SPTBN1-FLT3 in Atypical Chronic Myeloid Leukemia Transforms Ba/F3 Cells to IL-3 Independence and Is Sensitive to Both Tyrosine Kinase Inhibitors and Immunotherapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2009-2009
Author(s):  
Francis H. Grand ◽  
Sameena Iqbal ◽  
Lingyan Zhang ◽  
Nigel H. Russell ◽  
Andrew Chase ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Unrelated Donor ◽  
Rt Pcr

Abstract We have identified a patient who presented with BCR-ABL negative chronic myeloid leukemia (CML) and an acquired 46XX, t(2;13;2;21) (p13;q12;q33;q11.2) in all bone marrow metaphases examined. Fluorescence in situ hybridization (FISH) using probes flanking the FLT3 gene at 13q12 suggested that this gene was disrupted. 5′-RACE PCR using primers to the region of FLT3 encoding the tyrosine kinase domain identified a novel in-frame mRNA fusion between exon 3 of SPTBN1 (spectrin, beta, non-erythrocytic 1 isoform 2, NM 178313) on chromosome 2p16 and exon 13 of FLT3 (NM 004119). Juxtaposition of SPTBN1 and FLT3 was confirmed by two color FISH and amplification of the genomic DNA breakpoint confirmed a fusion between intron 3 of SPTBN1 and intron 12 of FLT3. The SPTBN1-FLT3 fusion gene is predicted to be translated into a 570 amino acid chimeric protein that retains two coiled-coil domains from SPTBN1 and 424 amino acids from FLT3, including the entire tyrosine kinase domain. Since the t(2;13) is readily visible by cytogenetic analysis but has not been reported previously it seems likely that SPTBN1-FLT3 is uncommon. However to test if FLT3 might be involved more widely in BCR-ABL negative CML we analysed 40 cases by RT-PCR. Two cases were positive for the FLT3 internal tandem duplication (ITD) but mutation of residue D835 was not observed. Expression of the SPTBN1-FLT3 fusion transformed the interleukin 3 (IL-3)-dependent cell line Ba/F3 to growth factor independence and was accompanied by constitutive phosphorylation of the fusion protein and the downstream substrate ERK1/2. The growth of transformed cells was inhibited in a dose-dependent fashion by SU11567 and PKC142, but not by imatinib mesylate. The patient was initially treated with hydroxyurea and subsequently underwent an unrelated donor bone marrow transplant. She relapsed cytogenetically at 4 years but responded to donor lymphocyte infusion (DLI), achieving sustained cytogenetic and molecular (nested RT-PCR) remission. We conclude that SPTBN1-FLT3 is a rare abnormality in BCR-ABL negative CML that is responsive to both targeted signal transduction therapy and immunotherapy by DLI.

A Novel Compound Inhibits Chronic Myeloid Leukemia Via Upregulating Apoptosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5559-5559
Author(s):  
Jiajia Xin ◽  
Dandan Yin ◽  
Wei Fu ◽  
Hui-Jie Zhang ◽  
Yaozhen Chen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Leukemic Cells ◽  
Hematopoietic Stem

Abstract Chronic myeloid leukemia (CML) is a myeloid proliferative disorder mainly result from chimeric protein BCR-ABL1 encoded by a fusion gene at the t(9;22) (q34;q11) chromosomal translocation. Intrinsically, this recombined protein results in an increased tyrosine kinase (TK) activity that directly related to hematopoietic stem cell malignant proliferation. Consequently, the drugs derived from tyrosine kinase inhibitors (TKI) have been developed as an infective therapy, and greatly improved patients survival in clinic. Unfortunately, single TKI administration led to toxicities or tolerance in long-term treated CML patients. Even worse is, about 5% CML patients were not caused by bcr-abl gene mutation. Thus better medicines are badly needed to compensate CML therapy. Herein, we investigated the undefined function of a biscoumarins. The new synthesized compound exhibited a null toxicity on HUVECs but intensive toxicity on K562 leukemic cells. Subsequent results demonstrated that it efficiently inhibited the expansion of human CML cell line and bone marrow cells of SCL-tTA-BCL/ABL transgenic model mice via increased apoptosis. Critically, we also showed that CD34+ bone marrow leukemic cells collected from patients underwent more apoptosis after treated by the biscoumarins derivate. To extend these results into vivo, we observed a prolonged survival of bcr-abl transgenic mice treated by derivate mono-therapy or combination with imatinib compared to those of untreated or imatinib-treated CML mice. All together, these results indicated that this biscoumarins derivate may have novel potential as a therapeutic agent against CML. Disclosures No relevant conflicts of interest to declare.

BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet

Blood ◽  
2011 ◽  
Vol 118 (5) ◽  
pp. 1208-1215 ◽  
Author(s):  
Simona Soverini ◽  
Andreas Hochhaus ◽  
Franck E. Nicolini ◽  
Franz Gruber ◽  
Thoralf Lange ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Mutation Analysis ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase

AbstractMutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection.

scholarly journals Droplet Digital PCR Phasing (DROP-PHASE): A Novel Method for Straightforward Detection of BCR-ABL1 Compound Mutations in Tyrosine Kinase Inhibitors Resistant Chronic Myeloid Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4660-4660 ◽  
Author(s):  
Pascal Vannuffel ◽  
Luana Bavaro ◽  
Friedel Nollet ◽  
Asena Aynaci ◽  
Margherita Martelli ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Digital Pcr ◽  
Kinase Domain ◽  
Droplet Digital Pcr ◽  
Drop Phase

Chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL) are, respectively, a myeloproliferative and a lymphoproliferative neoplasm that can be characterized by the chimeric fusion oncogene BCR-ABL1. Tyrosine Kinase Inhibitors (TKI) are the standard therapy for patients with CML/ALL. However, mutations of the BCR-ABL1 kinase domain constitute a major cause of treatment failure in CML and ALL receiving TKI therapy. While 2nd and 3rd generation TKI have proven their efficacy against mutated BCR-ABL1-mediated clonal expansion, the presence of compound mutations can produce high level of resistance to these TKIs. Even the last addition to the TKI armamentarium, ponatinib, remains ineffective against some BCR-ABL1 compound mutations (Zabriskie, M.S., et al., BCR-ABL1 Compound Mutations Combining Key Kinase Domain Positions Confer Clinical Resistance to Ponatinib in Ph Chromosome-Positive Leukemia. Cancer Cell, 2014. 26(3):p.428-442). Therefore, the distinction between compound (different mutations present on 1 unique malignant clone) and polyclonal mutations (different mutations present on 2 or more different clones) is of great clinical importance in order to select the most suitable treatment and to estimate outcomes. The objective of this study is to determine in a straightforward way whether BCR-ABL1 mutations discovered by Next Generation Sequencing are compound mutations or polyclonal mutations. A simple proof-of-concept experiment was first performed by using 3 synthetic oligonucleotides (gBlocks, IDT) mimicking the presence of compound mutations versus polyclonal mutations in resistant leukemia cells. The first oligo harbored the M237I mutation, the second oligo mutations E255K, E279K, V299L, T315I, F359V, A380S, H396R, S417Y, F459K and F486S and the third one contained all the mutations. Dual-color probes assays have been set up to target specifically 2 different mutations. Mixtures of 2 oligonucleotides harboring 1 mutation each versus 1 oligonucleotide harboring 2 mutations have been compared by performing duplex droplet digital PCR (ddPCR) reactions on the Bio-Rad ddPCR QX200 System. Linkage detection is based on the observation that the presence of 2 targets on the same DNA molecule increases the number of double-positive droplets relative to the number expected due to chance. Automatic linkage evaluation was made by the QuantaSoft Software and mathematical calculations refer to (Regan, J.F., et al., A rapid molecular approach for chromosomal phasing. PLoS One, 2015. 10(3): p. e0118270). The first experiment successfully validated the detection of mutations residing on two different oligonucleotides (polyclonal mutations) versus mutations on the same molecule (compound mutations). When performing serial dilutions of 2 oligonucleotides containing different mutations, a sensitivity of 10%:90% was achieved with a good linearity (r2=0.97). Mixing experiment also showed that ddPCR phasing could distinguish between a mixture of compound and polyclonal mutations versus and the sole presence of polyclonal mutations at the same sensitivity and linearity levels. Moreover, no influence of the genomic distance between mutations (from position 255 to position 562) was observed. The strategy was further applied to 20 clinical samples from CML/ALL patients characterized by multiple resistance mutations. Drop-phase is a rapid (< 4 hours), scalable (100 samples), technically easy to perform and cost-effective method. This strategy will help to identify compound mutations in patients with TKI-resistant CML/ALL and allow to modulate the patient's drug strategy and to prevent progression and therapeutic failure. Disclosures Vannuffel: Incyte: Consultancy. Soverini:Incyte: Consultancy.

