Rb Function Research Articles

Identification of highly penetrant Rb-related synthetic lethal interactions in triple negative breast cancer

Although defects in the RB1 tumour suppressor are one of the more common driver alterations found in triple-negative breast cancer (TNBC), therapeutic approaches that exploit this have not been identified. By integrating molecular profiling data with data from multiple genetic perturbation screens, we identified candidate synthetic lethal (SL) interactions associated with RB1 defects in TNBC. We refined this analysis by identifying the highly penetrant effects, reasoning that these would be more robust in the face of molecular heterogeneity and would represent more promising therapeutic targets. A significant proportion of the highly penetrant RB1 SL effects involved proteins closely associated with RB1 function, suggesting that this might be a defining characteristic. These included nuclear pore complex components associated with the MAD2 spindle checkpoint protein, the kinase and bromodomain containing transcription factor TAF1, and multiple components of the SCFSKP Cullin F box containing complex. Small-molecule inhibition of SCFSKP elicited an increase in p27Kip levels, providing a mechanistic rationale for RB1 SL. Transcript expression of SKP2, a SCFSKP component, was elevated in RB1-defective TNBCs, suggesting that in these tumours, SKP2 activity might buffer the effects of RB1 dysfunction.

Cancer reversion with oocyte extracts is mediated by cell cycle arrest and induction of tumour dormancy.

Inducing stable control of tumour growth by tumour reversion is an alternative approach to cancer treatment when eradication of the disease cannot be achieved. The process requires re-establishment of normal control mechanisms that are lost in cancer cells so that abnormal proliferation can be halted. Embryonic environments can reset cellular programmes and we previously showed that axolotl oocyte extracts can reprogram breast cancer cells and reverse their tumorigenicity. In this study, we analysed the gene expression profiles of oocyte extract-treated tumour xenografts to show that tumour reprogramming involves cell cycle arrest and acquisition of a quiescent state. Tumour dormancy is associated with increased P27 expression, restoration of RB function and downregulation of mitogen-activated signalling pathways. We also show that the quiescent state is associated with increased levels of H4K20me3 and decreased H4K20me1, an epigenetic profile leading to chromatin compaction. The epigenetic reprogramming induced by oocyte extracts is required for RB hypophosphorylation and induction of P27 expression, both occurring during exposure to the extracts and stably maintained in reprogrammed tumour xenografts. Therefore, this study demonstrates the value of oocyte molecules for inducing tumour reversion and for the development of new chemoquiescence-based therapies.

Therapeutic Challenge with a CDK 4/6 Inhibitor Induces an RB-Dependent SMAC-Mediated Apoptotic Response in Non-Small Cell Lung Cancer.

Purpose: The retinoblastoma tumor suppressor (RB), a key regulator of cell-cycle progression and proliferation, is functionally suppressed in up to 50% of non-small cell lung cancer (NSCLC). RB function is exquisitely controlled by a series of proteins, including the CyclinD-CDK4/6 complex. In this study, we interrogated the capacity of a CDK4/6 inhibitor, palbociclib, to activate RB function.Experimental Design and Results: We employed multiple isogenic RB-proficient and -deficient NSCLC lines to interrogate the cytostatic and cytotoxic capacity of CDK 4/6 inhibition in vitro and in vivo We demonstrate that while short-term exposure to palbociclib induces cellular senescence, prolonged exposure results in inhibition of tumor growth. Mechanistically, CDK 4/6 inhibition induces a proapoptotic transcriptional program through suppression of IAPs FOXM1 and Survivin, while simultaneously augmenting expression of SMAC and caspase-3 in an RB-dependent manner.Conclusions: This study uncovers a novel function of RB activation to induce cellular apoptosis through therapeutic administration of a palbociclib and provides a rationale for the clinical evaluation of CDK 4/6 inhibitors in the treatment of patients with NSCLC. Clin Cancer Res; 24(6); 1402-14. ©2018 AACR.

Shift of zero-dispersion wavelength in bent optical fibers.

The understanding of how bending modifies the dispersion of optical fibers, in particular, the zero-dispersion wavelength (λ0), is essential in the development of compact nonlinear optical devices such as parametric amplifiers, wavelength converters, soliton lasers and frequency comb generators. Typically, substantial variations in the parametric gain and/or conversion efficiency are significant for changes in λ0 of ~0.1 nm, which occur for variations on the bending radius (Rb) of 1 cm or less. Measuring λ0 as a function of bending radius (Rb) is challenging, as it requires detecting changes < 0.1 nm and in short fibers. By using a method based on four-wave mixing (FWM) generated by an incoherent-pump with relatively broad spectrum and a weak laser, we report measurements of λ0 as a function of Rb in a dispersion-shifted fiber with <0.1 nm accuracy on λ0. This method is sensitive enough to measure small variations in λ0 of ~0.04 nm in very short fibers (~20 m). We observe that λ0 increases by 12 nm when Rb is decreased from 10 cm to 1 cm, and a change of 1 nm is obtained for Rb = 3 cm. We also present numerical simulations of the bent fiber that are in good agreement with our measurements, and help us to explain the observations and to predict how high-order dispersion is modified with bending. This study can provide insights for dispersion engineering, in which bending could be used as a tuning, equalization, or tailoring mechanism for λ0, which can be used in the development of compact nonlinear optical devices based on fibers or other bent-waveguide structures.

