Functional characterization of D-type cyclins involved in cell division in rice.

BACKGROUND: D-type cyclins (CYCD) regulate the cell cycle G1/S transition and are thus closely involved in cell cycle progression. However, little is known about their functions in rice. RESULTS: We identified 14 CYCD genes in the rice genome and confirmed the presence of characteristic cyclin domains in each. The expression of the OsCYCD genes in different tissues was investigated. Most OsCYCD genes were expressed at least in one of the analyzed tissues, with varying degrees of expression. Ten OsCYCD proteins could interact with both retinoblastoma-related protein (RBR) and A-type cyclin-dependent kinases (CDKA) forming holistic complexes, while OsCYCD3;1, OsCYCD6;1, and OsCYCD7;1 bound only one component, and OsCYCD4;2 bound to neither protein. Interestingly, all OsCYCD genes except OsCYCD7;1, were able to induce tobacco pavement cells to re-enter mitosis with different efficiencies. Transgenic rice plants overexpressing OsCYCD2;2, OsCYCD6;1, and OsCYCD7;1 (which induced cell division in tobacco with high-, low-, and zero-efficiency, respectively) were created. Higher levels of cell division were observed in both the stomatal lineage and epidermal cells of the OsCYCD2;2- and OsCYCD6;1-overexpressing plants, with lower levels seen in OsCYCD7;1-overexpressing plants. CONCLUSIONS: The distinct expression patterns and varying effects on the cell cycle suggest different functions for the various OsCYCD proteins. Our findings will enhance understanding of the CYCD family in rice and provide a preliminary foundation for the future functional verification of these genes.

Analysis of E2F1 single-nucleotide polymorphisms reveals deleterious non-synonymous substitutions that disrupt E2F1-RB protein interaction in cancer.

Cancer is a medical condition that is caused by the abnormal growth and division of cells, leading to the formation of tumors. The E2F1 and RB pathways are critical in regulating cell cycle, and their dysregulation can contribute to the development of cancer. In this study, we analyzed experimentally reported SNPs in E2F1 and assessed their effects on the binding affinity with RB. Out of 46, nine mutations were predicted as deleterious, and further analysis revealed four highly destabilizing mutations (L206W, R232C, I254T, A267T) that significantly altered the protein structure. Molecular docking of wild-type and mutant E2F1 with RB revealed a docking score of -242 kcal/mol for wild-type, while the mutant complexes had scores ranging from -217 to -220 kcal/mol. Molecular simulation analysis revealed variations in the dynamics features of both mutant and wild-type complexes due to the acquired mutations. Furthermore, the total binding free energy for the wild-type E2F1-RB complex was -64.89 kcal/mol, while those of the L206W, R232C, I254T, and A267T E2F1-RB mutants were -45.90 kcal/mol, -53.52 kcal/mol, -55.67 kcal/mol, and -61.22 kcal/mol, respectively. Our study is the first to extensively analyze E2F1 gene mutations and identifies candidate mutations for further validation and potential targeting for cancer therapeutics.

Genetic alterations that deregulate RB and PDGFRA signaling pathways drive tumor progression in IDH2-mutant astrocytoma.

In IDH-mutant astrocytoma, IDH2 mutation is quite rare and biological mechanisms underlying tumor progression in IDH2-mutant astrocytoma remain elusive. Here, we report a unique case of IDH2 mutant astrocytoma, CNS WHO grade 3 that developed tumor progression. We performed a comprehensive genomic and epigenomic analysis for primary and recurrent tumors and found that both tumors harbored recurrent IDH2R172K and TP53R248W mutation with CDKN2A/B hemizygous deletion. We also found amplifications of CDK4 and MDM2 with PDGFRA gain in the recurrent tumor and upregulated protein expressions of these genes. We further developed, for the first time, a xenograft mouse model of IDH2R172K and TP53R248W mutant astrocytoma from the recurrent tumor, but not from the primary tumor. Consistent with parent recurrent tumor cells, amplifications of CDK4 and MDM2 and PDGFRA gain were found, while CDKN2A/B was identified as homozygous deletion in the xenografts, qualifying for integrated diagnosis of astrocytoma, IDH2-mutant, CNS WHO grade 4. Cell viability assay found that CDK4/6 inhibitor and PDGFR inhibitor potently decreased cell viability in recurrent tumor cells, as compared to primary tumor cells. These findings suggest that gene alterations that activate retinoblastoma (RB) signaling pathways and PDGFR may drive tumor progression and xenograft formation in IDH2-mutant astrocytoma, which is equivalent to progressive IDH1-mutant astrocytoma. Also, our findings suggest that these genomic alterations may represent therapeutic targets in IDH2-mutant astrocytoma.

