David Pellman - US grants

DESCRIPTION: The goal of this project is to understand the mechanism of chromosome segregation in yeast. Defining this process will be essential for understanding the basis for the defects in chromosome segregation observed in cancer cells. Our approach is to use the facile genetics of yeast to identify genes required for anaphase, determine the molecular function of the cloned genes, and define the cell cycle control of the encoded proteins. This laboratory has identified Ase1, a novel spindle component that localizes to the midzone of the anaphase spindle. The analysis of ase1 mutants suggests that Ase1 plays a key role in maintaining the interaction between the two halves of the anaphase spindle. Recently, we have found that the expression of Ase1 is strikingly similar to that of yeast B-type cyclins. ASE1 is both coordinately transcribed with B-type cyclins and is also abruptly degraded at the end of mitosis. In both yeast cells and Xenopus egg extracts we have found that the cell cycle-specific degradation of Ase1 is mediated by the Anaphase Promoting Complex (APC), the regulatory apparatus that targets cyclins for ubiquitination and subsequent degradation. Our observations on Ase1 regulation suggest a mechanism for how different stages of mitosis might be ordered: stage-specific events may be dependent upon the regulated expression of rate-limiting components. To characterize what may be the first of a number of cyclin-like spindle proteins in yeast, we propose experiments to determine the molecular function of Ase1, to characterize its cell cycle-specific regulation, and to characterize genes that are required with ASE1 for chromosome segregation.

DESCRIPTION (provided by applicant): The goal of this project is to comprehensively understand the molecular mechanisms leading to the positioning of the mitotic spindle in the budding yeast Saccaromyces cerevisiae. This process underlies asymmetric cell division and development in many cell types. Spindle positioning involves the interaction of astral microtubules (MTs) with polarized sites on the cell cortex. In many cases, this process also requires the actin cytoskeleton. The capture of MT at the cell cortex shares similarities with the capture of MTs at the kinetochore and some proteins are clearly involved in both processes. Thus, understanding mechanisms for spindle positioning may also impact upon the maintenance of genome stability. Our studies therefore address mechanisms that impact on human health through implications for development and cancer. Our approach utilizes genetic, biochemical, and imaging experiments. It also benefits from our recently solved structure of one component of the system, the Bnil FH2 domain. Building on our previous work to characterize proteins associated with the plus ends of MTs and proteins involved and aspects of actin assembly that are required for spindle orientation we propose experiments with the following aims: Aim1: How is the force generated to orient preanaphase spindles? Aim 2: How is dynein targeted to its site of action and how is dynein activity regulated? Aim 3: How is formin-dependent actin assembly regulated in vivo?

DESCRIPTION (provided by applicant): All cancer therapies must be based on differences between tumor cells and the normal tissue from which they arise. One obvious, but largely unexploited, difference between tumor cells and normal cells is aneuploidy. Aneuploidy (abnormal chromosome number), including near polyploidy (increased genome sets), is a prominent feature of most cancers yet the consequences of this alteration of the genome is poorly understood. Aneuploidy can either be due to abnormal numbers of whole chromosomes, which originate from mitotic chromosome segregation errors, or to structural rearrangements of chromosomes, which originate from DNA breaks and recombination. This laboratory has focused on whole chromosome aneuploidy because it is one of the most mysterious aspects of tumor biology; there has been a one hundred year debate over whole chromosome aneuploidy: Is it beneficial, detrimental, or a completely neutral passenger phenomenon during tumor development? The uncertainty about the contribution of aneuploidy to tumorigenesis is largely fueled by the paucity of mechanisms explaining how aneuploidy impacts tumorigenesis or the properties of mature cancers. This grant will address two key questions about aneuploidy and cancer. In the first two aims, I will characterize potential mechanisms by which whole chromosome aneuploidy could initiate/promote cancer. The first aim will address whether chromosome segregation errors can produce DNA damage. A series of imaging and biochemical experiments will test the hypothesis that micronuclei, generated by whole chromosome mis-segregation, have abnormal chromatin and/or nuclear architecture that leads to defective DNA replication and chromosome breaks. The second aim will define the role of a specific recurrent aneuploidy - extra copies of chromosome 8 - in tumor development. Although polysomy occurs in many tumor types, the experiments will focus on the development of acute myelogenous leukemia because of methods to generate myeloid progenitor cells that do or do not contain polysomy 8. In the third Aim, we will determine if polysomy 8 causes vulnerabilities that can be exploited to discover new cancer drug targets. This third aim follows directly from previous work under this grant that defined 'ploidy-specific lethality', where genes that are not essential in normal diploid cells become essential in polyploid cells or extra centrosome-containing cells that have a chromosomal instability phenotype.

