A Therapy for Most with Cystic Fibrosis.

TRIKAFTA is the third drug approved by the FDA that rescues defects caused by the major mutation F508del. It is superior to its predecessors that were approved for patients who are homozygous for F508del because TRIKAFTA is also effective in CF patients who harbor only one copy of this mutation.

A Small Molecule Targeting Mutagenic Translesion Synthesis Improves Chemotherapy.

Intrinsic and acquired drug resistance and induction of secondary malignancies limit successful chemotherapy. Because mutagenic translesion synthesis (TLS) contributes to chemoresistance as well as treatment-induced mutations, targeting TLS is an attractive avenue for improving chemotherapeutics. However, development of small molecules with high specificity and in vivo efficacy for mutagenic TLS has been challenging. Here, we report the discovery of a small-molecule inhibitor, JH-RE-06, that disrupts mutagenic TLS by preventing recruitment of mutagenic POL ζ. Remarkably, JH-RE-06 targets a nearly featureless surface of REV1 that interacts with the REV7 subunit of POL ζ. Binding of JH-RE-06 induces REV1 dimerization, which blocks the REV1-REV7 interaction and POL ζ recruitment. JH-RE-06 inhibits mutagenic TLS and enhances cisplatin-induced toxicity in cultured human and mouse cell lines. Co-administration of JH-RE-06 with cisplatin suppresses the growth of xenograft human melanomas in mice, establishing a framework for developing TLS inhibitors as a novel class of chemotherapy adjuvants.

Driving glioblastoma to drink.

Attempts to treat cancer with drugs that target mutated proteins have met with mixed success. By screening for compounds that alter the phenotype of glioblastoma cells-an aggressive brain tumor-Kitambi et al. identify a potential new treatment of the disease and shed light on an unusual cell death mechanism.

RETRACTED: Vulnerability of glioblastoma cells to catastrophic vacuolization and death induced by a small molecule.

Glioblastoma multiforme (GBM) is the most aggressive form of brain cancer with marginal life expectancy. Based on the assumption that GBM cells gain functions not necessarily involved in the cancerous process, patient-derived glioblastoma cells (GCs) were screened to identify cellular processes amenable for development of targeted treatments. The quinine-derivative NSC13316 reliably and selectively compromised viability. Synthetic chemical expansion reveals delicate structure-activity relationship and analogs with increased potency, termed Vacquinols. Vacquinols stimulate death by membrane ruffling, cell rounding, massive macropinocytic vacuole accumulation, ATP depletion, and cytoplasmic membrane rupture of GCs. The MAP kinase MKK4, identified by a shRNA screen, represents a critical signaling node. Vacquinol-1 displays excellent in vivo pharmacokinetics and brain exposure, attenuates disease progression, and prolongs survival in a GBM animal model. These results identify a vulnerability to massive vacuolization that can be targeted by small molecules and point to the possible exploitation of this process in the design of anticancer therapies.

Proteome dynamics at broken replication forks reveal a distinct ATM-directed repair response suppressing DNA double-strand break ubiquitination.

Cells have evolved an elaborate DNA repair network to ensure complete and accurate DNA replication. Defects in these repair machineries can fuel genome instability and drive carcinogenesis while creating vulnerabilities that may be exploited in therapy. Here, we use nascent chromatin capture (NCC) proteomics to characterize the repair of replication-associated DNA double-strand breaks (DSBs) triggered by topoisomerase 1 (TOP1) inhibitors. We reveal profound changes in the fork proteome, including the chromatin environment and nuclear membrane interactions, and identify three classes of repair factors according to their enrichment at broken and/or stalled forks. ATM inhibition dramatically rewired the broken fork proteome, revealing that ataxia telangiectasia mutated (ATM) signalling stimulates DNA end resection, recruits PLK1, and concomitantly suppresses the canonical DSB ubiquitination response by preventing accumulation of RNF168 and BRCA1-A. This work and collection of replication fork proteomes provide a new framework to understand how cells orchestrate homologous recombination repair of replication-associated DSBs.

EGFR activation limits the response of liver cancer to lenvatinib.