Pharmacological induction of translational readthrough of nonsense mutations in the retinoblastoma (RB1) gene.

The retinoblastoma protein (Rb) is encoded by the RB1 tumor suppressor gene. Inactivation of RB1 by inherited or somatic mutation occurs in retinoblastoma and various other types of tumors. A significant fraction (25.9%) of somatic RB1 mutations are nonsense substitutions leading to a premature termination codon (PTC) in the RB1 coding sequence and expression of truncated inactive Rb protein. Here we show that aminoglycoside G418, a known translational readthrough inducer, can induce full-length Rb protein in SW1783 astrocytoma cells with endogenous R579X nonsense mutant RB1 as well as in MDA-MB-436 breast carcinoma cells transiently transfected with R251X, R320X, R579X or Q702X nonsense mutant RB1 cDNA. Readthrough was associated with increased RB1 mRNA levels in nonsense mutant RB1 cells. Induction of full-length Rb protein was potentiated by the cereblon E3 ligase modulator CC-90009. These results suggest that pharmacological induction of translational readthrough could be a feasible strategy for therapeutic targeting of tumors with nonsense mutant RB1.

Non-canonical pathway for Rb inactivation and external signaling coordinate cell-cycle entry without CDK4/6 activity.

Cyclin-dependent kinases 4 and 6 (CDK4/6) are critical for initiating cell proliferation by inactivating the retinoblastoma (Rb) protein. However, mammalian cells can bypass CDK4/6 for Rb inactivation. Here we show a non-canonical pathway for Rb inactivation and its interplay with external signals. We find that the non-phosphorylated Rb protein in quiescent cells is intrinsically unstable, offering an alternative mechanism for initiating E2F activity. Nevertheless, this pathway incompletely induces Rb-protein loss, resulting in minimal E2F activity. To trigger cell proliferation, upregulation of mitogenic signaling is required for stabilizing c-Myc, thereby augmenting E2F activity. Concurrently, stress signaling promotes Cip/Kip levels, competitively regulating cell proliferation with mitogenic signaling. In cancer, driver mutations elevate c-Myc levels, facilitating adaptation to CDK4/6 inhibitors. Differentiated cells, despite Rb-protein loss, maintain quiescence through the modulation of c-Myc and Cip/Kip levels. Our findings provide mechanistic insights into an alternative model of cell-cycle entry and the maintenance of quiescence.

RBL2-E2F-GCN5 guide cell fate decisions during tissue specification by regulating cell-cycle-dependent fluctuations of non-cell-autonomous signaling.

The retinoblastoma family proteins (RBs) and E2F transcription factors are cell-autonomous regulators of cell-cycle progression, but they also impact fate choice in addition to tumor suppression. The range of mechanisms involved remains to be uncovered. Here, we show that RBs, particularly RBL2/p130, repress WNT ligands such as WNT4 and WNT8A, thereby directing ectoderm specification between neural crest to neuroepithelium. RBL2 achieves this function through cell-cycle-dependent cooperation with E2Fs and GCN5 on the regulatory regions of WNT loci, which direct neuroepithelial versus neural crest specification by temporal fluctuations of WNT/ß-catenin and DLL/NOTCH signaling activity. Thus, the RB-E2F bona fide cell-autonomous axis controls cell fate decisions, and RBL2 regulates field effects via WNT ligands. This reveals a non-cell-autonomous function of RBL2-E2F in stem cell and tissue progenitor differentiation that has broader implications for cell-cycle-dependent cell fate specification in organogenesis, adult stem cells, tissue homeostasis, and tumorigenesis.

