D1 Overexpression Research Articles

Doxycycline-Mediated Control of Cyclin D2 Overexpression in Human-Induced Pluripotent Stem Cells.

Previous studies have demonstrated that when the cyclin D2 (CCND2), a cell-cycle regulatory protein, is overexpressed in human-induced pluripotent stem cells (hiPSCs), cardiomyocytes (CMs) differentiated from these CCND2-overexpressing hiPSCs can proliferate after transplantation into infarcted hearts, which significantly improves the cells' potency for myocardial regeneration. However, persistent CM proliferation could lead to tumor growth or the development of arrhythmogenic complications; thus, the goal of the current study was to generate a line of hiPSCs in which CCND2 overexpression could be tightly controlled. First, we transfected hiPSCs with vectors coding for a doxycycline-inducible Tet-On transactivator and S. pyogenes dCas9 fused to the VPR activation domain; then, the same hiPSCs were engineered to express guide RNAs targeting the CCND2 promotor. Thus, treatment with doxycycline (dox) activated dCas9-VPR expression, and the guide RNAs directed dCas9-VPR to the CCND2 promoter, which activated CCND2 expression. Subsequent experiments confirmed that CCND2 expression was dox-dependent in this newly engineered line of hiPSCs (doxCCND2-hiPSCs): CCND2 protein was abundantly expressed after 48 h of treatment with dox and declined to near baseline level ~96 h after dox treatment was discontinued.

An immature, dedifferentiated, and lineage-deconstrained cone precursor origin of N-Myc–initiated retinoblastoma

Most retinoblastomas develop from maturing cone precursors in response to biallelic RB1 loss and are dependent on cone maturation-related signaling. Additionally, ∼2% lack RB1 mutations but have MYCN amplification (MYCNA), N-Myc protein overexpression, and more rapid and invasive growth, yet the MYCNA retinoblastoma cell of origin and basis for its responses to deregulated N-Myc are unknown. Here, using explanted cultured retinae, we show that ectopic N-Myc induces cell cycle entry in cells expressing markers of several retinal types yet induces continuous proliferation and tumorigenesis only in cone precursors. Unlike the response to RB1 loss, both immature cone arrestin-negative (ARR3-) and maturing ARR3+ cone precursors proliferate, and maturing cone precursors rapidly dedifferentiate, losing ARR3 as well as L/M-opsin expression. N-Myc-overexpressing retinal cells also lose cell lineage constraints, occasionally coexpressing the cone-specific RXRγ with the rod-specific NRL or amacrine-specific AP2α and widely coexpressing RXRγ with the progenitor and Müller cell-specific SOX9 and retinal ganglion cell-specific BRN3 and GAP43. Mechanistically, N-Myc induced Cyclin D2 and CDK4 overexpression, pRB phosphorylation, and SOX9-dependent proliferation without a retinoma-like stage that characterizes pRB-deficient retinoblastoma, despite continuous p16INK4A expression. Orthotopic xenografts of N-Myc-overexpressing retinal cells formed tumors with retinal cell marker expression similar to those in MYCN-transduced retinae and MYCNA retinoblastomas in patients. These findings demonstrate the MYCNA retinoblastoma origin from immature and lineage-deconstrained cone precursors, reveal their opportunistic use of an undifferentiated retinal progenitor cell feature, and illustrate that different cancer-initiating mutations cooperate with distinct developmental stage-specific cell signaling circuitries to drive retinoblastoma tumorigenesis.

Immunohistochemical Expression of Cyclin D1 in Oral Lichen Planus

Background: Many studies have reported that a condition initially classified as oral lichen planus (OLP) has a variable chance of developing cancer progression over time, although these results are still questionable. Cyclin D1 controls the mitotic cell cycle's transition from G1 to S. Cyclin D1 has a significant function in carcinogenesis due to its critical involvement in cell cycle control. Cyclin D1 deregulation or overexpression may result in shortened G1 phase, enhanced cell replication, and decreased reliance on growth regulators, causing disruption in conventional cell cycle progression and the progression of cancer. The aim of the current study was to evaluate the expression of Cyclin D1 in histopathologically confirmed OLP samples and to predict the rate of malignant transformation in these lesions by comparing the level of expression with some clinical parameters. Materials and Methods: The immunohistochemical procedure was used to examine the expression of cyclin D1 on paraffin-embedded sections of (40) OLP lesions and (10) specimens of normal oral mucosa. We used the SPSS software version (18.0) to do the statistical analysis in this work. Results: There is a very significant difference between the people investigated (P = 0.01), which is statistically significant. CyclinD1 was found in all of the oral lichen planus cases in the research (100%), but it was also found (80%) in the normal oral mucosa. There was also no statistically significant difference between the clinical types of OLP and the Cyclin D1 scoring (P = 0.942), which revealed that the reticular type was the most frequent (7) in mild scoring, whereas the erosive type was the most frequent in severe scoring, which accounted for the majority of the cases. Conclusion: This research found that higher cyclinD1 expression increases the risk of cancer and many of the markers should be tested. Furthermore, larger sample numbers, improved designs, and gene-reduction studies should be performed. OLP patients should be continuously monitored for OSCC progression.