Hepatocellular carcinoma (HCC)-the most common form of liver cancer-is an aggressive malignancy with few effective treatment options1. Lenvatinib is a small-molecule inhibitor of multiple receptor tyrosine kinases that is used for the treatment of patients with advanced HCC, but this drug has only limited clinical benefit2. Here, using a kinome-centred CRISPR-Cas9 genetic screen, we show that inhibition of epidermal growth factor receptor (EGFR) is synthetic lethal with lenvatinib in liver cancer. The combination of the EGFR inhibitor gefitinib and lenvatinib displays potent anti-proliferative effects in vitro in liver cancer cell lines that express EGFR and in vivo in xenografted liver cancer cell lines, immunocompetent mouse models and patient-derived HCC tumours in mice. Mechanistically, inhibition of fibroblast growth factor receptor (FGFR)  by lenvatinib treatment leads to feedback activation of the EGFR-PAK2-ERK5 signalling axis, which is blocked by EGFR inhibition. Treatment of 12 patients with advanced HCC who were unresponsive to lenvatinib treatment with the combination of lenvatinib plus gefitinib (trial identifier NCT04642547) resulted in meaningful clinical responses. The combination therapy identified here may represent a promising strategy for the approximately 50% of patients with advanced HCC who have high levels of EGFR.

Cancer-stromal cell interactions in breast cancer brain metastases induce glycocalyx-mediated resistance to HER2-targeting therapies.

Brain metastatic breast cancer is particularly lethal largely due to therapeutic resistance. Almost half of the patients with metastatic HER2-positive breast cancer develop brain metastases, representing a major clinical challenge. We previously described that cancer-associated fibroblasts are an important source of resistance in primary tumors. Here, we report that breast cancer brain metastasis stromal cell interactions in 3D cocultures induce therapeutic resistance to HER2-targeting agents, particularly to the small molecule inhibitor of HER2/EGFR neratinib. We investigated the underlying mechanisms using a synthetic Notch reporter system enabling the sorting of cancer cells that directly interact with stromal cells. We identified mucins and bulky glycoprotein synthesis as top-up-regulated genes and pathways by comparing the gene expression and chromatin profiles of stroma-contact and no-contact cancer cells before and after neratinib treatment. Glycoprotein gene signatures were also enriched in human brain metastases compared to primary tumors. We confirmed increased glycocalyx surrounding cocultures by immunofluorescence and showed that mucinase treatment increased sensitivity to neratinib by enabling a more efficient inhibition of EGFR/HER2 signaling in cancer cells. Overexpression of truncated MUC1 lacking the intracellular domain as a model of increased glycocalyx-induced resistance to neratinib both in cell culture and in experimental brain metastases in immunodeficient mice. Our results highlight the importance of glycoproteins as a resistance mechanism to HER2-targeting therapies in breast cancer brain metastases.

Mitapivat versus Placebo for Pyruvate Kinase Deficiency.

BACKGROUND: Pyruvate kinase deficiency is a rare, hereditary, chronic condition that is associated with hemolytic anemia. In a phase 2 study, mitapivat, an oral, first-in-class activator of erythrocyte pyruvate kinase, increased the hemoglobin level in patients with pyruvate kinase deficiency. METHODS: In this global, phase 3, randomized, placebo-controlled trial, we evaluated the efficacy and safety of mitapivat in adults with pyruvate kinase deficiency who were not receiving regular red-cell transfusions. The patients were assigned to receive either mitapivat (5 mg twice daily, with potential escalation to 20 or 50 mg twice daily) or placebo for 24 weeks. The primary end point was a hemoglobin response (an increase from baseline of ≥1.5 g per deciliter in the hemoglobin level) that was sustained at two or more scheduled assessments at weeks 16, 20, and 24. Secondary efficacy end points were the average change from baseline in the hemoglobin level, markers of hemolysis and hematopoiesis, and the change from baseline at week 24 in two pyruvate kinase deficiency-specific patient-reported outcome measures. RESULTS: Sixteen of the 40 patients (40%) in the mitapivat group had a hemoglobin response, as compared with none of the 40 patients in the placebo group (adjusted difference, 39.3 percentage points; 95% confidence interval, 24.1 to 54.6; two-sided P<0.001). Patients who received mitapivat had a greater response than those who received placebo with respect to each secondary end point, including the average change from baseline in the hemoglobin level. The most common adverse events were nausea (in 7 patients [18%] in the mitapivat group and 9 patients [23%] in the placebo group) and headache (in 6 patients [15%] and 13 patients [33%], respectively). Adverse events of grade 3 or higher occurred in 10 patients (25%) who received mitapivat and 5 patients (13%) who received placebo. CONCLUSIONS: In patients with pyruvate kinase deficiency, mitapivat significantly increased the hemoglobin level, decreased hemolysis, and improved patient-reported outcomes. No new safety signals were identified in the patients who received mitapivat. (Funded by Agios Pharmaceuticals; ACTIVATE ClinicalTrials.gov number, NCT03548220.).

PfCRT mutations conferring piperaquine resistance in falciparum malaria shape the kinetics of quinoline drug binding and transport.

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ.

