Resistance-conferring Mutations Research Articles

Abstract 3660: Crenolanib: A next generation FLT3 inhibitor

Abstract Every year in the US there are approximately 12,000 new cases of acute myeloid leukemia (AML). Amongst these patients, 25% have internal tandem duplications (ITDs) of the type III receptor tyrosine kinase, FLT3, causing constitutive activation of the receptor. This mutation has been associated with a poor survival rate, thus making the FLT3/ITD an important target in the treatment of AML. Treatment with small molecule tyrosine kinase inhibitors (TKIs) has shown high response rates in AML patients.1 However, this response is often temporary due to the development of resistance-conferring point mutations.2 Furthermore, QTc prolongation has proven to be a dose-limiting toxicity with some FLT3 TKIs.1 Crenolanib is a TKI with activity against PDGFR and FLT3 and is currently being tested in trials involving patients with a variety of solid tumors. We have determined that crenolanib has potent activity in vitro against FLT3. Crenolanib is cytotoxic to the FLT3/ITD-expressing leukemia cell lines Molm14 and MV411, with an IC50 of 7 nM and 8 nM, respectively. In immunoblots, crenolanib inhibited phosphorylation of both the wild type FLT3 receptor (in SEMK2 cells) and the FLT3/ITD receptor (in Molm14 cells) in culture medium with an IC50 of 1-3 nM. Importantly, the IC50 of crenolanib against the D835Y mutated form of FLT3 was 8.8 nM in culture medium. Using primary AML patient samples, we determined that crenolanib was cytotoxic to FLT3/ITD-expressing samples in a manner comparable to agents such as sorafenib and AC220. Furthermore, crenolanib had cytotoxic activity against primary samples that were obtained from patients who had developed D835 resistance mutations while receiving FLT3 TKIs. In vitro, the IC50 of crenolanib for inhibition of FLT3/ITD in plasma was found to be 34 nM, indicating a relatively low degree of plasma protein binding. From pharmacokinetic studies of crenolanib in solid tumor patients, steady state trough plasma levels of roughly 500 nM were found to be safe and tolerable, suggesting that crenolanib could potentially inhibit the target in vivo as effectively as AC220. Unlike AC220, crenolanib has no significant activity against c-KIT, which may be an advantage in that myelosuppression can be avoided. Furthermore, there was no evidence of QTc prolongation in patients treated with crenolanib. In summary, crenolanib offers a number of advantages over other FLT3 TKIs. Clinical trials of crenolanib in AML patients with FLT3 activating mutations are planned.

Effect of Mutation and Genetic Background on Drug Resistance in Mycobacterium tuberculosis

Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter -15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter -15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.

Cellulose microfibril crystallinity is reduced by mutating C-terminal transmembrane region residues CESA1 A903V and CESA3 T942I of cellulose synthase

The mechanisms underlying the biosynthesis of cellulose in plants are complex and still poorly understood. A central question concerns the mechanism of microfibril structure and how this is linked to the catalytic polymerization action of cellulose synthase (CESA). Furthermore, it remains unclear whether modification of cellulose microfibril structure can be achieved genetically, which could be transformative in a bio-based economy. To explore these processes in planta, we developed a chemical genetic toolbox of pharmacological inhibitors and corresponding resistance-conferring point mutations in the C-terminal transmembrane domain region of CESA1(A903V) and CESA3(T942I) in Arabidopsis thaliana. Using (13)C solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction, we show that the cellulose microfibrils displayed reduced width and an additional cellulose C4 peak indicative of a degree of crystallinity that is intermediate between the surface and interior glucans of wild type, suggesting a difference in glucan chain association during microfibril formation. Consistent with measurements of lower microfibril crystallinity, cellulose extracts from mutated CESA1(A903V) and CESA3(T942I) displayed greater saccharification efficiency than wild type. Using live-cell imaging to track fluorescently labeled CESA, we found that these mutants show increased CESA velocities in the plasma membrane, an indication of increased polymerization rate. Collectively, these data suggest that CESA1(A903V) and CESA3(T942I) have modified microfibril structure in terms of crystallinity and suggest that in plants, as in bacteria, crystallization biophysically limits polymerization.

