Nonviral CRISPR/Cas9 mutagenesis for streamlined generation of mouse lung cancer models.

Functional analysis in mouse models is necessary to establish the involvement of a set of genetic variations in tumor development. A modeling platform to facilitate and cost-effectively analyze the role of multiple genes in carcinogenesis would be valuable. Here, we present an innovative strategy for lung mutagenesis using CRISPR/Cas9 ribonucleoproteins delivered via cationic polymers. This approach allows the simultaneous inactivation of multiple genes. We validate the effectiveness of this system by targeting a group of tumor suppressor genes, specifically Rb1, Rbl1, Pten, and Trp53, which were chosen for their potential to cause lung tumors, namely small cell lung carcinoma (SCLC). Tumors with histologic and transcriptomic features of human SCLC emerged after intratracheal administration of CRISPR/polymer nanoparticles. These tumors carried loss-of-function mutations in all four tumor suppressor genes at the targeted positions. These findings were reproduced in two different pure genetic backgrounds. We provide a proof of principle for simplified modeling of lung tumorigenesis to facilitate functional testing of potential cancer-related genes.

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.

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107.

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine protein phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R.' Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether, these data provide key insights into PP2A/B55α's mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α's role in multiple cellular processes.

p107 mediated mitochondrial function controls muscle stem cell proliferative fates.

Muscle diseases and aging are associated with impaired myogenic stem cell self-renewal and fewer proliferating progenitors (MPs). Importantly, distinct metabolic states induced by glycolysis or oxidative phosphorylation have been connected to MP proliferation and differentiation. However, how these energy-provisioning mechanisms cooperate remain obscure. Herein, we describe a mechanism by which mitochondrial-localized transcriptional co-repressor p107 regulates MP proliferation. We show p107 directly interacts with the mitochondrial DNA, repressing mitochondrial-encoded gene transcription. This reduces ATP production by limiting electron transport chain complex formation. ATP output, controlled by the mitochondrial function of p107, is directly associated with the cell cycle rate. Sirt1 activity, dependent on the cytoplasmic glycolysis product NAD+, directly interacts with p107, impeding its mitochondrial localization. The metabolic control of MP proliferation, driven by p107 mitochondrial function, establishes a cell cycle paradigm that might extend to other dividing cell types.

Germline genetic contribution to the immune landscape of cancer.

Understanding the contribution of the host's genetic background to cancer immunity may lead to improved stratification for immunotherapy and to the identification of novel therapeutic targets. We investigated the effect of common and rare germline variants on 139 well-defined immune traits in ∼9000 cancer patients enrolled in TCGA. High heritability was observed for estimates of NK cell and T cell subset infiltration and for interferon signaling. Common variants of IFIH1, TMEM173 (STING1), and TMEM108 were associated with differential interferon signaling and variants mapping to RBL1 correlated with T cell subset abundance. Pathogenic or likely pathogenic variants in BRCA1 and in genes involved in telomere stabilization and Wnt-ß-catenin also acted as immune modulators. Our findings provide evidence for the impact of germline genetics on the composition and functional orientation of the tumor immune microenvironment. The curated datasets, variants, and genes identified provide a resource toward further understanding of tumor-immune interactions.

Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice.

DREAM (Dp, Rb-like, E2F, and MuvB) is a transcriptional repressor complex that regulates cell proliferation, and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we used an assembly-defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys but not the brain or bone marrow. Using laser-capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting apoA-IV amyloidosis (AApoAIV). AApoAIV is a recently described form, whereby WT apoA-IV has been shown to predominate in amyloid plaques. We determined by ChIP that DREAM directly regulated Apoa4 and that the histone variant H2AZ was reduced from the Apoa4 gene body in DREAM's absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of nongenetic amyloid subtypes.

Characterization and functional analysis of SMAD2 regulation in hair follicle cycle in Angora rabbits.