Dopamine Negatively Regulates Insulin Secretion Through Activation of D1-D2 Receptor Heteromer.

There is increasing evidence that dopamine (DA) functions as a negative regulator of glucose-stimulated insulin secretion; however, the underlying molecular mechanism remains unknown. Using total internal reflection fluorescence microscopy, we monitored insulin granule exocytosis in primary islet cells to dissect the effect of DA. We found that D1 receptor antagonists rescued the DA-mediated inhibition of glucose-stimulated calcium (Ca2+) flux, thereby suggesting a role of D1 in the DA-mediated inhibition of insulin secretion. Overexpression of D2, but not D1, alone exerted an inhibitory and toxic effect that abolished the glucose-stimulated Ca2+ influx and insulin secretion in β-cells. Proximity ligation and Western blot assays revealed that D1 and D2 form heteromers in β-cells. Treatment with a D1-D2 heteromer agonist, SKF83959, transiently inhibited glucose-induced Ca2+ influx and insulin granule exocytosis. Coexpression of D1 and D2 enabled β-cells to bypass the toxic effect of D2 overexpression. DA transiently inhibited glucose-stimulated Ca2+ flux and insulin exocytosis by activating the D1-D2 heteromer. We conclude that D1 protects β-cells from the harmful effects of DA by modulating D2 signaling. The finding will contribute to our understanding of the DA signaling in regulating insulin secretion and improve methods for preventing and treating diabetes.

HOXD3 Up-regulating KDM5C Promotes Malignant Progression of Diffuse Large B-Cell Lymphoma by Decreasing p53 Expression

Background:Diffuse large B-cell lymphoma is a type of B-cell non-Hodgkin lymphoma with a high incidence. About one-third of patients are resistant or eventually relapse. The prognosis for patients with relapsed/resistant diffuse large B-cell lymphoma who need salvage therapy is not optimistic.Aims:To explore whether homebox D3 binding to lysine (K)-specific demethylase 5C promoted malignant progression of diffuse large B-cell lymphoma by decreasing p53 expression.Study Design:Cell culture study.Methods:The mRNA and protein expression of lysine (K)-specific demethylase 5C and homebox D3 in cells were respectively detected by real-time quantitative polymerase chain reaction analysis and Western blot. Real-time quantitative polymerase chain reaction analysis and Western blot were also applied to determine the transfection effects of shRNA-KDM5C or OeHOXD3 in OCI-Ly7 cells. After transfection, the cell viability, proliferation, and apoptosis were respectively analyzed by Cell Counting Kit-8 assay, EdU staining, and acridine orange—ethidium bromide staining. The interaction between homebox D3 and lysine (K)-specific demethylase 5C promoter was verified by the dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay.Results:Lysine (K)-specific demethylase 5C mRNA expression (HBL1 2.84 ± 0.29; SUDHL4 3.53 ± 0.21; OCI-Ly8 4.06 ± 0.24; OCI-Ly7 5.03 ± 0.28 vs. GM12878 1.00 ± 0.07; all P < .001) and protein expression (HBL1 1.52 ± 0.06; SUDHL4 1.77 ± 0.10; OCI-Ly8 2.34 ± 0.07; OCI-Ly7 2.78 ± 0.07 vs. GM12878 1.00 ± 0.07; all P < .001) in DLBCL cells were higher than that in GM12878 cells and showed the highest in OCI-Ly7 cells. Homebox D3 mRNA (OCI-Ly7 3.85 ± 0.17 vs. GM12878 1.00 ± 0.05; P < .001) and protein (OCI-Ly7 1.73 ± 0.10 vs. GM12878 1.00 ± 0.06; P < .001) expression were also highly expressed in OCI-Ly7 cells. Moreover, down-regulation of lysine (K)-specific demethylase 5C suppressed the viability and proliferation and enhanced the apoptosis of OCI-Ly7 cells. Knockdown of lysine (K)-specific demethylase 5C decreased the B-cell lymphoma 2 expression while increased the expression of Bax, cleaved caspase 3, cytochrome C, p53, and p21. The transcription factor homebox D3 was confirmed to interact with the lysine (K)-specific demethylase 5C promoter. Homebox D3 overexpression could reverse the regulating effect of down-regulation of lysine (K)-specific demethylase 5C on the OCI-Ly7 cells.Conclusion:Homebox D3 up-regulating lysine (K)-specific demethylase 5C promotes malignant progression of diffuse large B-cell lymphoma by decreasing p53 expression.

