Retinoblastoma Tumor Suppressor Gene Research Articles

Pathogenic Mechanisms Activated by NSD2 Translocations in Multiple Myeloma

The NSD2 gene is overexpressed in multiple cancers and is associated with cancer cell proliferation, invasion, and metastasis. NSD2 gene is universally present in multiple myeloma (MM) patients with t(4;14) (p16;q32) translocation, a high-risk subtype. NSD2 is characterized by different breakpoints. NSD2Δ3 breakpoint is associated with shorter survival than other breakpoints. NSD2Δ3 contains non-coding RNA ACA11 within intron 18. Additionally, 80% of t(4;14) MM patients lack the retinoblastoma tumor suppressor gene (RB1). We hypothesize that ACA11 cooperates with RB1 loss in MM pathogenesis. Also, we hypothesize that NSD2Δ3 increases protein expression driving myeloma growth. To overexpress NSD2WT, snoACA11, and NSD2Δ3 in cells, we cloned NSD2WT, and NSD2Δ3 plasmid constructs in a murine stem cell virus (MSCV)-IRES- GFP vector. We isolated primary B cells from RB1 wild-type and knock-out mice by CD43 microbeads and transduced those cells using a virus containing ACA11 and NSD2WT. ACA11 overexpression significantly increased B cell proliferation in Rb1dKO mice. This hyperproliferative phenotype is induced by reactive oxygen species (ROS) levels. However, we did not find a further increase in B cell proliferation with co-overexpression of NSD2WT and ACA11 in Rb1dKO mice. We demonstrated increased protein stability by finding higher NSD2Δ3 protein expression compared to NSD2WT protein expression in HEK293T cells. These results support our hypothesis and demonstrate oncogenic role of ACA11 in t(4;14) MM. Future work will be done to determine the cell biological effects of NSD2Δ3 and establish its role in myeloma pathogenesis. Completion of this study will advance understanding of this epigenetic NSD2 gene and potentially develop targeted drugs for NSD2, providing more individualized treatment options for these patients. University of Iowa Department of Internal Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tissue-Based Diagnostic Biomarkers of Aggressive Variant Prostate Cancer: A Narrative Review.

Prostate cancer (PC) is a common malignancy among elderly men, characterized by great heterogeneity in its clinical course, ranging from an indolent to a highly aggressive disease. The aggressive variant of prostate cancer (AVPC) clinically shows an atypical pattern of disease progression, similar to that of small cell PC (SCPC), and also shares the chemo-responsiveness of SCPC. The term AVPC does not describe a specific histologic subtype of PC but rather the group of tumors that, irrespective of morphology, show an aggressive clinical course, dictated by androgen receptor (AR) indifference. AR indifference represents an adaptive response to androgen deprivation therapy (ADT), driven by epithelial plasticity, an inherent ability of tumor cells to adapt to their environment by changing their phenotypic characteristics in a bi-directional way. The molecular profile of AVPC entails combined alterations in the tumor suppressor genes retinoblastoma protein 1 (RB1), tumor protein 53 (TP53), and phosphatase and tensin homolog (PTEN). The understanding of the biologic heterogeneity of castration-resistant PC (CRPC) and the need to identify the subset of patients that would potentially benefit from specific therapies necessitate the development of prognostic and predictive biomarkers. This review aims to discuss the possible pathophysiologic mechanisms of AVPC development and the potential use of emerging tissue-based biomarkers in clinical practice.

Combined inhibition of ACLY and CDK4/6 reduces cancer cell growth and invasion.

The use of small molecule kinase inhibitors, which target specific enzymes that are overactive in cancer cells, has revolutionized cancer patient treatment. To treat some types of breast cancer, CDK4/6 inhibitors, such as palbociclib, have been developed that target the phosphorylation of the retinoblastoma tumor suppressor gene. Acquired resistance to CDK4/6 inhibitors may be due to activation of the AKT pro‑survival signaling pathway that stimulates several processes, such as growth, metastasis and changes in metabolism that support rapid cell proliferation. The aim of the present study was to investigate whether targeting ATP citrate lyase (ACLY), a downstream target of AKT, may combine with CDK4/6 inhibition to inhibit tumorigenesis. The present study determined that ACLY is activated in breast and pancreatic cancer cells in response to palbociclib treatment and AKT mediates this effect. Inhibition of ACLY using bempedoic acid used in combination with palbociclib reduced cell viability in a panel of breast and pancreatic cancer cell lines. This effect was also observed using breast cancer cells grown in 3D cell culture. Mechanistically, palbociclib inhibited cell proliferation, whereas bempedoic acid stimulated apoptosis. Finally, using Transwell invasion assays and immunoblotting, the present study demonstrated that ACLY inhibition blocked cell invasion, when used alone or in combination with palbociclib. These data may yield useful information that could guide the development of future therapies aimed at the reduction of acquired resistance observed clinically.

