Rb Function Research Articles

RNA-seq of 272 gliomas revealed a novel, recurrent PTPRZ1-MET fusion transcript in secondary glioblastomas

Studies of gene rearrangements and the consequent oncogenic fusion proteins have laid the foundation for targeted cancer therapy. To identify oncogenic fusions associated with glioma progression, we catalogued fusion transcripts by RNA-seq of 272 gliomas. Fusion transcripts were more frequently found in high-grade gliomas, in the classical subtype of gliomas, and in gliomas treated with radiation/temozolomide. Sixty-seven in-frame fusion transcripts were identified, including three recurrent fusion transcripts: FGFR3-TACC3, RNF213-SLC26A11, and PTPRZ1-MET (ZM). Interestingly, the ZM fusion was found only in grade III astrocytomas (1/13; 7.7%) or secondary GBMs (sGBMs, 3/20; 15.0%). In an independent cohort of sGBMs, the ZM fusion was found in three of 20 (15%) specimens. Genomic analysis revealed that the fusion arose from translocation events involving introns 3 or 8 of PTPRZ and intron 1 of MET. ZM fusion transcripts were found in GBMs irrespective of isocitrate dehydrogenase 1 (IDH1) mutation status. sGBMs harboring ZM fusion showed higher expression of genes required for PIK3CA signaling and lowered expression of genes that suppressed RB1 or TP53 function. Expression of the ZM fusion was mutually exclusive with EGFR overexpression in sGBMs. Exogenous expression of the ZM fusion in the U87MG glioblastoma line enhanced cell migration and invasion. Clinically, patients afflicted with ZM fusion harboring glioblastomas survived poorly relative to those afflicted with non-ZM-harboring sGBMs (P < 0.001). Our study profiles the shifting RNA landscape of gliomas during progression and reveled ZM as a novel, recurrent fusion transcript in sGBMs.

Regulation of cell transformation by Rb-controlled redox homeostasis.

Rb is a tumor suppressor, and regulates various biological progresses, such as cell proliferation, development, metabolism and cell death. In the current study, we show that Rb knockout in 3T3 cells leads to oxidative redox state and low mitochondrial membrane potential by regulating mitochondrial activity. Our results indicate that Rb plays an important role in controlling redox homeostasis. More importantly, the functions of Rb in modulating cell proliferation, death and transformation are, at least in part, mediated by its controlling cellular redox state. In addition, our results also suggest that the cellular redox state possibly determines various biological activities, including cell survival, death and transformation, where Rb is functioning as a regulator of redox homeostasis.

A combined device for the joint study of the surface tensions and electron work functions of liquid-metal melts at low temperatures

The design and test data of the combined device, which is intended to simultaneously measure the surface tensions and electron work functions of metals and alloys in static high-vacuum conditions in a temperature range of 60–400 K, are given. The measurements of the electron work function of pure Na, K, Rb, and Cs and near-eutectic alloys of the ternary Na-K-Cs system showed high reliability and accuracy of the obtained data.

Deregulation of p53 and RB Transcriptional Control Leads to Overexpression of DNA Methyltransferases in Lung Cancer

