Reversibility of the Resistance to Lenalidomide and Pomalidomide and Absence of Cross-Resistance in a Murine Model of MM

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 134-134 ◽  
Author(s):  
Enrique M Ocio ◽  
Diego Fernández-Lázaro ◽  
Laura San-Segundo ◽  
Lorena González-Méndez ◽  
Montserrat Martín-Sánchez ◽  
...  
Keyword(s):  
Research Funding ◽  
Tumor Volume ◽  
Ex Vivo ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Continuous Treatment ◽  
Cross Resistance ◽  
Initial Growth ◽  
To Receive

Abstract Abstract 134 Introduction: The introduction of novel agents has improved the outcome of MM patients, but MM is still considered an incurable disease and the emergence of resistance is the main responsible for this situation. The unraveling of the resistance mechanisms would help to design novel therapeutic strategies (combinations or sequencing treatments) to overcome this problem. Material and Methods: We have developed an in vivo model of acquired resistance to antimyeloma agents based in a model of subcutaneous plasmocytoma (MM1S) in CB17-SCID mice. For this purpose, mice were treated with Lenalidomide (25 mg/Kg) + Dex (1 mg/Kg) (LD), Pomalidomide (5 mg/Kg) + Dex (PD) or vehicle control, and after a period of initial sensitivity, tumors developed resistance to the administered combination. At this moment selected sensitive and resistant tumors were excised to analyze apoptosis, signaling pathways and gene expression profiling (GEP) changes. Moreover, some of these mice bearing resistant tumors were switched to receive the alternative combination (that is LD-PD or PD- LD), and, in selected mice, a second change of treatment was performed after secondary resistance to receive again the initial treatment (LD-PD-LD or PD-LD-PD). In order to evaluate TTP and to define the moment to change treatment we considered progression when tumor volume reached 1.700 mm3. Results: Both combinations (LD & PD) controlled the initial growth of the tumors, with a higher potency for the PD combination. Tumor volume reached 500 mm3 at a median of 8, 42 & 53 days for control, LD and PD respectively (p=0.01 and p=0.001). Nevertheless, after 30 days of continuous treatment, and despite maintaining the administration of the drugs, tumors started growing, and, once the tumors had reached 500 mm3, their growth kinetic was similar for the treated mice (despite still being treated) as compared to the untreated mice, indicating the emergence of complete resistance. This resistance was also confirmed ex vivo by in vitro culture with the corresponding drugs. In order to test the presence of cross-resistance, mice bearing big tumors resistant to LD or PD and already growing in an exponential phase (volume of 1.700 mm3), were at this point treated with the alternative combination. This sequential treatment change induced tumor stabilization and even a decrease of tumor volume. Again PD was significantly more potent at overcoming LD resistance as compared to the alternative situation, and this was verified both in terms of tumor growth inhibition (p=0.005) and in terms of time to progression (median of 16 vs 27 days for LD and PD respectively. p=0.004). Furthermore, mice that had been treated with LD-PD or PD-LD and had developed resistance to both combinations were again treated with the initial combination, and, surprisingly, they were again sensitive, indicating the reversibility of the acquired resistance. Similarly to previous experiments, PD was again significantly more potent than LD. This reversibility was also confirmed ex vivo after culture of the cells in medium without drugs. In order to investigate the resistance mechanisms, cells extracted from sensitive and resistant tumors were analyzed by Western Blot. Treatment with LD and PD induced a downregulation of pERK 1/2, nevertheless, when these cells developed resistance a very significant increase of pERK 1/2 levels (even higher than the basal levels) was observed. Moreover, these resistant cells also showed an upregulation of p-MEK, p-RAF and RAS. In this same line, the MEK inhibitor PD-98059 potentiated the in vitro activity of LD or PD in fresh MM1S cells, with high synergism (CI<0.1). Finally, changes in GEP were evaluated in extracted tumors. A significant change was observed in cells from tumors sensitive to PD and LD (with a higher gene deregulation with the former combination), as compared to untreated ones. Interestingly, when tumors became resistant, most of these changes disappeared and the GEP partially returned to a profile similar to that of active untreated tumors. Conclusion: The data presented would support treatment with alternative IMIDs if resistance was developed to one of them (specially PD was significantly more potent at rescuing resistance to LD), or even the retreatment with the same IMID after a wash up period. Moreover this study supports the evaluation of combinations of IMIDs with agents that abrogate the ERK pathway in order to increase efficacy or avoid resistance. Disclosures: Quintana: Celgene: Employment. García:Celgene: Employment. Pandiella:Celgene: Research Funding. San Miguel:Celgene: Consultancy, Research Funding.

