Ivosidenib in Chinese patients with relapsed or refractory isocitrate dehydrogenase 1 mutated acute myeloid leukemia: a registry study.

Ivosidenib, an isocitrate dehydrogenase 1 (IDH1) inhibitor, has demonstrated clinical benefits in a pivotal study (AG120-C-001) in patients with IDH1-mutated (mIDH1) acute myeloid leukemia (AML). A registry study (CS3010-101: NCT04176393) was conducted to assess the pharmacokinetic (PK) characteristics, safety, and efficacy of ivosidenib in Chinese patients with relapsed or refractory (R/R) mIDH1 AML. Patients received ivosidenib 500 mg once daily for 28-day cycles until disease progression. Ten subjects underwent intensive PK/progressive disease (PD) assessments. All subjects had the clinical response assessed at screening, every 28 days through month 12, and then every 56 days. Between November 12, 2019, and April 2, 2021, 30 patients were enrolled; 26 (86.7%) had de novo AML and 18 (60.0%) were transfusion-dependent at baseline. Following single and repeated doses of ivosidenib, median time to maximum plasma concentration (T max) was 4.0 and 2.0 hours, respectively. The inter-individual variability of pharmacokinetic exposure was moderate to high (coefficient of variation [CV], 25%-53%). No obvious accumulation was observed after repeated doses at cycle 2 day 1. Regarding the clinical response, the CR + CRh rate was 36.7% (95% confidence interval [CI]: 19.9%-56.1%), the median duration of CR + CRh was 19.7 months (95% CI: 2.9 months-not reached [NR]), and median duration of response (DoR) was 14.3 months (95% CI: 6.4 months-NR). Consistent clinical benefits and safety of ivosidenib were consistently observed at the final data cutoff with median follow-up time 26.0 months, as compared with primary data cutoff, and the data from Chinese R/R mIDH1 AML patients were also consistent with results from pivotal study.

A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 T cells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.

Identification and characterization of yellow stripe-like genes in maize suggest their roles in the uptake and transport of zinc and iron.

BACKGROUND: Yellow Stripe-Like (YSL) proteins are involved in the uptake and transport of metal ions. They play important roles in maintaining the zinc and iron homeostasis in Arabidopsis, rice (Oryza sativa), and barley (Hordeum vulgare). However, proteins in this family have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: In this study, we identified 19 ZmYSLs in the maize genome and analyzed their structural features. The results of a phylogenetic analysis showed that ZmYSLs are homologous to YSLs of Arabidopsis and rice, and these proteins are divided into four independent branches. Although their exons and introns have structural differences, the motif structure is relatively conserved. Analysis of the cis-regulatory elements in the promoters indicated that ZmYSLs might play a role in response to hypoxia and light. The results of RNA sequencing and quantitative real-time PCR analysis revealed that ZmYSLs are expressed in various tissues and respond differently to zinc and iron deficiency. The subcellular localization of ZmYSLs in the protoplast of maize mesophyll cells showed that they may function in the membrane system. CONCLUSIONS: This study provided important information for the further functional analysis of ZmYSL, especially in the spatio-temporal expression and adaptation to nutrient deficiency stress. Our findings provided important genes resources for the maize biofortification.

ZmGluTR1 is involved in chlorophyll biosynthesis and is essential for maize development.

Chlorophyll is the most important carrier of photosynthesis in plants and is therefore vital for plant growth and development. Synthesis of 5-aminolevulinic acid (ALA) is initiated and catalyzed by glutamyl-tRNA reductase (GluTR) and is the rate-limiting step in chlorophyll biosynthesis. GluTR is controlled by several regulating factors. Although many studies have investigated the structure and function of GluTR in plants, the maize (Zea mays L.) GluTR has not yet been reported. Here, we isolated and identified the first loss-of-function mutant of GluTR in plants from a maize mutagenic population. The stop-gain mutation in ZmGluTR1 resulted in leaf etiolation throughout the growing season. The level of intermediates of chlorophyll biosynthesis and photosynthetic pigments decreased markedly and abnormal chloroplast structure was also observed in the mutants. Further analysis revealed that the deletion of carboxyl terminal (C-terminal) led to premature transcription termination and this hindered the interaction with FLUORESCENT (FLU), thereby influencing the stability of mutated ZmGluTR1 and leading to abolish interaction with GluTR-binding protein (GluBP). Moreover, mutations in the catalytic domain or nicotinamide adenine dinucleotide phosphate (NADPH) binding domain were lethal under normal growth conditions. These results indicate that ZmGluTR1 plays a fundamental role in chlorophyll biosynthesis and maize development.