Fluoroquinolone Resistance in Helicobacter pylori: Role of Mutations at Position 87 and 91 of GyrA on the Level of Resistance and Identification of a Resistance Conferring Mutation in GyrB

Fluoroquinolone-containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87 and 91 of GyrA among fluoroquinolone-resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Norfloxacin-resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin-resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.

Molecular Evolution of Respiratory Syncytial Virus Fusion Gene, Canada, 2006–2010

To assess molecular evolution of the respiratory syncytial virus (RSV) fusion gene, we analyzed RSV-positive specimens from 123 children in Canada who did or did not receive RSV immunoprophylaxis (palivizumab) during 2006–2010. Resistance-conferring mutations within the palivizumab binding site occurred in 8.7% of palivizumab recipients and none of the nonrecipients.

A Fluoroquinolone Resistance Associated Mutation in gyrA Affects DNA Supercoiling in Campylobacter jejuni

The prevalence of fluoroquinolone (FQ)-resistant Campylobacter has become a concern for public health. To facilitate the control of FQ-resistant (FQR) Campylobacter, it is necessary to understand the impact of FQR on the fitness of Campylobacter in its natural hosts as understanding fitness will help to determine and predict the persistence of FQR Campylobacter. Previously it was shown that acquisition of resistance to FQ antimicrobials enhanced the in vivo fitness of FQR Campylobacter. In this study, we confirmed the role of the Thr-86-Ile mutation in GyrA in modulating Campylobacter fitness by reverting the mutation to the wild-type (WT) allele, which resulted in the loss of the fitness advantage. Additionally, we determined if the resistance-conferring GyrA mutations alter the enzymatic function of the DNA gyrase. Recombinant WT gyrase and mutant gyrases with three different types of mutations (Thr-86-Ile, Thr-86-Lys, and Asp-90-Asn), which are associated with FQR in Campylobacter, were generated in E. coli and compared for their supercoiling activities using an in vitro assay. The mutant gyrase with the Thr-86-Ile change showed a greatly reduced supercoiling activity compared with the WT gyrase, while other mutant gyrases did not show an altered supercoiling. Furthermore, we measured DNA supercoiling within Campylobacter cells using a reporter plasmid. Consistent with the results from the in vitro supercoiling assay, the FQR mutant carrying the Thr-86-Ile change in GyrA showed much less DNA supercoiling than the WT strain and the mutant strains carrying other mutations. Together, these results indicate that the Thr-86-Ile mutation, which is predominant in clinical FQR Campylobacter, modulates DNA supercoiling homeostasis in FQR Campylobacter.

A novel asymmetric-loop molecular beacon-based two-phase hybridization assay for accurate and ­high-throughput detection of multiple drug resistance-conferring point mutations in Mycobacterium tuberculosis

SummaryThe accurate and high-throughput detection of drug resistance-related multiple point mutations remains a challenge. Although the combination of molecular beacons with bio-immobilization technology, such as microarray, is promising, its application is difficult due to the ineffective immobilization of molecular beacons on the chip surface. Here, we propose a novel asymmetric-loop molecular beacon in which the loop consists of 2 parts. One is complementary to a target, while the other is complementary to an oligonucleotide probe immobilized on the chip surface. With this novel probe, a two-phase hybridization assay can be used for simultaneously detecting multiple point mutations. This assay will have advantages, such as easy probe availability, multiplex detection, low background, and high-efficiency hybridization, and may provide a new avenue for the immobilization of molecular beacons and high-throughput detection of point mutations.

Following evolution of bacterial antibiotic resistance in real time

A new study reports the development of the 'morbidostat', a device that allows for continuous culture of bacteria under a constant drug selection pressure using computer feedback control of antibiotic concentration. This device, together with bacterial whole-genome sequencing, allowed the authors to follow the evolution of resistance-conferring mutations in Escherichia coli populations in real time, providing support for deterministic evolution of resistance in some situations.