scholarly journals Trial in Progress: Glad-AML - a Randomized, Phase 2 Trial of Glasdegib with Two Standard Decitabine Regimens for Older Patients with Newly-Diagnosed, Poor-Risk Acute Myeloid Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 29-29
Author(s):  
Rory M. Shallis ◽  
Nikolai A. Podoltsev ◽  
Thomas Prebet ◽  
Amer M. Zeidan
Keyword(s):  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Intensive Therapy ◽  
Phase 2 ◽  
Newly Diagnosed ◽  
Poor Risk ◽  
To Receive ◽  
Randomized Phase 2

Background: Acute myeloid leukemia (AML) blasts often highly-express IHH, GLI1, GLI2, and other elements of the Hedgehog (HH) pathway, which induces downstream pro-survival gene expression. HH pathway overactivation in AML blasts is associated with chemoresistance in vitro and ex vivo and has associated with worse survival in post-hoc clinical analyses. Glasdegib is an oral antagonist of the HH pathway activating element Smoothened (SMO) that is able to stall cell-cycle progression of AML leukemic stem cells in vitro and ex vivo. Glasdegib in combinations with low-dose cytarabine (LDAC) is a recent FDA-approved option for intensive therapy-ineligible patients (pts). However, LDAC is not often used in the United States and may not be the optimal glasdegib partner, which may be a hypomethylating agent (HMA) like azacitidine (AZA). We previously-presented the favorable clinical trial data for the combination of glasdegib and AZA in older and mostly ELN poor-risk disease pts (NCT02367456). The HMAs AZA and decitabine (DEC) are related, but not equivalent. Many hematologists prefer DEC for different reasons including experience, shorter therapy duration when given over 5 days (DEC5), and insurance coverage. Ten-day decitabine (DEC10) has shown promising activity in pts with AML, specifically TP53-mutated disease. Based on promising early data for glasdegib in combination with DEC5, the possibility of favorable responses with DEC10 in pts with poor-risk AML, and the experience that some intensive therapy-ineligible pts cannot accept the risks of myelosuppression and toxicity with prolonged venetoclax (VEN) administration, we proposed a study of glasdegib in combination with either DEC5 or DEC10 for pts ineligible for or not willing to proceed with intensive therapy or HMA-VEN. Study Design and Methods: GLAD-AML is a multi-center, randomized phase 2 study (NCT03798678) designed to evaluate the efficacy of glasdegib in combination with either DEC5 or DEC10 in pts with newly-diagnosed poor-risk AML who either refuse or are ineligible for intensive therapy. Key inclusion criteria are a morphologically-confirmed diagnosis of AML according to WHO 2016 classification with poor-risk disease as defined by the cytogenetic or molecular abnormalities (excluding FLT3-mutated AML). Key exclusion criteria are prior therapy for myeloid malignancy as well as candidacy and willingness to receive intensive therapy. Pts are randomized 1:1 to receive wither DEC5 or DEC10 in combination with glasdegib, which is administered at the starting dose of 100 mg orally once daily and continuously on 28-day cycles (Figure 1). A Simon two-stage design will be employed. Assuming a historical CR rate of 17% for DEC5 based on the DACO-16 trial, a minimum acceptable CR rate of 25% (given the possible additional toxicity from glasdegib) and a predicted minimum CR rate of 47% (the alternative hypothesis) for pts accrued to either arm, a minimum of 23 evaluable pts per arm will be accrued to test for a statistically-significant difference with an 80% power and a 90% confidence interval (a type 1 error rate, alpha one-sided of 0.05). The primary objective of this study is to evaluate the toxicity and safety profiles of glasdegib/DAC5 and glasdegib/DAC10. Other secondary objectives include determining EFS, RFS, OS, duration of response and mutation clearance. Intended exploratory endpoints include the measurement and detection of any changes in the amount of bone marrow aspirate mRNA expression or protein quantification of the Hh pathway-related proteins SMO, GLI1, and GLI2 via mRNA isolation with reverse transcription-polymerase chain reaction and monoclonal antibody-assisted immunohistochemistry, respectively. The proposed study will "pick the winner" among the two above-mentioned therapies as a step towards establishing a new, more effective standard-of-care for AML pts with poor-risk disease including therapy-related and TP53-mutated AML who are ineligible for intensive therapy and risk averse to VEN combination therapy. The trial is ongoing. The work was in part supported by the DeLuca Center for Innovation in Hematology Research at Yale Cancer Center and The Frederick A. Deluca Foundation. Disclosures Podoltsev: Incyte: Consultancy, Honoraria; Agios Pharmaceuticals: Consultancy, Honoraria; Blueprint Medicines: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; AI Therapeutics: Research Funding; Kartos Therapeutics: Research Funding; Alexion: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Research Funding; Astex Pharmaceuticals: Research Funding; Boehringer Ingelheim: Research Funding; Astellas Pharma: Research Funding; Daiichi Sankyo: Research Funding; Sunesis Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; CTI biopharma: Consultancy, Honoraria, Research Funding; Samus Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding. Prebet:Jazz Pharmaceuticals: Consultancy, Research Funding. Zeidan:Celgene / BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Taiho: Consultancy, Honoraria; ADC Therapeutics: Research Funding; Aprea: Research Funding; Astex: Research Funding; Daiichi Sankyo: Consultancy, Honoraria; Cardinal Health: Consultancy, Honoraria; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Seattle Genetics: Consultancy, Honoraria; MedImmune/Astrazeneca: Research Funding; BeyondSpring: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Ionis: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria; Acceleron: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Leukemia and Lymphoma Society: Other; CCITLA: Other; Cardiff Oncology: Consultancy, Honoraria, Other. OffLabel Disclosure: The abstract presents the rationale for the use of glasdegib, a Hedgehog pathway inhibitor, in combination with decitabine, a combination that is not FDA approved at present.

