Udit N. Verma

Histone H3 phosphorylation by ikk-α is critical for cytokine-induced gene expression

Cytokine-induced activation of the IκB kinases (IKK) IKK-α and IKK-β is a key step involved in the activation of the NF-κB pathway. Gene-disruption studies of the murine IKK genes have shown that IKK-β, but not IKK-α, is critical for cytokine-induced IκB degradation. Nevertheless, mouse embryo fibroblasts deficient in IKK-α are defective in the induction of NF-κB-dependent transcription. These observations raised the question of whether IKK-α might regulate a previously undescribed step to activate the NF-κB pathway that is independent of its previously described cytoplasmic role in the phosphorylation of IκBα. Here we show that IKK-α functions in the nucleus to activate the expression of NF-κB-responsive genes after stimulation with cytokines. IKK-α interacts with CREB-binding protein and in conjunction with Rel A is recruited to NF-κB-responsive promoters and mediates the cytokine-induced phosphorylation and subsequent acetylation of specific residues in histone H3. These results define a new nuclear role of IKK-α in modifying histone function that is critical for the activation of NF-κB-directed gene expression.

Enhanced Chemosensitivity to Irinotecan by RNA Interference-Mediated Down-Regulation of the Nuclear Factor-κB p65 Subunit

In preclinical tumor models, inhibition of nuclear factor-κB (NF-κB) has been associated with increased sensitivity to chemotherapeutic agents such as irinotecan (CPT-11). This is based on the fact that a variety of chemotherapy agents such as CPT-11 activate NF-κB to result in the expression of genes such as c-IAP1 and c-IAP2 that might be responsible for the inhibition of chemotherapy-induced apoptosis. In this study, RNA interference [small interfering RNA (siRNA)] was used to down-regulate the NF-κB p65 subunit in the HCT116 colon cancer cell line, and its role, in the presence and absence of CPT-11, was assessed on cell growth and apoptosis. Reduction of endogenous p65 by siRNA treatment significantly impaired CPT-11-mediated NF-κB activation, enhanced apoptosis, and reduced colony formation in soft agar. Furthermore, the in vivo administration of p65 siRNA reduced HCT116 tumor formation in xenograft models in the presence but not the absence of CPT-11 administration. These data indicate that the administration of siRNA directed against the p65 subunit of NF-κB can effectively enhance in vitro and in vivo sensitivity to chemotherapeutic agents.

Protein Phosphatase 2Cβ Association with the IκB Kinase Complex Is Involved in Regulating NF-κB Activity

The NF-κB pathway is important in the control of the immune and inflammatory response. One of the critical events in the activation of this pathway is the stimulation of the IκB kinases (IKKs) by cytokines such as tumor necrosis factor-α and interleukin-1. Although the mechanisms that modulate IKK activation have been studied in detail, much less is known about the processes that down-regulate its activity following cytokine treatment. In this study, we utilized biochemical fractionation and mass spectrometry to demonstrate that protein phosphatase 2Cβ (PP2Cβ) can associate with the IKK complex. PP2Cβ association with the IKK complex led to the dephosphorylation of IKKβ and decreased its kinase activity. The binding of PP2Cβ to IKKβ was decreased at early times post-tumor necrosis factor-α treatment and was restored at later times following treatment with this cytokine. Experiments utilizing siRNA directed against PP2Cβ demonstrated an in vivo role for this phosphatase in decreasing IKK activity at late times following cytokine treatment. These studies are consistent with the ability of PP2Cβ to down-regulate cytokine-induced NF-κB activation by altering IKK activity.

