pRb Pathway Research Articles

Methylation of KSHV vCyclin by PRMT5 contributes to cell cycle progression and cell proliferation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA virus that encodes numerous cellular homologs, including cyclin D, G protein-coupled protein, interleukin-6, and macrophage inflammatory proteins 1 and 2. KSHV vCyclin encoded by ORF72, is the homolog of cellular cyclinD2. KSHV vCyclin can regulate virus replication and cell proliferation by constitutively activating cellular cyclin-dependent kinase 6 (CDK6). However, the regulatory mechanism of KSHV vCyclin has not been fully elucidated. In the present study, we identified a host protein named protein arginine methyltransferase 5 (PRMT5) that interacts with KSHV vCyclin. We further demonstrated that PRMT5 is upregulated by latency-associated nuclear antigen (LANA) through transcriptional activation. Remarkably, knockdown or pharmaceutical inhibition (using EPZ015666) of PRMT5 inhibited the cell cycle progression and cell proliferation of KSHV latently infected tumor cells. Mechanistically, PRMT5 methylates vCyclin symmetrically at arginine 128 and stabilizes vCyclin in a methyltransferase activity-dependent manner. We also show that the methylation of vCyclin by PRMT5 positively regulates the phosphorylate retinoblastoma protein (pRB) pathway. Taken together, our findings reveal an important regulatory effect of PRMT5 on vCyclin that facilitates cell cycle progression and proliferation, which provides a potential therapeutic target for KSHV-associated malignancies.

Mesenchymal Chondrosarcoma from Diagnosis to Clinical Trials.

Mesenchymal chondrosarcoma (MCS) is a rare subtype of chondrosarcoma with a poor prognosis. Although these tumors are sensitive to radiotherapy/chemotherapy, the standard treatment for localized MCS is only surgical resection, and there are no established treatment guidelines for patients with advanced and metastatic MCS. Due to the low incidence of MCS, the pathology of these tumors is still unknown, and other therapeutic options are lacking. Some studies show the potential role of the PDGF/PPI3K/AKT, PKC/RAF/MEK/ERK, and pRB pathways, and BCL2 overexpression in the pathogenesis of MCS. These findings provide an opportunity to use protein kinases and BCL2 inhibitors as potential therapy in MCS. In this review, we summarize the current knowledge about MCS diagnosis and treatment options. We show the immunological and molecular biomarkers used in the diagnosis of MCS. In addition, we discuss the known prognostic and predictive factors in MCS. Finally, we present the novel trends, including targeted therapies and ongoing clinical trials using protein kinase inhibitors and the death receptor 5 (DR5) agonist, which may be the focus of future MCS treatment studies.

Increased expression of HPV-E7 oncoprotein correlates with a reduced level of pRb proteins via high viral load in cervical cancer

Human Papillomavirus (HPV) is the most common cause of sexually transmitted diseases and causes a wide range of pathologies including cervical carcinoma. Integration of the HR-HPV DNA into the host genome plays a crucial role in cervical carcinoma. An alteration of the pRb pathways by the E7 proteins is one of the mechanisms that’s account for the transforming capacity of high-risk papillomavirus. For the proper understanding of the underline mechanism of the progression of the disease, the present study investigate the correlation of concentration of host pRb protein, viral E7 oncoprotein and viral load in early and advanced stages of cervical carcinoma. It was found that the viral load in early stages (stage I and II) was less (log10 transformed mean value 2.6 and 3.0) compared to advanced stages (stage III and IV) (Log10 transformed value 5.0 and 5.8) having high expression of HPV E7 onco-protein and reduced level of pRb protein, signifying the role of viral load and expression level of E7 oncoprotein in the progression of cervical cancer.

Pro-efferocytosis biomimetic nanocomplexes for targeted atherosclerosis therapy through promoting macrophage re-polarization and inhibiting senescence