Sucrose promotes D53 accumulation and tillering in rice.

Shoot branching is regulated by multiple signals. Previous studies have indicated that sucrose may promote shoot branching through suppressing the inhibitory effect of the hormone strigolactone (SL). However, the molecular mechanisms underlying this effect are unknown. Here, we used molecular and genetic tools to identify the molecular targets underlying the antagonistic interaction between sucrose and SL. We showed that sucrose antagonizes the suppressive action of SL on tillering in rice and on the degradation of D53, a major target of SL signalling. Sucrose inhibits the gene expression of D3, the orthologue of the Arabidopsis F-box MAX2 required for SL signalling. Overexpression of D3 antagonizes sucrose inhibition of D53 degradation and enables the SL inhibition of tillering under high sucrose. Sucrose prevents SL-induced degradation of D14, the SL receptor involved in D53 degradation. In contrast to D3, D14 overexpression enhances D53 protein levels and sucrose-induced tillering, even in the presence of SL. Our results show that sucrose inhibits SL response by affecting key components of SL signalling and, together with previous studies reporting the inhibition of SL synthesis by nitrate and phosphate, demonstrate the central role played by SLs in the regulation of plant architecture by nutrients.

Aberrant overexpression of transcription factor Forkhead box D1 predicts poor prognosis and promotes cancer progression in HNSCC

ObjectivesForkhead box D1, the core transcription factor member of FOX family, has gradually seen as a key cancerous regulatory. However, its expression and carcinogenicity in head and neck squamous cell carcinoma (HNSCC) have not been reported yet. This study was to investigate its expression pattern, clinicopathological significance and biological roles in HNSCC.MethodsHNSCC data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) was used to indicate the detailed expression pattern and outcome association of FOXD1, while Western Blot assay to detect FOXD1 level in a panel of HNSCC cell lines as well as immunocytochemistry to explore FOXD1 protein abundance and sublocation. Series of siRNA-mediated FOXD1 knock-down experiments to assess the proliferation, migration, invasion and anti- apoptosis ability after FOXD1 down-regulation. Bioinformatic analysis to find out which biological function and cancer-related pathways of FOXD1 associated genes involved in.ResultsFOXD1 mRNA was significantly overexpressed in TCGA-HNSCC, GSE6631, GSE12452, GSE25099 and GSE30784. Besides, IHC results shown that nuclear location FOXD1 protein was significantly higher in primary HNSCC specimens from cohort involved in this study. Also, FOXD1 abundance was significantly correlated with cervical node metastasis and poor over-all/disease-free survival after combination analysis with patient pathological information. siRNA-mediated FOXD1 knock-down significantly inhibited cell proliferation, migration and invasion and induced apoptosis in HNSCC cells. Further analysis of GSEA, GO and KEGG showed that FOXD1 expression was significantly associated with oncological function and cancer-related pathways.ConclusionsTaken together, our study implies that the potential oncogene, FOXD1, facilitates oncological behavior who can be identified as a brand-new HNSCC biomarker with diagnostic and prognostic significance.

Cyclin D2 overexpression drives B1a-derived MCL-like lymphoma in mice.

Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with poor long-term overall survival. Currently, MCL research and development of potential cures is hampered by the lack of good in vivo models. MCL is characterized by recurrent translocations of CCND1 or CCND2, resulting in overexpression of the cell cycle regulators cyclin D1 or D2, respectively. Here, we show, for the first time, that hematopoiesis-specific activation of cyclin D2 is sufficient to drive murine MCL-like lymphoma development. Furthermore, we demonstrate that cyclin D2 overexpression can synergize with loss of p53 to form aggressive and transplantable MCL-like lymphomas. Strikingly, cyclin D2-driven lymphomas display transcriptional, immunophenotypic, and functional similarities with B1a B cells. These MCL-like lymphomas have B1a-specific B cell receptors (BCRs), show elevated BCR and NF-κB pathway activation, and display increased MALT1 protease activity. Finally, we provide preclinical evidence that inhibition of MALT1 protease activity, which is essential for the development of early life-derived B1a cells, can be an effective therapeutic strategy to treat MCL.

