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The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIα regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIα levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur. (Cancer Res 2006; 66(21): 10603-12)

Supplementary Figure 1 from PRKAR1A Inactivation Leads to Increased Proliferation and Decreased Apoptosis in Human B Lymphocytes

Supplementary Table 1 from PRKAR1A Inactivation Leads to Increased Proliferation and Decreased Apoptosis in Human B Lymphocytes

Supplementary Figure 1 Legend from PRKAR1A Inactivation Leads to Increased Proliferation and Decreased Apoptosis in Human B Lymphocytes

Every sixth person in the world dies due to cancer, making it the second leading severe cause of death after cardiovascular diseases. According to WHO, cancer claimed nearly 10 million deaths in 2020. The most common types of cancers reported have been breast (lung, colon and rectum, prostate cases), skin (non-melanoma) and stomach. In addition to surgery, the most widely used traditional types of anti-cancer treatment are radio- and chemotherapy. However, these do not distinguish between normal and malignant cells. Additional treatment methods have evolved over time for early detection and targeted therapy of cancer. However, each method has its limitations and the associated treatment costs are quite high with adverse effects on the quality of life of patients. Use of individual atoms or a cluster of atoms (nanoparticles) can cause a paradigm shift by virtue of providing point of sight sensing and diagnosis of cancer. Nanoparticles (1–100 nm in size) are 1000 times smaller in size than the human cell and endowed with safer relocation capability to attack mechanically and chemically at a precise location which is one avenue that can be used to destroy cancer cells precisely. This review summarises the extant understanding and the work done in this area to pave the way for physicians to accelerate the use of hybrid mode of treatments by leveraging the use of various nanoparticles.

Growth factor receptors (GFRs) and receptor tyrosine kinases (RTK) are groups of proteins mediating a plethora of physiological processes, including cell growth, proliferation, survival, differentiation and migration. Under certain circumstances, expression of GFRs and subsequently their downstream kinase signaling are deregulated by genetic, epigenetic, and somatic changes leading to uncontrolled cell division in many human diseases, most notably cancer. Cancer cells rely on growth factors to sustain the increasing need to cell division and metabolic reprogramming through cancer-associated activating mutations of their receptors (i.e., GFRs). In this review, we highlight the recent advances of selected GFRs and their ligands (growth factors) in cancer with emphasis on structural and functional differences. We also interrogate how overexpression and/or hyperactivation of GFRs contribute to cancer initiation, development, progression, and resistance to conventional chemo- and radiotherapies. Novel approaches are being developed as anticancer agents to target growth factor receptors and their signaling pathways in different cancers. Here, we illustrate how the current knowledge of GFRs biology, and their ligands lead to development of targeted therapies to inhibit and/or block the activity of growth factors, GFRs and downstream kinases to treat diseases such as cancer.

The insulin-like growth factor 1 receptor (IGF-1R) has been suggested to be involved in hepatocyte differentiation. Human hepatocyte cancer cells and stem cells are known to express IGF-1R whereas normal hepatocytes do not. In the present study we optimized a differentiation protocol and verified the different stages by established markers. The expression levels of IGF-1R and major downstream signaling proteins during differentiation from human embryonic stem cells (hESC) to mature hepatocytes were investigated. We could only demonstrate a minor decrease in IGF-1R expression during endodermal differentiation compared to hESC, but declined substantially (>50%) after hepatic lineage commitment during the hepatocyte specification and maturation stages. This downregulation was paralleled by an upregulation of ERK 1/2, AKT and insulin substrate-1. Neither inhibition nor activation of IGF-1R had any essential effect on endoderm differentiation of human embryonic stem cells. Therefore, our data suggest that IGF-1R downregulation may have a regulatory impact after initiation of hepatic lineage commitment.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

Picropodophyllin (PPP) is an anticancer drug undergoing clinical development in NSCLC. PPP has been shown to suppress IGF-1R signaling and to induce a G2/M cell cycle phase arrest but the exact mechanisms remain to be elucidated. The present study identified an IGF-1-independent mechanism of PPP leading to pro-metaphase arrest. The mitotic block was induced in human cancer cell lines and in an A549 xenograft mouse but did not occur in normal hepatocytes/mouse tissues. Cell cycle arrest by PPP occurred in vitro and in vivo accompanied by prominent CDK1 activation, and was IGF-1R-independent since it occurred also in IGF-1R-depleted and null cells. The tumor cells were not arrested in G2/M but in mitosis. Centrosome separation was prevented during mitotic entry, resulting in a monopolar mitotic spindle with subsequent prometaphase-arrest, independent of Plk1/Aurora A or Eg5, and leading to cell features of mitotic catastrophe. PPP also increased soluble tubulin and decreased spindle-associated tubulin within minutes, indicating that it interfered with microtubule dynamics. These results provide a novel IGF-1R-independent mechanism of antitumor effects of PPP.

PURPOSE: Cyclin-dependent kinase 2 (CDK2) is critically involved in cell cycling and has been proposed as a potential cancer target. It remains largely elusive whether CDK2 targeting alters the tumor cell radiosensitivity. MATERIALS AND METHODS: CDK2(-/-) and wild type (WT) mouse embryonic fibroblasts (MEF) as well as six human head and neck squamous cell carcinoma (HNSCC) cell lines (SAS, FaDu, Cal-33, HSC-4, UTSCC-5, UTSCC-8) were used. Upon CDK2 knockdown using small interfering technology, colony formation, DNA double-strand breaks (DSB), cell cycle distribution and expression and phosphorylation of major proteins regulating cell cycle and DNA damage repair were examined. RESULTS: CDK2(-/-) MEF and CDK2 HNSCC knockdown cell cultures were more radiosensitive than the corresponding controls. Repair of DSB was attenuated under CDK2 knockout or knockdown. In contrast to data in MEF, combined CDK2 knockdown with irradiation showed no cell cycling alterations in SAS and FaDu cultures. Importantly, CDK2 knockdown failed to radiosensitize SAS and FaDu when cultured in a more physiological three-dimensional (3D) extracellular matrix environment. CONCLUSIONS: Our findings suggest that targeting of CDK2 radiosensitizes HNSCC cells growing as monolayer. Additional studies performed under more physiological conditions are warranted to clarify the potential of CDK2 as target in radiotherapy.