Germline HPF1 retrogene insertion in RB1 gene involved in cancer predisposition.

About half of the human genome is composed of repeated sequences derived from mobile elements, mainly retrotransposons, generally without pathogenic effect. Familial forms of retinoblastoma are caused by germline pathogenic variants in RB1 gene. Here, we describe a family with retinoblastoma affecting a father and his son. No pathogenic variant was identified after DNA analysis of RB1 gene coding sequence and exon-intron junctions. However, RB1 mRNA analysis showed a chimeric transcript with insertion of 114 nucleotides from HPF1 gene inside RB1 gene. This chimeric transcript led to an insertion of 38 amino acids in functional domain of retinoblastoma protein. Subsequent DNA analysis in RB1 intron 17 revealed the presence of a full-length HPF1 retrogene insertion in opposite orientation. Functional assay shows that this insertion has a deleterious impact on retinoblastoma protein function. This is the first report of a full-length retrogene insertion involved in human Mendelian disease leading to a chimeric transcript and a non-functional chimeric protein. Some retrogene insertions may be missed by standard diagnostic genetic testing, so contribution of retrogene insertions to human disease may be underestimated. The increasing use of whole genome sequencing in diagnostic settings will help to get a more comprehensive view of retrogenes.

Rbf/E2F1 control growth and endoreplication via steroid-independent Ecdysone Receptor signalling in Drosophila prostate-like secondary cells.

In prostate cancer, loss of the tumour suppressor gene, Retinoblastoma (Rb), and consequent activation of transcription factor E2F1 typically occurs at a late-stage of tumour progression. It appears to regulate a switch to an androgen-independent form of cancer, castration-resistant prostate cancer (CRPC), which frequently still requires androgen receptor (AR) signalling. We have previously shown that upon mating, binucleate secondary cells (SCs) of the Drosophila melanogaster male accessory gland (AG), which share some similarities with prostate epithelial cells, switch their growth regulation from a steroid-dependent to a steroid-independent form of Ecdysone Receptor (EcR) control. This physiological change induces genome endoreplication and allows SCs to rapidly replenish their secretory compartments, even when ecdysone levels are low because the male has not previously been exposed to females. Here, we test whether the Drosophila Rb homologue, Rbf, and E2F1 regulate this switch. Surprisingly, we find that excess Rbf activity reversibly suppresses binucleation in adult SCs. We also demonstrate that Rbf, E2F1 and the cell cycle regulators, Cyclin D (CycD) and Cyclin E (CycE), are key regulators of mating-dependent SC endoreplication, as well as SC growth in both virgin and mated males. Importantly, we show that the CycD/Rbf/E2F1 axis requires the EcR, but not ecdysone, to trigger CycE-dependent endoreplication and endoreplication-associated growth in SCs, mirroring changes seen in CRPC. Furthermore, Bone Morphogenetic Protein (BMP) signalling, mediated by the BMP ligand Decapentaplegic (Dpp), intersects with CycD/Rbf/E2F1 signalling to drive endoreplication in these fly cells. Overall, our work reveals a signalling switch, which permits rapid growth of SCs and increased secretion after mating, independently of previous exposure to females. The changes observed share mechanistic parallels with the pathological switch to hormone-independent AR signalling seen in CRPC, suggesting that the latter may reflect the dysregulation of a currently unidentified physiological process.

Seeing is believing: the impact of RB on nuclear organization.

The retinoblastoma tumor suppressor (RB) prevents G1 to S cell cycle transition by inhibiting E2F activity. This function requires that RB remains un- or underphosphorylated (the so-called active forms of RB). Recently, we showed that active forms of RB cause widespread changes in nuclear architecture that are visible under a microscope. These phenotypes did not correlate with cell cycle arrest or repression of the E2F transcriptional program, but appeared later, and were associated with the appearance of autophagy or in IMR-90 cells with senescence markers. In this perspective, we describe the relative timing of these RB-induced events and discuss the mechanisms that may underlie RB-induced chromatin dispersion. We consider the relationship between RB-induced dispersion, autophagy, and senescence and the potential connection between dispersion and cell cycle exit.

