Development Of Clinical Resistance Research Articles

Human visceral leishmaniasis and polymorphisms in interleukin-coding genes: a systematic review.

Visceral leishmaniasis (VL) is a neglected disease that is typical of tropical and subtropical parts of the world and is caused by the trypanosomatid Leishmania donovani complex. This disease is a multifactorial condition that involves parasitic, environmental, and immunogenetic characteristics. Genetic changes in genes encoding cytokines may be associated with changes in their expression and, consequently, with the development of clinical resistance or susceptibility to the disease. This systematic review and meta-analysis aimed to assess whether single nucleotide polymorphisms (SNPs) in interleukin genes influence the clinical consequences of visceral leishmaniasis infection. To this end, we carried out a systematic review and meta-analysis with structured searches in the EMBASE, PubMed, Scopus, SciELO, and Web of Science databases without time restrictions. Two independent reviewers examined the studies, performed data extraction, and assessed quality by assigning scores. If there were any discrepancies, a third reviewer with more experience was consulted. After the screening process, 28 articles were included in the systematic review and 9 in the final analysis of the meta-analysis. Statistical analyses were carried out using various genetic models. The odds ratio (OR) and corresponding 95% confidence intervals (CIs) were calculated to estimate the associations. Overall, the main clinical outcomes were classified as not associated or associated when they presented susceptibility, resistance, risk, or protective factors for the development of the disease. Associations between IFN-γ +874T/A polymorphisms in the dominant model (OR 1.64, 95% CI 1.13-2.38, I2 = 0%, p < 0.01) and heterozygous model (OR 1.72, 95% CI 1.15-2.57, I2 = 0%, p < 0.01) and IL-18 -137G/C in the recessive model (OR 1.33, 95% CI 1.02-1.71, I2 = 9%, p = 0.03) and VL were observed. For the IL-10 gene SNPs, there was no significant association. Our findings suggest that SNPs in the IFN-γ and IL-18 genes may be associated with the risk of developing VL.

Novel Agents in Chronic Lymphocytic Leukemia: New Combination Therapies and Strategies to Overcome Resistance.

Simple SummaryNowadays, many patients with chronic lymphocytic leukemia (CLL) are treated with so-called novel agents, including BTK inhibitors, Bcl-2 inhibitors and PI3K inhibitors. As CLL is a chronic disease, most patients will relapse on or after treatment with these drugs and various mechanisms behind this resistance to novel agents have been described. In this review, we present the current evidence on resistance to novel agents, discuss approaches to prevent its development and provide guidance on the treatment of patients who have already acquired resistance.The approval of Bruton’s tyrosine kinase (BTK) inhibitors such as ibrutinib and acalabrutinib and the Bcl-2 inhibitor venetoclax have revolutionized the treatment of chronic lymphocytic leukemia (CLL). While these novel agents alone or in combination induce long lasting and deep remissions in most patients with CLL, their use may be associated with the development of clinical resistance. In this review, we elucidate the genetic basis of acquired resistance to BTK and Bcl-2 inhibition and present evidence on resistance mechanisms that are not linked to single genomic alterations affecting these target proteins. Strategies to prevent resistance to novel agents are discussed in this review with a special focus on new combination therapies.

Mutant RNA polymerase can reduce susceptibility to antibiotics via ppGpp-independent induction of a stringent-like response.

BackgroundMutations in RNA polymerase (RNAP) can reduce susceptibility to ciprofloxacin in Escherichia coli, but the mechanism of transcriptional reprogramming responsible is unknown. Strains carrying ciprofloxacin-resistant (CipR) rpoB mutations have reduced growth fitness and their impact on clinical resistance development is unclear.ObjectivesTo assess the potential for CipRrpoB mutations to contribute to resistance development by estimating the number of distinct alleles. To identify fitness-compensatory mutations that ameliorate the fitness costs of CipRrpoB mutations. To understand how CipRrpoB mutations reprogramme RNAP.Methods E. coli strains carrying five different CipRrpoB alleles were evolved with selection for improved fitness and characterized for acquired mutations, relative fitness and MICCip. The effects of dksA mutations and a ppGpp0 background on growth and susceptibility phenotypes associated with CipRrpoB alleles were determined.ResultsThe number of distinct CipRrpoB mutations was estimated to be >100. Mutations in RNAP genes and in dksA can compensate for the fitness cost of CipRrpoB mutations. Deletion of dksA reduced the MICCip for strains carrying CipRrpoB alleles. A ppGpp0 phenotype had no effect on drug susceptibility.ConclusionsCipRrpoB mutations induce an ppGpp-independent stringent-like response. Approximately half of the reduction in ciprofloxacin susceptibility is caused by an increased affinity of RNAP to DksA while the other half is independent of DksA. Stringent-like response activating mutations might be the most diverse class of mutations reducing susceptibility to antibiotics.

