Phosphorylation of Receptor Tyrosine Kinase ROR1 At Tyrosine 641, 646 and Serine 652 residues Might Be of Importance for the Survival of CLL Leukemic Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3885-3885
Author(s):  
Mohammad Hojjat-Farsangi ◽  
Amir Hossein Daneshmanesh ◽  
Martin Norin ◽  
Åsa Sandin ◽  
Abdul Salam Khan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Tyrosine Kinase ◽  
Western Blot ◽  
Tyrosine Kinases ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Kinase Domain ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Tyrosine Kinase Domain

Abstract Abstract 3885 Background: Receptor tyrosine kinases (RTK) play crucial roles for different normal cellular processes as cell proliferation/differentiation, apoptosis and survival, as well as for the malignant phenotype of many types of cancer. ROR1, as a member of twenty RTKs families, has important functions during normal embryogenesis. ROR1 has shown to be overexpressed in chronic lymphocytic leukemia (CLL), mantle cell lymphoma and other hematological malignancies, as well as in solid tumors. ROR1 inhibition in CLL cells and cell lines with high expression of ROR1 induced specific apoptosis of the cells. In this study, we investigated the effects of an anti-ROR1 mAb against the extracellular CRD domain for specific dephosphorylation at the tyrosine kinase domain of ROR1 in CLL cells. The CRD domain contains a frizzle receptor, which is considered to be the ligand-binding region for e.g. Wnt5a. Wnt5a has been suggested to stimulate growth of CLL cells. Aims: To investigate phosphorylation of tyrosine and serine residues, within the tyrosine kinase domain of ROR1, of importance for survival of CLL cells. Methods: Bioinformatic analysis of the ROR1 structure revealed that three amino acid residues in the tyrosine kinase domain might be critically phosphorylated. Based on this prediction, a 19 amino acid long peptide, phosphorylated at two tyrosine (tyrosine 641, 646) and one serine (serine 652) residues was designed and used for immunization of rabbits. An anti-phospho-ROR1 (pROR1) polyclonal antibody (pAb) with high titers of anti-pROR1 pAb was purified, using phospho-peptide affinity chromatography. The specificity of anti-pROR1 pAb was determined by ELISA, immunoprecipitation (IP) and western blot experiments. An anti-ROR1 mAb (IgG) (CRD 1D8 clone) was used to analyse the effects on ROR1 phosphorylation in CLL cells at tyrosine 641, 646 and serine 652 residues preceding apoptosis. ROR1 phosphorylation was investigated by western blot and IP of ROR1 probed with anti-pROR1 pAb, from untreated and CLL cells treated with the anti-CRD 1D8 mAb. Quantitative intracellular staining of ROR1 by flowcytometry in time kinetics experiment after treatment with anti-CRD 1D8 mAb was also used to check phosphorylation of ROR1. Annexin V/PI staining (flowcytometry), MTT assay, PARP and caspase 8 cleavage as well as MCL-1 protein (western blot) were used for detection of apoptosis. To investigate phosphorylation and localization of 64–130 kDa ROR1 isoforms in various compartments of CLL cells, lysates were prepared from the nucleus and cytoplasmic proteins of CLL cells. Results: Two tyrosine (641, 646) residues and one serine (652) residue of the tyrosine kinase domain were phosphorylated in CLL cells. As previously described (Mellstedt et al, Abstract No: 1771, 53th ASH annals meeting, 2011), the 64, 105 and 130 kDa ROR1 isoforms were shown to be constitutively phosphorylated at tyrosine and serine residues in CLL leukemic cells. Treatment of CLL cells with an anti-ROR1 mAb against the CRD domain induced rapid dephosphorylation of ROR1 at tyrosine 641, 646 and serine 652 residues within 20 min and gradually increased up to 4 hours. The phosphorylated 64 kDa ROR1 isoform was localized to the nucleus of CLL cells and probably represents an intracellular part of ROR1, while the ROR1 130 kDa isoform was presented both in cytoplasm and nucleus of CLL cells. Conclusion: Our data show that the ROR1 molecule is phosphorylated at tyrosine 641, 646 and serine 652 residues. The presence of 64 and 130 kDa ROR1 isoforms in the nucleus of CLL cells may suggest a role of these isoforms as transcription factors. Collectively, the data might suggest that phosphorylated ROR1 may be an important protein for the growth of CLL cells as well as an interesting structure to target in a therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

