FLT-3 Activity and Its Response to Drugs Can Be Determined in AML Blast Cells by FLT-3 Phosphorylation Status Using Flow Cytometry.

Abstract One of the most common molecular defects identified in acute myeloid leukemia (AML) patients is an activating mutation of FLT3 tyrosine kinase. The identification of activated FLT3 as a contributor to the cause and progression of much leukemia has led to its consideration as a potential target for therapy. Since small molecule FLT3 kinase inhibitors are actually in clinical trials; a robust and standardized method for screening of FLT3 receptor activation is necessary. We evaluated the expression level of FLT3 receptor (CD135) by Facs analysis. We developed a flow cytometry method to measure FLT3 phosphorylation (P-FLT3) in samples with <10 (e)5 cells. The amount of P-FLT3 in the samples was determined as the mean fluorescence intensity (MFI). The P-FLT3 status of the treated samples was expressed as a percentage of the untreated control (100%). The method was first validated in FLT3 wild-type (HL-60) and mutant (MV4-11/ITD+) as well as FLT3 negative (K562) cell lines. The method also provides to be reproducible with samples AML from patients. Analysis was performed after exposure to drugs, in vitro and in vivo. In response to increasing drugs concentration (CEP-701 and SU11657) there was a linear reduction in P-FLT3. The results validate a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry, and enable an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.

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Targeting USP47 overcomes tyrosine kinase inhibitor resistance and eradicates leukemia stem/progenitor cells in chronic myelogenous leukemia

Nature Communications ◽

10.1038/s41467-020-20259-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hu Lei ◽

Han-Zhang Xu ◽

Hui-Zhuang Shan ◽

Meng Liu ◽

Ying Lu ◽

...

Keyword(s):

Tyrosine Kinase ◽

Progenitor Cells ◽

Chronic Myelogenous Leukemia ◽

Drug Targets ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Myelogenous Leukemia ◽

Tki Resistance

AbstractIdentifying novel drug targets to overcome resistance to tyrosine kinase inhibitors (TKIs) and eradicating leukemia stem/progenitor cells are required for the treatment of chronic myelogenous leukemia (CML). Here, we show that ubiquitin-specific peptidase 47 (USP47) is a potential target to overcome TKI resistance. Functional analysis shows that USP47 knockdown represses proliferation of CML cells sensitive or resistant to imatinib in vitro and in vivo. The knockout of Usp47 significantly inhibits BCR-ABL and BCR-ABLT315I-induced CML in mice with the reduction of Lin−Sca1+c-Kit+ CML stem/progenitor cells. Mechanistic studies show that stabilizing Y-box binding protein 1 contributes to USP47-mediated DNA damage repair in CML cells. Inhibiting USP47 by P22077 exerts cytotoxicity to CML cells with or without TKI resistance in vitro and in vivo. Moreover, P22077 eliminates leukemia stem/progenitor cells in CML mice. Together, targeting USP47 is a promising strategy to overcome TKI resistance and eradicate leukemia stem/progenitor cells in CML.

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TEL/PDGFβR Induces Hematologic Malignancies in Mice That Respond to a Specific Tyrosine Kinase Inhibitor

Blood ◽

10.1182/blood.v93.5.1707 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1707-1714 ◽

Cited By ~ 80

Author(s):

Michael H. Tomasson ◽

Ifor R. Williams ◽

Robert Hasserjian ◽

Chirayu Udomsakdi ◽

Shannon M. McGrath ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Tyrosine Kinases ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Transgenic Animals ◽

Myeloid Lineage ◽

Protein Tyrosine

Abstract The TEL/PDGFβR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFβR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFβR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFβR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFβR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFβR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFβR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase–mediated malignancies both early in the course of disease and after the development of additional transforming mutations.

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MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia

Scientific Reports ◽

10.1038/s41598-019-54901-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 3

Author(s):

María Luz Morales ◽

Alicia Arenas ◽

Alejandra Ortiz-Ruiz ◽

Alejandra Leivas ◽

Inmaculada Rapado ◽

...