scholarly journals DDRE-07. DIPG HARBOUR ALTERATIONS TARGETABLE BY MEK INHIBITORS, WITH ACQUIRED RESISTANCE MECHANISMS OVERCOME BY COMBINATORIAL INHIBITION

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii62-ii62
Author(s):  
Elisa Izquierdo ◽  
Diana Carvalho ◽  
Alan Mackay ◽  
Sara Temelso ◽  
Jessica K R Boult ◽  
...  
Keyword(s):  
Drug Exposure ◽  
Mapk Pathway ◽  
Synergistic Effects ◽  
Acquired Resistance ◽  
Mek Inhibitor ◽  
Resistance Mechanisms ◽  
Genetic Alterations ◽  
Mesenchymal Transition

Abstract The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples, which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

scholarly journals Substantiation inEnterococcus faecalisof Dose-Dependent Resistance and Cross-Resistance to Pore-Forming Antimicrobial Peptides by Use of a Polydiacetylene-Based Colorimetric Assay

2010 ◽  
Vol 77 (3) ◽  
pp. 786-793 ◽  
Author(s):  
Jitender Mehla ◽  
S. K. Sood
Keyword(s):  
Colorimetric Assay ◽  
Physiological Activity ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Common Mechanism ◽  
Membrane Phospholipids ◽  
Mechanism Of Resistance ◽  
Colorimetric Response ◽  
Dose Dependent

ABSTRACTA better understanding of the antimicrobial peptide (AMP) resistance mechanisms of bacteria will facilitate the design of effective and potent AMPs. Therefore, to understand resistance mechanisms and forin vitroassessment, variants ofEnterococcus faecalisthat are resistant to different doses of the fungal AMP alamethicin (Almr) were selected and characterized. The resistance developed was dose dependent, as both doses of alamethicin and degrees of resistance were colinear. The formation of bacterial cell aggregates observed in resistant cells may be the prime mechanism of resistance because overall, a smaller cell surface in aggregated cells is exposed to AMPs. Increased rigidity of the membranes of Almrvariants, because of their altered fatty acids, was correlated with limited membrane penetration by alamethicin. Thus, resistance developed against alamethicin was an adaptation of the bacterial cells through changes in their morphological features and physiological activity and the composition of membrane phospholipids. The Almrvariants showed cross-resistance to pediocin, which indicated that resistance developed against both AMPs may share a mechanism, i.e., an alteration in the cell membrane. High percentages of colorimetric response by both AMPs against polydiacetylene/lipid biomimetic membranes of Almrvariants confirmed that altered phospholipid and fatty acid compositions were responsible for acquisition of resistance. So far, this is the only report of quantification of resistance and cross-resistance using anin vitrocolorimetric approach. Our results imply that a single AMP or AMP analog may be effective against bacterial strains having a common mechanism of resistance. Therefore, an understanding of resistance would contribute to the development of a single efficient, potent AMP against resistant strains that share a mechanism of resistance.