Atherosclerosis is chronic inflammatory disease tightly related with defective efferocytosis of apoptotic cells (ACs) in the plaques. The strategy of efferocytosis reactivation using Atorvastatin (AT) and Metformin (Met) has attracted increasing attention for atherosclerosis treatment. Although these drug-based therapies could stimulate the phagocytic clearance of ACs, the rapid drug clearance and poor bioavailability limited their efficacy. In this study, by co-loading AT and Met into poly(lactic-co-glycolic) acid nanoparticles (PLGA NPs), we developed a biomimetic nanocomplexes for targeted atherosclerosis treatment. Macrophages membrane-coated nanocomplexes efficiently avoided the clearance of NPs from immune system and achieved targeting ability to the atherosclerotic plaques. Moreover, modified hyaluronic acid (HA) delivered NPs into the dysfunctional efferocytic cells of plaques, and achieved the plaques-macrophages-targeted therapy. In vitro results indicated that the biomimetic nanocomplexes reactivated efferocytosis by facilitating macrophages polarization from M1 to M2 phenotype through enhancing ERK5/MerTK pathway and inhibiting cellular senescence through inhibiting p53-p16/pRB pathway. In vivo study indicated that this biomimetic nanocomplexes accumulated in the atherosclerotic plaques, resulting in reduction of necrotic core area and protection against plaque rupture by reactivating efferocytosis. These findings indicated that reactivation of efferocytosis by AT and Met co-delivered nanocomplexes provided a novel promising strategy for targeted atherosclerosis treatment.

Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation

Cells can irreversibly exit the cell cycle and become senescent to safeguard against uncontrolled proliferation. While the p53-p21 and p16-Rb pathways are thought to mediate senescence, they also mediate reversible cell cycle arrest (quiescence), raising the question of whether senescence is actually reversible or whether alternative mechanisms underly the irreversibility associated with senescence. Here, we show that senescence is irreversible and that commitment to and maintenance of senescence are mediated by irreversible MYC degradation. Senescent cells start dividing when a non-degradable MYC mutant is expressed, and quiescent cells convert to senescence when MYC is knocked down. In early oral carcinogenesis, epithelial cells exhibit MYC loss and become senescent as a safeguard against malignant transformation. Later stages of oral premalignant lesions exhibit elevated MYC levels and cellular dysplasia. Thus, irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation, but bypassing this degradation may allow tumor cells to escape during cancer initiation.

20. Regulation of HPV oncogene expression

Human papillomaviruses (HPVs) are the aetiological agent of carcinomas at several anatomic subsites and are responsible for 5% of the worldwide cancer burden. Continued expression of the E6 and E7 viral proteins that target the p53 and pRb pathways respectively are essential for maintenance of the transformed phenotype. Targeting kinases critical to the cellular pathways that drive E6/ E7 expression may represent novel therapies specific for HPV-associated cancers. This study aimed to identify a novel kinase that is a potential targeted therapy for HPV-associated malignancy.

Simultaneous Analysis of the p16 Gene and Protein in Canine Lymphoma Cells and Their Correlation with pRb Phosphorylation.

Simple SummaryLymphoma is one of the most frequently diagnosed malignancies in dogs. The most common epigenetic alteration is gene methylation. Methylation of the p16 gene leads to decreased expression of its protein. The p16 protein inhibits the activity of cyclin-dependent kinase, as a negative control of the cell cycle to prevent phosphorylation of the retinoblastoma (pRb) protein. The methylation of the p16 gene has been reported in canine lymphomas, however, p16 protein expression has not been examined in previous studies. In this study, the gene and protein expression of p16, and phosphorylation of pRb, were examined simultaneously in canine lymphoma/leukemia cell lines treated with or without a demethylation drug in vitro. We identified the hypermethylation of the p16 gene, the decreased expression of p16 protein and the hyperphosphorylation of pRb in four out of eight cell lines. Furthermore, we revealed that the expression of the p16 protein was more stable than that of the p16 gene and more closely related to the phosphorylation of pRb. In conclusion, the p16 protein expression is suggested as a promising biomarker for canine lymphoma cells, and the p16–pRb pathway could be a target for the better treatment of canine lymphomas.Cyclin-dependent kinase inhibitor p16 (CDKN2A) primarily functions as a negative regulator of the retinoblastoma protein (pRb) pathway to prevent pRb phosphorylation, thus playing a critical role in cell cycle arrest. In canine lymphoma cells, methylation due to inactivation of the p16 gene has been reported. However, its protein expression has not been examined in previous studies. In our in vitro study, the gene and protein expression of p16 and phosphorylated pRb were examined simultaneously in eight canine lymphoma and leukemia cell lines (17-71, CLBL-1, GL-1, CLC, CLGL-90, Ema, Nody-1, and UL-1). Methylation of the p16 gene was also explored using the demethylation drug 5-Aza-2′-deoxycytidine (5-Aza). After 5-Aza treatment, p16 gene and protein expression increased and pRb phosphorylation decreased, suggesting that both hypermethylation of the p16 gene and pRb hyperphosphorylation occurred in four out of eight cell lines (CLBL-1, CLC, Nody-1, and UL-1). Moreover, the estimation of p16’s protein expression was better than that of p16’s mRNA expression because the expression of the protein was more stable than those of the gene, and highly related to the phosphorylation of pRb. These results revealed that p16’s protein expression could be a promising biomarker for canine lymphoma cells.

