Systematic omics analysis identifies CCR6 as a therapeutic target to overcome cancer resistance to EGFR inhibitors.

Epidermal growth factor receptor inhibitors (EGFRi) have exhibited promising clinical outcomes in the treatment of various cancers. However, their widespread application has been limited by low patient eligibility and the emergence of resistance. Leveraging a multi-omics approach (>1000 cancer cell lines), we explored molecular signatures linked to EGFRi responsiveness and found that expression signatures involved in the estrogen response could recapitulate cancer cell dependency on EGFR, a phenomenon not solely attributable to EGFR-activating mutations. By correlating genome-wide function screening data with EGFRi responses, we identified chemokine receptor 6 (CCR6) as a potential druggable target to mitigate EGFRi resistance. In isogenic cell models, pharmacological inhibition of CCR6 effectively reversed acquired EGFRi resistance, disrupting mitochondrial oxidative phosphorylation, a cellular process commonly associated with therapy resistance. Our data-driven strategy unveils drug-response biomarkers and therapeutic targets for resistance, thus potentially expanding EGFRi applicability and efficacy.

Crosstalk between Signaling Pathways and Energy Metabolism in Pluripotency.

The sequential change from totipotency to multipotency occurs during early mammalian embryo development. However, due to the lack of cellular models to recapitulate the distinct potency of stem cells at each stage, their molecular and cellular characteristics remain ambiguous. The establishment of isogenic naïve and primed pluripotent stem cells to represent the pluripotency in the inner cell mass of the pre-implantation blastocyst and in the epiblast from the post-implantation embryo allows the understanding of the distinctive characteristics of two different states of pluripotent stem cells. This review discusses the prominent disparities between naïve and primed pluripotency, including signaling pathways, metabolism, and epigenetic status, ultimately facilitating a comprehensive understanding of their significance during early mammalian embryonic development.

Epigenetic repression of CHCHD2 enhances survival from single cell dissociation through attenuated Rho A kinase activity.

During in vitro culture, human pluripotent stem cells (hPSCs) often acquire survival advantages characterized by decreased susceptibility to mitochondrial cell death, known as "culture adaptation." This adaptation is associated with genetic and epigenetic abnormalities, including TP53 mutations, copy number variations, trisomy, and methylation changes. Understanding the molecular mechanisms underlying this acquired survival advantage is crucial for safe hPSC-based cell therapies. Through transcriptome and methylome analysis, we discovered that the epigenetic repression of CHCHD2, a mitochondrial protein, is a common occurrence during in vitro culture using enzymatic dissociation. We confirmed this finding through genetic perturbation and reconstitution experiments in normal human embryonic stem cells (hESCs). Loss of CHCHD2 expression conferred resistance to single cell dissociation-induced cell death, a common stress encountered during in vitro culture. Importantly, we found that the downregulation of CHCHD2 significantly attenuates the activity of Rho-associated protein kinase (ROCK), which is responsible for inducing single cell death in hESCs. This suggests that hESCs may survive routine enzyme-based cell dissociation by downregulating CHCHD2 and thereby attenuating ROCK activity. These findings provide insights into the mechanisms by which hPSCs acquire survival advantages and adapt to in vitro culture conditions.

Partial in vivo reprogramming enables injury-free intestinal regeneration via autonomous Ptgs1 induction.

Tissue regeneration after injury involves the dedifferentiation of somatic cells, a natural adaptive reprogramming that leads to the emergence of injury-responsive cells with fetal-like characteristics. However, there is no direct evidence that adaptive reprogramming involves a shared molecular mechanism with direct cellular reprogramming. Here, we induced dedifferentiation of intestinal epithelial cells using OSKM (Oct4, Sox2, Klf4, and c-Myc) in vivo. The OSKM-induced forced dedifferentiation showed similar molecular features of intestinal regeneration, including a transition from homeostatic cell types to injury-responsive-like cell types. These injury-responsive-like cells, sharing gene signatures of revival stem cells and atrophy-induced villus epithelial cells, actively assisted tissue regeneration following damage. In contrast to normal intestinal regeneration involving Ptgs2 induction, the OSKM promotes autonomous production of prostaglandin E2 via epithelial Ptgs1 expression. These results indicate prostaglandin synthesis is a common mechanism for intestinal regeneration but involves a different enzyme when partial reprogramming is applied to the intestinal epithelium.

