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The cell and gene therapy product AGT103-T was designed to restore the Gag-specific CD4+ T cell response in persons with chronic HIV disease who are receiving antiretroviral therapy. This autologous, genetically engineered cell product is under investigation in a Phase 1 clinical trial (NCT03215004). Trial participants were conditioned with cyclophosphamide approximately 1 week before receiving a one-time low (< 10 9 genetically modified CD4+ T cells) or high (≥10 9 genetically modified CD4+ T cells) dose of AGT103-T, delivering between 2 and 21 million genetically modified cells per kilogram (kg) body weight. There were no serious adverse events (SAEs) and all adverse events (AEs) were mild. Genetically modified AGT103-T cells were detected in most of the participant blood samples collected 6 months after infusion, which was the last scheduled monitoring visit. Peripheral blood mononuclear cells (PBMC) collected after cell product infusion were tested to determine the abundance of Gag-specific T cells as a measure of objective responses to therapy. Gag-specific CD4+ T cells were detected in all treated individuals and were substantially increased by 9 to 300-fold compared to baseline, by 14 days after cell product infusion. Gag-specific CD8+ T cells were increased by 1.7 to 10-fold relative to baseline, by 28 days after cell product infusion. Levels of Gag-specific CD4+ T cells remained high (~2 to 70-fold higher relative to baseline) throughout 3-6 months after infusion. AGT103-T at low or high doses was safe and effective for improving host T cell immunity to HIV. Further studies, including antiretroviral treatment interruption, are warranted to evaluate the product's efficacy in HIV disease., Clinical Trial Registration: www.clinicaltrials.gov, identifier: NCT03215004., Competing Interests: Authors HL, TL, and CP are shareholders in American Gene Technologies International, Inc. Author JG is a shareholder and current employee of American Gene Technologies, International, Inc. Authors PK and JB received funding for clinical trial costs from American Gene Technologies International, Inc. Authors HL and CP are current employees of Viriom Inc. Author NM is a current employee of BioNTech., (Copyright © 2022 Muvarak, Li, Lahusen, Galvin, Kumar, Pauza and Bordon.)

Human Vγ9Vδ2 T cells are attractive candidates for cancer immunotherapy due to their potent capacity for tumor recognition and cytolysis of many tumor cell types. However, efforts to deploy clinical strategies for Vγ9Vδ2 T cell cancer therapy are hampered by insufficient potency. We are pursuing an alternate strategy of modifying tumors to increase the capacity for Vγ9Vδ2 T cell activation, as a means for strengthening the anti-tumor response by resident or ex vivo manufactured Vγ9Vδ2 T cells. Vγ9Vδ2 T cells are activated in vitro by non-peptidic antigens including isopentenyl pyrophosphate (IPP), a substrate of farnesyl diphosphate synthase (FDPS) in the pathway for biosynthesis of isoprenoids. In an effort to improve in vivo potency of Vγ9Vδ2 T cells, we reduced FDPS expression in tumor cells using a lentivirus vector encoding a short-hairpin RNA that targets FDPS mRNA (LV-shFDPS). Prostate (PC3) or hepatocellular carcinoma (Huh-7) cells transduced with LV-shFDPS induced Vγ9Vδ2 T cell stimulation in vitro , resulting in increased cytokine expression and tumor cell cytotoxicity. Immune deficient mice implanted with LV-shFDPS transduced tumor cells showed dramatic responses to intraperitoneal injection of Vγ9Vδ2 T cells with strong suppression of tumor growth. In vivo potency was increased by transducing tumor cells with a vector expressing both shFDPS and human IL-2. Tumor suppression by Vγ9Vδ2 T cells was dose-dependent with greater effects observed in mice injected with 100% LV-shFDPS transduced cells compared to mice injected with a mixture of 50% LV-shFDPS transduced cells and 50% control (no vector) tumor cells. Delivery of LV-shFDPS by intratumoral injection was insufficient to knockdown FDPS in the majority of tumor cells, resulting in insignificant tumor suppression by Vγ9Vδ2 T cells. Thus, Vγ9Vδ2 T cells efficiently targeted and suppressed tumors expressing shFDPS in mouse xenotransplant models. This proof-of-concept study demonstrates the potential for suppression of genetically modified tumors by human Vγ9Vδ2 T cells and indicates that co-expression of cytokines may boost the anti-tumor effect., Competing Interests: The authors M-LL, TL, and LX are employed by American Gene Technologies and HL and CDP are former employees of American Gene Technologies and currently employed by Viriom, Inc., (Copyright © 2022 Liou, Lahusen, Li, Xiao and Pauza.)