Interim survival analysis of the randomized phase III GEMSTONE-302 trial: sugemalimab or placebo plus chemotherapy as first-line treatment for metastatic NSCLC.

The randomized, double-blinded, multi-center, phase III GEMSTONE-302 ( NCT03789604 ) study evaluated the efficacy and safety of sugemalimab versus placebo in combination with chemotherapy as first-line treatment for metastatic non-small-cell lung cancer (NSCLC). In this study, 479 treatment-naive patients with stage IV squamous or non-squamous NSCLC without known EGFR sensitizing mutations, ALK, ROS1 or RET fusions were randomized (2:1) to receive 1,200 mg of sugemalimab (n = 320) or placebo (n = 159) every 3 weeks in combination with platinum-based chemotherapy for up to four cycles, followed by maintenance therapy with sugemalimab or placebo for squamous NSCLC and sugemalimab or placebo plus pemetrexed for non-squamous NSCLC. Placebo-treated patients could cross over to receive sugemalimab monotherapy on disease progression. The primary endpoint was investigator-assessed progression-free survival (PFS) and the secondary endpoints included overall survival (OS) and objective response rate. Sugemalimab plus chemotherapy has demonstrated significant PFS prolongation in the primary analysis as reported previously. As of 22 November 2021, the prespecified interim OS analysis showed significant improvement with the addition of sugemalimab to chemotherapy (median OS = 25.4 versus 16.9 months; hazard ratio = 0.65; 95% confidence interval = 0.50-0.84; P = 0.0008). Sugemalimab plus chemotherapy provided superior PFS and OS compared to placebo plus chemotherapy, supporting the use of sugemalimab as a first-line treatment option for metastatic NSCLC.

Co-expression of transcription factors ZmC1 and ZmR2 establishes an efficient and accurate haploid embryo identification system in maize.

Because of their high efficiency during chromosome doubling, immature haploid maize (Zea mays L.) embryos are useful for doubled haploid production. The R1-nj marker is commonly used in doubled haploid breeding and has improved the efficiency of haploid identification. However, its effectiveness is limited by genetic background and environmental factors. We addressed this technical challenge by developing an efficient and accurate haploid embryo identification marker through co-expression of two transcription factor genes (ZmC1 and ZmR2) driven by the embryo-aleurone-specific bidirectional promoter PZmBD1 ; these factors can activate anthocyanin biosynthesis in the embryo and aleurone layer during early seed development. We developed a new haploid inducer, Maize Anthocyanin Gene InduCer 1 (MAGIC1), by introducing the transgenes into the haploid inducer line CAU6. MAGIC1 could identify haploids at 12 days after pollination, which is nine days earlier than CAU6. Importantly, MAGIC1 increased haploid identification accuracy to 99.1%, compared with 88.3% for CAU6. In addition, MAGIC1 could effectively overcome the inhibition of anthocyanin synthesis in some germplasms. Furthermore, an upgraded anthocyanin marker was developed from ZmC1 and ZmR2 to generate MAGIC2, which could identify haploids from diploids due to differential anthocyanin accumulation in immature embryos, coleoptiles, sheaths, roots, leaves, and dry seeds. This haploid identification system is more efficient and accurate than the conventional R1-nj-based method, and it simplifies the haploid identification process. Therefore, this system provides technical support for large-scale doubled haploid line production.

Mediation of Zinc and Iron Accumulation in Maize by ZmIRT2, a Novel Iron-Regulated Transporter.