In Silico Discovery of 5'-Modified 7-Deoxy-7-ethynyl-4'-thioadenosine as a HASPIN Inhibitor and Its Synergistic Anticancer Effect with the PLK1 Inhibitor.

Despite genetic perturbations resulting in embryo lethality for most mitotic kinases, loss of the histone H3 mitotic kinase HASPIN reveals no adverse effect in mice models, establishing HASPIN as a promising target for anticancer therapy. However, developing a HASPIN inhibitor from conventional pharmacophores poses a technical challenge as this atypical kinase shares slight similarities with eukaryotic protein kinases. Chemically modifying a cytotoxic 4'-thioadenosine analogue through high genotoxicity yielded several novel nongenotoxic kinase inhibitors. In silico apporoaches utilizing transcriptomic and chemical similarities with known compounds and KINOMEscan profiles unveiled the HASPIN inhibitor LJ4827. LJ4827's specificity and potency as a HASPIN inhibitor were verified through in vitro kinase assay and X-ray crystallography. HASPIN inhibition by LJ4827 reduced histone H3 phosphorylation and impeded Aurora B recruitment in cancer cell centromeres but not in noncancer cells. Through transcriptome analysis of lung cancer patients, PLK1 was determined as a druggable synergistic partner to complement HASPIN inhibition. Chemical or genetic PLK1 perturbation with LJ4827 effectuated pronounced lung cancer cytotoxicity in vitro and in vivo. Therefore, LJ4827 is a novel anticancer therapeutic for selectively impeding cancer mitosis through potent HASPIN inhibition, and simultaneous HASPIN and PLK1 interference is a promising therapeutic strategy for lung cancer.

MutSα and MutSß as size-dependent cellular determinants for prime editing in human embryonic stem cells.

Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and ex vivo stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Considering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs. Through homozygous knockout (KO) of MMR pathway key proteins MSH2, MSH3, and MSH6, we reveal that MutSα and MutSß determine PE efficiency in an editing size-dependent manner. Notably, MSH2 perturbation disrupted both MutSα and MutSß complexes, dramatically escalating PE efficiency from base mispair to 10 bases, up to 50 folds. Similarly, impaired MutSα by MSH6 KO improved editing efficiency from single to three base pairs, while defective MutSß by MSH3 KO heightened efficiency from three to 10 base pairs. Thus, the size-dependent effect of MutSα and MutSß on prime editing implies that MMR is a vital PE efficiency determinant in hPSCs and highlights the distinct roles of MutSα and MutSß in its outcome.

Gene editing with 'pencil' rather than 'scissors' in human pluripotent stem cells.

Owing to the advances in genome editing technologies, research on human pluripotent stem cells (hPSCs) have recently undergone breakthroughs that enable precise alteration of desired nucleotide bases in hPSCs for the creation of isogenic disease models or for autologous ex vivo cell therapy. As pathogenic variants largely consist of point mutations, precise substitution of mutated bases in hPSCs allows researchers study disease mechanisms with "disease-in-a-dish" and provide functionally repaired cells to patients for cell therapy. To this end, in addition to utilizing the conventional homologous directed repair system in the knock-in strategy based on endonuclease activity of Cas9 (i.e., 'scissors' like gene editing), diverse toolkits for editing the desirable bases (i.e., 'pencils' like gene editing) that avoid the accidental insertion and deletion (indel) mutations as well as large harmful deletions have been developed. In this review, we summarize the recent progress in genome editing methodologies and employment of hPSCs for future translational applications.

TPX2 Amplification-Driven Aberrant Mitosis in Culture Adapted Human Embryonic Stem Cells with gain of 20q11.21.

