TTT-3002 Is Active Against Relapsed/Refractory AML Patient Bone Marrow With FLT3 TKI-Resistance Mutations At Residue D835

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3818-3818
Author(s):  
Hayley S Ma ◽  
Bao Nguyen ◽  
Mark Levis ◽  
Allen B Williams ◽  
Donald Small
Keyword(s):  
Drug Resistance ◽  
Clinical Trials ◽  
Ex Vivo ◽  
Point Mutations ◽  
Site Directed Mutagenesis ◽  
Resistance Mutations ◽  
Patient Sample ◽  
Tki Resistance ◽  
Selection Of

Abstract Over 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FLT3, either internal tandem duplication (ITD) or point mutations (FLT3/PM). FLT3/ITD mutations in particular confer a poor prognosis, and thus several FLT3 tyrosine kinase inhibitors (TKIs) have been developed and are currently in clinical trials. However, many have failed due to insufficient achievement of FLT3 kinase inhibition in vivo. This is thought to be due to insufficient potency, reduced activity against FLT3/PM, and/or selection for resistance-conferring point mutations in FLT3/ITD. Therefore, the search for novel FLT3 TKIs that overcome some of these resistance mechanisms that result in persistent FLT3 activation is necessary to improve the cure rate for this disease. We have previously reported on TTT-3002, a novel TKI that possesses the most potent activity against FLT3 phosphorylation, with IC50s of 100-250pM. Here we characterize the activity of this compound against TKI-resistant AML. The phenomenon of drug resistance associated with selection of FLT3 point mutations is being observed with increasing frequency as higher levels of inhibition by FLT3 TKIs are achieved in clinical trials of FLT3/ITD AML patients. In order to predict the ability of TTT-3002 to successfully treat some of these resistance mutations, we used site-directed mutagenesis to generate a series of FLT3/ITD TKI resistant cell lines. We directly compared the activity of TTT-3002 to other FLT3 TKIs currently in clinical trials including CEP-701, sorafenib, AC220 and PKC412. TTT-3002 remained the most potent inhibitor of all six FLT3/ITD TKI resistance mutants screened, with viability IC50 values of less than 1nM (with the single exception of Ba/F3-G697R/ITD, IC50=11nM). It was thus able to effectively inhibit FLT3 with TKI resistance mutations against which other TKIs are ineffective. Therefore, this compound’s broad spectrum of activity against FLT3/ITD TKI resistance mutations may enable it to successfully treat FLT3/ITD AML patients who have become resistant to other FLT3 TKIs. To examine this directly, human AML samples were obtained from patients that developed resistance to sorafenib or AC220 while on clinical trials and presented with a dual D835/ITD mutation or D835 point mutation alone at the time of relapse. These blasts were evaluated for their ex vivo sensitivity to TTT-3002, sorafenib and AC220, with a diagnostic FLT3/ITD+ patient sample for comparison. Encouragingly, we observed significant effects on proliferation, induction of apoptosis, and cell signaling when cells were treated with TTT-3002. AC220 was moderately active against FLT3/ITD+ blasts and one patient sample with a D835/ITD mutation, and sorafenib was active against only the diagnostic FLT3/ITD sample, but none of the three relapse samples. Therefore, TTT-3002 maintains activity against human AML patient samples that are resistant in clinical trials to the FLT3 TKIs sorafenib and AC220. To account for the patterns of drug resistance observed for different FLT3 TKIs in our study, we modeled the positions of staurosporine-like (TTT-3002, CEP-701 and PKC412) and sorafenib-like inhibitors (sorafenib and AC220) bound to FLT3. Examination of amino-acid substitutions in FLT3 known to confer resistance to TKIs from clinical trials shows that substituting arginine for glycine at position 697 likely leads to steric clashes with FLT3 TKIs. It has been proposed that the F691 residue forms a stabilizing π-π stacking interaction with AC220, and thus a mutation to a Leu accounts for reduced binding affinity for this TKI. TTT-3002 is predicted to bind FLT3 without making direct contact with the F691 residue, and thus is unaffected by mutations at this site accounting for its continued potency. The closer proximity of F691 and N676 to sorafenib compared to staurosporine likely underlies the trend for substitutions at these sites to confer resistance to sorafenib-like but not staurosporine-like inhibitors. In summary, TTT-3002 has the potential for increased clinical applicability due to its ability to target not only FLT3/ITD and FLT3/PM AML patients but also patients who have developed resistance to other FLT3 TKIs through the selection of resistance mutations within the FLT3/ITD allele. These preclinical studies further support the entry of TTT-3002 into human clinical trials. Disclosures: Levis: Ambit: Consultancy.