Identification of a point mutation associated with pyrethroid resistance in the para-type sodium channel of Triatoma infestans, a vector of Chagas’ disease

The voltage-gated sodium channel is the target site of pyrethroid insecticides. Point mutations in the domain II region of the channel have been implicated in pyrethroid resistance of several insect species. We identified the sequence of domain II from the para sodium channel in Rhodnius prolixus, a vector of Chagas’ disease. With this information, we cloned and sequenced the domain II of the sodium channel from the other main Chagas’ disease vector: Triatoma infestans. We also identified the presence of a resistance-conferring mutation (L1014F) in a pyrethroid-resistant population of T. infestans from Argentina, and present a PCR-based method to detect this mutation in individuals from field populations. These findings have important implications for the implementation of strategies for resistance management, and for the rational design of campaigns for the control of Chagas’ disease transmission.

Impaired Fitness and Transmission of Macrolide-Resistant Campylobacter jejuni in Its Natural Host

Campylobacter jejuni is a major zoonotic pathogen transmitted to humans via the food chain and is prevalent in chickens, a natural reservoir for this pathogenic organism. Due to the importance of macrolide antibiotics in clinical therapy of human campylobacteriosis, development of macrolide resistance in Campylobacter has become a concern for public health. To facilitate the control of macrolide-resistant Campylobacter, it is necessary to understand if macrolide resistance affects the fitness and transmission of Campylobacter in its natural host. In this study we conducted pairwise competitions and comingling experiments in chickens using clonally related and isogenic C. jejuni strains, which are either susceptible or resistant to erythromycin (Ery). In every competition pair, Ery-resistant (Ery(r)) Campylobacter was consistently outcompeted by the Ery-susceptible (Ery(s)) strain. In the comingling experiments, Ery(r) Campylobacter failed to transmit to chickens precolonized by Ery(s) Campylobacter, while isogenic Ery(s) Campylobacter was able to transmit to and establish dominance in chickens precolonized by Ery(r) Campylobacter. The fitness disadvantage was linked to the resistance-conferring mutations in the 23S rRNA. These findings clearly indicate that acquisition of macrolide resistance impairs the fitness and transmission of Campylobacter in chickens, suggesting that the prevalence of macrolide-resistant C. jejuni will likely decrease in the absence of antibiotic selection pressure.

Mycobacterium Tuberculosis Genetic Diversity and Drug Resistance Conferring Mutations in the Democratic Republic of the Congo

Background: The Democratic Republic of the Congo (DRC) belongs to the 22 tuberculosis (TB) high-burden countries and to the 27 high-burden multidrug-resistant (MDR)-TB countries. To date, there are no data on the genetic diversity of Mycobacterium tuberculosis in the DRC. Objective: To describe the genetic diversity and the distribution of drug resistance conferring mutations of clinical M. tuberculosis isolates from the DRC. Design: We analysed consecutive M. tuberculosis single patient isolates cultured in 2010 at the laboratory of the National TB Control Programme in Kinshasa. Setting: National TB Control Programme in Kinshasa, DRC. Results: Isolates from 50 patients with pulmonary TB were analysed, including 45 patients (90%) who failed treatment. All isolates belonged to the Euro-American lineage (main phylogenetic Lineage 4). Six different spoligotype families were observed within this lineage, including LAM (20 patients, 40%), T (15 patients; 30%), U (4 patients; 8%), S (3 patients; 6%), Haarlem (2 patients; 4%), and X (1 patient; 2%). No M. africanum strains were observed. The most frequently detected drug resistance-conferring mutations were rpoB S531L and katG S315T1. Various other mutations, including previously unreported mutations, were detected. Conclusions: The Euro-American lineage dominates in the DRC, with substantial variation in spoligotype families. This study fills an important gap on the molecular map of M. tuberculosis in sub-Saharan Africa.

Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

The development of resistance to direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) can compromise therapy. However, mechanisms that determine prevalence and frequency of resistance-conferring mutations remain elusive. Here, we studied the fidelity of the HCV RNA-dependent RNA polymerase NS5B in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo. Enzyme kinetic measurements revealed unexpectedly high error rates (approximately 10(-3) per site) for G:U/U:G mismatches. The strong preference for G:U/U:G mismatches over all other mistakes correlates with a mutational bias in favor of transitions over transversions. Deep sequencing of HCV RNA samples isolated from 20 treatment-naïve patients revealed an approximately 75-fold difference in frequencies of the two classes of mutations. A stochastic model based on these results suggests that the bias toward transitions can also affect the selection of resistance-conferring mutations. Collectively, the data provide strong evidence to suggest that the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.

Transmission of Resistant HIV Type 1 Variants and Epidemiological Chains in Italian Newly Diagnosed Individuals

Transmission of HIV-1 and drug resistance continue to occur at a considerable level in Italy, influenced mainly by changes in modality of infection. However, the long period of infectivity makes difficult the interpretation of epidemiological networks, based on epidemiological data only. We studied 510 naive HIV-1-infected individuals, of whom 400 (78.4%) were newly diagnosed patients with an unknown duration of infection (NDs), with the aim of identifying sexual epidemiological networks and transmitted drug resistance (TDR) over a 7-year period. Clusters were identified by Bayesian methods for 412 patients with B subtype; 145 individuals (35.2%) clustered in 34 distinct clades. Within epidemiological networks males were 93.1% (n=135); the same proportion of patients has been infected by the sexual route; 62.1% (n=90) were men having sex with men (MSM) of whom 67.8% (n=61) were NDs. Among heterosexuals (n=44), males were predominant (79.5%, n=35) and 77.3% (n=34) were NDs. TDR in clusters was 11.7 % (n=17), of whom 76.5% (n=13) was found in MSM. TDR was predominantly associated with NRTI resistance in individuals with chronic infection (n=11). A high prevalence of epidemiological networks has been found in the metropolitan area of Milan, indicating a high frequency of transmission events. The cluster analysis of networks suggested that the source of new infections was mainly represented by males and MSM who have long lasting HIV-1 infection. Notably, the prevalence of resistance-conferring mutations was higher in chronically infected patients, carrying mainly resistance to thymidine analogs, the backbone of first antiretroviral (ARV) generation. Intervention strategies of public health are needed to limit HIV-1 transmission and the associated TDR.

Analysis of in Vitro Activity of the Clinically-Active ABL/FLT3 Inhibitor Ponatinib (AP24534) Against AC220-Resistant FLT3-ITD Mutants