Targeted pharmacologic inhibition of S-phase kinase-associated protein 2 (SKP2) mediated cell cycle regulation in lung and other RB-Related cancers: A brief review of current status and future prospects.

Small cell lung cancer (SCLC) often exhibits Rb deficiency, TRß and p130 deletion, and SKP2 amplification, suggesting TRß inactivation and SKP2 activation. It is reported that SKP2 targeted therapy is effective in some cancers in vitro and in vivo, but it is not reported for the treatment of SCLC and retinoblastoma. SKP2 is the synthetic lethal gene in SCLC and retinoblastoma, so SKP2 can be used for targeted therapy in SCLC and retinoblastoma. RB1 knockout mice develop several kinds of tumors, but Rb1 and SKP2 double knockout mice are healthy, suggesting that SKP2 targeted therapy may have significant effects on Rb deficient cancers with less side effects, and if successful in SCLC and retinoblastoma in vitro and in animal model, such compounds may be promising for the clinical treatment of SCLC, retinoblastoma, and variety of Rb deficient cancers. Previously our studies showed that retinoblastomas exhibit retinal cone precursor properties and depend on cone-specific thyroid hormone receptor ß2 (TRß2) and SKP2 signaling. In this study, we sought to suppress SCLC and retinoblastoma cell growth by SKP2 inhibitors as a prelude to targeted therapy in vitro and in vivo. We knocked down TRß2 and SKP2 or over-expressed p27 in SCLC and retinoblastoma cell lines to investigate SKP2 and p27 signaling alterations. The SCLC cell lines H209 as well as retinoblastoma cell lines Y79, WERI, and RB177 were treated with SKP2 inhibitor C1 at different concentrations, following which Western blotting, Immunostaining, and cell cycle kinetics studies were performed to study SKP2 and p27 expression ubiquitination, to determine impact on cell cycle regulation and growth inhibition. TRß2 knockdown in Y79, RB177 and H209 caused SKP2 downregulation and degradation, p27 up-regulation, and S phase arrest, whereas, SKP2 knockdown or p27 over-expression caused p27 accumulation and G1-S phase arrest. In the cell lines Y79, WERI, RB177, and H209 treatment with C1 caused SKP2 ubiquitination and degradation, p27 de-ubiquitination and accumulation, and cell growth arrest. SKP2 inhibitor C1 significantly suppressed retinoblastoma as well as SCLC cell growth by SKP2 degradation and p27 accumulation. In vivo study also showed inhibition of tumor growth with C1 treatment. Potential limitations of the success of such a therapeutic approach and its translational application in human primary tumors, and alternative approaches to overcome such limitations are briefly discussed for the treatment of retinoblastoma, SCLC and other RB-related cancers.

Defining triple-negative breast cancer with neuroendocrine differentiation (TNBC-NED).

Primary breast neuroendocrine (NE) neoplasms are uncommon, and definitions harbor controversy. We retrospectively collected 73 triple-negative breast cancers (TNBC) and evaluated NE biomarker expression along with p53 aberrant staining (which correlates with TP53 gene mutation) and Rb protein loss by immunohistochemistry. In the study cohort, we found 11 (15%) cases of TNBC with neuroendocrine differentiation (TNBC-NED) showing positivity for one or more NE markers (synaptophysin/chromogranin/insulinoma-associated protein 1 [INSM1]). We also identified one separate small cell neuroendocrine carcinoma. Histologic types for these 11 TNBC-NED cases were as follows: 8 invasive ductal carcinoma (IDC) not otherwise specified (NOS), 2 IDC with apocrine features, 1 IDC with solid papillary features. INSM1 had the highest positivity and was seen in all 11 carcinomas. Seven (64%) cases showed p53 aberrant staining, 6 (55%) had Rb protein loss, while 6 (55%) had p53/Rb co-aberrant staining/protein loss. TNBC-NED was associated with Rb protein loss (p < 0.001), as well as p53/Rb co-aberrant staining/protein loss (p < 0.001). In 61 cases negative for NE markers, 37 (61%) showed p53 aberrant staining, while 5 (8%) had Rb protein loss. We also analyzed genomic and transcriptomic data from The Cancer Genome Atlas (TCGA) PanCancer Atlas of 171 basal/TNBC patients. Transcriptomic analysis revealed mRNA expression of RB1 to be correlated negatively with SYN1 mRNA expression (p = 0.0400) and INSM1 mRNA expression (p = 0.0106) in this cohort. We would like to highlight the importance of these findings. TNBC-NED is currently diagnosed as TNBC, and although it overlaps morphologically with TNBC without NED, the unique p53/Rb signature highlights a genetic overlap with NE carcinomas of the breast.