Iron (Fe) is an essential micronutrient for plant growth. Iron-regulated transporters (IRTs) play important roles in Fe2+ uptake and transport in strategy I plants. Maize (Zea mays) belongs to a strategy II plant, in which mugineic acid (MA)-Fe3+ uptake is mainly carried out by Yellow Stripe 1 (YS1). However, ZmIRT1 was previously identified by our laboratory. In this study, we isolated a novel gene from maize (ZmIRT2), which is highly homologous to OsIRT2 and ZmIRT1. ZmIRT2 was expressed in roots and anther and was induced by Fe and zinc (Zn) deficiencies. ZmIRT2-GFP fusion protein localized to the plasma membrane and endoplasmic reticulum. ZmIRT2 reversed growth defects involving Zn and Fe uptake in mutant yeast. ZmIRT2 overexpression in maize led to elevated Zn and Fe levels in roots, shoots and seeds of transgenic plants. Transcript levels of ZmIRT1 were elevated in roots, while levels of YS1 were reduced in shoots of ZmIRT2 transgenic plants. Our results imply that ZmIRT2 may function solely with ZmIRT1 to mediate Fe uptake in roots. ZmIRT1, ZmIRT2 and ZmYS1 may function in a cooperative manner to maintain Zn and Fe homeostasis in ZmIRT2 overexpressing plants. Furthermore, ZmIRT2 could be used in fortification efforts to elevate Zn and Fe levels in crop plants.

Pentatricopeptide repeat protein CNS1 regulates maize mitochondrial complex III assembly and seed development.

Mitochondrial function relies on the assembly of electron transport chain complexes, which requires coordination between proteins encoded by the mitochondrion and those of the nucleus. Here, we cloned a maize (Zea mays) cytochrome c maturation FN stabilizer1 (CNS1) and found it encodes a pentatricopeptide repeat (PPR) protein. Members of the PPR family are widely distributed in plants and are associated with RNA metabolism in organelles. P-type PPR proteins play essential roles in stabilizing the 3'-end of RNA in mitochondria; whether a similar process exists for stabilizing the 5'-terminus of mitochondrial RNA remains unclear. The kernels of cns1 exhibited arrested embryo and endosperm development, whereas neither conventional splicing deficiency nor RNA editing difference in mitochondrial genes was observed. Instead, most of the ccmFN transcripts isolated from cns1 mutant plants were 5'-truncated and therefore lacked the start codon. Biochemical and molecular data demonstrated that CNS1 is a P-type PPR protein encoded by nuclear DNA and that it localizes to the mitochondrion. Also, one binding site of CNS1 located upstream of the start codon in the ccmFN transcript. Moreover, abnormal mitochondrial morphology and dramatic upregulation of alternative oxidase genes were observed in the mutant. Together, these results indicate that CNS1 is essential for reaching a suitable level of intact ccmFN transcripts through binding to the 5'-UTR of the RNAs and maintaining 5'-integrity, which is crucial for sustaining mitochondrial complex III function to ensure mitochondrial biogenesis and seed development in maize.

Maize Interveinal Chlorosis 1 links the Yang Cycle and Fe homeostasis through Nicotianamine biosynthesis.

The Yang cycle is involved in many essential metabolic pathways in plant growth and development. As extended products of the Yang cycle, the function and regulation network of ethylene and polyamines are well characterized. Nicotianamine (NA) is also a product of this cycle and works as a key metal chelator for iron (Fe) homeostasis in plants. However, interactions between the Yang cycle and NA biosynthesis remain unclear. Here, we cloned maize interveinal chlorosis 1 (mic1), encoding a 5'-methylthioadenosine nucleosidase (MTN), that is essential for 5'-methylthioadenosine (MTA) salvage and NA biosynthesis in maize (Zea mays). A single base G-A transition in the fourth exon of mic1 causes a Gly to Asp change, resulting in increased MTA, reduced Fe distribution, and growth retardation of seedlings. Knockout of ZmMIC1 but not its paralog ZmMTN2 by CRISPR/Cas9 causes interveinal chlorosis, indicating ZmMIC1 is mainly responsible for MTN activity in maize. Transcriptome analysis showed a typical response of Fe deficiency. However, metabolic analysis revealed dramatically reduced NA content in mic1, suggesting NA biosynthesis was impaired in the mutant. Exogenous application of NA transiently reversed the interveinal chlorosis phenotype of mic1 seedlings. Moreover, the mic1 mutant overexpressing a NA synthase gene not only recovered from interveinal chlorosis and growth retardation but was also fertile. These findings provide a link between the Yang cycle and NA biosynthesis, which highlights an aspect of Fe homeostasis regulation in maize.

Genome-wide analysis of the NAAT, DMAS, TOM, and ENA gene families in maize suggests their roles in mediating iron homeostasis.

