proteins remain subject to regulation. We resolve this paradox, demonstrating that BCR-ABL1 kinase activity is regulated by RIN1, an ABL SH3/SH2 binding protein. BCR-ABL1 activity was increased by RIN1 overexpression and decreased by RIN1 silencing. Moreover, Rin1−/− bone marrow cells were not transformed by BCR-ABL1, ETV6-ABL1 or BCR-ABL1T315I, a patient-derived drug-resistant mutant, as judged by growth factor independence. Rescue by ectopic RIN1 verified a cell autonomous mechanism of collaboration with BCR-ABL1 during transformation. Sensitivity to the ABL kinase inhibitor imatinib was increased by RIN1 silencing, consistent with RIN1 stabilization of an activated BCR-ABL1 conformation having reduced drug affinity. The dependence on activation by RIN1 to unleash full catalytic and cell transformation potential reveals a previously unknown vulnerability that could be exploited for treatment of leukemic cases driven by ABL translocations. The findings suggest that RIN1 targeting could be efficacious for imatinib-resistant disease and might complement ABL kinase inhibitors in first-line therapy.

FIP1L1-PDGFRα Imposes Eosinophil Lineage Commitment on Hematopoietic Stem/Progenitor Cells

Although leukemogenic tyrosine kinases (LTKs) activate a common set of downstream molecules, the phenotypes of leukemia caused by LTKs are rather distinct. Here we report the molecular mechanism underlying the development of hypereosinophilic syndrome/chronic eosinophilic leukemia by FIP1L1-PDGFRalpha. When introduced into c-Kit(high)Sca-1(+)Lineage(-) cells, FIP1L1-PDGFRalpha conferred cytokine-independent growth on these cells and enhanced their self-renewal, whereas it did not immortalize common myeloid progenitors in in vitro replating assays and transplantation assays. Importantly, FIP1L1-PDGFRalpha but not TEL-PDGFRbeta enhanced the development of Gr-1(+)IL-5Ralpha(+) eosinophil progenitors from c-Kit(high)Sca-1(+)Lineage(-) cells. FIP1L1-PDGFRalpha also promoted eosinophil development from common myeloid progenitors. Furthermore, when expressed in megakaryocyte/erythrocyte progenitors and common lymphoid progenitors, FIP1L1-PDGFRalpha not only inhibited differentiation toward erythroid cells, megakaryocytes, and B-lymphocytes but aberrantly developed eosinophil progenitors from megakaryocyte/erythrocyte progenitors and common lymphoid progenitors. As for the mechanism of FIP1L1-PDGFRalpha-induced eosinophil development, FIP1L1-PDGFRalpha was found to more intensely activate MEK1/2 and p38(MAPK) than TEL-PDGFRbeta. In addition, a MEK1/2 inhibitor and a p38(MAPK) inhibitor suppressed FIP1L1-PDGFRalpha-promoted eosinophil development. Also, reverse transcription-PCR analysis revealed that FIP1L1-PDGFRalpha augmented the expression of C/EBPalpha, GATA-1, and GATA-2, whereas it hardly affected PU.1 expression. In addition, short hairpin RNAs against C/EBPalpha and GATA-2 and GATA-3KRR, which can act as a dominant-negative form over all GATA members, inhibited FIP1L1-PDGFRalpha-induced eosinophil development. Furthermore, FIP1L1-PDGFRalpha and its downstream Ras inhibited PU.1 activity in luciferase assays. Together, these results indicate that FIP1L1-PDGFRalpha enhances eosinophil development by modifying the expression and activity of lineage-specific transcription factors through Ras/MEK and p38(MAPK) cascades.