BACKGROUND: Despite highly effective machinery for the maintenance of genome integrity in human embryonic stem cells (hESCs), the frequency of genetic aberrations during in-vitro culture has been a serious issue for future clinical applications. METHOD: By passaging hESCs over a broad range of timepoints (up to 6 years), the isogenic hESC lines with different passage numbers with distinct cellular characteristics, were established. RESULT: We found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs) with normal copy number. Through high-resolution genome-wide approaches and transcriptome analysis, we found that culture adapted-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2, a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stabilization, misaligned chromosomes, and polyploidy. CONCLUSION: These studies suggest that the increased transcription of TPX2 in culture adapted hESCs could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

Transition Substitution of Desired Bases in Human Pluripotent Stem Cells with Base Editors: A Step-by-Step Guide.

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

TPX2 prompts mitotic survival via the induction of BCL2L1 through YAP1 protein stabilization in human embryonic stem cells.

Genetic alterations have been reported for decades in most human embryonic stem cells (hESCs). Survival advantage, a typical trait acquired during long-term in vitro culture, results from the induction of BCL2L1 upon frequent copy number variation (CNV) at locus 20q11.21 and is one of the strongest candidates associated with genetic alterations that occur via escape from mitotic stress. However, the underlying mechanisms for BCL2L1 induction remain unknown. Furthermore, abnormal mitosis and the survival advantage that frequently occur in late passage are associated with the expression of BCL2L1, which is in locus 20q11.21. In this study, we demonstrated that the expression of TPX2, a gene located in 20q11.21, led to BCL2L1 induction and consequent survival traits under mitotic stress in isogenic pairs of hESCs and human induced pluripotent stem cells (iPSCs) with normal and 20q11.21 CNVs. High Aurora A kinase activity by TPX2 stabilized the YAP1 protein to induce YAP1-dependent BCL2L1 expression. A chemical inhibitor of Aurora A kinase and knockdown of YAP/TAZ significantly abrogated the high tolerance to mitotic stress through BCL2L1 suppression. These results suggest that the collective expression of TPX2 and BCL2L1 from CNV at loci 20q11.21 and a consequent increase in YAP1 signaling promote genome instability during long-term in vitro hESC culture.

Label-free and non-destructive identification of naïve and primed embryonic stem cells based on differences in cellular metabolism.

Pluripotent stem cells (PSCs) exist in naïve or primed states based on their origin. For in vitro culture, these PSCs require different supplements and growth factors. However, owing to their similar phenotypic features, identifying both cell types without harming cellular functions is challenging. This study reports an electrochemical method that enables simple, label-free, and non-destructive detection of naïve embryonic stem cells (ESCs) derived from mouse ESCs, based on the differences in cellular metabolism. Two major metabolic pathways to generate adenosine triphosphate (ATP)-glycolysis and oxidative phosphorylation (OXPHOS)-were blocked, and it was found that mitochondrial energy generation is the origin of the strong electrochemical signals of naïve ESCs. The number of ESCs is quantified when mixed with primed ESCs or converted from naïve-primed switchable metastable ESCs. The mouse PSCs derived from doxycycline-inducible mouse embryonic fibroblasts (MEFs) are also sensitively identified among other cell types such as unconverted MEFs and primed PSCs. The developed sensing platform operates in a non-invasive and label-free manner. Thus, it can be useful in the development of stem cell-derived therapeutics.

The Korean undiagnosed diseases program phase I: expansion of the nationwide network and the development of long-term infrastructure.