Abstract 930 Background:Activating mutations in FLT3 are detected in approximately 30 percent of adult acute myeloid leukemia (AML) cases, most commonly in-tandem duplication (ITD) events. Ponatinib (AP24534) is a potent inhibitor of ABL and FLT3 that has shown clinical activity in CML as well as in a limited experience with FLT3-ITD-positive acute myeloid leukemia (AML) with 2/7 kinase inhibitor-naïve AML patients achieving CRi in a phase I study (Talpaz et al, ASCO 2011, abstract #6518). We recently reported that the FLT3 inhibitor AC220, which has achieved an interim composite CR rate of 43% in a phase II study in relapsed/refractory AML (Cortes, et al, EHA 2011, abstract #1019) is vulnerable to a limited number of resistance-conferring kinase domain mutations in vitro (Smith et al, AACR 2011, abstract #4737). Mutations at 2 of these residues, F691 and D835, have been identified in 9/9 FLT3-ITD+ patients who relapsed after achieving clearance of bone marrow blasts on AC220 (Smith et al, ASH 2011, submitted). Notably, AC220-resistant mutations were found to confer cross-resistance to sorafenib. Given that ponatinib retains activity against a wide range of TKI-resistant BCR-ABL mutants, we sought to test its activity against AC220-resistant mutants. Results:We assessed the activity of ponatinib against highly AC220-resistant FLT3-ITD mutations F691I/L, D835V/Y and Y842C/H. Ponatinib potently suppressed the growth of Ba/F3 cells transformed with native FLT3-ITD with an inhibitory concentration 50 (IC50) of 2 nM. Ba/F3 cells transformed with the “gatekeeper” F691I mutation retained sensitivity to ponatinib at a similar concentration (5 nM). This substitution is analogous to the BCR-ABL T315I mutation, which is clinically sensitive to ponatinib. The remaining AC220-resistant FLT3-ITD mutants, including the gatekeeper F691L substitution, conferred a substantial degree of relative cross-resistance to ponatinib. Mutations at the activation loop residue D835 confer the highest degree of resistance. In an effort to identify other substitutions in FLT3-ITD that are capable of conferring ponatinib resistance in vitro, we employed an in vitro mutagenesis strategy (Azam et al, Cell, 2003) of FLT3-ITD in varying concentrations of ponatinib. In a preliminary analysis, we have identified resistance-causing substitutions at D835, as well as other residues in the FLT3 activation loop including D839 and N841. Interestingly, one of these substitutions, D835N, confers resistance to ponatinib but not to AC220.A molecular analysis of the ponatinib/FLT3 complex based on the crystal structure of ABL/ponatinib was performed and revealed that isoleucine substitution at the FLT3 “gatekeeper” residue (F691I), as with the T315I mutation in Abl, does not creates substantial steric clash with ponatinib. In contrast, the leucine substitution, (F691L), is more bulky than isoleucine and may cause more steric hindrance. In addition, as leucine is more rigid than isoleucine, more energy would be required for the F691L mutation to adopt a conformation compatible with ponatinib binding. As ponatinib binds to the “DFG-out” inactive conformation of FLT3, activation loop mutations such as D835V/Y and Y842C/H likely destabilize the activation loop conformation of FLT3 required for ponatinib binding. Conclusions:Our studies demonstrate that gatekeeper and, in particular, activation loop substitutions in FLT3-ITD, confer a high degree of cross-resistance to the clinically-active FLT3 TKIs described to date. Ponatinib retains in vitro activity against the gatekeeper mutation FLT3-ITD/F691I, which confers a high degree of in vitro resistance to AC220, but leucine substitution at this residue notably confers a higher degree of resistance to ponatinib. Activation loop mutations confer substantial degrees of in vitro cross-resistance to ponatinib and are predicted to confer clinical resistance. Select substitutions at D835 appear to confer selective resistance to ponatinib. Categorization of the spectrum of resistance-conferring mutations may facilitate a more personalized approach toward patients with FLT3-ITD+ AML treated with clinically-active TKI therapy. Disclosures:Off Label Use: Investigational agent ponatinib will be discussed. Zhu:Ariad Pharmaceuticals: Employment. Shah:Ariad: Consultancy, Research Funding; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding.