DSF/Cu2+ inhibits colony formation and induces apoptosis in AML cell lines. THP-1 cells were treated with several doses of either DSF/Cu2+, BTZ or Ara-C. Viability was measured at 72 h by Trypan Blue dye exclusion assay. Viable cells were seeded in Methocult and colonies counted after 10 days. Viability and number of colonies are presented as percent of untreated control (A). CMY cells were exposed to increasing concentrations of DSF/Cu2+ for 24 h and apoptosis was assessed using Annexin V (x-axis) and propidium idodide (PI) (y-axis). Results are represented in four quadrants. For all treatment options, the bottom left quadrant represents live cells [Annexin V(−)/propidium iodide (PI) (−)], the bottom right quadrant represents the percentage of cells in early apoptosis [Annexin V(+)/PI (−)], the upper right quadrant represents the percentage of cells in late apoptosis [Annexin V (+)/PI (+)] and the upper left quadrant represents the percentage of cells in necrosis [Annexin V (−)/PI (+)]. (PPTX 223 kb)

Structure of the PSMB5 gene and mRNA (A). DNA chromatogram showing the location of PSMB5 Q62P mutation in exon 2 (B). 3D protein structure of the PSMB5 Q62P performed using Swiss Prot database, showing the location of the PSMB5 Q62P on the alpha helix (blue arrow). A previously reported PSMB5 mutation (Cys63) is shown (C). (PPTX 419Â kb)

Background Children with Down syndrome (DS) have increased risk for developing AML (DS-AMKL), and they usually experience severe therapy-related toxicities compared to non DS-AMKL. Refractory/relapsed disease has very poor outcome, and patients would benefit from novel, less toxic, therapeutic strategies that overcome resistance. Relapse/resistance are linked to cancer stem cells with high aldehyde dehydrogenase (ALDH) activity. The purpose of the present work was to study less toxic alternative therapeutic agents for relapsed/refractory DS-AMKL. Methods Fourteen AML cell lines including the DS-AMKL CMY and CMK from relapsed/refractory AML were used. Cytarabine (Ara-C), bortezomib (BTZ), disulfiram/copper (DSF/Cu2+) were evaluated for cytotoxicity, depletion of ALDH-positive cells, and resistance. BTZ-resistant CMY and CMK variants were generated by continuous BTZ treatment. Cell viability was assessed using CellTiter-Glo®, ALDH activity by ALDELUORTM, and proteasome inhibition by western blot of ubiquitinated proteins and the Proteasome-Glo™ Chymotrypsin-Like (CT-like) assay, apoptosis by Annexin V Fluos/Propidium iodide staining, and mutations were detected using PCR, cloning and sequencing. Results Ara-C-resistant AML cell lines were sensitive to BTZ and DSF/Cu2+. The Ara-C-resistant DS-AMKL CMY cells had a high percentage of ALDHbright “stem-like” populations that may underlie Ara-C resistance. One percent of these cells were still resistant to BTZ but sensitive to DSF/Cu2+. To understand the mechanism of BTZ resistance, BTZ resistant (CMY-BR) and (CMK-BR) were generated. A novel mutation PSMB5 Q62P underlied BTZ resistance, and was associated with an overexpression of the β5 proteasome subunit. BTZ-resistance conferred increased resistance to Ara-C due to G1 arrest in the CMY-BR cells, which protected the cells from S-phase damage by Ara-C. CMY-BR and CMK-BR cells were cross-resistant to CFZ and MG-132 but sensitive to DSF/Cu2+. In this setting, DSF/Cu2+ induced apoptosis and proteasome inhibition independent of CT-like activity inhibition. Conclusions We provide evidence that DSF/Cu2+ overcomes Ara-C and BTZ resistance in cell lines from DS-AMKL patients. A novel mutation underlying BTZ resistance was detected that may identify BTZ-resistant patients, who may not benefit from treatment with CFZ or Ara-C, but may be responsive to DSF/Cu2+. Our findings support the clinical development of DSF/Cu2+ as a less toxic efficacious treatment approach in patients with relapsed/refractory DS-AMKL. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0493-5) contains supplementary material, which is available to authorized users.

Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compounds have been isolated from mushrooms. The aim of the present work was to study the anticancer effects of schizophyllan (SCH), a β-d-glucan extracted from the mushroom Schizophyllum commune alone or in combination with tamoxifen (TAM) on 7, 12 Dimethylbenz(α)anthracene (DMBA)-induced carcinomas in mice. We isolated SCH from S. commune. Female mice received DMBA, SCH, DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH or vehicles. We studied mice survival, tumour incidence, histopathology, oestrogen receptor (ER) expression, cell proliferation by immunohistochemical detection of proliferating cell nuclear antigen (PCNA), apoptosis by TUNEL assay, as well as caspase-3 expression. DMBA treatment resulted in mammary and hepatocellular carcinomas (HCC). Both SCH and TAM reduced the incidence of DMBA-induced mammary tumours by 85 and 75 %, respectively, and equally decreased the PCNA labelling index relative to DMBA. TAM treatment increased the incidence of- and PCNA index in HCCs relative to DMBA, while SCH suppressed these effects. TAM was more effective than SCH in the induction of apoptosis in both mammary and hepatic carcinomas. Caspase-3 levels correlated with the apoptotic index in most experimental groups. Only one dose of SCH had similar therapeutic effects against DMBA-induced mammary carcinomas as 4 weeks of TAM treatment. This coupled with the ability of SCH to suppress hepatic lesions associated with TAM treatment provides the rationale for further investigating the combined therapeutic effects of TAM+SCH in preclinical models of ER-positive breast cancer, as well as in liver cancer.

Hepatocellular carcinoma (HCC) contributes to 14.8% of all cancer mortality in Egypt, which has a high prevalence of hepatitis C virus (HCV). We have previously shown alterations in the insulin-like growth factor-1 (IGF-1) receptor signalling pathway during experimental hepatocarcinogenesis. The aim of this study was to determine whether serum levels of IGF-1, IGF-2 and IGFBP-3 can be used to discriminate between HCC and the stages of hepatic dysfunction in patients with liver cirrhosis assessed by the Child-Pugh (CP) score, and to correlate these levels with HCC stages. We recruited 241 subjects to the present study; 79 with liver cirrhosis, 62 with HCV-induced HCC and 100 age-matched controls. Results showed that serum levels of IGF-1, IGF-2 and IGFBP-3 were reduced significantly in cirrhosis and HCC patients in comparison to the controls, and that this reduction negatively correlated with the CP scores. However, only IGFBP-3 levels showed significant negative correlation with α-fetoprotein levels. The reduction in IGF-1 and IGFBP-3 but not IGF-2 levels was significant in HCC in comparison to patients with cirrhosis. None of the parameters significantly correlated with the HCC stage. IGFBP-3 levels discriminated between cirrhosis and HCC at a sensitivity of 87%, a specificity of 80% and a cut-off value of

Patients with relapsed/ refractory AML have an overall survival rate Citation Format: Patrick Pirrotte, Victoria David, Ritin Sharma, Daniel H. Wai, Cristina Tognon, Brian Druker, Eiman A. Aleem. Functional proteomics to reveal distinct signature for acute myeloid leukemia subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2705.

Patients with Langerhans cell histiocytosis (LCH) harbor BRAF V600E and activating mutations of MAP2K1/MEK1 in 50% and 25% of cases, respectively. We evaluated a patient with treatment-refractory LCH for mutations in the RAS-RAF-MEK-ERK pathway and identified a novel mutation in the MAP2K1 gene resulting in a p.L98_K104 > Q deletion and predicted to be auto-activating. During treatment with the MEK inhibitor trametinib, the patient's disease showed significant progression. In vitro characterization of the MAP2K1 p.L98_K104 > Q deletion confirmed its effect on cellular activation of the ERK pathway and drug resistance.

