Deep RNA Sequencing Revealed Fusion Junctional Heterogeneity May Predict Crizotinib Treatment Efficacy in ALK-Rearranged NSCLC.

INTRODUCTION: Gene fusion variants in ALK-rearranged NSCLC may predict patient outcomes, but previous results have been inconclusive. Fusion isoforms coexisting in the same tumor may affect the efficacy of targeted therapy, but they have not been investigated. METHODS: Patients with ALK-rearranged NSCLC who received crizotinib treatments were recruited. Precrizotinib tumor tissues were analyzed by the anchored multiplex polymerase chain reaction for targeted RNA sequencing. Kaplan-Meier and Cox regression were used to compare overall and progression-free survivals. RESULTS: Of the 51 studied subjects, EML4-ALK variant types v1, v2, v3, and others were detected in 23 (45.1%), five (9.8%), 19 (37.3%), and four patients (7.8%), respectively. Multiple EML4-ALK RNA isoforms were detected in 24 tumors (47.1%), and single isoform in 27 (52.9%). Most of the v3 tumors (16 of 19) harbored both v3a and v3b RNA isoforms. Multiple isoforms were also detected in eight non-v3 tumors (33.3% of all 24 multiple isoforms; five v1, two v5', and one v2). Compared with patients with single isoform, those with multiple isoforms had worse progression-free (hazard ratio and 95% confidence interval: 2.45 [1.06-5.69]) and overall (hazard ratio [95% confidence interval]: 3.74 [1.26-11.13]) survivals after adjusting for potential confounders including variant type. Using the patient-derived H2228 cells known to express v3a and v3b, our single-cell polymerase chain reaction detected either v3a or v3b in most single cells. Treatment of H2228 cells by three ALK inhibitors revealed increased ratios of v3a-to-v3b expression over time. CONCLUSIONS: Intratumoral EML4-ALK isoforms may predict the efficacy of targeted therapy in ALK-rearranged NSCLC. Temporal changes of intratumoral fusion isoforms may result from differential selection pressures that a drug might have on one isoform over another. Larger studies on fusion heterogeneity using RNA sequencing are warranted.

Combinatorial Treatment of Human Cardiac Engineered Tissues With Biomimetic Cues Induces Functional Maturation as Revealed by Optical Mapping of Action Potentials and Calcium Transients.

Although biomimetic stimuli, such as microgroove-induced alignment (µ), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined µ-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-ß signaling was downregulated; the TGF-ß receptor agonist and antagonist TGF-ß1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also improves the use of engineered cardiac tissues for in vitro drug screening and disease modeling.

Correlation between frataxin expression and contractility revealed by in vitro Friedreich's ataxia cardiac tissue models engineered from human pluripotent stem cells.

BACKGROUND: Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by a non-coding mutation in the first intron of the frataxin (FXN) gene that suppresses its expression. Compensatory hypertrophic cardiomyopathy, dilated cardiomyopathy, and conduction system abnormalities in FRDA lead to cardiomyocyte (CM) death and fibrosis, consequently resulting in heart failure and arrhythmias. Murine models have been developed to study disease pathology in the past two decades; however, differences between human and mouse physiology and metabolism have limited the relevance of animal studies in cardiac disease conditions. To bridge this gap, we aimed to generate species-specific, functional in vitro experimental models of FRDA using 2-dimensional (2D) and 3-dimensional (3D) engineered cardiac tissues from FXN-deficient human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-hvCMs) and to compare their contractile and electrophysiological properties with healthy tissue constructs. METHODS: Healthy control and FRDA patient-specific hPSC-hvCMs were derived by directed differentiation using a small molecule-based protocol reported previously. We engineered the hvCMs into our established human ventricular cardiac tissue strip (hvCTS) and human ventricular cardiac anisotropic sheet (hvCAS) models, and functional assays were performed on days 7-17 post-tissue fabrication to assess the electrophysiology and contractility of FRDA patient-derived and FXN-knockdown engineered tissues, in comparison with healthy controls. To further validate the disease model, forced expression of FXN was induced in FXN-deficient tissues to test if disease phenotypes could be rescued. RESULTS: Here, we report for the first time the generation of human engineered tissue models of FRDA cardiomyopathy from hPSCs: FXN-deficient hvCTS displayed attenuated developed forces (by 70-80%) compared to healthy controls. High-resolution optical mapping of hvCAS with reduced FXN expression also revealed electrophysiological defects consistent with clinical observations, including action potential duration prolongation and maximum capture frequency reduction. Interestingly, a clear positive correlation between FXN expression and contractility was observed (ρ > 0.9), and restoration of FXN protein levels by lentiviral transduction rescued contractility defects in FXN-deficient hvCTS. CONCLUSIONS: We conclude that human-based in vitro cardiac tissue models of FRDA provide a translational, disease-relevant biomimetic platform for the evaluation of novel therapeutics and to provide insight into FRDA disease progression.