IκB Kinase α Regulates Subcellular Distribution and Turnover of Cyclin D1 by Phosphorylation

IκB kinases (IKKs), IKKα and IKKβ, with a regulatory subunit IKKγ/NEMO constitute a high molecular weight IKK complex that regulates NF-κB activity. Although IKKα and IKKβ share structural and biochemical similarities, IKKα has been shown to have distinct biological roles. Here we show that IKKα plays a critical role in regulating cyclin D1 during the cell cycle. Analysis of IKKα-/- mouse embryo fibroblast cells showed that cyclin D1 is overexpressed and localized in the nucleus compared with parental mouse embryo fibroblasts. IKKα associates with and phosphorylates cyclin D1. Analysis on cyclin D1 mutants demonstrated that IKKα phosphorylates cyclin D1 at Thr286. Reconstitution of IKKα in knockout cells leads to nuclear export and increased degradation of cyclin D1. Further, RNAi-mediated knockdown of IKKα results in similar changes as observed in IKKα-/- cells. These results suggest a novel role of IKKα in regulating subcellular localization and proteolysis of cyclin D1 by phosphorylation of cyclin D1 at Thr286, the same residue earlier found to be a target for glycogen synthase kinase-3β-induced phosphorylation.

Phase 2 study of preoperative radiation with concurrent capecitabine, oxaliplatin, and bevacizumab followed by surgery and postoperative 5-fluorouracil, leucovorin, oxaliplatin (FOLFOX), and bevacizumab in patients with locally advanced rectal cancer: ECOG 3204.

Recent studies have demonstrated the feasibility of combining oxaliplatin with 5‐fluorouracil (5‐FU) or capecitibine and radiation therapy. The addition of bevacizumab to chemotherapy improves overall survival for metastatic disease. We initiated a phase 2 trial to evaluate preoperative capecitabine, oxaliplatin, and bevacizumab with radiation therapy followed by surgery and postoperative 5‐FU, leucovorin, oxaliplatin (FOLFOX) and bevacizumab for locally advanced rectal cancer.

An Open-Label Clinical Trial Evaluating Safety and Pharmacokinetics of Two Dosing Schedules of Panitumumab in Patients with Solid Tumors

PURPOSE This study evaluated safety, pharmacokinetics, and efficacy of 2 dose schedules and 2 infusion times of panitumumab in patients with advanced solid malignancies. PATIENTS AND METHODS This phase I multicenter, open-label study sequentially enrolled patients with advanced solid tumors refractory to standard therapy, or for which no standard therapy exists, to receive panitumumab 6 mg/kg every 2 weeks or 9 mg/kg every 3 weeks. Patients receiving panitumumab every 2 weeks received either all infusions over 60 minutes or a 60-minute infusion for the first dose followed by 30-minute infusions if the first infusion was well tolerated. Patients in the every-3-week cohort received 60-minute infusions. Safety outcomes included the incidence of adverse events and antipanitumumab antibody formation. Pharmacokinetic properties were determined. Efficacy endpoints included response rate and duration of response. RESULTS Eighty-six patients were enrolled; 84 (98%) received panitumumab. Treatment-related adverse events occurred in 90% of patients. Safety profiles were similar between patients receiving 30-minute (n = 20) and 60-minute (n = 43) infusions every 2 weeks and patients receiving panitumumab every 3 weeks (n = 21). Panitumumab exposure at steady state increased dose proportionally, and peak serum concentrations were similar in patients receiving either 30- or 60-minute infusions every 2 weeks. Objective responses were seen in 4 patients (5%) with colon, rectal, esophageal, and bladder cancers. CONCLUSION Similar drug exposures and safety profiles were observed in patients receiving panitumumab 6 mg/kg every 2 weeks with either 30- or 60-minute infusions and antitumor activity was seen in some patients. Exposure increased approximately dose proportionally at steady state.