The loss of p53 induces spontaneous tumors in mice, and p53 mutations are found in approximately 50% of human tumors. These tumors are generally caused by a number of events, including genomic instability, checkpoint defects, mitotic defects, deregulation of transcriptional targets, impaired apoptosis, and G(1) deregulation or a combination of these effects. In order to determine the role of proteins involved in G(1) control in tumorigenesis, we focused on Cdk2 and Cdk4, two cyclin-dependent kinases that in association with cyclin E and cyclin D promote the G(1)/S phase transition. We analyzed the consequence of loss of Cdk2 in p53-null animals by generating Cdk2(-/-) p53(-/-) mice. These mice are viable and developed spontaneous tumors, predominantly lymphoblastic lymphomas, similar to p53(-/-) mice. In contrast, the genotypes Cdk4(-/-) p53(-/-) were mostly lethal, with few exceptions, and Cdk2(-/-) Cdk4(-/-) p53(-/-) mice die during embryogenesis at embryonic day 13.5. To study the oncogenic potential, we generated mouse embryonic fibroblasts (MEFs) and found that p53(-/-), Cdk2(-/-) p53(-/-), Cdk4(-/-) p53(-/-), and Cdk2(-/-) Cdk4(-/-) p53(-/-) MEFs grew at similar rates without entering senescence. Ras-transformed MEFs of these genotypes were able to form colonies in vitro and induce tumors in nude mice. Our results suggest that tumorigenicity mediated by p53 loss does not require either Cdk2 or Cdk4, which necessitates considering the use of broad-spectrum cell cycle inhibitors as a means of effective anti-Cdk cancer therapy.

Hematologic malignancies represent the fourth most frequently diagnosed cancer in economically developed countries. In hematologic malignancies normal hematopoiesis is interrupted by uncontrolled growth of a genetically altered stem or progenitor cell (HSPC) that maintains its ability of self-renewal. Cyclin-dependent kinases (CDKs) not only regulate the mammalian cell cycle, but also influence other vital cellular processes, such as stem cell renewal, differentiation, transcription, epigenetic regulation, apoptosis, and DNA repair. Chromosomal translocations, amplification, overexpression and altered CDK activities have been described in different types of human cancer, which have made them attractive targets for pharmacological inhibition. Mouse models deficient for one or more CDKs have significantly contributed to our current understanding of the physiological functions of CDKs, as well as their roles in human cancer. The present review focuses on selected cell cycle kinases with recent emerging key functions in hematopoiesis and in hematopoietic malignancies, such as CDK6 and its role in MLL-rearranged leukemia and acute lymphocytic leukemia, CDK1 and its regulator WEE-1 in acute myeloid leukemia (AML), and cyclin C/CDK8/CDK19 complexes in T-cell acute lymphocytic leukemia. The knowledge gained from gene knockout experiments in mice of these kinases is also summarized. An overview of compounds targeting these kinases, which are currently in clinical development in various solid tumors and hematopoietic malignances, is presented. These include the CDK4/CDK6 inhibitors (palbociclib, LEE011, LY2835219), pan-CDK inhibitors that target CDK1 (dinaciclib, flavopiridol, AT7519, TG02, P276-00, terampeprocol and RGB 286638) as well as the WEE-1 kinase inhibitor, MK-1775. The advantage of combination therapy of cell cycle inhibitors with conventional chemotherapeutic agents used in the treatment of AML, such as cytarabine, is discussed.

Depending upon the cellular and physiologic context, the overexpression of the MYC proto-oncogene results in rapid cell growth, proliferation, induction of apoptosis and/or proliferative arrest. What determines the precise consequences upon MYC activation is not clear. We have found that cyclin-dependent kinase 2 (CDK2) is required by MYC to induce apoptosis. MYC-induced apoptosis was suppressed in mouse embryonic fibroblasts (MEF) knocked out for Cdk2 or normal human fibroblasts (NHF) upon expression of the CDK2 inhibitor p27 or treated with RNAi directed at CDK2. Knockout of Cdk2 did not prevent MYC from inducing p53 and Bim. The inhibition of CDK2 did not prevent apoptosis induced by the DNA damaging agent etoposide. Our results surprisingly suggest that CDK2 defines whether MYC induction causes apoptosis.

Interleukin (IL)-7 is required for survival and homeostatic proliferation of T lymphocytes. The survival effect of IL-7 is primarily through regulation of Bcl-2 family members; however, the proliferative mechanism is unclear. It has not been determined whether the IL-7 receptor actually delivers a proliferative signal or whether, by promoting survival, proliferation results from signals other than the IL-7 receptor. We show that in an IL-7–dependent T cell line, cells protected from apoptosis nevertheless underwent cell cycle arrest after IL-7 withdrawal. This arrest was accompanied by up-regulation of the cyclin-dependent kinase inhibitor p27Kip1 through a posttranslational mechanism. Overexpression of p27Kip1 induced G1 arrest in the presence of IL-7, whereas knockdown of p27Kip1 by small interfering RNA promoted S phase entry after IL-7 withdrawal. CD4 or CD8 T cells transferred into IL-7–deficient hosts underwent G1 arrest, whereas 27Kip1-deficient T cells underwent proliferation. We observed that IL-7 withdrawal activated protein kinase C (PKC)θ and that inhibition of PKCθ with a pharmacological inhibitor completely blocked the rise of p27Kip1 and rescued cells from G1 arrest. The conventional pathway to breakdown of p27Kip1 is mediated by S phase kinase-associated protein 2; however, our evidence suggests that PKCθ acts via a distinct, unknown pathway inducing G1 arrest after IL-7 withdrawal from T cells. Hence, IL-7 maintains T cell proliferation through a novel pathway of p27Kip1 regulation.