Keyword(s):

Drug Resistance ◽

Acute Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Resistance Pathway ◽

Acute Myeloid

AbstractFMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

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Erratum: Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro-in vivo correlations

British Journal of Cancer ◽

10.1038/bjc.2017.217 ◽

2017 ◽

Vol 117 (5) ◽

pp. e3-e3

Author(s):

S Hu ◽

R H J Mathijssen ◽

P de Bruijn ◽

S D Baker ◽

A Sparreboom

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors

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The Discovery of Novel BCR-ABL Tyrosine Kinase Inhibitors Using a Pharmacophore Modeling and Virtual Screening Approach

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.649434 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ting-Ting Huang ◽

Xin Wang ◽

Shao-Jia Qiang ◽

Zhen-Nan Zhao ◽

Zhuo-Xun Wu ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Pharmacophore Modeling ◽

Myelogenous Leukemia ◽

Lipinski’S Rule ◽

Abl Tyrosine Kinase ◽

Pharmacophore Models

Chronic myelogenous leukemia (CML) typically results from a reciprocal translocation between chromosomes 9 and 22 to produce the bcr-abl oncogene that when translated, yields the p210 BCR-ABL protein in more than 90% of all CML patients. This protein has constitutive tyrosine kinase activity that activates numerous downstream pathways that ultimately produces uncontrolled myeloid proliferation. Although the use of the BCR-ABL tyrosine kinase inhibitors (TKIs), such as imatinib, nilotinib, dasatinib, bosutinib, and ponatinib have increased the overall survival of CML patients, their use is limited by drug resistance and severe adverse effects. Therefore, there is the need to develop novel compounds that can overcome these problems that limit the use of these drugs. Therefore, in this study, we sought to find novel compounds using Hypogen and Hiphip pharmacophore models based on the structures of clinically approved BCR-ABL TKIs. We also used optimal pharmacophore models such as three-dimensional queries to screen the ZINC database to search for potential BCR-ABL inhibitors. The hit compounds were further screened using Lipinski’s rule of five, ADMET and molecular docking, and the efficacy of the hit compounds was evaluated. Our in vitro results indicated that compound ZINC21710815 significantly inhibited the proliferation of K562, BaF3/WT, and BaF3/T315I leukemia cells by inducing cell cycle arrest. The compound ZINC21710815 decreased the expression of p-BCR-ABL, STAT5, and Crkl and produced apoptosis and autophagy. Our results suggest that ZINC21710815 may be a potential BCR-ABL inhibitor that should undergo in vivo evaluation.

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Disruption of Wnt/β-Catenin Signaling Exerts Antileukemia Activity and Sensitizes with Tyrosine Kinase Inhibitors in FLT3 Mutated AML In Vivo

Blood ◽

10.1182/blood.v128.22.2828.2828 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2828-2828

Author(s):

Xuejie Jiang ◽

Po Yee Mak ◽

Hong Mu ◽

Duncan Mak ◽

Qi Zhang ◽

...

Keyword(s):

Flow Cytometry ◽

Tyrosine Kinase ◽

Survival Time ◽

Nuclear Localization ◽

In Vivo Imaging ◽

Median Survival Time ◽

Research Funding ◽

Kinase Inhibitors ◽

Flt3 Mutations

Abstract Wnt/β-catenin signaling is associated with pathogenesis of AML and required for establishment of leukemic stem cells. FLT3 mutations are frequently observed in AML and predict poor clinic outcomes. Aberrant activation of FLT3 signaling stabilizes β-catenin and increases its nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKIs) are used to treat FLT3-mutated AML, but their effects are limited due to primary or secondary resistance to TKIs. We previously reported (ASH 2015) that disrupting Wnt/b-catenin signaling by C-82, a selective β-catenin/cAMP binding protein antagonist in combination with FLT3 inhibitors had a synergistic cytotoxicity in vitro in FLT3-mutated AML cells and AML stem/progenitor cells through inhibiting nuclear localization of β-catenin and suppressing the expression of β-catenin target proteins including survivin, CD44, c-Myc, and cyclin D1; several frizzled family receptors/co-receptors; Wnt ligands; and FLT3 downstream signaling proteins. The synergy was also observed in TKI-resistant FLT3 mutated cells. In this study, we evaluated the antileukemia effect of combined inhibition of β-catenin and FLT3 signaling in vivo in immunodeficient mice xenografted with FLT3-ITD mutated cells either from a cell line or from an AML patient. Molm13-GFP/Luc cells were injected into NOD-SCID IL2RγNull (NSG) mice. Once engraftment was confirmed by in vivo imaging, mice were treated with PRI-724 (C-82 pro-drug), sorafenib, or both. Treatment with PRI-724 or sorafenib decreased leukemia burden, and the combination was the most effective as assessed by in vivo imaging, flow cytometric measurement of human CD45+ cells in blood, and bone marrow (BM) and spleen H&E staining. The mice in PRI-724 (19 days, P = 0.025) or sorafenib (28 days, P = 0.0002) treated group had significantly longer median survival time than the control group (17 days), and the combined treatment further prolonged the survival time (30.5 days) (P = 0.0005, combination vs RPI-724; P = 0.0056, combination vs sorafenib). CyTOF and SPADE tree analysis showed a great reduction of human CD45+ cells and decreased expression of β-catenin, CD44, c-Myc, survivin, p-FLT3, p-ERK, p-AKT, and p-STAT5 in BM cells of the combination treated mice. Cells from a FLT3-ITD mutated AML patient sample collected from patient-derived xenograft (spleen) were injected into NOD-SCID IL2RγNull-3/GM/SF (NSGS) mice. After engraftment was confirmed by flow cytometry, mice were treated as above. Leukemia burden was decreased by sorafenib or PRI-724 treatment, as determined by flow cytometry measurement of human CD45+ cells in blood, BM, and spleen samples, but did not reach statistical significance. The combination significantly enhanced the antileukemia effect. PRI-724 (31 days, P = 0.008) or sorafenib (48 days, P = 0.0003) significantly prolonged median survival time compared with control (29 days), and the combination further extended the survival time (54 days) (P = 0.0005, combination vs RPI-724; P = 0.0067, combination vs sorafenib). Our in vivo study further demonstrates that disruption of Wnt/b-catenin signaling exerts antileukemia activity and sensitizes with TKIs in FLT3 mutated AML. These findings provide a rationale for clinic development of combined inhibition of Wnt/β-catenin and FLT3 signaling to overcome resistance and improve outcomes in AML patients with FLT3 mutations. Disclosures Konopleva: Cellectis: Research Funding; Calithera: Research Funding. Carter:PRISM Pharma/Eisai: Research Funding.