Activation, infection, and eventual depletion of human immunodeficiency virus (HIV)-specific cluster of differentiation 4 (CD4) T cells are the crucial pathogenetic events in acquired immunodeficiency syndrome (AIDS). We developed a cell and gene therapy to reconstitute HIV-specific CD4 T cells and prevent their destruction by HIV. Antigen-specific CD4 T cells will provide helper functions to support antiviral cytotoxic T lymphocyte (CTL) function and the production of virus-specific antibodies. However, ex vivo expansion of HIV-specific CD4 T cells is poor and previous gene therapies focused on bulk CD4 T cells without enriching for an antigen-specific subset. We developed a method for manufacturing autologous CD4 + T cell products highly enriched with Gag-specific T cells. Rare Gag-specific CD4 T cells in peripheral blood mononuclear cells (PBMCs) were increased nearly 1,000-fold by stimulating PBMC with Gag peptides, followed by depleting nontarget cells and transducing with lentivirus vector AGT103 to protect against HIV-mediated depletion and inhibit HIV release from latently infected cells. The average percentage of HIV-specific CD4 cells in the final products was 15.13%, and the average yield was 7 × 10 8 cells. The protocol for clinical-scale manufacturing of HIV-specific and HIV-resistant CD4 T cells is an important step toward effective immunotherapy for HIV disease., (© 2020 The Author(s).)

Human gamma delta T cells have extraordinary properties including the capacity for tumor cell killing. The major gamma delta T cell subset in human beings is designated Vγ9Vδ2 and is activated by intermediates of isoprenoid biosynthesis or aminobisphosphonate inhibitors of farnesyldiphosphate synthase. Activated cells are potent for killing a broad range of tumor cells and demonstrated the capacity for tumor reduction in murine xenotransplant tumor models. Translating these findings to the clinic produced promising initial results but greater potency is needed. Here, we review the literature on gamma delta T cells in cancer therapy with emphasis on the Vγ9Vδ2 T cell subset. Our goal was to examine obstacles preventing effective Vγ9Vδ2 T cell therapy and strategies for overcoming them. We focus on the potential for local activation of Vγ9Vδ2 T cells within the tumor environment to increase potency and achieve objective responses during cancer therapy. The gamma delta T cells and especially the Vγ9Vδ2 T cell subset, have the potential to overcome many problems in cancer therapy especially for tumors with no known treatment, lacking tumor-specific antigens for targeting by antibodies and CAR-T, or unresponsive to immune checkpoint inhibitors. Translation of amazing work from many laboratories studying gamma delta T cells is needed to fulfill the promise of effective and safe cancer immunotherapy.

The breast cancer associated gene 1 (BRCA1) contains 3 domains: an N-terminal RING domain with ubiquitin E3 ligase activity, C-terminal BRCT protein interaction domain and a central region. RING and BRCT domains are well characterized, yet the function of the central region remains unclear. In this study, we identified an essential DNA binding region (DBR: 421-701 amino acids) within the central region of human BRCA1, and found that BRCA1 brings DNA together and preferably binds to splayed-arm DNA in a sequence-independent manner. To investigate the biological role of the DBR, we generated mouse ES cells, which lack the DBR (ΔDBR) by using the TALEN method. The ΔDBR cells exhibited decreased survival as compared to the wild type (WT) cells treated with a PARP inhibitor, however they have an intact ability to conduct DNA repair mediated by homologous recombination (HR). The ΔDBR cells continued to incorporate more EdU in the presence of hydroxyurea (HU), which causes replication stress and exhibited reduced viability than the WT cells. Moreover, phosphorylation of CHK1, which regulates the intra-S phase checkpoint, was moderately decreased in ΔDBR cells. These data suggest that DNA binding by BRCA1 affects the stability of DNA replication folks, resulting in weakened intra-S-phase checkpoint control in the ΔDBR cells. The ΔDBR cells also exhibited an increased number of abnormal chromosome structures as compared with WT cells, indicating that the ΔDBR cells have increased genetic instability. Thus, we demonstrated that the DBR of BRCA1 modulates genetic stability through the intra-S-phase checkpoint activated by replication stress.