SOX11 and CD43 Biomarkers in Mantle Cell Lymphoma

Recently, mantle cell lymphoma (MCL) has been recognized as a heterogeneous clinical and biological entity, as molecular genetics highlighted different pathways of cell survival and progression. Histological diagnosis may be challenging due to atypical MCL variants, immunophenotyping or absence of cyclin D1 (D1), encountered in 10% of cases. SOX11 is a neural transcription factor, involved in blocking mature B cell differentiation and its overexpression is associated with poor prognosis. It is overexpressed in most conventional MCL (cMCL) and in MCL lacking D1 overexpression, but not detected in other mature B cell lymphomas or normal lymphoid cells. Recently, its overexpression has been considered as a specific biomarker for MCL diagnosis.Development of novel targeted therapy approaches renders monitoring of minimal residual disease (MRD) essential to assess treatment effectiveness. Amongst methods developed to monitor MRD, allele-specific oligonucleotide quantitative PCR for BCL1 or IgH, is the most sensitive and commonly used approach. Given the role of SOX11 and BCL1 in the pathophysiology of MCL, we developed an easy set up single step quantitative PCR (qPCR) to assess both markers simultaneously, which was tested on a large panel of MCL patients at diagnosis and MRD monitoring.A reverse transcription qPCR protocol was carried out to quantify SOX11, cyclin D1 and D2 overexpression simultaneously, using specific primers, Taqman probes and B2M house keeping gene (LC480, Roche). RNA was extracted from peripheral blood (PB), bone marrow (BM) and lymph nodes (LN). Sensitivity was determined using 6 fold dilutions starting with 1:10 from 30 subjects with normal lymphocytes. A retrospective study on a total of 170 samples for 145 patients between 2002 and 2017 was conducted. Samples were chosen based on morphology, immunophenotyping and histopathology if available. All samples were CD20+, 162 were CD5+ and 165 were CD10-. All patients had provided an informed written consent in accordance with the declaration of Helsinki. We tested 93 patients with MCL at diagnosis, 79 cMCL and 14 indolent mantle cell lymphoma (iMCL). To assess specificity 52 patients with other lymphoid proliferations CD5/20+, CD10- were studied. 20 samples from 5 patients SOX11+/D1+ were available for MRD monitoring.Sensitivity of the method achieved 1.10-4. At diagnosis, tumor cells ranged between 11-97% of total lymphocytes in PB and 1-91% in BM. SOX11 and D1 overexpression was correlated with the tumor burden. 79/79 cMCL patients (100%) overexpressed SOX11 and D1 (SOX11+/D1+), 2 cMCL were CD5-, SOX11+/ D1+ and 2 cMCL were CD10+, SOX11 +/ D1+. All 14 iMCL patients were (SOX11-/D1+). Sox 11 was not detected in normal lymphocytes or in other lymphoproliferative diseases. 5 patients presented with the blastoid variant, overexpressed SOX11, but only 4 overexpressed D1 and tumor infiltration rate at diagnosis was over 83% in PB. MRD follow up showed negativity of both markers for 2 patients at complete remission. SOX11 overexpression was detected before cyclin D1 in 2 patients at relapse, prior to clinical manifestations. Persistent SOX11 and D1 overexpression was observed for 1 splenctomized patient. Time to treatment was found to be 0.6 vs 18 months in 18 cMCL and 12 iMCL patients respectively (p= 0.0006). CD43 was evaluated for 27 patients, 4 blastoid variants and 23 cMCL. 100% of blastoid variants were positive for CD43, but only 43.4% of cMCL were positive for CD43 (p=0.0368). Overall survival curve for CD43 positive patients showed a median of 2.3 years survival (p=0.0285). This was not reached for the CD43 negative MCL.We developed a feasible single step sensitive method to quantify SOX11, D1 and D2 in MCL which allows differential diagnosis with other lymphoid proliferations. A parallel correlation was found between SOX11, D1 and clinical status of MCL during diagnosis and follow up. As histopathology is unsuitable for MRD evaluation, both markers could be used to assess MRD, though SOX11 is preferred to D1 as it lacks the D1 background overexpression. Considering the higher sensitivity of molecular MRD compared to conventional evaluating procedures, its integration in guided treatment strategies is warranted. To the best of our knowledge, this is the first report stating the association of CD43 with adverse prognosis in MCL which might be interesting as a prognostication marker and a target for a novel immunotherapy development. [Display omitted] DisclosuresTroussard:ROCHE: Honoraria; GILEAD: Honoraria; JANSSEN: Honoraria; ABBVIE: Honoraria.

