Rb Pathway Research Articles

RB-independent activity of Cdk4/6 in bladder cancer.

e16011Background: MIBC is treated, in most cases, by cystectomy and platinum-based chemotherapy. Nonetheless, the metastatic spreading occurs very often with fatal consequences. Various targeted therapies are being clinically and preclinically tested for BC management. MIBC have been associated with loss of cell cycle control. Defects in the RB pathway are a major mechanisms of cell cycle deregulation in MIBC. RB1 mutations were reported in 13% of TCGA samples, although significant mutations also were noted in CDKN1A, CDKN2A, and CCND3. The CDK 4/6 complex combines with cyclin D1 to inhibit RB activity. Therapeutic targeting of this complex improves outcome in ER positive advanced breast cancer. Methods: Palbociclib (PD-0332991) is a cdk4/6 inhibitor currently tested for the treatment of other malignancies characterized by the presence of a functional RB1 gene. A series of BC cell lines of known genomic characteristics and differing in their RB1 status, were tested for their sensitivity to Palbociclib. Re...

Relationship between cancer mutations and parameter sensitivity in Rb pathway

It has long been known that formation of all sorts of tumors is largely owing to the genomic variations. Oncogenic mutations are often found focused on one or more important pathways which indicate that it is meaningful to investigate oncogenic mutations and oncogenic mechanisms from the point of view of biological network. Recently, we found that in apoptosis pathway of mammalian cell, mutations that cause large variations on the bifurcation point are more probably oncogenic mutations. Here, we used the Rb-E2F pathway in mammalian cell in response to growth factor as another example to verify this correlation. To conduct this study, nonlinear dynamics equations that describe the behavior of the Rb-E2F pathway was first constructed. Then we identified sensitive parameters which have a great influence on the system’s bifurcation point. And we found that the sensitive parameters are highly related to high-frequency oncogenic mutations after comparing the results of parameter sensitivity analysis with profile of known cancer mutations. Moreover, the position of bifurcation point rather than concentration of a certain protein is a better measurement to determine biological network's function. Our results further confirm that nonlinear dynamics analysis of biological networks is an important way to understand oncogenesis. And the analysis method can become a powerful tool to understand and analyze the function of biological network.

Induction of Therapeutic Senescence in Vemurafenib-Resistant Melanoma by Extended Inhibition of CDK4/6.

Dysregulation of the p16-cyclin D1-CDK4/6-Rb pathway occurs frequently in melanoma; however, the therapeutic efficacy of CDK4/6 inhibition remains to be critically evaluated. We demonstrate that CDK4/6 inhibition inhibits melanoma progression through induction of senescence. Palbociclib, a specific CDK4/6 inhibitor, rapidly induces cell cycle arrest within 24 hours and continued exposure for 8 days or longer induces senescence. The induction of senescence correlates with inhibition of mTOR and more specifically mTORC1 signaling. Vemurafenib, a specific BRAF(V600E) inhibitor, has significant clinical efficacy in BRAF(V600E)-positive melanomas, but its impact is hampered by a rapid acquisition of resistance. Strikingly, we found that vemurafenib-resistant tumors remain sensitive to palbociclib, suggesting that initial treatment with vemurafenib followed by palbociclib with or without mTOR inhibitors might provide an avenue to overcome recurrence of vemurafenib-resistant metastatic disease. Taken together, these results support palbociclib as a promising therapeutic for treatment of melanoma. Cancer Res; 76(10); 2990-3002. ©2016 AACR.

Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia.

The DDX41 gene, encoding a DEAD-box type ATP-dependent RNA helicase, is rarely but reproducibly mutated in myeloid diseases. The acquired mutation in DDX41 is highly concentrated at c.G1574A (p.R525H) in the conserved motif VI located at the C-terminus of the helicase core domain where ATP interacts and is hydrolyzed. Therefore, it is likely that the p.R525H mutation perturbs ATPase activity in a dominant-negative manner. In this study, we screened for the DDX41 mutation of CD34-positive tumor cells based on mRNA sequencing and identified the p.R525H mutation in three cases among 23 patients. Intriguingly, these patients commonly exhibited acute myeloid leukemia (AML) with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits the growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia cell lines suggest a role for DDX41 in preribosomal RNA processing, in which the expression of the p.R525H mutant causes a certain ribosomopathy phenotype in hematopoietic cells by suppressing MDM2-mediated RB degradation, thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells. Age-dependent epigenetic alterations or other somatic changes might collaborate with the mutation to causeAML.