Functional dissection of p56lck, a protein tyrosine kinase which mediates interleukin-2-induced activation of the c-fos gene

1994 ◽  
Vol 14 (9) ◽  
pp. 5812-5819
Author(s):  
H Shibuya ◽  
K Kohu ◽  
K Yamada ◽  
E L Barsoumian ◽  
R M Perlmutter ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Protein Tyrosine Kinase ◽  
Gene Activation ◽  
Interleukin 2 ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Amino Acid Residues ◽  
Protein Tyrosine ◽  
Responsive Element

Members of the newly identified receptor family for cytokines characteristically lack the intrinsic protein tyrosine kinase domain that is a hallmark of other growth factor receptors. Instead, accumulating evidence suggests that these receptors utilize nonreceptor-type protein tyrosine kinases for downstream signal transduction by cytokines. We have shown previously that the interleukin-2 receptor beta-chain interacts both physically and functionally with a Src family member, p56lck, and that p56lck activation leads to induction of the c-fos gene. However, the mechanism linking p56lck activation with c-fos induction remains unelucidated. In the present study, we systematically examined the extent of c-fos promoter activation by expression of a series of p56lck mutants, using a transient cotransfection assay. The results define a set of the essential amino acid residues that regulate p56lck induction of the c-fos promoter. We also provide evidence that the serum-responsive element and sis-inducible element are both targets through which p56lck controls c-fos gene activation.

No Evidence for Constitutively Activated FLT3 in Juvenile Myelo-Monocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4915-4915
Author(s):  
Andrica C.H. de Vries ◽  
Ronald W. Stam ◽  
Pauline Schneider ◽  
Charlotte M. Niemeyer ◽  
Elisabeth R. van Wering ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Leukemic Cells ◽  
Tyrosine Kinase Domain ◽  
Mrna Levels ◽  
Monocytic Leukemia ◽  
Real Time Quantitative Pcr ◽  
Flt3 Inhibitor ◽  
Flt3 Mutations

Abstract Activating FLT3 mutations have been identified as prognostic factors in multiple myeloid malignancies. Recent studies have demonstrated that ligand-independent activation of FLT3 can also result from overexpression of wild-type FLT3. In addition, ligand-dependent activation has been observed in leukemic cells co-expressing FLT3 ligand (FLT3L), resulting in autocrine FLT3 signaling which is independent of FLT3 mutations. In Juvenile Myelo-Monocytic Leukemia (JMML), FLT3 internal tandem duplications (FLT3/ITDs) mutations affecting the tyrosine kinase domain (TKD) are rare. However, no data are yet available on the frequency of expression levels of FLT3 and FLT3L in JMML. If activated FLT3 occurs in JMML, one could imagine that these patients might benefit from treatment with small molecule FLT3 inhibitors, especially as to date the curative treatment of JMML is limited to allogeneous bone marrow transplantation. In 36 JMML patients FLT3 and FLT3L mRNA levels were assessed using real-time quantitative PCR (Taqman). Furthermore these samples were screened for the presence of activating FLT3/ITDs and FLT3/TKD mutations. MTT assays were performed to assess the response of JMML cells to the known FLT3 inhibitor PKC412 (Novartis). FLT3 appeared to be expressed only at basal levels and FLT3L expression was very low. In none of the 36 JMML samples FLT3/ITDs or TDK mutations were found, consistent with the observation that PKC412 was not cytotoxic in JMML samples (n=12), in contrast to leukemic cells of children with ALL which carried an activated FLT3. These data suggest that constitutively activated FLT3 does not occur in JMML. Therefore targeting FLT3 by tyrosine kinase inhibitors like PKC412 is unlikely to be effective in JMML.