Hair follicle (HF) development is characterized by periodic growth cycles regulated by numerous factors. We previously showed that SMAD2 might be involved in the HF growth cycle in Angora rabbits. However, its extra role in the HF growth and development remains obscure. In this study, we cloned the complete coding sequence (CDS) of the Angora rabbit SMAD2 gene. Within SMAD2 CDS, we identified the open reading frame (ORF) had a length of 1314 bp and encoding 437 amino acids. Bioinformatics analyses revealed that the SMAD2 protein is unstable and hydrophilic, and predominatelylocalizesin the cell nucleus. We identified that SMAD2 expression was elevated in the telogen phase of the during HF cycle. The knockdown and overexpression of SMAD2 could regulate HF growth and development related genes, such as WNT2, FGF2, and LEF1.Furthermore, SMAD2 may upregulate TGF-ß signaling pathway-related genes, including TFDP1, E2F4, and RBL1. In conclusion, our results indicate that SMAD2 plays a vital role in HF development by regulating the TGF-ß signaling pathway.

RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma in Xenopus tropicalis.

Alterations of the retinoblastoma and/or the p53 signaling network are associated with specific cancers such as high-grade astrocytoma/glioblastoma, small-cell lung cancer (SCLC), choroid plexus tumors, and small-cell pancreatic neuroendocrine carcinoma (SC-PaNEC). However, the intricate functional redundancy between RB1 and the related pocket proteins RBL1/p107 and RBL2/p130 in suppressing tumorigenesis remains poorly understood. Here we performed lineage-restricted parallel inactivation of rb1 and rbl1 by multiplex CRISPR/Cas9 genome editing in the true diploid Xenopus tropicalis to gain insight into this in vivo redundancy. We show that while rb1 inactivation is sufficient to induce choroid plexus papilloma, combined rb1 and rbl1 inactivation is required and sufficient to drive SC-PaNEC, retinoblastoma and astrocytoma. Further, using a novel Li-Fraumeni syndrome-mimicking tp53 mutant X. tropicalis line, we demonstrate increased malignancy of rb1/rbl1-mutant glioma towards glioblastoma upon concomitant inactivation of tp53. Interestingly, although clinical SC-PaNEC samples are characterized by abnormal p53 expression or localization, in the current experimental models, the tp53 status had little effect on the establishment and growth of SC-PaNEC, but may rather be essential for maintaining chromosomal stability. SCLC was only rarely observed in our experimental setup, indicating requirement of additional or alternative oncogenic insults. In conclusion, we used CRISPR/Cas9 to delineate the tumor suppressor properties of Rbl1, generating new insights in the functional redundancy within the retinoblastoma protein family in suppressing neuroendocrine pancreatic cancer and glioma/glioblastoma.

CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer.

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.

RB, p130 and p107 differentially repress G1/S and G2/M genes after p53 activation.

Cell cycle gene expression occurs in two waves. The G1/S genes encode factors required for DNA synthesis and the G2/M genes contribute to mitosis. The Retinoblastoma protein (RB) and DREAM complex (DP, RB-like, E2F4 and MuvB) cooperate to repress all cell cycle genes during G1 and inhibit entry into the cell cycle. DNA damage activates p53 leading to increased levels of p21 and inhibition of cell cycle progression. Whether the G1/S and G2/M genes are differentially repressed by RB and the RB-like proteins p130 and p107 in response to DNA damage is not known. We performed gene expression profiling of primary human fibroblasts upon DNA damage and assessed the effects on G1/S and G2/M genes. Upon p53 activation, p130 and RB cooperated to repress the G1/S genes. In addition, in the absence of RB and p130, p107 contributed to repression of G1/S genes. In contrast, G2/M genes were repressed by p130 and p107 after p53 activation. Furthermore, repression of G2/M genes by p107 and p130 led to reduced entry into mitosis. Our data demonstrates specific roles for RB, p130-DREAM, and p107-DREAM in p53 and p21 mediated repression of cell cycle genes.

Differential development of large-cell neuroendocrine or small-cell lung carcinoma upon inactivation of 4 tumor suppressor genes.