Sequential Targeting of Retinoblastoma and DNA Synthesis Pathways Is a Therapeutic Strategy for Sarcomas That Can Be Monitored in Real Time.

Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell-cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1,043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective two-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1 phase. Upon drug removal, cells synchronously traversed to S phase, where a second treatment with S-phase targeted agents (gemcitabine or Wee1 kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be noninvasively monitored using real-time positron emission tomography imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA-damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with noninvasive functional imaging and serum biomarkers as indicators of response and cell cycling. SIGNIFICANCE: An innovative sequential therapeutic strategy targeting Rb, followed by treatment with agents that perturb DNA synthesis pathways, results in synergistic killing of Rb-positive sarcomas that can be noninvasively monitored.

SETDB1 Modulates Degradation of Phosphorylated RB and Anticancer Efficacy of CDK4/6 Inhibitors.

Retinoblastoma (RB) protein can exert tumor suppressor functions even when it becomes phosphorylated. It is thus essential to understand how phosphorylated RB (p-RB) expression and function are regulated. Here, we demonstrated that RING finger domain protein TRIM28 bound and promoted ubiquitination and degradation of CDK4/6-phosphorylated RB protein. SETDB1, a known TRIM28 binding partner, protected p-RB from degradation through the binding of methylated RB by its Tudor domain independent of its methyltransferase activity. SETDB1 was found to be frequently overexpressed due to gene amplification and positively correlated with p-RB in prostate cancer patient specimens. Inhibition of SETDB1 expression using a gene-specific antisense oligonucleotide (ASO) reduced tumor growth but accelerated RB protein degradation, limiting the therapeutic efficacy. However, coadministration of the CDK4/6 inhibitor palbociclib blocked ASO-induced RB degradation and resulted in a much greater cancer-inhibitory effect than each inhibitor alone both in vitro and in vivo. This study identified CDK4/6-dependent, TRIM28-mediated proteasomal degradation as a mechanism of RB inactivation and reveals SETDB1 as a key inhibitor of this process. Our findings suggest that combined targeting of SETDB1 and CDK4/6 represents a viable approach for the treatment of cancers with SETDB1 gene amplification or overexpression. SIGNIFICANCE: The identification of a role for TRIM28 and SETDB1 in regulating CDK4/6-phosphorylated RB stability uncovers a combination strategy using CDK4/6 and SETDB1 inhibition to decrease RB degradation and inhibit cancer growth.

PP2A-B55 and its adapter proteins IER2 and IER5 regulate the activity of RB family proteins and the expression of cell cycle-related genes.

The retinoblastoma (RB) tumour suppressor protein regulates cell proliferation, motility, differentiation and apoptosis. The phosphorylation state of RB is modulated by kinases and phosphatases, and RB exhibits phosphorylation-sensitive interactions with E2F family transcription factors. Here, we characterize RB dephosphorylation by protein phosphatase 2A (PP2A). The growth factor-inducible immediate early response (IER) proteins IER2 and IER5 possess an adapter-like function in which IER proteins bind to both PP2A and its target proteins and enhance PP2A activity towards the proteins. IER2 interacts with RB and facilitates dephosphorylation of RB at T821/T826 by PP2A. In IER2 knockdown cells, elevated phosphorylation of RB resulted in reduced binding of RB to the promoters and derepression of cyclin D1 and p21. IER5 binds to both RB and RB-like 1 (p107/RBL1), enhances dephosphorylation of these proteins by PP2A and represses the expression of various cell cycle-related genes. However, IER2-regulated dephosphorylation at T821/T826 is not necessary for the repression function of RB in cell mobility-related gene expression. Our data identify PP2A adapter proteins as critical regulators of RB family proteins and suggest that the phosphorylation status of RB differentially affects gene expression.