Interleukin-2-activated hematopoietic stem cell transplantation for breast cancer: investigation of dose level with clinical correlates

Incubating hematopoietic stem cells with IL-2 in vitro for 24 h generates cytotoxic T cells. When infused into patients, these cells may stimulate a graft-versus-tumor (GVT) effect. This clinical trial was designed to assess the ability of IL-2 activated peripheral blood stem cells (PBSC) to reconstitute hematopoiesis, to investigate dose levels and dose-limiting toxicities of IL-2, and to evaluate clinical results and preliminary laboratory effects using a combination of IL-2-activated autologous PBSC followed by IL-2 after transplantation. Sixty-one women with stage II–IV breast cancer were treated. After the administration of carboplatin (200 mg/m2/day for 3 days) and cyclophosphamide (2 g/m2/day for 3 days), patients received autologous PBSC that were cultured in IL-2 for 24 h followed by parenteral administration of IL-2 beginning the day of transplantation. Three escalating doses of IL-2 were evaluated with increasing duration up to 4 weeks. Of the 57 patients receiving IL-2 after tranplantation, 19 patients (33.3%) were unable to complete the planned course of IL-2 therapy due to persistent fevers (n = 9), diarrhea (n = 2), pulmonary capillary leak syndrome (n = 3), development of a rash (n = 1), atrial fibrillation (n = 1), or patient’s request (n = 3). One death occurred during hospitalization. Engraftment of neutrophils occurred on day 11.5 (mean; range 8–21 days) and platelets on day 11.7 (mean; range 7–33 days). The maximal tolerated dose of IL-2 was 6 × 105 IU/m2/day for 4 weeks. Disease-free survival rates for all stages were comparable to current reports in the literature. Preliminary laboratory evaluations include FACScan analysis of the IL-2 activated PBSC demonstrating an increased percentage of CD3+, CD25+, HLA-DR+ T cells. Phenotypically similar cells were present in peripheral blood samples of patients when tested 15 days after transplantation. This study demonstrates successful engraftment with IL-2-activated PBSC after high-dose chemotherapy for women with stage II–IV breast cancer. The regimen is feasible and, although toxicities are common, they are manageable and correlate with increasing dose and duration of IL-2.

Immune reconstitution following bone marrow transplantation

Bone marrow transplantation (BMT) is increasingly being used for a variety of indications including hematological malignancies and some solid tumors. Most patients undergoing BMT have illnesses associated with compromised immune status [107, 124]. The immunodeficiency is further compounded by treatment of the condition and more so by the preparative regimens for BMT itself [76]. For a variable period of time, irrespective of the type of bone marrow graft [40], both innate and acquired arms of the immune system remain profoundly suppressed. Recovery is usually protracted, and it may take several months to years before a fully functioning immune system develops [6, 143]. In allogeneic BMT, the donor-derived lymphohemopoietic cells ultimately reconstitute a functioning immune system [64], while in autologous BMT, the hosts own immune system undergoes a new process of ontogeny [23, 115]. The recovering immune system is confronted with several inter-related events, which ultimately affect the tempo of immunological recovery. Residual immunity in the host is sometimes capable of mounting an effective allogeneic response and can contribute to graft rejection [15, 21]. Graft-versus-host disease (GVHD), strategies directed at its prevention or treatment [35, 37, 91], and certain infections [82] all affect immunological recovery. The graft-versus-leukemia (GVL) effect plays a role in eradication of minimal residual disease, reducing chances of relapse [23, 46, 59, 120]. Immune reconstitution in patients receiving T-cell-depleted syngeneic or autologous grafts follows similar kinetic patterns. In this review, first an attempt has been made to outline the available informa-

Phase II Trial of Preoperative Radiation With Concurrent Capecitabine, Oxaliplatin, and Bevacizumab Followed by Surgery and Postoperative 5-Fluorouracil, Leucovorin, Oxaliplatin (FOLFOX), and Bevacizumab in Patients With Locally Advanced Rectal Cancer: 5-Year Clinical Outcomes ECOG-ACRIN Cancer Research Group E3204