Inhibition of Apoptosome Formation by Suppression of Hsp90β Phosphorylation in Tyrosine Kinase-Induced Leukemias

Constitutively active tyrosine kinases promote leukemogenesis by increasing cell proliferation and inhibiting apoptosis. However, mechanisms underlying apoptotic inhibition have not been fully elucidated. In many settings, apoptosis occurs by mitochondrial cytochrome c release, which nucleates the Apaf-1/caspase-9 apoptosome. Here we report that the leukemogenic kinases, Bcr-Abl, FLT3/D835Y, and Tel-PDGFRbeta, all can inhibit apoptosome function. In cells expressing these kinases, the previously reported apoptosome inhibitor, Hsp90beta, bound strongly to Apaf-1, preventing cytochrome c-induced Apaf-1 oligomerization and caspase-9 recruitment. Hsp90beta interacted weakly with the apoptosome in untransformed cells. While Hsp90beta was phosphorylated at Ser 226/Ser 255 in untransformed cells, phosphorylation was absent in leukemic cells. Expression of mutant Hsp90beta (S226A/S255A), which mimics the hypophosphorylated form in leukemic cells, conferred resistance to cytochrome c-induced apoptosome activation in normal cells, reflecting enhanced binding of nonphosphorylatable Hsp90beta to Apaf-1. In Bcr-Abl-positive mouse bone marrow cells, nonphosphorylatable Hsp90beta expression conferred imatinib (Gleevec) resistance. These data provide an explanation for apoptosome inhibition by activated leukemogenic tyrosine kinases and suggest that alterations in Hsp90beta-apoptosome interactions may contribute to chemoresistance in leukemias.

Absence of SKP2 expression attenuates BCR-ABL–induced myeloproliferative disease

AbstractBCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCFSKP2 (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G1 arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2V617F, and TEL-PDGFRβ, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL–infected SKP2−/− marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2+/+ marrow. SKP2−/− leukemic cells demonstrated higher levels of nuclear p27 than SKP2+/+ counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL–induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCFSKP2 may be therapeutically useful.

FIP1L1/PDGFRα Imposes Commitment towards Eosinophil Lineage on Hematopoietic Stem/Progenitor Cells by Modifying the Expression and Function of Lineage Specific Transcription Factors.

Constitutively activated tyrosine kinases (TKs) cause various types of leukemia. For example, BCR/ABL and TEL/PDGFRβ (T/PRβ) are the causative genes of CML and CMML, respectively. However, these leukemogenic TKs (LTKs) activate common sets of downstream molecules such as Ras/MAPK, PI3-K/Akt, and STATs. So, it remains unknown how disease phenotypes (i.e., the lineage and differentiation stage of leukemic cells and the clinical course of the patient) are determined by LTKs. One possibility is that LTK by itself has a potential to promote the development of leukemic cells specific for each LTK. Alternatively, these phenotypes might originate from the lineage and/or differentiation stage of leukemic stem cells, in which each LTK was initially generated. To characterize these mechanisms, in this study, we utilized FIP1L1/PDGFRα (F/PRα), a causative gene of hyper eosinophilic syndrome (HES)/chronic eosiinophilic leukemia (CEL). First, we transduced F/PRα and T/PRβ into murine c-KithighSca-1+Lin− (KSL) cells using the retrovirus system. As a result, both LTKs enabled KSLs to grow under factor-deprived conditions. Next, we examined the effects of these LTKs on the development of Gr1+IL-5 receptor(R)+ eosinophil progenitors (EoPs) from KSL cells. After 6-day cultures with SCF, TPO, IL-6, and FLT3L, 52% of F/PRα-transfected KSL cells became to be Gr1+IL-5R+, while only 4% and 15% of the cultured cells were Gr1+IL-5R+ in mock- and T/PRβ-transfected KSL cells, respectively. Similarly, F/PRα preferentially developed EoPs from common myeloid progenitor (CMP)s. Furthermore, when expressed in megakaryocyte/erythrocyte progenitor (MEP)s and common lymphoid progenitor (CLP)s, F/PRα inhibited the development of erythroid cells, megakaryocytes and B lymphocytes, respectively. Importantly, F/PRα but not T/PRβ aberrantly developed EoPs from MEPs and CLPs. However, F/PRα was not able to immortalize CMP, granulocyte/monocyte progenitor (GMP), MEP, or CLP. These results suggest that, although F/PRα can reprogram these progenitors towards Eo lineage, it must be generated in hematopoietic stem cells to cause HES/CEL. Next, we expressed chimeric LTKs (F/PRβ and T/PRα) and two activated forms of PRα each harboring point mutation detected in gastrointestinal stromal tumors (GIST) (PRαV561D and PRαD842V) in KSL cells. As a result, all these TKs enabled KSL cells to proliferate under factor-deprived conditions. However, only T/PRα promoted the development of EoP from KSL cells, indicating that specific activity derived from chimeric PRα is necessary to cause HES/CEL. Finally, we examined the effects of F/PRα on the expression and function of lineage specific transcription factors by RT-PCR analysis and luciferase assays. When expressed in KSL cells, F/PRα augmented the expression of C/EBPα and GATA-2 and decreased PU.1 expression in KSL cells as early as 24h prior to the apparent development of EoPs as compared with mock and T/PRβ. Furthermore, we found that F/PRα and its downstream Ras but not STAT5 or PI3-K inhibited the activity of PU.1, but not of GATA-2 or C/EBPα. Together, these results suggest that F/PRα selectively induces EoP from hematopoietic stem/progenitor cells by modifying the expression and function of lineage specific transcription factors.