Abstract P5-04-14: Tracking ESR1 mutation clonal evolution in breast cancer using in situ mutation detection

Abstract Background: Approximately 70% of breast cancers (BCs) are estrogen receptor positive (ER+). Not all ER+ cancers respond to endocrine therapy (ET) and many eventually develop acquired resistance. Next-generation sequencing (NGS) has shown ESR1 mutations (ESRMs) are present in 10-50% of recurrent/metastatic cancers treated with aromatase inhibitors (AIs). Many of these mutations are located in the ligand-binding domain of ER, so they can lead to constitutive activation. This suggests ESRMs are a major mechanism of acquired resistance to endocrine therapy (ET) and numerous studies have shown a link between ESRMs and reduced sensitivity to 2nd line ET. The aim of this project was to investigate the incidence and clonal evolution of common ESRMs in BCs resistant to multiple sequential ETs using NGS, as well as novel PCR and in situ mutation detection methods. Methods: We have optimised an allele-specific real-time PCR (rtPCR) assay and an in situ mutation detection method (ER-ISMD) for the assessment of ESRMs. Both have been designed to identify a missense gain-of-function D538G mutation with a single nucleotide-resolution in formalin-fixed paraffin-embedded (FFPE) BC tissues. Two unique cohorts have been studied: (A) 20 post-menopausal women (PMW) with ER+ BC who acquired resistance to letrozole and were treated with up to 4 subsequent lines of ET. Serial RNA and DNA from 3-5 cancer samples per patient (58 samples from 20 patients) were analysed by Ribo0-RNAseq, DNA exome sequencing, rtPCR and ER-ISMD. (B) 150 PMW with ER+ BC who developed local (n=79), lymph node (n=59) or distant (n=12) recurrences on 1st line adjuvant letrozole, anastrozole or tamoxifen. Of these, 48 patients developed subsequent recurrences on 2nd line ET. Tissue samples from each recurrence and matched primary BC were collected. Results: In cohort A, 5/20 patients (20%) had expansion of a D538G ESR1 mutation clone at time of resistance 1st line ET (3:letrozole, 1:anastrozole, 1:tamoxifen). The mutant allele frequency (MAF) increased further in the 4 BCs treated with 2nd line ET (2:tamoxifen, 2:exemestane) and further still in the 1 BC who received 3rd line exemestane. 0/6 patients with ESRM responded to subsequent ET. Allele-specific rtPCR and ER-ISMD have been used to validate these findings and also identified low frequency ESRM clones in the sequential samples prior to the development of clinical resistance, that were not reported by NGS. Both methods have also been applied to screen tissues from patients in cohort B, where ESRMs have also been identified in recurrent samples. Complete analysis is currently ongoing. Conclusions: · ESRMs develop and expand in some BCs as a mechanism for acquired resistance to ET and are associated with a lack of response to subsequent standard ETs. · Allele-specific rtPCR can detect ESRMs and is more cost-effective and easier to use than NGS for ER mutation analysis. · Some ESRMs predate clinical resistance. · ER-ISMD is a novel approach that allows for identification and visualisation of the distribution of mutant clones in morphologically intact FFPE tissue. · ER-ISMD has the potential to become a clinically useful tool to help direct the use of 2nd line ET in routine care. Citation Format: Martinez-Perez C, Turnbull AK, Tanioka M, Fernando A, Renshaw L, Keys J, Wheless A, Garrett A, Parker J, He X, Sims AH, Carey LA, Perou CM, Dixon JM. Tracking ESR1 mutation clonal evolution in breast cancer using in situ mutation detection [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-14.

Abstract P3-03-03: An acquired HER2 T798I gatekeeper mutation induces resistance to neratinib in a patient with HER2 mutant-driven breast cancer

