The aims of the Molecular Therapeutics Unit (MTU) is the identification of novel biochemical pathways altered in head and neck cancer (HNSCC) leading to the discovery of small molecules which may modulate molecular events important for oral carcinogenesis, and thus may represent novel chemotherapeutic agents. Furthermore, the unit focuses in the preclinical assessment of biochemical and molecular parameters modulated by these small molecules, which may help to monitor the effects on tumor samples in patients with head and neck cancer receiving these novel treatment modalities. Finally, selected signaling agents developed in the unit are tested in early clinical trials in patients with advanced HNSCC and other malignancies for """"""""proof of principle testing"""""""", in order to assess whether the preclinical effects observed occur also in humans; novel effects observed in human trials are, then, investigated in in vitro and in vivo models in our unit (reverse translation). Basic Aspects: Flavopiridol is a novel cdk inhibitor with antiproliferative activity in in vitro and in vivo models including HNSCC. Initial studies from our lab demonstrated that flavopiridol can induce cell cycle arrest, apoptosis, differentiation, modulate transcription and has anti-angiogenic properties. How exactly flavopiridol produces these effects is still unknown. We are studying the exact mechanism(s) by which flavopiridol promotes cell cycle arrest and apoptosis. UCN-01 is a novel protein kinase C (PKC) inhibitor with antitumor in vitro and in vivo models with unique pharmacological features including induction of cell cycle arrest, apoptosis, and abrogation of G2 checkpoint imposed by DNA-damaging agents. We performed last year, in collaboration with NCI, the first Phase I trial of UCN-01 in patients with refractory neoplasms. In collaboration with Silvio Gutkind's group, we have studied the effects of UCN-01 in HNSCC models. This report will appear shortly in Clinical Cancer Research. UCN-01 potently inhibited the growth of all HNSCC lines tested. The antiproliferative effect observed was associated with G1/S arrest and apoptosis. The G1/S arrest was associated with depletion of cyclin D3, accumulation of p21waf1/p27kip1 , leading to loss in G1 cdk activity. Moreover, in vivo xenograft studies using the HN12 HNSCC model demonstrated a significant antitumor response with UCN-01 treatment. Tissues obtained from these animals revealed clear evidence of loss in cyclin D3 with upregulation of p27kip1. Moreover, apoptosis was also observed. These results suggest that HNSCC patients may be a good population for testing UCN-01 in the Phase II clinical setting. Moreover, some of these cell cycle markers may, indeed, help us in the biochemical assessment of UCN-01 in tissues from patients in UCN-01 clinical trials. Perifosine (per) is a novel alkylphospholipid with antitumor properties, derived from Miltefosine, an approved drug in Europe for the treatment of cutaneous malignancies. The exact mechanism of action is still unknown. In collaboration with Silvio Gutkind's group, we have studied the cell cycle effects of Perifosine in HNSCC models. This effort was published recently in Cancer Research. The antiproliferative effect observed in HNSCC was associated with G1/S and G2/M arrest and apoptosis. The cell cycle arrest observed was associated with increase in p21waf1 protein levels. Moreover, the increased p21waf1 associated with cdk2 and cdk1 kinase, leading to loss in cdk activity. Finally, the cell cycle arrest was dependent on p21waf1, as cells devoid of p21waf1 (HCT116 p21waf1-/-) were refractory to perifosine cell cycle effects. Follow up studies on the effects of Per in p21waf1 biology is being undertaken in my unit. We have established that the accumulation of p21 is due to transcriptional activation of the p21 promoter. Moreover, we have determined that the minimal promoter region required for Per effects is identical to the region required for ras to activate p21 promoter. This region contains several sp1 sites. Mutations in one specific site abolished the activation by ras or Per. Finally, chemical or genetic inhibitors of MEK, downstream kinase for ras (PD98059 or MEKaa, respectively) abolished p21 transactivation, concluding that Per activates the p21 promoter via activation of MEK and modulation of Sp1. This effort is being submitted for publication. Translational/Clinical Aspects: The first Phase I trial of bolus flavopiridol in cancer patients was recently accepted for publication and will be published shortly in the Journal of Clinical Oncology. Based on the encouraging results of this trial, we opened a protocol entitled """"""""Phase II trial of daily bolus flavopiridol for five consecutive days in patients with recurrent/metastatic squamous cell carcinoma of the Head and Neck (SCCHN"""""""" in collaboration with NCI and NIDCD. Thus far, we have treated only two patients. Accrual to this trial is improving and we anticipate treating several patients in the next few months. Moreover, in an effort to understand the main side effect of flavopiridol in humans, flu-like syndrome, we prospectively determined the plasma pro-inflammatory cytokines in patients receiving infusional flavopiridol. We demonstrated that there was a dose and time induction of plasma IL-6. Moreover, bone marrow aspirates (before and during infusion) from some patients revealed that the induction of IL-6 occurred in Non-T bone marrow cells. These results will be published shortly in Clinical Cancer Research. Our unit is studying the mechanism(s) by which flavopiridol may induce this pro-inflammatory cytokine. This information will help us in ameliorating this side effect in future trials, conducted by the unit, in patients receiving flavopiridol. We are completing a Phase I trial of perifosine, in collaboration with NCI. Preliminary translational studies in patients receiving this agent demonstrated that , similar to what was observed in in vitro models, a clear induction of p21waf1 at the protein/mRNA level was observed in oral keratinocytes and lymphocytes obtained from these patients. Based on our preclinical data with perifosine in HNSCC cell lines, we are considering to open a Phase 2 trial of this agent in patients with HNSCC. In our branch, we have started collaboration with NCI to determine the pharmacodynamic effects of small novel molecules in oral keratinocytes in patients enrolled in clinical trials at the Clinical Center. An NIDCR protocol to compare the genomics and proteomics in oral keratinocytes and lymphocytes in patients receiving novel small molecules is being reviewed at the NIDCR IRB. We are in the process of developing a new clinical/translational program at NIDCR for the diagnosis, prognosis and treatment of premalignant oral lesions (leukoplakia). Protocols involving small molecules for the treatment and prevention of leukoplakia are under review. In collaboration with NCI, we are participating in collaborative effort to study prospectively a large cohort of patients afflicted with Fanconi Anemia, a genetic disorder with high incidence of HNSCC. Patients with Fanconi anemia who survive the hematological complications succumb, in the majority of cases, due to HNSCC. We are evaluating these patients to prospectively determine the molecular and biochemical factors responsible for the malignant transformation.
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