C15-153: Randomized phase IB/II study of enzalutamide with and without ribociclib in patients with metastatic castrate resistant, chemotherapy naïve prostate cancer that retains RB expression.

TPS384 Background: Current data suggests RB function is often attenuated in tumors through hyperphosphorylation; thus, RB activity can be “re-awakened” in RB+ tumors by suppressing key kinases that phosphorylate RB (CDK4/6). Recent findings identified perturbations of the RB tumor suppressor as a major mechanism of androgen receptor (AR) deregulation and castration-resistant prostate cancer (CRPC) formation. Our preclinical studies indicate RB+ tumors are exquisitely responsive to CDK4/6 inhibitors, revealing tumor-specific cytostatic and cytotoxic effects. These analyses strongly support the use of CDK4/6 inhibitors to activate the RB pathway, and impede the transition to CRPC. We hypothesize the CDK4/6 inhibitor ribociclib (ribo) in combination with enzalutamide (enz) in patients (pts) with progressive metastatic CRPC (mCRPC) that retains RB expression will significantly increase the efficacy of enz. Methods: This is a phase IB/II study of pts with mCRPC with RB+ tumors who have not received chemotherapy. Since ribo and enz have not been combined together in pts, the standard doses of enz and a traditional 3x3 dose escalation schema will be used in phase I to establish the safe dose of the combination; this dose will be used in phase II. Since both drugs are metabolized via the liver, plasma concentrations of ribo and enz will be monitored. To determine if ribo will improve the clinical activity in pts treated with enz, a randomized (1:1) phase II study using enz as the control arm vs. the combination will follow. This study will evaluate an early clinical activity readout, thus the primary phase II endpoint will be a proportion of pts with ≥ 50% PSA decline at 12 weeks. Secondary endpoints are rPFS, PSA PFS, OS and safety. If there is clinical activity noted, further randomized testing will be required to establish ribo clinical benefit in combination with enz. 5 sites are participating, managed by the Prostate Cancer Clinical Trials Consortium. Dose Levels 1 and 2 have been completed without any DLTs in the phase I portion of the study. A review of Dose Level 3 is planned for October 2017. Clinical trial information: NCT02555189.

Aurora-A overexpression is linked to development of aggressive teratomas derived from human iPS cells.

The discovery of human induced pluripotent stem cells (hiPSCs) is a promising advancement in the field of regenerative and personalized medicine. Expression of SOX2, KLF4, OCT4 and MYC transcription factors induces the nuclear reprogramming of somatic cells into hiPSCs that share striking similarities with human embryonic stem cells (hESCs). However, several studies have demonstrated that hESCs and hiPSCs could lead to teratoma formation in vivo, thus limiting their current clinical applications. Aberrant cell cycle regulation of hESCs is linked to centrosome amplification, which may account, for their enhanced chromosomal instability (CIN), and thus increase their tumorigenicity. Significantly, the tumor suppressor p53 plays a key role as a ‘guardian of reprogramming’, safeguarding genomic integrity during hiPSC reprogramming. Nevertheless, the molecular mechanisms leading to development of CIN during reprogramming and increased tumorigenic potential of hiPSCs remains to be fully elucidated. In the present study, we analyzed CIN in hiPSCs derived from keratinocytes and established that chromosomal and mitotic aberrations were linked to centrosome amplification, Aurora-A overexpression, abrogation of p53-mediated G1/S cell cycle checkpoint and loss of Rb tumor-suppressor function. When hiPSCs were transplanted into the kidney capsules of immunocompromised mice, they developed high-grade teratomas characterized by the presence of cells that exhibited non-uniform shapes and sizes, high nuclear pleomorphism and centrosome amplification. Significantly, ex vivo cells derived from teratomas exhibited high self-renewal capacity that was linked to Aurora-A kinase activity and gave rise to lung metastasis when injected into the tail vein of immunocompromised mice. Collectively, these findings demonstrated a high risk for malignancy of hiPSCs that exhibit Aurora-A overexpression, loss of Rb function, centrosome amplification and CIN. Based on these findings, we proposed that Aurora-A-targeted therapy could represent a promising prophylactic therapeutic strategy to decrease the likelihood of CIN and development of aggressive teratomas derived from hiPSCs.

Identification of Head and Neck Cancer Subtypes Based on Human Papillomavirus Presence and E2F-Regulated Gene Expression.

