Biodiversity Medicine: New Horizon and New Opportunity for Cancer.

SUMMARY: Accessibility to standard of care remains a challenge to patients in low- and middle-income countries (LMIC), hampering efforts to alleviate the burden of cancer and to improve overall health outcomes. In response to this pressing global health care issue, we propose here a new strategy to create affordable, easily accessible, and effective therapeutic solutions to address this inequity in cancer treatment: the use of science-based biodiversity medicine as an alternative to modern drug therapy, in which we will leverage and combine high-throughput omics technologies with artificial intelligence, to study local biodiversity, their potential anticancer properties, and short- and long-term clinical response and outcomes.

Cholangiocarcinoma: Recent Advances in Molecular Pathobiology and Therapeutic Approaches.

Cholangiocarcinomas (CCA) pose a complex challenge in oncology due to diverse etiologies, necessitating tailored therapeutic approaches. This review discusses the risk factors, molecular pathology, and current therapeutic options for CCA and explores the emerging strategies encompassing targeted therapies, immunotherapy, novel compounds from natural sources, and modulation of gut microbiota. CCA are driven by an intricate landscape of genetic mutations, epigenetic dysregulation, and post-transcriptional modification, which differs based on geography (e.g., for liver fluke versus non-liver fluke-driven CCA) and exposure to environmental carcinogens (e.g., exposure to aristolochic acid). Liquid biopsy, including circulating cell-free DNA, is a potential diagnostic tool for CCA, which warrants further investigations. Currently, surgical resection is the primary curative treatment for CCA despite the technical challenges. Adjuvant chemotherapy, including cisplatin and gemcitabine, is standard for advanced, unresectable, or recurrent CCA. Second-line therapy options, such as FOLFOX (oxaliplatin and 5-FU), and the significance of radiation therapy in adjuvant, neoadjuvant, and palliative settings are also discussed. This review underscores the need for personalized therapies and demonstrates the shift towards precision medicine in CCA treatment. The development of targeted therapies, including FDA-approved drugs inhibiting FGFR2 gene fusions and IDH1 mutations, is of major research focus. Investigations into immune checkpoint inhibitors have also revealed potential clinical benefits, although improvements in survival remain elusive, especially across patient demographics. Novel compounds from natural sources exhibit anti-CCA activity, while microbiota dysbiosis emerges as a potential contributor to CCA progression, necessitating further exploration of their direct impact and mechanisms through in-depth research and clinical studies. In the future, extensive translational research efforts are imperative to bridge existing gaps and optimize therapeutic strategies to improve therapeutic outcomes for this complex malignancy.

Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies.

OBJECTIVES: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. DESIGN: Integrative multiomics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining and single-cell spatial transcriptomics were used to explore the immunogenicity of diverse CCA. RESULTS: We identified three distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumour growth in in vitro and in vivo models. The first group (ESTRO), with mostly fluke-positive CCAs, displayed activation in estrogen signalling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive to oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intratumour differences in AA mutation load were correlated to intratumour variation of different immune cell populations. CONCLUSION: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumourigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits.

Enhancer remodeling activates NOTCH3 signaling to confer chemoresistance in advanced nasopharyngeal carcinoma.

Acquired resistance to chemotherapy is one of the major causes of mortality in advanced nasopharyngeal carcinoma (NPC). However, effective strategies are limited and the underlying molecular mechanisms remain elusive. In this study, through transcriptomic profiling analysis of 23 tumor tissues, we found that NOTCH3 was aberrantly highly expressed in chemoresistance NPC patients, with NOTCH3 overexpression being positively associated with poor clinical outcome. Mechanistically, using an established NPC cellular model, we demonstrated that enhancer remodeling driven aberrant hyperactivation of NOTCH3 in chemoresistance NPC. We further showed that NOTCH3 upregulates SLUG to induce chemo-resistance of NPC cells and higher expression of SLUG have poorer prognosis. Genetic or pharmacological perturbation of NOTCH3 conferred chemosensitivity of NPC in vitro and overexpression of NOTCH3 enhanced chemoresistance of NPC in vivo. Together, these data indicated that genome-wide enhancer reprogramming activates NOTCH3 to confer chemoresistance of NPC, suggesting that targeting NOTCH3 may provide a potential therapeutic strategy to effectively treat advanced chemoresistant NPC.