Improvements of the productivity and saccharification efficiency of the cellulolytic \u03b2-glucosidase D2-BGL in Pichia pastoris via directed evolution

Backgroundβ-Glucosidases are essential for cellulose hydrolysis by catalyzing the final cellulolytic degradation of cello-oligomers and cellobiose to glucose. D2-BGL is a fungal glycoside hydrolase family 3 (GH3) β-glucosidase isolated from Chaetomella raphigera with high substrate affinity, and is an efficient β-glucosidase supplement to Trichoderma reesei cellulase mixtures for the saccharification of lignocellulosic biomass.ResultsWe have carried out error-prone PCR to further increase catalytic efficiency of wild-type (WT) D2-BGL. Three mutants, each with substitution of two amino acids on D2-BGL, exhibited increased activity in a preliminary mutant screening in Saccharomyces cerevisiae. Effects of single amino acid replacements on catalysis efficiency and enzyme production have been investigated by subsequent expression in Pichia pastoris. Substitution F256M resulted in enhancing the tolerance to substrate inhibition and specific activity, and substitution D224G resulted in increasing the production of recombinant enzyme. The best D2-BGL mutant generated, Mut M, was constructed by combining beneficial mutations D224G, F256M and Y260D. Expression of Mut M in Pichia pastoris resulted in 2.7-fold higher production of recombinant protein, higher Vmax and greater substrate inhibition tolerance towards cellobiose relative to wild-type enzyme. Surprisingly, Mut M overexpression induced the ER unfolded protein response to a level lower than that with WT D2 overexpression in P. pastoris. When combined with the T. reesei cellulase preparation Celluclast 1.5L, Mut M hydrolyzed acid-pretreated sugarcane bagasse more efficiently than WT D2.ConclusionsD2-BGL mutant Mut M was generated successfully by following directed evolution approach. Mut M carries three mutations that are not reported in other directed evolution studies of GH3 β-glucosidases, and this mutant exhibited greater tolerance to substrate inhibition and higher Vmax than wild-type enzyme. Besides the enhanced specific activity, Mut M also exhibited a higher protein titer than WT D2 when it was overexpressed in P. pastoris. Our study demonstrates that both catalytic efficiency and productivity of a cellulolytic enzyme can be enhanced via protein engineering.

Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction.

Human induced pluripotent stem cells with normal (wild-type) or upregulated (overexpressed) levels of CCND2 (cyclin D2) expression were differentiated into cardiomyocytes (CCND2WTCMs or CCND2OECMs, respectively) and injected into infarcted pig hearts. Acute myocardial infarction was induced by a 60-minute occlusion of the left anterior descending coronary artery. Immediately after reperfusion, CCND2WTCMs or CCND2OECMs (3×107 cells each) or an equivalent volume of the delivery vehicle was injected around the infarct border zone area. The number of the engrafted CCND2OECMs exceeded that of the engrafted CCND2WTCMs from 6- to 8-fold, rising from 1 week to 4 weeks after implantation. In contrast to the treatment with the CCND2WTCMs or the delivery vehicle, the administration of CCND2OECM was associated with significantly improved left ventricular function, as revealed by magnetic resonance imaging. This correlated with reduction of infarct size, fibrosis, ventricular hypertrophy, and cardiomyocyte apoptosis, and increase of vascular density and arterial density, as per histologic analysis of the treated hearts. Expression of cell proliferation markers (eg, Ki67, phosphorylated histone 3, and Aurora B kinase) was also significantly upregulated in the recipient cardiomyocytes from the CCND2OECM-treated than from the CCND2WTCM-treated pigs. The cell proliferation rate and the hypoxia tolerance measured in cultured human induced pluripotent stem cell cardiomyocytes were significantly greater after treatment with exosomes isolated from the CCND2OECMs (CCND2OEExos) than from the CCND2WTCMs (CCND2WTExos). As demonstrated by our study, CCND2OEExos can also promote the proliferation activity of postnatal rat and adult mouse cardiomyocytes. A bulk miRNA sequencing analysis of CCND2OEExos versus CCND2WTExos identified 206 and 91 miRNAs that were significantly upregulated and downregulated, respectively. Gene ontology enrichment analysis identified significant differences in the expression profiles of miRNAs from various functional categories and pathways, including miRNAs implicated in cell-cycle checkpoints (G2/M and G1/S transitions), or the mechanism of cytokinesis. We demonstrated that enhanced potency of CCND2OECMs promoted myocyte proliferation in both grafts and recipient tissue in a large mammal acute myocardial infarction model. These results suggest that CCND2OECMs transplantation may be a potential therapeutic strategy for the repair of infarcted hearts.