Effect of ROR1-targeting small molecules on chronic lymphocytic leukemia (CLL) cells.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 6557-6557 ◽  
Author(s):  
Hakan Mellstedt ◽  
Salam Khan ◽  
Jan Vågberg ◽  
Styrbjörn Byström ◽  
Johan Schultz ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Small Molecules ◽  
High Throughput Screening ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Parp Cleavage ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Substrate Peptide

6557 Background: There is a great interest to develop targeting drugs for CLL, both MAbs and small molecules, to improve the outcome for the disease. ROR1, a receptor tyrosine kinase, is overexpressed on CLL cells but not on normal cells. ROR1 is constitutively phosphorylated and siRNA transfection induces leukemic cell death. The aim of the study is to produce small molecules inhibiting the cytoplasmic tyrosine kinase activity of ROR1, which could induce specific killing of leukemic cells. Methods: A high-throughput screening assay in 384-format has been developed measuring phosphorylation of a substrate peptide by human recombinant intracellular ROR-1 kinase domain. A collection of 80.000 small molecules has been screened generating two chemical series. Novel leads have been synthesized and structure-activity-relationship has been developed using the assay. Results: Three compounds were selected (KAN0173631, KAN0438063, KAN0438175T). Freshly isolated leukemic CLL cells were used as targets in addition to PBMC from healthy donors to test cytotoxicity. The three compounds induced specific apoptosis of CLL cells (Annexin V/PI and MTT) both at 24 and 48h. Efficacy index (EI), i.e. killing of leukemic cells in relation to normal PBMC was favourable. The most promising drug KAN0438063 had an EI of 40 i.e. killed 40 times more CLL cells than PBMC at an IC50 of 10µM. The compounds induced PARP cleavage and cleavage of caspases 8 and 9 as well as down regulation of Mcl-1 and Bcl-xl (Western Blot). ROR1 was dephosphorylated (Western Blot). The selective apoptotic effect was compared to other small molecules targeting non-ROR1 structures in CLL (PCI-32765, CAL-101, R406, R788, STK-156485, STK-156133) and our compounds were significantly more effective (EI) (p<0.001). Conclusions: We have developed effective and selective series of compounds targeting ROR1 with promising ADMET properties. ROR1 targeting small molecules might also be effective for other tumor cells expressing ROR1 as has been noted for e.g. pancreatic carcinoma cells. Our model molecules will be further optimized and tested in animal tumor models. These molecules represent the first small molecules targeting ROR1 – a “survival factor” in CLL.

New insights into the mechanisms of hematopoietic cell transformation by activated receptor tyrosine kinases

Blood ◽  
2010 ◽  
Vol 116 (14) ◽  
pp. 2429-2437 ◽  
Author(s):  
Federica Toffalini ◽  
Jean-Baptiste Demoulin
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Cell Transformation ◽  
Chromosomal Rearrangements ◽  
Point Mutations ◽  
Large Cell ◽  
Kinase Domain ◽  
Leukemic Cells

Abstract A large number of alterations in genes encoding receptor tyrosine kinase (RTK), namely FLT3, c-KIT, platelet-derived growth factor (PDGF) receptors, fibroblast growth factor (FGF) receptors, and the anaplastic large cell lymphoma kinase (ALK), have been found in hematopoietic malignancies. They have drawn much attention after the development of tyrosine kinase inhibitors. RTK gene alterations include point mutations and gene fusions that result from chromosomal rearrangements. In both cases, they activate the kinase domain in the absence of ligand, producing a permanent signal for cell proliferation. Recently, this simple model has been refined. First, by contrast to wild-type RTK, many mutated RTK do not seem to signal from the plasma membrane, but from various locations inside the cell. Second, their signal transduction properties are altered: the pathways that are crucial for cell transformation, such as signal transducer and activator of transcription (STAT) factors, do not necessarily contribute to the physiologic functions of these receptors. Finally, different mechanisms prevent the termination of the signal, which normally occurs through receptor ubiquitination and degradation. Several mutations inactivating CBL, a key RTK E3 ubiquitin ligase, have been recently described. In this review, we discuss the possible links among RTK trafficking, signaling, and degradation in leukemic cells.