Preclinical characterization of WB737, a potent and selective STAT3 inhibitor, in natural killer/T-cell lymphoma.

Natural killer/T-cell lymphoma (NKTL) is an uncommon malignancy with poor prognosis and limited therapeutic options. Activating mutations of signal transducer and activator of transcription 3 (STAT3) are frequently found in patients with NKTL, suggesting that targeted inhibition of STAT3 is a potential therapeutic option for this disease. Here, we have developed a small molecule drug WB737 as a novel and potent STAT3 inhibitor that directly binds to the STAT3-Src homology 2 domain with high affinity. In addition, the binding affinity of WB737 to STAT3 is 250-fold higher than STAT1 and STAT2. Interestingly, WB737 is more selective for NKTL with STAT3-activating mutations in terms of growth inhibition and apoptotic induction when compared with Stattic. Mechanistically, WB737 inhibits both canonical and noncanonical STAT3 signaling via suppression of STAT3 phosphorylation at Tyr705 and Ser727, respectively, thereby inhibiting the expression of c-Myc and mitochondria-related genes. Moreover, WB737 inhibited STAT3 more potently than Stattic, resulting in a significant antitumor effect with undetectable toxicity, followed by almost complete tumor regression in an NKTL xenograft model harboring a STAT3-activating mutation. Taken together, these findings provide preclinical proof-of-concept for WB737 as a novel therapeutic strategy for the treatment of NKTL patients with STAT3-activating mutations.

EZH2 mediated metabolic rewiring promotes tumor growth independently of histone methyltransferase activity in ovarian cancer.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2), the key catalytic subunit of polycomb repressive complex 2 (PRC2), is overexpressed and plays an oncogenic role in various cancers through catalysis-dependent or catalysis-independent pathways. However, the related mechanisms contributing to ovarian cancer (OC) are not well understood. METHODS: The levels of EZH2 and H3K27me3 were evaluated in 105 OC patients by immunohistochemistry (IHC) staining, and these patients were stratified based on these levels. Canonical and noncanonical binding sites of EZH2 were defined by chromatin immunoprecipitation sequencing (ChIP-Seq). The EZH2 solo targets were obtained by integrative analysis of ChIP-Seq and RNA sequencing data. In vitro and in vivo experiments were performed to determine the role of EZH2 in OC growth. RESULTS: We showed that a subgroup of OC patients with high EZH2 expression but low H3K27me3 exhibited the worst prognosis, with limited therapeutic options. We demonstrated that induction of EZH2 degradation but not catalytic inhibition profoundly blocked OC cell proliferation and tumorigenicity in vitro and in vivo. Integrative analysis of genome-wide chromatin and transcriptome profiles revealed extensive EZH2 occupancy not only at genomic loci marked by H3K27me3 but also at promoters independent of PRC2, indicating a noncanonical role of EZH2 in OC. Mechanistically, EZH2 transcriptionally upregulated IDH2 to potentiate metabolic rewiring by enhancing tricarboxylic acid cycle (TCA cycle) activity, which contributed to the growth of OC. CONCLUSIONS: These data reveal a novel oncogenic role of EZH2 in OC and identify potential therapeutic strategies for OC by targeting the noncatalytic activity of EZH2.

PBRM1-deficient PBAF complexes target aberrant genomic loci to activate the NF-κB pathway in clear cell renal cell carcinoma.