Abstract ERBB2, the gene encoding HER2, is mutated in 2-4% of breast cancers. The HER2 irreversible tyrosine kinase inhibitor (TKI) neratinib has shown clinical activity against breast cancer cells harboring HER2 activating mutations. Here, we report for the first time an acquired gatekeeper HER2T798I mutation in a patient with HER2-mutant breast cancer after an initial exceptional response to neratinib. A patient with ER+/PR+/HER2-negative invasive lobular breast cancer progressing on standard therapy was found to harbor a L869R kinase domain mutation in HER2. HER2L869R is homologous to the known activating mutation EGFRL861R/Q. MCF10A breast epithelial cells expressing HER2L869R displayed enhanced HER2-mediated signaling and were resistant to lapatinib and trastuzumab but sensitive to neratinib. The patient was enrolled in the phase II SUMMIT trial (NCT01953926) and treated with neratinib, achieving a partial response lasting 16 months before developing progression. Next gen sequencing of DNA from both a new skin metastasis and plasma cell-free DNA (cfDNA) identified HER2L869R (8.7% cfDNA), whereas a novel HER2T798I mutation was detected only in plasma at 1.3%. Deep sequencing of pre-therapy tumor tissue and plasma did not detect HER2T798I, suggesting that this mutation arose upon resistance. HER2T798I has not been reported in TCGA, COSMIC, or among plasma samples from 17,345 cancer patients subjected to digital DNA sequencing using the Guardant360 assay. HER2T798I is homologous to the EGFRT790M, KITT670I and BCR-ABLT315I gatekeeper mutations known to mediate resistance to erlotinib/gefitinib and imatinib. To examine if HER2T798I mediates resistance to neratinib, we employed biochemical and biological assays and molecular modeling of wild-type (WT) HER2 and HER2T798I. Structural modeling showed the increased bulk of the isoleucine at position 798 would result in a steric clash with neratinib, thus reducing drug binding. We stably expressed HER2WT, HER2T798I, HER2L869R and HER2L869R/T798I in MCF10A cells and NR6 mouse fibroblasts. Neratinib (10-100 nM) blocked HER2-mediated signaling in cells expressing HER2WT or HER2L869R but did not in cells expressing HER2T798I. The EGFR irreversible TKI osimertinib (100 nM), which isselective for mutant EGFR (including EGFRT790M) and approved for treatment of NSCLC expressing EGFRT790M, failed to inhibit HER2WT, HER2L869R or HER2T798I. In contrast, either the EGFR/HER2 irreversible TKI afatinib or AZ5104, a metabolite of osimertinib, strongly blocked signaling induced by HER2WT, HER2L869R or HER2T798I. Cells expressing HER2T798M displayed a significantly higher IC50 to neratinib than cells expressing HER2WT, whereas afatinib or AZ5014 were very active against all cells (IC50&amp;lt;10 nM). Conclusions: The acquisition of a T798I gatekeeper mutation in HER2 upon development of clinical resistance to neratinib in a breast cancer with an initial activating mutation in HER2 strongly suggests that HER2L869R is a driver mutation. We speculate that HER2T798I may arise as a secondary mutation following response to effective HER2 TKIs in other cancers with HER2 activating mutations. Certain irreversible EGFR inhibitors may be effective in patients with HER2-driven breast cancer resistant to neratinib. Citation Format: Hanker AB, Red Brewer M, Sheehan JH, Koch JP, Lanman R, Hyman DM, Cutler, Jr. RE, Lalani AS, Cross D, Lovly CM, Meiler J, Arteaga CL. An acquired HER2 T798I gatekeeper mutation induces resistance to neratinib in a patient with HER2 mutant-driven breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-03.

Biocompatible silver nanoparticles reduced from Anethum graveolens leaf extract augments the antileishmanial efficacy of miltefosine

Despite the existence of chemotherapy, there is no effective cure for leishmaniasis. In the light of recommended therapeutic regimen is attributed for toxicity and development of clinical resistance, exploration of an efficient method of drug delivery could be one of the option in reducing the dosage and toxicity of drugs. This work is aimed in such fashion to study the enhanced antileishmanial activity of miltefosine with silver-nanoparticles (AgNPs) synthesized by using Anethum graveolens (dill) leaf extract as reducing agent. AgNPs were synthesized in a single step process and characterized by UV-visible, X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FTIR) to understand the crystal structure and functional groups on their surface. TEM analysis showed that the synthesized AgNPs are of an average size of 35 nm. By performing MTT assay, we found that, AgNPs (between 20 and 100 μM) are biocompatible in nature through pertaining >80% viability of macrophages. Furthermore, AgNPs alone (50 μM) have not shown antileishmanial effect on promastigote stage of Leishmania parasite but in combination with miltefosine (12.5 μM and 25 μM), it magnifies the leishmanicidal effect of miltefosine by ∼2-folds (i.e. AgNPs cut down the IC50 of miltefosine about to half). Scanning electron microscopic (SEM) observation for morphological aberration and genomic DNA fragmentation in promastigotes confirmed the enhanced effect of meltefosine in combination with AgNPs (50 μM AgNPs plus 12.5 μM miltefosine). Similarly, this combination has likely shown a slight augmentation (p = 0.057) of miltefosine (2.5 μM) leishmanicidal efficacy on amastigote stage of the parasite in infected human macrophages by reducing their intracellular growth.