Molecular Mechanisms of Drug Resistance in Plasmodium falciparum Malaria.

Understanding and controlling the spread of antimalarial resistance, particularly to artemisinin and its partner drugs, is a top priority. Plasmodium falciparum parasites resistant to chloroquine, amodiaquine, or piperaquine harbor mutations in the P. falciparum chloroquine resistance transporter (PfCRT), a transporter resident on the digestive vacuole membrane that in its variant forms can transport these weak-base 4-aminoquinoline drugs out of this acidic organelle, thus preventing these drugs from binding heme and inhibiting its detoxification. The structure of PfCRT, solved by cryogenic electron microscopy, shows mutations surrounding an electronegative central drug-binding cavity where they presumably interact with drugs and natural substrates to control transport. P. falciparum susceptibility to heme-binding antimalarials is also modulated by overexpression or mutations in the digestive vacuole membrane-bound ABC transporter PfMDR1 (P. falciparum multidrug resistance 1 transporter). Artemisinin resistance is primarily mediated by mutations in P. falciparum Kelch13 protein (K13), a protein involved in multiple intracellular processes including endocytosis of hemoglobin, which is required for parasite growth and artemisinin activation. Combating drug-resistant malaria urgently requires the development of new antimalarial drugs with novel modes of action.

Structure and drug resistance of the Plasmodium falciparum transporter PfCRT.

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.

Targeting NG2 relieves the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitors.

BRAFV600E represents a constitutively active onco-kinase and stands as the most prevalent genetic alteration in thyroid cancer. However, the clinical efficacy of small-molecule inhibitors targeting BRAFV600E is often limited by acquired resistance. Here, we find that nerve/glial antigen 2 (NG2), also known as chondroitin sulfate proteoglycan 4 (CSPG4), is up-regulated in thyroid cancers, and its expression is increased with tumor progression in a BRAFV600E-driven thyroid cancer mouse model. Functional studies show that NG2 knockout almost does not affect tumor growth, but significantly improves the response of BRAF-mutant thyroid cancer cells to BRAF inhibitor PLX4720. Mechanistically, the blockade of ERK-dependent feedback by BRAF inhibitor can activate receptor tyrosine kinase (RTK) signaling, causing the resistance to this inhibitor. NG2 knockout attenuates the PLX4720-mediated feedback activation of several RTKs, improving the sensitivity of BRAF-mutant thyroid cancer cells to this inhibitor. Based on this finding, we propose and demonstrate an alternative strategy for targeting NG2 to effectively treat BRAF-mutant thyroid cancers by combining multiple kinase inhibitor (MKI) Sorafenib or Lenvatinib with PLX4720. Thus, this study uncovers a new mechanism in which NG2 contributes to the resistance of BRAF-mutant thyroid cancer cells to BRAF inhibitor, and provides a promising therapeutic option for BRAF-mutant thyroid cancers.

Pharmacological Improvement of Cystic Fibrosis Transmembrane Conductance Regulator Function Rescues Airway Epithelial Homeostasis and Host Defense in Children with Cystic Fibrosis.

Rationale: Pharmacological improvement of cystic fibrosis transmembrane conductance regulator (CFTR) function with elexacaftor/tezacaftor/ivacaftor (ETI) provides unprecedented improvements in lung function and other clinical outcomes in patients with cystic fibrosis (CF). However, ETI effects on impaired mucosal homeostasis and host defense at the molecular and cellular levels in the airways of patients with CF remain unknown. Objectives: To investigate effects of ETI on the transcriptome of nasal epithelial and immune cells from children with CF at the single-cell level. Methods: Nasal swabs from 13 children with CF and at least one F508del allele aged 6 to 11 years were collected at baseline and 3 months after initiation of ETI, subjected to single-cell RNA sequencing, and compared with swabs from 12 age-matched healthy children. Measurements and Main Results: Proportions of CFTR-positive cells were decreased in epithelial basal, club, and goblet cells, but not in ionocytes, from children with CF at baseline and were restored by ETI therapy to nearly healthy levels. Single-cell transcriptomics revealed an impaired IFN signaling and reduced expression of major histocompatibility complex classes I and II encoding genes in epithelial cells of children with CF at baseline, which was partially restored by ETI. In addition, ETI therapy markedly reduced the inflammatory phenotype of immune cells, particularly of neutrophils and macrophages. Conclusions: Pharmacological improvement of CFTR function improves innate mucosal immunity and reduces immune cell inflammatory responses in the upper airways of children with CF at the single-cell level, highlighting the potential to restore epithelial homeostasis and host defense in CF airways by early initiation of ETI therapy.