Palbociclib Induces the Apoptosis of Lung Squamous Cell Carcinoma Cells via RB-Independent STAT3 Phosphorylation.

Lung squamous cell carcinoma (LUSC) treatment response is poor and treatment alternatives are limited. Palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, has recently been approved for hormone receptor-positive breast cancer patients and applied in multiple preclinical models, but its use for LUSC therapy remains elusive. Here, we investigated whether palbociclib induced cell apoptosis and dissected the underlying mechanism in LUSC. We found that palbociclib induced LUSC cell apoptosis through inhibition of Src tyrosine kinase/signal transducers and activators of transcription 3 (STAT3). Interestingly, palbociclib reduced STAT3 signaling in LUSC cells interfered by retinoblastoma tumor-suppressor gene (RB), suggesting that pro-apoptosis effect of palbociclib was independent of classic CDK4/6-RB signaling. Furthermore, palbociclib could suppress IL-1β and IL-6 expression, and therefore blocked Src/STAT3 signaling, which were rescued by either recombinant human IL-1β or IL-6. Moreover, Myc mediated the sensitivity of LUSC cells to palbociclib. Our discoveries demonstrated that palbociclib induces apoptosis of LUSC cells through the Src/STAT3 axis in an RB-independent manner, and provided a reliable experimental basis of clinical studies in LUSC patients.

Retraction Note: MicroRNA-215 enhances invasion and migration by targeting retinoblastoma tumor suppressor gene 1 in high-grade glioma.

Retraction Note: MicroRNA-215 enhances invasion and migration by targeting retinoblastoma tumor suppressor gene 1 in high-grade glioma.

Hyperbranched-star PEI-g-PEG as a nonviral vector with efficient uptake and hypotoxicity for retinoblastoma gene therapy application

Retinoblastoma (RB) is the main pediatric intraocular tumor in children. It is caused by the mutation of tumor suppressor gene RB transcriptional corepressor 1 (RB1). Herein, poly(ethylene glycol) grafted branched polyethyleneimine (hyperbranched-star PEI-g-PEG) was taken as the polycationic gene carrier for preparing the nonviral vectors with efficient uptake and hypotoxicity for potential retinoblastoma gene therapy application. The PEI-g-PEG can condense the genes effectively and the cationic, nano-scaled complexes with PEG shells were obtained by optimization the composition proportions. The cell behaviors investigations indicated that the PEGylation of PEI can greatly decrease the cytotoxicity, and such gene vector has efficient endocytosis effects. The gene transfection results indicated that different functional proteins can be expressed in the retinoblastoma cells when using the functional genes were encoded in the PEI-g-PEG based nonviral gene vectors. These results imply that the hyperbranched-star PEG-g-PEI is a promising nonviral carrier for gene delivery in retinoblastoma.

Role of MicroRNAs in Neuroendocrine Prostate Cancer.

Therapy-induced neuroendocrine prostate cancer (t-NEPC/NEPC) is an aggressive variant of prostate cancer (PCa) that frequently emerges in castration-resistant prostate cancer (CRPC) under the selective pressure of androgen receptor (AR)-targeted therapies. This variant is extremely aggressive, metastasizes to visceral organs, tissues, and bones despite low serum PSA, and is associated with poor survival rates. It arises via a reversible trans-differentiation process, referred to as ‘neuroendocrine differentiation’ (NED), wherein PCa cells undergo a lineage switch and exhibit neuroendocrine features, characterized by the expression of neuronal markers such as enolase 2 (ENO2), chromogranin A (CHGA), and synaptophysin (SYP). The molecular and cellular mechanisms underlying NED in PCa are complex and not clearly understood, which contributes to a lack of effective molecular biomarkers for diagnosis and therapy of this variant. NEPC is thought to derive from prostate adenocarcinomas by clonal evolution. A characteristic set of genetic alterations, such as dual loss of retinoblastoma (RB1) and tumor protein (TP53) tumor suppressor genes and amplifications of Aurora kinase A (AURKA), NMYC, and EZH2, has been reported to drive NEPC. Recent evidence suggests that microRNAs (miRNAs) are important epigenetic players in driving NED in advanced PCa. In this review, we highlight the role of miRNAs in NEPC. These studies emphasize the diverse role that miRNAs play as oncogenes and tumor suppressors in driving NEPC. These studies have unveiled the important role of cellular processes such as the EMT and cancer stemness in determining NED in PCa. Furthermore, miRNAs are involved in intercellular communication between tumor cells and stromal cells via extracellular vesicles/exosomes that contribute to lineage switching. Recent studies support the promising potential of miRNAs as novel diagnostic biomarkers and therapeutic targets for NEPC.