scholarly journals Detection of Multiple Recurrent Novel BCL2 Mutations Co-Occurring with BCL2 Gly101Val in Patients with Chronic Lymphocytic Leukemia on Long Term Venetoclax

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 171-171 ◽  
Author(s):  
Piers Blombery ◽  
Ella Thompson ◽  
Tamia Nguyen ◽  
Xiangting Chen ◽  
Michelle McBean ◽  
...  
Keyword(s):  
Research Funding ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Drug Binding ◽  
Cellular Systems ◽  
Clinical Relapse ◽  
Resistance Mutations ◽  
Recurrent Mutations ◽  
Eliza Hall Institute ◽  
Hall Institute

The BCL2 Gly101Val mutation may be acquired in patients with chronic lymphocytic leukaemia (CLL) treated with venetoclax (VEN), leading to reduced drug binding affinity and secondary resistance. In the majority of patients, the Gly101Val mutation is subclonal within the CLL compartment consistent with the presence of alternative resistance mechanisms in CLL cells not harboring the Gly101Val mutation. To date, two Gly101Val mutated patients have been identified with co-existing candidate resistance mechanisms in Gly101Val non-mutated cells; one with BCL-XL over-expression (Blombery et al, Cancer Discov., 2019) and another with a second subclonal candidate BCL2 resistance mutation - Asp103Tyr (Tausch et al, Haematologica 2019). Given the possibility of additional resistance mechanisms, we investigated patients with progressive CLL on VEN harboring the Gly101Val mutation for the presence of additional acquired resistance mutations in BCL2. Ten patients with progressive CLL on VEN with Gly101Val mutations were identified by sensitive allele-specific droplet digital PCR. To further assess for alternative BCL2 mutations in this cohort we performed ultra-deep amplicon-based next generation sequencing (NGS) (median depth ~50,000X) targeting BCL2. An amplicon variant caller (Canary) specifically designed for low level variant calling was used (Doig et al, BMC Bioinformatics, 2017). To achieve enhanced specificity we performed digital NGS with PCR error-correction using unique molecular indexes (UMI) (QiaSEQ Targeted DNA Panel). Given the high GC content of BCL2 we also used hybridization-based NGS using a custom targeted panel (Blombery et al, BJH 2017) combined with a sensitive unpaired variant caller (GATK4/Mutect2). In 7/10 (70%) patients, BCL2 mutations in addition to the Gly101Val were detected. Recurrent mutations (detected in more than one patient) were Asp103Tyr, Asp103Glu, Arg107_Arg110dup, and Val156Asp. All additional recurrent mutations were confirmed to be absent prior to commencing VEN (sensitivity 1% variant allele frequency[VAF]). Phase-analysis of NGS reads was consistent with the presence of the additional recurrent mutations on different alleles (and therefore cells, assuming heterozygosity) to both each other and to Gly101Val. Multiple addition recurrent mutations were observed in patients in the cohort with one patient harboring three recurrent mutations in addition to the Gly101Val (Asp103Tyr, Asp103Glu, Val156Asp). In multiple patients in the cohort, the VAF of non-Gly101Val mutations exceeded that of the Gly101Val mutation. Importantly, in all patients a significant (albeit variable) proportion of CLL cells were found to be BCL2 wild-type consistent with the presence of as yet unidentified resistance mechanisms unrelated to BCL2 mutations. In one patient, two additional non-recurrent mutations were observed (Ala113Gly and Arg129Leu) in addition to Gly101Val and Val156Asp. Again, all four mutations in this patient were observed to be in mutually exclusive NGS reads. Strikingly, all of the recurrent acquired BCL2 mutated residues identified in our cohort are situated in the BCL2 binding groove that binds VEN (Figure 1). The Asp103 codon in the P4 pocket is critical for VEN binding through hydrogen bonding between its sidechain and the azaindole moiety of VEN. The Asp103Glu mutation is noteworthy given that the equivalent residue to Asp103 in BCL-XL is a Glu, which reduces VEN binding to BCL-XL. The Val156 mutation situated at the base of the P2 pocket is close to the chlorophenyl moiety of VEN and a change to Asp in this position may disrupt VEN binding. Ongoing binding experiments and modeling in cellular systems will further elucidate the mechanism and contributions of these new recurrent mutations to VEN resistance. In summary, we have extended the landscape of acquired candidate resistance mutations occurring in patients treated with VEN to include four novel recurrent BCL2 mutations. Moreover, our data are consistent with the emerging observation of multiple acquired resistance mechanisms operating in different CLL cells in a single patient contributing to an "oligoclonal" pattern of clinical relapse on VEN therapy. Figure 1 - BCL2 protein structure surface bound to venetoclax (VEN) in orange. The Asp103Tyr, Asp103Glu and Val156Asp mutation sites are shown in red and Arg107_Arg110dup region in blue Disclosures Blombery: Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Seymour:Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; BeiGene: Research Funding.