14-3-3 Integrates Pro-Survival Signals in Hematopoietic Cells Transformed by Diverse Leukemogenic Tyrosine Kinases, and Represents a Common Target.

Constitutively activated tyrosine kinases associated with recurrent chromosomal abnormalities play an essential role in the pathogenesis and disease progression of a variety of hematopoietic malignancies. Selective tyrosine kinase inhibitors such as imatinib are effective in treating some forms of leukemia such as t(9;22) CML associated with expression of BCR-ABL fusion tyrosine kinase. However, they are not curative and clinical resistance may develop, prompting the design of alternate and/or complementary therapeutic strategies. To better understand the signaling properties of constitutively activated tyrosine kinases associated with different hematopoietic malignancies, we examined whether BCR-ABL, FLT3-ITD, NPM-ALK, TEL-PDGFbetaR, TEL-FGFR3 and ZNF198-FGFR1 activate the same set of signaling pathways. We found that they all activated AKT and MAPK signaling pathways. Activated AKT resulted in phosphorylation of FOXO3a at Thr-32 but not BAD at Ser-136, whereas activated MAPK led to phosphorylation of BAD at Ser-112. These phosphorylated residues subsequently sequestered the pro-apoptotic FOXO3a and BAD to 14-3-3, suggesting that 14-3-3 integrates pro-survival signals from AKT and MAPK pathways. We utilized a peptide-based 14-3-3 competitive antagonist, R18 to disrupt 14-3-3/ligand association. Expression of R18 effectively induced apoptosis in hematopoietic Ba/F3 cells transformed by these tyrosine kinases with significantly enhanced sensitivity compared to the control Ba/F3 cells. Moreover, doxycycline-induced expression of R18 significantly attenuated the disease latency and penetrance in mice induced by intravenous injection of representative ZNF198-FGFR1-transformed Ba/F3 cells. Co-immunoprecipitation experiments indicate that induced R18 expression disrupted interaction between 14-3-3 and FOXO3a, but not 14-3-3/BAD association. R18 induced apoptosis by rescuing the nuclear localization of FOXO3a and up-regulating FOXO3a transcription targets Bim and p27 in cells expressing ZNF198-FGFR1. Furthermore, fluorescent confocal microscopy revealed that expression of R18 generally resumed FOXO3a nuclear localization in cells transformed by the spectrum of diverse leukemogenic tyrosine kinases. Together, these data support a model that 14-3-3 functions as a general integrator of pro-survival signals in hematopoietic transformation induced by diverse leukemogenic fusion/mutant tyrosine kinases. Disrupting 14-3-3/ligand association may be a common and effective therapeutic strategy for hematopoietic neoplasms associated with these tyrosine kinases.