DNA damage elicits a cellular signaling response that initiates cell cycle arrest and DNA repair. Here, we find that DNA damage triggers a critical block in glutamine metabolism, which is required for proper DNA damage responses. This block requires the mitochondrial SIRT4, which is induced by numerous genotoxic agents and represses the metabolism of glutamine into tricarboxylic acid cycle. SIRT4 loss leads to both increased glutamine-dependent proliferation and stress-induced genomic instability, resulting in tumorigenic phenotypes. Moreover, SIRT4 knockout mice spontaneously develop lung tumors. Our data uncover SIRT4 as an important component of the DNA damage response pathway that orchestrates a metabolic block in glutamine metabolism, cell cycle arrest, and tumor suppression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Members of sirtuin family regulate multiple critical biological processes, yet their role in carcinogenesis remains controversial. To investigate the physiological functions of SIRT2 in development and tumorigenesis, we disrupted Sirt2 in mice. We demonstrated that SIRT2 regulates the anaphase-promoting complex/cyclosome activity through deacetylation of its coactivators, APC(CDH1) and CDC20. SIRT2 deficiency caused increased levels of mitotic regulators, including Aurora-A and -B that direct centrosome amplification, aneuploidy, and mitotic cell death. Sirt2-deficient mice develop gender-specific tumorigenesis, with females primarily developing mammary tumors, and males developing more hepatocellular carcinoma (HCC). Human breast cancers and HCC samples exhibited reduced SIRT2 levels compared with normal tissues. These data demonstrate that SIRT2 is a tumor suppressor through its role in regulating mitosis and genome integrity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The oncogene amplified in breast cancer 1 (AIB1) is a nuclear receptor coactivator that plays a major role in the progression of various cancers. We previously identified a splice variant of AIB1 called AIB1-Δ4 that is overexpressed in breast cancer. Using mass spectrometry, we define the translation initiation of AIB1-Δ4 at Met(224) of the full-length AIB1 sequence and have raised an antibody to a peptide representing the acetylated N terminus. We show that AIB1-Δ4 is predominantly localized in the cytoplasm, although leptomycin B nuclear export inhibition demonstrates that AIB1-Δ4 can enter and traffic through the nucleus. Our data indicate an import mechanism enhanced by other coactivators such as p300/CBP. We report that the endogenously and exogenously expressed AIB1-Δ4 is recruited as efficiently as full-length AIB1 to estrogen-response elements of genes, and it enhances estrogen-dependent transcription more effectively than AIB1. Expression of an N-terminal AIB1 protein fragment, which is lost in the AIB1-Δ4 isoform, potentiates AIB1 as a coactivator. This suggests a model whereby the transcriptional activity of AIB1 is squelched by a repressive mechanism utilizing the N-terminal domain and that the increased coactivator function of AIB1-Δ4 is due to the loss of this inhibitory domain. Finally, we show, using Scorpion primer technology, that AIB1-Δ4 expression is correlated with metastatic capability of human cancer cell lines.

Glucose homeostasis in mammals is mainly regulated by insulin signaling. It was previously shown that SIRT6 mutant mice die before 4 weeks of age, displaying profound abnormalities, including low insulin, hypoglycemia, and premature aging. To investigate mechanisms underlying the pleiotropic phenotypes associated with SIRT6 deficiency, we generated mice carrying targeted disruption of SIRT6. We found that 60% of SIRT6(-/-) animals had very low levels of blood glucose and died shortly after weaning. The remaining animals, which have relatively higher concentrations of glucose, survived the early post-weaning lethality, but most died within one year of age. Significantly, feeding the mice with glucose-containing water increased blood glucose and rescued 83% of mutant mice, suggesting that the hypoglycemia is a major cause for the lethality. We showed that SIRT6 deficiency results in more abundant membrane association of glucose transporters 1 and 4, which enhances glucose uptake. We further demonstrated that SIRT6 negatively regulates AKT phosphorylation at Ser-473 and Thr-308 through inhibition of multiple upstream molecules, including insulin receptor, IRS1, and IRS2. The absence of SIRT6, consequently, enhances insulin signaling and activation of AKT, leading to hypoglycemia. These data uncover an essential role of SIRT6 in modulating glucose metabolism through mediating insulin sensitivity.

Global DNA hypomethylation at CpG islands coupled with local hypermethylation is a hallmark for breast cancer, yet the mechanism underlying this change remains elusive. In this study, we showed that DNMT1, which encodes a methylation maintenance enzyme, is a transcriptional target of BRCA1. BRCA1 binds to the promoter of the DNMT1 gene through a potential OCT1 site and the binding is required for maintaining a transcriptional active configuration of the promoter in both mouse and human cells. We further demonstrated that impaired function of BRCA1 leads to global DNA hypomethylation, loss of genomic imprinting, and an open chromatin configuration in several types of tissues examined in a BRCA1 mutant mouse model at premaligant stages. BRCA1 deficiency is also associated with significantly increased expression levels of several protooncogenes, including c-Fos, Ha-Ras, and c-Myc, with a higher expression in tumors, while premalignant mammary epithelial cells displayed an intermediate state between tumors and controls. In human clinical samples, reduced expression of BRCA1 correlates with decreased levels of DNMT1, and reduced methylation of CpG islands. Thus, BRCA1 prevents global DNA hypomethylation through positively regulating DNMT1 expression, and this provides one of mechanisms for BRCA1-associated breast cancer formation.