scholarly journals Evaluation of Patient-Derived Xenografts for Modeling Outcome of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3759-3759
Author(s):  
Abdulmohsen M Alruwetei ◽  
Hernan Carol ◽  
Rosemary Sutton ◽  
Glenn M Marshall ◽  
Richard B Lock
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Clinical Outcome ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Nsg Mice ◽  
Relapse Prediction ◽  
Pediatric All ◽  
Selection Of

Abstract Introduction: Children with acute lymphoblastic leukemia (ALL) are stratified at diagnosis based on molecular/cytogenetic characteristics and their response to initial treatment to receive risk-adapted multi-agent chemotherapy. The majority of ALL patients are stratified as Intermediate Risk (IR) and present with moderate levels of minimal residual disease (MRD<5x104) after receiving induction therapy, although an unacceptably high proportion of these patients relapse. The lack of specific prognostic features makes it difficult to predict the response of IR patients to treatment. The early identification of patients who are destined to relapse would facilitate improvements in tailored treatments for IR ALL patients. Recent progress in the development of patient-derived xenografts (PDXs) in immune-deficient mice represents an opportunity to improve relapse prediction in ALL patients. The aims of this study were to: (1) optimize the engraftment conditions of IR pediatric ALL samples to predict patient response to treatment; and, (2) to assess the development and mechanisms of therapy-induced drug resistance. Methods: Two pairs of IR pediatric ALL patients were matched based on clinical and genetic features, except that one patient from each pair relapsed early while the other remains relapse-free (ALL-Rel and ALL-CR1, respectively). Three parameters were varied in establishing PDXs by inoculating one million bone marrow (BM) derived biopsy cells collected at diagnosis into groups of 4 mice: (1) mouse strain (NOD/SCID vs. NSG); (2) site of inoculation (intravenous vs. intra-femoral); and (3) early treatment of mice with a 2-week induction chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Leukemia engraftment was monitored weekly based on the proportion of human versus mouse CD45+ cells in the murine PB, and the median times to engraftment were compared according to patient outcome. The median time to engraft was also compared between the VXL-treated and non-treated groups. PDXs harvested from mice were compared for ex vivo sensitivity to single agent vincristine, dexamethasone and L-asparaginase. PDX gene expression profiles were also compared to identify pathways associated with evasion of VXL treatment in vivo. Results: The efficiency of engraftment was greater for NSG mice (29/32 mice engrafted) versus NOD/SCID mice (20/32 mice), and primary ALL cells also engrafted significantly faster in NSG mice (median time to engraft 71.1 days) compared with NOD/SCID mice (83.5 days) (P < 0.01), with no apparent difference associated with clinical outcome. Intrafemoral inoculation did not improve the efficiency or speed of engraftment compared with intravenous inoculation, nor predicted clinical outcome. However, PDX responses to VXL induction chemotherapy reflected the clinical outcome of the patients from whom they were derived; those derived from the 2 ALL-Rel patients exhibited in vivo drug resistance (leukemia growth delay of 1 and 6.2 days) compared with those derived from the 2 ALL-CR1 patients (34.7 and >119.8 days). Further, ex vivo analysis showed that the PDXs derived from the ALL-Rel patients exhibited resistance to vincristine or L-asparaginase compared with those derived from the ALL-CR1 cases. Moreover, the in vivo VXL treatment of an ALL-CR1 PDX resulted in selection of cells that exhibited vincristine resistance. Gene expression profiling revealed significant up-regulation of microtubule associated proteins (MAPs) and tubulin isotypes (alpha and beta) in vincristine-resistant PDXs. Genes that were significantly upregulted in vincristine resistant PDXs with a false discovery rate (FDR) < 0.05 and P value < 0.02 include TUBB6, TUBA1A, TUBA1B, MAP1S, TUBA3D and TBCA. The increased expression of genes that affect microtubule functions suggest that changes in microtubule dynamics and/or stability led to decreased sensitivity to antimicrotubule agents. Conclusions: In vivo selection of PDXs with an induction-type regimen of chemotherapeutic drugs may lead to improved relapse prediction and identify novel mechanisms of drug resistance in IR pediatric ALL. Support: Steven Walter Foundation; NHMRC Australia, APP1057746 Disclosures No relevant conflicts of interest to declare.

