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Jan Cools - One of the best experts on this subject based on the ideXlab platform.

  • use of crispr cas genome editing in ba f3 cells to generate the FIP1L1 pdgfra and nup214 abl1 fusion genes
    Blood, 2014
    Co-Authors: Marlies Vanden Bempt, Nicole Mentens, Olga Gielen, Charles E. De Bock, Ellen Geerdens, Sofie Demeyer, Jan Cools
    Abstract:

    CRISPR/Cas genome editing is a powerful tool to precisely induce chromosomal breaks and to modify genes of interest. Cas9, an RNA-guided DNA endonuclease derived from Streptococcus pyogenes, is able to generate double stranded breaks (DSBs) in the genomic locus to where it is directed by its guide RNA (gRNA) component. The DSBs are subsequently repaired by one of the two main host repair mechanisms: the error-prone Non-homologous end joining (NHEJ) pathway or the very specific Homology-directed repair (HDR) pathway. We aimed to use CRISPR/Cas genome editing to generate the FIP1L1-Pdgfra and Nup214-Abl1 fusion genes by inducing chromosomal rearrangements in the interleukin-3 dependent Ba/F3 cell line. Prior to generating the chromosomal rearrangements, we optimized CRISPR/Cas genome editing in Ba/F3 cells, by targeting Cas9 to exon 24 of CD45, a cell surface transmembrane protein, of which inactivation can be easily detected by flow cytometry. Electroporation of Ba/F3 cells with plasmids expressing Cas9 and the specific guide RNA led to efficient inactivation of the CD45 gene, as measured by flow cytometry (30% of the cells showed loss of CD45 expression). The use of the Cas9 nickase variant led to an increased efficiency of CD45 inactivation with 58% of the cells showing loss of CD45 expression. We then extended these studies to assess the efficiency of homology-directed repair to introduce a specific mutation, using a single strand donor template to generate a premature stop codon in exon 24 of CD45. The successful introduction of the novel stop codon in CD45 was confirmed by PCR amplification of the targeted exon followed by massive parallel sequencing (MiSeq, Illumina) and we observed this endogenous mutation in 80% of the Ba/F3 clones. Having optimised the use and efficiency of CRISPR/Cas in Ba/F3 cells, we aimed to introduce double stranded breaks simultaneously in the genes FIP1L1 and Pdgfra to generate a cell based model for the FIP1L1-PDGFRA fusion gene as observed in chronic eosinophilic leukemia. Double strand breaks were introduced in FIP1L1 exon 23, 31, 32 or 34 together with simultaneous breaks in Pdgfra exon 12, both located on mouse chromosome 5. Upon IL3 removal, cells harbouring the deletion and fusion gene were able to survive, grow and form colonies in semi-solid medium, as was shown before for Ba/F3 cells transduced with retroviral vectors expressing FIP1L1-PDGFRA. The presence of the deletion was confirmed by PCR, and fusion protein expression was detected by Western blotting. A fusion between exon 1 of FIP1L1 and exon 12 of Pdgfra could also transform the cells, which confirmed earlier findings that the transforming capacities of the fusion protein are independent of FIP1L1 and dependent on the interruption of the juxtamembrane region of PDGFRA. The expression and phosphorylation levels of FIP1L1-Pdgfra were compared between the CRISPR/Cas generated Ba/F3 cells and retrovirally transduced cells overexpressing FIP1L1-PDGFRA. As expected, retrovirally transduced cells showed a much higher protein expression level of FIP1L1-PDGFRA, and much stronger phosphorylation compared to the CRISPR/Cas generated cells, in which the endogenous FIP1L1 promoter is used to drive the expression of the fusion protein. We also observed a difference in sensitivity to inhibition by imatinib, a kinase inhibitor with strong activity against PDGFRA. The same strategy was followed to generate a fusion between Nup214 and Abl1, as observed in a subset of T-cell acute lymphoblastic leukemia cases. Ba/F3 cells harbouring the Nup214-Abl1 fusion gene were able to survive and grow independent of IL3. The presence of the fusion gene was confirmed by PCR, and fusion protein expression was detected by Western blotting. Taken together, these data show that CRISPR/Cas induced chromosomal translocations in cells more faithfully recapitulate gene expression levels and sensitivity to chemotherapeutics when compared to retroviral transduction based expression of an oncogene. In conclusion, we have now designed and implemented an optimised platform to use CRISPR/Cas genome editing in Ba/F3 cells and measure gRNA efficacy by massive parallel sequencing. Our data confirm that the CRISPR/Cas genome editing system can be used to generate chromosomal rearrangements in Ba/F3 cells and provides a method to generate improved cell based models for the study of oncogenic tyrosine kinases. Disclosures No relevant conflicts of interest to declare.

  • Use of Crispr/Cas Genome Editing in Ba/F3 Cells to Generate the FIP1L1-Pdgfra and Nup214-Abl1 Fusion Genes
    Blood, 2014
    Co-Authors: Marlies Vanden Bempt, Nicole Mentens, Olga Gielen, Charles E. De Bock, Ellen Geerdens, Sofie Demeyer, Jan Cools
    Abstract:

    CRISPR/Cas genome editing is a powerful tool to precisely induce chromosomal breaks and to modify genes of interest. Cas9, an RNA-guided DNA endonuclease derived from Streptococcus pyogenes, is able to generate double stranded breaks (DSBs) in the genomic locus to where it is directed by its guide RNA (gRNA) component. The DSBs are subsequently repaired by one of the two main host repair mechanisms: the error-prone Non-homologous end joining (NHEJ) pathway or the very specific Homology-directed repair (HDR) pathway. We aimed to use CRISPR/Cas genome editing to generate the FIP1L1-Pdgfra and Nup214-Abl1 fusion genes by inducing chromosomal rearrangements in the interleukin-3 dependent Ba/F3 cell line. Prior to generating the chromosomal rearrangements, we optimized CRISPR/Cas genome editing in Ba/F3 cells, by targeting Cas9 to exon 24 of CD45, a cell surface transmembrane protein, of which inactivation can be easily detected by flow cytometry. Electroporation of Ba/F3 cells with plasmids expressing Cas9 and the specific guide RNA led to efficient inactivation of the CD45 gene, as measured by flow cytometry (30% of the cells showed loss of CD45 expression). The use of the Cas9 nickase variant led to an increased efficiency of CD45 inactivation with 58% of the cells showing loss of CD45 expression. We then extended these studies to assess the efficiency of homology-directed repair to introduce a specific mutation, using a single strand donor template to generate a premature stop codon in exon 24 of CD45. The successful introduction of the novel stop codon in CD45 was confirmed by PCR amplification of the targeted exon followed by massive parallel sequencing (MiSeq, Illumina) and we observed this endogenous mutation in 80% of the Ba/F3 clones. Having optimised the use and efficiency of CRISPR/Cas in Ba/F3 cells, we aimed to introduce double stranded breaks simultaneously in the genes FIP1L1 and Pdgfra to generate a cell based model for the FIP1L1-PDGFRA fusion gene as observed in chronic eosinophilic leukemia. Double strand breaks were introduced in FIP1L1 exon 23, 31, 32 or 34 together with simultaneous breaks in Pdgfra exon 12, both located on mouse chromosome 5. Upon IL3 removal, cells harbouring the deletion and fusion gene were able to survive, grow and form colonies in semi-solid medium, as was shown before for Ba/F3 cells transduced with retroviral vectors expressing FIP1L1-PDGFRA. The presence of the deletion was confirmed by PCR, and fusion protein expression was detected by Western blotting. A fusion between exon 1 of FIP1L1 and exon 12 of Pdgfra could also transform the cells, which confirmed earlier findings that the transforming capacities of the fusion protein are independent of FIP1L1 and dependent on the interruption of the juxtamembrane region of PDGFRA. The expression and phosphorylation levels of FIP1L1-Pdgfra were compared between the CRISPR/Cas generated Ba/F3 cells and retrovirally transduced cells overexpressing FIP1L1-PDGFRA. As expected, retrovirally transduced cells showed a much higher protein expression level of FIP1L1-PDGFRA, and much stronger phosphorylation compared to the CRISPR/Cas generated cells, in which the endogenous FIP1L1 promoter is used to drive the expression of the fusion protein. We also observed a difference in sensitivity to inhibition by imatinib, a kinase inhibitor with strong activity against PDGFRA. The same strategy was followed to generate a fusion between Nup214 and Abl1, as observed in a subset of T-cell acute lymphoblastic leukemia cases. Ba/F3 cells harbouring the Nup214-Abl1 fusion gene were able to survive and grow independent of IL3. The presence of the fusion gene was confirmed by PCR, and fusion protein expression was detected by Western blotting. Taken together, these data show that CRISPR/Cas induced chromosomal translocations in cells more faithfully recapitulate gene expression levels and sensitivity to chemotherapeutics when compared to retroviral transduction based expression of an oncogene. In conclusion, we have now designed and implemented an optimised platform to use CRISPR/Cas genome editing in Ba/F3 cells and measure gRNA efficacy by massive parallel sequencing. Our data confirm that the CRISPR/Cas genome editing system can be used to generate chromosomal rearrangements in Ba/F3 cells and provides a method to generate improved cell based models for the study of oncogenic tyrosine kinases. Disclosures No relevant conflicts of interest to declare.

  • The tyrosine phosphatase SHP2 is required for cell transformation by the receptor tyrosine kinase mutants FIP1L1-PDGFRα and PDGFRα D842V
    Molecular oncology, 2014
    Co-Authors: Laura A. Noël, Jan Cools, Florence A. Arts, Carmen P. Montano-almendras, Luk Cox, Olga Gielen, Federica Toffalini, Catherine Y. Marbehant, Jean-baptiste Demoulin
    Abstract:

    Abstract Activated forms of the platelet derived growth factor receptor alpha (PDGFRα) have been described in various tumors, including FIP1L1-PDGFRα in patients with myeloproliferative diseases associated with hypereosinophilia and the PDGFRαD842V mutant in gastrointestinal stromal tumors and inflammatory fibroid polyps. To gain a better insight into the signal transduction mechanisms of PDGFRα oncogenes, we mutated twelve potentially phosphorylated tyrosine residues of FIP1L1-PDGFRα and identified three mutations that affected cell proliferation. In particular, mutation of tyrosine 720 in FIP1L1-PDGFRα or PDGFRαD842V inhibited cell growth and blocked ERK signaling in Ba/F3 cells. This mutation also decreased myeloproliferation in transplanted mice and the proliferation of human CD34+ hematopoietic progenitors transduced with FIP1L1-PDGFRα. We showed that the non-receptor protein tyrosine phosphatase SHP2 bound directly to tyrosine 720 of FIP1L1-PDGFRα. SHP2 knock-down decreased proliferation of Ba/F3 cells transformed with FIP1L1-PDGFRα and PDGFRαD842V and affected ERK signaling, but not STAT5 phosphorylation. Remarkably, SHP2 was not essential for cell proliferation and ERK phosphorylation induced by the wild-type PDGF receptor in response to ligand stimulation, suggesting a shift in the function of SHP2 downstream of oncogenic receptors. In conclusion, our results indicate that SHP2 is required for cell transformation and ERK activation by mutant PDGF receptors.