BACKGROUND: Nicotianamine (NA), 2'-deoxymugineic acid (DMA), and mugineic acid (MA) are chelators required for iron uptake and transport in plants. Nicotianamine aminotransferase (NAAT), 2'-deoxymugineic acid synthase (DMAS), transporter of MAs (TOM), and efflux transporter of NA (ENA) are involved in iron uptake and transport in rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare); however, these families have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: Here, we identified 5 ZmNAAT, 9 ZmDMAS, 11 ZmTOM, and 2 ZmENA genes by genome mining. RNA-sequencing and quantitative real-time PCR analysis revealed that these genes are expressed in various tissues and respond differently to high and low iron conditions. In particular, iron deficiency stimulated the expression of ZmDMAS1, ZmTOM1, ZmTOM3, and ZmENA1. Furthermore, we determined protein subcellular localization by transient expression of green fluorescent protein fusions in maize mesophyll protoplasts. ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1 localized in the cytoplasm, whereas ZmTOMs and ZmENAs targeted to plasma and tonoplast membranes, endomembranes, and vesicles. CONCLUSIONS: Our results suggest that the different gene expression profiles and subcellular localizations of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA family members may enable specific regulation of phytosiderophore metabolism in different tissues and under different external conditions, shedding light on iron homeostasis in maize and providing candidate genes for breeding iron-rich maize varieties.

ZmGRAS11, transactivated by Opaque2, positively regulates kernel size in maize.

Although the genetic basis for endosperm development in maize (Zea mays) has been well studied, the mechanism for coordinating grain filling with increasing kernel size remains elusive. Here, we report that increased kernel size was selected during modern breeding and identify a novel DELLA-like transcriptional regulator, ZmGRAS11, which positively regulates kernel size and kernel weight in maize. We find that Opaque2, a core transcription factor for zein protein and starch accumulation, transactivates the expression of ZmGRAS11. Our data suggest that the Opaque2-ZmGRAS11 module mediates synergistic endosperm enlargement with grain filling.

Metabolic engineering of astaxanthin-rich maize and its use in the production of biofortified eggs.

Production of the high-value carotenoid astaxanthin, which is widely used in food and feed due to its strong antioxidant activity and colour, is less efficient in cereals than in model plants. Here, we report a new strategy for expressing ß-carotene ketolase and hydroxylase genes from algae, yeasts and flowering plants in the whole seed using a seed-specific bidirectional promoter. Engineered maize events were backcrossed to inbred maize lines with yellow endosperm to generate progenies that accumulate astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than those in previous reports (16.2-16.8 mg/kg DW) in cereals. A feeding trial with laying hens indicated that they could take up astaxanthin from the maize and accumulate it in egg yolks (12.10-14.15 mg/kg) without affecting egg production and quality, as observed using astaxanthin from Haematococcus pluvialis. Storage stability evaluation analysis showed that the optimal conditions for long-term storage of astaxanthin-rich maize are at 4 °C in the dark. This study shows that co-expressing of functional genes driven by seed-specific bidirectional promoter could dramatically boost astaxanthin biosynthesis in every parts of kernel including embryo, aleurone layer and starch endosperm other than previous reports in the starch endosperm only. And the staple crop maize could serve as a cost-effective plant factory for reliably producing astaxanthin.

The thick aleurone1 Gene Encodes a NOT1 Subunit of the CCR4-NOT Complex and Regulates Cell Patterning in Endosperm.

Maize (Zea mays) thick aleurone1 (thk1-R) mutants form multiple aleurone layers in the endosperm and have arrested embryogenesis. Prior studies suggest that thk1 functions downstream of defective kernel1 (dek1) in a regulatory pathway that controls aleurone cell fate and other endosperm traits. The original thk1-R mutant contained an ∼2-Mb multigene deletion, which precluded identification of the causal gene. Here, ethyl methanesulfonate mutagenesis produced additional alleles, and RNA sequencing from developing endosperm was used to identify a candidate gene based on differential expression compared with the wild-type progenitor. Gene editing confirmed the gene identity by producing mutant alleles that failed to complement existing thk1 mutants and that produced multiple-aleurone homozygous phenotypes. Thk1 encodes a homolog of NEGATIVE ON TATA-LESS1, a protein that acts as a scaffold for the CARBON CATABOLITE REPRESSION4-NEGATIVE ON TATA-LESS complex. This complex is highly conserved and essential in all eukaryotes for regulating a wide array of gene expression and cellular activities. Maize also harbors a duplicate locus, thick aleurone-like1, which likely accounts for the ability of thk1 mutants to form viable cells. Transcriptomic analysis indicated that THK1 regulates activities involving cell division, signaling, differentiation, and metabolism. Identification of thk1 provides an important new component of the DEK1 regulatory system that patterns cell fate in endosperm.