The cyclin-dependent kinase inhibitor p27(Kip1) is known as a negative regulator of cell-cycle progression and as a tumour suppressor. Cdk2 is the main target of p27 (refs 2, 3) and therefore we hypothesized that loss of Cdk2 activity should modify the p27(-/-) mouse phenotype. Here, we show that although p27(-/-) Cdk2(-/-) mice developed ovary tumours and tumours in the anterior lobe of the pituitary, we failed to detect any functional complementation in p27(-/-) Cdk2(-/-) double-knockout mice, indicating a parallel pathway regulated by p27. We observed elevated levels of S phase and mitosis in tissues of p27(-/-) Cdk2(-/-) mice concomitantly with elevated Cdc2 activity in p27(-/-) Cdk2(-/-) extracts. p27 binds to Cdc2, cyclin B1, cyclin A2, or suc1 complexes in wild-type and Cdk2(-/-) extracts. In addition, cyclin E binds to and activates Cdc2. Our in vivo results provide strong evidence that Cdc2 may compensate the loss of Cdk2 function.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and young adults. The five-year survival rate for RMS has hardly improved over the last three decades despite intensive and toxic chemotherapy, radiotherapy with surgery. Therefore, novel treatment approaches are required to change these outcomes. RMS has two major subtypes, embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS, the more aggressive subtype, is characterized by translocations that fuse two transcription factor-encoding genes; creating novel PAX3/PAX7-FOXO1 fusion proteins. The PAX3-FOXO1 is highly expressed in the G2 phase of the cell cycle, allowing the cell to divide following a sustained checkpoint arrest despite DNA damage induced by chemotherapy, suggesting that PAX3-FOXO1 may enhance the survival of tumor cells in response to chemotherapy. Many cell cycle regulators are altered in RMS, including CDK2, CDK4 and p53. Furthermore, RMS cells are highly dependent on the insulin-like growth factor -1 receptor (IGF-1R) signaling, however, IGF-1R targeting was not successful in the clinic. Therefore, targeting key cell cycle regulators individually or in combination with IGF-1R inhibition may expand the available therapeutic options for RMS. Purpose: The goal of the present study was to investigate the cytotoxicity of 15 small molecule inhibitors targeting the IGF-1R and cell cycle regulators in RMS cell lines and to determine potential mechanisms of drug sensitivity or resistance. Methods and Results: Seven RMS cell lines including ERMS and ARMS were studied. The IC50 values were determined for the following targeting compounds: linsitinib, BMS-754807 and picropodophyllin (PPP) (IGF-1R), riobociclib and palbociclib (CDK4/6), dinaciclib and flavopiridol (pan CDK inhibitors), BS-181HCL (CDK7), MK-1775 (WEE-1), MK-8776 (CHK1), alisertib (AURKA) and volasertib (PLK1). The most potent compounds with IC50< 10 nM were dinaciclib and volasertib. The ARMS cell lines were resistant to alisertib in comparison to the ERMS cell lines. Most cell lines were sensitive to flavopiridol, MK-1775, BMS-754807 and PPP with IC50< 100 nM, and relatively resistant to BS-181 HCL, linsitinib, R0-3306 and MK-8776 with IC50 (1-70 μM). Palbociclib and BMS-754807 showed a synergistic effect in some RMS cell lines. Ongoing studies are focusing on determining the mechanisms of interaction of these two compounds through studying cell cycle, apoptosis, and mRNA and protein expression of key regulators in the IGF-1R and RB pathways. Conclusion: These data demonstrate that dinaciclib, volasertib, flavopiridol, MK-1775, BMS-754807 and PPP are highly cytotoxic in RMS cell lines. The CDK4/6 inhibitor palbociclib may sensitize selected RMS cell lines to IGF-1R inhibitors. Targeting selected cell cycle regulators individually, or in combination with IGF-1R inhibitors may thus provide an efficacious treatment approach to be further validated in RMS patients with poor outcome. Citation Format: Justin Montoya, David W. Lee, Eiman Aleem. Sensitivity and resistance to cell cycle and IGF-1R inhibitors in rhabdomyosarcoma [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 2349. doi:10.1158/1538-7445.AM2017-2349

The mRNA expression of protein phosphatase inhibitor-1 (inhibitor-1) in rat liver was demonstrated using highly sensitive semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Quantification by real-time RT-PCR (LightCycler technology) yielded the same copy number of inhibitor-1 mRNA in total rat liver and isolated hepatocytes (12 copies per cell). This novel finding shows that rat liver expresses indeed inhibitor-1 mRNA, albeit in low amounts. The low copy number explains why the mRNA had not been detected by Northern blotting so far. For comparison, about 425 copies/cell were detected in brain and 2500 copies/cell in skeletal muscle from rat. The full-length coding sequence of rat liver inhibitor-1 was cloned and sequenced, 100% homology with the muscle cDNA was obtained, indicating the expression of the same gene in liver and muscle. In vitro transcription and translation yielded a protein (Mr ~ 30 kDa) which could be detected with a specific antibody by immunoblotting. This indicates an intact open reading frame of inhibitor-1 in rat liver. Immunoblotting of liver extract yielded a very weak band which comigrated with the inhibitor-1 proteins from muscle and brain. It is concluded that mRNA expression of inhibitor-1 may have implications for the regulation of protein phosphatase-1 (PP1) in rat liver.

It has long been believed that Cdk2 and its activator cyclin E play essential roles in the progression of the mitotic cell cycle. However, recent studies using knockout mouse models revealed that neither Cdk2 nor cyclin E are essential in vivo. The purpose of this Perspective is to compare both Cdk2 and cyclin E knockout mice models and to discuss potential mechanisms driving the cell cycle in the absence of Cdk2 or cyclin E. Particular emphasis is placed on possible non-catalytic roles of cyclin E, the expression and activity of the second cyclin binding partner of Cdk2, cyclin A, as well as on the expression and degradation of the Cdk2 inhibitor p27Kip1 in the absence of Cdk2.

Background: The type 1 insulin-like growth factor receptor (IGF-1R) and its signaling components promote cell proliferation, survival, and development of the malignant phenotype. Recently, the IGF-1R has been shown to translocate to the nucleus; however its nuclear functions are not fully elucidated. The proliferating cell nuclear antigen (PCNA) is a nuclear protein that coordinates DNA replication, and is involved in DNA damage repair and in the DNA damage tolerance (DDT) pathways. DDT allows the cell to replicate over polymerase-blocking lesions. DDT mechanisms include translesion DNA synthesis (TLS), and template switching, and the ubiquitination status of PCNA is crucial in these processes. Mono-ubiquitination of PCNA by Rad6 (E2)-Rad18 (E3) activates TLS. The mono-ubiquitin can be further extended though K63-linkages and the non degradative K63 polyubiquitination of PCNA by Ubc13 (E2)-SHPRH/HLTF (E3) leads to template switching. Purpose: The purpose of the present work was to study the functions of nIGF-1R in human embryonic stem cells (hESCs) and in the IGF-1R knockout (igf1r -/-) (R-) and the R- cells overexpressing human IGF1R (R+) mouse embryonic fibroblasts (MEFs), using a mass-spectrometry based approach. Methods and Results: Using subcellular protein fractionation (Qiagen Qproteome Cell Compartment Kit, Qiagen, Germany) and western blot (WB), as well as immunofluorescence, we detected the expression of the nIGF-1R in hESCs, human fibroblasts, MEFs, and in cancer cell lines. To investigate potential functions of nIGF-1R in hESCs, nIGF-1R binding partners were identified using co-immunoprecipitation (Co-IP) and mass spectrometry (MS) (hybrid LTQ-Orbitrap Velos mass spectrometer, Thermo Scientific). Multiple protein candidates were identified including PCNA. IGF-1R-PCNA association was confirmed by Co-IP/immunoblot and by in situ proximity ligation assay (PLA) (OLINK Bioscience, Uppsala, Sweden) to morphologically demonstrate the nuclear localization of the complex. The association of nIGF-1R with PCNA could only be detected in hESCs, MEFs and in human fibroblast cell lines, but not in any of the seven cancer cell lines used. Using a phospho-tyrosine antibody we detected the phosphorylation of PCNA immunoprecipitated from hESCs and R+ but not from R- MEFs or cancer cell lines. Using an in vitro kinase assay, recombinant IGF-1R kinase domain and His-tag recombinant PCNA, we showed that IGF-1R directly phosphorylates PCNA. Using LC-MS/MS and site directed mutagenesis, 3 tyrosine phosphorylation sites (Y60,133,250) were identified. Although PCNA ubiquitination was detected by WB of PCNA immunoprecipitated from R+ MEFs, PCNA protein stability was not affected. The ubiquitination pattern resembled the K63-linked polyubiquitination. Using Co-IP/WB, Ubc13 and SHPRH/HLTF were found to be associated with PCNA in R+ but not in R- cells. Similar results were also achieved for the mono-ubiquitin Rad18 E3 ligase and the main TLS polymerase DNA polymerase. Results were confirmed in hESCs but not in cancer cell lines. Further, PCNA ubiquitination was increased after DNA damage using UV irradiation in R+ MEFs and DNA replication was delayed after methyl methanesulfonate (MMS) treatment, while replication fork stalling in R- cells was highly significant (DNA-fiber labeling assay). Inhibition or knockout of IGF-1R abrogated PCNA phosphorylation, ubiquitination, progress through S-phase of cell cycle, or restart of DNA synthesis after inducing DNA damage. Conclusions: Our results demonstrate a novel role of nIGF-1R in regulating the DNA damage tolerance pathway through PCNA phosphorylation and ubiquitination, potentially maintaining genomic stability in in hESC and in normal fibroblasts. In contrast, nIGF-1R does not interact with PCNA in cancer cell lines. Citation Format: Ahmed Waraky, Yingbo Lin, Eiman Aleem, Olle Larsson. The nuclear IGF-1R regulates DNA damage tolerance through tyrosine phosphorylation of PCNA in human embryonic stem cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr B16.