scholarly journals Comparison of tyrosine kinase domain properties for the neurotrophin receptors TrkA and TrkB

2020 ◽  
Vol 477 (20) ◽  
pp. 4053-4070
Author(s):  
Stephen C. Artim ◽  
Anatoly Kiyatkin ◽  
Mark A. Lemmon
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Catalytic Efficiency ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Signaling Dynamics ◽  
Cellular Context ◽  
And Function ◽  
High Degree

The tropomyosin-related kinase (Trk) family consists of three receptor tyrosine kinases (RTKs) called TrkA, TrkB, and TrkC. These RTKs are regulated by the neurotrophins, a class of secreted growth factors responsible for the development and function of neurons. The Trks share a high degree of homology and utilize overlapping signaling pathways, yet their signaling is associated with starkly different outcomes in certain cancers. For example, in neuroblastoma, TrkA expression and signaling correlates with a favorable prognosis, whereas TrkB is associated with poor prognoses. To begin to understand how activation of the different Trks can lead to such distinct cellular outcomes, we investigated differences in kinase activity and duration of autophosphorylation for the TrkA and TrkB tyrosine kinase domains (TKDs). We find that the TrkA TKD has a catalytic efficiency that is ∼2-fold higher than that of TrkB, and becomes autophosphorylated in vitro more rapidly than the TrkB TKD. Studies with mutated TKD variants suggest that a crystallographic dimer seen in many TrkA (but not TrkB) TKD crystal structures, which involves the kinase-insert domain, may contribute to this enhanced TrkA autophosphorylation. Consistent with previous studies showing that cellular context determines whether TrkB signaling is sustained (promoting differentiation) or transient (promoting proliferation), we also find that TrkB signaling can be made more transient in PC12 cells by suppressing levels of p75NTR. Our findings shed new light on potential differences between TrkA and TrkB signaling, and suggest that subtle differences in signaling dynamics can lead to substantial shifts in the cellular outcome.

scholarly journals Efficient CD28 signalling leads to increases in the kinase activities of the TEC family tyrosine kinase EMT/ITK/TSK and the SRC family tyrosine kinase LCK

1998 ◽  
Vol 330 (3) ◽  
pp. 1123-1128 ◽  
Author(s):  
Spencer GIBSON ◽  
Ken TRUITT ◽  
Yiling LU ◽  
Ruth LAPUSHIN ◽  
Humera KHAN ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
T Cell Activation ◽  
Tyrosine Kinases ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Cell Receptor ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain

Optimal T cell activation requires crosslinking of the T cell receptor (TCR) concurrently with an accessory receptor, most efficiently CD28. Crosslinking of CD28 leads to increased interleukin 2 (IL2) production, inhibition of anergy and prevention of programmed cell death. Crosslinking of CD28 leads to rapid increases in tyrosine phosphorylation of specific intracellular substrates including CD28 itself. Since CD28 does not encode an intrinsic tyrosine kinase domain, CD28 must activate an intracellular tyrosine kinase(s). Indeed, crosslinking of CD28 increases the activity of the intracellular tyrosine kinases EMT/ITK and LCK. The phosphatidylinositol 3-kinase (PI3K) and GRB2 binding site in CD28 is dispensable for optimal IL2 production in Jurkat T cells. We demonstrate herein that murine Y170 (equivalent to human Y173) in CD28 is also dispensable for activation of the SRC family tyrosine kinase LCK and the TEC family tyrosine kinase EMT/ITK. In contrast, the distal three tyrosines in CD28 are required for optimal IL2 production as well as for optimal activation of the LCK and EMT/ITK tyrosine kinases. The distal three tyrosines of CD28, however, are not required for recruitment of PI3K to CD28. Furthermore, PI3K is recruited to CD28 in JCaM1 cells which lack LCK and in which EMT/ITK is not activated by ligation of CD28. Thus optimal activation of LCK or EMT/ITK is not obligatory for recruitment of PI3K to CD28 and thus is also not required for tyrosine phosphorylation of the YMNM motif in CD28. Taken together the data indicate that the distal three tyrosines in CD28 are integral to the activation of LCK and EMT/ITK and for subsequent IL2 production.