  • ponatinib is active against imatinib resistant mutants of FIP1L1 pdgfra and kit and against fgfr1 derived fusion kinases
    Leukemia, 2012
    Co-Authors: Els Lierman, Jan Cools, Sanne Smits, Bart Dewaele, Maria Debiecrychter, Peter Vandenberghe
    Abstract:

    Ponatinib is active against imatinib-resistant mutants of FIP1L1-PDGFRA and KIT, and against FGFR1-derived fusion kinases

  • Ponatinib Is Active Against the CUX1-FGFR1 Fusion Kinase and Against Imatinib Resistance Mutations of the FIP1L1-PDGFRα Fusion Kinase and of KIT,
    Blood, 2011
    Co-Authors: Els Lierman, Jan Cools, Sanne Smits, Barbara Dewaele, Maria Debiec-rychter, Peter Vandenberghe
    Abstract:

    Abstract 3848 Imatinib (IM) was initially developed as a small molecule inhibitor of the BCR-ABL1 kinase, but also potently inhibits other oncogenic kinases, such as PDGFRβ and PDGFRα fusion proteins. IM has revolutionized the treatment of chronic myeloid leukemia (CML) and other neoplasms, but the development of IM resistant mutations has emerged as an important problem, triggering a search for novel compounds that overcome resistance. A leading third generation candidate is ponatinib, a novel multikinase inhibitor with potent activity towards BCR-ABL1, KIT, FGFR1, PDGFRα and other kinases. Importantly, ponatinib also targets numerous IM resistant BCR-ABL1 kinase domain mutations including the panresistant T315I mutation. We investigated the effect of this compound on IM resistant kinase mutations in lymphoid/myeloid neoplasms associated with eosinophilia and rearrangements of PDGFRα and FGFR1, as well as KIT associated malignancies. Ba/F3 cells were used expressing either FIP1L1-PDGFRα, the IM resistant FIP1L1-PDGFRα-T674I mutant, the panresistant FIP1L1-PDGFRα-D842V mutant, or the novel CUX1-FGFR1 fusion. In addition, several KIT mutants were investigated. The growth of FIP1L1-PDGFRα and IM resistant FIP1L1-PDGFRα-T674I mutant expressing cells was strongly inhibited by ponatinib with IC50 values of 0,6 nM and 9 nM respectively. Also the panresistant FIP1L1-PDGFRα-D842V mutant and the novel CUX1-FGFR1 fusion responded well to ponatinib treatment with 50% growth inhibition at 154 nM and 56 nM respectively. IL3-driven growth of Ba/F3 cells was resistant to ponatinib (IC50: 2 μM). Western blot analysis confirmed the direct effect of ponatinib on the auto-phosphorylation of the PDGFRα and FGFR1 fusion proteins, as well as on the downstream signaling protein STAT5. Finally, we investigated several KIT single and double mutants and preliminary data indicate an inhibitory effect of ponatinib towards several KIT mutants. In conclusion, our results demonstrate the in vitro activity of ponatinib against IM resistant mutants of the FIP1L1-PDGFRα fusion kinase, against the CUX1-FGFR1 fusion kinase as well as against IM resistant KIT mutations. Our data indicate that ponatinib, which is currently under investigation in phase II clinical trials for IM resistant CML, may also be active against neoplasms driven by FGFR1, PDGFR or KIT kinase activity, and able to overcome IM resistance in these malignancies. Disclosures: No relevant conflicts of interest to declare.

Jingxuan Pan - One of the best experts on this subject based on the ideXlab platform.

  • Antitumor activity of S116836, a novel tyrosine kinase inhibitor, against imatinib-resistant FIP1L1-PDGFRα-expressing cells.
    Oncotarget, 2014
    Co-Authors: Yingying Shen, Ke Ding, Xiaomei Ren, Zhang Zhang, Deping Wang, Jingxuan Pan
    Abstract:

    // Yingying Shen 1,* , Xiaomei Ren 2,* , Ke Ding 2 , Zhang Zhang 2 , Deping Wang 2 and Jingxuan Pan 1,3,4 1 Department of Pathophysiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China 2 Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China 3 State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China 4 Collaborative Innovation Center for Cancer Medicine, State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Guangzhou, China * These authors contributed equally to this work Correspondence: Jingxuan Pan, email: // Keywords : PDGFRα, T674I, tyrosine kinase inhibitor, imatinib, resistance, S116836, apoptosis, Bim Received : February 20, 2014 Accepted : June 10, 2014 Published : June 11, 2014 Abstract The FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRα) fusion oncogene is the driver factor in a subset of patients with hypereosinophilic syndrome (HES)/chronic eosinophilic leukemia (CEL). Most FIP1L1-PDGFRα-positive patients respond well to the tyrosine kinase inhibitor (TKI) imatinib. Resistance to imatinib in HES/CEL has been described mainly due to the T674I mutation in FIP1L1-PDGFRα, which is homologous to the imatinib-resistant T315I mutation in BCR-ABL. Development of novel TKIs is imperative to overcome resistance to imatinib. We synthesized S116836, a novel TKI. In this study, we evaluated the antitumor activity of S116836 in FIP1L1-PDGFRα-expressing cells. The results showed that S116836 potently inhibited PDGFRα and its downstream signaling molecules such as STAT3, AKT, and Erk1/2. S116836 effectively inhibited the growth of the WT and T674I FIP1L1-PDGFRα-expressing neoplastic cells in vitro and in nude mouse xenografts. Moreover, S116836 induced intrinsic pathway of apoptosis as well as the death receptor pathway, coincided with up-regulation of the proapoptotic BH3-only protein Bim-EL through the Erk1/2 pathway. In conclusion, S116836 is active against WT and T674I FIP1L1-PDGFRα-expressing cells, and may be a prospective agent for the treatment of HES/CEL.

  • Ponatinib efficiently kills imatinib-resistant chronic eosinophilic leukemia cells harboring gatekeeper mutant T674I FIP1L1-PDGFRα: roles of Mcl-1 and β-catenin.
    Molecular cancer, 2014
    Co-Authors: Yanli Jin, Ke Ding, Mengzhu Xue, Xiaoke Shi, Chengyan Wang, Jingxuan Pan
    Abstract:

    Background: T674I FIP1L1-PDGFRα in a subset of chronic eosinophilic leukemia (CEL) is a gatekeeper mutation that is resistant to many tyrosine kinase inhibitors (TKIs) (e.g., imatinib, nilotinib and dasatinib), similar to T315I Bcr-Abl. Therefore, novel TKIs effective against T674I FIP1L1-PDGFRα are needed. Ponatinib (AP24534) is a novel orally bioavailable TKI against T315I Bcr-Abl, but it is not clear whether ponatinib is effective against T674I FIP1L1-PDGFRα. The purpose of this study was to examine the effect of ponatinib on T674I FIP1L1-PDGFRα. Methods: Molecular docking analysis in silico was performed. The effects of ponatinib on PDGFRα signaling pathways, apoptosis and cell cycling were examined in EOL-1, BaF3 cells expressing either wild type (WT) or T674I FIP1L1-PDGFRα .T he in vivo antitumor activity of ponatinib was evaluated with xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice models. Results: Molecular docking analysis revealed that ponatinib could bind to the DFG (Asp-Phe-Gly)-out state of T674I PDGFRα. Ponatinib potently inhibited the phosphorylation of WT and T674I FIP1L1-PDGFRα and their downstream signaling molecules (e.g., Stat3, Stat5). Ponatinib strikingly inhibited the growth of both WT and T674I FIP1L1-PDGFRα-carrying CEL cells (IC50 :0 .004– 2.5 nM). It induced apoptosis in CEL cells with caspase-3-dependent cleavage of Mcl-1, and inhibited tyrosine phosphorylation of β-catenin to decrease its stability and pro-survival functions. In vivo, ponatinib abrogated the growth of xenografted BaF3-T674I FIP1L1-PDGFRα cells in nude mice. Conclusions: Ponatinib is a pan-FIP1L1-PDGFRα inhibitor, and clinical trials are warranted to investigate its efficacy in imatinib-resistant CEL.

  • The Conformational Control Inhibitor of Tyrosine Kinases DCC-2036 Is Effective for Imatinib-Resistant Cells Expressing T674I FIP1L1-PDGFRα
    PloS one, 2013
    Co-Authors: Yingying Shen, Xiaoke Shi, Jingxuan Pan
    Abstract:

    The cells expressing the T674I point mutant of FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1-PDGFRα) in hypereosinophilics syndrome (HES) are resistant to imatinib and some second-generation tyrosine kinase inhibitors (TKIs). There is a desperate need to develop therapy to combat this acquired drug resistance. DCC-2036 has been synthesized as a third-generation TKI to combat especially the Bcr-Abl T315I mutant in chronic myeloid leukemia. This study evaluated the effect of DCC-2036 on FIP1L1-PDGFRα-positive cells, including the wild type (WT) and the T674I mutant. The in vitro effects of DCC-2036 on the PDGFRα signal pathways, proliferation, cell cycling and apoptosis of FIP1L1-PDGFRα-positive cells were investigated, and a nude mouse xenograft model was employed to assess the in vivo antitumor activity. We found that DCC-2036 decreased the phosphorylated levels of PDGFRα and its downstream targets without apparent effects on total protein levels. DCC-2036 inhibited proliferation, and induced apoptosis with MEK-dependent up-regulation of the pro-apoptotic protein Bim in FIP1L1-PDGFRα-positive cells. DCC-2036 also exhibited in vivo antineoplastic activity against cells with T674I FIP1L1-PDGFRα. In summary, FIP1L1-PDGFRα-positive cells are sensitive to DCC-2036 regardless of their sensitivity to imatinib. DCC-2036 may be a potential compound to treat imatinib-resistant HES.

  • Triptolide abrogates oncogene FIP1L1-PDGFRα addiction and induces apoptosis in hypereosinophilic syndrome
    Cancer science, 2009
    Co-Authors: Yanli Jin, Qi Chen, Bo Chen, Jingxuan Pan
    Abstract:

    The pathogenesis of hypereosinophilic syndrome (HES) in some patients is highly dependent on FIP1-Like-1 (FIP1L1)–platelet-derived growth factor receptor alpha (PDGFRα), which can generate sustained activation signaling to maintain a cell malignant phenotype. HES usually shows good response to the tyrosine kinase inhibitor imatinib, but mutations in FIP1L1-PDGFRα (e.g. T674I) can confer acquired resistance to imatinib. An alternative therapeutic strategy other than with tyrosine kinase inhibitors is needed to overcome acquired drug resistance. We hypothesized that switching off the crucial chimeric oncoprotein FIP1L1-PDGFRα on which HES cells depend, should have deleterious effects on the cancer cells. We used low concentrations of triptolide, a transcription inhibitor, to shut down the expression of FIP1L1-PDGFRα. EOL-1 cells and BaF3 cells expressing wild-type or T674I FIP1L1-PDGFRα were treated with triptolide, and signaling pathways, cell cycling, and apoptosis were analyzed by RT-PCR, immunoblotting, and flow cytometry, respectively. The results revealed that at nanomolar concentrations triptolide decreased the levels of mRNA and protein of FIP1L1-PDGFRα and the growth of the neoplastic cells, regardless of the mutational status of PDGFRα. Triptolide also downregulated the signaling molecules Stat3, Akt, and Erk1/2, which are downstream from PDGFRα, and induced G1 cell-cycle arrest. Triptolide time- and dose-dependently induced apoptosis by decreasing the anti-apoptotic proteins Mcl-1 and Bcl-XL,triggering the intrinsic apoptotic pathway. In conclusion, triptolide has potent activity against malignant cells in HES bearing FIP1L1-PDGFRα, regardless of its mutational status that confer acquired resistance to imatinib. Our results suggest that triptolide may be a promising agent in the treatment of HES. (Cancer Sci 2009; 00: 000–000)

  • The novel tyrosine kinase inhibitor EXEL-0862 induces apoptosis in human FIP1L1-PDGFR-α-expressing cells through caspase-3-mediated cleavage of Mcl-1
    Leukemia, 2007
    Co-Authors: Jingxuan Pan, Alfonso Quintás-cardama, Taghi Manshouri, Francis J. Giles, Peter Lamb, A Tefferi, Jorge E. Cortes, Hagop M. Kantarjian, Srdan Verstovsek
    Abstract:

    The FIP1-like-1 (FIP1L1)-platelet-derived growth factor receptor-alpha (FIP1L1-PDGFR-α) fusion kinase causes hypereosinophilic syndrome (HES) in a defined subset of patients. Imatinib mesylate is a potent inhibitor of ABL but also of PDGFR-α, and has been associated with durable hematologic responses in patients with HES. However, development of mutations in the tyrosine kinase domain may hamper the activity of tyrosine kinase inhibitors (TKIs), which suggests that novel agents are warranted to prevent or overcome resistance. We evaluated the efficacy of the novel TKI EXEL-0862 in FIP1L1-PDGFR-α-expressing cell lines and in cells from a patient with HES harboring the FIP1L1-PDGFR-α gene. EXEL-0862 inhibited the proliferation of EOL-1 and imatinib-resistant T674I FIP1L1-PDGFR-α-expressing cells and resulted in potent inhibition of the phosphorylation of PDGFR-α and downstream proteins STAT3 and Erk1/2, both in vitro and ex vivo. Moreover, EXEL-0862 induced apoptotic death in EOL-1 cells and imatinib-resistant T674I FIP1L1-PDGFR-α-expressing cells, and resulted in significant downregulation of the antiapoptotic protein Mcl-1 through a caspase-dependent mechanism. Our data establish EXEL-0862 as a solid candidate for the targeted treatment of patients with FIP1L1-PDGFR-α-positive HES.

Peter Vandenberghe - One of the best experts on this subject based on the ideXlab platform.

  • ponatinib is active against imatinib resistant mutants of FIP1L1 pdgfra and kit and against fgfr1 derived fusion kinases
    Leukemia, 2012
    Co-Authors: Els Lierman, Jan Cools, Sanne Smits, Bart Dewaele, Maria Debiecrychter, Peter Vandenberghe
    Abstract:

    Ponatinib is active against imatinib-resistant mutants of FIP1L1-PDGFRA and KIT, and against FGFR1-derived fusion kinases

  • Ponatinib Is Active Against the CUX1-FGFR1 Fusion Kinase and Against Imatinib Resistance Mutations of the FIP1L1-PDGFRα Fusion Kinase and of KIT,
    Blood, 2011
    Co-Authors: Els Lierman, Jan Cools, Sanne Smits, Barbara Dewaele, Maria Debiec-rychter, Peter Vandenberghe
    Abstract:

    Abstract 3848 Imatinib (IM) was initially developed as a small molecule inhibitor of the BCR-ABL1 kinase, but also potently inhibits other oncogenic kinases, such as PDGFRβ and PDGFRα fusion proteins. IM has revolutionized the treatment of chronic myeloid leukemia (CML) and other neoplasms, but the development of IM resistant mutations has emerged as an important problem, triggering a search for novel compounds that overcome resistance. A leading third generation candidate is ponatinib, a novel multikinase inhibitor with potent activity towards BCR-ABL1, KIT, FGFR1, PDGFRα and other kinases. Importantly, ponatinib also targets numerous IM resistant BCR-ABL1 kinase domain mutations including the panresistant T315I mutation. We investigated the effect of this compound on IM resistant kinase mutations in lymphoid/myeloid neoplasms associated with eosinophilia and rearrangements of PDGFRα and FGFR1, as well as KIT associated malignancies. Ba/F3 cells were used expressing either FIP1L1-PDGFRα, the IM resistant FIP1L1-PDGFRα-T674I mutant, the panresistant FIP1L1-PDGFRα-D842V mutant, or the novel CUX1-FGFR1 fusion. In addition, several KIT mutants were investigated. The growth of FIP1L1-PDGFRα and IM resistant FIP1L1-PDGFRα-T674I mutant expressing cells was strongly inhibited by ponatinib with IC50 values of 0,6 nM and 9 nM respectively. Also the panresistant FIP1L1-PDGFRα-D842V mutant and the novel CUX1-FGFR1 fusion responded well to ponatinib treatment with 50% growth inhibition at 154 nM and 56 nM respectively. IL3-driven growth of Ba/F3 cells was resistant to ponatinib (IC50: 2 μM). Western blot analysis confirmed the direct effect of ponatinib on the auto-phosphorylation of the PDGFRα and FGFR1 fusion proteins, as well as on the downstream signaling protein STAT5. Finally, we investigated several KIT single and double mutants and preliminary data indicate an inhibitory effect of ponatinib towards several KIT mutants. In conclusion, our results demonstrate the in vitro activity of ponatinib against IM resistant mutants of the FIP1L1-PDGFRα fusion kinase, against the CUX1-FGFR1 fusion kinase as well as against IM resistant KIT mutations. Our data indicate that ponatinib, which is currently under investigation in phase II clinical trials for IM resistant CML, may also be active against neoplasms driven by FGFR1, PDGFR or KIT kinase activity, and able to overcome IM resistance in these malignancies. Disclosures: No relevant conflicts of interest to declare.

  • FIP1L1 pdgfr alpha d842v a novel panresistant mutant emerging after treatment of FIP1L1 pdgfr alpha t674i eosinophilic leukemia with single agent sorafenib
    Leukemia, 2009
    Co-Authors: Els Lierman, Jan Cools, Peter Marynen, Lucienne Michaux, Pascal Pierre, Els Beullens, Peter Vandenberghe
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1–PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRα T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRα D842V mutant. An N-Nitroso-N-ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRα D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRα T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.

  • FIP1L1-PDGFR|[alpha]| D842V, a novel panresistant mutant, emerging after treatment of FIP1L1-PDGFR|[alpha]| T674I eosinophilic leukemia with single agent sorafenib
    Leukemia, 2009
    Co-Authors: Els Lierman, Jan Cools, Peter Marynen, Lucienne Michaux, Pascal Pierre, Els Beullens, Peter Vandenberghe
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1–PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRα T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRα D842V mutant. An N-Nitroso-N-ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRα D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRα T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.