PBRM1 encodes an accessory subunit of the PBAF SWI/SNF chromatin remodeller, and the inactivation of PBRM1 is a frequent event in kidney cancer. However, the impact of PBRM1 loss on chromatin remodelling is not well examined. Here we show that, in VHL-deficient renal tumours, PBRM1 deficiency results in ectopic PBAF complexes that localize to de novo genomic loci, activating the pro-tumourigenic NF-κB pathway. PBRM1-deficient PBAF complexes retain the association between SMARCA4 and ARID2, but have loosely tethered BRD7. The PBAF complexes redistribute from promoter proximal regions to distal enhancers containing NF-κB motifs, heightening NF-κB activity in PBRM1-deficient models and clinical samples. The ATPase function of SMARCA4 maintains chromatin occupancy of pre-existing and newly acquired RELA specific to PBRM1 loss, activating downstream target gene expression. Proteasome inhibitor bortezomib abrogates RELA occupancy, suppresses NF-κB activation and delays growth of PBRM1-deficient tumours. In conclusion, PBRM1 safeguards the chromatin by repressing aberrant liberation of pro-tumourigenic NF-κB target genes by residual PBRM1-deficient PBAF complexes.

Aberrant JAK-STAT signaling-mediated chromatin remodeling impairs the sensitivity of NK/T-cell lymphoma to chidamide.

BACKGROUND: Natural killer/T-cell lymphoma (NKTL) is a rare type of aggressive and heterogeneous non-Hodgkin's lymphoma (NHL) with a poor prognosis and limited therapeutic options. Therefore, there is an urgent need to exploit potential novel therapeutic targets for the treatment of NKTL. Histone deacetylase (HDAC) inhibitor chidamide was recently approved for treating relapsed/refractory peripheral T-cell lymphoma (PTCL) patients. However, its therapeutic efficacy in NKTL remains unclear. METHODS: We performed a phase II clinical trial to evaluate the efficacy of chidamide in 28 relapsed/refractory NKTL patients. Integrative transcriptomic, chromatin profiling analysis and functional studies were performed to identify potential predictive biomarkers and unravel the mechanisms of resistance to chidamide. Immunohistochemistry (IHC) was used to validate the predictive biomarkers in tumors from the clinical trial. RESULTS: We demonstrated that chidamide is effective in treating relapsed/refractory NKTL patients, achieving an overall response and complete response rate of 39 and 18%, respectively. In vitro studies showed that hyperactivity of JAK-STAT signaling in NKTL cell lines was associated with the resistance to chidamide. Mechanistically, our results revealed that aberrant JAK-STAT signaling remodels the chromatin and confers resistance to chidamide. Subsequently, inhibition of JAK-STAT activity could overcome resistance to chidamide by reprogramming the chromatin from a resistant to sensitive state, leading to synergistic anti-tumor effect in vitro and in vivo. More importantly, our clinical data demonstrated that combinatorial therapy with chidamide and JAK inhibitor ruxolitinib is effective against chidamide-resistant NKTL. In addition, we identified TNFRSF8 (CD30), a downstream target of the JAK-STAT pathway, as a potential biomarker that could predict NKTL sensitivity to chidamide. CONCLUSIONS: Our study suggests that chidamide, in combination with JAK-STAT inhibitors, can be a novel targeted therapy in the standard of care for NKTL. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02878278. Registered 25 August 2016, https://clinicaltrials.gov/ct2/show/NCT02878278.

RAD21 amplification epigenetically suppresses interferon signaling to promote immune evasion in ovarian cancer.

Prevalent copy number alteration is the most prominent genetic characteristic associated with ovarian cancer (OV) development, but its role in immune evasion has not been fully elucidated. In this study, we identified RAD21, a key component of the cohesin complex, as a frequently amplified oncogene that could modulate immune response in OV. Through interrogating the RAD21-regulated transcriptional program, we found that RAD21 directly interacts with YAP/TEAD4 transcriptional corepressors and recruits the NuRD complex to suppress interferon (IFN) signaling. In multiple clinical cohorts, RAD21 overexpression is inversely correlated with IFN signature gene expression in OV. We further demonstrated in murine syngeneic tumor models that RAD21 ablation potentiated anti-PD-1 efficacy with increased intratumoral CD8+ T cell effector activity. Our study identifies a RAD21-YAP/TEAD4-NuRD corepressor complex in immune modulation, and thus provides a potential target and biomarker for precision immunotherapy in OV.

Therapeutic potential of tucidinostat, a subtype-selective HDAC inhibitor, in cancer treatment.