Cancer is a disease that can be characterized by overexpression of key oncogenic drivers that support tumor development and maintenance. In many instances, these oncogenic drivers are ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. We investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific oncogenic drivers, such as c-Myc and Bcl-2, used by tumor cells for growth and survival. We tested the activity of two GQIDs, GQC-05 and GSA-1103, on cell growth of a panel of eight pediatric and eight adult acute myeloid leukemia (AML) cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 μM for GQC-05 and from 40 nM to 2 μM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-Myc and/or Bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of the highly sensitive line MV-4-11 with GQC-05 showed a 3-4 fold decrease in expression of c-Myc and Bcl-2 mRNA. Furthermore, GQC-05 treatment resulted in decreased levels of both c-Myc and Bcl-2 proteins. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures. Citation Format: Megan Turnidge, Justin J. Montoya, Apurvi Patel, David W. Lee, Eiman Aleem, Daniel H. Wai, Laurence H. Hurley, Vijay Gokhale, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of c-Myc and Bcl-2 in AML cells using G-quadruplex interacting drugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3766.

Background Hypoxia is characteristic of almost all types of solid tumors and is an important component of the bone marrow microenvironment. It is an adverse prognostic indicator in cancer as it is associated with tumor progression and chemoresistance. Cells may adapt to hypoxia in different ways; including growth arrest, stimulation of angiogenic factors, inhibition of apoptosis, induction of the ER stress program, or modification of the cellular energy metabolism. YM155 inhibits survivin gene expression by suppressing promoter activity and subsequent gene expression. The role of hypoxia and its clinical implications in childhood cancer remain largely unknown. Therefore, studying the drug response of cancer cells within a hypoxic environment is critical for the accurate prediction of patients’ response to therapy. The aims of the present study were (1) to perform screening of the effects of 5 molecularly targeted compounds on a large panel of pediatric cancer cell lines including acute myeloid leukemia (AML), Ewings sarcoma (ES), osteosarcoma (OS), and neuroblastoma (NB), under normoxia and hypoxia, and (2) to determine the potential mechanisms of drug resistance under hypoxia. Methods and Results In the present study the IC50 values were determined for the following targeting agents: linsitinib, picropodophylin (insulin-like growth factor-1 receptor), YM155 (survivin), AZD 8055 (mTOR), dovitinib (FLT3, c-kit, FGF4), and Palbociclib (CDK4/CDK6) in a panel of 15 AML, 4 ES, 2 OS and 2 NB cell lines using Cell Titer Glo assay. The effect of hypoxia was differential. It depended on the cell line and the compound used. However, hypoxia induced universal resistance to YM155 in all cell lines tested. YM155 had significant cytotoxicity in AML cell lines (IC50 between 2.5-1775 nM) in normoxia, however, the IC50 values in hypoxia were in the micromolar range, with an increase of > 100 fold. The effect of hypoxia on YM155 response in ES cell lines was less pronounced with (IC50s between 1-10 nM) in normoxia compared to (IC50 between 6 and 350 nM) in hypoxia. We studied the effect of hypoxia on the cell cycle using propidium iodide and flow cytometry in MV4-11 AML cells. Treatment with YM155 for 24 h in normoxia induced a pronounced decrease in the percentage of cells in S phase and G2/M phase and an increase in the sub-G1 phase. However, no change in cell cycle phases was detected under hypoxia. Ongoing studies are focusing on determining the mechanisms of hypoxia-induced YM155 resistance through studying the expression of proteins regulating hypoxia, cell cycle, and apoptosis using western blot. Conclusion These data demonstrate that hypoxia induced different effects in a panel of pediatric cancer cell lines. However, it conferred pronounced resistance to YM155 in AML and ES cell lines. Hypoxia is likely a key factor in programming treatment resistance and, thus, relapse and poor outcomes. Citation Format: Justin Montoya, Kieran Finch, Jan Simper, Robert J. Arceci, Eiman A. Aleem. Hypoxia selectively confers resistance to the survivin-targeting compound YM155 in acute myeloid leukemia and Ewings sarcoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2791.

Background: Acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children. Treatment options for relapsed or refractory AML are limited. Bortezomib, a well-established proteasome inhibitor, is used in AML therapy. It binds to the beta 5 subunit (PSMB5) of the 26S proteasome and inhibits the chymotrypsin (CT)-like activity. Although patients develop resistance to bortezomib, the mechanism is not fully elucidated. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor used to treat alcoholism without major side effects. DS has anticancer cytotoxicity that is in part copper (Cu2+)-dependent (DS/Cu2+). One of the reported mechanisms of action of DS is through proteasome inhibition. We previously demonstrated that both DS/Cu2+ and bortezomib are cytotoxic in a panel of AML cell lines. In the present study, we focused on two acute megakaryoblastic leukemia cell lines derived from patients with Down syndrome (CMY and CMK), one of the cell lines is resistant to cytarabine (CMY), a key drug used in AML treatment. Down syndrome children with leukemia are more prone to suffer from significant toxicity to chemotherapy; therefore, the need for novel treatment options for this category of patients is warranted. Purpose: The purpose of the present study was to develop an in vitro model of bortezomib resistance in CMY and CMK cell lines, to understand the mechanism of resistance, and to identify drugs that are cytotoxic in the bortezomib-resistant cell lines. Methods and Results: The bortezomib-resistant cell lines CMY and CMK were generated by exposure to stepwise increasing concentrations of bortezomib over a period of 9 months up to a concentration of 200 nM in CMY, and 100 nM in CMK cells. Viability was determined using Cell Titer Glo assay. CMY B200nM was 70 fold resistant to bortezomib with decreased inhibition of CT-like activity compared to the parent CMY cell line. Similar results were observed in the CMK cell line. An A362C mutation in PSMB5 causing a change from glutamine to proline was found in the bortezomib resistant CMY cell line. In order to confirm that this mutation caused bortezomib resistance, the PSMB5 (A362C) from CMY B200nM was cloned into the pcDNA3.1 vector and transfected into HEK 293A cells. This caused a two-fold higher bortezomib resistance than the corresponding control. The bortezomib-resistant AML cell lines were cross-resistant to the proteasome inhibitors carfilzomib and MG132 compared to the parent cell line, but were sensitive to DS/Cu2+. Moreover, the PSMB5 mRNA levels and the beta 5-subunit protein levels increased with bortezomib resistance , as evaluated by quantitative real-time RT-PCR and western blot, respectively. Although DS/Cu2+ induced ubiquitination similar to bortezomib, no comparable inhibition of CT-like activity was observed, suggesting a different mechanism of proteasome inhibition. Conclusions: The present results show that (1) the A362C mutation in the PSMB5 may underlie bortezomib-resistance in the cytarabine-resistant CMY cell line. This may provide an explanation for resistance to bortezomib in AML patients, which we hope to assess in the ongoing COG AML trial in which patients are randomized to receive bortezomib plus chemotherapy versus chemotherapy alone, and (2) DS/Cu2+ sensitivity may provide an effective and less toxic treatment approach for AML to be tested in the Down syndrome group of patients with poor outcome following relapse. Citation Format: Ranjan Bista, Oliver Pepper, David W. Lee, David A. Azorsa, Robert J. Arceci, Eiman Aleem. Bortezomib-resistant pediatric acute myeloid leukemia cell lines derived from Down syndrome patients are sensitive to disulfiram. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B02.