Serial monoclonal antibody administration demonstrates therapeutic effects via induction of apoptosis in HPV related cervical cancer

Abstract Introduction All cervical cancer is caused by the persistent expression of human papillomavirus (HPV) E6 and E7 oncoproteins that interfere with p53 and Rb pathways, respectively. Virally specific treatments form an excellent basis for highly specific, low toxicity therapies. Methods Serial doses of anti-HPV 16/18 E6 C1P5 (Abcam) were compared against Cisplatin in the treatment of human cervical cancer derived tumors (CaSki). C1P5 and Cisplatin were administered on days 1, 4, and 7 after CaSki tumor size reached 3–5mm on the flank of athymic nude mice. Measurements were performed every 3 days during the 40 day observation period. After which the tumors were procured, paraffin embedded, and stained with H&E. SiHa cells which compare to HPV copies in patient explants (previously published) were used in mechanistic studies. Dual staining flow cytometry with PI and Annexin was performed to evaluate cell death and MTT assay was used to confirm findings. Results Tumor growth was inhibited in all treatment groups compared to untreated controls (p <0.05). No statistically significant differences between C1P5 treatment groups were noted when compared to cisplatin. However, on H&E we noted increased necrosis proportional to the number of doses: 1 dose 30%, 2 doses 60%, and 3 doses 85%. Dual-staining of Annexin and PI on flow cytometry demonstrated increased apoptosis as a result of C1P5 administration suggesting this as the underlying mechanism of increased necrosis. These results were corroborated by MTT assay (p<0.05). Conclusion Virally specific therapy is promising for the treatment of women with cervical cancer. To extend the paucity of options, further mechanistic understanding must be developed to derive maximal therapeutic benefit.

The Oncolytic Adenovirus VCN-01 as Therapeutic Approach Against Pediatric Osteosarcoma.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Despite aggressive chemotherapy, more than 30% of patients do not respond and develop bone or lung metastasis. Oncolytic adenoviruses engineered to specifically destroy cancer cells are a feasible option for osteosarcoma treatment. VCN-01 is a replication-competent adenovirus specifically engineered to replicate in tumors with a defective RB pathway, presents an enhanced infectivity through a modified fiber and an improved distribution through the expression of a soluble hyaluronidase. The aim of this study is to elucidate whether the use of VCN-01 would be an effective therapeutic strategy for pediatric osteosarcoma. We used osteosarcoma cell lines established from patients with metastatic disease (531MII, 678R, 588M, and 595M) and a commercial cell line (143B). MTT assays were carried out to evaluate the cytotoxicity of VCN-01. Hexon assays were used to evaluate the replication of the virus. Western blot analysis was performed to assess the expression levels of viral proteins and autophagic markers. The antitumor effect of VCN-01 was evaluated in orthotopic and metastatic osteosarcoma murine animal models. This study found that VCN-01, a new generation genetically modified oncolytic adenovirus, administered locally or systemically, had a potent antisarcoma effect in vitro and in vivo in mouse models of intratibial and lung metastatic osteosarcoma. Moreover, VCN-01 administration showed a safe toxicity profile. These results uncover VCN-01 as a promising strategy for osteosarcoma, setting the bases to propel a phase I/II trial for kids with this disease. Clin Cancer Res; 22(9); 2217-25. ©2015 AACR.

Genetic biomarkers of drug response for small-molecule therapeutics targeting the RTK/Ras/PI3K, p53 or Rb pathway in glioblastoma.