  • FIP1L1-PDGFRα D842V, a Novel Panresistant Mutant, Emerges after Treatment of FIP1L1-PDGFRα T674I Eosinophilic Leukemia with Single Agent Sorafenib
    Blood, 2008
    Co-Authors: Peter Vandenberghe, Els Lierman, Peter Marynen, Lucienne Michaux, Pascal Pierre, Jan Cools
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions, or other genetic lesions. Most FIP1L1-PDGFRA positive patients enjoy durable complete molecular responses to low-dose imatinib, but resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease. Sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, has been shown to be active against this mutant in vitro. We explored a case of FIP1L1-PDGFRα T674I CEL in blast crisis that was treated with sorafenib as single agent. A partial hematological response was induced, but three months later, progression to blast crisis again occurred. At this time point, sequencing of FIP1L1-PDGFRA revealed the presence of a novel FIP1L1-PDGFRα D842V mutant, while FIP1L1-PDGFRα T674I was no longer detected. The sensitivity of this mutant to different inhibitors was further explored using FIP1L1-PDGFRα D842V transformed Ba/F3 cells. The growth of FIP1L1-PDGFRα D842V transformed Ba/F3 cells was highly resistant to sorafenib and PKC412, in addition to imatinib and dasatinib (IC 50 ≥ 1000 nM for imatinib, sorafenib and dasatinib; IC 50 of PKC412 not reached due to toxicity above 500 nM). Consistent with these dose response curves, FIP1L1-PDGFRα D842V cells did not undergo apoptosis when cultured in 500 nM sorafenib, imatinib or dasatinib, while 30% of FIP1L1-PDGFRα cells did under the same conditions. Analysis of FIP1L1-PDGFRα autophosphorylation and phosphorylation of the downstream signaling proteins ERK1 and ERK2 confirmed that the D842V mutant protein was not inhibited by sorafenib, imatinib or dasatinib at concentrations up to 1000 nM, in contrast to FIP1L1-PDGFRα itself or the T674I mutant. Intriguingly, FIP1L1-PDGFRα D842V cells are significantly less sensitive to dasatinib than PDGFRα D842V expressing cells. Finally, an ENU-mutagenesis screen indeed identified this mutant as a major sorafenib resistant mutant. In summary, this case represents the fifth reported case of acquired resistance to imatinib in FIP1L1-PDGFRA positive CEL. Our data illustrate the efficacy of sorafenib against FIP1L1-PDGFRα T674I as a single agent in vivo . Yet, selection of secondary sorafenib resistant clones is likely to occur, as has also been observed in imatinib resistant CML treated with second line tyrosine kinase inhibitors. While FIP1L1-PDGFRα D842V is a novel panresistant mutation in CEL, the PDGFRα D842V mutation is a known activating mutation of PDGFRα and causes primary imatinib resistance in a small percentage of gastro-intestinal stromal tumors. The homologous KIT D816V mutation in systemic mastocytosis is also associated with imatinib resistance. Of note, the latter two mutations respond better to dasatinib than FIP1L1-PDGFRα D842V. Our observation highlights the difficult challenge of treating resistant mutations and provides a basis for further proactive development of inhibitors with activity against sorafenib resistance mutants.

Lucienne Michaux - One of the best experts on this subject based on the ideXlab platform.

  • FIP1L1 pdgfr alpha d842v a novel panresistant mutant emerging after treatment of FIP1L1 pdgfr alpha t674i eosinophilic leukemia with single agent sorafenib
    Leukemia, 2009
    Co-Authors: Els Lierman, Jan Cools, Peter Marynen, Lucienne Michaux, Pascal Pierre, Els Beullens, Peter Vandenberghe
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1–PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRα T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRα D842V mutant. An N-Nitroso-N-ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRα D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRα T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.

  • FIP1L1-PDGFRα D842V, a novel panresistant mutant, emerging after treatment of FIP1L1-PDGFRα T674I eosinophilic leukemia with single agent sorafenib
    Leukemia, 2009
    Co-Authors: E Lierman, Lucienne Michaux, Pascal Pierre, Els Beullens, P Marynen, J Cools, P Vandenberghe
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1–PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRα T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRα D842V mutant. An N -Nitroso- N -ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRα D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRα T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.

  • FIP1L1-PDGFR|[alpha]| D842V, a novel panresistant mutant, emerging after treatment of FIP1L1-PDGFR|[alpha]| T674I eosinophilic leukemia with single agent sorafenib
    Leukemia, 2009
    Co-Authors: Els Lierman, Jan Cools, Peter Marynen, Lucienne Michaux, Pascal Pierre, Els Beullens, Peter Vandenberghe
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1–PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRα T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRα D842V mutant. An N-Nitroso-N-ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRα D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRα T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.

  • FIP1L1-PDGFRα D842V, a Novel Panresistant Mutant, Emerges after Treatment of FIP1L1-PDGFRα T674I Eosinophilic Leukemia with Single Agent Sorafenib
    Blood, 2008
    Co-Authors: Peter Vandenberghe, Els Lierman, Peter Marynen, Lucienne Michaux, Pascal Pierre, Jan Cools
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions, or other genetic lesions. Most FIP1L1-PDGFRA positive patients enjoy durable complete molecular responses to low-dose imatinib, but resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease. Sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, has been shown to be active against this mutant in vitro. We explored a case of FIP1L1-PDGFRα T674I CEL in blast crisis that was treated with sorafenib as single agent. A partial hematological response was induced, but three months later, progression to blast crisis again occurred. At this time point, sequencing of FIP1L1-PDGFRA revealed the presence of a novel FIP1L1-PDGFRα D842V mutant, while FIP1L1-PDGFRα T674I was no longer detected. The sensitivity of this mutant to different inhibitors was further explored using FIP1L1-PDGFRα D842V transformed Ba/F3 cells. The growth of FIP1L1-PDGFRα D842V transformed Ba/F3 cells was highly resistant to sorafenib and PKC412, in addition to imatinib and dasatinib (IC 50 ≥ 1000 nM for imatinib, sorafenib and dasatinib; IC 50 of PKC412 not reached due to toxicity above 500 nM). Consistent with these dose response curves, FIP1L1-PDGFRα D842V cells did not undergo apoptosis when cultured in 500 nM sorafenib, imatinib or dasatinib, while 30% of FIP1L1-PDGFRα cells did under the same conditions. Analysis of FIP1L1-PDGFRα autophosphorylation and phosphorylation of the downstream signaling proteins ERK1 and ERK2 confirmed that the D842V mutant protein was not inhibited by sorafenib, imatinib or dasatinib at concentrations up to 1000 nM, in contrast to FIP1L1-PDGFRα itself or the T674I mutant. Intriguingly, FIP1L1-PDGFRα D842V cells are significantly less sensitive to dasatinib than PDGFRα D842V expressing cells. Finally, an ENU-mutagenesis screen indeed identified this mutant as a major sorafenib resistant mutant. In summary, this case represents the fifth reported case of acquired resistance to imatinib in FIP1L1-PDGFRA positive CEL. Our data illustrate the efficacy of sorafenib against FIP1L1-PDGFRα T674I as a single agent in vivo . Yet, selection of secondary sorafenib resistant clones is likely to occur, as has also been observed in imatinib resistant CML treated with second line tyrosine kinase inhibitors. While FIP1L1-PDGFRα D842V is a novel panresistant mutation in CEL, the PDGFRα D842V mutation is a known activating mutation of PDGFRα and causes primary imatinib resistance in a small percentage of gastro-intestinal stromal tumors. The homologous KIT D816V mutation in systemic mastocytosis is also associated with imatinib resistance. Of note, the latter two mutations respond better to dasatinib than FIP1L1-PDGFRα D842V. Our observation highlights the difficult challenge of treating resistant mutations and provides a basis for further proactive development of inhibitors with activity against sorafenib resistance mutants.