Retinoblastoma Expression and Targeting by CDK4/6 Inhibitors in Small Cell Lung Cancer.

The canonical model of "small cell lung cancer" (SCLC) depicts tumors arising from dual inactivation of TP53 and RB1. However, many genomic studies have persistently identified tumors with no RB1 mutations. Here, we examined RB1 protein expression and function in SCLC. RB1 expression was examined by IHC analysis of 62 human SCLC tumors. These studies showed that ∼14% of SCLC tumors expressed abundant RB1 protein, which is associated with neuroendocrine gene expression and is enriched in YAP1 expression, but no other lineage proteins that stratify SCLC. SCLC cells and xenograft tumors with RB1 protein expression were sensitive to growth inhibition by the CDK4/6 inhibitor palbociclib, and this inhibition was shown to be dependent on RB1 expression by CRISPR knockout. Furthermore, a patient with biopsy-validated wild-type RB1 SCLC who received the CDK4/6 inhibitor abemaciclib demonstrated a dramatic decrease in mutant TP53 ctDNA allelic fraction from 62.1% to 0.4% and decreased tumor mass on CT scans. Importantly, IHC of the diagnostic biopsy specimen showed RB1 positivity. Finally, we identified a transcriptomics-based RB1 loss-of-function signature that discriminates between SCLC cells with or without RB1 protein expression and validated it in the patient who was responsive to abemaciclib, suggesting its potential use to predict CDK4/6 inhibitor response in patients with SCLC. Our study demonstrates that RB1 protein is an actionable target in a subgroup of SCLC, a cancer that exhibits no currently targetable mutations.

Simultaneous depletion of RB, RBL1 and RBL2 affects endoderm differentiation of human embryonic stem cells.

RB is a well-known cell cycle regulator controlling the G1 checkpoint. Previous reports have suggested that it can influence cell fate decisions not only by regulating cell proliferation and survival but also by interacting with transcription factors and epigenetic modifiers. However, the functional redundancy of RB family proteins (RB, RBL1 and RBL2) renders it difficult to investigate their roles during early development, especially in human. Here, we address this problem by generating human embryonic stem cells lacking RB family proteins. To achieve this goal, we first introduced frameshift mutations in RBL1 and RBL2 genes using the CRISPR/Cas9 technology, and then integrated the shRNA-expression cassette to knockdown RB upon tetracycline treatment. The resulting RBL1/2_dKO+RB_iKD cells remain pluripotent and efficiently differentiate into the primary germ layers in vitro even in the absence of the RB family proteins. In contrast, we observed that subsequent differentiation into foregut endoderm was impaired without the expression of RB, RBL1 and RBL2. Thus, it is suggested that RB proteins are dispensable for the maintenance and acquisition of cell identities during early development, but they are essential to generate advanced derivatives after the formation of primary germ layers. These results also indicate that our RBL1/2_dKO+RB_iKD cell lines are useful to depict the detailed molecular roles of RB family proteins in the maintenance and generation of various cell types accessible from human pluripotent stem cells.

The Rb/E2F axis is a key regulator of the molecular signatures instructing the quiescent and activated adult neural stem cell state.

Long-term maintenance of the adult neurogenic niche depends on proper regulation of entry and exit from quiescence. Neural stem cell (NSC) transition from quiescence to activation is a complex process requiring precise cell-cycle control coordinated with transcriptional and morphological changes. How NSC fate transitions in coordination with the cell-cycle machinery remains poorly understood. Here we show that the Rb/E2F axis functions by linking the cell-cycle machinery to pivotal regulators of NSC fate. Deletion of Rb family proteins results in activation of NSCs, inducing a transcriptomic transition toward activation. Deletion of their target activator E2Fs1/3 results in intractable quiescence and cessation of neurogenesis. We show that the Rb/E2F axis mediates these fate transitions through regulation of factors essential for NSC function, including REST and ASCL1. Thus, the Rb/E2F axis is an important regulator of NSC fate, coordinating cell-cycle control with NSC activation and quiescence fate transitions.