Glioblastoma is the most deadly and frequently occurring primary malignant tumor of the central nervous system. Genomic studies have shown that mutated oncogenes and tumor suppressor genes in glioblastoma mainly occur in three pathways: the RTK/Ras/PI3K signaling, the p53 and the Rb pathways. In this review, we summarize the modulatory effects of genetic aberrations in these three pathways to drugs targeting these specific pathways. We also provide an overview of the preclinical efforts made to identify genetic biomarkers of response and resistance. Knowledge of biomarkers will finally promote patient stratification in clinical trials, a prerequisite for trial design in the era of precision medicine.

Targeting CDK4/6 in patients with cancer.

The cyclin D-cyclin dependent kinase (CDK) 4/6-inhibitor of CDK4 (INK4)-retinoblastoma (Rb) pathway controls cell cycle progression by regulating the G1-S checkpoint. Dysregulation of the cyclin D-CDK4/6-INK4-Rb pathway results in increased proliferation, and is frequently observed in many types of cancer. Pathway activation can occur through a variety of mechanisms, including gene amplification or rearrangement, loss of negative regulators, epigenetic alterations, and point mutations in key pathway components. Due to the importance of CDK4/6 activity in cancer cells, CDK4/6 inhibitors have emerged as promising candidates for cancer treatment. Moreover, combination of a CDK4/6 inhibitor with other targeted therapies may help overcome acquired or de novo treatment resistance. Ongoing studies include combinations of CDK4/6 inhibitors with endocrine therapy and phosphatidylinositol 3-kinase (PI3K) pathway inhibitors for hormone receptor-positive (HR+) breast cancers, and with selective RAF and MEK inhibitors for tumors with alterations in the mitogen activated protein kinase (MAPK) pathway such as melanoma. In particular, the combination of CDK4/6 inhibitors with endocrine therapy, such as palbociclib's recent first-line approval in combination with letrozole, is expected to transform the treatment of HR+ breast cancer. Currently, three selective CDK4/6 inhibitors have been approved or are in late-stage development: palbociclib (PD-0332991), ribociclib (LEE011), and abemaciclib (LY2835219). Here we describe the current preclinical and clinical data for these novel agents and discuss combination strategies with other agents for the treatment of cancer.

Abstract OT2-01-02: Phase III study of ribociclib (LEE011) in combination with fulvestrant for the treatment of postmenopausal patients (pts) with hormone receptor-positive (HR+), HER2-negative (HER2–) advanced breast cancer (aBC) who have received no or only one line of prior endocrine treatment: MONALEESA-3

