The stem cell niche transcription factor ETHYLENE RESPONSE FACTOR 115 participates in aluminum-induced terminal differentiation in Arabidopsis roots.

Aluminum-dependent stoppage of root growth requires the DNA damage response (DDR) pathway including the p53-like transcription factor SUPPRESSOR OF GAMMA RADIATION 1 (SOG1), which promotes terminal differentiation of the root tip in response to Al dependent cell death. Transcriptomic analyses identified Al-induced SOG1-regulated targets as candidate mediators of this growth arrest. Analysis of these factors either as loss-of-function mutants or by overexpression in the als3-1 background shows ERF115, which is a key transcription factor that in other scenarios is rate-limiting for damaged stem cell replenishment, instead participates in transition from an actively growing root to one that has terminally differentiated in response to Al toxicity. This is supported by a loss-of-function erf115 mutant raising the threshold of Al required to promote terminal differentiation of Al hypersensitive als3-1. Consistent with its key role in stoppage of root growth, a putative ERF115 barley ortholog is also upregulated following Al exposure, suggesting a conserved role for this ATR-dependent pathway in Al response. In contrast to other DNA damage agents, these results show that ERF115 and likely related family members are important determinants of terminal differentiation of the root tip following Al exposure and central outputs of the SOG1-mediated pathway in Al response.

Arabidopsis casein kinase 2 triggers stem cell exhaustion under Al toxicity and phosphate deficiency through activating the DNA damage response pathway.

Aluminum (Al) toxicity and inorganic phosphate (Pi) limitation are widespread chronic abiotic and mutually enhancing stresses that profoundly affect crop yield. Both stresses strongly inhibit root growth, resulting from a progressive exhaustion of the stem cell niche. Here, we report on a casein kinase 2 (CK2) inhibitor identified by its capability to maintain a functional root stem cell niche in Arabidopsis thaliana under Al toxic conditions. CK2 operates through phosphorylation of the cell cycle checkpoint activator SUPPRESSOR OF GAMMA RADIATION1 (SOG1), priming its activity under DNA-damaging conditions. In addition to yielding Al tolerance, CK2 and SOG1 inactivation prevents meristem exhaustion under Pi starvation, revealing the existence of a low Pi-induced cell cycle checkpoint that depends on the DNA damage activator ATAXIA-TELANGIECTASIA MUTATED (ATM). Overall, our data reveal an important physiological role for the plant DNA damage response pathway under agriculturally limiting growth conditions, opening new avenues to cope with Pi limitation.

Safe and rapid radial hemostasis achieved using a novel topical hemostatic patch: Results of a first-in-human pilot study using hydrophobically modified polysaccharide-chitosan.

BACKGROUND: The transradial approach (TRA) for catheter interventions decreases vascular complications and bleeding versus transfemoral approach. Reducing time to hemostasis and preventing radial artery occlusion (RAO) following TRA are important and incompletely realized aspirations. OBJECTIVES: This first-in-human study sought to evaluate the efficacy of a novel, topically applied compound (hydrophobically modified polysaccharide-chitosan, hm-P) plus minimal required pneumatic compression, to achieve rapid radial arterial hemostasis in post-TRA procedures compared with de facto standards. MATERIALS AND METHODS: About 50 adult patients undergoing 6 French diagnostic TRA procedures were prospectively enrolled. At procedure completion, a topical hm-P impregnated patch was placed over the dermotomy and TR Band (TRB) compression was applied to the access site. This patch was used as part of a novel rapid deflation protocol with a primary outcome of time to hemostasis. Photographic and vascular ultrasound evaluation of the radial artery was performed to evaluate the procedural site. RESULTS: Time to hemostasis was 40.5 min (IQR: 38-50 min) with the majority of patients (n = 39, 78%) not requiring reinflation. Patients with bleeding requiring TRB reinflation were more likely to have low body weight and liver dysfunction, with absence of hypertension and LV dysfunction. The rate of RAO was 0% with predischarge radial artery patency documented in all patients using vascular ultrasound. One superficial hematoma was noted. No late bleeding events or cutaneous reactions were reported in the study follow-up. CONCLUSIONS: Topical application of hm-P in conjunction with pneumatic compression was safe and resulted in rapid and predictable hemostasis at the arterial puncture site.