BackgroundOverexpression of DNA 5’-cytosine-methyltransferases (DNMTs), which silence genes including tumor suppressor genes, is involved in many cancers. However, the mechanism of DNMT overexpression remains mostly unclear. ObjectivesThe tumor suppressor p53 and RB proteins, which repress gene transcription, are often inactivated by gene mutation or overexpression of the E3 ubiquitin ligase MDM2 in cancer cells. Therefore, this study aims to examine whether the transcriptional regulation of DNMT1, 3A and 3B genes is controlled by p53/RB/MDM2 pathway in lung cancer using cell and clinical studies. MethodsThe interaction and regulation between p53 or RB with DNMT1, 3A and 3B promoters were detected by promoter activity assay and chromatin immunoprecipitation. Transcriptional repression of DNMT1, 3A and 3B by p53 and RB was confirmed using siRNA-mediated knockdown or overexpression of p53/RB. Immunohistochemistry for p53, RB, MDM2, and DNMTs protein expression and DNA sequencing for p53 mutation were performed on tumor tissues from lung cancer patients. Resultsp53 and RB suppressed the promoter activity and mRNA/protein expression of DNMT1, 3A and 3B through binding to their promoter. The suppression could be attenuated by knocking down of RB and p53. Importantly, co-transfection of both p53 and RB expression constructs showed additional inhibition of DNMT protein expression. In clinical study, functional RB and p53 inversely correlated with DNMT1, 3A and 3B expressions in samples from lung cancer patients (P=0.016~0.024). Lung cancer patients with low RB expression or p53 mutation resulting in DNMTs overexpression showed promoter hypermethylation in multiple tumor suppressor genes. Patients with normal expression of DNMT3A, RB and MDM2 was associated with better prognosis compared to other patients (P=0.049). ConclusionsThis study provides cell and clinical evidence that p53 and RB pathways transcriptionally repress DNMT expression. Normal expression of DNMT3A, RB and MDM2 proteins can be a biomarker for good prognosis in lung cancer.

Retinoblastoma protein (RB) interacts with E2F3 to control terminal differentiation of Sertoli cells.

The retinoblastoma protein (RB) is essential for normal cell cycle control. RB function depends, at least in part, on interactions with the E2F family of DNA-binding transcription factors (E2Fs). To study the role of RB in the adult testis, a Sertoli cell (SC)-specific Rb knockout mouse line (SC-RbKO) was generated using the Cre/loxP recombination system. SC-RbKO mice exhibited an age-dependent testicular atrophy, impaired fertility, severe SC dysfunction, and spermatogenic defects. Removal of Rb in SC induced aberrant SC cycling, dedifferentiation, and apoptosis. Here we show that E2F3 is the only E2F expressed in mouse SCs and that RB interacts with E2F3 during mouse testicular development. In the absence of RB, the other retinoblastoma family members p107 and p130 began interacting with E2F3 in the adult testes. In vivo silencing of E2F3 partially restored the SC maturation and survival as well as spermatogenesis in the SC-RbKO mice. These results point to RB as a key regulator of SC function in adult mice and that the RB/E2F3 pathway directs SC maturation, cell cycle quiescence, and RB protects SC from apoptosis.

LIMD1 antagonizes E2F1 activity and cell cycle progression by enhancing Rb function in cancer cells

Tumour suppressor genes restrain inappropriate cell growth and division, as well as stimulate cell death to maintain tissue homeostasis. Loss of function leads to abnormal cellular behaviour, including hyperproliferation of cell and perturbation of cell cycle regulation. LIMD1 is a tumour suppressor gene located at chromosome 3p21.3, a region commonly deleted in many solid malignancies. LIMD1 interacts with retinoblastoma (Rb) and is involved in Rb-mediated downregulation of E2F1-target genes. However, the role of LIMD1 in cell cycle regulation remains unclear. We propose that LIMD1 induces cell cycle arrest, utilising Rb-E2F1 axis, and show that ectopic expression of LIMD1 in A549 cells results in hypo-phosphorylation that potentiates Rb function, which correlates with downregulation of E2F1. In agreement with these observations, LIMD1 overexpression retards cell cycle progression and blocks S-phase entry, as cells accumulate in G0/G1 phase and have reduced incorporation of BrdU. Most significantly, LIMD1-dependent effects on Rb function and cell cycle are reversed on depletion of endogenous LIMD1, underscoring its centrality in Rb-mediated cell cycle regulation. Hence, our findings provide new insight into cell cycle control by Rb-LIMD1 nexus.