scholarly journals Mechanisms of Disease Progression and Resistance to Tyrosine Kinase Inhibitor Therapy in Chronic Myeloid Leukemia: An Update

2019 ◽  
Vol 20 (24) ◽  
pp. 6141 ◽  
Author(s):  
Luana Bavaro ◽  
Margherita Martelli ◽  
Michele Cavo ◽  
Simona Soverini
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Disease Progression ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Fusion Gene ◽  
Primary Resistance ◽  
Secondary Resistance ◽  
Dismal Prognosis

Chronic myeloid leukemia (CML) is characterized by the presence of the BCR-ABL1 fusion gene, which encodes a constitutive active tyrosine kinase considered to be the pathogenic driver capable of initiating and maintaining the disease. Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, some patients may not respond (primary resistance) or may relapse after an initial response (secondary resistance). In a small proportion of cases, development of resistance is accompanied or shortly followed by progression from chronic to blastic phase (BP), characterized by a dismal prognosis. Evolution from CP into BP is a multifactorial and probably multistep phenomenon. Increase in BCR-ABL1 transcript levels is thought to promote the onset of secondary chromosomal or genetic defects, induce differentiation arrest, perturb RNA transcription, editing and translation that together with epigenetic and metabolic changes may ultimately lead to the expansion of highly proliferating, differentiation-arrested malignant cells. A multitude of studies over the past two decades have investigated the mechanisms underlying the closely intertwined phenomena of drug resistance and disease progression. Here, we provide an update on what is currently known on the mechanisms underlying progression and present the latest acquisitions on BCR-ABL1-independent resistance and leukemia stem cell persistence.

Recurrent bone marrow aplasia secondary to nilotinib in a patient with chronic myeloid leukemia: A case report

2012 ◽  
Vol 18 (4) ◽  
pp. 440-444 ◽  
Author(s):  
Prathima Prodduturi ◽  
Anamarija M Perry ◽  
Patricia Aoun ◽  
Dennis D Weisenburger ◽  
Mojtaba Akhtari
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Chronic Phase ◽  
Bone Marrow Aplasia ◽  
Line Therapy ◽  
Grade 3

Nilotinib is a potent tyrosine kinase inhibitor of breakpoint cluster region-abelson (BCR-ABL), which has been approved as front-line therapy for newly diagnosed chronic myeloid leukemia in chronic phase and as second-line therapy after imatinib failure in chronic or accelerated phase chronic myeloid leukemia. Tyrosine kinase inhibitors have been associated with myelosuppression and grade 3 or grade 4 cytopenias are not uncommon in chronic myeloid leukemia patients treated with these drugs. There are a few reports of imatinib-associated bone marrow aplasia, but to our knowledge only one reported case of bone marrow aplasia associated with nilotinib. Herein, we report a 49-year-old male patient with chronic phase chronic myeloid leukemia, who developed severe bone marrow aplasia due to nilotinib. Possible mechanisms for this significant adverse drug reaction are discussed along with a review of literature.