Histone deacetylase (HDAC) is one of the most characterized epigenetic modifiers, modulating chromatin structure and gene expression, which plays an important role in cell cycle, differentiation and apoptosis. Dysregulation of HDAC promotes cancer progression, thus inhibitors targeting HDACs have evidently shown therapeutic efficacy in multiple cancers. Tucidinostat (formerly known as chidamide), a novel subtype-selective HDAC inhibitor, inhibits Class I HDAC1, HDAC2, HDAC3, as well as Class IIb HDAC10. Tucidinostat is approved in relapsed or refractory (R/R) peripheral T-cell lymphoma (PTCL), advanced breast cancer and R/R adult T-cell leukemia-lymphoma (ATLL). Compared with other HDAC inhibitors, tucidinostat shows notable antitumor activity, remarkable synergistic effect with immunotherapy, and manageable toxicity. Here, we comprehensively summarize recent advances in tucidinostat as both monotherapy and a regimen of combination therapy in both hematological and solid malignancies in clinic. Further studies will endeavor to identify more combination strategies with tucidinostat and to identify specific clinical biomarkers to predict the therapeutic effect.

Therapeutic and immunomodulatory potential of pazopanib in malignant phyllodes tumor.

Malignant phyllodes tumors (PT) are rare aggressive fibroepithelial neoplasms with high metastatic potential and lack effective therapy. We established a patient-derived xenograft (PDX) and cell line model (designated MPT-S1) of malignant PT which demonstrated clinical response to pazopanib. Whole exome sequencing identified somatic mutations in TP53, RB1, MED12, and KMT2D. Immunohistochemistry and genomic profiles of the tumor, PDX and cell line were concordant. In keeping with clinical observation, pazopanib reduced cell viability in a dose-dependent manner and evoked apoptosis, and led to significant abrogation of in vivo tumor growth. Whole transcriptomic analysis revealed that pazopanib decreased expression of genes involved in oncogenic and apoptosis signaling. We also observed decreased expression of ENPP1, with known roles in cancer invasion and metastasis, as well as STING pathway upregulation. Accordingly, pazopanib induced micronuclei formation, and evoked phospho-TBK1 and PD-L1 expression. In an additional cohort of malignant PT (n = 14), six (42.9%) showed comparable or higher levels of ENPP1 relative to MPT-S1, highlighting its potential role as a therapeutic target. In conclusion, we established MPT-S1, a new PDX and cell line model, and provided evidence for the clinical efficacy of pazopanib in malignant PT.

Rare Occurrence of Aristolochic Acid Mutational Signatures in Oro-Gastrointestinal Tract Cancers.

BACKGROUND: Aristolochic acids (AAs) are potent mutagens commonly found in herbal plant-based remedies widely used throughout Asian countries. PATIENTS AND METHODS: To understand whether AA is involved in the tumorigenesis of the oro-gastrointestinal tract, we used whole-exome sequencing to profile 54 cases of four distinct types of oro-gastrointestinal tract cancer (OGITC) from Taiwan. RESULTS: A diverse landscape of mutational signatures including those from DNA mismatch repair and reactive oxygen species was observed. APOBEC mutational signatures were observed in 60% of oral squamous cell carcinomas. Only one sample harbored AA mutational signatures, contradictory to prior reports of cancers from Taiwan. The metabolism of AA in the liver and urinary tract, transient exposure time, and high cell turnover rates at OGITC sites may explain our findings. CONCLUSION: AA signatures in OGITCs are rare and unlikely to be a major contributing factor in oro-gastrointestinal tract tumorigenesis.