Cancer is primarily a disease characterized by aberrant gene expression that is manifested by the overexpression of key genes that support tumor development and maintenance. In many instances, oncogenic drivers are frequently ‘undruggable’ because of structural challenges, the inability to effectively inhibit high concentrations of overexpressed proteins, and the development of drug resistance mutations. An alternative therapeutic approach is to directly inhibit gene transcription by targeting unique secondary DNA structures, called G-quadruplexes, that are associated with subsets of promoters. Our laboratory investigated the activity of a class of compounds termed G-quadruplex Interacting Drugs (GQIDs) that are able to shut down the expression of specific genes used by tumor cells for growth and survival. We tested the activity of two GQIDs GQC-05 and GSA-1103 on cell growth of a panel of eight pediatric and eight adult AML cell lines. Drug dose response analysis showed IC50 values ranging from approximately 10 nM to 1 µM for GQC-05 and from 40 nM to 2 µM for GSA-1103. The AML cell lines had different sensitivities to each GQID indicating a different mechanism of action for each compound. Three AML cell lines that were highly resistant to cytarabine were among the more sensitive to GQC-05. Four cell lines sensitive to GQC-05 had high expression of either c-myc and/or bcl-2, both of which are potential targets of these two GQIDs. Furthermore, treatment of cells with the GQC-05 decreased expression of c-myc and bcl-2. These studies will help define a novel approach of repressing key drivers of leukemia by targeting selective promoter structures. Validation of such an approach for AML will have important implications for testing this therapeutic approach to other childhood cancers in order to improve the length and quality of survival. Citation Format: Apurvi Patel, Justin J. Montoya, Megan Turnidge, Marya Sabir, Daniel H. Wai, David W. Lee, Vijay Gokhale, Eiman Aleem, Laurence J. Hurley, Robert J. Arceci, David O. Azorsa. Targeting promoter regions of oncogenic drivers in pediatric AML cells using G-quadruplex Interacting Drugs (GQIDs). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B24.

β-glucans belong to a group of polysaccharides located in the cell wall of bacteria, fungi including mushrooms, as well as cereals such as barley and oats. All β-glucans are glucose polymers linked together by a (β 1-3) linear β-glycosidic chain core and they differ by their length and branching structures. They are considered biological response modifiers with immunomodulatory and health beneficial effects including anticancer properties. Few studies using purified β- glucans were performed, but their anticancer potential was demonstrated mainly through studies using extracts from mushrooms, yeast or other sources which contain β-glucan as a key component. Their anticancer effects were demonstrated mainly in in vitro and in vivo experimental systems but fewer studies from human populations are available. β-glucans have been used as adjuvant therapy in clinical trials, mainly in the Far East, with a positive effect on patients'survival and quality of life. The mechanism of action is suggested to be through its stimulation of the immune system. This review focuses on human studies; clinical trials and epidemiological data assessing the efficacy and safety of mushroom-derived β- glucans in cancer treatment and prevention. The potential direct effects of β-glucans on cancer cells are also described.

Although colorectal cancer can be successfully treated by conventional strategies such as chemo/radiotherapy and surgery, a substantial number of cases, in particular those with liver metastases, remain incurable. Therefore, novel treatment approaches are warranted. The IGF-1R and its ligands, mainly IGF-1 and IGF-2, have been suggested to play pivotal roles in proliferation, survival and migration of adenocarcinoma cells of the colon/rectum. Therefore, interference with IGF-1R-mediated signaling may represent a therapeutic option for this malignancy. In this study, semi-quantitative RT-PCR analyses of 48 paired, colorectal cancer patient samples showed significant overexpression of tumor IGF-1R and IGF-2 mRNA. There was also an overexpression of MMP-7, which was significantly correlated with histopathological parameters. Based on these findings, the effect of the IGF-1R-inhibitory cyclolignan picropodophyllin (PPP) was assessed in the four colon carcinoma cell lines HT-29, HCT-116, DLD-1 and CaCO-2. PPP strongly and dose-dependently inhibited proliferation and migration in all cell lines. However, when exposed to 0.5 µM PPP, only HT-29 showed a net decrease of viable cells as compared with the cell number at the beginning of the experiment, a finding that coincided with decreased expression/phosphorylation of IGF-1R, AKT and ERK. This cell line also exhibited PPP-induced downregulation of MMP-7 and MMP-9. Similar to the DLD-1 and HCT-116 cell lines, HT-29 also showed substantial cell detachment in response to PPP. Although a net reduction of cells by PPP seems to require a synchronized downregulation of IGF-1R, AKT and ERK1/2, part of the antitumor effect may be explained by other, possibly IGF-1R-unrelated mechanism(s). Such a multitude of inhibitory effects of PPP in colon cancer cells together with its low toxicity in vivo makes it a promising drug candidate in the treatment of this disease.