Abstract 5788: Genomic and transcriptomic profiles of DNA damage response genes in acute myeloid leukemia

Abstract The DNA damage response (DDR) pathway is frequently deregulated in cancer and it represent an attractive therapeutic opportunity. In acute myeloid leukemia (AML), different mechanisms of DDR deregulation have been identified, but a systematic investigation on DDR alterations is missing. To understand how the DDR pathways contribute to leukemogenesis, we studied the gene expression and mutational profiles of 274 DDR genes by analysing 539 AML cases profiled by whole genome (WGS) and RNA sequencing. WGS data were used to identify mutations in genes of the DDR and in a panel of genes known to be mutated in AML (n=73). Transcriptomic data were analysed through unsupervised clustering, differential expression and enrichment analysis. We detected 150 single nucleotide variants (SNVs) in 130 patients (24%, average 0.3 SNVs/case). Genes mutated in more than 1% of cases were ATM, BLM, BRCA2, POLG and POLQ. The most frequently altered pathway was the homologous recombination/Fanconi Anemia (HR) pathway (29%), followed by the genes that coordinates the DDR pathway (20%). We detected a trend toward mutual exclusivity between mutations in TP53 and mutations in genes of HR pathway or the genes that coordinates the DDR pathway (adj-p <0.02). To further investigate the interplay between TP53 mutations and the HR pathway, we analysed the expression profiles of HR genes in 539 patients. We identified two groups of patients having higher (HR-high) or lower (HR-low) expression levels of HR genes. A panel of 5 genes was able to discriminate patients between the two groups (BRCA1, RAD54B, RMI2, UBE2T and XRCC2; AUC=0.9). Enrichment analysis on differentially expressed genes and gene set enrichment analysis showed that the cell cycle pathway, together with the G2/M transition/mitotic phase, E2F targets and the fatty acid metabolism pathways were upregulated in HR-high patients, while the pRB, EZH2, RPS14 and HOXA9 pathways were downregulated. Moreover, we observed that AML expressing CBFB-MYH1, RUNX1-RUNXT1 or carrying RAD21 mutations had higher chances to express lower levels of HR genes (HR-low), while patients with STAG2, SRSF2, U2AF1, FLT3-ITD alterations had higher chances of having higher expression of HR genes (p<0.05). NPM1-mutated cases without FLT3-ITD clustered within the HR-low profile (adj-p<0.05), while TP53 mutated cases tended to cluster in the HR-high group, although statistical significance was not reached. In conclusion, our data showed the presence of alterations in the DDR pathway that might be the reflection of driver events in AML. Functional studies will elucidate the functional impact of these alterations. The results suggested the presence of a therapeutic window that might be exploited with DDR inhibitors in molecularly-defined subgroups of patients. Supported by the Torsten Haferlach-Leukämiediagnostik-Stiftung and AIRC IG 2019 (project 23810). Citation Format: Antonella Padella, Stephan Hutter, Wencke Walter, Constance Baer, Irene Azzali, Andrea Ghelli Luserna Di Rorà, Martina Ghetti, Lorenzo Ledda, Matteo Paganelli, Claudia Haferlach, Wolfgang Kern, Giorgia Simonetti, Giovanni Martinelli, Torsten Haferlach. Genomic and transcriptomic profiles of DNA damage response genes in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5788.

Securin overexpression correlates with the activated Rb/E2F1 pathway and histone H3 epigenetic modifications in raw areca nut-induced carcinogenesis in mice