  • clinical and molecular features of FIP1L1 pdfgra chronic eosinophilic leukemias
    Leukemia, 2004
    Co-Authors: Peter Vandenberghe, Marc Boogaerts, Lucienne Michaux, Iwona Wlodarska, Pierre Zachee, D Vanstraelen, M-c Herregods, A. Van Hoof, D Selleslag, Florence Roufosse
    Abstract:

    Detection of the FIP1L1-PDGFRA fusion gene or the corresponding cryptic 4q12 deletion supports the diagnosis of chronic eosinophilic leukemia (CEL) in patients with chronic hypereosinophilia. We retrospectively characterized 17 patients fulfilling WHO criteria for idiopathic hypereosinophilic syndrome (IHES) or CEL, using nested RT-PCR and interphase fluorescence in situ hybridization (FISH). Eight had FIP1L1-PDGFRA (+) CEL, three had FIP1L1-PDGFRA (-) CEL and six had IHES. FIP1L1-PDGFRA (+) CEL responded poorly to steroids, hydroxyurea or interferon-alpha, and had a high probability of eosinophilic endomyocarditis (n = 4) and disease-related death (n = 4). In FIP1L1-PDGFRA (+) CEL, palpable splenomegaly was present in 5/8 cases, serum vitamin B-12 was always markedly increased, and marrow biopsies revealed a distinctively myeloproliferative aspect. Imatinib induced rapid complete hematological responses in 4/4 treated FIP1L1-PDGFRA (+) cases, including one female, and complete molecular remission in 2/3 evaluable cases. In the female patient, 1 log reduction of FIP1L1-PDGFRA copy number was reached as by real-time quantitative PCR (RQ-PCR). Thus, correlating IHES/CEL genotype with phenotype, FIP1L1-PDGFRA (+) CEL emerges as a homogeneous clinicobiological entity, where imatinib can induce molecular remission. While RT-PCR and interphase FISH are equally valid diagnostic tools, the role of marrow biopsy in diagnosis and of RQ-PCR in disease and therapy monitoring needs further evaluation.

Peter Marynen - One of the best experts on this subject based on the ideXlab platform.

  • FIP1L1 pdgfr alpha d842v a novel panresistant mutant emerging after treatment of FIP1L1 pdgfr alpha t674i eosinophilic leukemia with single agent sorafenib
    Leukemia, 2009
    Co-Authors: Els Lierman, Jan Cools, Peter Marynen, Lucienne Michaux, Pascal Pierre, Els Beullens, Peter Vandenberghe
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1–PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRα T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRα D842V mutant. An N-Nitroso-N-ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRα D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRα T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.

  • FIP1L1-PDGFR|[alpha]| D842V, a novel panresistant mutant, emerging after treatment of FIP1L1-PDGFR|[alpha]| T674I eosinophilic leukemia with single agent sorafenib
    Leukemia, 2009
    Co-Authors: Els Lierman, Jan Cools, Peter Marynen, Lucienne Michaux, Pascal Pierre, Els Beullens, Peter Vandenberghe
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions or other genetic lesions. Most FIP1L1–PDGFRA positive patients enjoy durable and complete molecular responses to low-dose imatinib (Glivec/Gleevec). However, resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease, and sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, is active against this mutant in vitro. We describe a case of FIP1L1-PDGFRα T674I CEL in blast crisis that responded to sorafenib (Nexavar). However, this clinical response was short-lived because of the rapid emergence of a FIP1L1-PDGFRα D842V mutant. An N-Nitroso-N-ethylurea-mutagenesis screen indeed identified this mutant as a major sorafenib-resistant mutant. In vitro, the novel FIP1L1-PDGFRα D842V mutant is highly resistant to sorafenib, imatinib, dasatinib (Sprycell) and PKC412 (Midostaurin). Thus, sorafenib is clinically active in imatinib-resistant FIP1L1-PDGFRα T674I CEL, but the rapid emergence of other mutants may limit the response duration. The identification of new PDGFR inhibitors will be required to overcome resistance by this D842V mutant.