Validation of FLT3-ITD As a Therapeutic Target in Human Acute Myeloid Leukemia

Abstract 937 Background. Activating in-tandem duplication (ITD) mutations within the FLT3 juxtamembrane region are detected in ∼25% of adult acute myeloid leukemia (AML) cases and portend a poor prognosis. AC220 (quizartinib) is a potent selective investigational FLT3/KIT inhibitor with encouraging preliminary clinical activity, as evidenced by a composite complete remission rate of 43% in 53 chemotherapy refractory/relapsed AML patients evaluable for an interim analysis of the ongoing phase II study in FLT3-ITD+ relapsed/refractory AML (Cortes et al, EHA 2011 abstract 1019). Many patients who initially respond ultimately suffer disease progression. We sought to utilize the clinical efficacy of AC220 to determine if FLT3-ITD is a valid therapeutic target in human AML. A saturation mutagenesis strategy identified amino acid substitutions at three amino acid residues (F691, D835, Y842) in FLT3-ITD that confer a high degree of resistance to AC220 in vitro. Of these, substitutions at F691 and D835 conferred the greatest degree of relative resistance. Results. To assess the validity of FLT3-ITD as a therapeutic target in AML, we analyzed paired pretreatment and relapse samples obtained from a cohort of 9 FLT3-ITD-positive AML patients enrolled in the exploratory part of the ongoing phase II study of AC220 in relapsed/refractory AML (clinicaltrials.gov NCT00989261) who relapsed after initially achieving morphologic clearance of bone marrow blasts to <5% on treatment. Through RT-PCR subcloning and sequencing of the FLT3 kinase domain, we observed evolution of one or more acquired kinase domain mutations on the FLT3-ITD containing allele in all 9 cases at relapse. Clinically detected mutations were restricted to the “gatekeeper” residue F691 and/or the activation loop residue D835 (F691L, n=3, D835Y, n=4; D835V, n=2, D835F, n=1). Each of these mutations successfully transformed Ba/F3 cells to IL-3 independence and conferred substantial resistance to AC220 in cell growth and cell-based biochemical assays. All of the AC220-resistant mutations conferred substantial in vitro cross-resistance to sorafenib, which has been reported to harbor clinical activity in a small number of FLT3-ITD-associated AML cases.In an expanded analysis of genomic DNA samples from 30 patients enrolled in the exploratory part of the Phase II study who discontinued study drug for any reason, we observed the occurrence of acquired mutations in the kinase domain (D835 and F691) in a total of 10 of 30 (33%) patients at the off study timepoint. One patient had a D835Y mutation prior to going on AC220.Molecular docking studies were undertaken to provide mechanistic insights into the structural basis of resistance conferred by AC220-resistant mutations. These studies revealed that AC220 likely binds strongly to the DFG-out inactive FLT3 kinase domain. AC220 directly interacts with the gatekeeper residue F691, explaining the drug-resistance associated with the F691L mutation. Mutations at D835 and Y842 may potentially de-stabilize the inactive conformation, and result in a more active, AC220 binding-deficient FLT3 kinase. Indeed, substitutions at these residues are known to activate FLT3 in the absence of an ITD mutation.To more precisely assess the frequency and identity of resistance-conferring mutations at relapse, we analyzed a subset of samples using single molecule real-time (SMRT™; Pacific Biosciences, Menlo Park, CA) sequencing, which can generate reads of sufficient length to enable focused interrogation of the kinase domain of FLT3-ITD alleles. With this assay, more than 350 reads of >1000 nucleotides were reliably obtained. Analyses of pretreatment and relapse samples from four patients confirmed the presence of resistance-conferring FLT3-ITD kinase domain mutations at F691 or D835 at relapse in 36–60% of FLT3-ITD sequence reads. Additionally, this method detected polyclonal resistance in two of the four samples assessed. Conclusions. Our studies validate FLT3-ITD as a therapeutic target in a proportion of AML cases, and demonstrate that the clinical activity of AC220 is mediated by FLT3-ITD inhibition. AC220-resistant FLT3-ITD gatekeeper and activation loop mutations identified in clinical samples from relapsing patients represent high-value therapeutic targets for next-generation FLT3 inhibitors. Disclosures:Off Label Use: AC220 is an investigational agent and has no approved drug indication in AML. Chin:Pacific Biosciences: Employment. Hunt:Ambit Biosciences: Employment. Levis:Ambit Biosciences, Inc: Consultancy. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.

PLX3397 Is An Investigational Selective FLT3 Inhibitor That Retains Activity Against the Clinically-Relevant FLT3-ITD/F691L “Gatekeeper” Mutation in Vitro