scholarly journals Targeting Venetoclax-Resistant CLL By Bcl-XL Degradation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2252-2252
Author(s):  
Daisy Diaz Rohena ◽  
Janani Ravikrishnan ◽  
Chaomei Liu ◽  
Guangrong Zheng ◽  
Daohong Zhou ◽  
...  
Keyword(s):  
Western Blot ◽  
Research Funding ◽  
Annexin V ◽  
Phosphatidyl Serine ◽  
Mitochondrial Pathway ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Response Curves ◽  
Combining Data ◽  
Treatment Naïve

Abstract Chronic lymphocytic leukemia (CLL) is the most prevalent leukemia in adults and is associated with resistance to apoptosis. Clinical responses are dramatically improved with the use of targeted therapies such as venetoclax, a selective inhibitor of the anti-apoptotic protein Bcl-2, compared to chemoimmunotherapy. However, some patients can ultimately develop resistant CLL. Bcl-xL overexpression has been implicated in multiple mechanisms of venetoclax resistance, representing a switch that confers cell survival despite Bcl-2 inhibition. Mechanisms of venetoclax resistance include mutated Bcl-2 (ex. G101V), switches in survival dependence to alternative BCL2 family members, and microenvironmental interactions (Reviewed in Bose et al., 2017). Moreover, disease progression on venetoclax is accelerated in CLL patients with aberrant p53 , and our laboratory identified Bcl-xL as one of the main contributors to the oncogenic abilities of mutant p53.Thus, Bcl-xL is a promising target for re-sensitizing CLL to apoptosis, but since it is crucial for platelet viability, direct targeting has not been clinically successful. New Bcl-xL PROTAC degraders provide an exciting opportunity to target Bcl-xL in venetoclax-resistant CLL. Due to differences in the expression of VHL E3 ligase in cancer cells versus platelets, these degraders have shown potency and selectivity to target this protein for degradation in multiple Bcl-xL-dependent malignancies (He, Y. et al., 2020). Our results show that exposing primary CLL samples (n=4) from treatment-naïve patients to the Bcl-xL degrader DT2216 is associated with apoptosis at concentrations known to be non-toxic to platelets (EC50 = 162 nM at 18 hr treatment). Furthermore, the dual Bcl-xL/Bcl-2 degrader PZ18753B was synthesized with enhanced potency against Bcl-xL and Bcl-2 co-dependent cancers. Our results confirm enhanced potency in primary cells from treatment naïve patients with CLL (EC50 = 7.602 nM at 18 hr treatment), compared to DT2216. Also, the degraders exhibit a similar kinetic profile at 10-fold concentrations (n=5, t1/2 = 2.3 hr for 1 uM DT2216, and t1/2 = 3.1 hr for 100 nM PZ18753B). For these results, apoptosis via the mitochondrial pathway was studied by flow cytometry, assessing extracellular exposure of phosphatidyl serine and mitochondrial depolarization by Annexin V and TMRM staining, respectively. Consistently, as confirmed by Western Blot, PZ18753B is more potent at degrading Bcl-xL than DT2216, achieving 50% degradation in RS4;11 leukemic cells at 46 hr treatment at a concentration of 0.86 nM, compared to 7.83 nM for DT2216. Comparing dose-response curves showing apoptosis by AnnexinV/TMRM staining and protein degradation by Western Blot, we confirmed that Bcl-xL degradation precedes cell death. Given that Bcl-xL overexpression confers a survival advantage to CLL cells, we hypothesize that venetoclax-resistant CLL cells