  • FIP1L1-PDGFRα D842V, a Novel Panresistant Mutant, Emerges after Treatment of FIP1L1-PDGFRα T674I Eosinophilic Leukemia with Single Agent Sorafenib
    Blood, 2008
    Co-Authors: Peter Vandenberghe, Els Lierman, Peter Marynen, Lucienne Michaux, Pascal Pierre, Jan Cools
    Abstract:

    Chronic eosinophilic leukemia (CEL) is a rare myeloproliferative neoplasm characterized by the FIP1L1-PDGFRA fusion gene, variant PDGFRA fusions, or other genetic lesions. Most FIP1L1-PDGFRA positive patients enjoy durable complete molecular responses to low-dose imatinib, but resistance mediated by a T674I mutation in the ATP-binding pocket of PDGFRA has been reported in advanced disease. Sorafenib, a potent inhibitor of RAF-1, B-RAF, VEGFR and PDGFR, has been shown to be active against this mutant in vitro. We explored a case of FIP1L1-PDGFRα T674I CEL in blast crisis that was treated with sorafenib as single agent. A partial hematological response was induced, but three months later, progression to blast crisis again occurred. At this time point, sequencing of FIP1L1-PDGFRA revealed the presence of a novel FIP1L1-PDGFRα D842V mutant, while FIP1L1-PDGFRα T674I was no longer detected. The sensitivity of this mutant to different inhibitors was further explored using FIP1L1-PDGFRα D842V transformed Ba/F3 cells. The growth of FIP1L1-PDGFRα D842V transformed Ba/F3 cells was highly resistant to sorafenib and PKC412, in addition to imatinib and dasatinib (IC 50 ≥ 1000 nM for imatinib, sorafenib and dasatinib; IC 50 of PKC412 not reached due to toxicity above 500 nM). Consistent with these dose response curves, FIP1L1-PDGFRα D842V cells did not undergo apoptosis when cultured in 500 nM sorafenib, imatinib or dasatinib, while 30% of FIP1L1-PDGFRα cells did under the same conditions. Analysis of FIP1L1-PDGFRα autophosphorylation and phosphorylation of the downstream signaling proteins ERK1 and ERK2 confirmed that the D842V mutant protein was not inhibited by sorafenib, imatinib or dasatinib at concentrations up to 1000 nM, in contrast to FIP1L1-PDGFRα itself or the T674I mutant. Intriguingly, FIP1L1-PDGFRα D842V cells are significantly less sensitive to dasatinib than PDGFRα D842V expressing cells. Finally, an ENU-mutagenesis screen indeed identified this mutant as a major sorafenib resistant mutant. In summary, this case represents the fifth reported case of acquired resistance to imatinib in FIP1L1-PDGFRA positive CEL. Our data illustrate the efficacy of sorafenib against FIP1L1-PDGFRα T674I as a single agent in vivo . Yet, selection of secondary sorafenib resistant clones is likely to occur, as has also been observed in imatinib resistant CML treated with second line tyrosine kinase inhibitors. While FIP1L1-PDGFRα D842V is a novel panresistant mutation in CEL, the PDGFRα D842V mutation is a known activating mutation of PDGFRα and causes primary imatinib resistance in a small percentage of gastro-intestinal stromal tumors. The homologous KIT D816V mutation in systemic mastocytosis is also associated with imatinib resistance. Of note, the latter two mutations respond better to dasatinib than FIP1L1-PDGFRα D842V. Our observation highlights the difficult challenge of treating resistant mutations and provides a basis for further proactive development of inhibitors with activity against sorafenib resistance mutants.

  • Activation of FIP1L1-PDGFRα requires disruption of the juxtamembrane domain of PDGFRα and is FIP1L1-independent
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Elizabeth H Stover, Cedric Folens, Nicole Mentens, Peter Marynen, D. Gary Gilliland, Jing Chen, Benjamin H. Lee, Ifor R. Williams, Jan Cools
    Abstract:

    Genetic abnormalities that result in expression of chimeric tyrosine kinase proteins such as BCR-ABL1 and ETV6-PDGFRβ are common causes of hematopoietic malignancies. The paradigm for constitutive activation of these fusion tyrosine kinases is enforced homodimerization by self-association domains present in the fusion partner proteins. The unique interstitial deletion on chromosome 4q12 that leads to expression of the FIP1L1-PDGFRα fusion tyrosine kinase was recently identified as a cause of chronic eosinophilic leukemia. In this report, we demonstrate that FIP1L1 is completely dispensable for PDGFRα activation in vitro and in vivo. Instead, truncation of PDGFRα between two conserved tryptophan residues in the juxtamembrane (JM) domain is required for kinase activation and transforming potential of FIP1L1-PDGFRα. The presence of a complete JM domain in FIP1L1-PDGFRα is inhibitory, but this autoinhibition can be overcome by enforced homodimerization. Similar effects of the JM domain in the context of PDGFRβ were observed. These results suggest that disruption of the autoinhibitory JM domain is an alternative, dimerization-independent mechanism by which chimeric tyrosine kinases are constitutively activated and induce leukemogenesis.

  • Sorafenib is a potent inhibitor of FIP1L1-PDGFRα and the imatinib-resistant FIP1L1-PDGFRα T674I mutant
    Blood, 2006
    Co-Authors: Els Lierman, Elizabeth H Stover, Cedric Folens, Nicole Mentens, Helen Van Miegroet, Werner Scheers, Marc Boogaerts, Peter Vandenberghe, Peter Marynen, Jan Cools
    Abstract:

    The FIP1L1-PDGFRA oncogene is a common cause of chronic eosinophilic leukemia (CEL), and encodes an activated tyrosine kinase that is inhibited by imatinib. FIP1L1-PDGFRA–positive patients with CEL respond to low-dose imatinib therapy, but resistance due to acquired T674I mutation has been observed. We report here the identification of sorafenib as a potent inhibitor of the FIP1 like 1–platelet-derived growth factor receptor alpha (FIP1L1-PDGFRα) (T674I) mutant. Sorafenib inhibited the proliferation of FIP1L1-PDGFRα and FIP1L1-PDGFRα(T674I)–transformed Ba/F3 cells and induced apoptosis of the EOL-1 cell line at a low nanomolar concentration. Western blot analysis confirmed that these effects were due to a direct effect on FIP1L1-PDGFRα and FIP1L1-PDGFRα(T674I). Sorafenib was recently approved for the treatment of renal cell carcinoma. Our data suggest that low doses of sorafenib could be efficient for the treatment of FIP1L1-PDGFRA–positive CEL and could be used to overcome resistance to imatinib associated with the T674I mutation.