A multi-level response to DNA damage induced by aluminium.

Aluminium (Al) ions are one of the primary growth-limiting factors for plants on acid soils, globally restricting agriculture. Despite its impact, little is known about Al action in planta. Earlier work has indicated that, among other effects, Al induces DNA damage. However, the loss of major DNA damage response regulators, such SOG1, partially suppressed the growth reduction in plants seen on Al-containing media. This raised the question whether Al actually causes DNA damage and, if so, how. Here, we provide cytological and genetic data corroborating that exposure to Al leads to DNA double-strand breaks. We find that the Al-induced damage specifically involves homology-dependent (HR) recombination repair. Using an Al toxicity assay that delivers higher Al concentrations than used in previous tests, we find that sog1 mutants become highly sensitive to Al. This indicates a multi-level response to Al-induced DNA damage in plants.

Al-Tolerant Barley Mutant hvatr.g Shows the ATR-Regulated DNA Damage Response to Maleic Acid Hydrazide.

ATR, a DNA damage signaling kinase, is required for cell cycle checkpoint regulation and detecting DNA damage caused by genotoxic factors including Al3+ ions. We analyzed the function of the HvATR gene in response to chemical clastogen-maleic acid hydrazide (MH). For this purpose, the Al-tolerant barley TILLING mutant hvatr.g was used. We described the effects of MH on the nuclear genome of hvatr.g mutant and its WT parent cv. "Sebastian", showing that the genotoxic effect measured by TUNEL test and frequency of cells with micronuclei was much stronger in hvatr.g than in WT. MH caused a significant decrease in the mitotic activity of root cells in both genotypes, however this effect was significantly stronger in "Sebastian". The impact of MH on the roots cell cycle, analyzed using flow cytometry, showed no differences between the mutant and WT.

Aluminum-Dependent Terminal Differentiation of the Arabidopsis Root Tip Is Mediated through an ATR-, ALT2-, and SOG1-Regulated Transcriptional Response.

By screening for suppressors of the aluminum (Al) hypersensitive Arabidopsis thaliana mutant als3-1, it was found that mutational loss of the Arabidopsis DNA damage response transcription factor SUPPRESSOR OF GAMMA RESPONSE1 (SOG1) confers increased Al tolerance similar to the loss-of-function mutants for the cell cycle checkpoint genes ATAXIA TELANGIECTASIA AND RAD3 RELATED (ATR) and ALUMINUM TOLERANT2 (ALT2). This suggests that Al-dependent terminal differentiation of the root tip is an active process resulting from activation of the DNA damage checkpoint by an ATR-regulated pathway, which functions at least in part through SOG1. Consistent with this, ATR can phosphorylate SOG1 in vitro. Analysis of SOG1's role in Al-dependent root growth inhibition shows that sog1-7 prevents Al-dependent quiescent center differentiation and endoreduplication in the primary root tip. Following Al exposure, SOG1 increases expression of several genes previously associated with DNA damage, including BRCA1 and PARP2, with gel-shift analysis showing that SOG1 can physically associate with the BRCA1 promoter in vitro. Al-responsive expression of these SOG1-regulated genes requires ATR and ALT2, but not ATAXIA TELANGIECTASIA MUTATED, thus demonstrating that in response to chronic Al exposure, ATR, ALT2, and SOG1 function together to halt root growth and promote terminal differentiation at least in part in a transcription-dependent manner.

SUV2, which encodes an ATR-related cell cycle checkpoint and putative plant ATRIP, is required for aluminium-dependent root growth inhibition in Arabidopsis.

A suppressor mutagenesis screen was conducted in order to identify second site mutations that could reverse the extreme hypersensitivity to aluminium (Al) seen for the Arabidopsis mutant, als3-1. From this screen, it was found that a loss-of-function mutation in the previously described SUV2 (SENSITIVE TO UV 2), which encodes a putative plant ATRIP homologue that is a component of the ATR-dependent cell checkpoint response, reversed the als3-1 phenotype. This included prevention of hallmarks associated with als3-1 including Al-dependent terminal differentiation of the root tip and transition to endoreduplication. From this analysis, SUV2 was determined to be required for halting cell cycle progression and triggering loss of the quiescent centre (QC) following exposure to Al. In conjunction with this, SUV2 was found to have a similar role as ATR, ALT2 and SOG1 in Al-dependent stoppage of root growth, all of which are required for promotion of expression of a suite of genes that likely are part of an Al-dependent DNA damage transcriptional response. This work argues that these Al response factors work together to detect Al-dependent damage and subsequently activate a DNA damage response pathway that halts the cell cycle and subsequently promotes QC differentiation and entrance into endocycling.