Alterations in the RB Pathway With Inactivation of RB1 Characterize Glioblastomas With a Primitive Neuronal Component.

A primitive neuronal component is a feature of some glioblastomas but defining molecular alterations of this histologic variant remains uncertain. We performed next-generation sequencing of 1500 tumor related genes on tissue from 9 patients with glioblastoma with a primitive component (G/PN) and analyzed 27 similar cases from the Cancer Genome Atlas (TCGA) dataset. Alterations in the RB pathway were identified in all of our patients' tumors and 81% of TCGA tumors with the retinoblastoma tumor suppressor gene (RB1) commonly affected. Although RB1 mutations were observed in some conventional glioblastomas, the allelic fractions of these mutations were significantly higher in tumors with a primitive neuronal component in both our and TCGA cohorts (median, 72% vs 25%, p < 0.001 and 80% vs 40%, p < 0.02, respectively). Further, in 78% of patients in our cohort, RB expression was lost by immunohistochemistry. Our findings indicate that alterations in the RB pathway are common in G/PNs and suggest that inactivation of RB1 may be a driving mechanism for the phenotype.

The two-hit theory hits 50.

Few ideas in cancer genetics have been as influential as the “two-hit” theory of tumor suppressors. This idea was introduced in 1971 by Al Knudson in a paper in the Proceedings of the National Academy of Science and forms the basis for our current understanding of the role of mutations in cancer. In this theoretical discussion proposing a genetic basis for retinoblastoma, a childhood cancer of the retina, Knudson posited that these tumors arise from two inactivating mutations, targeting both alleles of a putative tumor suppressor gene. While this work built on earlier proposals that cancers are the result of mutations in more than one gene, it was the first to propose a plausible mechanism by which single genes that are affected by germ-line mutations in heritable cancers could also cause spontaneous, nonheritable tumors when mutated in somatic tissues. Remarkably, Knudson described the existence and properties of a retinoblastoma tumor suppressor gene a full 15 years before the gene was cloned.

The DREAM complex represses growth in response to DNA damage in Arabidopsis

The DNA of all organisms is constantly damaged by physiological processes and environmental conditions. Upon persistent damage, plant growth and cell proliferation are reduced. Based on previous findings that RBR1, the only Arabidopsis homolog of the mammalian tumor suppressor gene retinoblastoma, plays a key role in the DNA damage response in plants, we unravel here the network of RBR1 interactors under DNA stress conditions. This led to the identification of homologs of every DREAM component in Arabidopsis, including previously not recognized homologs of LIN52. Interestingly, we also discovered NAC044, a mediator of DNA damage response in plants and close homolog of the major DNA damage regulator SOG1, to directly interact with RBR1 and the DREAM component LIN37B. Consistently, not only mutants in NAC044 but also the double mutant of the two LIN37 homologs and mutants for the DREAM component E2FB showed reduced sensitivities to DNA-damaging conditions. Our work indicates the existence of multiple DREAM complexes that work in conjunction with NAC044 to mediate growth arrest after DNA damage.

Molecular Genetics Mutations of Retinoblastoma

Retinoblastoma is a cancer that arises because both copies of the RB1 gene that normally suppresses retinoblastoma are lost from a developing retinal cell in fetuses, babies, and young children. Retinoblastoma is the prototype genetic cancer in one or both eyes of young children, most retinoblastomas are initiated by bial­lelic mutation of the retinoblastoma tumor suppressor gene, RB1, in a developing retinal cell. All those with bilateral retinoblastoma have heri­table cancer, although 95% have not inherited the RB1 mutation. Non­heritable retinoblastoma is always unilateral, with 98% caused by loss of both RB1 alleles from the tumor, whereas 2% have normal RB1 in tumors initiated by amplification of the MYCN oncogene. A rare subset of retinoblastoma is initiated by somatic amplification of the MYCN oncogene in a predisposing retinal cell. The retinoblastoma protein (pRB), encoded by RB1, is an important transcription factor.