Human papillomavirus (HPV) is present in a subset of head and neck squamous cell carcinomas (HNSCCs). The cell cycle regulatory Rb-E2F pathway is a major target of HPV and is perturbed by these viruses in cell culture and animal models, as well as in human tumors. In this study, we examined differences in the Rb-E2F pathway displayed by HPV-positive (HPV+) and HPV-negative (HPV-) HNSCC tumors. We created a computational approach that effectively categorizes gene expression as unchanged, downregulated, or upregulated by comparing the gene's mRNA levels in the tumor to the corresponding mRNA levels across normal tissue samples. Our findings suggest that there are three major HNSCC subtypes, defined by differences in the presence of HPV and in E2F-regulated gene expression. Most HPV+ HNSCC tumors show upregulation of E2F-regulated genes, which is consistent with inactivation of Rb by the virus-encoded E7 protein. In contrast, many HPV- HNSCCs show little or no change in the Rb-E2F pathway. However, we also identified a set of tumors that show alterations in the Rb-E2F pathway in the absence of HPV. Thus, one class of HPV- HNSCCs arise without significant alterations of the Rb-E2F pathway, while a second class of tumors appear to deregulate this pathway independently of the presence of HPV. IMPORTANCE Cancer is a complex disease that can be caused by a multitude of factors. HNSCC is complicated because some of these cancers are clearly associated with HPV, while others have no viral involvement. Determining the pathways that are commonly altered in both types of HNSCC, as well as those that are unique to viral and nonviral tumors, is important for a basic understanding of how these cancers arise and progress and critical to the development of targeted therapies. In this work, we show that all HPV-associated tumors have increased expression of E2F target genes, indicating that the tumor suppressor function of Rb is blocked. Importantly, Rb is also inhibited in a subset of nonviral tumors, suggesting that mutations present in these cancers mimic the action of the HPV E6 and E7 oncogenes.

Determination of thermodynamic function of the Rb–Te(IV)–O system using DSC

The standard molar heat capacity measurements on compounds Rb2TeO3 (s), Rb2Te2O5 (s) and Rb2Te4O9 (s) in the Rb–Te(IV)–O system were taken using differential scanning calorimeter in the temperature range 325–675 K. The compounds were synthesized by solid-state route and characterized using X-ray diffraction technique. The molar heat capacity values were least squares analyzed, and the dependence of the molar heat capacity with temperature can be given as, $$ \begin{aligned} C^{\text{o}}_{\text{p,m}} \left( {{\text{Rb}}_{2} {\text{TeO}}_{3} , \, s,T} \right) \, /{\text{J K}}^{ - 1} {\text{mol}}^{ - 1} & = 156.00 + 0.02 \, \left( {T/{\text{ K}}} \right){-}4.12 \times 10^{6} \left( {T \, /{\text{K}}} \right)^{ - 2} \\ C^{\text{o}}_{\text{p,m}} \left( {{\text{Rb}}_{2} {\text{Te}}_{2} {\text{O}}_{5} , \, s,T} \right) \, /{\text{J K}}^{ - 1} {\text{mol}}^{ - 1} &= 216.50 + 0.03 \, \left( {T/{\text{ K}}} \right){-}2.52 \times 10^{6} \left( {T \, /{\text{K}}} \right)^{ - 2} \\ C^{\text{o}}_{\text{p,m}} \left( {{\text{Rb}}_{2} {\text{Te}}_{4} {\text{O}}_{9} , \, s,T} \right) \, /{\text{J K}}^{ - 1} {\text{mol}}^{ - 1} &= 351.80 + 0.04 \, \left( {T/{\text{ K}}} \right){-}3.52 \times 10^{6} \left( {T \, /{\text{K}}} \right)^{ - 2} \\ \end{aligned} $$ From these data, thermodynamic functions such as enthalpy increment (H T o − H 298.15 o ), entropy (S T o ) and Gibbs energy functions (fef) were evaluated.