Under various conditions, mammals have the ability to maintain serum glucose concentration within a narrow range. SIRT1 plays an important role in regulating gluconeogenesis and fat metabolism; however, the underlying mechanisms remain elusive. Here, we show that SIRT1 forms a complex with FOXO3a and NRF1 on the SIRT6 promoter and positively regulates expression of SIRT6, which, in turn, negatively regulates glycolysis, triglyceride synthesis, and fat metabolism by deacetylating histone H3 lysine 9 in the promoter of many genes involved in these processes. Liver-specific deletion of SIRT6 in mice causes profound alterations in gene expression, leading to increased glycolysis, triglyceride synthesis, reduced beta oxidation, and fatty liver formation. Human fatty liver samples exhibited significantly lower levels of SIRT6 than did normal controls. Thus, SIRT6 plays a critical role in fat metabolism and may serve as a therapeutic target for treating fatty liver disease, the most common cause of liver dysfunction in humans., (2010 Elsevier Inc. All rights reserved.)

AIB1 (amplified in breast cancer 1), also called SRC-3 and NCoA-3, is a member of the p160 nuclear receptor co-activator family and is considered an important oncogene in breast cancer. Increased AIB1 levels in human breast cancer have been correlated with poor clinical prognosis. Overexpression of AIB1 in conjunction with members of the epidermal growth factor receptor (EGF/HER) tyrosine kinase family, such as HER2, is associated with resistance to tamoxifen therapy and decreased disease-free survival. A number of functional studies in cell culture and in rodents indicate that AIB1 has a pleiotropic role in breast cancer. Initially AIB1 was shown to have a role in the estrogen-dependent proliferation of breast epithelial cells. However, AIB1 also affects the growth of hormone-independent breast cancer and AIB1 levels are limiting for IGF-1-, EGF- and heregulin-stimulated biological responses in breast cancer cells and consequently the PI3 K/Akt/mTOR and other EGFR/HER2 signaling pathways are controlled by changes in AIB1 protein levels. The cellular levels and activity of AIB1 are in turn regulated at the levels of transcription, mRNA stability, post-translational modification, and by a complex control of protein half life. In particular, AIB1 activity as well as its half-life is modulated through a number of post-translational modifications including serine, threonine and tyrosine phosphorylation via kinases that are components of multiple signal transduction pathways. This review summarizes the possible mechanisms of how dysregulation of AIB1 at multiple levels can lead to the initiation and progression of breast cancer as well as its role as a predictor of response to breast cancer therapy, and as a possible therapeutic target.

The steroid receptor coactivator amplified in breast cancer 1 (AIB1) as well as epidermal growth factor receptor (EGFR) family members are frequently overexpressed in epithelial tumors, and their expression is associated with poor prognosis. However, a direct role of AIB1 in EGF signaling has not been determined. To address this, we reduced endogenous AIB1 levels using RNA interference in lung, breast, and pancreatic cancer cell lines. We found that a knockdown of AIB1 levels resulted in a loss of the growth response of these cell lines to EGF. Further analysis revealed that the depletion of AIB1 reduced tyrosine phosphorylation of EGFR at multiple residues both at autophosphorylation and Src kinase phosphorylation sites. AIB1 knockdown did not affect tyrosine phosphorylation of the receptor tyrosine kinases, platelet-derived growth factor receptor and HER3, or overall tyrosine phosphorylation of cellular proteins. However, EGF-dependent phosphorylation of HER2 was decreased. EGFR levels and membrane trafficking were not changed by AIB1 depletion, but there was less recruitment of Src homology 2 domain-containing proteins to the EGFR. This led to a substantial reduction in EGF-induced phosphorylation of signal transducers and activators of transcription 5 and c-Jun NH(2)-terminal kinase but no significant change in the activation of AKT. Vanadate treatment of cells revealed that the reduction in EGFR tyrosine phosphorylation is dependent in part on changes in cellular phosphatase activity. We propose that a portion of the oncogenic effect of AIB1 could be through control of EGFR and HER2 activity and subsequent modulation of cellular signaling pathways.

