scholarly journals Gene expression in circulating tumor cells reveals a dynamic role of EMT and PD-L1 during osimertinib treatment in NSCLC patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aliki Ntzifa ◽  
Areti Strati ◽  
Galatea Kallergi ◽  
Athanasios Kotsakis ◽  
Vassilis Georgoulias ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Resistance Mechanisms ◽  
Theoretical Background ◽  
Strong Positive Correlation ◽  
Twist 1 ◽  
Egfr Mutant ◽  
Nsclc Patients ◽  
Cell Profile

AbstractLiquid biopsy is a tool to unveil resistance mechanisms in NSCLC. We studied changes in gene expression in CTC-enriched fractions of EGFR-mutant NSCLC patients under osimertinib. Peripheral blood from 30 NSCLC patients before, after 1 cycle of osimertinib and at progression of disease (PD) was analyzed by size-based CTC enrichment combined with RT-qPCR for gene expression of epithelial (CK-8, CK-18, CK-19), mesenchymal/EMT (VIM, TWIST-1, AXL), stem cell (ALDH-1) markers, PD-L1 and PIM-1. CTCs were also analyzed by triple immunofluorescence for 45 identical blood samples. Epithelial and stem cell profile (p = 0.043) and mesenchymal/EMT and stem cell profile (p = 0.014) at PD were correlated. There was a strong positive correlation of VIM expression with PIM-1 expression at baseline and increased PD-L1 expression levels at PD. AXL overexpression varied among patients and high levels of PIM-1 transcripts were detected. PD-L1 expression was significantly increased at PD compared to baseline (p = 0.016). The high prevalence of VIM positive CTCs suggest a dynamic role of EMT during osimertinib treatment, while increased expression of PD-L1 at PD suggests a theoretical background for immunotherapy in EGFR-mutant NSCLC patients that develop resistance to osimertinib. This observation merits to be further evaluated in a prospective immunotherapy trial.

ctDNA resistance landscape of lazertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI).

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. 9601-9601
Author(s):  
Ji-Youn Han ◽  
Myung-Ju Ahn ◽  
Sang-We Kim ◽  
Ki Hyeong Lee ◽  
Eun Kyung Cho ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Resistance Mechanisms ◽  
Third Generation ◽  
Egfr Amplification ◽  
Phase 2 Trial ◽  
Egfr Mutant ◽  
Nsclc Patients ◽  
In Cis ◽  
Egfr Tki ◽  
Egfr Tkis

9601 Background: While EGFR mutant ( EGFRm) non-small cell lung cancer (NSCLC) patients usually experience improved clinical benefit with EGFR TKIs, most eventually progress. Understanding mechanisms of resistance (MoR) may allow for more personalized treatment. Lazertinib is an irreversible third generation EGFR TKI for which MoR are unknown. Obtaining sufficient tumor tissue for genotyping at progression is often difficult. Therefore, we utilized plasma ctDNA from patients treated with lazertinib to explore MoR. Methods: Plasma samples from 47 NSCLC patients in the phase 2 trial of lazertinib (NCT03046992) were collected at screening and progressive disease (PD) and underwent ctDNA NGS of 74 genes using Guarant360. All patients were positive for an EGFR Ex19del or L858R ( EGFRm) and T790M by tissue testing at screening. Acquired, nonsynonymous, characterized mutations detected in a PD sample but not in the screening sample from the respective patient were considered putative MoR, excluding aneuploidy. Patients with detectable plasma EGFRm and/or T790M at screening were evaluable. Results: ctDNA was detected in 47 (100%) screening samples and 43/45 (96%) PD samples (two failed sequencing). An EGFRm was detected in 85% of patients at screening (n = 40), 38 of which had PD ctDNA results and were included in analysis. T790M was detected in 30 patients at screening and subsequently not detected at PD in 21 of these patients, 55% of all 38 included patients. Among the ten patients with T790M detected at PD, on-target MoR were detected in 7 (18% of all included patients) including EGFR C797S (n = 3, 8%), EGFR amplification (n = 3, 8%), and EGFR T854A (n = 1, 3%). All C797S were in cis with T790M. No on-target MoR were detected in patients without T790M detected at PD. Off-target MoR were seen in 34% of patients (13/38) including mutations in PIK3CA (13%; 2 E545K, 2 E542K, 1 E81K), ERBB2 (5%; 1 D769H, 1 V777L), KRAS (3%; 1 G12C), and BRAF (3%; 1 G469A). Gene amplifications were detected in CCND1 (n = 1, 3%) , CCNE1 (n = 2, 5%) , ERBB2 (n = 1, 3%) , FGFR1 (n = 1, 3%) , MET (n = 4, 11%) , and PIK3CA (n = 1, 3%), with some patients having multiple MoR. Conclusions: The spectrum of MoR identified in this cohort of patients treated with lazertinib is similar to that reported in other third generation EGFR TKIs, but with some differences in frequencies. The most common resistance mechanisms are T790M loss and PIK3CA alterations which may address the mechanism of action. Our findings suggest putative MoR of lazertinib and show that ctDNA NGS is an effective way to identify MoR in patients progressing on targeted therapy. Clinical trial information: NCT03046992 .

