Novel imatinib-sensitive PDGFRA-activating point mutations in hypereosinophilic syndrome induce growth factor independence and leukemia-like disease

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2935-2943 ◽  
Author(s):  
Christian Elling ◽  
Philipp Erben ◽  
Christoph Walz ◽  
Marie Frickenhaus ◽  
Mirle Schemionek ◽  
...  
Keyword(s):  
Growth Factor ◽  
Hypereosinophilic Syndrome ◽  
Point Mutations ◽  
Imatinib Treatment ◽  
Presumptive Diagnosis ◽  
Activating Mutations ◽  
Transforming Activity ◽  
Selection For ◽  
Constitutive Phosphorylation

Abstract The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA–negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and—on selection for high expressors—also H650Q and R748G mutants induced growth factor–independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA–negative HES patients.

Novel PDGFRA Point Mutations in Hypereosinophilic Syndrome Induce Growth Factor Independence and Leukemia in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 767-767
Author(s):  
Christian Elling ◽  
Philipp Erben ◽  
Christoph Walz ◽  
Marie Frickenhaus ◽  
Mirle Schemionek ◽  
...  
Keyword(s):  
Growth Factor ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Hypereosinophilic Syndrome ◽  
Point Mutations ◽  
Fusion Genes ◽  
Double Mutation ◽  
Expression Levels

Abstract Abstract 767 Considerable progress has been achieved in our understanding of the pathogenesis of hypereosinophilic syndrome (HES) and chronic eosinophilic leukemia (CEL) by identification of constitutively activated tyrosine kinase fusion genes, e.g. FIP1L1-PDGFRA or ETV6-PDGFRB. However, the overall incidence of those fusion genes in HES/CEL is below 15%, and the molecular pathogenesis of the remaining cases remains elusive. We therefore established generic quantitative RT-PCR assays (RQ-PCR) to detect overexpression of 3'-regions of PDGFRA or PDGFRB as a possible indicator of an underlying fusion gene or point mutation. Patients with known fusion genes involving PDGFRA (n=5, 51 patients) or PDGFRB (n=5; 7 patients) showed significantly increased normalized expression levels compared to 191 patients with fusion gene-negative eosinophilia or healthy individuals (PDGFRA/ABL: 0.73 vs. 0.0066 vs. 0.0064, p<0.0001; PDGFRB/ABL: 196 vs. 3.8 vs. 5.85, p<0.0001). In all patients with significantly increased expression levels who were negative for fusion genes, functionally relevant regions of PDGFRA were sequenced. Several novel mutations (R481G, I562M, H570R, M628T, L705P, G729D) as well as a double mutation (H650Q and R748G) were identified. When cloned into 32D cells, M628T, H650Q, and R748G mutants separately induced growth factor-independent proliferation and clonogenic growth, and this was associated with constitutive phosphorylation of downstream targets STAT5, ERK, and AKT. Low doses of imatinib antagonized all of these effects in vitro. M628T and R748G but not H650Q 32D cell mutants induced acute leukemia after injection into congenic C3H/HeJ mice, similar to FIP1L1-PDGFRA. Interestingly, these two mutants showed a significantly higher propensity to invade the lymph nodes than the FIP1L1-PDGFRA fusion. Oral administration of imatinib to injected mice significantly decreased leukemic growth in vivo and significantly prolonged survival of the recipients. In conclusion, we demonstrate that novel point mutations of the PDGFRA gene found in patients with HES/CEL induce growth factor independence and leukemia in vitro and in vivo and suggest that these patients may benefit from treatment with imatinib. *CE, PE, AR, and SK contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Distinct Leukemogenic Activity and Imatinib Responsiveness of a BCR-PFGFRα Fusion Tyrosine Kinase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3634-3634 ◽  
Author(s):  
L. Cristina Gavrilescu ◽  
Nicholas C.P. Cross ◽  
Richard A. Van Etten
Keyword(s):  
Bone Marrow ◽  
Cellular Proliferation ◽  
Chromosomal Rearrangements ◽  
Hypereosinophilic Syndrome ◽  
Growth Factor Receptor ◽  
Imatinib Treatment ◽  
Myeloproliferative Disease ◽  
Imatinib Resistance ◽  
Inhibition Of Proliferation