Evidencia de afatinib en pacientes que progresan a un tratamiento de primera línea

After description of the importance of EGFR mutations in non-small cell lung cancer and confirmation that tyrosine-kinase inhibitors are more beneficial than chemotherapy in patients with EGFR+ tumours, treatment with one of these drugs has become the standard recommendation. Despite this advance, patients continue to progress and consequently there is a need to search for alternative treatments.Some studies have analysed afatinib activity after first-generation TKI therapy, as well as its administration in combination with conventional chemotherapy. Afatinib produces significant response rates and progression-free survival times after the development of clinical resistance, which are independent of the presence of the T790M resistance mutation and can be attributed to continued pan-HER inhibition. In addition to the initial clinical trial, LUX-LUNG-1, data are available from the use of afatinib in routine clinical practice, within extended use programs. Overall, response rates of between 7 and 15% can be expected with a duration of approximately 24 months and a median progression-free time of about 4 months. A study combining afatinib with cetuximab has obtained a high response rate.Afatinib toxicity in second-line treatment is similar to that appears when the drug is used as first-line therapy (mainly mucocutaneous and diarrhoea) and can be managed with routine measures.In conclusion, afatinib should be considered as a treatment option in patients with EGFR mutations who show disease progression after a first tyrosine-kinase inhibitor.

Abstract P3-05-07: RAD1901, a novel oral, selective estrogen receptor degrader (SERD) with single agent efficacy in an ER+ primary patent derived ESR1 mutant xenograft model

Abstract Despite advances in the treatment of metastatic breast cancer, many women eventually relapse with more aggressive forms of endocrine-resistant disease. Mutations in the ESR1 gene encoding the estrogen receptor (ER) have recently emerged as a potential mechanism for the development of clinical resistance to conventional anti-estrogen therapies, such as fulvestrant. To overcome some of the pharmacokinetic limitations and intramuscular administration challenges associated with fulvestrant endocrine therapy and to combat the development of resistance, there is a significant need for the development of more durable and more effective ER-targeted therapies. Here, we begin to describe and characterize the preclinical efficacy of RAD1901, a novel, orally bioavailable small-molecule SERD, with significant therapeutic potential for treatment of breast cancer. RAD1901 selectively binds to and degrades the ER and is a potent antagonist of ER-positive breast cancer cell proliferation. Importantly, RAD1901 demonstrated profound tumor growth inhibition in MCF-7 xenograft models when compared to fulvestrant and tamoxifen. Importantly, RAD1901 also demonstrated marked single agent efficacy in a primary patient-derived xenograft (PDx) model harboring the ESR1 Y537S mutation indicating the utility of this SERD against clinically relevant ER mutants. Further biochemical binding studies and co-crystallization experiments of RAD1901 bound to the ER further confirms the ability of RAD1901 to bind to both mutant and wild type forms of the ER. RAD1901 is currently undergoing clinical testing in postmenopausal women with ER-positive advanced breast cancer. Citation Format: Garner F, Brown J, Katzenellenbogen J, Lyttle CR, Hattersley G. RAD1901, a novel oral, selective estrogen receptor degrader (SERD) with single agent efficacy in an ER+ primary patent derived ESR1 mutant xenograft model. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-05-07.

ABCB1 haplotypes but not individual SNPs predict for optimal response/failure in Egyptian patients with chronic-phase chronic myeloid leukemia receiving imatinib mesylate

Imatinib mesylate (IM) has so far been the standard of care for treating chronic myeloid leukemia (CML), but the initial striking efficacy of this drug has been overshadowed by the development of clinical resistance, which may in part be caused by pharmacogenetic variability. The ATP-binding cassette, subfamily B, member 1 (ABCB1) gene codes for P-glycoprotein (P-gp), a membrane-bound efflux transporter known to affect the pharmacokinetics of many drugs. IM is a substrate of the P-gp-mediated efflux. ABCB1 single nucleotide polymorphisms (SNPs) have been reported as modulators of ABCB1-mediated transport, affecting IM's bioavailability and consequently the treatment outcome of IM therapy. We aimed to examine the association between ABCB1 SNPs and the likelihood of achieving optimal response in IM-treated CML patients. Three ABCB1 SNPs (C1236T, G2677T, and C3435T) were genotyped in 100 Egyptian patients with CML undergoing IM therapy using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The optimal response rate did not differ significantly between C1236T, G2677T, or C3435T genotypes (P > 0.05). Optimal response rate was significantly different among patients with the CGC, TTT, TGC, CGT, TGT, CTC, CTT, and TTC haplotypes (P = 0.023). The 1236T-2677G-3435T haplotype was significantly associated with lower probability of achieving optimal response (P = 0.001). ABCB1 SNPs haplotype analysis should be taken into account in an attempt to get clearer insights into who is likely to respond optimally to IM for identifying CML patients who may not respond optimally to standard-dose IM therapy and potentially need an individualized therapeutic approach.