scholarly journals Gilteritinib overcomes lorlatinib resistance in ALK-rearranged cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hayato Mizuta ◽  
Koutaroh Okada ◽  
Mitsugu Araki ◽  
Jun Adachi ◽  
Ai Takemoto ◽  
...  
Keyword(s):  
Gene Rearrangement ◽  
Kinase Inhibitors ◽  
Inhibitory Effect ◽  
In Vivo Model ◽  
Resistance Mutations ◽  
Lung Cancer Patients ◽  
Short Period ◽  
Tki Resistance

AbstractALK gene rearrangement was observed in 3%–5% of non-small cell lung cancer patients, and multiple ALK-tyrosine kinase inhibitors (TKIs) have been sequentially used. Multiple ALK-TKI resistance mutations have been identified from the patients, and several compound mutations, such as I1171N + F1174I or I1171N + L1198H are resistant to all the approved ALK-TKIs. In this study, we found that gilteritinib has an inhibitory effect on ALK-TKI–resistant single mutants and I1171N compound mutants in vitro and in vivo. Surprisingly, EML4-ALK I1171N + F1174I compound mutant-expressing tumors were not completely shrunk but regrew within a short period of time after alectinib or lorlatinib treatment. However, the relapsed tumor was markedly shrunk after switching to the gilteritinib in vivo model. In addition, gilteritinib was effective against NTRK-rearranged cancers including entrectinib-resistant NTRK1 G667C-mutant and ROS1 fusion-positive cancer.

scholarly journals In Vivo Evidence for TonB Dimerization

2003 ◽  
Vol 185 (19) ◽  
pp. 5747-5754 ◽  
Author(s):  
Annette Sauter ◽  
S. Peter Howard ◽  
Volkmar Braun
Keyword(s):  
Single Point ◽  
Point Mutations ◽  
Ferric Citrate ◽  
Site Directed Mutagenesis ◽  
Toxin Gene ◽  
Transmembrane Region ◽  
Dimer Formation ◽  
Citrate Transport ◽  
Hybrid Proteins

ABSTRACT TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and φ80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P ctx ::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P ctx ::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

scholarly journals Characterization of Resistance to the Nonnucleoside NS5B Inhibitor Filibuvir in Hepatitis C Virus-Infected Patients

2011 ◽  
Vol 56 (3) ◽  
pp. 1331-1341 ◽  
Author(s):  
Philip J. F. Troke ◽  
Marilyn Lewis ◽  
Paul Simpson ◽  
Katrina Gore ◽  
Jennifer Hammond ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Phenotypic Resistance ◽  
Number Of Patients ◽  
Chronic Hcv ◽  
Selection Of

ABSTRACTFilibuvir (PF-00868554) is an investigational nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural 5B (NS5B) RNA-dependent RNA polymerase currently in development for treating chronic HCV infection. The aim of this study was to characterize the selection of filibuvir-resistant variants in HCV-infected individuals receiving filibuvir as short (3- to 10-day) monotherapy. We identified amino acid M423 as the primary site of mutation arising upon filibuvir dosing. Through bulk cloning of clinical NS5B sequences into a transient-replicon system, and supported by site-directed mutagenesis of the Con1 replicon, we confirmed that mutations M423I/T/V mediate phenotypic resistance. Selection in patients of an NS5B mutation at M423 was associated with a reduced replicative capacityin vitrorelative to the pretherapy sequence; consistent with this, reversion to wild-type M423 was observed in the majority of patients following therapy cessation. Mutations at NS5B residues R422 and M426 were detected in a small number of patients at baseline or the end of therapy and also mediate reductions in filibuvir susceptibility, suggesting these are rare but clinically relevant alternative resistance pathways. Amino acid variants at position M423 in HCV NS5B polymerase are the preferred pathway for selection of viral resistance to filibuvirin vivo.

scholarly journals Plasmodium falciparum DHODH inhibitor complexes reveal flexible binding site