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SECOND-GENERATION TYROSINE KINASE INHIBITORS (TKI) AS SALVAGE THERAPY FOR RESISTANT OR INTOLERANT PATIENTS TO PRIOR TKIs

Mediterranean Journal of Hematology and Infectious Diseases ◽

10.4084/mjhid.2014.003 ◽

2014 ◽

Vol 6 (1) ◽

pp. e2014003 ◽

Cited By ~ 13

Author(s):

Massimo Breccia ◽

Giuliana Alimena

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Second Generation ◽

Kinase Inhibitors ◽

Clinical Activity ◽

Third Generation ◽

Discontinue Therapy ◽

Third Line

With the advent of target therapies, imatinib became the mainstay for treatment of chronic myeloid leukemia. However, despite the brilliant results obtained with this drug, more than 30% of patients discontinue therapy in long-term due to several reasons, including failure and/or intolerance. Second-generation tyrosine kinase inhibitors (TKIs) are more potent drugs and have expanded inhibition against a broad spectrum of mutations resistant to imatinib. Both nilotinib and dasatinib have demonstrated in vitro and in vivo clinical activity against different types of mutations and various forms of resistance. However, patients with T315I mutation do not obtain an advantage from these drugs and a third generation inhibitor ponatinib, a pan-BCR drug, was tested with significant results. In this review, we report the results of second- and third-generation TKIs tested as second or third line therapy in patients resistant and/or intolerant to previous inhibitors.

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Different Classes of ABL1 Fusions Activate Different Downstream Signalling Nodes

Blood ◽

10.1182/blood-2018-99-117844 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2628-2628

Author(s):