scholarly journals The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer

2019 ◽  
Author(s):  
Seth A. Wander ◽  
Ofir Cohen ◽  
Xueqian Gong ◽  
Gabriela N. Johnson ◽  
Jorge Buendia-Buendia ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Acquired Resistance ◽  
Metastatic Breast ◽  
Resistance Mechanisms ◽  
Akt Activation ◽  
Hormone Receptor Positive ◽  
Ras Activation ◽  
Whole Exome ◽  
Clinical Resistance

AbstractClinical resistance mechanisms to CDK4/6 inhibitors in HR+ breast cancer have not been clearly defined. Whole exome sequencing of 59 tumors with CDK4/6i exposure revealed multiple candidate resistance mechanisms including RB1 loss, activating alterations in AKT1, RAS, AURKA, CCNE2, ERBB2, and FGFR2, and loss of ER expression. In vitro experiments confirmed that these alterations conferred CDK4/6i resistance. Cancer cells cultured to resistance with CDK4/6i also acquired RB1, KRAS, AURKA, or CCNE2 alterations, which conferred sensitivity to AURKA, ERK, or CHEK1 inhibition. Besides inactivation of RB1, which accounts for ∼5% of resistance, seven of these mechanisms have not been previously identified as clinical mediators of resistance to CDK4/6 inhibitors in patients. Three of these—RAS activation, AKT activation, and AURKA activation—have not to our knowledge been previously demonstrated preclinically. Together, these eight mechanisms were present in 80% of resistant tumors profiled and may define therapeutic opportunities in patients.SignificanceWe identified eight distinct mechanisms of resistance to CDK4/6 inhibitors present in 80% of resistant tumors profiled. Most of these have a therapeutic strategy to overcome or prevent resistance in these tumors. Taken together, these findings have critical implications related to the potential utility of precision-based approaches to overcome resistance in many patients with HR+ MBC.

scholarly journals Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis

Cells ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 2450
Author(s):  
Lucy Urwin ◽  
Katarzyna Okurowska ◽  
Grace Crowther ◽  
Sanhita Roy ◽  
Prashant Garg ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Bacterial Species ◽  
Resistance Mechanisms ◽  
Bacterial Keratitis ◽  
Corneal Surface ◽  
Corneal Infection ◽  
Infection Models

Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.

scholarly journals Nitazoxanide, a Potential Drug for Eradication ofHelicobacter pylori with No Cross-Resistance to Metronidazole

1998 ◽  
Vol 42 (11) ◽  
pp. 2836-2840 ◽  
Author(s):  
Francis Mégraud ◽  
Alessandra Occhialini ◽  
Jean François Rossignol
Keyword(s):  
Pilot Study ◽  
Dna Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Acquired Resistance ◽  
Anaerobic Bacteria ◽  
Antimicrobial Properties ◽  
Cross Resistance ◽  
Treatment Regimens ◽  
H Pylori