[unreadable] DESCRIPTION (provided by applicant): This proposal focuses on cytoskeletal mechanisms necessary for normal mitotic exit: cytokinesis and spindle disassembly. The proposal builds on three series of novel findings. First, in budding yeast we discovered a mechanism by which the Polo kinase (Cdc5) controls cytokinesis. In late mitosis, Cdc5/Polo targets guanine nucleotide exchange factors (GEFs) for the small G-protein Rho1 (human RhoA) to the division site. The GEFs, in turn, recruit the small G-protein Rho1. Rho1 targeting and activation are essential for the recruitment of formins, actin nucleators required for contractile actin ring (CAR) assembly in all eukaryotes. Similar mechanisms were recently shown to explain the role of human Polo-like kinase in CAR assembly in human cells. Second, we have unpublished evidence that the yeast formin Bni1 is regulated by a novel mechanism involving regulated proteolysis. Third, we have defined the biochemical properties of a key regulator of spindle disassembly. Kip3, a member of the Kinesin 8 family of proteins, is both a plus end directed MT motor and a plus end-specific MT depolymerase. We defined new mechanisms regulating the Kip3 depolymerase activity which may be central to controlling spindle dynamics during late mitosis. We now propose experiments to (1) define the mechanism of Rho1 targeting to the division site and the mechanism controlling global Rho1 activation during mitotic exit; (2) Define the mechanisms and biological role of regulated formin degradation; (3) Characterize novel Kip3/Kinesin 8 regulatory mechanisms and define their role in spindle disassembly and microtubule dynamics. The proposed research is relevant to cancer biology because cytokinesis failure is known to promote tumorigenesis. PUBLIC HEALTH RELEVANCE: This proposal will define mechanisms that mediate changes in the cytoskeleton necessary for mitotic exit: cytokinesis and disassembly of the mitotic spindle. The results will be relevant to cancer biology because abnormal cytokinesis is known to promote tumorigenesis. [unreadable] [unreadable] [unreadable]

PROJECT SUMMARY/ABSTRACT The primary goal of this proposal is to elucidate the mechanism of a newly discovered mutational process called chromothripsis. Chromothripsis generates rapid karyotype evolution in cancer, congenital disease, and other contexts. Chromothripsis is characterized by extensive genomic rearrangements and an oscillating pattern of DNA copy number levels, all surprisingly restricted to one or a few chromosomes. We have made significant recent progress in defining a mechanism for chromothripsis. We showed that intact chromosomes missegregated into aberrant cancer-associated nuclear structures called micronuclei (MN) develop extensive DNA damage. This led us to propose that the physical isolation of chromosomes into MN could cause chromothripsis. Recently, we developed a method (Look-Seq) to combine live cell imaging and single cell genome sequencing, enabling us to recreate chromothripsis in the laboratory and directly demonstrate that it can originate from disrupted MN. We established that chromothripsis could arise by fragmentation and reassembly of MN chromosomes. These fragments can also circularize, potentially providing a mechanism for forming double minute chromosomes, major vehicles for oncogene amplification in cancer. The Look-Seq approach now positions the laboratory to attack the key mechanistic questions in the field: the timing and order of chromosome fragmentation and reassembly, the mechanism of MN chromosome fragmentation; and how it is reassembled. We also propose a series of experiments to define the contribution of DNA replication errors in generating localized chromosome rearrangements. Finally, we propose experiments to elucidate the mechanistic basis for two major sources of oncogene amplification in cancer.

Cancer Cell Biology Program Project Summary / Abstract The mission of the Cancer Cell Biology Program is to facilitate basic cancer research and accelerate the application of basic science discoveries in the clinic. The Program has three Specific Aims: 1) To elucidate the pathophysiology of cancer; 2) To support the development and dissemination of new technologies for cancer research; and 3) To facilitate the clinical translation of basic scientific discoveries. The program has 97 members, representing seven DF/HCC institutions and 17 academic departments. In 2014 peer-reviewed grant funding attributed to the Program was $23.8 million in total costs from the NCI and $31.9 million from other sponsors. During the current funding period, Cancer Cell Biology Program members published 1,769 cancer- relevant papers. Of these 24% were inter-institutional, 9% were intra-programmatic, and 34% were inter- programmatic collaborations between two or more DF/HCC members. Overall, when counted once, 27% of DF/HCC publications were inter-programmatic collaborations.