Nuclear Translocation of Heme Oxygenase-1 Confers Resistance to Imatinib in Chronic Myeloid Leukemia Cells

Identification of imatinib mesylate as a potent inhibitor of the Abl kinase and the subsequent findings that this compound displays growth inhibitory and pro-apoptotic effects in Bcr-Abl+ cells, has deeply conditioned CML treatment. Unfortunately the initial striking efficacy of this drug has been overshadowed by the development of clinical resistance. A wide variety of molecular mechanisms can underlie such resistance mechanisms. In the recent years, heme oxygenase-1 (HO-1) expression has been reported as an important protective endogenous mechanism against physical, chemical and biological stress and this cytoprotective role has already been demonstrated for several solid tumors and acute leukemias. The aim of the present study was to investigate the effect of HO-1 expression on cell proliferation and apoptosis in chronic myeloid leukemia cells, K562 and LAMA-84 cell lines following imatinib treatment. Cells were incubated for 24h with Imatinib (1 μM) alone or in combination with Hemin (10μM), an inducer of HO-1. In addition, cells were also treated with HO byproducts, bilirubin and carbon monoxide (CO), or with a protease inhibitor (Ed64) to inhibit HO-1 nuclear translocation. Pharmacological induction of HO-1 was able to overcome the effect of imatinib. The cytoprotective effect of HO-1 was further confirmed after silencing HO-1 by siRNA. Interestingly, neither bilirubin nor CO was able to protect cells from Imatinib-induced toxicity. By contrast, the protective effect of HO-1 was mitigated by the addition of E64d, preventing HO-1 nuclear translocation. Finally, imatinib was able to increase the formation of cellular reactive oxygen species (ROS) and this effect was reversed by HO-1 induction or the addition of N-acetylcisteine (NAC). In conclusion, the protective effect of HO-1 on imatinib-induced cytotoxicity does not involve its enzymatic byproducts, but rather the nuclear translocation of HO-1 following proteolytic cleavage.

Abstract PD01-02: Increased expression of phosporylated mTOR in metastatic breast tumors compared to primary tumors in patients who received adjuvant endocrine therapy

Abstract BACKGROUND: Activation of the PI3K pathway as an adaptive change in response to estrogen depletion results in acquired endocrine therapy resistance in vitro. Metastatic breast cancer patients with previous exposure to endocrine therapy do derive substantial benefit from the addition of an mTOR inhibitor to endocrine therapy compared to endocrine therapy alone. This suggests that compensatory PI3K pathway activation might play a role in the development of clinical resistance to endocrine therapy. We evaluated the activation of the downstream PI3K pathway protein mTOR in primary breast tumors and matched metastatic lesions. METHODS: From a previously described series of breast cancer patients from whom both primary tumor tissue as well as metastatic tumor tissue was collected, we selected estrogen receptor α (ERα) positive patients who had received endocrine therapy (N = 34) and who did not receive endocrine therapy (N = 37). The difference in expression of phosphorylated mTOR (p-mTOR) between primary and metastatic tumor was assessed by immunohistochemistry, which was scored by using the proportion of tumor cells with sub-membranous p-mTOR expression. We assessed whether this p-mTOR difference between primary and metastatic tumor was associated with clinico-pathological factors (location of metastasis, lymph node status, T-stage, grade, progesterone receptor status) or varied between patients who did and did not receive endocrine therapy, using Mann-Whitney tests. In addition we performed a linear regression model including the same clinico-pathological factors. RESULTS: In patients who had received endocrine therapy we observed an increase in p-mTOR expression in metastatic tumor lesions compared to the primary tumor (median difference 45%). This was significantly different from the mTOR changes observed in patients who did not receive endocrine therapy (median difference between metastatic and primary lesion 0 %) (p = 0.003). The difference remained significant in the multivariate regression model (p = 0.001). None of the other factors was significantly associated with a differential p-mTOR change. CONCLUSION: In patients who received previous adjuvant endocrine therapy, the increase in p-mTOR expression in metastatic tumor lesions compared to the primary tumor is significantly higher than in patients who did not receive endocrine therapy. Compensatory activation of the PI3K pathway might therefore be a clinically relevant resistance mechanism resulting in secondary endocrine therapy resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-02.