Temperature dependent atomic transport properties of liquid Rb

A simple analytical model for atomic motion is used to obtain velocity autocorrelation function (VACF) for liquid Rb. The modified empty-core potential due to Hasegawa et al., which represents the orthogonalisation effect due to s-core states in such sp-bonded metals, is used for electron–ion interaction. The potential parameter rc is determined at different temperatures from the knowledge of structure factor. We find quantitative explanation for the density and temperature dependence of VACF and self-diffusion coefficients. The coherent behaviour of liquid Rb in terms of the dynamic structure factor employing viscoelastic theory has also been studied. Intrinsic temperature effects have been studied through damping term in the pair potential. The predicted results for VACF, cosine power spectrum, mean square displacement, diffusion and viscosity coefficients have been compared with recent available molecular dynamics (MD) data and a good agreement has been achieved.

Epigallocatechin-3-Gallate Prevents Autoimmune-Associated Down- Regulation of p21 in Salivary Gland Cells Through a p53-Independent Pathway

The submandibular salivary glands of non-obese diabetic (NOD) mice, a model for Sjogren's syndrome and type-1 diabetes, show an elevated level of proliferating cell nuclear antigen (PCNA), a protein involved in cell proliferation and repair of DNA damage. We reported previously that epigallocatechin-3-gallate (EGCG), the most abundant green tea catechin, normalizes the PCNA level. PCNA's activity can be regulated by the cyclin-dependent kinase inhibitor p21, which is also important for epithelial cell differentiation. In turn, expression of p21 and PCNA are partially regulated by Rb phosphorylation levels. EGCG was found to modulate p21 expression in epithelial cells, suggesting that EGCG-induced p21 could be associated with down-regulation of PCNA in vivo. The current study examined the protein levels of p21 and p53 (which can up-regulate p21) in NOD mice fed with either water or EGCG, and the effect of EGCG on p21 and p53 in cell line models with either normal or defective Rb. In NOD mice, the p21 level was low, and EGCG normalized it. In contrast to HSG cells with functional Rb, negligible expression of p21 in NS-SVAC cells that lack Rb was not altered by EGCG treatment. Inhibition of p53 by siRNA demonstrated that p21 and p53 were induced independently in HSG cells by a physiological concentration range of EGCG, suggesting p53 could be an important but not conditional factor associated with p21 expression. In conclusion, PCNA and p21 levels are altered inversely in the NOD model for SS and in HSG cells, and warrant further study as candidate new markers for salivary dysfunction associated with xerostomia. Induction of p21 by EGCG could provide clinically useful normalization of salivary glands by promoting differentiation and reducing PCNA levels.

P53 suppresses carcinoma progression by inhibiting mTOR pathway activation

Genetic alterations in human cancers and murine models indicate that Rb and p53 have critical tumor suppressive functions in retinoblastoma, a tumor of neural origin, and neuroendocrine tumors including small cell lung cancer and medullary thyroid cancer (MTC). Rb inactivation is the initiating lesion in retinoblastoma and current models propose that induction of apoptosis is a key p53 tumor suppressive function. Genetic studies in mice, however, indicate that other undefined p53 tumor suppressive functions are operative in vivo. How p53 loss cooperates with Rb inactivation to promote carcinogenesis is also not fully understood. In the current study, genetically engineered mice were generated to determine the role of Rb and p53 in MTC pathogenesis and test the hypothesis that p53 suppresses carcinogenesis by inhibiting mTOR signaling. Conditional Rb ablation resulted in thyroid tumors mimicking human MTC, and additional p53 loss led to rapid tumor progression. p53 suppressed tumorigenesis by inhibiting cell cycle progression, but did not induce apoptosis. On the contrary, p53 loss led to increased apoptosis that had to be overcome for tumor progression. mTOR activity was markedly increased in p53 deficient tumors and rapamycin treatment suppressed tumor cell growth identifying mTOR inhibition as a critical p53 tumor suppressive function. Rapamycin treatment did not result in AKT/MAPK activation providing evidence that this feedback mechanism operative in other cancers is not a general response to mTORC1 inhibition. Together, these studies provide mechanistic links between genetic alterations and aberrant signaling pathways critical in carcinogenesis, and identify essential Rb and p53 tumor suppressive functions in vivo.