Modulation of chromatin remodeling proteins SMYD1 and SMARCD1 promotes contractile function of human pluripotent stem cell-derived ventricular cardiomyocyte in 3D-engineered cardiac tissues.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have the ability of differentiating into functional cardiomyocytes (CMs) for cell replacement therapy, tissue engineering, drug discovery and toxicity screening. From a scale-free, co-expression network analysis of transcriptomic data that distinguished gene expression profiles of undifferentiated hESC, hESC-, fetal- and adult-ventricular(V) CM, two candidate chromatin remodeling proteins, SMYD1 and SMARCD1 were found to be differentially expressed. Using lentiviral transduction, SMYD1 and SMARCD1 were over-expressed and suppressed, respectively, in single hESC-VCMs as well as the 3D constructs Cardiac Micro Tissues (CMT) and Tissue Strips (CTS) to mirror the endogenous patterns, followed by dissection of their roles in controlling cardiac gene expression, contractility, Ca2+-handling, electrophysiological functions and in vitro maturation. Interestingly, compared to independent single transductions, simultaneous SMYD1 overexpression and SMARCD1 suppression in hESC-VCMs synergistically interacted to increase the contractile forces of CMTs and CTSs with up-regulated transcripts for cardiac contractile, Ca2+-handing, and ion channel proteins. Certain effects that were not detected at the single-cell level could be unleashed under 3D environments. The two chromatin remodelers SMYD1 and SMARCD1 play distinct roles in cardiac development and maturation, consistent with the notion that epigenetic priming requires triggering signals such as 3D environmental cues for pro-maturation effects.

Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.

BACKGROUND: Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to ß-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in ß-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs. METHODS AND RESULTS: First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant, respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase, Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly, Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally, similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However, Ad-PLB altered neither the global transcriptome nor ICa,L, implicating a PLB-specific effect. CONCLUSIONS: Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to ß-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses.

A simple, cost-effective but highly efficient system for deriving ventricular cardiomyocytes from human pluripotent stem cells.

Self-renewable human pluripotent stem cells (hPSCs) serve as a potential unlimited ex vivo source of human cardiomyocytes (CMs) for cell-based disease modeling and therapies. Although recent advances in directed differentiation protocols have enabled more efficient derivation of hPSC-derived CMs with an efficiency of ∼50%-80% CMs and a final yield of ∼1-20 CMs per starting undifferentiated hPSC, these protocols are often not readily transferrable across lines without first optimizing multiple parameters. Further, the resultant populations are undefined for chamber specificity or heterogeneous containing mixtures of atrial, ventricular (V), and pacemaker derivatives. Here we report a highly cost-effective and reproducibly efficient system for deriving hPSC-ventricular cardiomyocytes (VCMs) from all five human embryonic stem cell (HES2, H7, and H9) and human induced PSC (hiPSC) (reprogrammed from human adult peripheral blood CD34(+) cells using nonintegrating episomal vectors) lines tested. Cardiogenic embryoid bodies could be formed by the sequential addition of BMP4, Rho kinase inhibitor, activin-A, and IWR-1. Spontaneously contracting clusters appeared as early as day 8. At day 16, up to 95% of cells were cTnT(+). Of which, 93%, 94%, 100%, 92%, and 92% of cardiac derivatives from HES2, H7, H9, and two iPSC lines, respectively, were VCMs as gauged by signature ventricular action potential and ionic currents (INa(+)/ICa,L(+)/IKr(+)/IKATP(+)); Ca(2+) transients showed positive chronotropic responses to ß-adrenergic stimulation. Our simple, cost-effective protocol required the least amounts of reagents and time compared with others. While the purity and percentage of PSC-VCMs were comparable to a recently published protocol, the present yield and efficiency with a final output of up to 70 hPSC-VCMs per hPSC was up to 5-fold higher and without the need of performing line-specific optimization. These differences were discussed. The results may lead to mass production of hPSC-VCMs in bioreactors.