2014 ◽  
Vol 70 (a1) ◽  
pp. C1793-C1793
Author(s):  
Paul Rowland ◽  
Onkar SINGH ◽  
Leila Ross ◽  
Francisco Gamo ◽  
Maria Lafuente-Monasterio ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Binding Site ◽  
Crystal Structures ◽  
Point Mutations ◽  
Crystal Form ◽  
Drug Binding ◽  
Binding Modes ◽  
Resistance Mutations ◽  
Drug Binding Site

Malaria is a preventable and treatable disease, yet annually there are still hundreds of thousands of malaria-related deaths. The disease is caused by infection with mosquito-borne Plasmodium parasites. With hundreds of millions of cases each year there is a very high potential for drug resistance and this has compromised many existing therapies. One target under investigation is the enzyme dihydroorotate dehydrogenase (DHODH) which catalyses the rate-limiting step of pyrimidine biosynthesis and is an essential enzyme in the malaria parasite. There are currently several Plasmodium-selective DHODH inhibitors under development. To investigate the potential for drug resistance against DHODH inhibitors in vitro resistance selections were carried out using known inhibitors from different structural classes [1]. These studies identified point mutations in the drug binding site which lead to reduced sensitivity to the inhibitors, and in some cases increased sensitivity to a different inhibitor, suggesting a novel combination therapy approach to combat resistance. To help understand the significance of the inhibitor binding site mutations we determined the crystal structures of P. falciparum DHODH in complex with the inhibitors Genz-669178, IDI-6253 and IDI-6273. Co-crystallisation experiments led to a new crystal form in each case. Here we describe the crystal structures, the binding modes of the inhibitors and the great flexibility of the binding site, which is able to adjust to accommodate different inhibitor series. The structural role of the resistance mutations is also discussed.

scholarly journals Barrier-Independent, Fitness-Associated Differences in Sofosbuvir Efficacy against Hepatitis C Virus

2016 ◽  
Vol 60 (6) ◽  
pp. 3786-3793 ◽  
Author(s):  
Isabel Gallego ◽  
Julie Sheldon ◽  
Elena Moreno ◽  
Josep Gregori ◽  
Josep Quer ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Drug Sensitivity ◽  
Specific Resistance ◽  
Antiviral Agents ◽  
Resistance Mutations ◽  
Coding Region ◽  
High Fitness ◽  
Selection Of

Sofosbuvir displays a high phenotypic barrier to resistance, and it is a component of several combination therapies for hepatitis C virus (HCV) infections. HCV fitness can be a determinant of decreased sensitivity to direct-acting antiviral agents such as telaprevir or daclatasvir, but fitness-dependent decreased drug sensitivity has not been established for drugs with a high phenotypic barrier to resistance. Low- and high-fitness HCV populations and biological clones derived from them were used to infect Huh-7.5 hepatoma cells. Sofosbuvir efficacy was analyzed by measuring virus progeny production during several passages and by selection of possible sofosbuvir resistance mutations determined by sequencing the NS5B-coding region of the resulting populations. Sofosbuvir exhibited reduced efficacy against high-fitness HCV populations, without the acquisition of sofosbuvir-specific resistance mutations. A reduced sofosbuvir efficacy, similar to that observed with the parental populations, was seen for high-fitness individual biological clones. In independently derived high-fitness HCV populations or clones passaged in the presence of sofosbuvir, M289L was selected as the only substitution in the viral polymerase NS5B. In no case was the sofosbuvir-specific resistance substitution S282T observed. High HCV fitness can lead to decreased sensitivity to sofosbuvir, without the acquisition of specific sofosbuvir resistance mutations. Thus, fitness-dependent drug sensitivity can operate with HCV inhibitors that display a high barrier to resistance. This mechanism may underlie treatment failures not associated with selection of sofosbuvir-specific resistance mutations, linked toin vivofitness of pretreatment viral populations.