The steroid receptor coactivator oncogene, amplified in breast cancer 1 (AIB1; also known as ACTR/RAC-3/TRAM-1/SRC-3/p/CIP), is amplified and overexpressed in a variety of epithelial tumors. AIB1 has been reported to have roles in both steroid-dependent and steroid-independent transcription during tumor progression. In this report, we describe that the cellular levels of AIB1 are controlled through regulated proteasomal degradation. We found that serum withdrawal or growth in high cell density caused rapid degradation of AIB1 protein, but not mRNA, in immortalized cell lines. Proteasome inhibitors prevented this process, and high molecular weight ubiquitylated species of AIB1 were detected. Nuclear export was required for proteasomal degradation of AIB1 and involved the ubiquitin ligase, E6AP. AIB1/E6AP complexes were detected in cellular extracts, and reduction of cellular E6AP levels with E6AP short interfering RNA prevented proteasomal degradation of AIB1. Conversely, overexpression of E6AP promoted AIB1 degradation. The COOH terminus of AIB1 interacted with E6AP in vitro and deletion of this region in AIB1 rendered it resistant to degradation in cells. From our results, we propose a model whereby signals promoted by changes in the cellular milieu initiate E6AP-mediated proteasomal degradation of AIB1 and thus contribute to the control of steady-state levels of this protein.

The majority of human neoplasms have aberrations in the retinoblastoma pathway due to hyperactivation of cyclin-dependent kinases (CDK). Based on this observation, novel small molecules, such as flavopiridol and UCN-01, are being developed and are currently being tested in the clinic. Efforts to develop CDK modulators led us to the discovery of a novel class of CDK inhibitors, the paullones [Cancer Res 1999;59:2566]. Initial studies demonstrated that paullones inhibit CDKs in vitro, thereby blocking cell-cycle progression. However, the exact mechanism for the antiproliferative effects of paullones was never explored. In this report, we demonstrate for the first time that the most potent paullone, alsterpaullone (Alp), induced apoptosis and promoted loss in clonogenicity in the Jurkat cell line. Alp caused early activation of both caspase-8 and -9, leading to cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Moreover, apoptosis by Alp was not associated with loss in anti-apoptotic proteins such as XIAP or BCL-XL. Pre-incubation with cell-permeable inhibitors z-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethylketone and benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (ZVAD) blocked Alp-induced apoptosis. Moreover, the general caspase inhibitor ZVAD blocked the cleavage and activation of most caspases tested except caspase-9. Studies of mitochondrial membrane potential also demonstrated that Alp is able to disrupt mitochondrial potential in the presence of ZVAD, suggesting that the activation of caspase-9 by Alp follows mitochondrial perturbation. Pre-incubation of Jurkat cells with ZVAD did not prevent the depletion of cyclin D3, loss of CDK, or cell-cycle arrest by Alp. In summary, these experiments suggest that Alp activates caspase-9 via mitochondrial perturbation. Active caspase-9 cleaves and activates caspase-8 and caspase-3, leading to apoptosis. In the presence of the general caspase inhibitor ZVAD, the cell-cycle effects of Alp are unaltered while apoptosis is blocked, suggesting that the CDK effects of Alp are not sufficient for Alp-induced apoptosis. Additional studies with paullones are warranted to further characterize their preclinical effects and to explore their potential use in the clinical setting., (Published 2003 Wiley-Liss, Inc.)