Dominant gain-of-function mutations in transmembrane domain III of ERS1 and ETR1 suggest a novel role for this domain in regulating the magnitude of ethylene response in Arabidopsis.

Prior work resulted in identification of an Arabidopsis mutant, eer5-1, with extreme ethylene response in conjunction with failure to induce a subset of ethylene-responsive genes, including AtEBP. EER5, which is a TREX-2 homolog that is part of a nucleoporin complex, functions as part of a cryptic aspect of the ethylene signaling pathway that is required for regulating the magnitude of ethylene response. A suppressor mutagenesis screen was carried out to identify second site mutations that could restore the growth of ethylene-treated eer5-1 to wild-type levels. A dominant gain-of-function mutation in the ethylene receptor ETHYLENE RESPONSE SENSOR 1 (ERS1) was identified, with the ers1-4 mutation being located in transmembrane domain III at a point nearly equivalent to the previously described etr1-2 mutation in the other Arabidopsis subfamily I ethylene receptor, ETHYLENE RESPONSE 1 (ETR1). Although both ers1-4 and etr1-2 partially suppress the ethylene hypersensitivity of eer5-1 and are at least in part REVERSION TO ETHYLENE SENSITIVITY 1 (RTE1)-dependent, ers1-4 was additionally found to restore the expression of AtEBP in ers1-4;eer5-1 etiolated seedlings after ethylene treatment in an EIN3-dependent manner. Our work indicates that ERS1-regulated expression of a subset of ethylene-responsive genes is related to controlling the magnitude of ethylene response, with hyperinduction of these genes correlated with reduced ethylene-dependent growth inhibition.

The Arabidopsis cell cycle checkpoint regulators TANMEI/ALT2 and ATR mediate the active process of aluminum-dependent root growth inhibition.

Aluminum (Al) toxicity is a global issue that severely limits root growth in acidic soils. Isolation of suppressors of the Arabidopsis thaliana Al-hypersensitive mutant, als3-1, resulted in identification of a cell cycle checkpoint factor, ALUMINUM TOLERANT2 (ALT2), which monitors and responds to DNA damage. ALT2 is required for active stoppage of root growth after Al exposure, because alt2 loss-of-function mutants fail to halt root growth after Al exposure, do not accumulate CyclinB1;1 in the root tip, and fail to force differentiation of the quiescent center. Thus, alt2-1 mutants are highly tolerant of Al levels that are severely inhibitory to the wild type. The alt2-1 allele is a loss-of-function mutation in a protein containing a putative DDB1-binding WD40 motif, previously identified as TANMEI, which is required for assessment of DNA integrity, including monitoring of DNA crosslinks. alt2-1 and atr loss-of-function mutants, the latter of which affects the cell cycle checkpoint ATAXIA TELANGIECTASIA-MUTATED AND RAD3-RELATED, are severely sensitive to DNA crosslinking agents and have increased Al tolerance. These results suggest that Al likely acts as a DNA-damaging agent in vivo and that Al-dependent root growth inhibition, in part, arises from detection of and response to this damage by TANMEI/ALT2 and ATR, both of which actively halt cell cycle progression and force differentiation of the quiescent center.

Team-based Care for Patients Hospitalized with Heart Failure.

Hospitalizations for acute heart failure (HF) and subsequent readmissions have received increased attention because of the burden they place on patients, providers, and the health care system. These hospitalizations represent a significant portion of the total cost of HF care and health care in general. Although much of the care of the patient with HF occurs outside of the hospital, the genesis of the programs that attempt to limit repeat hospitalizations begin in the impatient setting. By using evidence-based guidelines, interdisciplinary teams, and comprehensive discharge planning, costly readmissions can be reduced and outcomes improved.