Highlights of This Issue

The EWS/FLI (EF) fusion protein is well known as an aberrant transcription factor that drives oncogenesis in Ewing sarcoma by inducing widespread transcriptional reprogramming. In this issue, Tanner and colleagues explore the previously unstudied role of EF in driving metabolic misregulation. Silencing of EF resulted in significant changes in the global metabolic profile and glucose utilization. Additionally, it was demonstrated that EF is required for high expression of de novo serine synthesis and 1-carbon metabolism pathways, which correlated with worse patient survival. These findings expand the understanding of Ewing sarcoma biology and offer potential new avenues of therapeutic development.Incurable castration-resistant prostate cancer (CRPC) is often accompanied by unrestrained androgen receptor (AR) signaling driven by (currently undruggable) constitutively active, C-terminally truncated AR variants (e.g., AR-V7); which retain the potent transactivating N-terminal domain of AR. Transcriptional activities of AR and variants are enhanced by select coactivators (e.g., Vav3) with elevated expression in CRPC. Magani and colleagues demonstrate that Vav3 (via its DH domain) interacts with the N-termini of AR and AR-V7. Vav3 DH domain overexpression disrupts coactivator interactions with and transcriptional activities of AR-V7 and AR, decreasing CRPC aggressiveness. This study identifies AR N-terminal coactivator interactions as exploitable vulnerabilities in CRPC.Spontaneous nuclear envelope (NE) rupture has been observed in several cancer cell lines but does not occur in untransformed human cells. NE rupture can lead to mixing of nuclear and cytoplasmic factors and may compromise genome integrity. Yang and colleagues have identified the tumor suppressor proteins Rb and p53 as suppressors of NE rupture. Depletion of either Rb or p53 increased the frequency of NE rupture in primary cells. This finding reveals novel protective functions of Rb and p53 and provides a mechanistic explanation for the occurrence of NE rupture in cancer cells.Glioblastoma (GBM) is an invasive and malignant brain cancer. Heparan sulfate proteoglycans (HSPG), present on cell surfaces and in the extracellular matrix (ECM), regulate cell signaling and cell-microenvironment interactions. Heparan sulfate (HS) expression and enzymes that modulate their function are altered in GBM, but the actual HS content and structure are unknown. Using liquid chromatography-mass spectrometry (LC/MS), Tran and colleagues define the patterns of HS structure and their modification in patient-derived tumorspheres. Using this approach, a novel role is uncovered for the HS-modifying enzyme heparanase in GBM invasion. These results suggest that HS-interacting factors are potential therapeutic targets in GBM.

HPV16 E7 Genetic Conservation Is Critical to Carcinogenesis

Although most cervical human papillomavirus type16 (HPV16) infections become undetectable within 1-2 years, persistent HPV16 causes half of all cervical cancers. We used a novel HPV whole-genome sequencing technique to evaluate an exceptionally large collection of 5,570 HPV16-infectedcase-control samples to determine whether viral genetic variation influences risk of cervical precancer and cancer. We observed thousands of unique HPV16 genomes; very few women shared the identical HPV16 sequence, which should stimulate a careful re-evaluation of the clinical implications of HPV mutation rates, transmission, clearance, and persistence. In case-control analyses, HPV16 in the controls had significantly more amino acid changing variants throughout the genome. Strikingly, E7 was devoid of variants in precancers/cancers compared to higher levels in the controls; we confirmed this in cancers from around the world. Strict conservation of the 98 amino acids of E7, which disrupts Rb function, is critical for HPV16 carcinogenesis, presenting a highly specific target for etiologic and therapeutic research.

Misidentified Human Gene Functions with Mouse Models: The Case of the Retinoblastoma Gene Family in Senescence

Although mice models rank among the most widely used tools for understanding human genetics, biology, and diseases, differences between orthologous genes among species as close as mammals are possible, particularly in orthologous gene pairs in which one or more paralogous (i.e., duplicated) genes appear in the genomes of the species. Duplicated genes can possess overlapping functions and compensate for each other. The retinoblastoma gene family demonstrates typical composite functionality in its three member genes (i.e., RB1, RB2/P130, and P107), all of which participate in controlling the cell cycle and associated phenomena, including proliferation, quiescence, apoptosis, senescence, and cell differentiation. We analyzed the role of the retinoblastoma gene family in regulating senescence in mice and humans. Silencing experiments with each member of the gene family in mesenchymal stromal cells (MSCs) and fibroblasts from mouse and human tissues demonstrated that RB1 may be indispensable for senescence in mouse cells, but not in human ones, as an example of species specificity. Furthermore, although RB2/P130 seems to be implicated in maintaining human cell senescence, the function of RB1 within any given species might differ by cell type, as an example of cell specificity. For instance, silencing RB1 in mouse fibroblasts induced a reduced senescence not observed in mouse MSCs. Our findings could be useful as a general paradigm of cautions to take when inferring the role of human genes analyzed in animal studies and when examining the role of the retinoblastoma gene family in detail.

Ginsenoside Rb1 inhibits hypoxia-induced epithelial-mesenchymal transition in ovarian cancer cells by regulating microRNA-25.

Metastasis frequently occurs in advanced ovarian cancer, which not only leads to substantial mortality but also becomes a major challenge to effective treatment. Epithelial-mesenchymal transition (EMT) is a key mechanism facilitating cancer metastasis. Targeting the EMT process with more efficacious and less toxic agents to prevent metastasis is of significant therapeutic value for ovarian cancer treatment. The anti-EMT function and mechanism of ginsenoside Rb1, a monomer composition extracted from the traditional Chinese herb Panax ginseng or P. notoginseng, was investigated in the present study. Western blotting demonstrated that treatment with ginsenoside Rb1 antagonized hypoxia-induced E-cadherin downregulation and vimentin upregulation in SKOV3 and 3AO human ovarian cancer cells. Wound healing assays and in vitro migration assays indicated that ginsenoside Rb1 weakened hypoxia-enhanced cell migration ability. Furthermore, it was demonstrated that microRNA (miR)-25 is upregulated by hypoxia in ovarian cancer cells, which was attenuated by ginsenoside Rb1 treatment. Additionally, forced expression of miR-25 in ovarian cancer cells was identified to not only trigger EMT, but also block the suppressive effects of ginsenoside Rb1 on hypoxia-induced EMT by negatively targeting the E-cadherin transactivator, EP300. In conclusion, ginsenoside Rb1 may reverse hypoxia-induced EMT by abrogating the suppression of miR-25 on EP300 and E-cadherin, which suggests that ginsenoside Rb1 may be a potential therapeutic candidate for the treatment of ovarian cancer.