BACKGROUND: Phase I of the Korean Undiagnosed Diseases Program (KUDP), performed for 3 years, has been completed. The Phase I program aimed to solve the problem of undiagnosed patients throughout the country and develop infrastructure, including a data management system and functional core laboratory, for long-term translational research. Herein, we share the clinical experiences of the Phase I program and introduce the activities of the functional core laboratory and data management system. RESULTS: During the program (2018-2020), 458 patients were enrolled and classified into 3 groups according to the following criteria: (I) those with a specific clinical assessment which can be verified by direct testing (32 patients); (II) those with a disease group with genetic and phenotypic heterogeneity (353 patients); and (III) those with atypical presentations or diseases unknown to date (73 patients). All patients underwent individualized diagnostic processes based on the decision of an expert consortium. Confirmative diagnoses were obtained for 242 patients (52.8%). The diagnostic yield was different for each group: 81.3% for Group I, 53.3% for Group II, and 38.4% for Group III. Diagnoses were made by next-generation sequencing for 204 patients (84.3%) and other genetic testing for 35 patients (14.5%). Three patients (1.2%) were diagnosed with nongenetic disorders. The KUDP functional core laboratory, with a group of experts, organized a streamlined research pipeline covering various resources, including animal models, stem cells, structural modeling and metabolic and biochemical approaches. Regular data review was performed to screen for candidate genes among undiagnosed patients, and six different genes were identified for functional research. We also developed a web-based database system that supports clinical cohort management and provides a matchmaker exchange protocol based on a matchbox, likely to reinforce the nationwide clinical network and further international collaboration. CONCLUSIONS: The KUDP evaluated the unmet needs of undiagnosed patients and established infrastructure for a data-sharing system and future functional research. The advancement of the KUDP may lead to sustainable bench-to-bedside research in Korea and contribute to ongoing international collaboration.

Activation of the HSP27-AKT axis contributes to gefitinib resistance in non-small cell lung cancer cells independent of EGFR mutations.

PURPOSE: Although epidermal growth factor receptor (EGFR)-activating mutations in non-small cell lung cancer (NSCLC) usually show sensitivity to first-generation EGFR-tyrosine kinase inhibitors (TKIs), most patients relapse because of drug resistance. Heat shock protein 27 (HSP27) has been reported to be involved in the resistance of EGFR-TKIs, although the underlying mechanism is unclear. Here, we explore the mechanisms of HSP27-mediated EGFR TKI resistance and propose novel therapeutic strategies. METHODS: To determine the mechanism of HSP27 associated gefitinib resistance, differences were assessed using gefitinib-sensitive and -resistant NSCLC cell lines. In vivo xenograft experiments were conducted to elucidate the combinatorial effects of J2, a small molecule HSP27 inhibitor, and gefitinib. Analyses of human NSCLC tissues and PDX tissues were also used for comparison of HSP27 and phosphorylated AKT expression. RESULTS: Large-scale cohort analysis of NSCLC cases revealed that HSP27 expression correlated well with the incidence of EGFR mutations and affected patient survival. Increased pAKT and HSP27 was observed in gefitinib-resistant cells compared with gefitinib-sensitive cells. Moreover, increased phosphorylation of HSP27 by gefitinib augmented its protein stability and potentiated its binding activity with pAKT, which resulted in increased gefitinib resistance. However, in gefitinib-sensitive cells, stronger binding activity between EGFR and HSP27 was observed. Moreover, these phenomena occurred regardless of EGFR mutation including secondary mutations, such as T790M. AKT knockdown switched HSP27-pAKT binding to HSP27-EGFR, which promoted gefitinib sensitivity in gefitinib-resistant cells. Functional inhibition of HSP27 yielded sensitization to gefitinib in gefitinib-resistant cells by inhibiting the interaction between HSP27 and pAKT. CONCLUSIONS: Our results indicate that combination of EGFR-TKIs with HSP27 inhibitors may represent a good strategy to overcome resistance to EGFR-TKIs, especially in cancers exhibiting AKT pathway activation.

Dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells.

BACKGROUND: The requirement of the Mek1 inhibitor (iMek1) during naïve pluripotency maintenance results from the activation of the Mek1-Erk1/2 (Mek/Erk) signaling pathway upon leukemia inhibitory factor (LIF) stimulation. METHODS: Through a meta-analysis of previous genome-wide screening for negative regulators of naïve pluripotency, Ptpn11 (encoding the Shp2 protein, which serves both as a tyrosine phosphatase and putative adapter), was predicted as one of the key factors for the negative modulation of naïve pluripotency through LIF-dependent Jak/Stat3 signaling. Using an isogenic pair of naïve and primed mouse embryonic stem cells (mESCs), we demonstrated the differential role of Shp2 in naïve and primed pluripotency. RESULTS: Loss of Shp2 increased naïve pluripotency by promoting Jak/Stat3 signaling and disturbed in vivo differentiation potential. In sharp contrast, Shp2 depletion significantly impeded the self-renewal of ESCs under primed culture conditions, which was concurrent with a reduction in Mek/Erk signaling. Similarly, upon treatment with an allosteric Shp2 inhibitor (iShp2), the cells sustained Stat3 phosphorylation and decoupled Mek/Erk signaling, thus iShp2 can replace the use of iMek1 for maintenance of naïve ESCs. CONCLUSIONS: Taken together, our findings highlight the differential roles of Shp2 in naïve and primed pluripotency and propose the usage of iShp2 instead of iMek1 for the efficient maintenance and establishment of naïve pluripotency.