Abstract 16488: Exploring the Functional Role of Exosomes in Stem Cell-mediated Cardiac Repair

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Jennifer K Lang ◽  
Rebecca F Young ◽  
Hashmat Ashraf ◽  
John M Canty
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Information Transfer ◽  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Cardiac Repair ◽  
In Vitro Assays ◽  
Collagen Iii ◽  
Human Cardiac Fibroblasts

Numerous experimental and clinical studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium. Paracrine signaling by CDC secreted exosomes is hypothesized to be the principal mediator of improved function, however the mechanism of exosome uptake, cell specificity, and information transfer has yet to be elucidated. Our aim was to define the role of physiologically secreted human CDC-derived exosomes on cardiac fibroblasts, endothelial cells and cardiomyocytes by employing a knockdown (KD) strategy against nSMase2 and Rab27a, two genes crucial in exosome secretion. Methods: Human CDCs (hCDCs) were grown from atrial tissue biopsy explant culture and characterized by flow cytometry and immunocytochemistry. CDC-derived exosomes were isolated and characterized by particle size, immunoblot analysis and electron microscopy. Protein concentration of exosome preparations was quantified by Bradford microassay. nSMase2 and Rab27a shRNAi lentivirus was generated and used to create stable CDC knockdown lines. Co-culture transwell in vitro assays were designed to assess target cell proliferation, migration, angiogenesis, and fibrotic gene expression. Results: hCDCs expressed CD105, CD90, GATA4 and Nkx2.5, and were CD45 and cardiac troponin T negative. hCDC-derived exosomes were immunoreactive for CD63 and HSP90 and negative for Cyctochrome c and EEA1. shRNAi KD of nSMase2 and Rab27a resulted in successful blockade of exosome release from hCDCs. HUVECs co-cultured with scrambled shRNAi CDCs versus shRNAi nSMase2 KD CDCs showed increased angiogenesis and migration without affecting proliferation. While TGF-β stimulation of human cardiac fibroblasts significantly increased collagen I (COLI) and collagen III (COLIII) gene expression, there was no significant decrease in COLI or COLIII after treatment with exosomes. Conclusions: Secretion of hCDC-derived exosomes was effectively inhibited by nSMase2 and Rab27a lentiviral KD. Exosome release contributed to the angiogenic and pro-migratory effects of hCDCs but did not play a role in the fibrotic gene expression of human cardiac fibroblasts, suggesting a role for physiological exosome secretion in stem cell-mediated cardiac repair.

scholarly journals INPP4B is a Biomarker of Poor Prognosis in AML Which is Associated with EVI1 Overexpression and a LSC Signature

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3929-3929
Author(s):  
Irakli Dzneladze ◽  
John F Woolley ◽  
Youqi Han ◽  
Mark Sharobim ◽  
Ayesha Rashid ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Differential Expression ◽  
Cell Lines ◽  
Colony Formation ◽  
Promoter Region ◽  
Akt Signaling ◽  
Hematopoietic Stem ◽  
Hematopoietic Lineage