can be targeted with the new Bcl-xL degraders. We studied the potency of the degraders against RS4;11 leukemic cells which express wildtype (WT) Bcl-2, or also overexpress G101V mutant Bcl-2. This is one of the mutations associated with venetoclax resistance and, combining data from various cohorts, it was identified in 23% venetoclax-resistant patients (Blombery et al., 2019 and 2020; Tausch et al., 2019; Weiss et al., 2019). PZ18753B induced apoptosis of RS4;11 Bcl-2 WT cells within 46 hrs (EC50 = 11.1 nM) as well as in RS4;11 cells expressing Bcl-2 G101V mutation (EC50 = 109.1 nM). PZ18753B degraded both Bcl-xL and Bcl-2 in RS4;11 WT cells effectively. In RS4;11 Bcl-2 G101V cells, it did not affect Bcl-2 but retained potent Bcl-xL degradation (86% vs 75% at 1 uM in WT vs mutant Bcl-2 cell lines, respectively). Interestingly, DT2216 did show ability to degrade G101V mutant Bcl-2 at the highest concentrations tested (Ex. 36% and 33%, respectively, at 1 uM). We also demonstrated that CLL cells from chemo-naïve patients depend primarily on Bcl-2 for survival, as measured by their responses to BH3 mimetic peptides in a BH3 profiling assay. However, venetoclax-resistant CLL cells undergo a shift in dependence to alternative Bcl-2 family proteins, such as Bcl-xL and Mcl-1, as a mechanism for resistance to apoptosis. Thus, resistant CLL represents an excellent setting in which to continue testing the efficacy of these potent Bcl-xL degraders, to overcome resistance to Bcl-2 inhibitors. Disclosures Zheng: Dialectic Therapeutics: Other: Co-founder and stakeholder. Inventor of Bcl-xL PROTACS disclosed in this study.. Zhou: Dialectic Therapeutics: Other: Co-founder and stakeholder. Inventor of Bcl-xL PROTACS disclosed in this study.. Zhang: Dialectic Therapeutics: Other: Co-founder and stakeholder. Co-inventor of Bcl-xL PROTACs disclosed in this study.. Wierda: Oncternal Therapeutics, Inc.: Research Funding; Genentech: Research Funding; Juno Therapeutics: Research Funding; Xencor: Research Funding; KITE Pharma: Research Funding; Sunesis: Research Funding; GSK/Novartis: Research Funding; Janssen: Research Funding; Loxo Oncology, Inc.: Research Funding; Acerta Pharma Inc.: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Gilead Sciences: Research Funding; Karyopharm: Research Funding; Miragen: Research Funding; Cyclacel: Research Funding; AstraZeneca: Research Funding; Genzyme Corporation: Consultancy; AbbVie: Research Funding. Jain: Incyte: Research Funding; Servier: Honoraria, Research Funding; Precision Biosciences: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; Beigene: Honoraria; Cellectis: Honoraria, Research Funding; Pfizer: Research Funding; Fate Therapeutics: Research Funding; Aprea Therapeutics: Research Funding; Genentech: Honoraria, Research Funding; ADC Therapeutics: Honoraria, Research Funding; Adaptive Biotechnologies: Honoraria, Research Funding; TG Therapeutics: Honoraria; Janssen: Honoraria; AbbVie: Honoraria, Research Funding; Pharmacyclics: Research Funding. Woyach: AbbVie Inc, Loxo Oncology Inc, a wholly owned subsidiary of Eli Lilly & Company: Research Funding; AbbVie Inc, ArQule Inc, Janssen Biotech Inc, AstraZeneca, Beigene: Other: Advisory Committee; Gilead Sciences Inc: Other: Data & Safety; AbbVie Inc, ArQule Inc, AstraZeneca Pharmaceuticals LP, Janssen Biotech Inc, Pharmacyclics LLC, an AbbVie Company,: Consultancy.