Background: Flavopiridol is a flavonoid with antiproliferative effects mediated, in part, by inhibition of cyclin-dependent kinases. Clinical manifestations in a previous Phase I trial in patients with refractory malignancies treated with a 72-h flavopiridol infusion included a proinflammatory syndrome consisting of fever, fatigue, and "local" tumor pain with concomitant alterations in plasma acute-phase reactant proteins., Purpose: The aim of this study was to determine whether the proinflammatory syndrome observed in this trial was associated with modulation of plasma cytokines., Methods: Patients receiving flavopiridol (n = 76) had serial plasma samples drawn preinfusion and during the infusion for evaluation of interleukin (IL)-6, IL-10, IL-12, granulocyte macrophage colony-stimulating factor, basic-fibroblast growth factor, transforming growth factor-beta, and tumor necrosis factor-alpha levels by standard ELISA assays. The Wilcoxon signed rank test was used to test the significance of the difference between the baseline (time 0) plasma cytokine levels compared with the values of each subsequent data collection time points (8, 24, 48, and 72 h)., Results: There was a significant and sustained increase in plasma IL-6 levels at all time points when compared with baseline values. Paired values were used in the statistical analysis. Median plasma (interquartile range) values of IL-6 were elevated from 15.5 (9-52) pg/ml at baseline to 23 (4-48) pg/ml (P < 0.01) at 8 h; from 15 (2-48) pg/ml at baseline to 46 (21-105) pg/ml (P < 0.001) at 24 h; from 16 (9-52) pg/ml at baseline to 61 (32-170) pg/ml (P < 0.001) at 48 h; and from 15.5 (6-48) pg/ml to 68 (40-200) pg/ml (P < 0.001) at 72 h. Significance was maintained even when adjusted for multiple comparisons. The relative increase in IL-6 concentration was dose-dependent. Moreover, IL-6 elevation had a direct correlation with flavopiridol peak plasma concentration, flavopiridol area under the curve, and plasma C-Reactive protein levels. A significant decrease in plasma granulocyte macrophage colony-stimulating factor occurred at the 8-h sampling point: 50 pg/ml (interquartile range 10-205 pg/ml, P < 0.01) when compared with baseline plasma levels and 71 pg/ml (interquartile range 5-152 pg/ml, P < 0.01). No changes in the other pro or anti-inflammatory cytokines were observed. Immunohistochemistry studies in bone marrow aspirates from a prospective group of patients in this trial demonstrated approximately 4-fold induction of IL-6 (compared with baseline), mostly in non-T cells., Conclusion: Biochemical analysis of plasma in patients undergoing infusional flavopiridol found a significant dose-dependent induction of IL-6. IL-6 elevation could be a marker for the process leading to the appearance of the proinflammatory syndrome observed in patients treated with infusional flavopiridol. The mechanism(s) underlying IL-6 induction and its significance are still unknown but may influence strategies to modulate flavopiridol's clinical effects.

Altered and deregulated cyclin-dependent kinase (cdk) activity is now believed to play a major role in the pathogenesis of head and neck squamous cell carcinomas (HNSCC), thus providing a suitable cellular target for therapeutic intervention. UCN-01 (7-hydroxy-staurosporine), a known protein kinase C and cdk modulator, demonstrates antiproliferative and antitumor properties in many experimental tumor models and may represent a potential candidate to test in HNSCC. In this study, UCN-01 displayed potent antiproliferative properties (IC50 of approximately 17-80 nM) in HNSCC cells. Cell cycle analysis revealed that UCN-01 treatment of HNSCC cells for 24 h leads to a G1 block with a concomitant loss of cells in S and G2-M and the emerging sub-G1 cell population, confirmed to be apoptotic by terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. Additional in vitro studies demonstrated a G1 arrest that was preceded by depletion in cyclin D3, elevation of p21(WAF1) and p27(KIP1) leading to a loss in activity of G1 cdks (cdk2, cdk4), and reduction in pRb phosphorylation. Antitumor properties of UCN-01 were also assessed in vivo by treating HN12 xenografts (7.5 mg/kg/i.p./daily) with UCN-01 for 5 consecutive days. Total sustained abolition of tumor growth (P < 0.00001) was obtained with only one cycle of UCN-01 treatment. Terminal deoxynucleotidyl transferase-mediated nick end labeling staining of xenograft samples revealed a higher incidence of apoptosis in treated tissues when compared with control. Additional tissue analysis demonstrated that elevated p27(KIP1) with minimal increase in p21(WAF1) and reduced cyclin D3 levels were readily detected in those animals treated with UCN-01, similar to those observed in HNSCC cells. Thus, UCN-01 exhibits both in vitro and in vivo antitumor properties in HNSCC models, and these effects are associated with a decrease in cyclin D3 and an increase in p27(KIP1) protein levels, thus providing appropriate surrogate markers to follow treatment efficacy in vivo and, therefore, a suitable drug candidate for treating HNSCC patients.