CREBBP cooperates with the cell cycle machinery to attenuate chidamide sensitivity in relapsed/refractory diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) exhibits frequent inactivating mutations of the histone acetyltransferase CREBBP, highlighting the attractiveness of targeting CREBBP deficiency as a therapeutic strategy. In this study, we demonstrate that chidamide, a novel histone deacetylase (HDAC) inhibitor, is effective in treating a subgroup of relapsed/refractory DLBCL patients, achieving an overall response rate (ORR) of 25.0% and a complete response (CR) rate of 15.0%. However, the clinical response to chidamide remains poor, as most patients exhibit resistance, hampering the clinical utility of the drug. Functional in vitro and in vivo studies have shown that CREBBP loss of function is correlated with chidamide sensitivity, which is associated with modulation of the cell cycle machinery. A combinatorial drug screening of 130 kinase inhibitors targeting cell cycle regulators identified AURKA inhibitors, which inhibit the G2/M transition during the cell cycle, as top candidates that synergistically enhanced the antitumor effects of chidamide in CREBBP-proficient DLBCL cells. Our study demonstrates that CREBBP inactivation can serve as a potential biomarker to predict chidamide sensitivity, while combination of an AURKA inhibitor and chidamide is a novel therapeutic strategy for the treatment of relapsed/refractory DLBCL.

Targeting enhancer reprogramming to mitigate MEK inhibitor resistance in preclinical models of advanced ovarian cancer.

Ovarian cancer is characterized by aberrant activation of the mitogen-activated protein kinase (MAPK), highlighting the importance of targeting the MAPK pathway as an attractive therapeutic strategy. However, the clinical efficacy of MEK inhibitors is limited by intrinsic or acquired drug resistance. Here, we established patient-derived ovarian cancer models resistant to MEK inhibitors and demonstrated that resistance to the clinically approved MEK inhibitor trametinib was associated with enhancer reprogramming. We also showed that enhancer decommissioning induced the downregulation of negative regulators of the MAPK pathway, leading to constitutive ERK activation and acquired resistance to trametinib. Epigenetic compound screening uncovered that HDAC inhibitors could alter the enhancer reprogramming and upregulate the expression of MAPK negative regulators, resulting in sustained MAPK inhibition and reversal of trametinib resistance. Consequently, a combination of HDAC inhibitor and trametinib demonstrated a synergistic antitumor effect in vitro and in vivo, including patient-derived xenograft mouse models. These findings demonstrated that enhancer reprogramming of the MAPK regulatory pathway might serve as a potential mechanism underlying MAPK inhibitor resistance and concurrent targeting of epigenetic pathways and MAPK signaling might provide an effective treatment strategy for advanced ovarian cancer.

Targeting the IRAK1-S100A9 Axis Overcomes Resistance to Paclitaxel in Nasopharyngeal Carcinoma.

Novel strategies to treat late-stage nasopharyngeal carcinoma that often develop resistance to chemotherapy remains an unmet clinical demand. In this study, we identify the multi-kinase inhibitor pacritinib as capable of resensitizing the response to paclitaxel in an acquired resistance model. Transcriptome analysis of paclitaxel-sensitive and -resistant cell lines, as well as chemorefractory clinical samples, identified S100A9 as the top candidate gene suppressed by pacritinib and whose overexpression was significantly associated with paclitaxel resistance and poor clinical outcome. Moreover, both paclitaxel-resistant nasopharyngeal carcinoma cells and relapsed/metastatic clinical samples exhibited increased IRAK1 phosphorylation and demonstrated that pacritinib could abolish the IRAK1 phosphorylation to suppress S100A9 expression. Functional studies in both in vitro and in vivo models showed that genetic or pharmacologic blockade of IRAK1 overcame the resistance to paclitaxel, and combined treatment of pacritinib with paclitaxel exhibited superior antitumor effect. Together, these findings demonstrate an important role for the IRAK1-S100A9 axis in mediating resistance to paclitaxel. Furthermore, targeting of IRAK1 by pacritinib may provide a novel therapeutic strategy to overcome chemoresistance in nasopharyngeal carcinoma. SIGNIFICANCE: Deregulation of the IRAK1-S100A9 axis correlates with poor prognosis, contributes to chemoresistance in nasopharyngeal carcinoma, and can be targeted by pacritinib to overcome chemoresistance in nasopharyngeal carcinoma.

Functional characterisation guides classification of novel BAP1 germline variants.

