Randall Evans

Sorafenib induces apoptosis of AML cells via Bim-mediated activation of the intrinsic apoptotic pathway

Raf/MEK/Erk signaling is activated in the majority of acute myeloid leukemias (AMLs), providing rationale for targeting this pathway with therapeutic intent. We investigated growth-inhibitory and proapoptotic effects of sorafenib in AML. Our studies demonstrated that sorafenib significantly inhibited the phosphorylation levels of Raf downstream target proteins MEK1/2 and Erk, induced apoptosis and inhibited colony formation in AML cell lines and in primary AML samples. Mechanistically, treatment with sorafenib resulted in upregulation of proapoptotic Bim, accompanied by an increase in Bad, Bax and Bak protein levels and decreased Mcl-1, X-linked inhibitor of apoptosis and surviving levels, which mainly led to the activation of the intrinsic apoptotic pathway. Silencing of Bim protein expression significantly abrogated sorafenib-induced apoptosis, suggesting a critical function of Bim in the activation of the intrinsic mitochondrial pathway induced by sorafenib. Importantly, sorafenib also modulated phospho-Erk, Bim, Bax and Mcl-1 levels in samples procured from patients in an ongoing Phase I clinical trial of sorafenib in AML. Combination of sorafenib with cytarabine or the novel small molecule Bcl-2 inhibitor ABT-737 synergistically induced cell death in AML cell lines. Our results strongly suggest potential activity of sorafenib as a novel mechanism-based therapeutic agent in AML.

NudC Is Required for Plk1 Targeting to the Kinetochore and Chromosome Congression

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.

p53 Mutations in Hairless SKH-hr1 Mouse Skin Tumors Induced by a Solar Simulator

In this study, we investigated whether the spectrum of p53 mutations in skin tumors induced in hairless SKH‐hr1 mice by a solar simulator (290–400 nm) are similar to those found in skin tumors induced in C3H mice by UV radiation from unfiltered (250–400 nm) and Kodacelfiltered (290–400 nm) FS40 sunlamps. Analysis of tumor DNA for p53 mutations revealed that 14 of 16 (87.5%) SkH‐hr1 skin tumors induced by the solar simulator contained mutations. Single C → T transitions at dipyrimidine sequences located on the nontranscribed DNA strand were the most predominant type of p53 mutation. Remarkably, 52% of all p53 mutations in solar simulator‐induced SKH‐hr1 skin tumors occurred at codon 270, which is also a hotspot in C3H skin tumors induced by unfiltered and Kodacel‐filtered FS40 sunlamps. However, T → G transversions, which are hallmarks of UVA‐induced mutations, were not detected in any of the solar simulator‐induced skin tumors analyzed. These results demonstrate that the p53 mutation spectra seen in solar simulator‐induced SKH‐hr1 skin tumors are similar to those present in unfiltered and Kodacel‐filtered FS40 sunlamp‐induced C3H skin tumors. In addition, our data indicate that the UVA present in solar simulator radiation does not play a role in the induction of p53 mutations that contribute to skin cancer development.

Persistence of p53 mutations and resistance of keratinocytes to apoptosis are associated with the increased susceptibility of mice lacking the XPC gene to UV carcinogenesis

Like xeroderma pigmentosum (XP) patients, transgenic mice lacking nucleotide excision repair (NER) genes such as XPA and XPC are extremely susceptible to ultraviolet (UV)-induced skin cancer. Because the p53 gene is an important target for UV carcinogenesis and because the p53 protein modulates NER, we investigated the consequences of NER deficiency on UV-induced p53 mutations in XPC−/− mouse skin tumors. Thirty-eight (76%) of 50 UV-induced XPC−/− skin tumor analysed displayed C→T or CC→TT transitions at dipyrimidine sites on the untranscribed strand of the p53 gene. A major hot spot for p53 mutation occurred at codon 270, which is also a hot spot in UV-induced skin tumors from NER-proficient C3H and SKH-hr 1 mice. Interestingly, codon 270 mutations were induced in both XPC−/− and +/+ mouse skin after 1 week of UV irradiation, but the mutations persisted only in XPC−/− mouse skin after 3 – 4 weeks of chronic UV. The persistence of UV-induced p53 mutations in XPC−/− mouse skin was associated with decreased apoptosis and increased proliferation of keratinocytes, suggesting that these events may contribute to the accelerated development of UV-induced skin tumors in XPC−/− mice.

Targeted therapy of AML new concepts.

In aspergillus infections of the central nervous system (CNS) the mortality risk usually exceeds 90%. Data from case-reports and a recent retrospective study suggest that neurosurgical interventions, such as abscess resections, stereotactic drainages, or the use of intraventricular catheters, might improve the outcome in CNS aspergillosis. However, there is a lack of clear evidence supporting an extensive neurosurgical management in these patients. A major reason for the devastating prognosis in CNS aspergillosis is a poor penetration of antifungal drugs into the CNS, with the exception of voriconazole. Treatment with voriconazole results in measurable drug levels in the cerebrospinal fluid, which may exceed the minimal inhibitory concentration for aspergillus. Moreover, voriconazole brain tissue levels exceed those measured for other antifungal drugs. In a recent retrospective study, a complete or partial response occurred in 35% of patients who were treated with voriconazole for CNS aspergillosis with a survival rate of 31%. These data support the use of voriconazole in this clinical setting. An intense neurosurgical management and higher doses of voriconazole might further improve the outcome in CNS aspergillosis, but this needs to be evaluated in future studies.

Nuclear localization is required for induction of apoptotic cell death by the Rb-associated p84N5 death domain protein.

The mechanisms utilized to transduce apoptotic signals that originate from within the nucleus, in response to DNA damage for example, are not well understood. Identifying these mechanisms is important for predicting how tumor cells will respond to genotoxic radiation or chemotherapy. The Rb tumor suppressor protein can inhibit apoptosis triggered by DNA damage, but how it does so is unclear. We have previously characterized a death domain protein, p84N5, that specifically associates with an amino-terminal domain of Rb protein. The p84N5 death domain is required for its ability to trigger apoptotic cell death. Association with Rb protein inhibits p84N5-induced apoptosis suggesting that it may be a mediator of Rbs effects on apoptosis. Unlike other death domain-containing apoptotic signaling proteins, however, p84N5 is localized predominantly within the nucleus of interphase cells. Here we test whether p84N5 requires nuclear localization in order to trigger apoptosis. We identify the p84N5 nuclear localization signal and demonstrate that nuclear localization is required for p84N5-induced apoptosis. To our knowledge, this identifies p84N5 as the first death-domain containing apoptotic signaling protein that functions within the nucleus. By analogy to other death domain containing proteins, p84N5 may play some role in apoptotic signaling within the nucleus. Further, p84N5 is a potential mediator of Rb proteins effects on DNA damage induced apoptosis.