Rb Suppresses Collective Invasion, Circulation and Metastasis of Breast Cancer Cells in CD44-Dependent Manner

Basal-like breast carcinomas (BLCs) present with extratumoral lymphovascular invasion, are highly metastatic, presumably through a hematogenous route, have augmented expression of CD44 oncoprotein and relatively low levels of retinoblastoma (Rb) tumor suppressor. However, the causal relation among these features is not clear. Here, we show that Rb acts as a key suppressor of multiple stages of metastatic progression. Firstly, Rb suppresses collective cell migration (CCM) and CD44-dependent formation of F-actin positive protrusions in vitro and cell-cluster based lymphovascular invasion in vivo. Secondly, Rb inhibits the release of single cancer cells and cell clusters into the hematogenous circulation and subsequent metastatic growth in lungs. Finally, CD44 expression is required for collective motility and all subsequent stages of metastatic progression initiated by loss of Rb function. Altogether, our results suggest that Rb/CD44 pathway is a crucial regulator of CCM and metastatic progression of BLCs and a promising target for anti-BLCs therapy.

Cytoplasmic translocation of the retinoblastoma protein disrupts sarcomeric organization

Skeletal muscle degeneration is a complication arising from a variety of chronic diseases including advanced cancer. Pro-inflammatory cytokine TNF-α plays a pivotal role in mediating cancer-related skeletal muscle degeneration. Here, we show a novel function for retinoblastoma protein (Rb), where Rb causes sarcomeric disorganization. In human skeletal muscle myotubes (HSMMs), up-regulation of cyclin-dependent kinase 4 (CDK4) and concomitant phosphorylation of Rb was induced by TNF-α treatment, resulting in the translocation of phosphorylated Rb to the cytoplasm. Moreover, induced expression of the nuclear exporting signal (NES)-fused form of Rb caused disruption of sarcomeric organization. We identified mammalian diaphanous-related formin 1 (mDia1), a potent actin nucleation factor, as a binding partner of cytoplasmic Rb and found that mDia1 helps maintain the structural integrity of the sarcomere. These results reveal a novel non-nuclear function for Rb and suggest a potential mechanism of TNF-α-induced disruption of sarcomeric organization. DOI: http://dx.doi.org/10.7554/eLife.01228.001.

Abstract A87: Overcoming NSCLC to SCLC transformation as a resistance mechanism in EGFR mutant lung cancer.

Abstract The EGFR TKIs gefitinib and erlotinib produce dramatic responses in NSCLC patients with EGFR activating mutations, however, the efficacy of these drugs is severely limited by the development of acquired resistance. Recently, we and others have found that a subset of these cancers transform from NSCLC to SCLC as they acquire resistance to EGFR TKI. Here we report an initial characterization of the molecular mechanisms underlying the transformation from NSCLC to SCLC in samples and cell lines derived from resistant patients. Much like de novo SCLC, Rb and p53 are altered in the resistant tumors that transformed to SCLC, although loss of p53 and Rb function alone were insufficient to drive the transformation in NSCLC cell lines. In addition, we demonstrate that EGFR expression is lost in resistant samples, suggesting that in switching to SCLC these cancers become a cell type that is no longer dependent on EGFR activity and providing an explanation for lack of EGFR TKI sensitivity. Finally, we find that SCLC transformed cells are uniquely sensitive to the Bcl-2/Bcl-XL inhibitor Abt-263, offering a potential therapeutic option for these patients. Together, these results represent the first insights into the molecular and genetic details regarding this novel type of resistance to targeted therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A87. Citation Format: Matthew J. Niederst, Lecia V. Sequist, Elizabeth L. Lockerman, Carlotta Costa, Darrell R. Borger, Mari Mino-Kenudson, Anthony J. Iafrate, Jeffrey A. Engelman. Overcoming NSCLC to SCLC transformation as a resistance mechanism in EGFR mutant lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A87.

Abstract CN03-02: Conditionally reprogrammed cells for personalized medicine.