Further Preclinical Studies with APR-246, a Novel Anticancer Compound Currently in Clinical Trials for Hematological Malignancies and Prostate Cancer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3773-3773
Author(s):  
Nina Mohell ◽  
Charlotta Liljebris ◽  
Jessica Alfredsson ◽  
Ylva Lindman ◽  
Maria Uustalu ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cancer Cells ◽  
Cell Lines ◽  
Cancer Cell ◽  
Ex Vivo ◽  
Cancer Cell Lines ◽  
Solid Cancer ◽  
Dependent Manner

Abstract Abstract 3773 Poster Board III-709 Introduction The tumor suppressor protein p53 induces cell cycle arrest and/or apoptosis in response to various forms of cellular stress, through transcriptional regulation of a large number of down stream target genes. p53 is frequently mutated in cancer, and cancer cells carrying defects in the p53 protein are often more resistant to conventional chemotherapy. Thus, restoration of the wild type function to mutant p53 appears to be a new attractive strategy for cancer therapy. APR-246 is a novel small molecule quinuclidinone compound that has been shown to reactivate non-functional p53 and induce apoptosis. Although the exact molecular mechanism remains to be determined, recent results suggest that an active metabolite of APR-246 alkylates thiol groups in the core domain of p53, which promotes correct folding of p53 and induces apoptosis (Lambert et al., Cancer Cell 15, 2009). Currently, APR-246 is in Phase I/IIa clinical trials for hematological malignancies and prostate cancer. In the present abstract results from in vitro, ex vivo and in vivo preclinical studies with APR-246 are presented. Results The lead compound of APR-246, PRIMA-1 (p53 reactivation and induction of massive apoptosis), was originally identified by a cellular screening of the NCI library for low molecular weight compounds (Bykov et al., Nat. Med., 8, 2002). Further development and optimization of PRIMA-1 led to the discovery of the structural analog APR-246 (PRIMA-1MET), with improved drug like and preclinical characteristics. In in vitro experiments APR-246 reduced cell viability (WST-1 assay) in a large number of human cancer cell lines with various p53 status, including several leukemia (CCRF-CEM, CEM/VM-1, KBM3), lymphoma (U-937 GTP, U-937-vcr), and myeloma (RPMI 8226/S, 8226/dox40, 8226/LR5) cell lines, as well as many solid cancer cell lines, including osteosarcoma (SaOS-2, SaOS-2-His273,U-2OS), prostate (PC3, PC3-His175, 22Rv1), breast (BT474, MCF-7, MDA-MB-231), lung (H1299, H1299-His175) and colon cancer (HT-29). In human osteosarcoma cell lines APR-246 reduced cell viability and induced apoptosis (FLICA caspase assay) in a concentration dependent manner being more potent in the p53 mutant (SaOS-2-His273) than in the parental p53 null (SaOS-2) cells. The IC50 values (WST-1 assay) were 14 ± 3 and 27 ± 5 μM, respectively (n=35). In in vivo subcutaneous xenograft studies in SCID (severe combined immunodeficiency) mice APR-246 reduced growth of p53 mutant SaOS-2-His273 cells in a dose-dependent manner, when injected i.v. twice daily with 20 -100 mg/kg (64 – 76% inhibition). An in vivo anticancer effect of APR-246 was also observed in hollow-fiber test with NMRI mice using the acute myeloid leukemia (AML) cell line MV-4-11. An ex vivo cytotoxic effect of APR-246 and/or its lead compound PRIMA-1 has also been shown in primary cells from AML and CLL (chronic lymphocytic leukemia) patients, harbouring both hemizygously deleted p53 as well as normal karyotype (Nahi et al., Br. J. Haematol., 127, 2004; Nahi et al., Br. J. Haematol., 132, 2005; Jonsson-Videsater et al., abstract at this meeting). APR-246 was also tested in a FMCA (fluorometric microculture assay) test using normal healthy lymphocytes (PBMC) and cancer lymphocytes (CLL). It was 4-8 fold more potent in killing cancer cells than normal cells, indicating a favorable therapeutic index. This is in contrast to conventional cytostatics that often show negative ratio in this test. Furthermore, when tested in a well-defined panel of 10 human cancer cell lines consisting of both hematological and solid cancer cell lines, the cytotoxicity profile/activity pattern of APR-246 differed from common chemotherapeutic drugs (correlation coefficient less than 0.4), suggesting a different mechanism of action. Conclusion In relevant in vitro, in vivo and ex vivo cancer models, APR-246 showed unique pharmacological properties in comparison with conventional cytostatics, by being effective also in cancer cells with p53 mutations and by demonstrating tumor specificity. Moreover, in experimental safety/toxicology models required to start clinical trials, APR-246 was non toxic at the predicted therapeutic plasma concentrations. Thus, APR-246 appears to be a promising novel anticancer compound that may specifically target cancer cells in patients with genetic abnormality associated with poor prognosis. Disclosures: Mohell: Aprea AB: Employment. Liljebris:Aprea AB: Employment. Alfredsson:Aprea AB: Employment. Lindman:Aprea AB: Employment. Uustalu:Aprea AB: Employment. Wiman:Aprea AB: Co-founder, shareholder, and member of the board. Uhlin:Aprea AB: Employment.