Pathogenic role of delta 2 tubulin in bortezomib-induced peripheral neuropathy

The pathogenesis of chemotherapy-induced peripheral neuropathy (CIPN) is poorly understood. Here, we report that the CIPN-causing drug bortezomib (Bort) promotes delta 2 tubulin (D2) accumulation while affecting microtubule stability and dynamics in sensory neurons in vitro and in vivo and that the accumulation of D2 is predominant in unmyelinated fibers and a hallmark of bortezomib-induced peripheral neuropathy (BIPN) in humans. Furthermore, while D2 overexpression was sufficient to cause axonopathy and inhibit mitochondria motility, reduction of D2 levels alleviated both axonal degeneration and the loss of mitochondria motility induced by Bort. Together, our data demonstrate that Bort, a compound structurally unrelated to tubulin poisons, affects the tubulin cytoskeleton in sensory neurons in vitro, in vivo, and in human tissue, indicating that the pathogenic mechanisms of seemingly unrelated CIPN drugs may converge on tubulin damage. The results reveal a previously unrecognized pathogenic role for D2 in BIPN that may occur through altered regulation of mitochondria motility.

Expression of Cyclin D1 in oral squamous cell carcinoma and its correlation with histopathological differentiation

Cyclin D1 is a vital protein that has a widespread role in cell cycle regulations, providing control over G1 to S Phase transition and governing cell proliferation rates.Cyclin D1 overexpression has been reported in variety of tumors. Present study was carried out to the study the expression of cyclin D1 and its association with histopathological differentiation and stage of oral squamous cell carcinoma.48 formalin fixed paraffin embedded tissue blocks of biopsy specimens of oral squamous cell carcinoma were immunohistochemically evaluated for expression of cyclinD1.CyclinD1 expression was seen in 98.5% (46) cases of OSCC. It did not correlate with site and staging. CyclinD1 expression was seen in 94.6% of well differentiated tumor,100% in moderately differentiated and 100% in poorly differentiated oral squamous cell carcinoma, but it was not statistically significant(p=0.065).Relatively higher frequency of Cyclin D1 immuno-reactivity observed in patients with less differentiated tumours suggest inverse correlation of Cyclin D1 expression with histological differentiation of tumour.

Abstract A49: Combined targeting of the p53 and pRb pathway in neuroblastoma

Abstract Neuroblastomas account for approximately 15% of all childhood cancer deaths. Clinical complete remission is achieved in many stage 4 neuroblastoma patients, but the high risk of relapse and the accompanying treatment-resistant nature of these tumors is still a challenge. We have previously identified higher frequencies of mutations that affect both the p53 and the pRb pathway, such as the homozygous loss of CDKN2A or co-amplification of MDM2 and CDK4. In addition, both cyclin D1 and MDM2 overexpression is a common characteristic of primary neuroblastoma. These genes act upstream of pRb and p53 and hence play a major role in cell cycle regulation. To study whether these G1 checkpoint aberrations render cells more sensitive to CDK4 or MDM2 inhibition, we exposed a series of 11 cell lines to the most promising CDK4 (ribociclib, palbociclib, and abemaciclib) and MDM2 (SAR405838, HDM-201, and idasanutlin) inhibitors. These cell lines have well-defined mutational properties: homozygous CDKN2A deletion, co-amplification of CDK4 and MDM2, TP53 mutation, and MYCN amplification. Whereas sensitivity for the MDM2 inhibitors inversely correlated with TP53 mutation status, it did not correlate to other G1 checkpoint aberrations. Similarly, there was no clear correlation seen for the CDK4 inhibitors. To further explore this, we generated cell lines with inducible overexpression of CDK4, MDM2, or both. The overexpression did indeed not increase sensitivity to CDK4 or MDM2 inhibitors. Next, we were interested whether combined treatment with the most potent CDK4 and MDM2 inhibitors, abemaciclib and idasanutlin respectively, would be of added value. Cells were exposed to 10 different concentrations of both compounds and Bliss Independence values were calculated as a measure of synergy. Interestingly, combined treatment resulted in slight antagonism in the range of clinically relevant doses in most cell lines. This adverse effect was supported by cell cycle analyses, which showed a lower apoptotic fraction, as well as PARP and caspase 3 cleavage, after combined treatment, as opposed to MDM2 inhibition alone. As was previously suggested in the first clinical trials with CDK4 inhibitors, neither CDK4 nor CDKN2A status is a clear biomarker for CDK4 inhibitor sensitivity. Our results suggest that MDM2 and CDKN2A status also fail as biomarkers for MDM2 inhibitor sensitivity. Further testing is necessary to identify (other) biomarkers for these inhibitors. Moreover, the logical rationale to combine CDK4 and MDM2 inhibitors in neuroblastoma patients with both pRb and p53 pathway disturbances should be taken with precaution, as first results do not show a beneficial response. In vivo experiments to confirm this finding are currently ongoing. Citation Format: Nil Schubert, Linda Schild, Stijn van Oirschot, Kaylee Keller, Lindy Alles, Lindy Vernooij, Marloes Nulle, Emmy Dolman, Marlinde van den Boogaard, Jan Molenaar. Combined targeting of the p53 and pRb pathway in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A49.

Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

SummaryHuman pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

Predictive value of CCND1/cyclin D1 alterations in the malignant transformation of potentially malignant head and neck disorders: Systematic review and meta-analysis.

To evaluate published evidence on the predictive value of CCND1 amplification/cyclin D1 overexpression as malignant transformation risk markers in potentially malignant disorders (PMDs) of the head and neck. We searched PubMed, Embase, Web of Science, and Scopus for studies published before June 2018. We conducted a meta-analysis to quantify the impact of CCND1/cyclin D1 amplification/overexpression on malignant transformation of head and neck PMDs. Nine studies met inclusion criteria. Quantitative evaluation indicated strong statistically significant association between CCND1/cyclin D1 amplification/overexpression and the progression of head and neck PMD to head and neck squamous cell carcinoma (risk ratio [RR] = 2.04, 95% confidence interval [CI] = 1.37-3.03, P < .001, and RR = 2.27, 95% CI = 1.32-3.91, P = .003, respectively). We observed moderate heterogeneity among studies (I2 = 40.7%), and we cannot rule out small-study effects such as publication bias. The oral cavity subgroup showed the strongest association between CCND1/cyclin D1 amplification/overexpression and progression to cancer. CCND1/cyclin D1 amplification/overexpression is important to predict the malignant transformation risk of head and neck PMDs, especially oral PMDs.

CCND2 and CCND3 hijack immunoglobulin light-chain enhancers in cyclin D1− mantle cell lymphoma

AbstractMantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation resulting in overexpression of cyclin D1. However, a small subset of cyclin D1− MCL has been recognized, and approximately one-half of them harbor CCND2 translocations while the primary event in cyclin D1−/D2− MCL remains elusive. To identify other potential mechanisms driving MCL pathogenesis, we investigated 56 cyclin D1−/SOX11+ MCL by fluorescence in situ hybridization (FISH), whole-genome/exome sequencing, and gene-expression and copy-number arrays. FISH with break-apart probes identified CCND2 rearrangements in 39 cases (70%) but not CCND3 rearrangements. We analyzed 3 of these negative cases by whole-genome/exome sequencing and identified IGK (n = 2) and IGL (n = 1) enhancer hijackings near CCND3 that were associated with cyclin D3 overexpression. By specific FISH probes, including the IGK enhancer region, we detected 10 additional cryptic IGK juxtapositions to CCND3 (6 cases) and CCND2 (4 cases) in MCL that overexpressed, respectively, these cyclins. A minor subset of 4 cyclin D1− MCL cases lacked cyclin D rearrangements and showed upregulation of CCNE1 and CCNE2. These cases had blastoid morphology, high genomic complexity, and CDKN2A and RB1 deletions. Both genomic and gene-expression profiles of cyclin D1− MCL cases were indistinguishable from cyclin D1+ MCL. In conclusion, virtually all cyclin D1− MCLs carry CCND2/CCND3 rearrangements with immunoglobulin genes, including a novel IGK/L enhancer hijacking mechanism. A subset of cyclin D1−/D2−/D3− MCL with aggressive features has cyclin E dysregulation. Specific FISH probes may allow the molecular identification and diagnosis of cyclin D1− MCL.

Abstract 17089: Cyclin D2 Overexpression Enhances the Potency of Human Induced-Pluripotent Stem Cell-Derived Cardiomyocytes for Cardiac Repair in a Porcine Model of Myocardial Infarction

Introduction: Stem-cell-based therapies provide promise for the treatment of ischemic myocardial injury, but the number of cells that remain engrafted at the site of administration is exceptionally low, which is believed to limit the effectiveness of treatment. In this study, we investigated whether the number of engrafted human induced-pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) can be increased via overexpression of the cell-cycle activator cyclin D2 (CCND2) and if so, whether this increase is accompanied by improvements in myocardial recovery. Experiments were conducted in a large-animal (swine) model of myocardial injury. Methods and results: CCND2-overexpressing hiPSC-CMs (CCND2 OE CMs) were constructed via lentiviral transfection; then, myocardial infarction (MI) was surgically induced in swine, and the animals were randomly assigned to treatment with CCND2 OE CMs (i.e., the MI+CCND2 OE CM group), wild-type hiPSC-CMs (the MI+CCND2 WT CM group), or neither experimental treatment (the MI group); the cells were injected into five sites (6х10 6 cells/site) surrounding the infarct. Four weeks after MI, measurements of the heart-weight to bodyweight ratio (HW/BW), cardiac function (determined via magnetic resonance imaging), and vascularity (CD31 staining) were significantly better in MI+CCND2 OE CM animals than in the CCND2 WT CM group. HW/BW was also significantly lower in both cell-treatment groups than in MI animals, and CCND2 overexpression was associated with increases in both hiPSC-CM engraftment (mRNA levels) and cell-cycle activity (ki67 and phosphorylated histone 3 levels). Conclusion: Experiments in a large-animal MI model indicated that CCND2 overexpression in transplanted hiPSC-CMs was associated with increases in cell-cycle activity and the number of engrafted cells, as well as improvements in cardiac function, hypertrophy, and vascularity.