Abstract Background: Endocrine therapy (ET) is the predominant first-line treatment for HR+, HER2– aBC; however, resistance eventually occurs in a large number of cases. Dysregulation of the cyclin D–cyclin dependent kinase (CDK)4/6–inhibitor of CDK4 (INK4)–retinoblastoma (Rb) pathway has been associated with endocrine resistance. Inhibition of CDK4/6 may overcome such resistance and enhance the efficacy of existing endocrine regimens. Combination of ribociclib (a type of CDK4/6 inhibitor) with fulvestrant (a selective estrogen receptor downregulator) has demonstrated potent tumor regressions in preclinical HR+ breast cancer (BC) models. Design & Objectives: MONALEESA-3 (NCT02422615) is a randomized, double-blind, placebo-controlled study of oral ribociclib (600 mg QD on Days 1–21 of each 28-day cycle) in combination with fulvestrant (500 mg intramuscularly on Days 1 and 15 of Cycle 1 and Day 1 of each cycle thereafter) in postmenopausal pts with HR+, HER2– aBC. Pts may have newly diagnosed aBC that is treatment-naive, relapsed BC that has progressed at any time during or after (neo)adjuvant ET with no treatment for metastatic disease, relapsed BC that has progressed >12 months after adjuvant ET and then subsequently progressed after one line of ET for metastatic disease (with either an antiestrogen or an aromatase inhibitor), or newly diagnosed aBC that has progressed after one line of ET (with either an antiestrogen or an aromatase inhibitor). Pts must have measurable disease or at least one predominantly lytic bone lesion, and an Eastern Cooperative Oncology Group performance status (ECOG PS) of ≤1. Prior treatment with chemotherapy (except for [neo]adjuvant chemotherapy), fulvestrant, or any CDK4/6 inhibitor is prohibited. Randomization (2:1) to receive ribociclib plus fulvestrant (Arm A) or placebo plus fulvestrant (Arm B) is stratified by visceral disease status and previous ET. Tumor assessments will be performed every 8 weeks for the first 18 months and every 12 weeks thereafter until disease progression, death, withdrawal of consent, loss to follow-up, or patient/guardian decision. The primary endpoint is progression-free survival (PFS; local, RECIST 1.1); secondary endpoints include overall survival, PFS (central, RECIST 1.1), overall response rate, clinical benefit rate, time to response, duration of response, safety and tolerability, ECOG PS, pt-reported outcomes, and pharmacokinetics. Molecular alterations of genes associated with HR+ BC and the cyclin D–CDK4/6–INK4–Rb pathway will also be explored in tumor and blood samples. Statistical Methods: PFS between Arms A and B will be compared using a stratified log-rank test; 364 PFS events will be required to detect a hazard ratio of 0.67 with 90% power using the log-rank test and a 3-look (interim look futility only, interim look efficacy only, then final analysis) group sequential design at one-sided cumulative 2.5% level of significance. Target Accrual: Approximately 660 pts at 300 sites worldwide. Citation Format: Fasching PA, Jerusalem G, Pivot X, Martin M, De Laurentiis M, Blackwell K, Esteva FJ, Paquet-Luzy T, Tang Z, Lorenc KR, Slamon DJ. Phase III study of ribociclib (LEE011) in combination with fulvestrant for the treatment of postmenopausal patients (pts) with hormone receptor-positive (HR+), HER2-negative (HER2–) advanced breast cancer (aBC) who have received no or only one line of prior endocrine treatment: MONALEESA-3. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT2-01-02.

Abstract P3-14-01: Phase Ib/II study of ribociclib and alpelisib and letrozole in ER+, HER2– breast cancer: Safety, preliminary efficacy and molecular analysis

Abstract Background: The interaction between the PI3K/AKT/mTOR and cyclin D–CDK4/6–INK4–Rb pathways has been reported to play a critical role in ER-driven breast cancer (BC). Furthermore, in preclinical ER+ BC models, the combination of ribociclib (LEE011; LEE), alpelisib (BYL719; BYL), and letrozole (LET) has recently shown enhanced activity vs each agent alone. A Phase Ib/II, 3-arm study is investigating the combination of LEE, BYL, and LET in pts with ER+ advanced (a)BC (CLEE011X2107; NCT01872260). Here, we report on safety, preliminary efficacy, and molecular analysis focusing on Arm (A)3 (LEE + BYL + LET) of Phase Ib. Methods: Postmenopausal women with ER+, HER2– aBC received a fixed dose of 2.5 mg LET QD (continuous) with escalating doses of either oral LEE QD (3-wks-on/1-wk-off) or BYL QD (continuous), or both, in 28-day cycles. The primary objective is to determine the MTD and/or RP2D of each combination by assessing DLTs in Cycle 1 using an adaptive Bayesian Logistic Regression Model with overdose control principle. Secondary objectives include safety, PK, and preliminary efficacy assessments. Potential biomarkers predictive of response were assessed by next-generation sequencing in all patients, and immunohistochemistry in available tumor samples. Results: As of Mar 2, 2015, 41 pts received LEE + LET (A1), 21 pts received BYL + LET (A2), and 36 pts received LEE (300–500 mg) + BYL (200–250 mg) + LET (A3). Here, we present results from A3. The number of prior endocrine regimens for advanced disease was: 0 (14 pts); 1–2 (14 pts); 3–4 (7 pts); ≥5 (1 pt); 33% of pts had previously received PI3K/AKT/mTOR inhibitors for aBC. Fifteen pts discontinued treatment: 7 (19%) due to PD and 8 (22%) due to AEs. The most frequent study drug-related AEs (all grade >35%) were: nausea (all grade, 44%; G3/4, 6%), hyperglycemia (44%; 17%), neutropenia (42%; 22%), and fatigue (36%; 11%). The median duration of exposure was 8 weeks; 9 (25%) pts received treatment for ≥4 cycles. Of 27 evaluable pts, 2 (7%) pts had PR, 4 (15%) pts had unconfirmed PR, 6 (22%) pts had SD, 6 (22%) pts had non-CR non-PD (NCRNPD), and 5 (19%) pts had PD as best overall response. One pt with PR was treated for 9 cycles, had 5 lines of prior therapy including a PI3K/AKT/mTOR inhibitor, and had alterations in PIK3R1 and CDK4. The other pt with PR was treated for >9 cycles, had 3 lines of prior therapy, and had alterations in PIK3CA and CCND1. In A1, an increase from baseline to C1D15 in pAKT and pS6(235) was observed in 5 and 3 pts, respectively; importantly, there was a more consistent reduction in Ki67 in A3 vs A1 or A2. Conclusions: LET + LEE + BYL (A3) has an acceptable safety profile and demonstrates preliminary clinical activity in heavily pretreated pts with ER+, HER2– aBC. Preliminary data suggest CDK4/6 pathway activation is associated with improved response to triplet therapy. Importantly, the pathway analysis reported is supportive of a mechanistic combination effect whereby inhibition of the three pathways provides a sustained downregulation of Ki67, potentially preventing a feedback mechanism and hence delaying progression through therapy. Future randomized studies will compare LET + LEE or BYL with LET + LEE + BYL. Citation Format: Juric D, Ismail-Khan R, Campone M, García-Estévez L, Becerra C, De Boer R, Hamilton E, Mayer IA, Hui R, Lathrop KI, Pagani O, Asano S, Bhansali SG, Zhang V, Hewes B, Munster P. Phase Ib/II study of ribociclib and alpelisib and letrozole in ER+, HER2– breast cancer: Safety, preliminary efficacy and molecular analysis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-01.