We have identified six patients harbouring distinct germline BAP1 mutations. In this study, we functionally characterise known BAP1 pathogenic and likely benign germline variants out of these six patients to aid in the evaluation and classification of unknown BAP1 germline variants. We found that pathogenic germline variants tend to encode truncated proteins, show diminished expression of epithelial-mesenchymal transition (EMT) markers, are localised in the cytosol and have reduced deubiquitinase capabilities. We show that these functional assays are useful for BAP1 variant curation and may be added in the American College of Medical Genetics and Genomics (ACMG) criteria for BAP1 variant classification. This will allow clinicians to distinguish between BAP1 pathogenic and likely benign variants reliably and may aid to quickly benchmark newly identified BAP1 germline variants. Classification of novel BAP1 germline variants allows clinicians to inform predisposed patients and relevant family members regarding potential cancer risks, with appropriate clinical interventions implemented if required.

VHL Deficiency Drives Enhancer Activation of Oncogenes in Clear Cell Renal Cell Carcinoma.

Protein-coding mutations in clear cell renal cell carcinoma (ccRCC) have been extensively characterized, frequently involving inactivation of the von Hippel-Lindau (VHL) tumor suppressor. Roles for noncoding cis-regulatory aberrations in ccRCC tumorigenesis, however, remain unclear. Analyzing 10 primary tumor/normal pairs and 9 cell lines across 79 chromatin profiles, we observed pervasive enhancer malfunction in ccRCC, with cognate enhancer-target genes associated with tissue-specific aspects of malignancy. Superenhancer profiling identified ZNF395 as a ccRCC-specific and VHL-regulated master regulator whose depletion causes near-complete tumor elimination in vitro and in vivoVHL loss predominantly drives enhancer/superenhancer deregulation more so than promoters, with acquisition of active enhancer marks (H3K27ac, H3K4me1) near ccRCC hallmark genes. Mechanistically, VHL loss stabilizes HIF2α-HIF1ß heterodimer binding at enhancers, subsequently recruiting histone acetyltransferase p300 without overtly affecting preexisting promoter-enhancer interactions. Subtype-specific driver mutations such as VHL may thus propagate unique pathogenic dependencies in ccRCC by modulating epigenomic landscapes and cancer gene expression.Significance: Comprehensive epigenomic profiling of ccRCC establishes a compendium of somatically altered cis-regulatory elements, uncovering new potential targets including ZNF395, a ccRCC master regulator. Loss of VHL, a ccRCC signature event, causes pervasive enhancer malfunction, with binding of enhancer-centric HIF2α and recruitment of histone acetyltransferase p300 at preexisting lineage-specific promoter-enhancer complexes. Cancer Discov; 7(11); 1284-305. ©2017 AACR.See related commentary by Ricketts and Linehan, p. 1221This article is highlighted in the In This Issue feature, p. 1201.

TOPOISOMERASE1α Acts through Two Distinct Mechanisms to Regulate Stele and Columella Stem Cell Maintenance.

TOPOISOMERASE1 (TOP1), which releases DNA torsional stress generated during replication through its DNA relaxation activity, plays vital roles in animal and plant development. In Arabidopsis (Arabidopsis thaliana), TOP1 is encoded by two paralogous genes (TOP1α and TOP1ß), of which TOP1α displays specific developmental functions that are critical for the maintenance of shoot and floral stem cells. Here, we show that maintenance of two different populations of root stem cells is also dependent on TOP1α-specific developmental functions, which are exerted through two distinct novel mechanisms. In the proximal root meristem, the DNA relaxation activity of TOP1α is critical to ensure genome integrity and survival of stele stem cells (SSCs). Loss of TOP1α function triggers DNA double-strand breaks in S-phase SSCs and results in their death, which can be partially reversed by the replenishment of SSCs mediated by ETHYLENE RESPONSE FACTOR115 In the quiescent center and root cap meristem, TOP1α is epistatic to RETINOBLASTOMA-RELATED (RBR) in the maintenance of undifferentiated state and the number of columella stem cells (CSCs). Loss of TOP1α function in either wild-type or RBR RNAi plants leads to differentiation of CSCs, whereas overexpression of TOP1α mimics and further enhances the effect of RBR reduction that increases the number of CSCs Taken together, these findings provide important mechanistic insights into understanding stem cell maintenance in plants.