Harmonisation of resistance monitoring programmes in veterinary medicine: an urgent need in the EU?

Antimicrobial surveillance systems in Denmark (DANMAP), The Netherlands (MARAN), Spain (VAV) and Sweden (SVARM) as well as the European Antimicrobial Susceptibility Surveillance in Animals (EASSA) were reviewed. Data have been considered for extended-spectrum cephalosporins, fluoroquinolones and macrolides against food-borne and commensal bacteria. The greatest challenge arises from the lack of agreement between programmes on what is meant by resistance through the use of different interpretive criteria. Indeed, it is shown here that the extent of the differences depends on the antibacterial compound being investigated, the methodology and the interpretive criteria used. This emphasises a need to agree a definition for resistance and for epidemiological cut-off values and to consider harmonising the antimicrobials used in surveillance. This analysis of the data highlights the usefulness of using both epidemiological cut-off values and clinical resistance breakpoints for the purpose of detection of decreased susceptibility and development of clinical resistance, respectively. It is concluded that harmonisation in resistance monitoring programmes is needed since there is potential for data to be appropriately used within risk analysis, providing the opportunity to implement appropriate risk management steps as a response to the public health issues arising from changes in antibiotic resistance in food-borne pathogens and commensal organisms.

Drug discovery and assay development in the ubiquitin–proteasome system

The observation that tumour cells are more sensitive to pharmacological inhibition of the proteasome than normal cells has led to the development of the proteasome inhibitor bortezomib. To date, this is the only proteasome inhibitor that has been approved for clinical use. The clinical success of bortezomib, combined with the occurrence of adverse effects and the development of clinical resistance against this compound, has initiated the development of a broad range of second-generation proteasome inhibitors as well as of assays that can be used to establish a relationship between the extent and type of proteasome inhibition and the effectiveness of a particular drug. In the present paper, we discuss new strategies that may be used in the future to overcome drug resistance and to broaden the use of proteasome inhibitors for the treatment of both cancer and infectious and autoimmune disease.

Growth factor-induced resistance to tamoxifen is associated with a mutation of estrogen receptor α and its phosphorylation at serine 305

Estrogens play a crucial role in breast tumor growth, which is the rationale for the use of antiestrogens, such as tamoxifen, in women with estrogen receptor (ER)-alpha-positive breast cancer. However, hormone resistance is a major clinical problem. Altered growth factor signaling to the ERalpha pathway has been shown to be associated with the development of clinical resistance. We previously have identified a mutation that replaces arginine for lysine at residue 303 (K303R) of ERalpha, which confers hypersensitive growth in low levels of estrogen. To determine if the K303R mutation could participate in the evolution of hormone resistance, we generated MCF-7 breast cancer cells stably transfected with either wild-type (WT) or K303R ERalpha. We found that the mutation confers decreased sensitivity to tamoxifen in the presence of the growth factor heregulin, using anchorage-independent growth assays. K303R ERalpha-expressing cells were hypersensitive to growth factor signals. Our data suggest that phosphorylation of serine 305 within the hinge domain of ERalpha might play a key role in increasing ligand-independent activity of the mutant receptor. We hypothesize that the mutation adapts the receptor for enhanced bidirectional cross-talk with the HER2 growth factor receptor pathway, which then impacts on responsiveness to tamoxifen.

Anticipating Clinical Resistance to Target-Directed Agents

The deregulated tyrosine kinase activity of BCR-ABL is necessary and sufficient to induce chronic myelogenous leukemia (CML). This observation has paved the way for the development of small-molecule inhibitors specifically targeting the kinase activity of the BCR-ABL protein. Indeed, the amazing success of imatinib has revolutionized the whole area of targeted cancer therapeutics. However, enthusiasm for the striking efficacy of imatinib has been tempered by the development of clinical resistance. In essentially all cases, resistance results from kinase domain mutations and/or overexpression of the BCR-ABL gene. To overcome resistance, several novel BCR-ABL inhibitors have been developed and are in clinical trials, though it is inevitable that resistance to second-generation inhibitors will occur as well. Nonetheless, kinases represent an attractive target for therapeutic intervention in several diseases and, at present, some 50 different kinase inhibitors are in clinical trials. We anticipate that resistance to these compounds will follow mechanisms similar to those observed with imatinib. Resistance mutations cause their effect either by direct steric hindrance to drug binding or by allosterically modulating kinase dynamics. This review highlights the principal mechanisms underlying point mutations from these two different classes to confer drug resistance.