Abstract Chromosomal rearrangements of the platelet-derived growth factor receptor alpha (PDGFRA) gene on 4q12 have been described in a subset of patients with idiopathic hypereosinophilic syndrome/chronic eosinophilic leukemia (IHES/CEL). The most common, found in 12–17% of CEL patients, is the FIP1L1-PDGFRA fusion, resulting from a cytogenetically invisible deletion on 4q12 (Cools et al., NEJM2003; 348:1201; Griffin et al., PNAS2003; 100:7830). FIP1L1-PDGFRA+ CEL patients have an excellent response to low-dose imatinib therapy, often accompanied by complete molecular remission (Pardanani et al., Leuk. Res.2006; 30:965). Additional fusion partners of PDGFRA include KIF5B and CDK5RAP2 in CEL, and BCR in atypical chronic myeloid leukemia with eosinophilia and t(4;22)(q12;q11) (Baxter et al., Hum. Mol. Genet.2002; 11:1391). However, the oncogenic activity and imatinib responsiveness of these other activated PDGFRA alleles are unknown. In this study, we assessed the imatinib sensitivity of BCR-PDGFRα and FIP1L1-PDGFRα in hematopoietic cell lines, and compared their leukemogenic activity in a mouse retroviral bone marrow transduction/transplantation model. Like FIP1L1-PDGFRα, BCR-PDGFRα transformed Ba/F3 cells to become independent of IL-3 for survival and growth. In the absence of IL-3, FIP1L1-PDGFRα-expressing Ba/F3 cells were 30-fold more sensitive to imatinib than BCR-PDGFRα (IC50 = 3 nM vs. 90 nM) for inhibition of proliferation and induction of apoptosis. A FIP1L1-PDGFRα N659D mutant (Cools et al., Cancer Cell2003; 3:459) was relatively resistant to imatinib (IC50 = 200 nM), while the corresponding BCR-PDGFRα N659D mutant displayed increased imatinib resistance (IC50 > 2 μM). Inhibition of cellular proliferation correlated with decreased tyrosyl phosphorylation of the respective fusion kinase and of the downstream substrate PLCγ1. In leukemogenesis experiments, recipients of bone marrow transduced with retrovirus expressing either FIP1L1-PDGFRα or BCR-PDGFRα developed fatal myeloproliferative disease, with similar median and overall survival. This was accompanied by significant eosinophilia, relative to the disease induced by BCR-ABL in this model system (Li et al., Blood2001; 97:1442). Treatment of recipients of FIP1L1-PDGFRA- or BCR-PDGFRA-transduced marrow with imatinib (125 mg/kg/day by oral gavage for 20 days) suppressed leukocytosis and prolonged their survival. These results suggest that distinct signaling pathways activated by leukemogenic PDGFRα fusions in hematopoietic progenitors induce eosinophila in vivo. However, different fusion partners of PDGFRα can significantly influence the sensitivity of the fusion kinase to imatinib treatment.

A FLT3-targeted tyrosine kinase inhibitor is cytotoxic to leukemia cells in vitro and in vivo

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3885-3891 ◽  
Author(s):  
Mark Levis ◽  
Jeffrey Allebach ◽  
Kam-Fai Tse ◽  
Rui Zheng ◽  
Brenda R. Baldwin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Internal Tandem Duplication ◽  
Activating Mutations ◽  
Flt3 Inhibitor

Constitutively activating internal tandem duplication (ITD) and point mutations of the receptor tyrosine kinase FLT3 are present in up to 41% of patients with acute myeloid leukemia (AML). These FLT3/ITD mutations are likely to be important because their presence is associated with a poor prognosis. Both types of mutations appear to activate the tyrosine kinase activity of FLT3. We describe here the identification and characterization of the indolocarbazole derivative CEP-701 as a FLT3 inhibitor. This drug potently and selectively inhibits autophosphorylation of wild-type and constitutively activated mutant FLT3 in vitro in FLT3/ITD-transfected cells and in human FLT3-expressing myeloid leukemia–derived cell lines. We demonstrate that CEP-701 induces a cytotoxic effect on cells in a dose-responsive fashion that parallels the inhibition of FLT3. STAT5 and ERK1/2, downstream targets of FLT3 in the signaling pathway, are inhibited in response to FLT3 inhibition. In primary leukemia blasts from AML patients harboring FLT3/ITD mutations, FLT3 is also inhibited, with an associated cytotoxic response. Finally, using a mouse model of FLT3/ITD leukemia, we demonstrate that the drug inhibits FLT3 phosphorylation in vivo and prolongs survival. These findings form the basis for a planned clinical trial of CEP-701 in patients with AML harboring FLT3- activating mutations.