Abstract Acute myeloid leukemia (AML) is a highly heterogeneous cancer of the bone marrow. To better understand leukemogenesis and improve predictors of patient response to chemotherapy and overall survival (OS), we recently examined the role of inositol polyphosphate-4-phosphatase, type-II (INPP4B) in AML. Normally, INPP4B plays a role in PI3K/Akt signaling by regulating phosphorylation of phosphoinositides (PIs), critical membrane-bound second messenger molecules, by dephosphorylating the 4'-position of PI(3,4)P2 to generate PI(3)P. Because PI(3,4)P2, like PI(3,4,5)P3, is necessary for the activation Akt, INPP4B was first hypothesized and demonstrated to be a tumor suppressor protein, akin to PTEN, in several cancers including breast, prostate and ovarian. Recent work however, including our own study, has demonstrated a paradoxical tumor-promoting role of INPP4B in AML and estrogen receptor positive (ER+) breast cancer. Specifically, we demonstrated that INPP4Bhigh AML patients (25% of patients) had significantly shorter OS, lower response to induction therapy, and shorter event-free survival. Furthermore, INPP4B expression was found to be an independent prognostic marker for OS in AML outperforming FLT3-ITD and NPM1 mutation status. Overexpression of INPP4B in several AML cell lines results in enhanced colony formation potential, chemotherapy drug resistance, and increased proliferation. Though this previous work has shown that INPP4B plays a significant role in AML, it remains unclear why INPP4Bhigh AML differs from INPP4Blow AML, and what causes its upregulation. To address this question, we interrogated gene expression data from three independent datasets (n=942) to identify genes with differential expression between INPP4Bhigh and INPP4Blow AML. Gene expression analysis revealed that INPP4Bhigh AML was associated with differential expression of 233 genes. High INPP4B expression was associated with higher expression of genes related to the hematopoietic lineage, PI3K-Akt signaling, Jak-STAT signaling and ECM-receptor interaction pathways. Specifically, INPP4Bhigh AML has significantly higher expression of leukemic stem cell signature (LSC) genes CD34, RBPMS, GUCY1A3, KIAA0125, SOCS2, SPINK2, HTR1F, PPP1R16B, EVI1 (MECOM), DAPK1, BAALC, ABCB1, and PRKCH. Furthermore, INPP4B was found to be co-expressed with anti-apoptotic genes of the BCL2 family, namely BCL2 and BCL2L1. Further analysis revealed that INPP4B was co-expressed with the transcription factors EVI1, GATA2, NFATC2, ZEB1, GATA3 and ETS1, all of which having predicted binding sites within the INPP4B promoter region. Due to the observed enrichment of hematopoietic lineage/LSC genes in INPP4Bhigh AML, we wanted to validate the potential role of the EVI1 transcription factor in regulating INPP4B expression. Chromatin immunoprecipitation was used to demonstrate that EVI1 binding was enriched in the INPP4B promoter region of both EVI1high OCI/AML-4 and OCI/AML-6 cell lines. In addition, retroviral overexpression of EVI1 in EVI1low U937 cells resulted in subsequent upregulation of INPP4B transcript levels. Moreover, shRNA mediated knockdown of EVI1 in EVI1highOCI/AML-4 and UCSD-1 cells resulted in downregulation of INPP4B expression. Overall, our analysis reveals that INPP4Bhigh AML is characterized by upregulation of genes related to the hemotopoietic lineage, and LSC signature - consistent with the in vitro colony formation phenotype seen in INPP4B overexpressing AML cell lines. Furthermore, we demonstrate that one of the hematopoietic stem cell genes, EVI1 is a potentially key regulator of INPP4B expression in AML. Disclosures Jain: Roche Canada: Research Funding.

scholarly journals Ing4 Regulates Hematopoiesis through Suppression of NF-Kb

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 642-642
Author(s):  
Zanshe Thompson ◽  
Vera Binder ◽  
Michelle Ammerman ◽  
Ellen Durand ◽  
Leonard I. Zon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Target Gene ◽  
Target Genes ◽  
Zebrafish Embryos ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells. This activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In the absence of Ing4, there is a significant increase in NF-kB target gene expression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis, where Ing4 deficiency impairs hematopoietic stem cell (HSC) function, but enhances multipotent progenitor cell (MPP) regenerative capacity. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. To define the role of Ing4 in zebrafish HSPCs, we designed an anti-sense morpholino oligo against Ing4 and injected into zebrafish embryos at the single cell stage. Embryos were screened using in situ hybridizations for c-myb and runx1 expression, which are highly expressed in the aorta, gonad, mesonephros (AGM) region in the developing zebrafish embryo. We found that Ing4-deficient zebrafish embryos lose >90% of runx1+/c-myb+ cells in the AGM, demonstrating a lack of HSPC specification. Analysis of ephrinB2 expression showed normal specification of the aorta in Ing4 morphant embryos, signifying that the step of HSPC specification is affected in the absence of Ing4. Overexpression of human Ing4 in zebrafish embryos resulted in increased HSPC marker staining suggesting that normal expression levels of Ing4 are required for HSC specification. As Ing4 is an epigenetic regulator that binds specific gene loci, we examined the chromatin occupancy of Ing4 in human peripheral blood CD34+ progenitor cells. Using ChIP-seq for Ing4 in CD34+ cells, we show that Ing4 binds to many regulators of blood development including MYB, LMO2, RUNX1, and IKAROS, and several NF-kB target genes. In other tissues, Ing4 negatively regulates NF-kB, so accordingly, loss of Ing4 results in an overabundance of NF-kB signaling. To address NF-kB target gene expression in Ing4-deficient zebrafish embryos, we performed qPCR analysis at 36hpf. These assays showed an increase in the expression of a subset of NF-kB target genes (IKBKE, IL-19, IL-1b, IL-20R). Simultaneous knockdown of both Ing4 and RelA, through combined morpholino injections against both factors, resulted in the rescue of HSC marker expression in the aorta. These results suggest that NF-kB inhibition could remediate the loss of Ing4. A mouse model for Ing4 deficiency was generated to further evaluate the role of Ing4 in differentiated immune cells. These mice are developmentally normal but are hypersensitive to stimulation with LPS. Interestingly, we found that Ing4-/- mice showed skewed hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. Based on these exciting findings, we hypothesize that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Disclosures No relevant conflicts of interest to declare.