BCR-ABL Kinase Dynamics and Drug Resistance.

The aberrant signaling behavior caused by the expression of BCR-ABL is necessary and sufficient to cause chronic myeloid leukemia (CML), an observation which paved the way for the development of imatinib (GleevecTM), a small molecule inhibitor of the BCR-ABL kinase. Enthusiasm for the remarkable efficacy of imatinib has been tempered by the development of clinical resistance. The most common mechanisms for resistance are the development of kinase domain mutations and/or overexpression of the BCR-ABL gene, with mutations in the kinase accounting for ~90 % of all cases. The resistance-conferring lesions are found in regions of the kinase that are critical to its autoregulation, such as P-loop, C-helix, gatekeeper area, activation loop and the SH2-C-lobe interface. Mechanistically, these mutations effect either a steric blockade or a change in the dynamic equilibrium that favors the active kinase conformation that precludes imatinib binding. We have analyzed two dual Src-Abl kinase inhibitors, AP23464 and PD166326, against 58 BCR-ABL kinase variants conferring imatinib resistance. PD166326 binds to the Abl kinase domain in the open although enzymatically inactive conformation, while AP23464 targets the active conformation. Both of these compounds have effectively suppressed the cell growth of imatinib resistance variants, except for a recurrent mutation in the gatekeeper residue (T315I). The P-loop variants are more sensitive to AP23464 than PD166326. Interestingly, the imatinib resistant variants from the C-helix, hinge region, activation loop and SH2-C-lobe region, are hypersensitive to both compounds, as compared to native BCR-ABL. The BCR-ABL variants in the C-helix, gatekeeper area, and the activation loop are more sensitive to AP23464 than PD166326, while variants from the hinge region and the SH2-C-lobe interface are hypersensitive to PD166326. Altogether, these results define a differential requirement for a specific ABL conformation for drug binding of AP23464 and PD166326. In order to better understand their structure activity relationships and the patterns of resistance, we carried out an in-vitro mutagenesis-screen using different concentration of the drug either alone or in combination with imatinib. AP23464 mediates 2–3 time less resistance than PD166326. A higher concentration of all three compounds suppresses all resistance mutations, save for the notable exceptions, T315I and F317L/VandC. Resistance conferring mutations selected at 10–20 fold higher IC50 values are different. AP23464 efficiently suppresses the mutations from the P-loop (except E255K) and two mutations from the activation loop, while PD166326 remains refractory to the mutations in the C-helix and SH2-C-lobe interface. In combination with imatinib, AP23464 and PD166326 suppressed the emergence of most resistance mutations, with the notable exception of T315I. These in-vitro studies demonstrate that the combination of two or three different conformation specific inhibitors is needed to suppress the emergence of resistance. We are characterizing variants of AP23464 that we predict will show activity against the most challenging imatinib resistance mutant T315I.

Development and characterization of five cell models for chemoresistance studies of human ovarian carcinoma

Platinum agents and paclitaxel (taxol) are among the most effective drugs currently available for treatment of ovarian cancer. One of the hurdles with taxol and platinum- based therapy is the clinical development of resistance to these agents. To investigate the mechanism of drug resistance in human ovarian cancer, we developed and characterized 5 cell models for chemoresistance studies of cisplatin, carboplatin and taxol. We report in this study that these human ovarian carcinoma cell model systems include 2 models for cisplatin resistance, 2 models for carboplatin resistance, and 1 model for taxol resistance. The biological and biochemical characteristics of the models showed that (i), the IC50 values of the drugs for all these resistant cell models were 3 times (or more) higher than those for the parental tumor cells. There also exist varying degrees of cross-resistance to several other chemotherapeutic agents in these systems. Moreover, the intracellular drug accumulations in these cells were significantly reduced as compared to those in the parental cells. (ii), The proliferation rates of these resistant cells were markedly decreased. However, there were no obvious changes in cell cycle distribution in these model systems. (iii), Our results for the expression of a few major drug resistance-related genes revealed that the expression of p53, lrp-1 and mrp-1 was decreased, while the expression of pkc, topo I and topo II beta was increased in the resistant tumor cells as compared with the parental cells. In contrast, no significant alterations in gst-pi and topo II alpha expression were found. Interestingly, the levels of mdr-1 expression were elevated in some models, but were reduced in others, thus suggesting that different pathways are involved in the formation of drug resistance in different cell model systems, and that different mechanisms are responsible for the development of different drug resistances in tumor cells. Taken together, our findings indicate that these models may be potentially used to assess the biochemical and genetic mechanisms of drug resistance in human ovarian cancer and to identify new drug resistance-related genes.