Abstract 4165: SOX2 promotes lineage plasticity and antiandrogen resistance in TP53 and RB1 deficient prostate cancer

Abstract Some cancers evade targeted therapies through a mechanism known as lineage plasticity, whereby tumor cells acquire phenotypic characteristics of a cell lineage whose survival no longer depends on the drug target. Here we show, using in vitro and in vivo prostate cancer models, that these tumors can develop resistance to the antiandrogen drug enzalutamide by a phenotypic shift from androgen receptor (AR) dependent luminal epithelial cells to AR independent basal-like cells. This lineage plasticity is enabled by loss of TP53 and RB1 function, is mediated by increased expression of the reprogramming transcription factor SOX2 and can be reversed by restoring TP53 and RB1 function or by inhibiting SOX2 expression. Thus, mutations in tumor suppressor genes can create a state of increased cellular plasticity that, when challenged with antiandrogen therapy, promotes resistance through lineage switching. Citation Format: Ping Mu, Zeda Zhang, Matteo Benelli, Wouter Karthaus, Elizebeth Hoover, Chi-Chao Chen, John Wongvipat, Sheng-Yu Ku, Dong Gao, Zhen Cao, Neel Shah, Elizabeth Adams, Wassim Abida, Philip Watson, Davide Prandi, Chun-Hao Huang, Elisa de Stanchina, Scott Lowe, Leigh Ellis, Himisha Beltran, Mark Rubin, David Goodrich, Francesca Demichelis, Charles L. Sawyers. SOX2 promotes lineage plasticity and antiandrogen resistance in TP53 and RB1 deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4165. doi:10.1158/1538-7445.AM2017-4165

Abstract 2344: Beyond RB1: the pocket protein p130 mediates the effects of CDK4/6 inhibition in RB1-deficient urothelial carcinoma

Abstract Introduction: Urothelial carcinoma is characterized by a high incidence of molecular alterations in the CDKN2A-RB-E2F axis. Loss of the cyclin-dependent kinase inhibitor p16 encoded by CDKN2A or amplification of CDK4/6 is associated with sensitivity to CDK4/6 inhibitors in various cancers. Functional RB1 is believed to be canonically required for mediating the effects of CDK4/6 inhibition in this setting. However, muscle-invasive urothelial carcinomas harbor RB mutations and copy-number losses in 21% of patients. To extend the activity spectrum of CDK4/6 inhibitors to these RB1-deficient cancers, we investigated the role of pocket protein p130 encoded by the Retinoblastoma-Like 2 (RBL2) gene, in mediating the downstream effects of CDK4/6 inhibition. Methods: CRISPR-Cas9 was used for CDKN2A knockout in the 5637-urothelial cancer cell line with an RB-mutant background and in TCCSUP cell line with wild-type RB. CDKN2A- cells were subsequently treated with CDK4/6 inhibitor palbociclib. Levels of RB1, phospho-RB1 (at serine 807/ serine 811), p130 and phospho-p130 (specifically, the CDK4/6-dependent serine-672 phosphorylation) proteins were quantified using SDS-PAGE. Cell-cycle phase analysis was performed using flow cytometric measurement of BrdU and PI staining. Results: CRISPR-induced disruption of CDKN2A was validated using genomic PCR and targeted Miseq sequencing. p16 protein knockdown was validated using SDS-PAGE. CRISPR-induced CDKN2A knockdown resulted in increased cell proliferation and faster G1/S transition even in the absence of functional RB in the 5637-urothelial cancer cell line. A significant increase in the levels of phospho-p130 was observed after p16 knockdown without changes in the levels of unphosphorylated p130. TCCSUP cell line with functional RB demonstrated a similar increase in proliferation, faster G1/S transition and an increase in phospho-p130. Palbociclib was effective in reversing these changes in all the tested cell lines. Conclusions: the pocket protein p130 plays an important role in mediating downstream effects of CDK4/6 inhibition in urothelial cancer cell lines with both wild-type and more importantly, deficient RB. CDK4/6-dependent phosphorylation of p130 can effectively compensate for the functional loss of RB activating E2F family of transcription factors that control G1/S checkpoint. These findings potentially explain the lack of correlation between RB and response to CDK4/6 inhibitors in clinical trials. These results provide a mechanistic rationale for extending clinical trials of CDK4/6 inhibitors to patients with RB1-deficient tumors. Citation Format: Bishoy M. Faltas, Ethan Shelkey, Rebecca Meyer, Nathan Young, Mark A. Rubin. Beyond RB1: the pocket protein p130 mediates the effects of CDK4/6 inhibition in RB1-deficient urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2344. doi:10.1158/1538-7445.AM2017-2344