Overexpression of the maize γ-tocopherol methyltransferase gene (ZmTMT) increases α-tocopherol content in transgenic Arabidopsis and maize seeds.

The vitamin E family includes tocopherols and tocotrienols, which are essential lipid-soluble antioxidants necessary for human and livestock health. The seeds of many plant species, including maize, have high gamma (γ)-tocopherol but low alpha (α)-tocopherol contents; however, α-tocopherol is the most effective antioxidant. Therefore, it is necessary to optimize the tocopherol composition in plants. α-Tocopherol is synthesized from γ-tocopherol by γ-tocopherol methyltransferase (γ-TMT, VTE4) in the final step of the tocopherol biosynthetic pathway. In the present study, the full-length coding sequence (CDS) of γ-TMT was isolated from Zea mays, named ZmTMT. The ZmTMT CDS was 1059 bp in size, encoding 352 amino acids. Recombinant ZmTMT was expressed in Escherichia coli and the purified protein effectively converted γ-tocopherol into α-tocopherol in vitro. A comparison of enzyme activities showed that the activity of ZmTMT was higher than that of GmTMT2a (Glycine max) and AtTMT (Arabidopsis thaliana). Overexpression of ZmTMT increased the α-tocopherol content 4-5-fold in transgenic Arabidopsis and around 6.5-fold in transgenic maize kernels, and increased the α-/γ-tocopherol ratio to approximately 15 and 17, respectively. These results show that it is feasible to overexpress ZmTMT to optimize the tocopherol composition in maize; such a corn product might be useful in the feed industry in the near future.

Genome-scale mining of root-preferential genes from maize and characterization of their promoter activity.

BACKGROUND: Modification of root architecture and improvement of root resistance to stresses can increase crop productivity. Functional analyses of root-specific genes are necessary for root system improvement, and root-specific promoters enable research into the regulation of root development and genetic manipulation of root traits. Maize is an important crop species; however, little systematic mining of root-specific genes and promoters has been performed to date. RESULTS: Genomic-scale mining based on microarray data sets followed by transcript detection resulted in the identification of 222 root-specific genes. Gene Ontology enrichment analyses revealed that these 222 root-specific genes were mainly involved in responses to chemical, biotic, and abiotic stresses. Of the 222 genes, 33 were verified by quantitative reverse transcription polymerase chain reaction, and 31 showed root-preferential activity. About 2 kb upstream 5 of the 31 identified root-preferential genes were cloned from the maize genome as putative promoters and named p8463, p5023, p1534, p8531 and p6629. GUS staining of transgenic maize-derived promoter-GUS constructs revealed that the five promoters drove GUS expression in a root-preferential manner. CONCLUSIONS: We mined root-preferential genes and their promoters in maize and verified p8463, p5023, p1534, p8531 and p6629 as root-preferential promoters. Our research enables the identification of other tissue-specific genes and promoters in maize and other species. In addition, the five promoters may enable enhancement of target gene(s) of maize in a root-preferential manner to generate novel maize cultivars with resistance to water, fertilizer constraints, or biotic stresses.

Pharmacological Inhibition of TFF3 Enhances Sensitivity of CMS4 Colorectal Carcinoma to 5-Fluorouracil through Inhibition of p44/42 MAPK.