Abstract LB119: VRN08, a Selective Mps1 Kinase Inhibitor Induces Mitotic Catastrophe and Apoptosis Efficiently in Advanced Cancers Including Rb1-Deficient

Abstract Spindle assembly checkpoint (SAC) prevents mis-segregation of chromosome and allows accurate division in mitosis. Mps1 (also known as TTK) is one of the essential kinases for recruitment of SAC proteins after chromosome segregation. Failure of SAC induces chromosome instability, mitotic catastrophe, and ultimately apoptotic cell death and Mps1 is induced or activated in rapidly dividing tumor cells. Therefore, Mps1 has been considered an attractive anti-cancer drug target. VRN081569 is a potent type I kinase inhibitor of Mps1, which stabilizes Mps1 kinase domain in a closed conformation by forming a tight interface with its kinase domain. This structural stabilization appears to increase cellular potency for the induction of aneuploidy and apoptosis (~ 10 nM or less GI50 value). The loss of retinoblastoma tumor suppressor gene (Rb1), which is prevalent in advanced cancers, dysregulates cell cycle and promotes cell division. In particular, the resistance to CDK4/6 inhibitors has been reportedly mediated by loss of the Rb1 gene. We found that Rb1-negative cells express higher level of Mps1 than Rb1-positive cells and VRN081569 efficiently induce apoptosis of Rb1-negative Triple Negative Breast Cancer (TNBC) cell line MDA-MB-468 than Rb1-positive TNBC MDA-MB-231. Especially we confirmed that VRN081569 induces caspase 3/7 activity faster in Rb1-negative cells (~50-fold EC50 of caspase 3/7 activity compared to Rb1-positive cells), and cell cycle analysis has shown that our compound induces a dose-dependent dysregulation of mitosis, resulting in a decrease in the frequency of the G0/G1 phase, and also induces aneuploidy and apoptosis. Finally, we confirmed a combination effects with anti-microtubule agents BCL-2 inhibitor and PARP-1 inhibitor, suggesting that modulation of two different cellular processes may overcome the lack of single agent activity for TNBC. Citation Format: Hong Ryul Jung, Yeonsil Kim, Haelee Kim, Ji-Hye Kwon, Dong-hyuk Seo, Jung Beom Son, Nam Doo Kim, Daekwon Kim, Sunghwan Kim. VRN08, a Selective Mps1 Kinase Inhibitor Induces Mitotic Catastrophe and Apoptosis Efficiently in Advanced Cancers Including Rb1-Deficient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB119.

Retinoblastoma Related Gene RB2/P130 Are Rarely Mutated in Burkitt's Lymphoma from Brazil.

Human RB2/p130 is a member of the retinoblastoma (Rb) family which also includes the retinoblastoma tumor suppressor gene RB/p105 (RB1) and the retinoblastoma related gene, p107. RB1 and other members of Rb family, p107 and p130 perform overlapping, but not fully redundant functions in cell cycle regulation and cell growth inhibition. Moreover, all three Rb family members display suppressive activities, which result in cell cycle G1 phase blocking. It has been suggested that alterations in genes controlling cell cycle probably contribute to Burkitt's lymphoma pathogenesis, in association to the 8:14 translocation that leads to c-myc protein up regulation. Mutations in tumor suppressor gene RB2/p130 were detected in cell lines and primary tumors of patients with endemic Burkitt's lymphoma (eBL). In addition, RB2/p130 mutational inactivation has been reported as a common feature in lung and nasopharyngeal carcinomas. RB2/p130 gene mutation screening was performed in 26 BL tumor pediatric samples and in mononuclear cells from 2 healthy blood donors by single strand conformation polymorphism (PCR-SSCP) analysis. Exons 19, 20, 21 and 22 were amplified separately and denatured PCR products were heated at 95°C for 5 min, chilled on ice, and immediately electrophoresed in a native 10% polyacrilamide gel, with or without the addition of glycerol at 80 volts for 20–26 hours, and silver stained. In this series, 26 children were diagnosed as BL according to histopathologic and clinical criteria. The median age was 4.5 years (range: 2 to 9 years). There were 7 females and 19 males. According to the St Jude system 19% of patients had stage I/II and 81% stage III/IV disease. Burkitt's lymphoma HIV-associated was diagnosed only in 2 patients. All four exons (19–22) were analyzed by PCR-SSCP. No changes were found upon SSCP analysis. Our results suggest that mutations in exons 19–22 of the RB2/p130 are unlikely to be involved directly in the pathogenesis of Brazilian BL. In conclusion, the characterization of tumor cells continues to emphasize the important role of Rb gene in tumor suppression but the evidence linking p107 or p130 inactivation to tumor development remains limited and controversial in Burkitt's lymphomas and other malignant tumors.