A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis.

As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena.

Kinetic approach to pathway attenuation using XOMA 052, a regulatory therapeutic antibody that modulates interleukin-1beta activity.

Many therapeutic antibodies act as antagonists to competitively block cellular signaling pathways. We describe here an approach for the therapeutic use of monoclonal antibodies based on context-dependent attenuation to reduce pathologically high activity while allowing homeostatic signaling in biologically important pathways. Such attenuation is achieved by modulating the kinetics of a ligand binding to its various receptors and regulatory proteins rather than by complete blockade of signaling pathways. The anti-interleukin-1beta (IL-1beta) antibody XOMA 052 is a potent inhibitor of IL-1beta activity that reduces the affinity of IL-1beta for its signaling receptor and co-receptor but not for its decoy and soluble inhibitory receptors. This mechanism shifts the effective dose response of the cytokine so that the potency of IL-1beta bound by XOMA 052 is 20-100-fold lower than that of IL-1beta in the absence of antibody in a variety of in vitro cell-based assays. We propose that by decreasing potency of IL-1beta while allowing binding to its clearance and inhibitory receptors, XOMA 052 treatment will attenuate IL-1beta activity in concert with endogenous regulatory mechanisms. Furthermore, the ability to bind the decoy receptor may reduce the potential for accumulation of antibody.target complexes. Regulatory antibodies like XOMA 052, which selectively modulate signaling pathways, may represent a new mechanistic class of therapeutic antibodies.

Aluminum-dependent root-growth inhibition in Arabidopsis results from AtATR-regulated cell-cycle arrest.

Aluminum (Al) toxicity is a global problem severely limiting agricultural productivity in acid-soil regions comprising upwards of 50% of the world's arable land [1, 2]. Although Al-exclusion mechanisms have been intensively studied [3-9], little is known about tolerance to internalized Al, which is predicted to be mechanistically complex because of the plethora of predicted cellular targets for Al(3+)[2, 10]. An Arabidopsis mutant with Al hypersensitivity, als3-1, was found to represent a lesion in a phloem and root-tip-localized factor similar to the bacterial ABC transporter ybbm, with ALS3 likely responsible for Al transfer from roots to less-sensitive tissues [10-12]. To identify mutations that enhance mechanisms of Al resistance or tolerance, a suppressor screen for mutants that mask the Al hypersensitivity of als3-1 was performed [13]. Two allelic suppressors conferring increased Al tolerance were found to represent dominant-negative mutations in a factor required for monitoring DNA integrity, AtATR[14-17]. From this work, Al-dependent root-growth inhibition primarily arises from DNA damage coupled with AtATR-controlled blockage of cell-cycle progression and terminal differentiation because of loss of the root-quiescent center, with mutations that prevent response to this damage resulting in quiescent-center maintenance and sustained vigorous growth in an Al-toxic environment.

Aluminum or Low pH - Which Is the Bigger Enemy of Barley? Transcriptome Analysis of Barley Root Meristem Under Al and Low pH Stress.

Aluminum (Al) toxicity is considered to be the most harmful abiotic stress in acidic soils that today comprise more than 50% of the world's arable lands. Barley belongs to a group of crops that are most sensitive to Al in low pH soils. We present the RNA-seq analysis of root meristems of barley seedlings grown in hydroponics at optimal pH (6.0), low pH (4.0), and low pH with Al (10 µM of bioavailable Al3+ ions). Two independent experiments were conducted: with short-term (24 h) and long-term (7 days) Al treatment. In the short-term experiment, more genes were differentially expressed (DEGs) between root meristems grown at pH = 6.0 and pH = 4.0, than between those grown at pH = 4.0 with and without Al treatment. The genes upregulated by low pH were associated mainly with response to oxidative stress, cell wall organization, and iron ion binding. Among genes upregulated by Al, overrepresented were those related to response to stress condition and calcium ion binding. In the long-term experiment, the number of DEGs between hydroponics at pH = 4.0 and 6.0 were lower than in the short-term experiment, which suggests that plants partially adapted to the low pH. Interestingly, 7 days Al treatment caused massive changes in the transcriptome profile. Over 4,000 genes were upregulated and almost 2,000 genes were downregulated by long-term Al stress. These DEGs were related to stress response, cell wall development and metal ion transport. Based on our results we can assume that both, Al3+ ions and low pH are harmful to barley plants. Additionally, we phenotyped the root system of barley seedlings grown in the same hydroponic conditions for 7 days at pH = 6.0, pH = 4.0, and pH = 4.0 with Al. The results correspond to transcriptomic data and show that low pH itself is a stress factor that causes a significant reduction of root growth and the addition of aluminum further increases this reduction. It should be noted that in acidic arable lands, plants are exposed simultaneously to both of these stresses. The presented transcriptome analysis may help to find potential targets for breeding barley plants that are more tolerant to such conditions.