Increased expression of trefoil factor 3 (TFF3) has been reported in colorectal carcinoma (CRC), being correlated with distant metastasis and poor clinical outcomes. Amongst the CRC subtypes, mesenchymal (CMS4) CRC is associated with the worst survival outcome. Herein, the functional roles of TFF3 and the pharmacological inhibition of TFF3 by a novel specific small molecule TFF3 inhibitor-2-amino-4-(4-(6-fluoro-5-methylpyridin-3-yl)phenyl)-5-oxo-4H,5H-pyrano[3,2-c]chromene-3-carbonitrile (AMPC) in CMS4 CRC was explored. Forced expression of TFF3 in CMS4 CRC cells promoted cell proliferation, cell survival, foci formation, invasion, migration, cancer stem cell like behaviour and growth in 3D Matrigel. In contrast, siRNA-mediated depletion of TFF3 or AMPC inhibition of TFF3 in CMS4 CRC cells decreased oncogenic behaviour as indicated by the above cell function assays. AMPC also inhibited tumour growth in vivo. The TFF3-stimulated oncogenic behaviour of CMS4 CRC cells was dependent on TFF3 activation of the p44/42 MAPK (ERK1/2) pathway. Furthermore, the forced expression of TFF3 decreased the sensitivity of CMS4 CRC cells to 5-fluorouracil (5-FU); while depleted TFF3 expression enhanced 5-FU sensitivity in CMS4 CRC cells. 5-FU treatment induced TFF3 expression in CMS4 CRC cells. AMPC, when used in combination with 5-FU in CMS4 CRC cells exhibited a synergistic inhibitory effect. In summary, this study provides functional evidence for TFF3 as a therapeutic target in CMS4 CRC.

A novel small-molecule inhibitor of trefoil factor 3 (TFF3) potentiates MEK1/2 inhibition in lung adenocarcinoma.

TFF3 has been identified as a novel biomarker to distinguish between lung adenocarcinoma (ADC) and lung squamous-cell carcinoma (SCC). Herein, we determined the oncogenic functions of TFF3 and demonstrated the potential of pharmacological inhibition of TFF3 in lung ADC using a novel small-molecule inhibitor of TFF3 dimerization (AMPC). Forced expression of TFF3 in lung ADC cells enhanced cell proliferation and survival, increased anchorage-independent growth, cancer stem cell behavior, growth in 3D Matrigel, and cell migration and invasion. In contrast, depleted expression of TFF3 suppressed these cellular functions. Mechanistically, TFF3 exerted its oncogenic function through upregulation of ARAF and hence enhanced downstream activation of MEK1/2 and ERK1/2. Pharmacological inhibition of TFF3 by AMPC, resulted in markedly decreased cell survival, proliferation, 3D growth and foci formation, and impaired tumor growth in a xenograft mouse model. Moreover, the combination of various MEK1/2 inhibitors with AMPC exhibited synergistic inhibitory effects on lung ADC cell growth. In conclusion, this study provides the first evidence that TFF3 is a potent promoter of lung ADC progression. Targeting TFF3 with a novel small-molecule inhibitor alone or in combination with conventional MEK1/2 inhibitors are potential strategies to improve the outcome of lung ADC.

Inhibition of TFF3 Enhances Sensitivity-and Overcomes Acquired Resistance-to Doxorubicin in Estrogen Receptor-Positive Mammary Carcinoma.

Dose-dependent toxicity and acquired resistance are two major challenges limiting the efficacious treatment of mammary carcinoma (MC) with doxorubicin. Herein, we investigated the function of Trefoil Factor 3 (TFF3) in the sensitivity and acquired resistance of estrogen receptor positive (ER+) MC cells to doxorubicin. Doxorubicin treatment of ER+MC cells increased TFF3 expression. The depletion of TFF3 by siRNA or inhibition with a small molecule TFF3 inhibitor (AMPC) synergistically enhanced the efficacy of doxorubicin in ER+MC through the suppression of doxorubicin-induced AKT activation and enhancement of doxorubicin-induced apoptosis. Elevated expression of TFF3 and increased activation of AKT were also observed using a model of acquired doxorubicin resistance in ER+MC cells. AMPC partially re-sensitized the doxorubicin resistant cells to doxorubicin-induced apoptosis. Indeed, doxorubicin resistant ER + MC cells exhibited increased sensitivity to AMPC as a single agent compared to doxorubicin sensitive cells. In vivo, AMPC attenuated growth of doxorubicin sensitive ER+MC xenografts whereas it produced regression of xenografts generated by doxorubicin resistant ER+MC cells. Hence, TFF3 inhibition may improve the efficacy and reduce required doses of doxorubicin in ER+MC. Moreover, inhibition of TFF3 may also be an effective therapeutic strategy to eradicate doxorubicin resistant ER+MC.