Abstract 5861: Enhanced radiosenistivity of EGFR-TKI sensitive and resistant NSCLC cells by abemaciclib is mediated by altered DNA repair and metabolic pathways

Abstract Dysregulation of the p16(INK4a)-CyclinD-CDK4/6-Rb pathways in patients with NSCLC (Non-Small Cell Lung Cancer) is a rational therapeutic target. The current study investigated the radiosensitizing potential of a novel CDK4/6 inhibitor, LY2835219 (LY, Abemaciclib) in NSCLC cell lines with varied genomic context to identify genomic and metabolic biomarkers that are predictive of a response over conventional EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy. NSCLC cell lines were exposed to LY (0-10uM) for 24 hr immediately after 0-10 Gy radiation. Cell survival was assessed by clonogenic assay and cell cycle distribution was quantified by flow cytometry. Dose modifying factors (DMF) were calculated at 10% survival from radiation survival curves. Altered DNA repair pathways and metabolic profiling of cells post LY treatment was assessed by immunoblot and LC/MS mass spectrometry analysis. LY treatment enhanced radiosensitivity of EGFR-TKI sensitive (HCC827, PC9) and EGFR-TKI resistant (H820 and H1975) cell lines with DMF of 1.3 (±0.06), 1.4 (±0.30), 1.5 (±0.51) and 1.3 (±0.02), respectively. Values in the parenthesis indicates standard deviation. Wild type EGFR expressing cells (A549 and H460) also showed enhanced radiosensitivity by LY with DMF of 1.6 (±0.09) and 1.75(±0.15), respectively. Interestingly, no radiation enhancement by LY was observed for cells deficient in functional PTEN (H1650), Rb (H82) and p53 (H460 DNp53 and H1299) protein. Radiosensitization was also observed for cells made resistant to third generation EGFR-TKI, AZD9291. Flow cytometry analysis of majority of cell types exposed to LY exhibited 55% to 94% G1 arrest (depending on cell type). Mechanistically, the combinatorial treatment in radiosensitive cells showed elevated phosphorylated-γH2Ax. Combination treatment also reduced expression of ATR, ATM, DNA-PK, Rad51 and Chk-2 suggestive of reduced DNA repair compared to radiation alone. LY treatment brought major changes in the glycolysis/TCA/ total amino acids. LY increased significantly Acetyl-CoA, fumarate and malate, indicating enhanced oxidative phosphorylation. LY significantly elevated Uric acid levels suggestive of oxidative stress and elevated nucleotide degradation. Finally, administration of 100mg/kg LY2835219 for five days in combination with fractionated dose of radiation (3 Gy) significantly delayed tumor regrowth in H460 xenograft (p&amp;lt; 0.014). Collectively, our pre-clinical data indicates altered Rb, p53 and PTEN status are distinct predictive biomarkers of response for LY mediated radiosensitivity and provides an alternative therapeutic option in overcoming EGFR-TKI resistance in NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Sarwat Naz, William DeGraff, Anastasia Sowers, Rajani Choudhuri, Maria Wissler, Janet Gamson, John Cook, James B. Mitchell. Enhanced radiosenistivity of EGFR-TKI sensitive and resistant NSCLC cells by abemaciclib is mediated by altered DNA repair and metabolic pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5861. doi:10.1158/1538-7445.AM2017-5861

Effects of concomitant inactivation of p53 and pRb on response to doxorubicin treatment in breast cancer cell lines

Loss of TP53 and RB1 function have both been linked to poor response to DNA damaging drugs in breast cancer patients. We inactivated TP53 and/or RB1 by siRNA mediated knockdown in breast cancer cell lines varying with respect to ER/PgR and Her-2 status as well as TP53 and RB1 mutation status (MCF-7, T47D, HTB-122 and CRL2324) and determined effects on cell cycle arrest, apoptosis and senescence with or without concomitant treatment with doxorubicin. In T47D cells, we found the cell cycle phase distribution to be altered when inactivating TP53 (P=0.0003) or TP53 and RB1 concomitantly (P≤0.001). No similar changes were observed in MCF-7, HTB-122 or CRL2324 cells. While no significant change was observed for the CRL2324 cells upon doxorubicin treatment, MCF-7, T47D as well as HTB-122 cells responded to knockdown of TP53 and RB1 in concert, with a decrease in the fraction of cells in G1/G0-phase (P=0.042, 0.021 and 0.027, respectively). Inactivation of TP53 and/or RB1 caused no change in induction of apoptosis. Upon doxorubicin treatment, inactivation of TP53 or RB1 separately caused no induction of apoptosis in MCF-7 and HTB-122 cells; however, concomitant inactivation leads to a slightly reduced activation of apoptosis. Interestingly, upon doxorubicin treatment, concomitant inactivation of TP53 and RB1 caused a decrease in senescence in MCF-7 cells (P=0.027). Comparing the effects of concomitant knockdown on apoptosis and senescence, we observed a strong interaction (P=0.001). We found concomitant inactivation of TP53 and RB1 to affect various routes of response to doxorubicin treatment in breast cancer cells.