BackgroundRaw areca nut (RAN) consumption induces oral, esophageal and gastric cancers, which are significantly associated with the overexpression of pituitary tumor transforming gene 1/securin and chromosomal instability (CIN). An association of Securin/PTTG1 upregulation and gastric cancer in human was also demonstrated earlier. Since the molecular mechanism underlying securin upregulation remains unclear, this study intended to investigate the association of securin upregulation with the Rb-E2F1 circuit and epigenetic histone (H3) modification patterns both globally and in the promoter region of the securin gene.MethodsSix groups of mice were used, and in the treated group, each mouse consumed 1 mg of RAN extract with lime per day ad libitum in the drinking water for 60 days, after which the dose was increased by 1 mg every 60 days. Histopathological evaluation of stomach tissues was performed and securin expression was analysed by immunoblotting as well as by immunohistochemistry. ChIP-qPCR assays were performed to evaluate the recruitment of different histone modifications in the core promoter region of securin gene as well as its upstream and downstream regions.ResultsAll mice developed gastric cancer with securin overexpression after 300 days of feeding. Immunohistochemistry data revealed hyperphosphorylation of Rb and upregulation of E2F1 in the RAN-treated samples. Increased trimethylation of H3 lysine 4 and acetylation of H3 lysine 9 and 18 both globally and in the promoter region of the securin gene were observed by increasing the levels of lysine-N-methyltransferase 2A, lysine-acetyltransferase, EP-300 and PCAF after RAN treatment. ChIP-qPCR data revealed that the quantity of DNA fragments retrieved from the immunoprecipitated samples was maximum in the -83 to -192 region than further upstream and the downstream of the promoter for H3K4Me3, H3K9ac, H3K18ac and H3K9me3.ConclusionsRAN-mediated pRb-inactivation induced securin upregulation, a putative E2F1 target, by inducing misregulation in chromatin remodeling in its promoter region, which led to transcriptional activation and subsequent development of chromosomal instability. Therefore, present results have led to the hypothesis that RAN-induced changes in the epigenetic landscape, securin overexpression and subsequent elevation of chromosomal instability is probably byproducts of inactivation of the pRb pathway.

Targeted deletion of keratin 8 in intestinal epithelial cells disrupts tissue integrity and predisposes to tumorigenesis in the colon

Keratin 8 (K8) is the main intestinal epithelial intermediate filament protein with proposed roles for colonic epithelial cell integrity. Here, we used mice lacking K8 in intestinal epithelial cells (floxed K8 and Villin-Cre1000 and Villin-CreERt2) to investigate the cell-specific roles of intestinal epithelial K8 for colonocyte function and pathologies. Intestinal epithelial K8 deletion decreased K8 partner proteins, K18–K20, 75–95%, and the remaining keratin filaments were located at the colonocyte apical regions with type II K7, which decreased 30%. 2-Deoxy-2-[18F]-fluoroglucose positron emission tomography in vivo imaging identified a metabolic phenotype in the lower gut of the conditional K8 knockouts. These mice developed intestinal barrier leakiness, mild diarrhea, and epithelial damage, especially in the proximal colon. Mice exhibited shifted differentiation from enterocytes to goblet cells, displayed longer crypts and an increased number of Ki67 + transit-amplifying cells in the colon. Significant proproliferative and regenerative signaling occurred in the IL-22, STAT3, and pRb pathways, with minor effects on inflammatory parameters, which, however, increased in aging mice. Importantly, colonocyte K8 deletion induced a dramatically increased sensitivity to azoxymethane-induced tumorigenesis. In conclusion, intestinal epithelial K8 plays a significant role in colonocyte epithelial integrity maintenance, proliferation regulation and tumor suppression.Graphical abstract

Potential antiviral and anticancer effect of imidazoles and bridgehead imidazoles generated by HPV-Induced cervical carcinomas via reactivating the P53/ pRb pathway and inhibition of CA IX

Human papillomaviruses E6 and E7 oncoproteins are crucial to viral-induced cervical cancers and targeting E6 / E7 leads to safer and better treatment for cervical cancer. Hence, a simple and green solvent-free protocol was applied for the synthesis of novel imidazoles and bridgehead imidazoles (purine analogs) via versatile straightforward synthetic routes. All the synthesized compounds have been characterized by IR, 1H NMR, 13C NMR, mass spectroscopy, and elemental analyses and then have been estimated for their in vitro antiviral activity against HPV, genotype 18. Two compounds 4 and 12 were the most promising HR- HPV inhibitors with a percentage of 95.00 and 96.84 %, respectively. Both compounds demonstrated a substantial down-regulation of HPV oncoproteins E6 and E7 with up-regulation of tumor suppressor proteins, p53, and pRb, respectively using western blot technique. Furthermore, the cytotoxicity of compounds 4 and 12 against the cervical cancer Hela cell line was further examined. Compound 4 exhibited strong anticancer activity with IC50 0.08 μM, which is equivalent to 5-FU (IC50 0.09 μM). The cell cycle analysis was performed for investigating the potential mechanism of compound 4, resulting in a significant accumulation of the cell population in both pre-G1 and G0-G1 phases and arrest the cell cycle at G1 phase. Besides, compound 4 induced apoptosis, triggering cell death via increasing the early and late apoptotic rates by approximately 16 and 188 folds compared with the control. The most cytotoxic agent 4 revealed a remarkable inhibition of the targeted carbonic anhydrase IX enzyme with an IC50 value of 0.12 μM comparable to the standard drug. In addition, the ADME profiles of the most highly successful derivatives have been studied in order to determine their ability to be produced as good drug candidates.