Retinoblastoma tumor suppressor gene 1 enhances 5-Fluorouracil chemosensitivity through SDF-1/CXCR4 axis by regulating autophagy in gastric cancer

Due to lack of effective biomarkers for early diagnosis, most patients are diagnosed with advanced gastric cancer and have lower survival rates. 5-Fluorouracil (5-FU) is one of commonly used drugs for chemotherapy of gastric cancer, but drug resistance limits the wide application of agents. Retinoblastoma tumor suppressor gene 1 (RB1) is a key regulator in the progression of various human cancers, including gastric cancer. However, the effects of RB1 on chemosensitivity and the underlying mechanisms in gastric cancer (GC) are not clear. In this study, expressions of RB1 in GC cell lines were evaluated by RT-qPCR and western blot assay. CCK-8 was applied to examine the effect of 5-FU on cell viability. Meanwhile, IC50 values were calculated. The drug-resistance protein MDR1 and autophagy-related proteins were detected by western blot assay. Flow cytometry was used to detect cell cycle. The results showed that RB1 expressions were downregulated in GC cell lines and had significant differences between 5-FU resistance cell lines (SNU-620/5-FU and NUGC-3/5-FU) and non-resistance cell lines (SNU-620 and NUGC-3). Overexpression of RB1 enhanced 5-FU sensitivity of GC cells and caused cell cycle arrest in the S phase. Meanwhile, autophagy-related proteins were downregulated. Mechanistically, SDF-1/CXCR4 participated in the regulation of RB1 on cell autophagy. Autophagy activator, SDF-1 treatment and CXCR4 activation reversed the promoted effects of RB1 on 5-FU sensitivity in GC cells. In conclusion, our data revealed that RB1 was downregulated in GC cell lines. RB1 overexpression enhanced 5-FU chemosensitivity in GC cells by regulating cell autophagy via SDF-1/CXCR4 pathway. RB1 might serve as a promising therapeutic target of GC.

Piperlongumine promotes death of retinoblastoma cancer cells.

Retinoblastoma is the most common pediatric intraocular malignant tumor. While retinoblastoma initiation is triggered by the inactivation of both alleles of the retinoblastoma tumor suppressor gene (RB1) in the developing retina, tumor progression requires additional epigenetic changes, retinoblastoma genomes being quite stable. Although the management of RB has recently improved, new therapeutic agents are necessary to improve the treatment of advanced forms of retinoblastoma.In this report, we analyzed the pro-death effect of piperlongumine (PL), a natural compound isolated from Piper longum L., on two human retinoblastoma cell lines, WERI-Rb and Y79. The effects of PL on cell proliferation, cell death and cell cycle were investigated. PL effectively inhibited cell growth, impacted the cell cycle by decreasing the level of cyclins and CDK1 and increasing CDKN1A and triggered a caspase-3 independant cell death process in which reactive oxygen species (ROS) production is a major player. Indeed, PL toxicity in retinoblastoma cell lines was inhibited by a ROS scavenger N-acetyl-l-cysteine (NAC) treatment. These findings suggest that PL reduces tumor growth and induces cell death by regulating the cell cycle.

RB Regulates DNA Double Strand Break Repair Pathway Choice by Mediating CtIP Dependent End Resection.

Inactivation of the retinoblastoma tumor suppressor gene (RB1) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.

Effect of miR-215 on the Expression of Tumor Suppressor Gene Rb1 in Retinoblastoma Cell Lines.