Phase II trial of ribociclib and everolimus in p16 low anaplastic thyroid cancer (ATC).

TPS6098 Background: ATC is a rare thyroid cancer with a highly aggressive clinical course. Median OS is 3-4 months and 90% of patients die within 1 year of the diagnosis. There are no effective treatment options in metastatic disease. Targeted therapies to ALK and BRAF are occasionally associated with dramatic responses. Retinoblastoma (Rb) inhibits cell cycle progression and is inactivated by CDK 4/6, which is the target for ribociclib. Nearly all differentiated thyroid cancers are Rb negative, conversely nearly 100% of ATC expresses intact Rb which may be crucial for rapid cell cycle progression. Ribociclib is a CDK 4/6 inhibitors that slows cell cycle progression and DNA replication in tumors with functional Rb. The p16 protein similarly inhibits CDK4/6; if p16 levels are high Rb is phosphorylated and thus inactive. p16 is low in ATC, and from our comprehensive genomic analysis 30% of ATC lacks the p16 gene CDKN2a. Most ATC demonstrates PI3K/Akt/mTOR abnormalities, this pathway represents an attractive target in ATC. The mTOR inhibitor everolimus has shown promising efficacy in ATC cell lines, especially in those with TSC2 mutation whose wild type negatively regulates mTOR. Methods: The combination of everolimus and ribociclib targets mutations/abnormalities that are frequently seen in ATC, and is tolerable based on Phase I/II trial results in the latest arm of the biomarker/oncogene driven ATC Master Protocol . This open-label trial treats metastatic Rb+ ATC patients with p16-/ CDKN2a-. Treatment is ribociclib 400 mg + everolimus 5 mg QD. The primary endpoint is the overall response rate; secondary endpoints are PFS, OS, safety and toxicity. Exploratory objectives include if tissue biomarkers or mutations noted on Next Generation Sequencing correlate for enhanced/impaired response to combination therapy. Simon's two-stage design will be used with the null hypothesis that the true response rate is 5%, this will be tested against a one-sided alternative. In the first stage, 9 patients will be accrued. If no response in these 9 patients, the study will be stopped. Otherwise, 21 additional patients will be accrued for a total of 30. Clinical trial information: NCT02289144.

Circulating tumor cells (CTCs) N-terminal androgen receptor expression to identify patients (pts) with castrate resistant prostate cancer (CRPC) who are more sensitive to chemotherapy.

5034 Background: Loss of the retinoblastoma tumor suppressor (RB) function was identified as a major means to develop CRPC; the expression of the androgen receptor (AR) is under stringent RB control; and tumors devoid of RB function are hypersensitive to treatment with chemotherapy. Exploratory analysis evaluated baseline N-terminal AR expression in CTCs in men with chemotherapy-naïve CPRC and correlated to changes in PSA, leading us to inquire if this biomarker may identify pts sensitive to chemotherapy. Methods: In a multicenter phase II randomized trial of approved doses of abiraterone acetate/prednisone (AA-Arm 1) or combination AA and standard doses of cabazitaxel (AA/CBZ-Arm 2). Patients on AA received CBZ upon progression. Baseline CTCs were obtained on all pts and expression of N-terminal AR expression was performed by Epic Sciences. Positive AR N-terminal expression (AR+) was based on the presence of at least 1 CTC or CK- cell with AR N-terminal signal expression above the 3.0 positivity threshold. Serial PSAs were determined at baseline and every 3 weeks with routine labs and imaging every 12 weeks. Results: To date, 42 of 80 pts have been enrolled: 22 pts to AA, and 20 pts to AA/CBZ. Both regimens were well tolerated with 8/42 (19%) pts experiencing treatment-related grade 3 or 4 toxicities. Blood from 35 patients underwent CTC analysis. Seventy-seven percent of pts (27/35) had detectable CTCs; 11 of 35 pts (31%) had AR overexpression. Of the pts with AR+ CTCs, 1/5 pts treated with AA, and 5/6 pts treated with AA/CBZ had a PSA decline &gt; 50% from baseline. Conclusions: Real-time CTC analysis of N-terminal AR expression was feasible and data suggests that this may identify a cohort of pts who may benefit from the combination of CBZ with AA. Further studies are ongoing to evaluate whether cellular heterogeneity and RB expression in CTCs play a role in identifying pts who would benefit from chemotherapy. The trial is coordinated by the Prostate Cancer Clinical Trials Consortium, LLC and funded by Sanofi US Services Inc. and Prostate Cancer Foundation. Clinical trial information: NCT02218606.