Targeting Mir-548m-HDAC6 Axis Circumvents Stroma- Mediated Drug Resistance (CAM-DR) and Clonogenicity in Non-Hodgkin B-Cell Lymphomas

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5094-5094
Author(s):  
Tint Lwin ◽  
Xiaohong Zhao ◽  
Fengdong Cheng ◽  
Xinwei Zhang ◽  
Yizhuo Zhang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Lymph Node ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoma Cell ◽  
Histone Deacetylase 6 ◽  
B Cell Lymphomas ◽  
Non Hodgkin Lymphoma

Abstract Abstract 5094 A dynamic interaction occurs between the lymphoma cell and its microenvironment, with each profoundly influencing the behavior of the other. Here, we demonstrate that adhesion of mantle cell lymphoma (MCL) and other non-Hodgkin lymphoma cells to lymph node stromal cells, follicular dendritic cells (FDCs), confers drug resistance, enhances lymphoma cell clonogenicity, and is associated with induction of histone deacetylase 6 (HDAC6). Furthermore, stroma down-regulated miR-548m contributes to HDAC6 up-regulation, and HDAC6 is a key determinant for FDC-mediated lymphoma cell survival and colony formation. We further showed that stroma-mediated drug resistance and clonogenic growth are reversed by enforced expression of miR-548m and inhibition of HDAC6. The HDAC6-selective inhibitor tubastatin significantly enhances cell death, abolishes cell adhesion-mediated drug resistance, and suppresses clonogenicity and lymphoma growth suppression ex vivo and in vivo. Together, these data suggest that the lymphoma–stroma interaction in lymph node microenvironment directly impacts the biology of lymphoma through epigenetic regulation of miRNA and HDAC, with HDAC6 as a potential therapeutic target to overcome drug resistance in MCL and other B-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 5011-5015 ◽  
Author(s):  
Arghya Ray ◽  
Sandra W. Cowan-Jacob ◽  
Paul W. Manley ◽  
Jürgen Mestan ◽  
James D. Griffin
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Single Point ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Multiple Point ◽  
Resistance Mutations ◽  
Mutagenesis Study

Abstract Patients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 μM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate–resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

Transient in vivo selection of transduced peripheral blood cells using antifolate drug selection in rhesus macaques that received transplants with hematopoietic stem cells expressing dihydrofolate reductase vectors

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 796-803 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Aylin Bonifacino ◽  
Taihe Lu ◽  
Brian Agricola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Drug Resistance ◽  
Drug Treatment ◽  
Blood Cells ◽  
Rhesus Macaques ◽  
Peripheral Blood Cells ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Selection Of

Abstract One of the main obstacles for effective human gene therapy for hematopoietic disorders remains the achievement of an adequate number of genetically corrected blood cells. One approach to this goal is to incorporate drug resistance genes into vectors to enable in vivo selection of hematopoietic stem cells (HSCs). Although a number of drug resistance vectors enable HSC selection in murine systems, little is known about these systems in large animal models. To address this issue, we transplanted cells transduced with dihydrofolate resistance vectors into 6 rhesus macaques and studied whether selection of vector-expressing cells occurred following drug treatment with trimetrexate and nitrobenzylmercaptopurineriboside-phosphate. In some of the 10 administered drug treatment courses, substantial increases in the levels of transduced peripheral blood cells were noted; however, numbers returned to baseline levels within 17 days. Attempts to induce stem cell cycling with stem cell factor and granulocyte-colony stimulating factor prior to drug treatment did not lead to sustained enrichment for transduced cells. These data highlight an important species-specific difference between murine and nonhuman primate models for assessing in vivo HSC selection strategies and emphasize the importance of using drugs capable of inducing selective pressure at the level of HSCs.

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