Abstract 764 Background:Activating mutations in FLT3 are detected in approximately 30% of adult acute myeloid leukemia (AML) cases, most commonly involving internal tandem duplication (ITD) events. The clinically-active FLT3 inhibitor AC220 (quizartinib) was recently reported to be associated with a composite complete remission (CR) rate of 43% in relapsed/refractory FLT3-ITD+ AML patients in an interim analysis of a phase II study (Cortes, et al, EHA 2011, abstract #1019). AC220 is vulnerable in vitro to a limited number of resistance-conferring mutations in the FLT3 kinase domain (Smith et al, AACR 2011, abstract #4737). Mutations at two of these residues, F691 and D835, have been detected in 9/9 patients with AML with loss of response to AC220 analyzed to date (Smith et al, ASH 2011, submitted). Mutations at “gatekeeper” residues such as F691 have repeatedly surfaced as important mediators of clinical resistance to inhibitors of BCR-ABL, EGFR, ALK and KIT kinases. Identifying tyrosine kinase inhibitors that retain activity against these substitutions has proven challenging. PLX3397 is a potent and selective inhibitor of FMS, KIT and oncogenic FLT3 with an elimination half-life of 20 hours in humans. Steady-state plasma concentrations of 10–20 uM have been achieved in solid tumor patients enrolled in a phase I study, where minimal myelosuppression has been observed (Anthony et al, J Clin Oncol 29: 2011, suppl abstr 3093). Results:PLX3397 selectively inhibited the proliferation of the human FLT3-ITD+ AML cell lines MV4-11 and MOLM-14 with a 50% inhibitory concentration (IC50) in the submicromolar range (0.09–0.2 uM). PLX3397 inhibited phosphorylation of FLT3-ITD in cells with a dose response similar to the growth inhibition range. We next evaluated the ability of PLX3397 to inhibit the proliferation of Ba/F3 cells transformed with FLT3-ITD and AC220-resistant FLT3-ITD mutant isoforms F691L, D835V/Y, and Y842C/H. PLX3397 inhibited the proliferation of Ba/F3/FLT3-ITD cells (IC50 0.07 uM) at a concentration comparable to that needed to inhibit the growth of MV4-11. Encouragingly, PLX3397 retained activity against Ba/F3 cells expressing the clinically-relevant F691L gatekeeper mutation at a similar concentration (IC50 0.37 uM). Other AC220-resistant mutations evaluated conferred substantial cross-resistance to PLX3397 (IC50 >200x IC50 of FLT3-ITD alone for all mutations; range 14.3–130,000 uM). Western blot analysis of FLT3 autophosphorylation demonstrated dose responsiveness in the same concentration range as observed in cellular proliferation results for each FLT3-ITD mutant isoform. Based upon molecular docking studies, PLX3397 forms non-specific hydrophobic interactions with the gatekeeper side-chain, and thus is less sensitive to the F691L mutation than AC220, for which the combined effects of steric hindrance and polarity mismatch impede its binding to the FLT3-ITD/F691L. To further assess if PLX3397 warrants clinical evaluation as a FLT3 inhibitor in AML, we performed a modified plasma inhibitory assay by incubating MOLM-14 cells in either normal donor or AML patient plasma spiked with increasing concentrations of PLX3397 as well as unmanipulated, steady-state plasma samples from the solid tumor PLX3397 phase I trial. Using phospho-specific flow cytometry to evaluate FLT3 signaling through the downstream protein ribosomal S6, we observed near-maximal reductions in phospho-S6 in both normal and AML patient plasma containing >10 uM PLX3397 as well as plasma samples obtained from the solid tumor trial. Conclusions:PLX3397 harbors substantial promise for the treatment of FLT3-ITD+ AML. Our data demonstrate that potent FLT3 inhibitory concentrations are achieved in humans. Moreover, while mutations in the FLT3-ITD kinase domain represent an important cause of loss of response to clinically-active FLT3 inhibitors, PLX3397 retains activity against the FLT3-ITD/F691L “gatekeeper” mutation, which we have found to be a common cause of preclinical and acquired clinical resistance to AC220. A multi-site phase I/II study of PLX3397 in FLT3-ITD+ AML has been initiated. Disclosures:Off Label Use: Investigational agent PLX3397 will be discussed. Zhang:Plexxikon Inc.: Employment. Carroll:Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding. Shah:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Research Funding.

Winning the Arms Race by Improving Drug Discovery against Mutating Targets

Enzymes are often excellent drug targets. Yet drug pressure on an enzyme target often fosters the rise of cells with resistance-conferring mutations, some of which may compromise fitness and others that compensate to restore fitness. This review presents, first, a structural analysis of a diverse group of wild-type and mutant enzyme targets and, second, an in-depth analysis of five diverse targets to elucidate a broader perspective of the effects of resistance-conferring mutations on protein or organismal fitness. The structural analysis reveals that resistance-conferring mutations may introduce steric hindrance or eliminate critical interactions, as expected, but that they may also have indirect effects such as altering protein dynamics and enzyme kinetics. The structure-based development of the latest generation of inhibitors targeting HIV reverse transcriptase, P. falciparum and S. aureus dihydrofolate reductase, neuraminidase, and epithelial growth factor receptor (EGFR) tyrosine kinase, is highlighted to emphasize lessons that may be applied to future drug discovery to overcome mutation-induced resistance. Successful next-generation drugs tend to be more flexible and exploit a greater number of interactions mimicking those of the substrate with conserved residues.