Live isolation of naïve ESCs via distinct glucose metabolism and stored glycogen.

Naïve and primed pluripotent stem cells recapitulate the peri- and post-implantation development, respectively. Thus, investigation of distinct traits between each pluripotent stem cell type would shed light on early embryonic processes. Herein, by screening a fluorescent probe library, we found that intracellular glycogen led to specific reactivity to CDg4, a glycogen fluorescence sensor, in both human and mouse naïve embryonic stem cells (ESCs). The requirement of constant inhibition of Gsk3ß as well as high oxidative phosphorylation (OxPHOS) in naïve compared to primed ESCs was closely associated to high level of intracellular glycogen in naïve ESCs. Both capacity of OxPHOS and stored glycogen, rescued naïve ESCs by transient inhibition of glycolysis, which selectively eliminated primed ESCs. Additionally, naïve ESCs with active OxPHOS were enriched from a mixture with primed ESCs by high reactivity to ATP-Red1, a mitochondrial ATP fluorescence probe. These results indicate the active OxPHOS and high intracellular glycogen as a novel "biomarker" delineating metabolic remodeling during the transition of naïve pluripotency.

Multiple isogenic GNE-myopathy modeling with mutation specific phenotypes from human pluripotent stem cells by base editors.

Despite the great potential of disease modeling using human pluripotent stem cells (hPSCs) derived from patients with mutations, lack of an appropriate isogenic control hinders a precise phenotypic comparison due to the bias arising from the dissimilar genetic backgrounds between the control and diseased hPSCs. Herein, we took advantage of currently available base editors (BEs) to epitomize the isogenic disease model from hPSCs. Using this method, we established multiple isogenic GNE myopathy disease models that harbor point mutations on the GNE gene, including four different mutations found in GNE myopathy patients. Four different mutations in the epimerase or kinase domains of GNE revealed mutation-specific hyposialylation and hyposialylation dependent gene signature, which was closely correlated to pathological clinical phenotypes. GNE protein structure modeling based on the mutations, addressed these mutation-specific hyposialylation patterns. Furthermore, treatment with a drug candidate currently under clinical trials showed a mutation-specific drug response in GNE myopathy disease models. These data suggest that derivation of multiple isogenic disease models from hPSCs by using genome editing can enable translationally relevant studies on the pathophysiology of GNE myopathy and drug responses.

High expression of uracil DNA glycosylase determines C to T substitution in human pluripotent stem cells.

Precise genome editing of human pluripotent stem cells (hPSCs) is crucial not only for basic science but also for biomedical applications such as ex vivo stem cell therapy and genetic disease modeling. However, hPSCs have unique cellular properties compared to somatic cells. For instance, hPSCs are extremely susceptible to DNA damage, and therefore Cas9-mediated DNA double-strand breaks (DSB) induce p53-dependent cell death, resulting in low Cas9 editing efficiency. Unlike Cas9 nucleases, base editors including cytosine base editor (CBE) and adenine base editor (ABE) can efficiently substitute single nucleotides without generating DSBs at target sites. Here, we found that the editing efficiency of CBE was significantly lower than that of ABE in human embryonic stem cells (hESCs), which are associated with high expression of DNA glycosylases, the key component of the base excision repair pathway. Sequential depletion of DNA glycosylases revealed that high expression of uracil DNA glycosylase (UNG) not only resulted in low editing efficiency but also affected CBE product purity (i.e., C to T) in hESCs. Therefore, additional suppression of UNG via transient knockdown would also improve C to T base substitutions in hESCs. These data suggest that the unique cellular characteristics of hPSCs could determine the efficiency of precise genome editing.