Combined targeting of the p53 and pRb pathway in neuroblastoma does not lead to synergistic responses.

Despite intensive treatment protocols and recent advances, neuroblastomas still account for approximately 15% of all childhood cancer deaths. In contrast with adult cancers, p53 pathway inactivation in neuroblastomas is rarely caused by p53 mutationbut rather by altered MDM2 or p14ARF expression. Moreover, neuroblastomas are characterised by high proliferation rates, frequently triggered by pRb pathway dysfunction due to aberrant expression of cyclin D1, CDK4 or p16INK4a. Simultaneous disturbance of these pathways can occur via co-amplification of MDM2 and CDK4 or homozygous deletion of CDKN2A, which encodes both p14ARF and p16INK4a. We examined whether both single and combined inhibition of MDM2 and CDK4/6 is effective in reducing neuroblastoma cell viability. In our panel of ten cell lines with a spectrum of aberrations in the p53 and pRb pathway, idasanutlin and abemaciclib were the most potent MDM2 and CDK4/6 inhibitors, respectively. No correlation was observed between the genetic background and response to the single inhibitors. We confirmed this lack of correlation in isogenic systems overexpressing MDM2 and/or CDK4. In addition, combined inhibition did not result in synergistic effects. Instead, abemaciclib diminished the pro-apoptotic effect of idasanutlin, leading to slightly antagonistic effects. Invivo treatment with idasanutlin and abemaciclib led to reduced tumour growth compared with single drug treatment, but no synergistic response was observed. We conclude that p53 and pRb pathway aberrations cannot be used as predictive biomarkers for neuroblastoma sensitivity to MDM2 and/or CDK4/6 inhibitors. Moreover, we advise to be cautious with combining these inhibitors in neuroblastomas.

71TiP P16 protein expression as a predictive biomarker for response to neoadjuvant chemotherapy in osteosarcoma

Osteosarcoma is the most common primary malignancy of bone in children and young adults. Necrotic response in the tumor specimen after preoperative chemotherapy is a reproducible and reliable prognostic marker for survival in patients with OS. Pathologic tumor necrosis 90% or greater in the surgical specimen has been correlated with a 5-year survival of 75% to 90%. Although several molecular markers, including P-glycoprotein, Ki-67, p53, ezrin and HER2 have been associated with OS, a clear consensus has not emerged regarding the prognostic and predictive significance. The downregulation of p16 INK4A proteins is found in around 60% of high-grade osteosarcomas and plays an important role in growth of osteosarcoma cells by deregulation of both the pRb and p53 cell growth control pathways. Based on previously published studies, p16 protein negative expression on IHC is a predictive biomarker to poor pathologic response to neoadjuvant chemotherapy and is associated with a worse prognosis. p16 expression is correlated to the tumor biology and is not influenced by chemotherapy treatment. Thus, given the paucity of Indian data and lack of prospective studies, we aimed to investigate the role of P16 protein expression as a predictive biomarker in osteosarcoma. Aim: To investigate the relationship of p16 expression in pretreatment osteosarcoma tumors to pathologic necrotic response after neoadjuvant chemotherapy in a prospective pilot study. Inclusion criteria: Histologically proven cases of newly diagnosed osteosarcoma planned for neoadjuvant chemotherapy. Exclusion criteria: 1. Metastatic and relapsed osteosarcoma; 2. Upfront operated cases of osteosarcoma; 3. Prior therapy. Study procedure: Immuno-histochemistry for p16 will be conducted in core biopsy samples upfront before starting neoadjuvant chemotherapy in newly diagnosed case of osteosarcoma tumor cell necrosis rate will be calculated after neoadjuvant chemotherapy on surgical specimen. The grade of tumour cell necrosis rate will be classified using the Huvos necrosis grading system P16 correlation with good response to chemotherapy in terms of tumour necrosis will be evaluated. The author.