ABSTRACT Nitazoxanide, a thiazolide compound, and its desacetyl derivative, tizoxanide, have antimicrobial properties against anaerobic bacteria, as well as against helminths and protozoa. Because the treatment ofHelicobacter pylori infection may be jeopardized by metronidazole resistance, nitazoxanide and tizoxanide were tested in vitro against these bacteria. The MICs of these two compounds were determined by agar dilution and were compared to those of metronidazole. Exposure to subinhibitory concentrations of nitazoxanide was also carried out by the method of Szybalski (W. Szybalski and V. Bryson, J. Bacteriol. 64:489–499, 1952). The MICs of nitazoxanide and tizoxanide for 103 strains ranged from 0.25 to 8 μg/ml, with the MIC at which 50% of strains are inhibited (MIC50) being 1 μg/ml and the MIC90 being 4 μg/ml, and no resistant strain was detected, whereas strains resistant to metronidazole were detected. When 10 strains were successively subcultured on medium containing nitazoxanide, no significant change in the MICs of this compound was observed. A pilot study of nitazoxanide for the treatment of H. pyloriinfection was carried out with 86 patients in association with 20 mg of omeprazole. An eradication rate of 83% (95% confidence interval, 64% to 94%) was obtained in a per-protocol analysis in the group receiving 1 g of nitazoxanide orally twice daily, and a few side effects were observed. The failures could not be explained by the selection of resistant strains since the MICs of nitazoxanide were similar for six pairs of isolates (proven to be the same strain by random amplified polymorphic DNA analysis in four cases) cultured before and after the treatment failure. Nitazoxanide exhibits good antimicrobial activity against H. pylori without the problem of acquired resistance which is encountered with metronidazole and has been demonstrated to have a satisfactory effect in a dose-ranging pilot study. It is therefore a good candidate to be included in treatment regimens aimed at the eradication of H. pylori.

scholarly journals Dissecting Mechanisms of Melanoma Resistance to BRAF and MEK Inhibitors Revealed Genetic and Non-Genetic Patient- and Drug-Specific Alterations and Remarkable Phenotypic Plasticity

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 142 ◽  
Author(s):  
Mariusz L. Hartman ◽  
Malgorzata Sztiller-Sikorska ◽  
Anna Gajos-Michniewicz ◽  
Malgorzata Czyz
Keyword(s):  
Phenotypic Plasticity ◽  
Cell Lines ◽  
Mapk Pathway ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Genetic Alterations ◽  
Resistant Cell ◽  
Cross Resistance ◽  
Preclinical Model ◽  
Development Of Resistance

The clinical benefit of MAPK pathway inhibition in BRAF-mutant melanoma patients is limited by the development of acquired resistance. Using drug-naïve cell lines derived from tumor specimens, we established a preclinical model of melanoma resistance to vemurafenib or trametinib to provide insight into resistance mechanisms. Dissecting the mechanisms accompanying the development of resistance, we have shown that (i) most of genetic and non-genetic alterations are triggered in a cell line- and/or drug-specific manner; (ii) several changes previously assigned to the development of resistance are induced as the immediate response to the extent measurable at the bulk levels; (iii) reprogramming observed in cross-resistance experiments and growth factor-dependence restricted by the drug presence indicate that phenotypic plasticity of melanoma cells largely contributes to the sustained resistance. Whole-exome sequencing revealed novel genetic alterations, including a frameshift variant of RBMX found exclusively in phospho-AKThigh resistant cell lines. There was no similar pattern of phenotypic alterations among eleven resistant cell lines, including expression/activity of crucial regulators, such as MITF, AXL, SOX, and NGFR, which suggests that patient-to-patient variability is richer and more nuanced than previously described. This diversity should be considered during the development of new strategies to circumvent the acquired resistance to targeted therapies.

Cytokine-Mediated Signaling Is Suppressed in Myeloid Cells and Enhanced in Lymphatic Cells in Patients with Chronic Myeloid Leukemia (CML) — Partial Normalization with Tyrosine Kinase Inhibitors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2180-2180
Author(s):  
Sari Jalkanen ◽  
Satu Mustjoki ◽  
Kimmo Porkka ◽  
Jukka Vakkila
Keyword(s):  
T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Healthy Controls ◽  
Gm Csf ◽  
Single Cell Profiling ◽  
Treg Lymphocytes