High-grade neuroendocrine lung malignancies (large-cell neuroendocrine cell carcinoma, LCNEC, and small-cell lung carcinoma, SCLC) are among the most deadly lung cancer conditions with no optimal clinical management. The biological relationships between SCLC and LCNEC are still largely unknown and a current matter of debate as growing molecular data reveal high heterogeneity with potential therapeutic consequences. Here we describe murine models of high-grade neuroendocrine lung carcinomas generated by the loss of 4 tumor suppressors. In an Rbl1-null background, deletion of Rb1, Pten, and Trp53 floxed alleles after Ad-CMVcre infection in a wide variety of lung epithelial cells produces LCNEC. Meanwhile, inactivation of these genes using Ad-K5cre in basal cells leads to the development of SCLC, thus differentially influencing the lung cancer type developed. So far, a defined model of LCNEC has not been reported. Molecular and transcriptomic analyses of both models revealed strong similarities to their human counterparts. In addition, a 68Ga-DOTATOC-based molecular-imaging method provides a tool for detection and monitoring the progression of the cancer. These data offer insight into the biology of SCLC and LCNEC, providing a useful framework for development of compounds and preclinical investigations in accurate immunocompetent models.

Rb1/Rbl1/Vhl loss induces mouse subretinal angiomatous proliferation and hemangioblastoma.

Von Hippel-Lindau (Vhl) protein inhibits hypoxia-inducible factor (Hif), yet its deletion in murine retina does not cause the extensive angiogenesis expected with Hif induction. The mechanism is unclear. Here we show that retinoblastoma tumor suppressor (Rb1) constrains expression of Hif target genes in the Vhl-/- retina. Deleting Rb1 induced extensive retinal neovascularization and autophagic ablation of photoreceptors in the Vhl-/- retina. RNA-sequencing, ChIP, and reporter assays showed Rb1 recruitment to and repression of certain Hif target genes. Activating Rb1 by deleting cyclin D1 induced a partial defect in the retinal superficial vascular plexus. Unexpectedly, removing Vhl suppressed retinoblastoma formation in murine Rb1/Rbl1-deficient retina but generated subretinal vascular growths resembling retinal angiomatous proliferation (RAP) and retinal capillary hemangioblastoma (RCH). Most stromal cells in the RAP/RCH-like lesions were Sox9+, suggesting a Müller glia origin, and expressed Lgals3, a marker of human brain hemangioblastoma. Thus, the Rb family limit Hif target gene expression in the Vhl-/- retina, and removing this inhibitory signal generates new models for RAP and RCH.

Two novel lipases purified from rice bran displaying lipolytic and esterification activities.

In this study, two novel lipases, rice bran lipase 1 (RBL1) and rice bran lipase 2 (RBL2), were first identified in rice (Oryza sativa) bran. Through the purification by ammonium sulfate precipitation, ion-exchange chromatography and size-exclusion chromatography, RBL1 and RBL2 were purified to 36- and 339-fold with the final specific activity of 4.73 and 44.06 U/mg, respectively. The purified RBL1 and RBL2 had the molecular weight of 18.8 and 35.5 kDa, respectively. The Km values of RBL1 and RBL2 were 0.766 and 0.432 mM while catalytic efficiency (kcat/Km) values of RBL1 and RBL2 were 102.4 and 2559.3 s-1/mM, respectively. RBL1 and RBL2 both showed esterification activity, but had no transesterification activity. In a solvent-free system, RBL1 and RBL2 exhibited degree of esterification (ED) of 4.4% and 6.5%, respectively. These two novel lipases exerted great properties for their potentials in industrial applications. First, RBL1 and RBL2 showed both mild reaction pH of 7.0 and temperature of 35 °C and 50 °C, respectively. Secondly, they showed great tolerance to several organic solvents and detergents while RBL1 exhibited great pH stability across a very broad range of pH (pH 3-12). Lastly and most importantly, RBL1 and RBL2 both exhibited esterification activity better than a commercial lipase, Candida rugosa lipase (CRL), in a solvent-free system. In conclusion, two novel lipases, RBL1 and RBL2, are different from published native lipases in rice bran and may be alternative potential candidates of biocatalysts contributing to the development of diverse industrial application fields.

DREAM and RB cooperate to induce gene repression and cell-cycle arrest in response to p53 activation.