The eer5 mutation, which affects a novel proteasome-related subunit, indicates a prominent role for the COP9 signalosome in resetting the ethylene-signaling pathway in Arabidopsis.

An Arabidopsis mutant, eer5-1, which has an enhanced ethylene response in etiolated seedlings, including hypersensitivity and extreme exaggeration of response to ethylene, was isolated and characterized. As with other identified eer mutants, the enhanced response phenotype of eer5-1 was correlated with failure to induce appropriately a subset of ethylene-regulated genes, suggesting that proper ethylene-responsive gene expression is necessary for resetting the ethylene response pathway. eer5-1 represents a mutation that causes an amino acid substitution in a previously uncharacterized gene, which encodes a protein with a PAM [proteasome COP9 initiation factor (PCI/PINT)-associated module] domain similar to those found in components of the COP9 signalosome (CSN). Genetic analysis shows that manifestation of the eer5 mutant phenotype is solely dependent on ethylene signaling, as the ein2-5 eer5-1 double mutant was indistinguishable from ein2-5 in the presence of saturating ethylene concentrations. In contrast, the ein3-1 eer5-1 double mutant displayed characteristics of an enhanced ethylene response, and this suggests that EER5 regulates ethylene signaling independently of EIN3. Analysis of the EER5 protein indicates that it interacts with the C-terminus of EIN2 and with the CSN, suggesting that EER5 serves as a bridge between EIN2 and the modification or degradation of target proteins, including a proposed group of transcriptional repressors, as part of a resetting mechanism during or following ethylene signaling.

Congenital lymphocytic choriomeningitis virus infection: spectrum of disease.

OBJECTIVE: Lymphocytic choriomeningitis virus (LCMV) is a human pathogen and an emerging neuroteratogen. When the infection occurs during pregnancy, the virus can target and damage the fetal brain and retina. We examined the spectrum of clinical presentations, neuroimaging findings, and clinical outcomes of children with congenital LCMV infection. METHODS: Twenty children with serologically confirmed congenital LCMV infection were identified. The children underwent neuroimaging studies and were followed prospectively for up to 11 years. RESULTS: All children with congenital LCMV infection had chorioretinitis and structural brain anomalies. However, the presenting clinical signs, severity of vision disturbance, nature and location of neuropathology, and character and severity of brain dysfunction varied substantially among cases. Neuroimaging abnormalities included microencephaly, periventricular calcifications, ventriculomegaly, pachygyria, cerebellar hypoplasia, porencephalic cysts, periventricular cysts, and hydrocephalus. The combination of microencephaly and periventricular calcifications was the most common neuroimaging abnormality, and all children with this combination had profound mental retardation, epilepsy, and cerebral palsy. However, others had less severe neuroimaging abnormalities and better outcomes. Some children had isolated cerebellar hypoplasia, with jitteriness as their presenting sign and ataxia as their principal long-term neurological dysfunction. INTERPRETATION: Congenital LCMV infection can have diverse presenting signs, neuroimaging abnormalities, and clinical outcomes. In the companion article to this study, we utilize an animal model to show that the clinical and pathological diversity in congenital LCMV infection is likely due to differences in the gestational timing of infection.

Friedreich's Ataxia: Clinical Presentation of a Compound Heterozygote Child with a Rare Nonsense Mutation and Comparison with Previously Published Cases.