Identification of potent BRD4-BD1 inhibitors using classical and steered molecular dynamics based free energy analysis.

In the present work a combination of traditional and steered molecular dynamics based techniques were employed to identify potential inhibitors against the human BRD4 protein (BRD4- BD1); an established drug target for multiple illnesses including various malignancies. Quinoline derivatives that were synthesized in-house were tested for their potential as new BRD4-BD1 inhibitors. Initially molecular docking experiments were performed to determine the binding poses of BRD4-BD1 inhibitors. To learn more about the thermodynamics of inhibitor binding to the BRD4-BD1 active site, the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) free energy calculations were conducted afterwards. The findings of the MM-PBSA analysis were further reinforced by performing steered umbrella sampling simulations which revealed crucial details about the binding/unbinding process of the most potent quinoline derivatives at the BRD4-BD1 active site. We report a novel quinoline derivative which can be developed into a fully functional BRD4-BD1 inhibitor after experimental validation. The identified compound (4 g) shows better properties than the standard BRD4-BD1 inhibitors considered in the study. The study also highlights the crucial role of Gln78, Phe79, Trp81, Pro82, Phe83, Gln84, Gln85, Val87, Leu92, Leu94, Tyr97, Met105, Cys136, Asn140, Ile146 and Met149 in inhibitor binding. The study provides a possible lead candidate and key amino acids involved in inhibitor recognition and binding at the active site of BRD4-BD1 protein. The findings might be of significance to medicinal chemists involved in the development of potent BRD4-BD1 inhibitors.

Neratinib could be effective as monotherapy or in combination with trastuzumab in HER2-low breast cancer cells and organoid models.

BACKGROUND: Previous studies have suggested that patients with HER2-low breast cancers do not benefit from trastuzumab treatment although the reasons remain unclear. METHODS: We investigated the effect of trastuzumab monotherapy and its combination with different HER2 targeting treatments in a panel of breast cancer cell lines and patient-derived organoids (PDOs) using biochemical methods and cell viability assays. RESULTS: Compared to sensitive HER2 over-expressing (IHC3 + ) breast cancer cells, increasing doses of trastuzumab could not achieve IC50 in MDA-MB-361 (IHC 2 + FISH + ) and MDA-MB-453 (IHC 2 + FISH-) cells which showed an intermediate response to trastuzumab. Trastuzumab treatment induced upregulation of HER ligand release, resulting in the activation of HER receptors in these cells, which could account for their trastuzumab insensitivity. Adding a dual ADAM10/17 inhibitor to inhibit the shedding of HER ligands in combination with trastuzumab only showed a modest decrease in the cell viability of HER2-low breast cancer cells and PDOs. However, the panHER inhibitor neratinib was an effective monotherapy in HER2-low breast cancer cells and PDOs, and showed additive effects when combined with trastuzumab. CONCLUSION: This study demonstrates that neratinib in combination with trastuzumab may be effective in a subset of HER2-low breast cancers although further validation is required in a larger panel of PDOs and in future clinical studies.

Extended blood stage sensitivity profiles of Plasmodium cynomolgi to doxycycline and tafenoquine, as a model for Plasmodium vivax.

Testing Plasmodium vivax antimicrobial sensitivity is limited to ex vivo schizont maturation assays, which preclude determining the IC50s of delayed action antimalarials such as doxycycline. Using Plasmodium cynomolgi as a model for P. vivax, we determined the physiologically significant delayed death effect induced by doxycycline [IC50(96 h), 1,401 ± 607 nM]. As expected, IC50(96 h) to chloroquine (20.4 nM), piperaquine (12.6 µM), and tafenoquine (1,424 nM) were not affected by extended exposure.

Aurora-A inhibitor synergistically enhances the inhibitory effect of anlotinib on hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor with a global prevalence. In addition to the existing clinical guidelines, the effectiveness of anlotinib and Aurora-A inhibitors in treating HCC has also been demonstrated. However, Anlotinib, as an anti-angiogenesis therapy, has shown significant benefits in clinical trials but is limited by its single-agent treatment and the development of drug resistance. Aurora-A inhibitors are currently being tested in clinical trials but have limited efficacy. Combination therapy may offer clear advantages over monotherapy in this context. METHODS: In this study, we used HCC cell lines to investigate whether the combination of the two drugs could enhance their individual strengths and mitigate their weaknesses, thereby providing greater clinical benefits both in vitro and in vivo. RESULTS: Our findings confirmed that the Aurora-A inhibitor alisertib and anlotinib exhibited a time-dose-dependent inhibitory effect on HCC cells. In vitro cytological experiments demonstrated that the combination of the two drugs synergistically inhibited cell proliferation, invasion, and metastasis, while promoting cell apoptosis. Furthermore, we identified the underlying molecular mechanism by which the combination of the Aurora-A inhibitor alisertib and anlotinib inhibited HCC through the inhibition of the NF-ĸB signaling pathway. CONCLUSIONS: In summary, we have demonstrated the effectiveness of combining anlotinib with an Aurora-A inhibitor, which expands the potential applications of anlotinib in the clinical treatment of HCC in the future.