Emerging Roles for the INK4a/ARF (CDKN2A) Locus in Adipose Tissue: Implications for Obesity and Type 2 Diabetes

Besides its role as a cell cycle and proliferation regulator, the INK4a/ARF (CDKN2A) locus and its associated pathways are thought to play additional functions in the control of energy homeostasis. Genome-wide association studies in humans and rodents have revealed that single nucleotide polymorphisms in this locus are risk factors for obesity and related metabolic diseases including cardiovascular complications and type-2 diabetes (T2D). Recent studies showed that both p16INK4a-CDK4-E2F1/pRB and p19ARF-P53 (p14ARF in humans) related pathways regulate adipose tissue (AT) physiology and adipocyte functions such as lipid storage, inflammation, oxidative activity, and cellular plasticity (browning). Targeting these metabolic pathways in AT emerged as a new putative therapy to alleviate the effects of obesity and prevent T2D. This review aims to provide an overview of the literature linking the INK4a/ARF locus with AT functions, focusing on its mechanisms of action in the regulation of energy homeostasis.

Abstract A49: Combined targeting of the p53 and pRb pathway in neuroblastoma

Abstract Neuroblastomas account for approximately 15% of all childhood cancer deaths. Clinical complete remission is achieved in many stage 4 neuroblastoma patients, but the high risk of relapse and the accompanying treatment-resistant nature of these tumors is still a challenge. We have previously identified higher frequencies of mutations that affect both the p53 and the pRb pathway, such as the homozygous loss of CDKN2A or co-amplification of MDM2 and CDK4. In addition, both cyclin D1 and MDM2 overexpression is a common characteristic of primary neuroblastoma. These genes act upstream of pRb and p53 and hence play a major role in cell cycle regulation. To study whether these G1 checkpoint aberrations render cells more sensitive to CDK4 or MDM2 inhibition, we exposed a series of 11 cell lines to the most promising CDK4 (ribociclib, palbociclib, and abemaciclib) and MDM2 (SAR405838, HDM-201, and idasanutlin) inhibitors. These cell lines have well-defined mutational properties: homozygous CDKN2A deletion, co-amplification of CDK4 and MDM2, TP53 mutation, and MYCN amplification. Whereas sensitivity for the MDM2 inhibitors inversely correlated with TP53 mutation status, it did not correlate to other G1 checkpoint aberrations. Similarly, there was no clear correlation seen for the CDK4 inhibitors. To further explore this, we generated cell lines with inducible overexpression of CDK4, MDM2, or both. The overexpression did indeed not increase sensitivity to CDK4 or MDM2 inhibitors. Next, we were interested whether combined treatment with the most potent CDK4 and MDM2 inhibitors, abemaciclib and idasanutlin respectively, would be of added value. Cells were exposed to 10 different concentrations of both compounds and Bliss Independence values were calculated as a measure of synergy. Interestingly, combined treatment resulted in slight antagonism in the range of clinically relevant doses in most cell lines. This adverse effect was supported by cell cycle analyses, which showed a lower apoptotic fraction, as well as PARP and caspase 3 cleavage, after combined treatment, as opposed to MDM2 inhibition alone. As was previously suggested in the first clinical trials with CDK4 inhibitors, neither CDK4 nor CDKN2A status is a clear biomarker for CDK4 inhibitor sensitivity. Our results suggest that MDM2 and CDKN2A status also fail as biomarkers for MDM2 inhibitor sensitivity. Further testing is necessary to identify (other) biomarkers for these inhibitors. Moreover, the logical rationale to combine CDK4 and MDM2 inhibitors in neuroblastoma patients with both pRb and p53 pathway disturbances should be taken with precaution, as first results do not show a beneficial response. In vivo experiments to confirm this finding are currently ongoing. Citation Format: Nil Schubert, Linda Schild, Stijn van Oirschot, Kaylee Keller, Lindy Alles, Lindy Vernooij, Marloes Nulle, Emmy Dolman, Marlinde van den Boogaard, Jan Molenaar. Combined targeting of the p53 and pRb pathway in neuroblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A49.

Genetic analysis of the cooperative tumorigenic effects of targeted deletions of tumor suppressors Rb1, Trp53, Men1, and Pten in neuroendocrine tumors in mice.