The Spectrum of E2F in Liver Disease--Mediated Regulation in Biology and Cancer.

Uncoordinated cell growth is one of the fundamental concepts in carcinogenesis and occurs secondary to dysregulation of the cell cycle. The E2Fs are a large family of transcription factors and are key regulators of the cell cycle. The activation of E2Fs is intimately regulated by retinoblastoma 1 (RB1). The RB pathway has been implicated in almost every human malignancy. Recently there have been exciting developments in the E2F field using animal models to better understand the role of E2Fs in vivo. Genetic mouse models have proven essential in implicating E2Fs in hepatocellular carcinoma (HCC) and liver disease. In this review, the general structure and function of E2Fs as well as the role for E2Fs in the development of HCC and liver disease is evaluated. Specifically, what is known about E2Fs in human disease is explored in depth, and future directions are discussed.

Ras Regulates Rb via NORE1A

Mutations in the Ras oncogene are one of the most frequent events in human cancer. Although Ras regulates numerous growth-promoting pathways to drive transformation, it can paradoxically promote an irreversible cell cycle arrest known as oncogene-induced senescence. Although senescence has clearly been implicated as a major defense mechanism against tumorigenesis, the mechanisms by which Ras can promote such a senescent phenotype remain poorly defined. We have shown recently that the Ras death effector NORE1A plays a critical role in promoting Ras-induced senescence and connects Ras to the regulation of the p53 tumor suppressor. We now show that NORE1A also connects Ras to the regulation of a second major prosenescent tumor suppressor, the retinoblastoma (Rb) protein. We show that Ras induces the formation of a complex between NORE1A and the phosphatase PP1A, promoting the activation of the Rb tumor suppressor by dephosphorylation. Furthermore, suppression of Rb reduces NORE1A senescence activity. These results, together with our previous findings, suggest that NORE1A acts as a critical tumor suppressor node, linking Ras to both the p53 and the Rb pathways to drive senescence.