Repurposing of FDA approved kinase inhibitor bosutinib for mitigation of radiation induced damage via inhibition of JNK pathway.

Radiotherapy is a common modality for cancer treatment. However, it is often associated with normal tissue toxicity in 20-80% of the patients. Radioprotectors can improve the outcome of radiotherapy by selectively protecting normal cells against radiation toxicity. In the present study, compound libraries containing 54 kinase inhibitors and 80 FDA-approved drugs were screened for radioprotection of lymphocytes using high throughput cell analysis. A second-generation FDA-approved kinase inhibitor, bosutinib, was identified as a potential radioprotector for normal cells. The radioprotective efficacy of bosutinib was evinced from a reduction in radiation induced DNA damage, caspase-3 activation, DNA fragmentation and apoptosis. Oral administration of bosutinib protected mice against whole body irradiation (WBI) induced morbidity and mortality. Bosutinib also reduced radiation induced bone-marrow aplasia and hematopoietic damage in mice exposed to 4 Gy and 6 Gy dose of WBI. Mechanistic studies revealed that the radioprotective action of bosutinib involved interaction with cellular thiols and modulation of JNK pathway. The addition of glutathione and N-acetyl cysteine significantly reduced the radioprotective efficacy of bosutinib. Moreover, bosutinib did not protect cancer cells against radiation induced toxicity. On the contrary, bosutinib per se exhibited anticancer activity against human cancer cell lines. The results highlight possible use of bosutinib as a repurposable radioprotective agent for mitigation of radiation toxicity in cancer patients undergoing radiotherapy.

GCN2 kinase activation by ATP-competitive kinase inhibitors.

Small-molecule kinase inhibitors represent a major group of cancer therapeutics, but tumor responses are often incomplete. To identify pathways that modulate kinase inhibitor response, we conducted a genome-wide knockout (KO) screen in glioblastoma cells treated with the pan-ErbB inhibitor neratinib. Loss of general control nonderepressible 2 (GCN2) kinase rendered cells resistant to neratinib, whereas depletion of the GADD34 phosphatase increased neratinib sensitivity. Loss of GCN2 conferred neratinib resistance by preventing binding and activation of GCN2 by neratinib. Several other Food and Drug Administration (FDA)-approved inhibitors, such erlotinib and sunitinib, also bound and activated GCN2. Our results highlight the utility of genome-wide functional screens to uncover novel mechanisms of drug action and document the role of the integrated stress response (ISR) in modulating the response to inhibitors of oncogenic kinases.

Molecular markers of artemisinin resistance during falciparum malaria elimination in Eastern Myanmar.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum threatens global malaria elimination efforts. To contain and then eliminate artemisinin resistance in Eastern Myanmar a network of community-based malaria posts was instituted and targeted mass drug administration (MDA) with dihydroartemisinin-piperaquine (three rounds at monthly intervals) was conducted. The prevalence of artemisinin resistance during the elimination campaign (2013-2019) was characterized. METHODS: Throughout the six-year campaign Plasmodium falciparum positive blood samples from symptomatic patients and from cross-sectional surveys were genotyped for mutations in kelch-13-a molecular marker of artemisinin resistance. RESULT: The program resulted in near elimination of falciparum malaria. Of 5162 P. falciparum positive blood samples genotyped, 3281 (63.6%) had K13 mutations. The prevalence of K13 mutations was 73.9% in 2013 and 64.4% in 2019. Overall, there was a small but significant decline in the proportion of K13 mutants (p < 0.001). In the MDA villages there was no significant change in the K13 proportions before and after MDA. The distribution of different K13 mutations changed substantially; F446I and P441L mutations increased in both MDA and non-MDA villages, while most other K13 mutations decreased. The proportion of C580Y mutations fell from 9.2% (43/467) before MDA to 2.3% (19/813) after MDA (p < 0.001). Similar changes occurred in the 487 villages where MDA was not conducted. CONCLUSION: The malaria elimination program in Kayin state, eastern Myanmar, led to a substantial reduction in falciparum malaria. Despite the intense use of artemisinin-based combination therapies, both in treatment and MDA, this did not select for artemisinin resistance.