Epigenetic regulation of the electrophysiological phenotype of human embryonic stem cell-derived ventricular cardiomyocytes: insights for driven maturation and hypertrophic growth.

Epigenetic regulation is implicated in embryonic development and the control of gene expression in a cell-specific manner. However, little is known about the role of histone methylation changes on human cardiac differentiation and maturation. Using human embryonic stem cells (hESCs) and their derived ventricular (V) cardiomyocytes (CMs) as a model, we examined trimethylation of histone H3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3) on promoters of genes associated with cardiac electrophysiology, contraction, and Ca(2+) handling. To avoid ambiguities due to heterogeneous chamber-specific types, hESC-derived ventricular cardiomyocytes (VCMs) were selected by dual zeocin-GFP expression under the transcriptional control of the MLC2v promoter and confirmed electrophysiologically by its signature action potential phenotype. High levels of H3K4me3 are present on pluripotency genes in hESCs with an absence of H3K27me3. Human ESC-VCMS, relative to hESCs, were characterized by a profound loss of H3K27me3 and an enrichment of H3K4me3 marks on cardiac-specific genes, including MYH6, MYH7, MYL2, cTNT, and ANF. Gene transcripts encoding key voltage-gated ion channels and Ca(2+)-handling proteins in hESC-VCMs were significantly increased, which could be attributed to a distinct pattern of differential H3K4me3 and H3K27me3 profiles. Treatment of hESC-VCMs with the histone deacetylase inhibitor valproic acid increased H3K4me3 on gene promoters, induced hypertrophic growth (as gauged by cell volume and capacitance), and augmented cardiac gene expression, but it did not affect electrophysiological properties of these cells. Hence, cardiac differentiation of hESCs involves a dynamic shift in histone methylation, which differentially affects VCM gene expression and function. We conclude that the epigenetic state of hESC-VCMs is dynamic and primed to promote growth and developmental maturation, but that proper environmental stimuli with chromatin remodeling will be required to synergistically trigger global CM maturation to a more adult-like phenotype.

Histone H4 lysine 16 hypoacetylation is associated with defective DNA repair and premature senescence in Zmpste24-deficient mice.

Specific point mutations in lamin A gene have been shown to accelerate aging in humans and mice. Particularly, a de novo mutation at G608G position impairs lamin A processing to produce the mutant protein progerin, which causes the Hutchinson Gilford progeria syndrome. The premature aging phenotype of Hutchinson Gilford progeria syndrome is largely recapitulated in mice deficient for the lamin A-processing enzyme, Zmpste24. We have previously reported that Zmpste24 deficiency results in genomic instability and early cellular senescence due to the delayed recruitment of repair proteins to sites of DNA damage. Here, we further investigate the molecular mechanism underlying delayed DNA damage response and identify a histone acetylation defect in Zmpste24(-/-) mice. Specifically, histone H4 was hypoacetylated at a lysine 16 residue (H4K16), and this defect was attributed to the reduced association of a histone acetyltransferase, Mof, to the nuclear matrix. Given the reversible nature of epigenetic changes, rescue experiments performed either by Mof overexpression or by histone deacetylase inhibition promoted repair protein recruitment to DNA damage sites and substantially ameliorated aging-associated phenotypes, both in vitro and in vivo. The life span of Zmpste24(-/-) mice was also extended with the supplementation of a histone deacetylase inhibitor, sodium butyrate, to drinking water. Consistent with recent data showing age-dependent buildup of unprocessable lamin A in physiological aging, aged wild-type mice also showed hypoacetylation of H4K16. The above results shed light on how chromatin modifications regulate the DNA damage response and suggest that the reversal of epigenetic marks could make an attractive therapeutic target against laminopathy-based progeroid pathologies.