Characterization of the MM.1 human multiple myeloma (MM) cell lines: a model system to elucidate the characteristics, behavior, and signaling of steroid-sensitive and -resistant MM cells.

Multiple myeloma (MM) is a clonal B-lymphocyte malignancy, which is characterized by the accumulation of terminally differentiated antibody-producing cells in the bone marrow. Because current treatments offer only a median survival of 3 years, investigators continue to search for novel therapeutic strategies to combat the disease. Rational drug design is enhanced by understanding MM cell proliferation and key signaling pathways employed. In addition, a model system for preclinical evaluation of novel therapeutics is critical. Our laboratory has developed MM cell lines to study drug action and resistance, cell proliferation, and apoptosis. These cell lines are widely used in MM research. From a single MM patient, three separate cell lines were established that parallel the progression of the disease. These three cell lines, designated MM1.S, MM1.R(E), and MM1.R(L), can be distinguished on the basis of their sensitivity to steroid hormones such as glucocorticoids (GCs). Utilization of these cell lines to study the etiology of MM, effects of chemotherapeutic agents, and development of clinical resistance, will provide us with vital information for the evolution of new and more efficacious therapeutics. The aim of this review is to summarize the morphological, biochemical, and growth characteristics of these cells, and to review the results from investigations of the MM.1 signaling pathways. This information will enhance the study, treatment, and eventual eradication of MM.

Interferon-α Resistance in a Cutaneous T-Cell Lymphoma Cell Line Is Associated With Lack of STAT1 Expression

Interferon-alpha (IFNα) mediates its biological effects through activation of the JAK-STAT signaling pathway and it has been shown to be one of most effective therapeutic agents for a number of hematological malignancies, including cutaneous T-cell lymphoma (CTCL). Nevertheless, its efficacy is limited by the development of clinical resistance but the reasons for resistance in CTCL are unknown. Here, we report the development of an IFNα-resistant CTCL cell line (HUT78R), characterized by its ability to proliferate in high concentration of recombinant IFNα, which can be used as a model system to study IFN resistance. The levels of IFN receptor expression and binding affinity were found to be comparable between the parental sensitive (HUT78S) and resistant (HUT78R) cells. However, IFNα stimulation failed to induce interferon-stimulated gene factor 3 (ISGF3) complex formation in HUT78R cells. In addition, the expression of the IFN-inducible 2-5 OAS gene was significantly reduced in HUT78R cells, suggesting the presence of a defect in the Jak-STAT signaling pathway. Our results showed that the IFNα-activated form of a latent transcriptional factor STAT1 was not found in HUT78R cells, whereas activated STAT2 and STAT3 were clearly detectable. By Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, we found that HUT78R cells do not express any STAT1 protein or mRNA, suggesting the possibility of a null mutation in the STAT1 gene. Resistance to the growth inhibitory effect of IFNα in CTCL cells may result from lack of STAT1 expression.

Interferon-α Resistance in a Cutaneous T-Cell Lymphoma Cell Line Is Associated With Lack of STAT1 Expression

AbstractInterferon-alpha (IFNα) mediates its biological effects through activation of the JAK-STAT signaling pathway and it has been shown to be one of most effective therapeutic agents for a number of hematological malignancies, including cutaneous T-cell lymphoma (CTCL). Nevertheless, its efficacy is limited by the development of clinical resistance but the reasons for resistance in CTCL are unknown. Here, we report the development of an IFNα-resistant CTCL cell line (HUT78R), characterized by its ability to proliferate in high concentration of recombinant IFNα, which can be used as a model system to study IFN resistance. The levels of IFN receptor expression and binding affinity were found to be comparable between the parental sensitive (HUT78S) and resistant (HUT78R) cells. However, IFNα stimulation failed to induce interferon-stimulated gene factor 3 (ISGF3) complex formation in HUT78R cells. In addition, the expression of the IFN-inducible 2-5 OAS gene was significantly reduced in HUT78R cells, suggesting the presence of a defect in the Jak-STAT signaling pathway. Our results showed that the IFNα-activated form of a latent transcriptional factor STAT1 was not found in HUT78R cells, whereas activated STAT2 and STAT3 were clearly detectable. By Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses, we found that HUT78R cells do not express any STAT1 protein or mRNA, suggesting the possibility of a null mutation in the STAT1 gene. Resistance to the growth inhibitory effect of IFNα in CTCL cells may result from lack of STAT1 expression.