Alkylphospholipids (ALKs) are a novel class of antineoplastic compounds that display potent antiproliferative activity against several in vitro and in vivo human tumor models. However, the mechanism by which these agents exert this desired effect is still unclear. In this study, we investigated the effect of perifosine, a p.o.-bioavailable ALK, on the cell cycle kinetics of immortalized keratinocytes (HaCaT) as well as head and neck squamous carcinoma cells. All cells were sensitive to the antiproliferative properties of perifosine with an IC(50) of similar0.6-8.9 microM. Cell cycle arrest at the G(1)-S and G(2)-M boundaries was observed in HN12, HN30, and HaCaT cells independent of p53 function, and this effect was preceded by loss in cdc2 and cyclin-dependent kinase (cdk) 2 activity. Analysis of cdk complexes in vitro demonstrated that perifosine, up to 20 microM, did not directly interfere with these enzymes. However, aphidicolin-synchronized HN12 cells released in the presence of perifosine (10 microM) demonstrated increased expression of total p21(WAF1) and increased association of p21(WAF1) with cyclin-cdk complexes resulting in reduced cdc2 activity. HCT116 isogenic cell lines were used to assess the role of p21(WAF1) induction by perifosine. This compound (20 microM) induced both G(1)-S and G(2)-M cell cycle arrest, together with p21(WAF1) expression in both p53 wild-type and p53(-/-) clones. By contrast, p21(-/-) variants demonstrated no p21(WAF1) induction or cell cycle arrest. Similar results were obtained with other ALK congeners (miltefosine and edelfosine). These data, therefore, indicate that perifosine blocks cell cycle progression of head and neck squamous carcinoma cells at G(1)-S and G(2)-M by inducing p21(WAF1), irrespective of p53 function, and may be exploited clinically because the majority of human malignancies harbor p53 mutations.

Purpose: To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV)., Patients and Methods: Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed., Results: The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m(2)/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m(2)/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m(2)/d for 3 days included mean total plasma concentration of 36.4 microM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G(2) checkpoint., Conclusion: UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.

We sought to characterize the interactions of flavopiridol with members of the ATP-binding cassette (ABC) transporter family. Cells overexpressing multidrug resistance-1 (MDR-1) and multidrug resistance-associated protein (MRP) did not exhibit appreciable flavopiridol resistance, whereas cell lines overexpressing the ABC half-transporter, ABCG2 (MXR/BCRP/ABCP1), were found to be resistant to flavopiridol. Flavopiridol at a concentration of 10 microM was able to prevent MRP-mediated calcein efflux, whereas Pgp-mediated transport of rhodamine 123 was unaffected at flavopiridol concentrations of up to 100 microM. To determine putative mechanisms of resistance to flavopiridol, we exposed the human breast cancer cell line MCF-7 to incrementally increasing concentrations of flavopiridol. The resulting resistant subline, MCF-7 FLV1000, is maintained in 1,000 nM flavopiridol and was found to be 24-fold resistant to flavopiridol, as well as highly cross-resistant to mitoxantrone (675-fold), topotecan (423-fold), and SN-38 (950-fold), the active metabolite of irinotecan. Because this cross-resistance pattern is consistent with that reported for ABCG2-overexpressing cells, cytotoxicity studies were repeated in the presence of 5 microM of the ABCG2 inhibitor fumitremorgin C (FTC), and sensitivity of MCF-7 FLV1000 cells to flavopiridol, mitoxantrone, SN-38, and topotecan was restored. Mitoxantrone efflux studies were performed, and high levels of FTC-reversible mitoxantrone efflux were found. Northern blot and PCR analysis revealed overexpression of the ABCG2 gene. Western blot confirmed overexpression of ABCG2; neither P-glycoprotein nor MRP overexpression was detected. These results suggest that ABCG2 plays a role in resistance to flavopiridol.

We have investigated the effects of flavopiridol, a novel protein kinase inhibitor that is selective for cyclin-dependent kinases, on hypoxia-induced vascular endothelial growth factor (VEGF) expression in human monocytes. We found that hypoxia induces a time-dependent increase of VEGF mRNA expression and protein levels in human monocytes. Flavopiridol showed a minimal effect on the constitutive levels of VEGF mRNA but completely blocked hypoxia-induced VEGF mRNA and protein expression. The inhibitory effects of flavopiridol on VEGF mRNA induction also occurred in the presence of cycloheximide. The transcriptional activation of either a VEGF promoter-luciferase construct or a hypoxia-inducible factor 1 reporter plasmid was not affected by addition of flavopiridol in transient transfection experiments. In contrast, actinomycin D experiments demonstrated that flavopiridol dramatically decreased VEGF mRNA stability. These data provide the first evidence that flavopiridol can affect gene expression by altering mRNA stability. We propose that flavopiridol may interfere with one or more signaling events, leading to hypoxia-induced, protein kinase-modulated, RNA protein binding activity. An important clinical implication of our results is that flavopiridol, presently under investigation in clinical trials, might have antiangiogenic as well as direct antiproliferative effects.