A novel multi-CDK inhibitor P1446A-05 restricts melanoma growth and produces synergistic effects in combination with MAPK pathway inhibitors

ABSTRACTNearly 100% of melanomas have a defect in the p16INK4A:cyclin D-CDK4/6:RB pathway, leading to abnormal cell cycle control and unregulated cellular proliferation. Here, we report that P1446A-05, a novel multi-CDK inhibitor has significant inhibitory activity against cutaneous and uveal melanoma. Mechanistic studies revealed that P1446A-05 inhibits phosphorylation targets of CDK members, and induces cell cycle arrest and apoptosis irrespective of melanoma genotype or phenotype. Additionally, we show preclinical evidence that P1446A-05 can synergize with other small molecule inhibitors previously studied in melanoma. Collectively, these data demonstrate that targeting cell cycle and transcriptional CDKs with a small molecule multi-CDK inhibitor is a viable approach for developing novel anti-melanoma therapeutics.

Abstract IA08: An epigenome perspective of human tumor evolution

Abstract Cancers develop through a process of clonal evolution in which ongoing genetic and epigenetic diversification allows for repeated cycles of sub-clonal selection and expansion (Greaves and Maley, 2012; Nowell, 1976). As a result, human tumors can display substantial intratumoral heterogeneity, including discordant genetic alterations between the initial tumor and local recurrence or distant metastases (Gerlinger et al., 2012; Okosun et al., 2014; Wu et al., 2012; Yachida et al., 2010). As mutations accumulate over time, genomic profiling of spatially or temporally separated tumor samples can be used to reconstruct the evolutionary history and underlying clonal architectures of individual tumors (Gerlinger et al., 2014). In contrast to stable genetic events, epigenetic states are reversible and may change in response to the tumor microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. In low-grade glioma, the course of tumor evolution is particularly clinically significant. Low-grade gliomas are diffuse, infiltrative tumors that recur locally and may unpredictably undergo malignant progression to a higher grade with a worse prognosis (Sanai et al., 2011). Recurrences that progress to highly malignant WHO grade IV glioblastoma (GBM) acquire genetic alterations in the RB and AKT-mTOR pathways (Johnson et al., 2014; Louis, 2006). In fact, adjuvant treatment with alkylating chemotherapeutics such as temozolomide (TMZ) can induce hypermutation that emerges in recurrent tumors (Bodell et al, 2003; Hunter et al., 2006), and we recently linked treatment-associated driver mutations in these two pathways to malignant progression of grade II glioma to GBM (Johnson et al., 2014). The treatment associated malignant progression follows selection of tumor cells with epigenetic silencing of the DNA repair protein MGMT (van Thiujl et al, 2015). The critical role that epigenetic alterations play in the development and therapeutic response of gliomas is increasingly being appreciated (Fouse and Costello, 2009). Somatic mutations in IDH1 or IDH2 may be the first genetic driver in the development of many low-grade gliomas (Johnson et al., 2014; Lai et al., 2011; Watanabe et al., 2009). Genetic mutations in IDH genes induce a pattern of early epigenetic alterations known as the glioma CpG island methylator phenotype (G-CIMP) characterized by extensive remodeling of the DNA methylome (Hill et al., 2014; Noushmehr et al., 2010; Toyota et al., 1999; Turcan et al., 2012). The inactivation of other genes mutated in low-grade gliomas, such as ATRX (Jiao et al., 2012) and SMARCA4 (Johnson et al., 2014), is known to induce specific DNA methylation changes as well (Banine et al., 2005; Gibbons et al., 2000). Although there has been extensive characterization of tumor methylomes using a single sampling per tumor, little is known about intratumoral heterogeneity at the epigenetic level or of temporal evolution of the low-grade glioma methylome and its relationship to the genome. In this presentation, I will show that phylogenic analysis of spatial and temporal patterns of either reversible DNA methylation or irreversible somatic mutations independently yield remarkably similar evolutionary histories. I will also incorporate this inter-dependent evolution into a detailed model of brain tumorigenesis that extends from the first mutation and epimutations through tumor recurrence. Citation Format: Tali Mazor, Aleksandr Pankov, Jun Song, Joseph Costello. An epigenome perspective of human tumor evolution. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr IA08.

Malignant Peripheral Nerve Sheath Tumors.