Abstract CN03-02: Conditionally reprogrammed cells for personalized medicine.

P16 in HPV-associated cancers

Infections with high-risk human papillomaviruses (HPV) account for approximately 5% of all human cancers. Cervical cancer, in particular, is almost universally caused by “high-risk” HPV infections and these HPVs also cause a large fraction of other anogenital tract cancers as well as oral carcinomas. The viral E6 and E7 proteins are the universal oncogenic drivers of HPV-associated cancers, and persistent E6/E7 expression is necessary for the maintenance of the transformed state. Hence the HPV E6 and E7 proteins should represent excellent therapeutic targets. The HPV E6 and E7 proteins lack intrinsic enzymatic activities and attempts to discover small molecule inhibitors of E6 or E7 containing cellular protein complexes have been futile. HPV E6 and E7 function by associating with host cellular proteins, thereby functionally re-programming cellular signal transduction pathways. The resulting rewiring of cellular circuitry causes “addictions” to certain signaling pathways that are non-essential and/or redundant in normal cells. This in turn generates specific cellular vulnerabilities that may be more readily druggable. The article (McLaughlin-Drubin ME et al., Proc. Natl. Acad. Sci. USA. 2013; 110:16175-16180) identifies a surprising and potentially druggable vulnerability in the retinoblastoma tumor suppressor pathway caused by the high-risk HPV E7 protein. High-risk HPV E7 protein expression triggers a cellular defense response referred to as oncogene induced senescence (OIS), which is mediated by the p16INK4A (p16) tumor suppressor and executed by the retinoblastoma tumor suppressor protein, Rb. In normal cells, p16 expression is epigenetically silenced by polycomb repressive complexes. p16 inhibits cyclin dependent kinase 4 and 6 (CDK4/6). This causes accumulation of hypophosphorylated Rb, which in turn signals cell cycle arrest (Fig. ​(Fig.11 upper panel). E7 protein triggers p16. High-level p16 expression is an excellent biomarker for high-risk HPV-associated lesions and cancers. Cell cycle arrest and senescence in these tumors, however, is subverted by E7 targeting Rb for proteasomal degradation (Fig. ​(Fig.11 lower panel). p16 induction, in the absence of Rb (through Rb degradation by E7) causes a unique and surprising addiction to p16INK4A expression. Even though p16 is a tumor suppressor in most cellular contexts, it is essential for survival of cervical carcinoma lines. The iconoclastic “oncogenic” activity of p16 in HPV E7 expressing cells, like its more familiar tumor suppressor activity, is based on the ability to inhibit CDK4/6. Indeed, a human melanoma derived p16-insensitive CDK4 mutant also causes cell death when expressed in HPV E7-expressing cells. Hence, this oncogenic CDK4 mutant exhibits biological activities that are akin to those of a tumor suppressor in the context of a p16-expressing, Rb defective cell. These results imply that CDK4/6 activity is not tolerated in cells that lack Rb activity. One may hypothesize that there must be CDK4/6 substrates that cause cell death when they are phosphorylated in cells that lack Rb. Tumorigenic activities of the Rb pathway including CDK4/6 and p16 are context dependent. CDK4/CDK6 inhibition may be efficacious only in tumors that retain functional Rb. In contrast, CDK4/CDK6 inhibition in cancers that have suffered Rb mutations is necessary for tumor growth and survival. In such cases it may be therapeutically useful to activate rather than to inhibit CDK4/CDK6 activity. One way CDK4/6 activation may be achieved is by epigenetic silencing of p16 through inhibition of the KDM6B enzyme. Indeed the KDM6-selective small molecule inhibitor GSK-J4 induced cell death specifically in KDM6B/p16-addicted cell lines. This shows that the addiction of HPV-associated lesions and cancer to p16 expression creates a cellular vulnerability to KDM6B inhibition. De-repression of polycomb-silenced genes, such as p16, is a complex, multistep process. Hence, “epigenetic therapies” targeting KDM6B and potentially other enzymes that may also be rate limiting for maintaining the transcriptional competence of the p16 promoter may be efficacious in p16-addicted cancers. Figure 1 The retinoblastoma (Rb) tumor suppressor pathway It has been noted early on that p16 expression is confined to those tumors that contain pRB mutations, and a number of non HPV-associated cancer types, including some breast, prostate, lung and high-grade serous ovarian carcinomas express p16. It will be important to determine whether some of these tumors are similarly addicted to p16 expression and whether they are vulnerable to inhibition of KDM6B and/or other epigenetic enzymes that are necessary for p16 expression.