Targeted inhibition of FLT3 overcomes the block to myeloid differentiation in 32Dcl3 cells caused by expression of FLT3/ITD mutations

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 4154-4161 ◽  
Author(s):  
Rui Zheng ◽  
Alan D. Friedman ◽  
Donald Small
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Transform Growth Factor ◽  
Leukemic Cells ◽  
Tyrosine Kinase Domain ◽  
Enhancer Binding Protein ◽  
Dependent Cell ◽  
Targeted Inhibition ◽  
The Impact

Internal tandem duplication (ITD) mutations of the juxtamembrane domain–coding sequence of the FLT3 gene are found in up to 34% of patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. FLT3/ITDs result in constitutive activation of the tyrosine kinase domain and transform growth factor–dependent cell lines. FLT3 activation leads to antiapoptotic and proliferative signals, but little is known about the impact of FLT3/ITDs on differentiation. This study was designed to investigate the effect of FLT3/ITD expression on the differentiation of the 32Dcl3 (32D) myeloblastic cell line to neutrophils in response to granulocyte colony-stimulating factor (G-CSF). Expression of FLT3/ITD completely blocked morphologic differentiation and induction of myeloperoxidase (MPO), lysozyme, and CCAAT/enhancer-binding protein ε (C/EBPε) in response to G-CSF. Wild-type FLT3 and vector-transfected 32D cells were able to differentiate, although the maturation of FLT3-transfected cells was delayed by FLT3 ligand (FL) stimulation. CEP-701, a potent FLT3 tyrosine kinase inhibitor, overcame the morphologic block in differentiation caused by FLT3/ITD expression and allowed G-CSF induction of myeloid maturation markers. These findings suggest that blocking differentiation may be one of the mechanisms by which FLT3/ITDs contribute to leukemogenesis. CEP-701 and other FLT3 inhibitors may be useful for overcoming the block to differentiation (as well as the block to apoptosis) in the leukemic cells of patients with AML.

scholarly journals Secondary mutations as mediators of resistance to targeted therapy in leukemia

Blood ◽  
2015 ◽  
Vol 125 (21) ◽  
pp. 3236-3245 ◽  
Author(s):  
Naval Daver ◽  
Jorge Cortes ◽  
Farhad Ravandi ◽  
Keyur P. Patel ◽  
Jan A. Burger ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Tyrosine Kinase ◽  
Small Molecule ◽  
Myeloid Leukemia ◽  
Point Mutations ◽  
Kinase Domain ◽  
Lymphocytic Leukemia ◽  
Tyrosine Kinase Domain ◽  
Flt3 Inhibitors ◽  
Kinase Domain Mutations

AbstractThe advent of small molecule-based targeted therapy has improved the treatment of both acute and chronic leukemias. Resistance to small molecule inhibitors has emerged as a common theme. The most frequent mode of acquired resistance is the acquisition of point mutations in the kinase domain. FLT3 inhibitors have improved response rates in FLT3-mutated acute myeloid leukemia (AML). The occurrence of the ATP-binding site and activation loop mutations confers varying degrees of resistance to the individual FLT3 inhibitors. Second-generation FLT3 inhibitors such as crenolanib may overcome the resistance of these mutations. Furthermore, nonmutational mechanisms of resistance such as prosurvival pathways and bone marrow signaling may be upregulated in FLT3 inhibitor-resistant AML with secondary kinase domain mutations. More recently, point mutations conferring resistance to the Bruton tyrosine kinase inhibitor ibrutinib in chronic lymphocytic leukemia, arsenic trioxide in acute promyelocytic leukemia, and the BH3-mimetic ABT199 in lymphoma have been identified. In chronic myeloid leukemia, the emergence of tyrosine kinase domain mutations has historically been the dominant mechanism of resistance. The early identification of secondary point mutations and their downstream effects along with the development of second- or third-generation inhibitors and rationally designed small molecule combinations are potential strategies to overcome mutation-mediated resistance.

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