Lauren M Brown ◽

Hannah Huckstep ◽

Jarrod Sandow ◽

Ray C Bartolo ◽

Nadia Davidson ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Inhibitors ◽

Conflicts Of Interest ◽

Sh2 Domain ◽

Signalling Pathways ◽

Stable Isotope Labelling ◽

In Vivo Models ◽

Sh3 Domains

Abstract Background: Philadelphia-like acute lymphoblastic leukaemia (Ph-like ALL) is a high-risk subtype of ALL driven by a range of tyrosine kinase and cytokine receptor rearrangements. ABL1-class rearrangements (ABL1, ABL2, CSF1R and PDGFRB) account for 17% of Ph-like ALL cases in children, and are clinically important to identify as they can be therapeutically targeted with tyrosine kinase inhibitors (TKIs). While the p190 BCR-ABL1 fusion is well described, less is known about the function and downstream signalling by rare ABL1 fusions. We identified a rare ABL1 fusion, SFPQ-ABL1, in a paediatric B-ALL patient using RNA-sequencing. This fusion lacks the ABL1 Src-homology-3 (SH3) and part of the SH2 domain, which are retained in BCR-ABL1. Other ABL1 fusions, RCSD1-ABL1 and SNX2-ABL1, have a similar structure. In this work we have utilised phosphoproteomics and Stable Isotope Labelling by Amino Acids in Cell Culture (SILAC), as well as in vitro and in vivo models, to determine differential signalling pathways between SFPQ-ABL1 and BCR-ABL1. Methods: We cloned SFPQ-ABL1 from patient cDNA, and engineered SFPQ-ABL1 and BCR-ABL1 fusions to include or delete the SH2 and SH3 domains. We performed proliferation and viability assays to assess the ability of these fusions to transform Ba/F3 cells and test sensitivity to TKIs. We performed total phosphopeptide and phosphotyrosine enrichments and utilised mass spectrometry to identify the phosphoproteome activated by canonical SFPQ-ABL1 and BCR-ABL1. Over representation analysis was performed on phosphopeptides significantly differing between BCR-ABL and SFPQ-ABL (Log fold change cut-off > 2.5) using the Gene Ontology (GO) knowledge base under the biological process category. Furthermore, we compared the phosphoproteome of canonical SFPQ-ABL1 to SFPQ-ABL1 with the SH2 and SH3 domains reintroduced (SFPQ-ABL1+SH). We have also developed novel mouse models, using syngeneic transplantation, of SFPQ-ABL1 and SNX2-ABL1 driven leukaemia. Results: SFPQ-ABL1 expressing Ba/F3 cells are sensitive to cell death induced by TKIs that block ABL1. Interestingly, while SFPQ-ABL1 and BCR-ABL1 both effectively blocked apoptosis, SFPQ-ABL1 was less able to drive cytokine-independent proliferation. Phosphoproteomic analysis showed that BCR-ABL1 and SFPQ-ABL1 differentially activate downstream signalling pathways, including SH-binding proteins. Hierarchical clustering of phosphopeptides quantified from cells expressing canonical BCR-ABL1, SFPQ-ABL1, and SFPQ-ABL1+SH, demonstrated that BCR-ABL1 and SFPQ-ABL1+SH were more similar to each other than to SFPQ-ABL1. SFPQ-ABL1 expression resulted in phosphorylation of proteins involved in RNA processing, metabolism and splicing, suggesting that SFPQ region of SFPQ-ABL1 also contributes to signalling. Conclusions: In this study, we have utilised phosphoproteomics for the unbiased identification of signalling nodes that are required for the function of different classes of ABL fusions. We have developed novel in vitro and in vivo models to further understand how these fusions function to drive leukaemia. Our data also suggests that ABL1 fusion partners play a role beyond dimerization and transphosphorylation of the kinase domains in oncogenic signalling, but further study is needed to establish the contribution to leukaemogenesis. Establishing signalling pathways that are critical to the function of rare ABL1 fusions may inform clinical approaches to treating this disease. Disclosures No relevant conflicts of interest to declare.

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Intracellular delivery of anti-BCR/ABL antibody by PLGA nanoparticles suppresses the oncogenesis of chronic myeloid leukemia cells

Journal of Hematology & Oncology ◽

10.1186/s13045-021-01150-x ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Guoyun Jiang ◽

Zhenglan Huang ◽

Ying Yuan ◽

Kun Tao ◽

Wenli Feng

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Myeloid Leukemia ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Fusion Gene ◽

Intracellular Delivery ◽

Tyrosine Kinase Activity

Abstract Background The pathogenesis of chronic myeloid leukemia (CML) is the formation of the BCR/ABL protein, which is encoded by the bcr/abl fusion gene, possessing abnormal tyrosine kinase activity. Despite the wide application of tyrosine kinase inhibitors (TKIs) in CML treatment, TKIs drug resistance or intolerance limits their further usage in a subset of patients. Furthermore, TKIs inhibit the tyrosine kinase activity of the BCR/ABL oncoprotein while failing to eliminate the pathologenic oncoprotein. To develop alternative strategies for CML treatment using therapeutic antibodies, and to address the issue that antibodies cannot pass through cell membranes, we have established a novel intracellular delivery of anti-BCR/ABL antibodies, which serves as a prerequisite for CML therapy. Methods Anti-BCR/ABL antibodies were encapsulated in poly(d, l-lactide-co-glycolide) nanoparticles (PLGA NPs) by a double emulsion method, and transferrin was labeled on the surface of the nanoparticles (Ab@Tf-Cou6-PLGA NPs). The characteristics of nanoparticles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cellular uptake of nanoparticles was measured by flow cytometry (FCM). The effect of nanoparticles on the apoptosis and proliferation of CML cells was testified by FCM and CCK-8 assay. In addition, the anti-cancer impact of nanoparticles was evaluated in mouse models of CML. Results The results demonstrated that the Ab@Tf-Cou6-PLGA NPs functioned as an intracellular deliverer of antibodies, and exhibited an excellent effect on degrading BCR/ABL oncoprotein in CML cells via the Trim-Away pathway. Treatment with Ab@Tf-Cou6-PLGA NPs inhibited the proliferation and induced the apoptosis of CML cells in vitro as well as impaired the oncogenesis ability of CML cells in vivo. Conclusions In conclusion, our study indicated that this approach achieved safe and efficient intracellular delivery of antibodies and degraded BCR/ABL oncoprotein via the Trim-Away pathway, which provides a promising therapeutic strategy for CML patients, particularly those with TKI resistance.

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