Hoxa-10 and Cyclin D3 Overexpression in the Decidual Reaction in a Superovulation Protocol in Young Adult C57BL/6J Mice.

Following the performance of a superovulation protocol, multiple nodules were observed bilaterally in the uterine horns of 31 of 276 (11.2%) C57BL/6 J female mice aged 8.5 ± 0.6 (mean and standard error of mean) weeks. These lesions prevented embryo collection, and the uterine decidual reaction was suspected. Samples of pathological uteri (n = 20) and the normal genital tracts of donors treated with a similar superovulation protocol (control group, n = 10) were collected. Immunohistochemistry was performed to evaluate pancytokeratin, desmin, vimentin, progesterone receptor (PR), estrogen receptor α (ERα), Ki-67, cyclin D3 and c-Myc expression, as well as quantitative polymerase chain reaction to assess cyclin D3, Hoxa-10 and heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA expression. The uterine decidual reaction presented a high degree of structural organization and specifically affected the antimesometrial region of the endometrium. The abnormal decidual cells were large polygonal cells that were frequently polyploid or binucleated and strongly positive for desmin. Immunohistochemistry showed higher Ki-67 proliferation index and higher expression of PR and cyclin D3 in decidual cells in the antimesometrial aspect of the endometrium, compared to nondecidualized endometrial stromal cells in the mesometrial aspect of affected uteri, and compared to endometrial stromal cells in healthy uteri. High expression of cyclin D3 and Hoxa-10 mRNA was also observed in uteri affected by the decidual reaction. These results suggest that PR overexpression in endometrial stromal cells, likely due to high progesterone levels, triggers cyclin D3 and Hoxa-10 overexpression, which may be involved in the pathological mechanisms of the mouse uterine decidual reaction.

Kinetic mRNA Profiling in a Rat Model of Left-Ventricular Hypertrophy Reveals Early Expression of Chemokines and Their Receptors.

Left-ventricular hypertrophy (LVH), a risk factor for heart failure and death, is characterized by cardiomyocyte hypertrophy, interstitial cell proliferation, and leukocyte infiltration. Chemokines interacting with G protein-coupled chemokine receptors may play a role in LVH development by promoting recruitment of activated leukocytes or modulating left-ventricular remodeling. Using a pressure overload-induced kinetic model of LVH in rats, we examined during 14 days the expression over time of chemokine and chemokine receptor mRNAs in left ventricles from aortic-banded vs sham-operated animals. Two phases were clearly distinguished: an inflammatory phase (D3-D5) with overexpression of inflammatory genes such as il-1ß, tnfa, nlrp3, and the rela subunit of nf-kb, and a hypertrophic phase (D7-D14) where anp overexpression was accompanied by a heart weight/body weight ratio that increased by more than 20% at D14. No cardiac dysfunction was detectable by echocardiography at the latter time point. Of the 36 chemokines and 20 chemokine receptors analyzed by a Taqman Low Density Array panel, we identified at D3 (the early inflammatory phase) overexpression of mRNAs for the monocyte chemotactic proteins CCL2 (12-fold increase), CCL7 (7-fold increase), and CCL12 (3-fold increase), for the macrophage inflammatory proteins CCL3 (4-fold increase), CCL4 (2-fold increase), and CCL9 (2-fold increase), for their receptors CCR2 (4-fold increase), CCR1 (3-fold increase), and CCR5 (3-fold increase), and for CXCL1 (8-fold increase) and CXCL16 (2-fold increase). During the hypertrophic phase mRNA expression of chemokines and receptors returned to the baseline levels observed at D0. Hence, this first exhaustive study of chemokine and chemokine receptor mRNA expression kinetics reports early expression of monocyte/macrophage-related chemokines and their receptors during the development of LVH in rats, followed by regulation of inflammation as LVH progresses.