Malignant peripheral nerve sheath tumors (MPNST) are tumors derived from Schwann cells or Schwann cell precursors. Although rare overall, the incidence of MPNST has increased with improved clinical management of patients with the neurofibromatosis type 1 (NF1) tumor predisposition syndrome. Unfortunately, current treatment modalities for MPNST are limited, with no targeted therapies available and poor efficacy of conventional radiation and chemotherapeutic regimens. Many murine and zebrafish models of MPNST have been developed, which have helped to elucidate the genes and pathways that are dysregulated in MPNST tumorigenesis, including the p53, and the RB1, PI3K-Akt-mTOR, RAS-ERK and Wnt signaling pathways. Preclinical results have suggested that new therapies, including mTOR and ERK inhibitors, may synergize with conventional chemotherapy in human tumors. The discovery of new genome editing technologies, like CRISPR-cas9, and their successful application to the zebrafish model will enable rapid progress in the faithful modeling of MPNST molecular pathogenesis. The zebrafish model is especially suited for high throughput screening of new targeted therapeutics as well as drugs approved for other purposes, which may help to bring enhanced treatment modalities into human clinical trials for this devastating disease.

CDK6 mediates the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy.

MicroRNA-1 (miR-1) is approved involved in cardiac hypertrophy, but the underlying molecular mechanisms of miR-1 in cardiac hypertrophy are not well elucidated. The present study aimed to investigate the potential role of miR-1 in modulating CDKs-Rb pathway during cardiomyocyte hypertrophy. A rat model of hypertrophy was established with abdominal aortic constriction, and a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocytes (NRVCs). We demonstrated that miR-1 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes. Dual luciferase reporter assays revealed that miR-1 interacted with the 3'UTR of CDK6, and miR-1 was verified to inhibit CDK6 expression at the posttranscriptional level. CDK6 protein expression was observed increased in hypertrophic myocardium and hypertrophic cardiomyocytes. Morover, miR-1 mimic, in parallel to CDK6 siRNA, could inhibit PE-induced hypertrophy of NRVCs, with decreases in cell size, newly transcribed RNA, expressions of ANF and β-MHC, and the phosphorylated pRb. Taken together, our results reveal that derepression of CDK6 and activation of Rb pathway contributes to the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy.

PIK3CA(H1047R) Accelerates and Enhances KRAS(G12D)-Driven Lung Tumorigenesis.

KRAS-activating mutations drive human non-small cell lung cancer and initiate lung tumorigenesis in genetically engineered mouse (GEM) models. However, in a GEM model of KRAS(G12D)-induced lung cancer, tumors arise stochastically following a latency period, suggesting that additional events are required to promote early-stage tumorigenic expansion of KRAS(G12D)-mutated cells. PI3Kα (PIK3CA) is a direct effector of KRAS, but additional activation of PI3'-lipid signaling may be required to potentiate KRAS-driven lung tumorigenesis. Using GEM models, we tested whether PI3'-lipid signaling was limiting for the promotion of KRAS(G12D)-driven lung tumors by inducing the expression of KRAS(G12D) in the absence and presence of the activating PIK3CA(H1047R) mutation. PIK3CA(H1047R) expression alone failed to promote tumor formation, but dramatically enhanced tumorigenesis initiated by KRAS(G12D). We further observed that oncogenic cooperation between KRAS(G12D) and PIK3CA(H1047R) was accompanied by PI3Kα-mediated regulation of c-MYC, GSK3β, p27(KIP1), survivin, and components of the RB pathway, resulting in accelerated cell division of human or mouse lung cancer-derived cell lines. These data suggest that, although KRAS(G12D) may activate PI3Kα by direct biochemical mechanisms, PI3'-lipid signaling remains rate-limiting for the cell-cycle progression and expansion of early-stage KRAS(G12D)-initiated lung cells. Therefore, we provide a potential mechanistic rationale for the selection of KRAS and PIK3CA coactivating mutations in a number of human malignancies, with implications for the clinical deployment of PI3' kinase-targeted therapies.

Suppression of RAD21 Induces Senescence of MDA-MB-231 Human Breast Cancer Cells Through RB1 Pathway Activation Via c-Myc Downregulation.