Flavopiridol is a novel flavonoid that induces cell cycle arrest at different stages of the cell cycle because of the inhibition of cyclin-dependent kinases (cdks). In previous studies from our laboratory, (B. A. Carlson et al., Cancer Res., 56: 2973-2978, 1996), we observed that exposure of the MCF-7 breast carcinoma cell line to flavopiridol resulted in G1-S arrest, which was associated with the loss of cdk4 and cdk2 activity by 24 h of exposure. Along with this inhibition, flavopiridol decreased total cyclin-D protein levels in this cell line. In this work, we demonstrate that using isoform-specific antibodies, flavopiridol induces an early (by 6 h) decrease in cyclin D1 protein levels. This decline is followed by a decline in cyclin D3 with no effect on cyclin D2 or cyclin E levels by 10 h. Furthermore, at early time points (up to 8 h), the activity of cdk4 and the expression of endogenous phosphorylated retinoblastoma species from intact cells exposed to flavopiridol are unchanged. Thus, the decline in cdk4 activity and the induction of retinoblastoma hypophosphorylation follows cyclin D1 decline. Turnover studies demonstrate that the half-life of cyclin D1 (approximately 30 min) is not shortened in flavopiridol-exposed cells, and that the turnover of cdk4-bound cyclin D1 is unaltered. However, steady-state levels of cyclin D1 mRNA display a significant decrease by 4 h of flavopiridol treatment, with total disappearance by 8 h. This mRNA decline is not abrogated by the presence of cycloheximide. Furthermore, we have found that flavopiridol specifically represses the activity of the full-length cyclin D1 promoter linked to a luciferase reporter gene. In summary, we have found that the flavopiridol-induced decline in cyclin D1 is an early event, specific and, at least in part, due to the transcriptional repression of the cyclin D1 promoter. These results extend our understanding of flavopiridol's action to include regulation of cyclin D1 transcription.

Analysis of the National Cancer Institute Human Tumor Cell Line Anti-Cancer Drug Screen data using the COMPARE algorithm to detect similarities in the pattern of compound action to flavopiridol, a known inhibitor of cyclin-dependent kinases (CDKs), has suggested several possible novel CDK inhibitors. 9-Bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one, NSC-664704 (kenpaullone), is reported here to be a potent inhibitor of CDK1/cyclin B (IC50, 0.4 microM). This compound also inhibited CDK2/cyclin A (IC50, 0.68 microM), CDK2/cyclin E (IC50, 7.5 microM), and CDK5/p25 (IC50, 0.85 microM) but had much less effect on other kinases; only c-src (IC50, 15 microM), casein kinase 2 (IC50, 20 microM), erk 1 (IC50, 20 microM), and erk 2 (IC50, 9 microM) were inhibited with IC50s less than 35 microM. Kenpaullone acts by competitive inhibition of ATP binding. Molecular modeling indicates that kenpaullone can bind in the ATP binding site of CDK2 with residue contacts similar to those observed in the crystal structures of other CDK2-bound inhibitors. Analogues of kenpaullone, in particular 10-bromopaullone (NSC-672234), also inhibited various protein kinases including CDKs. Cells exposed to kenpaullone and 10-bromopaullone display delayed cell cycle progression. Kenpaullone represents a novel chemotype for compounds that preferentially inhibit CDKs.

Cyclin-dependent kinases (CDKs) have been recognized as key regulators of cell cycle progression. Alteration and deregulation of CDK activity are pathogenic hallmarks of neoplasia. Therefore, inhibitors or modulators would be of interest to explore as novel therapeutic agents in cancer, as well as other hyperproliferative disorders. Flavopiridol is a semisynthetic flavonoid that emerged from an empirical screening program as a potent antiproliferative agent that mechanistic studies demonstrated to directly inhibit CDKs 1, 2, and 4 as a competitive ATP site antagonist. Initial clinical trials have shown that concentrations that inhibit cell proliferation and CDK activity in vitro can be safely achieved in humans, and additional clinical trials will establish its clinical potential. To address the need for additional chemotypes that may serve as lead structures for drugs that would not have the toxicities associated with flavopiridol, compounds with a similar pattern of cell growth inhibitory activity in the National Cancer Institute's in vitro anticancer drug screen have been recognized by the computer-assisted pattern recognition algorithm COMPARE and then screened for anti-CDK activity in a biochemical screen. The benzodiazepine derivative NSC 664704 (7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one) was revealed by that approach as a moderately potent (IC50 0.4 microM) inhibitor of CDK2, which in initial experiments shows evidence of causing cell cycle redistribution in living cells. NSC 664704 is, therefore, a candidate for further structural optimization, guided in part by understanding of the ATP-binding site in CDK2. This approach represents one way of combining empirical screening information with structure-based design to derive novel candidate therapeutic agents directed against an important cellular target.