Rb Protein is Essential to the Senescence-Associated Heterochromatic Foci Formation Induced by HMGA2 in Primary WI38 Cells

Cellular senescence is an irreversible form of cell cycle arrest that provides a barrier to neoplastic transformation. The integrity of the Rb (Retinoblastoma) pathway is necessary for the formation of the senescence-associated heterochromatin foci (SAHF) that offers a molecular basis for the stability of the senescent state. Surprisingly, although high mobility group A2 protein (HMGA2) can promote tumorigenesis and inhibit Rb function in tumor cells, high-level expression of HMGA2 is sufficient to induce SAHF formation in primary cells. It therefore becomes significant to determine whether Rb protein is necessary in HMGA2-induced SAHF formation. In this study, we established the cellular senescence and SAHF assembly WI38 cell model by ectopic expression of HMGA2, in which typical senescent markers were seen, including notable upregulation of p53, p21 and p16, and elevated SA-β-galactosidase staining together with downregulation of E2F target genes. We then showed that the Rb pathway inhibitor E7 protein was able to partly abolish the ability of SAHF formation after HMGA2 expression in WI38 cells, indicating that Rb is a crucial factor for HMGA2-induced SAHF formation. However, Rb depletion did not completely rescue the cell growth arrest induced by HMGA2, suggesting that Rb is not an exclusive pathway for HMGA2-induced senescence in WI38 cells.

The Rb1 gene inhibits the viability of retinoblastoma cells by regulating homologous recombination

Retinoblastoma is a childhood ocular tumor caused by the inactivation of both alleles of the retinoblastoma gene (Rb1). Without Rb1 gene function, chromosomal aberrations are observed in retinoblastoma cells. The instability of the genome is closely associated with the repair of DNA double-strand breaks (DSBs). However, the precise molecular mechanism of action of Rb1 in DNA DSB repair remains unclear. Thus, in this study, we aimed to investigate whether the Rb1 gene affects DNA stability by assaying DNA DSB repair and also whether it regulates the proliferation of retinoblastoma cells. Rb1 immunofluorescence and RT-PCR were performed, demonstrating that the Rb1 gene is silenced in SO-Rb50 retinoblastoma cells, and the karyotype analysis of SO-Rb50 cells indicated that the loss of Rb1 function led to genomic instability; both numerical and structural chromosomal aberrations were observed in our study. In addition, the DNA DSB repair efficiency of the SO-Rb50 cells was measured by γ-H2AX immunofluorescence, a commonly used in situ marker of DNA DSBs, following exposure to ionizing radiation (IR) (2.5 and 5.0 Gy). We found that the DNA repair efficiency was significantly increased following IR-induced damage (P<0.01). However, there was no significant difference in DNA repair efficiency between the cells expressing exogenous Rb1 and the control (P>0.05). The assay for the screening of the effect of Rb1 on the sub-pathway of DNA DSB repair, non-homologous end joining (NHEJ) and homologous recombination (HR), indicated that Rb1 did not affect NHEJ activity, although it significantly promoted the HR pathway (HR levels increased by 2.46-fold) compared with the control (P<0.01). Furthermore, we found that the cell viability of the SO-Rb50 cells transfected with exogenous Rb1 was significantly inhibited (P<0.01) and cell cycle assay indicated that exogenous Rb1 induced S phase arrest (P<0.001) which also inhibited the proliferation of retinoblastoma cells (SO-Rb50) in vitro. Therefore, this study provides new insight into the mechanisms of action of the Rb1 gene in regulating the proliferation of retinoblastoma cells.