Most human cancers acquire mutations causing defects in the p53 signaling pathway. The tumor suppressor p53 becomes activated in response to genotoxic stress and is essential for arresting the cell cycle to facilitate DNA repair or to initiate apoptosis. p53-induced cell cycle-arrest is mediated by expression of the CDK inhibitor p21WAF1/Cip1, which prevents phosphorylation and inactivation of the pocket proteins RB, p130, and p107. In a hypophosphorylated state, pocket proteins bind to E2F factors forming RB-E2F and DREAM transcriptional repressor complexes. Here, we analyze the influence of RB and DREAM on p53-induced gene repression and cell-cycle arrest. We show that abrogation of DREAM function by knockout of the DREAM component LIN37 results in a reduced repression of cell-cycle genes. We identify the genes repressed by the p53-DREAM pathway and describe a set of genes that is downregulated by p53 independent of LIN37/DREAM. Most strikingly, p53-dependent repression of cell-cycle genes is completely abrogated in LIN37-/-;RB-/- cells leading to a loss of the G1/S checkpoint. Taken together, we show that DREAM and RB are key factors in the p53 signaling pathway to downregulate a large number of cell-cycle genes and to arrest the cell cycle at the G1/S transition.

Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix.

The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. However, the role of Rb phosphorylation by cyclin D-Cdk4,6 in cell-cycle progression is unclear because Rb can be phosphorylated by other cyclin-Cdks, and cyclin D-Cdk4,6 has other targets involved in cell division. Here, we show that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by cyclins E, A, and B. This helix-based docking mechanism is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. Our work conclusively demonstrates that the cyclin D-Rb interaction drives cell division and expands the diversity of known cyclin-based protein docking mechanisms.

A Longitudinal Study of the Association between Mammographic Density and Gene Expression in Normal Breast Tissue.

High mammographic density (MD) is associated with a 4-6 times increase in breast cancer risk. For post-menopausal women, MD often decreases over time, but little is known about the underlying biological mechanisms. MD reflects breast tissue composition, and may be associated with microenvironment subtypes previously identified in tumor-adjacent normal tissue. Currently, these subtypes have not been explored in normal breast tissue. We obtained biopsies from breasts of healthy women at two different time points several years apart and performed microarray gene expression analysis. At time point 1, 65 samples with both MD and gene expression were available. At time point 2, gene expression and MD data were available from 17 women, of which 11 also had gene expression data available from the first time point. We validated findings from our previous study; negative correlation between RBL1 and MD in post-menopausal women, indicating involvement of the TGFß pathway. We also found that breast tissue samples from women with a large decrease in MD sustained higher expression of genes in the histone family H4. In addition, we explored the previously defined active and inactive microenvironment subtypes and demonstrated that normal breast samples of the active subtype had characteristics similar to the claudin-low breast cancer subtype. Breast biopsies from healthy women are challenging to obtain, but despite a limited sample size, we have identified possible mechanisms relevant for changes in breast biology and MD over time that may be of importance for breast cancer risk and tumor initiation.

Involvement of the STAT5-cyclin D/CDK4-pRb pathway in ß-cell proliferation stimulated by prolactin during pregnancy.

During pregnancy, maternal pancreatic ß-cells undergo a compensatory expansion in response to the state of insulin resistance, where prolactin (PRL) plays a major role. Retinoblastoma protein (Rb) has been shown to critically regulate islet proliferation and function. The aim of the study was to explore the role of Rb in ß-cell mass expansion during pregnancy. Expression of pocket protein family and E2Fs were examined in mouse islets during pregnancy and in insulinoma cells (INS-1) stimulated by PRL. PRL-stimulated INS-1 cells were used to explore the signaling pathway that regulates Rb downstream of the PRL receptor. Pancreas-specific Rb-knockout (Rb-KO) mice were assessed to evaluate the in vivo function of Rb in ß-cell proliferation during pregnancy. During pregnancy, expression of Rb, phospho-Rb (p-Rb), p107, and E2F1 increased, while p130 decreased in maternal islets. With PRL stimulation, induction of Rb expression occurred mainly in the nucleus, while p-Rb was predominantly in the cytoplasm. Inhibition of STAT5 significantly restrained the expression of CDK4, Rb, p-Rb, and E2F1 in PRL-stimulated INS-1 cells with attenuation in cell cycle progression. Reduction of Rb phosphorylation by CDK4 inhibition blocked PRL-mediated proliferation of INS-1 cells. On the other hand, knockdown of Rb using siRNA led to an induction in E2F1 leading to cell cycle progression from G1 to S and G2/M phase, similar to the effects of PRL-mediated induction of p-Rb that led to cell proliferation. With Rb knockdown, PRL did not lead to further increase in cell cycle progression. Similarly, while Rb-KO pregnant mice displayed better glucose tolerance and higher insulin secretion, they had similar ß-cell mass and proliferation to wild-type pregnant controls, supporting the essential role of Rb suppression in augmenting ß-cell proliferation during pregnancy. Rb-E2F1 regulation plays a pivotal role in PRL-stimulated ß-cell proliferation. PRL promotes Rb phosphorylation and E2F1 upregulation via STAT5-cyclin D/CDK4 pathway during pregnancy.