Lack of Retinoblastoma Protein Shifts Tumor Metabolism from Glycolysis to OXPHOS and Allows the Use of Alternate Fuels.

Mutations in the RB1 locus leading to a loss of functional Rb protein cause intraocular tumors, which uniquely affect children worldwide. These tumors demonstrate rapid proliferation, which has recently been shown to be associated with an altered metabolic signature. We found that retinoblastoma tumors and in-vitro models lack Hexokinase 1 (HK1) and exhibit elevated fatty acid oxidation. We show that ectopic expression of RB1 induces HK1 protein in Rb null cells, and both RB1 and HK1 can mediate a metabolic switch from OXPHOS to glycolysis with increased pyruvate levels, reduced ATP production and reduced mitochondrial mass. Further, cells lacking Rb or HK1 can flexibly utilize glutamine and fatty acids to enhance oxidative phosphorylation-dependent ATP generation, as revealed by metabolic and biochemical assays. Thus, loss of Rb and HK1 in retinoblastoma reprograms tumor metabolic circuits to enhance the glucose-independent TCA (tricarboxylic acid) cycle and the intermediate NAD+/NADH ratios, with a subsequent increase in fatty-acid derived L-carnitine to enhance mitochondrial OXPHOS for ATP production instead of glycolysis dependence. We also demonstrate that modulation of the Rb-regulated transcription factor E2F2 does not result in any of these metabolic perturbations. In conclusion, we demonstrate RB1 or HK1 as critical regulators of the cellular bioenergetic profile and identify the altered tumor metabolism as a potential therapeutic target for cancers lacking functional Rb protein.

Retinoblastoma protein as an intrinsic BRD4 inhibitor modulates small molecule BET inhibitor sensitivity in cancer.

Bromodomain and extraterminal (BET) proteins including BRD4 play important roles in oncogenesis and immune inflammation. Here we demonstrate that cancer cells with loss of the retinoblastoma (RB) tumor suppressor became resistant to small molecule bromodomain inhibitors of BET proteins. We find that RB binds to bromodomain-1 (BD1) of BRD4, but binding is impeded by CDK4/6-mediated RB phosphorylation at serine-249/threonine-252 (S249/T252). ChIP-seq analysis shows RB knockdown increases BRD4 occupancy at genomic loci of genes enriched in cancer-related pathways including the GPCR-GNBIL-CREB axis. S249/T252-phosphorylated RB positively correlates with GNBIL protein level in prostate cancer patient samples. BET inhibitor resistance in RB-deficient cells is abolished by co-administration of CREB inhibitor. Our study identifies RB protein as a bona fide intrinsic inhibitor of BRD4 and demonstrates that RB inactivation confers resistance to small molecule BET inhibitors, thereby revealing a regulatory hub that converges RB upstream signaling onto BRD4 functions in diseases such as cancer.

Reconstruct Human Retinoblastoma In Vitro.

Human RB is pediatric cancer, which is lethal if no treatment is administered. As RB originates from cone precursors, which is relatively rare in rodent models, meanwhile regarding the interspecies differences between humans and rodents, a disease model derived from humans is more beneficial for uncovering the mechanisms of human RB and seeking the targets of therapy. Herein, the protocol describes the generation of two gene-edited hESC lines with a biallelic RB1 point mutation (RB1Mut/Mut) and an RB1 knockout mutation (RB1-/-), respectively. During the process of retinal development, the formation of RB is observed. The RB cell lines are also established by segregating from the RB organoids. Altogether, by differentiating the gene-edited hESC lines into the retinal organoids using a 2D and 3D combined differentiation protocol, we have successfully reconstructed the human RB in a dish and identified its cone-precursor origin. It would provide a helpful disease model for observing the retinoblastoma genesis, proliferation, and growth as well as further developing novel therapeutic agents.