The Role of Forkhead Transcription Factors FoxO1, FoxO3 and FoxO4 in Hematopoiesis and Leukemogenesis.

FoxO is a family of forkhead transcription factors that negatively regulate proliferation and survival signals in hematopoietic cells. We and others have previously shown that inhibition of the three members of this family (FoxO1, FoxO3 and FoxO4) by leukemogenic tyrosine kinase fusion genes results in enhanced proliferative and survival signaling in leukemic cells. For example, the transforming activities of the lymphoma associated NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) fusion, BCR-ABL, or FLT3-ITD, are mediated in part by inactivation of FoxO through phosphorylation and ubiquitin mediated degradation by constitutively active Akt (Gu TL, et al. Blood 2004), with subsequent induction of proliferative and survival signals. Furthermore, inhibition of FoxO is required for efficient transformation of hematopoietic cells. However, the roles of FoxO in adult hematopoiesis are unknown. We have initiated studies to examine the role of FoxO in the context of normal hematopoiesis and leukemogenesis using a triple conditional knockout mouse for each of the FoxO1, FoxO3 and FoxO4 alleles. The FoxO alleles are flanked by lox-P sites and conditional excision is mediated by Cre expression under the control of the interferon inducible Mx1 promoter. Based on the normal function of FoxO family members to repress proliferative and survival signals, we hypothesized that the deletion of FoxO subfamily members would lead to an enhanced proliferation and survival in the hematopoietic compartment, and might contribute to the development of a myeloproliferative and/or lymphoproliferative phenotype in vivo. Triple homozygous conditional FoxO knockout mice were generated in an Mx1-Cre background to allow for excision of the FoxO alleles in the hematopoietic stem cell compartment after treatment with pIpC. Complete excision of each of the three alleles in the hematopoietic compartment was confirmed. However, in contrast with our working hypothesis, we observed that loss of function of FoxO family members was associated with a relatively subtle hematopoietic phenotype with 12 months of follow-up. The phenotype includes a non-fatal mild myeloproliferative phenotype that is progressive over time and characterized by modest splenomegaly, extramedullary hematopoiesis and increased mature myeloid populations in bone marrow and spleen. In addition, there are subtle alterations in both B and T lymphoid cell populations, including a decrease in both immature and mature B cells in the spleen and bone marrow; and abnormalities of CD4+CD8+ double positive and CD4+ and CD8+ T cells in the thymus. Examination of stem and progenitor populations also revealed subtle differences in the HSC and CLP progenitor populations at 4 weeks post pIpC. Thus, these data indicate that complete loss of FoxO function in the adult hematopoietic compartment results in a relatively subtle hematopoietic phenotype. They further demonstrate that although inhibition of FoxO family members is required for efficient transformation of hematopoietic cells by leukemogenic fusion tyrosine kinases, loss of FoxO function alone is not sufficient to induce a leukemic phenotype.

Quantitative Proteomic Analysis Using Isobaric Protein Tags Enables Rapid Comparison of Changes in Transcript and Protein Levels in Transformed Cells

Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.

MOZ-TIF2, but Not BCR-ABL, Confers Properties of Leukemic Stem Cells to Committed Murine Hematopoietic Progenitors.