Friedreich's ataxia is a neurodegenerative disorder associated with a GAA trinucleotide repeat expansion in intron 1 of the frataxin (FXN) gene. It is the most common autosomal recessive cerebellar ataxia, with a mean age of onset at 16 years. Nearly 95-98% of patients are homozygous for a 90-1300 GAA repeat expansion with only 2-5% demonstrating compound heterozygosity. Compound heterozygous individuals have a repeat expansion in one allele and a point mutation/deletion/insertion in the other. Compound heterozygosity and point mutations are very rare causes of Friedreich's ataxia and nonsense mutations are a further rarity among point mutations. We report a rare compound heterozygous Friedrich's ataxia patient who was found to have one expanded GAA FXN allele and a nonsense point mutation in the other. We summarize the four previously published cases of nonsense mutations and compare the phenotype to that of our patient. We compared clinical information from our patient with other nonsense FXN mutations reported in the literature. This nonsense mutation, to our knowledge, has only been described once previously; interestingly the individual was also of Cuban ancestry. A comparison with previously published cases of nonsense mutations demonstrates some common clinical characteristics.

Phosphatidic acid binds to and inhibits the activity of Arabidopsis CTR1.

Phosphatidic acid (PA) has only recently been identified as an important eukaryotic lipid-signalling molecule. In plants, PA formation is triggered by various biotic and abiotic stresses, including wounding, pathogen attack, drought, salinity, cold, and freezing. However, few molecular targets of PA have been identified so far. One of the best characterized is Raf-1, a mammalian MAPKKK. Arabidopsis thaliana CTR1 (constitutive triple response 1) is one of the plant homologues of Raf-1 and functions as a negative regulator of the ethylene signalling pathway. Here, it is shown that PA binds CTR1 and inhibits its kinase activity. Using different PA-binding assays, the kinase domain of CTR1 (CTR1-K) was found to bind PA directly. Addition of PA resulted in almost complete inhibition of CTR1 kinase activity and disrupted the intramolecular interaction between CTR1-K and the CTR1 N-terminal regulatory domain. Additionally, PA blocked the interaction of CTR1 with ETR1, one of the ethylene receptors. The basic amino acid motif shown to be required for PA binding in Raf-1 is conserved in CTR1-K. However, mutations in this motif did not affect either PA-binding or PA-dependent inhibition of CTR1 activity. Subsequent deletion analysis of CTR1's kinase domain revealed a novel PA-binding region at the C-terminus of the kinase.

Modification of DNA Checkpoints to Confer Aluminum Tolerance.

Although aluminum (Al) toxicity represents a global agricultural problem, the biochemical targets for Al remain elusive. Recently identified Arabidopsis mutants with increased Al tolerance provide evidence of DNA as one of the main targets of Al. This insight could lead the way for novel strategies to generate Al-tolerant crop plants.

A Next-Generation TRK Kinase Inhibitor Overcomes Acquired Resistance to Prior TRK Kinase Inhibition in Patients with TRK Fusion-Positive Solid Tumors.

Larotrectinib, a selective TRK tyrosine kinase inhibitor (TKI), has demonstrated histology-agnostic efficacy in patients with TRK fusion-positive cancers. Although responses to TRK inhibition can be dramatic and durable, duration of response may eventually be limited by acquired resistance. LOXO-195 is a selective TRK TKI designed to overcome acquired resistance mediated by recurrent kinase domain (solvent front and xDFG) mutations identified in multiple patients who have developed resistance to TRK TKIs. Activity against these acquired mutations was confirmed in enzyme and cell-based assays and in vivo tumor models. As clinical proof of concept, the first 2 patients with TRK fusion-positive cancers who developed acquired resistance mutations on larotrectinib were treated with LOXO-195 on a first-in-human basis, utilizing rapid dose titration guided by pharmacokinetic assessments. This approach led to rapid tumor responses and extended the overall duration of disease control achieved with TRK inhibition in both patients.Significance: LOXO-195 abrogated resistance in TRK fusion-positive cancers that acquired kinase domain mutations, a shared liability with all existing TRK TKIs. This establishes a role for sequential treatment by demonstrating continued TRK dependence and validates a paradigm for the accelerated development of next-generation inhibitors against validated oncogenic targets. Cancer Discov; 7(9); 963-72. ©2017 AACR.See related commentary by Parikh and Corcoran, p. 934This article is highlighted in the In This Issue feature, p. 920.