Molecular mechanisms underlying RB protein function

Inactivation of the RB protein is one of the most fundamental events in cancer. Coming to a molecular understanding of its function in normal cells and how it impedes cancer development has been challenging. Historically, the ability of RB to regulate the cell cycle placed it in a central role in proliferative control, and research focused on RB regulation of the E2F family of transcription factors. Remarkably, several recent studies have found additional tumour-suppressor functions of RB, including alternative roles in the cell cycle, maintenance of genome stability and apoptosis. These advances and new structural studies are combining to define the multifunctionality of RB.

Abstract 3450: ABT-348 in combination with inhibition of CDK4/6 highlights a strategy to target RB mutant cells while sparing RB wild-type cells.

Abstract ABT-348 is a novel, potent and orally bioavailable inhibitor of the Aurora kinases as well as the VEGF and PDGF families of receptor tyrosine kinases. ABT-348 is broadly efficacious preclinically as a single agent against a wide range of tumor types and is currently in Phase I/II clinical trials. Several published studies have supported the potential utility of combining targeted anti-proliferative agents with cytotoxic chemotherapies. Herein, the efficacy of combining the neocytotoxic agent, ABT-348, with CDK4/6 inhibition in the treatment of solid tumor cells has been evaluated. Inhibition of CDK4/6 via siRNA elicits a potent cytostatic response in cells that harbor a wild-type, functional RB. We demonstrate that features of cellular senescence are induced in human tumor cells with siRNAs targeting both CDK4 and CDK6. Dual inhibition of CDK4 and CDK6 is required for antiproliferative activity in most cancer cell lines and this is not accompanied by induction of apoptosis. In RB wild-type cells, CDK4/6 inhibition antagonizes the activity of ABT-348 by inducing G0/G1 arrest. Conversely, CDK4/6 inhibition does not alter the therapeutic response of RB-deficient cells to ABT-348, indicating that the effects of ABT-348 are dependent on cells progressing into mitosis. A preponderance of evidence from previous publications and our studies indicate that CDK4/6 inhibition attenuates the cellular response to cytotoxic chemotherapies in RB wild-type cells providing a potential to expand the therapeutic window for ABT-348. Thus combination with ABT-348 and a CDK4/6 inhibitor may provide an opportunity to selectively target RB mutant cells. Citation Format: Jun Guo, Michael L. Curtin, Zehan Chen, Daniel H. Albert, Paul Tapang, Yujia Dai, Mia-Ha Bui, Peter Kovar, Michael R. Michaelides, Keith B. Glaser, Chris Tse, O. Jameel Shah. ABT-348 in combination with inhibition of CDK4/6 highlights a strategy to target RB mutant cells while sparing RB wild-type cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3450. doi:10.1158/1538-7445.AM2013-3450

Abstract 346: p53 mutation in Rb deficient neuroendocrine lung and thyroid cells results in high grade neuroendocrine carcinomas promoted by mTOR pathway activation .