The existence of leukemia stem cells has been demonstrated in acute myeloid and lymphoblastic leukemias (AML and ALL). The origins of these cells are unknown, but it has been suggested that they result from the transformation of adult hematopoietic stem cells (HSC). To challenge this hypothesis we tested the ability of representative leukemia oncogenes to transform committed myeloid progenitor cells that lack the capacity for self-renewal. Flow-sorted populations of common myeloid progenitors (CMP), and granulocyte-monocyte progenitors (GMP) were transduced with the fusion oncogenes MOZ-TIF2 and BCR-ABL, respectively and their self-renewal and leukemogenic potential were tested in in vitro and in vivo assays. Utilizing the same experimental design we were also able to address the poorly understood question of the contribution of the cell of transformation to the eventual leukaemia phenotype.In contrast to CMP or GMP transduced with BCR-ABL or non-transduced control cells, CMP or GMP that were retrovirally transduced with MOZ-TIF2 could be serially replated in methylcellulose cultures, and continuously propagated in liquid culture media containing IL-3. In further contrast, transplantation of CMP or GMP transduced with MOZ-TIF2 into recipient mice also resulted in an acute myeloid leukemia (AML). This leukaemia could be transplanted to secondary recipients, documenting the long-term self-renewal properties of the leukemic stem cells, yet in limiting dilution experiments did not cause disease below a transplanted cell dose of 1 x104 cells, suggesting that the probability of transferring leukemia to secondary recipients relates to the frequency of self-renewing leukemic stem cells within the total leukemic population. This in turn suggests that our retroviral bone marrow transduction and transplantation models have the same hierarchical organization of self-renewal as has been shown for human AML. The phenotype of the leukemias were virtually indistinguishable regardless of whether the initially transduced cell population was CMP, GMP or the control populations of whole bone marrow mononuclear cells or HSC, suggesting that MOZ-TIF2 may also have a dominant effect upon the eventual leukaemia phenotype.These observations indicate that MOZ-TIF2, but not BCR-ABL, can confer properties of leukemic stem cells to committed myeloid progenitors. Control experiments conducted with with MOZ-TIF2 point mutants that do not cause leukemia in the murine BMT system and with BCR-ABL, a fully active leukemogenic tyrosine kinase, were insufficient to cause in-vitro changes in self-renewal or leukaemia. Together, these data argue strongly that retroviral insertional mutagenesis alone cannot explain these results. However, we cannot exclude the possibility that an active MOZ-TIF2, but not BCR-ABL, can collaborate with mutations induced by retroviral mutagenesis to confer properties of leukemic stem cells to committed progenitors. These findings have important implications regarding the origin of leukemic stem cells, and provide tools for understanding the transcriptional programs that confer properties of self-renewal in malignant and non-malignant cells.

Dual-specific Src and Abl kinase inhibitors, PP1 and CGP76030, inhibit growth and survival of cells expressing imatinib mesylate–resistant Bcr-Abl kinases

The leukemogenic tyrosine kinase Bcr-Abl contains a highly conserved inhibitor-binding pocket (IBP), which serves as a binding site for imatinib mesylate. Mutations at the IBP may lead to resistance of the Abl kinase against imatinib mesylate. To examine the mechanisms of imatinib mesylate binding and resistance in more detail, we created several point mutations at amino acid positions 315 and 380 of Abl, blocking the access to the IBP and rendering Bcr-Abl imatinib mesylate-resistant. Moreover, introduction of a mutation destabilizing the inactive conformation of Abl (Asp276Ser/Glu279Ser) also led to imatinib mesylate resistance, suggesting that the inhibitor required inactivation of the kinase prior to binding. These Bcr-Abl mutants were then used to evaluate the binding mode and specificity of 2 compounds, PP1 and CGP76030, originally characterized as Src kinase inhibitors. Both compounds inhibited Bcr-Abl in a concentration-dependent manner by overlapping binding modes. However, in contrast to imatinib mesylate, PP1 and CGP76030 blocked cell growth and survival in cells expressing various inhibitor-resistant Abl mutants. Studies on the potential signaling mechanisms demonstrated that in cells expressing inhibitor-resistant Bcr-Abl mutants, PP1 and CGP76030 inhibited the activity of Src family tyrosine kinases and Akt but not signal transducer and activator of transcription-5 (STAT5) and JUN kinase (Jnk). The results suggest that the use of Src kinase inhibitors is a potential strategy to prevent or overcome clonal evolution of imatinib mesylate resistance in Bcr-Abl(+) leukemia.