SOX2 and Rb1 in esophageal small-cell carcinoma: their possible involvement in pathogenesis

Clinicopathological features and pathogenesis of esophageal small-cell carcinoma remain unclear. We hypothesized common cellular origin and pathogenesis in small-cell carcinoma of esophagus and lung associated with SOX2 overexpression and loss of Rb1. Expression of squamous-basal markers (CK5/6 and p40), glandular markers (CK18 and CEA), SOX2, and Rb1 were evaluated in 15 esophageal small-cell carcinomas, 46 poorly differentiated squamous cell carcinomas, and 88 small-cell lung carcinoma, as well as 16 embryonic esophagus. Esophageal small-cell carcinoma expressed higher levels of glandular markers and lower levels of squamous-basal markers than poorly differentiated squamous cell carcinoma. No significant differences were observed in immunohistochemistry profiles between small-cell carcinoma of the esophagus and the lung. SOX2 expression was high in esophageal small-cell carcinoma (70%±33% of nuclei), small-cell lung carcinoma (70%±26%), and the embryonic esophagus (75%±4%), and it was significantly lower in poorly differentiated squamous cell carcinoma (29%±28%). Rb1 expression was significantly lower in esophageal small-cell carcinoma (0.3%±1%), small-cell lung carcinoma (2%±6%), and the embryonic esophagus (7%±5%), and it was significantly higher in poorly differentiated squamous cell carcinoma (51%±24%). The immunohistochemistry profiles of small-cell carcinoma of the esophagus and the lung are highly similar. The loss of Rb1 function is a key contributor to the pathogenesis of both neoplasms. In addition, SOX2 overexpression observed in esophageal and lung small-cell carcinoma as well as in the embryonic esophagus indicated that esophageal small-cell carcinoma may arise from embryonic-like stem cells in the esophageal epithelium. The two distinct differentiation patterns (neuroendocrine and glandular) of esophageal small-cell carcinoma further support the fact that SOX2 has a pivotal role in the differentiation of pluripotent stem cells into esophageal small-cell carcinoma cells.

Abstract A24: BRM polymorphisms, part of a novel epigenetic mechanism, are predictive of cancer risk and clinic outcome in multiple cancer types

Abstract BRM polymorphisms lie with the promoter region of the anticancer gene and SWI/SNF catalytic subunit, Brahma (SMARCA2). These polymorphisms statistically correlate with loss of BRM expression in both cell lines and primary lung tumors and function as part of an epigenetic mechanism which underlies BRM reversible silencing. Specifically, these polymorphisms function as the binding site of at least two transcription factors (MEF2D and GATA3) and two HDACs (HDAC3 and HDAC9). These proteins form a complex which drives the reversible silencing of the BRM protein. As BRM can serve as an anticancer protein, in part because BRM and SWI/SNF is required for the normal function of TP53 and RB as well as multiple DNA repair mechanisms, the silencing of BRM potentiates cancer development in mice. As such, BRM polymorphisms are predictive of cancer risk for lung cancer (n=600), breast cancer (N=300), head/neck cancer (n=400), colon cancer (N=250) and lymphoma (N=300) with an odds ratio ranging from 1.8 to 2.3. As these polymorphisms occur more frequently in African Americans, the odds ratio (cancer risk) in African Americans for lung cancer (n=250) is higher (3.5-4.5) as compared to that observed in Caucasians (2-2.3) (N=600). Similarly, BRM polymorphisms have a higher predictive value in HPV positive head/neck cancer with an odds ratio of 3.2 compared with an odds ratio of 2.0 in HPV positive head/neck cancer. This is in part due to the fact that BRM along with the HPV E2 protein regulates the expression of the transforming HPV proteins E6/E7. Unlike other polymorphisms which impart cancer risk and are fixed (cannot be changed), the fact that BRM silencing is reversible by compounds such as Flavonoids and certain NSAIDs, the cancer risk imparted by these polymorphisms can, in theory, be reversed or nullified by changes in diet, thereby making these polymorphisms unique in this respect. As BRM polymorphisms and BRM expression is also tied to differentiation and cell adhesion, these polymorphisms are also predictive of a worse clinical outcome with a hazard ratio ranging from 3 to 10 in pancreatic, head/neck, lung, colon and esophageal cancers. Thus, BRM polymorphisms represent a novel factor which is predictive of cancer risk and clinical outcome in multiple cancer types. Furthermore, these polymorphisms can potentially explain the observed health disparities (higher lung cancer occurrence despite lower rate of tobacco usage) which occur in African Americans. Citation Format: Suhdir Rai, Jennifer Wang, Kit Man Wong, Xiaoping Qiu, Geoff Liu, David Reisman. BRM polymorphisms, part of a novel epigenetic mechanism, are predictive of cancer risk and clinic outcome in multiple cancer types. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A24.