scholarly journals Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo

1992 ◽  
Vol 12 (2) ◽  
pp. 609-618
Author(s):  
B J Mayer ◽  
P K Jackson ◽  
R A Van Etten ◽  
D Baltimore
Keyword(s):  
Tyrosine Kinases ◽  
Point Mutations ◽  
Sh2 Domain ◽  
Sh2 Domains ◽  
Phosphorylated Proteins ◽  
Abl Tyrosine Kinase ◽  
Transforming Activity ◽  
Src Homology

We have constructed a series of point mutations in the highly conserved FLVRES motif of the src homology 2 (SH2) domain of the abl tyrosine kinase. Mutant SH2 domains were expressed in bacteria, and their ability to bind to tyrosine-phosphorylated proteins was examined in vitro. Three mutants were greatly reduced in their ability to bind both phosphotyrosine itself and tyrosine-phosphorylated cellular proteins. All of the mutants that retained activity bound to the same set of tyrosine-phosphorylated proteins as did the wild type, suggesting that binding specificity was unaffected. These results implicate the FLVRES motif in direct binding to phosphotyrosine. When the mutant SH2 domains were inserted into an activated abl kinase and expressed in murine fibroblasts, decreased in vitro phosphotyrosine binding correlated with decreased transforming ability. This finding implies that SH2-phosphotyrosine interactions are involved in transmission of positive growth signals by the nonreceptor tyrosine kinases, most likely via the assembly of multiprotein complexes with other tyrosine-phosphorylated proteins.

scholarly journals Point mutations in the abl SH2 domain coordinately impair phosphotyrosine binding in vitro and transforming activity in vivo.

1992 ◽  
Vol 12 (2) ◽  
pp. 609-618 ◽  
Author(s):  
B J Mayer ◽  
P K Jackson ◽  
R A Van Etten ◽  
D Baltimore
Keyword(s):  
Tyrosine Kinases ◽  
Point Mutations ◽  
Sh2 Domain ◽  
Sh2 Domains ◽  
Phosphorylated Proteins ◽  
Abl Tyrosine Kinase ◽  
Transforming Activity ◽  
Src Homology

We have constructed a series of point mutations in the highly conserved FLVRES motif of the src homology 2 (SH2) domain of the abl tyrosine kinase. Mutant SH2 domains were expressed in bacteria, and their ability to bind to tyrosine-phosphorylated proteins was examined in vitro. Three mutants were greatly reduced in their ability to bind both phosphotyrosine itself and tyrosine-phosphorylated cellular proteins. All of the mutants that retained activity bound to the same set of tyrosine-phosphorylated proteins as did the wild type, suggesting that binding specificity was unaffected. These results implicate the FLVRES motif in direct binding to phosphotyrosine. When the mutant SH2 domains were inserted into an activated abl kinase and expressed in murine fibroblasts, decreased in vitro phosphotyrosine binding correlated with decreased transforming ability. This finding implies that SH2-phosphotyrosine interactions are involved in transmission of positive growth signals by the nonreceptor tyrosine kinases, most likely via the assembly of multiprotein complexes with other tyrosine-phosphorylated proteins.

scholarly journals Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

2017 ◽  
Vol 114 (40) ◽  
pp. E8448-E8457 ◽  
Author(s):  
Benedikt Bosbach ◽  
Ferdinand Rossi ◽  
Yasemin Yozgat ◽  
Jennifer Loo ◽  
Jennifer Q. Zhang ◽  
...  
Keyword(s):  
Binding Site ◽  
Point Mutations ◽  
Mek Inhibitor ◽  
Pi3k Pathway ◽  
Mapk Signaling ◽  
Oncogenic Signaling ◽  
Activating Mutations ◽  
Imatinib Resistant ◽  
Cells Of Cajal

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors.

Improvement of effectiveness in maize breeding

2006 ◽  
Vol 54 (3) ◽  
pp. 351-358 ◽  
Author(s):  
P. Pepó
Keyword(s):  
Recurrent Selection ◽  
Genetic Manipulation ◽  
Field Testing ◽  
Tissue Cultures ◽  
Maize Breeding ◽  
Breeding Programmes ◽  
Speed Up ◽  
Selection For

Plant regeneration via tissue culture is becoming increasingly more common in monocots such as maize (Zea mays L.). Pollen (gametophytic) selection for resistance to aflatoxin in maize can greatly facilitate recurrent selection and the screening of germplasm for resistance at much less cost and in a shorter time than field testing. In vivo and in vitro techniques have been integrated in maize breeding programmes to obtain desirable agronomic attributes, enhance the genes responsible for them and speed up the breeding process. The efficiency of anther and tissue cultures in maize and wheat has reached the stage where they can be used in breeding programmes to some extent and many new cultivars produced by genetic manipulation have now reached the market.

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