Cellular senescence impedes cancer progression by limiting uncontrolled cell proliferation. To identify new genetic events controlling senescence, we performed a small interfering RNA screening human cancer cells and identified a number of targets potentially involved in senescence of MDA-MB-231 human breast cancer cells. Importantly, we showed that knockdown of RAD21 resulted in the appearance of several senescent markers, including enhanced senescence-associated β-galactosidase activity and heterochromatin focus formation, as well as elevated p21 protein levels and RB1 pathway activation. Further biochemical analyses revealed that RAD21 knockdown led to the downregulation of c-Myc and its targets, including CDK4, a negative regulator of RB1, and blockedRB1 phosphorylation (pRB1), and the RB1-mediated transcriptional repression of E2F. Moreover, c-Myc downregulation was partially mediated by proteasome-dependent degradation within promyelocytic leukemia (PML) nuclear bodies, which were found to be highly abundant during RAD21 knockdown-induced senescence. Exogenous c-Myc reconstitution rescued cells from RAD21 silencing-induced senescence. Altogether, data arising from this study implicate a novel function of RAD21 in cellular senescence in MDA-MB-231 cells that is mainly dependent onRB1 pathway activation via c-Myc downregulation.

Screening of a kinase library reveals novel pro-senescence kinases and their common NF-κB-dependent transcriptional program.

Cellular senescence results in proliferation arrest and acquisition of hallmarks such as the Senescence-Associated Secretory Phenotype (SASP). Senescence is involved in regulating numerous physio-pathological responses, including embryonic development, cancer, and several aging-related diseases. Only a few kinases, centered on the RAS signaling pathway, have been identified as inducing premature senescence. About possible other senescence-regulating kinases and signaling pathways, practically little is known. By screening a library of activated kinases, we identified 33 kinases whose constitutive expression decreases cell proliferation and induces expression of senescence markers; p16 and SASP components. Focusing on some kinases showing the strongest pro-senescence effects, we observed that they all induce expression of SASP-component genes through activation of an NF-κB-dependent transcriptional program. Furthermore, inhibition of the p53 or Rb pathway failed to prevent the SASP-inducing effect of pro-senescence kinases. Inhibition of the NF-κB, p53, or Rb pathway proved insufficient to prevent kinase-triggered cell cycle arrest. We have thus identified a repertoire of novel pro-senescence kinases and pathways. These results will open new perspectives in the understanding on the role of cellular senescence in various physio-pathological responses.

GENO-05CHROMOTHRIPSIS IS A COMMON MECHANISM DRIVING GENOMIC REARRANGEMENTS IN GLIOBLASTOMA

Findings based on recent advances in next-generation sequence analysis suggest that, in some tumors, a single catastrophic event, termed chromothripsis, results in several simultaneous tumorigenic alterations. Previous studies have suggested that glioblastoma (GBM) may exhibit chromothripsis at a higher rate (39%) than other tumors (9%). Primary glioblastoma is an aggressive form of brain cancer that typically appears suddenly in older adults. With aggressive treatment, the median survival time is only 15 months. Their acute onset and widespread genomic instability indicates that chromothripsis may play a key role in their initiation and progression. GBMs are often characterized by EGFR amplification, CDKN2A and PTEN deletion, although approximately 20% of GBMs harbor additional amplifications in MDM2 or MDM4 with CDK4. We used the chromothripsis prediction tool, Shatterproof, in conjunction with a custom whole genome sequence analysis pipeline in order to generate putative regions of chromothripsis. The data derived from this study was further expanded on using fluorescence in situ hybridization (FISH) analysis and susceptibility studies with colony formation assays. We show that primary GBMs are associated with higher chromothripsis scores and establish a link between chromothripsis and gene amplification of receptor tyrosine kinases (RTKs), as well as modulators of the TP53 and RB1 pathways. Utilizing a newly introduced bioinformatic tool, we provide evidence that chromothripsis is associated with the formation of amplicons containing several oncogenes involved in key pathways that are likely essential for post-chromothriptic cell survival.