Abstract Small cell lung carcinoma (SCLC) and medullary thyroid carcinoma (MTC) are aggressive, rapidly metastatic neuroendocrine carcinomas that portend a dismal prognosis. Rb loss is detected in ≥ 90% of SCLC and p53 is mutated in ∼80% of cases providing evidence that Rb and p53 suppress SCLC. Extensive genetic evidence in mouse models also supports a critical role for Rb in suppressing MTC: however, the mechanisms promoting tumorigenesis in Rb and p53 deficient cells and their relation to therapeutic response remain unclear. In this study, a conditional SCLC and MTC mouse model was generated by targeting Rb and p53 ablation or mutant protein expression to lung and thyroid epithelial cells. Rb loss alone resulted in slowly progressing MTC and lung neuroendocrine hyperplasia. While p53 alteration alone was not sufficient for tumor initiation, p53 loss or mutation in Rb deficient cells led to MTC and SCLC with metastases, phenotypically mimicking human disease. Mice expressing p53 R172H (R175H in human), but not R270H (R273H in human), mutant protein had a gain-of-function phenotype as evidenced by decreased survival of Rb/p53R172H as compared to Rb/p53 ablated mice. No dominant negative effect was detected by comparing Rb/p53+/mutant to Rb/p53+/− mice. Mechanistic studies revealed p53 dependent mammalian target of rapamycin (mTOR) pathway activation in MTC without coincident AKT or MAPK pathway activation. Similar mTOR pathway activation was detected in Rb/p53 deficient and mutant lungs. Rapamycin dramatically inhibited mTOR pathway signaling in Rb/p53 deficient or mutant thyroid tumors and lungs in vivo as well as in MTC and SCLC tumor cells in culture. Interestingly, mTORC1 inhibition was not associated with feedback AKT or MAPK pathway activation in vitro or in vivo as previously reported for breast and prostate cancers. Rapamycin inhibited MTC and SCLC tumor cell growth demonstrating a functional role for mTOR activation in tumor progression. These studies identify distinct, cooperative Rb and p53 tumor suppressive functions. The oncogenic stimulus provided by Rb loss induces aberrant cell growth and leads to a p53 tumor suppressive response that inhibits tumor progression and mTOR dependent cell growth demonstrating that mTOR pathway inhibition is a critical p53 tumor suppressive function. Notably, rapamycin did not result in feedback MAPK or AKT pathway activation providing evidence this is not a general mechanism limiting therapeutic response to mTORC1 inhibition. Together these studies provide mechanistic links between genetic alterations and aberrant signaling pathways critical in carcinogenesis. In addition to providing mechanistic insights, this genetically engineered mouse model provides a robust preclinical platform to explore novel therapeutic strategies and define mechanisms underlying treatment response and resistance. Citation Format: Nagako Akeno, Ashley Miller, Ncik Levinsky, Kathryn A. Wikenheiser-Brokamp. p53 mutation in Rb deficient neuroendocrine lung and thyroid cells results in high grade neuroendocrine carcinomas promoted by mTOR pathway activation . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 346. doi:10.1158/1538-7445.AM2013-346

Abstract 5386: Investigating the role of N-terminal protein methylation in colon cancer progression.

Abstract We have recently identified the first mammalian N-terminal methyltransferase, NRMT. NRMT is a highly conserved, ubiquitously expressed nuclear enzyme that is predicted to methylate more than 300 target proteins, including the proven targets Retinoblastoma Protein (RB), DDB2, and the oncoprotein SET. N-terminal methylation has been shown to regulate protein-DNA interactions and has also been implicated in regulating protein stability. Multiple microarray analyses have shown that NRMT is overexpressed in human colon cancer, and our preliminary data indicate the more aggressive the cancer, the higher the NRMT expression. However, there are currently no commercially available inhibitors specific for this enzyme and very little is known about the downstream signaling consequences of NRMT misregulation. We have used in silico screening of the ZINC small molecule library to identify potential NRMT-specific inhibitors. Screening of the top 100 candidates produced 10 compounds that could inhibit NRMT methyltransferase activity in vitro. The top compound of these 10 can also inhibit NRMT methyltransferase activity in cell culture and specifically stops the growth of colon cancer cell lines that have high NRMT expression. We are currently working to optimize this small molecule inhibitor for stability and solubility, understand its downstream signaling consequences, and test its efficacy in a murine model system. We are especially interested in the potential of NRMT inhibitors as a treatment for patients with mutant kRAS. These patients are often insensitive to anti-EGFR therapy, but oncogenic RAS seems to require functional RB. Therefore, impairment of NRMT activity could also impair oncogenic RAS signaling through destabilization of RB. Data from this study will enhance our understanding of how colon cancer develops and could lead to novel treatments for the disease. Citation Format: John G. Tooley, John O. Trent, Christine E. Schaner Tooley. Investigating the role of N-terminal protein methylation in colon cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2013-5386