Background:Effect of miR-215 on the expression of tumor suppressor gene retinoblastoma (Rb)1 in Rb cell lines was investigated.Methods:A total of 128 patients were selected. The expression of miR-215 in cancer and adjacent healthy tissues of the 128 patients was detected by reverse transcription-quantitative PCR (RT-qPCR). HXO-Rb44 and Y79 cell lines were transfected with miR-215 analogs or miR-215 inhibitors, and the expression of Rb1 protein in the cell lines was detected by western blotting.Results:The expression of miR-215 in the adjacent healthy tissues of patients was significantly lower than that in cancer tissues (P<0.001). The expression of miR-215 in Y79 and HXO-Rb44 cells was significantly higher than that in APRE-19 cells (P<0.001). The expression of miR-215 in HXO-Rb44 cells was significantly higher than that in Y79 cells (P<0.001). The expression of miR-215 was statistically different from the degree of differentiation and nerve infiltration (P<0.05). The expression of Rb1 in cancer tissues was significantly lower than that in adjacent tissues (P<0.001), the expression of APRE-19 was significantly higher than that in Y79 and HXO-Rb44 cells (P<0.001), and the expression of Rb1 in HXO-Rb44 cells was significantly higher than that in Y79 cells (P<0.05). There was a negative correlation between miR-215 and Rb1 in the tissues of patients, and Rb1 expression decreased with the increase of miR-215 (r=-0.576, P<0.001).Conclusion:miR-215 is highly expressed in Rb cell lines, and is related to the clinicopathological features of this disease.

Variability in retinoblastoma genome stability is driven by age and not heritability.

Retinoblastoma (RB) is a childhood intraocular cancer initiated by biallelic inactivation of the RB tumor suppressor gene (RB1-/- ). RB can be hereditary (germline RB1 pathogenic allele is present) or non-hereditary. Somatic copy number alterations (SCNAs) contribute to subsequent tumorigenesis. Previous studies of only enucleated RB eyes have reported associations between heritability status and the prevalence of SCNAs. Herein, we use an aqueous humor (AH) liquid biopsy to investigate RB genomic profiles in the context of germline RB1 status, age, and International Intraocular Retinoblastoma Classification (IIRC) clinical grouping for both enucleated and salvaged eyes. Between 2014 and 2019, AH was sampled from a total of 54 eyes of 50 patients. Germline RB1 status was determined from clinical blood testing, and cell-free DNA from AH was analyzed for SCNAs. Of the 50 patients, 23 (46.0%; 27 eyes) had hereditary RB, and 27 (54.0%, 27 eyes) had non-hereditary RB. Median age at diagnosis was comparable between hereditary (13 ± 10 months) and non-hereditary (13 ± 8 months) eyes (P = 0.818). There was no significant difference in the prevalence or number of SCNAs based on (1) hereditary status (P > 0.56) or (2) IIRC grouping (P > 0.47). There was, however, a significant correlation between patient age at diagnosis, and (1) number of total SCNAs (r[52] = 0.672, P < 0.00001) and (2) number of highly-recurrent RB SCNAs (r[52] = 0.616, P < 0.00001). This evidence does not support the theory that specific molecular or genomic subtypes exist between hereditary and non-hereditary RB; rather, the prevalence of genomic alterations in RB eyes is strongly related to patient age at diagnosis.

Pan-cancer molecular analysis of the RB tumor suppressor pathway

The retinoblastoma tumor suppressor gene (RB1) plays a critical role in coordinating multiple pathways that impact cancer initiation, disease progression, and therapeutic responses. Here we probed molecular features associated with the RB-pathway across 31 tumor-types. While the RB-pathway has been purported to exhibit multiple mutually exclusive genetic events, only RB1 alteration is mutually exclusive with deregulation of CDK4/6 activity. An ER+ breast cancer model with targeted RB1 deletion was used to identify signatures of CDK4/6 activity and RB-dependency (CDK4/6-RB integrated signature). This signature was prognostic in tumor-types with gene expression features indicative of slower growth. Single copy loss on chromosome 13q encompassing the RB1 locus is prevalent in many cancers, yielding reduced expression of multiple genes in cis, and is inversely related to the CDK4/6-RB integrated signature supporting a cause-effect relationship. Genes that are positively and inversely correlated with the CDK4/6-RB integrated signature define new tumor-specific pathways associated with RB-pathway activity.

Generation of two H1 hESC sublines carrying deletions of RB1 exon 1/promoter in heterozygous or compound heterozygous state

Biallelic inactivation of the retinoblastoma tumor suppressor gene (RB1) causes formation of retinoblastoma, a retinal eye tumor occurring in early childhood. Using the CRISPR/Cas9 nickase system, exon 1 of RB1 was deleted, including the RB1 promoter. As a result, sublines were generated carrying deletions of RB1 exon 1/promoter on one or both alleles.