The multiple neoplasia syndrome Carney complex (CNC) is caused by heterozygote mutations in the gene, which codes for the RIα regulatory subunit (PRKAR1A) of protein kinase A. Inactivation of PRKAR1A and the additional loss of the normal allele lead to tumors in CNC patients and increased cyclic AMP signaling in their cells, but the oncogenetic mechanisms in affected tissues remain unknown. Previous studies suggested that PRKAR1A down-regulation may lead to increased mitogen-activated protein kinase (MAPK) signaling. Here, we show that, in lymphocytes with PRKAR1A-inactivating mutations, there is increased extracellular signal-regulated kinase (ERK) 1/2 and B-raf phosphorylation and MAPK/ERK kinase 1/2 and c-Myc activation, whereas c-Raf-1 is inhibited. These changes are accompanied by increased cell cycle rates and decreased apoptosis that result in an overall net gain in proliferation and survival. In conclusion, inactivation of PRKAR1A leads to widespread changes in molecular pathways that control cell cycle and apoptosis. This is the first study to show that human cells with partially inactivated RIα levels have increased proliferation and survival, suggesting that loss of the normal allele in these cells is not necessary for these changes to occur. (Cancer Res 2006; 66(21): 10603-12)

Cisplatin is one of the most effective chemotherapeutics, but its usefulness is limited by its toxicity to normal tissues, including cells of the kidney proximal tubule. The purpose of these studies was to determine the mechanism of cisplatin cytotoxicity. It was shown in vivo that cisplatin administration induces upregulation of the gene for the p21 cyclin-dependent kinase (cdk) inhibitor in kidney cells. This protein is a positive effector on the fate of cisplatin-exposed renal tubule cells in vivo and in vitro; adenoviral transduction of p21 completely protected proximal tubule cells from cisplatin toxicity. Herein is reported that cdk2 inhibitory drugs protect kidney cells in vivo and in vitro, that transduction of kidney cells in vitro with dominant-negative cdk2 also protected, and that cdk2 knockout cells were resistant to cisplatin. The cdk2 knockout cells regained cisplatin sensitivity after transduction with wild-type cdk2. It is concluded that cisplatin cytotoxicity depends on cdk2 activation and that the mechanism of p21 protection is by direct inhibition of cdk2. This demonstrated the involvement of a protein that previously was associated with cell-cycle progression with pathways of apoptosis. It also was demonstrated that this pathway of cisplatin-induced cell death can be interceded in vivo to prevent nephrotoxicity.

In yeast, a single cyclin-dependent kinase (Cdk) is able to regulate diverse cell cycle transitions (S and M phases) by associating with multiple stage-specific cyclins. The evolution of multicellular organisms brought additional layers of cell cycle regulation in the form of numerous Cdks, cyclins and Cdk inhibitors to reflect the higher levels of organismal complexity. Our current knowledge about the mammalian cell cycle emerged from early experiments using human and rodent cell lines, from which we built the current textbook model of cell cycle regulation. In this model, the functions of different cyclin/ Cdk complexes were thought to be specific for each cell cycle phase. In the last decade, studies using genetically engineered mice in which cell cycle regulators were targeted revealed many surprises. We discovered the in vivo functions of cell cycle proteins within the context of a living animal and whether they are essential for animal development. In this review, we discuss first the textbook model of cell cycle regulation, followed by a global overview of data obtained from different mouse models. We describe the similarities and differences between the phenotypes of different mouse models including embryonic lethality, sterility, hematopoietic, pancreatic, and placental defects. We also describe the role of key cell cycle regulators in the development of tumors in mice, and the implications of these data for human cancer. Furthermore, animal models in which two or more genes are ablated revealed which cell cycle regulators interact genetically and functionally complement each other. We discuss for example the interaction of cyclin D1 and p27 and the compensation of Cdk2 by Cdc2. We also focus on new functions discovered for certain cell cycle regulators such as the regulation of S phase by Cdc2 and the role of p27 in regulating cell migration. Finally, we conclude the chapter by discussing the limitations of animal models and to what extent can the recent findings be reconciled with the past work to come up with a new model for cell cycle regulation with high levels of redundancy among the molecular players. (Less)

It has been long believed that the cyclin-dependent kinase 2 (Cdk2) binds to cyclin E or cyclin A and exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a major role in mitosis. We now provide evidence that Cdc2 binds to cyclin E (in addition to cyclin A and B) and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2 can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle model and how results from knockout mice provide new evidence that refute this model. We focus on the roles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cell cycle regulation that accommodates these novel findings.

Background Despite toxic treatments, acute myeloid leukemia (AML) continues to have an overall poor prognosis of about 50% in children while significantly less in older adults. For patients with relapsed AML refractory to chemotherapy, there is little chance for survival. Therefore, the need for novel treatment modalities continues to be critically important. Disulfiram (DS) is an aldehyde dehydrogenase (ALDH) inhibitor that has been used for decades for the treatment of alcoholism without major side effects. While DS has been reported to have anticancer cytotoxicity that is in part copper (Cu2+)-dependent, the mechanisms are still not fully elucidated. The goal of the present study was to investigate whether DS has significant cytotoxicity against AML. Methods and Results In the present study the IC50 were determined for DS alone or in combination with 1 µM Cu2+ in a panel of 16 AML cell lines using Cell Titer Glo assay. DS/Cu2+ resulted in significant killing of all cell lines (IC50 between 20-75 nM) while the response to DS alone showed significant variation (IC50 from 20 nM - 7 µM). Cu2+ alone was not toxic. Cytarabine (Ara-C), a key drug in AML treatment, was compared with DS/Cu2+. The AML cell lines THP1 (acute monocytic leukemia), UT7-epo and CMY (acute megakaryoblastic leukemia) were profoundly resistant to Ara-C (IC50 between 1.2-2.4 µM) but showed extreme sensitivity to DS/Cu2+ (IC50 between 31-46 nM). Although DS is an inhibitor of ALDH, we demonstrated that the cytotoxicity of DS or DS/Cu2+ was not directly associated with the level of ALDH or its inhibition. AML cell lines were treated with different doses of DS or DS/Cu2+ and ALDH activity and cell death were determined by flow cytometry. The percentage of cell death was dose-dependent and occurred irrespective of ALDH activity in cell lines with high endogenous ALDH activity (MV-4-11, CMY, CMK and K562) compared to those with low or undetectable ALDH activity (Kasumi-1, Molm-13, NB4 and KG-1a). As DS-mediated cytotoxicity has been associated in previous reports with proteasome inhibition, the effects of DS and DS/Cu2+ were compared to Bortezumib. Western blot analysis showed that DS induced PARP cleavage similar to Bortezumib, suggesting that proteasome inhibition may be contributing to DS or DS/Cu2+ cytotoxicity. Conclusion These data demonstrate that multiple AML cell lines are sensitive to DS and to DS/Cu2+, including some that show extreme resistance to Ara-C, such as CMY from a patient with Down Syndrome. This DS sensitivity may provide a less toxic and efficacious treatment approach to be tested in this group of patients with poor outcome following relapse. The cytotoxic activity of DS does not appear to be solely dependent on ALDH inhibition. Ongoing studies are focusing on determining the mechanisms of cell death, and cytotoxic efficacy in in vitro and xenograft models using patient AML samples, with the goal of developing a clinical trial in patients with relapsed/refractory AML. Citation Format: Eiman A. Aleem, David O. Azorsa, Ranjan Bista, Oliver B. Pepper, David W. Lee, Daniel H. Wai, Robert J. Arceci. Repurposing disulfiram for treatment of relapsed/refractory acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3982. doi:10.1158/1538-7445.AM2014-3982