Genetic alterations of tumor suppressor genes (TSGs) are frequently observed to have cumulative or cooperative tumorigenic effects. We examined whether the TSGs Rb1, Trp53, Pten and Men1 have cooperative effects in suppressing neuroendocrine tumors (NETs) in mice. We generated pairwise homozygous deletions of these four genes in insulin II gene expressing cells using the Cre-LoxP system. By monitoring growth and examining the histopathology of the pituitary (Pit) and pancreas (Pan) in these mice, we demonstrated that pRB had the strongest cooperative function with PTEN in suppressing PitNETs and had strong cooperative function with Menin and TRP53, respectively, in suppressing PitNETs and PanNETs. TRP53 had weak cooperative function with PTEN in suppressing pituitary lesions. We also found that deletion of Pten singly led to prolactinomas in female mice, and deletion of Rb1 alone led to islet hyperplasia in pancreas. Collectively, our data indicated that pRB and PTEN pathways play significant roles in suppressing PitNETs, while the Menin-mediated pathway plays a significant role in suppressing PanNETs. Understanding the molecular mechanisms of these genes and pathways on NETs will help us understand the molecular mechanisms of neuroendocrine tumorigenesis and develop effective preclinical murine models for NET therapeutics to improve clinical outcomes in humans.

Establishment of Immortalized Laryngeal Epithelial Cells Transfected with Bmi1

Primary laryngeal epithelial cells are essential to exploring the mechanisms of laryngeal and voice disorders; however, they are difficult to study and apply because of their limited life span. The purpose of this study was to develop a stable and reliable in vitro model for the comprehensive study of the pathogenesis of laryngeal and voice diseases. The pLVTHM-Bmi1 plasmid was constructed and used to immortalize primary laryngeal epithelial cells by lentiviral infection. The expressions of Bmi1, human telomerase reverse transcriptase (hTERT), p53, and pRB pathway proteins were detected by western blotting. Functional characteristics of the immortalized cell lines were verified by cell senescence β-galactosidase staining, 5-ethynyl-2′-deoxyuridine cell proliferation test, and flow cytometry. We successfully introduced Bmi into human subglottic (hSG) cells and human ventricle (hV) cells. Both the human immortalized subglottic Bmi1 (hSG-Bmi1) cell line and the human immortalized ventricle Bmi1 (hV-Bmi1) cell line maintained normal epithelial morphology and divided successfully after more than 20 culture passages. As Bmi1 was overexpressed in these cells, the expression of human telomerase reverse transcriptase (hTERT) and phosphorylated Rb increased while p16 and p21 decreased. Following Bmi1-mediated immortalization, cell senescence decreased significantly, and cell proliferation was accelerated. Tumor formation was not observed for hSG, hV, or hSG-Bmi1, and hV-Bmi1 cells in nude mice. hSG-Bmi1 cells dominated by stratified squamous epithelium and hV-Bmi1 cells dominated by columnar cells were established. The new cell lines lay a foundation for the study of the pathogenic mechanisms of laryngeal and voice diseases.

Radiotherapy resistance in chondrosarcoma cells; a possible correlation with alterations in cell cycle related genes

BackgroundConventional chondrosarcomas are malignant cartilage tumors considered radioresistant. Nevertheless, retrospective series show a small but significant survival benefit for patients with locally advanced disease treated with radiotherapy. And, in daily practice when considered inoperable their irradiation is an accepted indication for proton beam radiotherapy. Therefore, we investigated the sensitivity of chondrosarcoma cell lines and -tissue samples towards radiotherapy and screened for biomarkers to identify predictors of radiosensitivity.MethodsProliferation and clonogenic assays were performed in chondrosarcoma cell lines after γ-radiation in combination with mutant IDH1 inhibitor AGI-5198. In addition, glutathione levels were measured using mass spectrometry. Chondrosarcoma tumor explants were irradiated after which γ-H2AX foci were counted. Mutation analysis was performed using the Ion AmpliSeq™ Cancer Hotspot Panel and immunohistochemical staining’s were performed for P-S6, LC-3B, P53, Bcl-2, Bcl-xl and Survivin. Results were correlated with the number of γ-H2AX foci.ResultsChondrosarcoma cell lines were variably γ-radiation resistant. No difference in radiosensitivity, nor glutathione levels was observed after treatment with AGI-5198. Irradiated chondrosarcoma patient tissue presented a variable increase in γ-H2AX foci compared to non-radiated tissue. Samples were divided into two groups, high and low radioresistant, based on the amount of γ-H2AX foci. All four highly resistant tumors exhibited mutations in the pRb pathway, while none of the less radioresistant tumors showed mutations in these genes.ConclusionsChondrosarcoma cell lines as well as primary tumors are variably radioresistant, particularly in case of a defective Rb pathway. Whether selection for radiotherapy can be based upon an intact Rb pathway should be further investigated.