Primary HIV-1 drug resistance in the C-terminal domains of viral reverse transcriptase among drug-naïve patients from Southern Brazil

BackgroundMajor and accessory drug resistance mutations have been recently characterized in the C-terminal RT subdomains of HIV-1, connection and RNase H. However, their presence in treatment-naïve patients infected with HIV-1 non-B subtypes remains largely unknown. ObjectivesTo characterize the patterns of primary resistance at the C-terminal RT subdomains of HIV-1 infecting subjects in the southern region of Brazil, where HIV-1 subtypes B and C co-circulate. Study designPlasma viral RNA was extracted from patients recently diagnosed for HIV infection (2005–2008). The protease and reverse transcriptase regions were PCR-amplified and sequenced. Infecting HIV subtypes were assigned by phylogenetic inference and drug resistance mutations were determined following the IAS consensus and recent reports on C-terminal RT mutations. ResultsThe major mutation to NNRTI T369I/V was found in 1.8% of patients, while A376S was present in another 8.3%. In the RNase H domain, the compensatory mutation D488E was more frequently observed in subtype C than in subtype B (p=0.038), while the inverse was observed for mutation Q547K (p<0.001). The calculated codon genetic barrier showed that 22% of subtype B isolates, but no subtype C, carried T360, requiring two transitions to change into the resistance mutation 360V. ConclusionsMajor resistance-conferring mutations to NNRTI were detected in 10% of RT connection domain viral sequences from treatment-naïve subjects. We showed for the first time that the presence of specific polymorphisms can constrain the acquisition of definite resistance mutations in the connection and RNase H subdomains of HIV-1 RT.

Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat

Chemical control of Septoria leaf blotch, caused by Mycosphaerella graminicola, is essential to ensure wheat yield and food security in most European countries. Mycosphaerella graminicola has developed resistance to several classes of fungicide and, with the efficacy of azoles gradually declining over time, new modes of action and/or improvements in host varietal resistance are urgently needed to ensure future sustainable disease control. Several new-generation carboxamide fungicides with broad-spectrum activity have recently been introduced into the cereal market. Carboxamides inhibit succinate dehydrogenase (Sdh) of the mitochondrial respiratory chain (complex II) but, because of their single-site specificity, these fungicides may be prone to resistance development. The objective of this study was to assess the risk of resistance development to different Sdh inhibitor (SDHI) fungicides in M. graminicola. UV mutagenesis was conducted to obtain a library of carboxin-resistant mutants. A range of SDHI resistance-conferring mutations was found in Sdh subunits B, C and D. Pathogenicity studies with a range of Sdh variants did not detect any fitness costs associated with these mutations. Most of the amino acid residues identified (e.g. B-S221P/T, B-H267F/L/N/Y, B-I269V and D-D129E/G/T) are directly involved in forming the cavity in which SDHI fungicides bind. Docking studies of SDHI fungicides in structural models of wild-type and mutated Sdh complexes also indicated which residues were important for the binding of different SDHI fungicides and showed a different binding for fluopyram. The predictive power of the model was also shown. Further diagnostic development, enabling the detection of resistant alleles at low frequencies, and cross-resistance studies will aid the implementation of anti-resistance strategies to prolong the cost-effectiveness and lifetime of SDHI fungicides.

Characterization of Mutations Conferring Extensive Drug Resistance to Mycobacterium tuberculosis Isolates in Pakistan

The increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosis in high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosis strains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the "hot-spot" regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrs genes in 50 XDR M. tuberculosis strains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosis strains would be undetected if only these common hot-spot regions were tested. The frequencies of resistance-conferring mutations were found to be comparable among all XDR M. tuberculosis strain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDR M. tuberculosis strains.