scholarly journals TEAD family transcription factors in development and disease

Development ◽  
2021 ◽  
Vol 148 (12) ◽  
Author(s):  
Laura Currey ◽  
Stefan Thor ◽  
Michael Piper
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Transcription Factors ◽  
Cell Biology ◽  
Hippo Pathway ◽  
Binding Motif ◽  
Lineage Specification ◽  
Intrinsic Cues ◽  
Hippo Signalling

ABSTRACT The balance between stem cell potency and lineage specification entails the integration of both extrinsic and intrinsic cues, which ultimately influence gene expression through the activity of transcription factors. One example of this is provided by the Hippo signalling pathway, which plays a central role in regulating organ size during development. Hippo pathway activity is mediated by the transcriptional co-factors Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which interact with TEA domain (TEAD) proteins to regulate gene expression. Although the roles of YAP and TAZ have been intensively studied, the roles played by TEAD proteins are less well understood. Recent studies have begun to address this, revealing that TEADs regulate the balance between progenitor self-renewal and differentiation throughout various stages of development. Furthermore, it is becoming apparent that TEAD proteins interact with other co-factors that influence stem cell biology. This Primer provides an overview of the role of TEAD proteins during development, focusing on their role in Hippo signalling as well as within other developmental, homeostatic and disease contexts.

scholarly journals The role of the stem cell epigenome in normal aging and rejuvenative therapy

2020 ◽  
Vol 29 (R2) ◽  
pp. R236-R247
Author(s):  
Jeyan Jayarajan ◽  
Michael D Milsom
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Adult Stem Cells ◽  
Cell Function ◽  
Biological Function ◽  
Functional Decline ◽  
Driving Factor ◽  
Key Factor

Abstract Adult stem cells are ultimately responsible for the lifelong maintenance of regenerating of tissues during both homeostasis and following injury. Hence, the functional attrition of adult stem cells is thought to be an important driving factor behind the progressive functional decline of tissues and organs that is observed during aging. The mechanistic cause underlying this age-associated exhaustion of functional stem cells is likely to be complex and multifactorial. However, it is clear that progressive remodeling of the epigenome and the resulting deregulation of gene expression programs can be considered a hallmark of aging, and is likely a key factor in mediating altered biological function of aged stem cells. In this review, we outline cell intrinsic and extrinsic mediators of epigenome remodeling during aging; discuss how such changes can impact on stem cell function; and describe how resetting the aged epigenome may rejuvenate some of the biological characteristics of stem cells.

Role of syndecan-1 and exogenous heparin in hepatoma sphere formation

2020 ◽  
Vol 98 (2) ◽  
pp. 112-119 ◽  
Author(s):  
Shih-Chiang Lin ◽  
Ching-Po Wu ◽  
TingTing Tseng ◽  
Yaoyun Jhang ◽  
Shao-Chen Lee
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Stem Cell ◽  
Cell Differentiation ◽  
Cd44 Expression ◽  
Tumor Sphere ◽  
Family Protein ◽  
Sphere Formation ◽  
Development Application

Glycosaminoglycan-modified proteoglycans play important roles in many cell activities, including cell differentiation and stem cell development. Tumor sphere formation ability is one of properties in cancer stem cells (CSCs). The correlation between CSC markers and proteoglycan remains to be clarified. Upon hepatoma sphere formation, expression of CSC markers CD13, CD90, CD133, and CD44, as well the syndecan family protein syndecan-1 (SDC1), increased as analyzed by PCR. Further examination by suppression of CD13 expression showed downregulation of SDC1 and CD44 gene expression, whereas suppression of SDC1 gene expression downregulated CD13 and CD44 gene expression. Suppression of SDC1 gene expression also suppressed sphere development, as analyzed by a novel sphereocrit assay to quantify the level of sphere formation. The heparin disaccharide components, but not those of chondroitin disaccharide, changed with hepatoma sphere development, revealing the increased levels of N-sulfation and 2-O-sulfation. These explained the inhibition of hepatoma sphere formation by exogenous heparin. In conclusion, we found that SDC1 affected CSC marker CD13 and CD44 expression. SDC1 proteoglycan and heparin components changed and affected hepatoma sphere development. Application of heparin mimics in reduction of hepatoma stem cells might be possible.

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