Phase 1 Study Evaluating the Association of the Cyclin-Dependent Kinase 4/6 Inhibitor Ribociclib and Cetuximab in Recurrent/Metastatic p16-Negative Squamous Cell Carcinoma of the Head and Neck.

Background: The majority of human papillomavirus (HPV)-negative squamous cell carcinoma of the head and neck (SCCHN) present upregulation of the epidermal growth factor receptor (EGFR) and frequent alterations in the cyclin D1-cyclin dependent kinase (CDK) 4/6 (CDK 4/6)-retinoblastoma protein (pRb) pathway, resulting in cell cycle progression and tumor proliferation. This study investigated the combination of ribociclib, an orally highly selective inhibitor of CDK 4/6, and cetuximab in recurrent and/or metastatic (R/M) SCCHN.Methods: A phase I trial using a 3 + 3 design was performed to determine the dose limiting toxicity (DLT) and maximum tolerated dose (MTD) of ribociclib with standard dose of weekly cetuximab in HPV-negative patients with R/M SCCHN. Ribociclib was administered orally (3 weeks on/1 week off) at dose level 1 of 400 mg daily and dose level 2 of 600 mg daily. The MTD of ribocilib was then further evaluated in an expansion cohort.Results: 10 patients were enrolled in the escalation trial. No DLTs were observed at dose level 1 (n = 3); at dose level 2, one patient was replaced due to rapid disease progression, and one patient out of six evaluable patients experienced a DLT (grade 4 thrombocytopenia >7 days). Ribociclib 600 mg daily was thus determined to be the MTD. Eleven additional patients were enrolled in the expansion cohort. Diarrhea (52%), rash (52%), fatigue (43%), nausea (33%), and mucositis (28%) were the most frequent grade 1–2 adverse events (AE). Neutropenia was the most frequent grade 3–4 AE (20%). Median progression-free survival (PFS) was 3.5 months (range 0.4–17.3 months) and median overall survival (OS) was 8.3 months (range 0.4–24.1 months). Among the 19 radiologically evaluable patients, two (10.5%) achieved a partial response and 11 (58%) had stable disease.Conclusions: The MTD of ribociclib is 600 mg daily when administered in combination with standard dose cetuximab for 3 weeks on and 1 week off. This combination was safe and showed efficacy. Further clinical trials should be conducted to evaluate the antitumor effects of this combination.Trial Information: ClinicalTrials.gov: NCT02429089; Eudract number 2014-005371-83.

DNA demethylation with 5-aza-2′-deoxycytidine induces the senescence-associated secretory phenotype in the immortal fish cell line, EPC

Fish cell lines are known to be immortal and do not show the signs of cellular senescence despite the absence of transformation. Furthermore, high telomerase activities responsible for maintenance of telomere length are detected in many organs in live fish, irrespective of fish age. On the other hand, although it is reported that cytosine methylation at CpG island shores decreases as zebrafish age, the relationship between DNA methylation and cellular senescence in fish has not been explored. In this study, we investigated the induction of cellular senescence and senescence-associated secretory phenotype (SASP) in a fathead minnow Pimephales promelas immortal cell line, Epithelioma papulosum cyprini (EPC) treated with the DNA demethylating agent 5-Aza-2′-deoxycytidine (5-Aza-dC). DNA demethylation by 10 μM of 5-Aza-dC caused cell growth arrest, morphological senescence-like phenotypes and induction of senescence-associated β-galactosidase (SA-β-gal) activity, likely due to a mitotic catastrophe caused by disruption of chromosome segregation. Furthermore, RT-qPCR analyses revealed significant up-regulation of senescence markers such as p53-p21 and p16-Rb pathways as well as several SASP factors in 5-Aza-dC treated cells. Meanwhile, although DNA demethylation suppressed the transcription of myc and its downstream target, telomerase reverse transcriptase (tert), telomerase activity was no more than modestly decreased. These results suggest that although DNA methylation may be involved in the suppression of cellular senescence, it not critical for the immortalization of the fish cell line.