p107 Deficiency Increases Energy Expenditure by Inducing Brown-Fat Thermogenesis and Browning of White Adipose Tissue.

SCOPE: The tumor suppressor p107, a pocket protein member of the retinoblastoma susceptibility protein family, plays an important role in the cell cycle and cellular adipocyte differentiation. Nonetheless, the mechanism by which it influences whole body Energy homeostasis is unknown. METHODS AND RESULTS: The phenotype of p107 knockout (KO) mixed-background C57BL6/129 mice phenotype is studied by focusing on the involvement of white and brown adipose tissue (WAT and BAT) in energy metabolism. It is shown that p107 KO mice are leaner and have high-fat diet resistence. This phenomenon is explained by an increase of energy expenditure. The higher energy expenditure is caused by the activation of thermogenesis and may be mediated by both BAT and the browning of WAT. Consequently, it leads to the resistance of p107 KO mice to high-fat diet effects, prevention of liver steatosis, and improvement of the lipid profile and glucose homeostasis. CONCLUSION: These data allowed the unmasking of a mechanism by which a KO of p107 prevents diet-induced obesity by increasing energy expenditure via increased thermogenesis in BAT and browning of WAT, indicating the relevance of p107 as a modulator of metabolic activity of both brown and white adipocytes. Therefore, it can be targeted for the development of new therapies to ameliorate the metabolic syndrome.

A transient DMSO treatment increases the differentiation potential of human pluripotent stem cells through the Rb family.

The propensity for differentiation varies substantially across human pluripotent stem cell (hPSC) lines, greatly restricting the use of hPSCs for cell replacement therapy or disease modeling. Here, we investigate the underlying mechanisms and demonstrate that activation of the retinoblastoma (Rb) pathway in a transient manner is important for differentiation. In prior work, we demonstrated that pre-treating hPSCs with dimethylsulfoxide (DMSO) before directed differentiation enhanced differentiation potential across all three germ layers. Here, we show that exposure to DMSO improves the efficiency of hPSC differentiation through Rb and by repressing downstream E2F-target genes. While transient inactivation of the Rb family members (including Rb, p107, and p130) suppresses DMSO's capacity to enhance differentiation across all germ layers, transient expression of a constitutively active (non-phosphorylatable) form of Rb increases the differentiation efficiency similar to DMSO. Inhibition of downstream targets of Rb, such as E2F signaling, also promotes differentiation of hPSCs. More generally, we demonstrate that the duration of Rb activation plays an important role in regulating differentiation capacity.

The Retinoblastoma (RB) Tumor Suppressor: Pushing Back against Genome Instability on Multiple Fronts.

The retinoblastoma (RB) tumor suppressor is known as a master regulator of the cell cycle. RB is mutated or functionally inactivated in the majority of human cancers. This transcriptional regulator exerts its function in cell cycle control through its interaction with the E2F family of transcription factors and with chromatin remodelers and modifiers that contribute to the repression of genes important for cell cycle progression. Over the years, studies have shown that RB participates in multiple processes in addition to cell cycle control. Indeed, RB is known to interact with over 200 different proteins and likely exists in multiple complexes. RB, in some cases, acts through its interaction with E2F1, other members of the pocket protein family (p107 and p130), and/or chromatin remodelers and modifiers. RB is a tumor suppressor with important chromatin regulatory functions that affect genomic stability. These functions include the role of RB in DNA repair, telomere maintenance, chromosome condensation and cohesion, and silencing of repetitive regions. In this review we will discuss recent advances in RB biology related to RB, partner proteins, and their non-transcriptional functions fighting back against genomic instability.