Background Acute myeloid leukemia (AML) is a complex and heterogeneous disease leading to a wide range of response variability to conventional therapy, excessive treatment related toxicity, and an overall poor outcome. The NCI supported Therapeutically Applicable Research to Generate Effective Treatments (TARGET) Initiative provided initial support for large-scale genomics to identify novel disease-associated genomic and epigenomic alterations that could be used to develop novel, targeted therapies for pediatric AML. Patients Diagnostic (Dx), remission (Rm), and relapse (Rel) samples were obtained from over 200 children with AML treated on the most recent Children's Oncology Group clinical trials. Data analyses were performed using Partek Genomics Suite 6.6 and Ingenuity Pathway Analysis. Results Methylation profiling (Illumina HumanMethylation27) identified 603 CpG sites significantly differentially methylated between Dx and Rm. In addition, 514 CpG sites were significantly differentially methylated between matched Rel and Rm (p Gene expression profiling (Affymetrix Human Gene 1.1 ST) revealed 1,142 significantly differentially expressed between Dx and normal bone marrow obatined from the blood bank. Similarly, there were 1,242 significantly differentially expressed genes between Rel and normal bone marrow (p Copy-number data analysis (Affymetrix Genome-Wide Human SNP 6.0) identified 180,379 significantly different regions in 254 Dx vs matched Rm. Similarly, 4,707 regions a significantly different in 49 Rel vs matched Rm (p Whole-genome sequencing (Complete Genomics, Inc) was performed on 100 cases with matched Dx and Rm pairs and on 86 cases with matched Dx, Rel, and Rm samples. The frequencies of either somatic or germline variants were not significantly different when comparing Dx vs. Rel samples. Pathway analysis compared 1,528 mutated genes in the pediatric (TARGET) cohort with 1,963 mutated genes in adult de novo AML (TCGA cohort). Of the 85 and 80 significantly (p Conclusion A combination of genomic approaches have identified genes and pathways that appear deregulated in pediatric AML. Many changes are specific to Dx or Rel sample groups, or to pediatric vs. adult AML. Further study into these genes and pathways may reveal candidate targets for therapeutic intervention. Citation Format: Daniel H. Wai, Rhonda E. Ries, Michael Considine, David W. Lee, Sun-Mei Liu, Nicole M. O'Connor, Oliver Pepper, Ranjan Bista, Eiman Aleem, David O. Azorsa, Tanja M. Davidsen, Toni Farley, Jaime Guidry-Auvil, Jason E. Farrar, Malcolm A. Smith, Daniela S. Gerhard, MIchael F. Ochs, Spyro Mousses, Soheil Meshinchi, Robert J. Arceci. Integrative genomic analyses on pediatric acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5583. doi:10.1158/1538-7445.AM2014-5583

Streptokinase (SK) is a therapeutically important thrombolytic agent. Cardiovascular disease is the first cause of adult death worldwide. In Egypt about 13% of the population die every year due to ischemic heart disease. In spite of this fact, there is no local production of cardiovascular therapeutics. We reported for the first time the expression of a recombinant SK from a local Streptococcus strain. When produced on industrial scale this r-SK may substantially contribute to reducing the costs of thrombolytic therapy in developing countries. In this study, a highly purified r-SK from Streptococcus sp. isolated from Egyptian pharyngitis patients was obtained. The isolated strain was partially identified using 16S rDNA sequencing and namedStreptococcus sp. SalMarEg. It was found to be phylogenetically related toStreptococcus pyogenes. Analysis of the obtained sequence showed high similarity with other SK genes. The protein expression in a prokaryotic system obtained a 47-kDa SK protein that could be purified using a single-step his-tagged affinity purification chromatography, with nearly 80% recovery. The clot lytic activities of both recombinant and commercial SK were similar, thus giving the basis to scale up this SK product in order to evaluate the possibilities of its commercialization in local and/or regional markets. Key words: Streptokinase, Streptococcus SalMarEg, thrombolytic agent,heterologous expression.

Protein phosphatase inhibitor-1 plays an important role in the regulation of glycogen metabolism through inhibition of protein phosphatase-1 activity, and it has been implicated in the regulation of cell growth. Using real-time quantitative RT-PCR, we studied the mRNA expression of inhibitor-1 in hepatocellular carcinomas induced in rats by oral administration of N-nitrosomorpholine, and in a non-tumorigenic liver cell line (C 1 I), that stores glycogen in excess during early passages. In late passages, glycogen is gradually lost concomitant with cell transformation. Our in vitro model included a tumorigenic subline of C 1 I cells that was obtained by chemically-induced neoplastic transformation using N-methyl-N'-nitro-N-nitrosoguanidine (C 1 I ct ), and does not store glycogen, as well as Morris hepatoma 3924A (MH3924A) cells. We found that in hepatocellular carcinomas, in the late glycogen-poor passages (C 1 I late ), and in the tumorigenic subline (C 1 I et ) of C 1 I cells, and in MH3924A cells the mRNA expression of inhibitor-1 is significantly increased. This increase in expression varied from 15 to 290-fold of that observed in normal liver. In contrast, in the early glycogen-storing passage of C 1 I cells (C 1 I early ) the level of inhibitor-1 mRNA was found to be slightly less than that of normal liver. Inhibitor-1 mRNA levels correlated with the degree of differentiation of HCCs. These results indicate that the expression of inhibitor-1 mRNA is tightly linked to tumor progression and to the process of liver cell transformation in vitro and is inversely correlated with the glycogen content of the cell.

Summary. – Selected commercial and/or local vineyards and nurseries in three different governorates of Egypt (Alexandria, El-Beheira and El-Menofia) were surveyed for symptoms indicative of infection by grape vine viruses. Leaf samples from red-fruited and white-fruited Vitis vinefera were tested for grapevine leafroll associated viruses (GLRaV-1, GLRaV-2, and GLRaV-3), grapevine viruses A and B (GVA, GVB), grapevine rupestris stem pitting virus (GRSPaV), grapevine fanleaf virus (GFLV), and grapevine fleck virus (GFKV) from early April to late October 2010. Incidence of these viruses was assessed by RT-PCR in 60 different samples. Selected amplicons were sequenced. While GVA was the most wide spread (30%), GLRaV-1, GVB, GFLV, and GFKV were not detected during the survey. However, GVA, GLRaV-2, GLRaV-3, and GRSPaV were detected in the form of single infection or in mixed infections of 2 to 4 viruses. Phylogenetic analysis was performed on all Egyptian isolates of GLRaV-2 (4), GLRaV-3 (7), GVA (3), and GRSPaV (6). GRSPaV was detected for the first time in Egypt. Phylogenetic analysis provided insights into the evolutionary relationship between the reported Egyptian isolates and other previously reported isolates.

Chloramphenicol has been associated with the development of aplastic anaemia. As it is still widely used in Egypt, we studied its effect on 100 Egyptian toads [Bufo regularis] given a dose of chloramphenicol of 5 mg/40 g body weight for 12 weeks. We found it induced numerous, severe ultrastructural changes in almost all types of leukocytes. These changes were similar to those induced by the chemical carcinogen 7, 12-dimethylbenz[a]anthracene in 100 toads used as the carcinogen control group, and similar to those in leukocytes reported in humans with leukaemia. We recommend regulations be applied on the use of this antibiotic in countries where it is still widely used.