LESSONS LEARNED The 5-year oncologic outcomes from the trial regimen were excellent. However, the neoadjuvant and surgical toxicity of this regimen was significant and was the primary reason for the low compliance with adjuvant systemic therapy.Due to the lack of an improvement in the pathologic complete response rate, the substantial associated toxicity, and the negative phase III trials of adjuvant bevacizumab in colon cancer, this regimen will not be pursued for further study. BACKGROUND The addition of bevacizumab to chemotherapy improves overall survival for metastatic colorectal cancer. We initiated a phase II trial to evaluate preoperative capecitabine, oxaliplatin, and bevacizumab with radiation therapy (RT) followed by surgery and postoperative 5-fluorouracil, leucovorin, oxaliplatin (FOLFOX), and bevacizumab for locally advanced rectal cancer. The purpose of this report is to describe the 5-year oncologic outcomes of this regimen. METHODS In a phase II Simon two-stage design study, we evaluated preoperative treatment with capecitabine (825 mg/m(2) b.i.d. Monday-Friday), oxaliplatin (50 mg/m(2) weekly), bevacizumab (5 mg/kg on days 1, 15, and 29), and RT (50.4 Gy). Surgery was performed by 8 weeks after RT. Beginning 8-12 weeks after surgery, patients received FOLFOX plus bevacizumab (5 mg/kg) every 2 weeks for 12 cycles (oxaliplatin stopped after 9 cycles). The primary endpoint was a pathologic complete response (path-CR) rate of 30%. Fifty-seven patients with resectable T3/T4 rectal adenocarcinoma were enrolled between 2006 and 2010. RESULTS Of 57 enrolled patients, 53 were eligible and included in the analysis. Forty-eight (91%) patients completed preoperative therapy, all of whom underwent curative surgical resection. Nine patients (17%) achieved path-CR. There were 29 worst grade 3 events, 8 worst grade 4 events, and 2 patient deaths, 1 of which was attributed to study therapy. Twenty-six patients (54%) began adjuvant chemotherapy. After a median follow-up period of 41 months, the 5-year overall survival (OS) rate for all patients was 80%. Only 2 patients experienced cancer recurrence: 1 distant (liver) and 1 loco-regional (pelvic lymph nodes), respectively. Both of these patients are still alive. The 5-year relapse-free survival rate was 81%. CONCLUSION Despite the path-CR primary endpoint of this trial not being reached, the 5-year OS and recurrence-free survival rates were excellent. However, the neoadjuvant and surgical toxicity of this regimen was significant and was the primary reason for the low compliance with adjuvant systemic therapy. Because of the lack of an improvement in the path-CR rate, the substantial associated toxicity, and the negative phase III trials of adjuvant bevacizumab in colon cancer, this regimen will not be pursued for further study.

Soluble and insoluble nickel compounds exert a differential inhibitory effect on cell growth through IKKα-dependent cyclin D1 down-regulation

It is well‐known that insoluble nickel compounds possess much more potent carcinogenic activities as compared with soluble nickel compounds. Although it is assumed that the different entry and clearance rate are responsible for the difference, the mechanisms underlying the different carcinogenic activities are still not well understood yet. In the present study, we found that exposure to soluble, but not insoluble nickel compounds, caused a significant inhibition of cell growth and G1/G0 cell cycle arrest, which was concomitant with a marked down‐regulation of cylin D1, an essential nuclear protein for controlling G1/S transition, while both soluble and insoluble nickel compounds showed similar effects on NFκB activation, HIF‐1α protein accumulation and TNF‐α transcription and CAP43 protein expression at same doses range. The down‐regulation of cyclin D1 is due to protein degradation rather than inhibition of transcription, because the nickel compounds treatment did not change cyclin D1 mRNA level, while MG132, the proteasome inhibitor, can rescue the degradation of cyclin D1 caused by soluble nickel compound. Moreover, the soluble nickel‐induced cyclin D1 degradation is dependent on its Thr286 residue and requires IKKα, but not HIF‐1α, which are both reported to be involved in cyclin D1 down‐regulation. Taken together, we demonstrate that soluble, but not insoluble nickel compound, is able to cause cyclin D1 degradation and a cell growth arrest in an IKKα‐dependent manner. Given the role of cyclin D1 and cell proliferation in carcinogenesis, we anticipate that the different effects of soluble and insoluble nickel compounds on cyclin D1 degradation and cell growth arrest may at least partially account for their different carcinogenic activities. J. Cell. Physiol. 218: 205–214, 2009.