Induced Pluripotent Stem Cells (iPSC) From Chronic Myeloid Leukemia: Study of BCR-ABL Addiction and Effect of Tyrosine Kinase Inhibitors,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3754-3754 ◽  
Author(s):  
Aurélie Bedel ◽  
Francois Moreau-Gaudry ◽  
Jean- Max Pasquet ◽  
Miguel Taillepierre ◽  
Éric Lippert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Blood Cells ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Leukemic Stem Cells ◽  
Rt Pcr ◽  
Reprogramming Factors

Abstract Abstract 3754 The tyrosine kinase inhibitors (TKI) such as imatinib, by suppressing BCR-ABL oncogene activity, are an effective therapy for chronic myeloid leukemia disease (CML). However, the majority of patients achieving remission with TKI continue have molecular evidence of persistent disease. In addition, we have reported that for patients who achieved a sustained complete molecular remission, 60% of them relapse after discontinuation of imatinib. Various mechanisms have been proposed to explain disease persistence and disease recurrence. One of the hypotheses is that primitive leukemic stem cells can survive in the presence of TKI. Little is known about the stem cells survival due to technical difficulties (small and poorly defined primary populations). Understanding the mechanisms by which these cells survive to TKI therapy will be critical to devising strategy aimed to their elimination. We propose to generate iPSC derived from CD34+ blood cells isolated from CML patient (CML-iPSC), as a model for study leukemic stem cells survival in the presence of TKI and study the mechanism of TKI resistance of the stem cells. Primary CD34+ CML patient cells were transduced by 2 excisable lentiviral vectors (both flanked by two LoxP sites), one expressing three reprogramming factors (OCT4-SOX2-KLF4) and another one with c-MYC and a shRNA against TP53. Twenty-one days after co-transduction, CML-iPSC colonies were picked and five iPS clones were characterized (expression of pluripotency markers by RT-PCR (DPPA4, NANOG, CRIPTO) and immunofluorescence (NANOG, SSEA-4, TRA1-60)). Efficiency of reprogrammation was low compared to cord blood CD34+ control cells (0.01% vs 0.1%, respectively), and delayed (21 days vs 14 days). Philadelphia chromosome (Ph) positive was observed in 4/5 clones after cytogenetic analysis. Expression of BCR-ABL (Western-blot and RT-PCR) was present at various levels. Interestingly, 1/5 clone was generated from non-leukemic cell (Ph negative) and was used as internal control for the following function assays. We used these 5 CML-iPSC clones to study their behavior in presence of TKI. All CML-iPSC clones survived to escalating concentration of imatinib (0 to 20μM) and ponatinib (0 to 50nM) for 6 days. To understand if the CML-iPSC survival was due to resistance or independence mechanisms, we performed western blot analysis of TKI targets. BCR-ABL activity was inhibited under TKI exposure (dephosphorylations of BCR/ABL and of Crkl). In order to check whether survival was due to the expression of reprogramming factors, we excised the gene cassettes by an Adenovirus expressing CRE recombinase. After proviral excision and subcloning, excised CML-iPSC continued to survive to TKI exposure. Taken together, these results demonstrate that CML-iPSC survival do not depend on BCR-ABL (oncogene independence). Upon induction of hematopoietic differentiation, CML-iPSC were able to efficiently generate progenitors of hematopoietic lineages (up to 40% of CD45+) and colony forming units in methylcellulose. TKI effect on iPSC-derived hematopoietic progenitors, to analyze the putative recovery of TKI sensibility compared to primitive CML blood cells from the same patient, are in progress. We conclude that reprogrammation of CD34BCR-ABL+ cells from CML patient is possible and that CML-iPSC lost the BCR-ABL dependency and became resistant to TKI. A specific differentiated epigenetic cell state is probably needed to maintain BCR-ABL dependency. CML-iPSC can be used to study mechanisms by which leukemic stem cells survive to TKI therapy and is a promising tool for testing and screening new therapeutic target reducing leukemic stem cell survival. Disclosures: Mahon: Novartis Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizzer: Honoraria.

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