Abstract Abstract 2180 Poster Board II-157 Introduction. Aberrant phosphorylation of the BCR-ABL1 tyrosine kinase (TK) is characteristic of chronic myeloid leukemia (CML). This oncoprotein interacts directly with intracellular signaling proteins, alters the responsiveness of cytokine receptors and regulates secretion of autocrine cytokines. Targeted inhibition of BCR-ABL1 with TK inhibitor (TKI) imatinib mesylate (IM) is the current standard treatment of CML. For overcoming IM resistance or intolerance, 2nd generation TKIs (nilotinib, dasatinib) with broader kinase inhibition profile have been approved for clinical use. Although in vitro results suggest that TKIs are immunosuppressive, no increases in opportunistic infections or secondary malignancies have been observed to date. In contrast, in some TKI-treated patients immunoactivation in the form of chronic lymphocytosis linked to excellent therapy responses has recently been shown. Dynamic monitoring of aberrant cytokine signaling pathways would aid in understanding and predicting the development of TKI-resistance or adverse/off-target effects. The aim of this study was to analyze the responsiveness of leukocytes to cytokine stimuli in CML patients at diagnosis and during TKI therapy using single-cell profiling of phosphoprotein networks by multiparameter flow cytometry. Patients and methods. The study consisted of 4 healthy controls, 6 CML patients at diagnosis, 6 IM patients and 5 dasatinib patients. Stimuli included GM-CSF, IL-2+IL-10+IFNα and IL-4+IL-6+IFNγ and they were added immeadately to freshly drawn whole blood ex vivo. The readout phosphoproteins were pERK1/2, pSTAT1, pSTAT3, pSTAT5a and pSTAT6 (with isotype controls), and were analyzed separately from granulocytes, monocytes, CD4+ CD25neg T helper cells (Th), CD4neg lymphocytes and CD4+CD25+ T cells including regulatory T-cells (Treg). Analysis was performed with heatmap function of Cytobank software (http://cytobank.stanford.edu/public/). Results. Unstimulated phosphoprotein levels reflecting the activation state of leukocytes in vivo did not differ between healthy controls and CML patients at diagnosis or during dasatinib therapy. Strikingly, in IM patients, baseline levels of pSTAT3 were relatively high indicating in vivo occurring activation of leukocytes in this patient group. We next studied ex vivo responsiveness of immune effector cells with cytokines and found clear differences between healthy controls and CML patients. At CML diagnosis. GM-CSF/pERK1+pSTAT5a, IFNa/pSTAT1,and IL-4/pSTAT6 (stimulus/readout) as well as pSTAT3 responses with all stimuli were suppressed in monocytes. In granulocytes, GM-CSF/pSTAT1 levels were diminished. In Th and Treg lymphocytes, IL-6/pSTAT3 responses were markedly pronounced, while IL-10/pSTAT3 responses were not affected when compared to healthy controls. Such difference was not observed in CD4neg lymphocytes. During TKI therapy. Most patients (9/11) were in cytogenetic remission at the time of analysis. The unresponsiveness of myeloid cells at diagnosis was restored by IM or dasatinib therapy in most, but not all patients. Similarly, in Th and Treg lymphocytes TKI-therapy normalized the enhanced IL-6/pSTAT3 responses that were evident at diagnosis. However, in Th and Treg cells pSTAT3 responses provoked by IL-10 were particularly prominent. Interestingly, one dasatinib patient with aberrant constant blood NK-lymphocytosis and monocytosis had uniquely strong IFNg/pSTAT1 and IL-4/pSTAT6 responses in monocytes. Furthermore, one patient who have stayed in persistent remission after IM discontinuation had exceptionally high pSTAT3 responses with all of stimuli used. Similar kind of signaling profile was unseen with the other patients and could reflect immunoactivation related to leukemia control. Conclusions. Dynamic single-cell profiling of signaling networks is feasible in CML patients and can be used to study mechanisms of aberrant immune reactivity in TKI-treated patients. The method could be particularly suitable for assessing candidate patients for TKI discontinuation. Although in vitro results suggest immunosuppressive effects of TKIs on lymphocytes, leukocytes ex vivo from patients were able to respond similarly to cytokine stimuli as in healthy controls. Disclosures: Mustjoki: BMS: Honoraria. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 580-580
Author(s):  
Mark Wunderlich ◽  
Mahesh Shrestha ◽  
Lin Kang ◽  
Eric Law ◽  
Vladimir Jankovic ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Cell Product ◽  
Equity Ownership ◽  
Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document