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Indiana University-Purdue University Indianapolis (IUPUI) / The Fanconi anemia (FA) pathway consists of 21 genes that maintain genomic stability and prevent cancer. Biallelic mutations within this network cause Fanconi anemia, an inherited bone marrow failure and cancer predisposition syndrome. Heterozygous inborn mutations in FA genes increase risk of breast/ovarian cancers, and somatic mutations occur in malignancies in non-Fanconi patients. Understanding the tumor suppressive functions of FA signaling is important for the study of Fanconi anemia, inherited cancers, and sporadic cancers. The FA network functions as a genome guardian throughout the cell cycle. In addition to the well-established roles of FA proteins in interphase DNA replication/repair, the FA pathway controls mitosis by regulating the spindle assembly checkpoint (SAC) to ensure proper chromosome segregation. The SAC consists of several tumor suppressors, including Mad2, and SAC impairment predisposes to aneuploidy and cancer. However, the in vivo contribution of SAC dysfunction to malignant transformation of FA-deficient cells remains unknown. Furthermore, the mechanisms by which FA proteins regulate the SAC are unclear. To test whether SAC dysfunction drives genomic instability and tumorigenesis in FA, we generated a novel FA-SAC model by intercrossing Fancc-/- and Mad2+/- mice. The intercrossed mice displayed heightened aneuploidy secondary to exacerbated SAC dysfunction. Importantly, these mice were prone to developing hematologic malignancies, particularly leukemia, faithfully recapitulating the clinical phenotype of Fanconi anemia. Upon establishing SAC dysfunction as a driver of tumorigenesis in FA, we next explored the mechanism by which FANCC regulates the SAC. We demonstrated that the mitotic kinase CDK1 phosphorylates FANCC to regulate subcellular localization and SAC function of FANCC during mitosis. Our study highlights the essential role of compromised chromosome segregation in the development of leukemia due to impaired FA signaling. This work furthers our knowledge of FANCC signaling at the SAC, and has implications for future use of mitotic-centered therapies for FA-associated tumors. / 2 years

Indiana University-Purdue University Indianapolis (IUPUI) / The ubiquitin-proteasome system is responsible for the targeted degradation of proteins within the cell. The 26S proteasome, which is the protease of this system, is a high molecular weight complex consisting of 33 subunits that arrange to form two smaller complexes the 19S regulatory particle (RP) and the 20S core particle (CP). The 19S RP can bind one or both ends of the 20S CP and is responsible for recognizing the ubiquitinated substrates. After recognition, the 19S RP will subsequently deubiquitinate, unfold, and translocate the substrates into the proteolytic 20S CP. The 20S CP consists of seven unique alpha and seven unique beta subunits that arrange into four stacked rings, with two alpha rings capping two beta rings. Assembly of the alpha(1-7)beta(1-7)beta(1-7)alpha(1-7) structure begins with the formation of an alpha ring and proceeds through specific assembly intermediates. This process is assisted by assembly chaperone proteins that promote on pathway interactions to efficiently construct the 20S CP. In this dissertation, three new findings are described which further characterize the proteasome assembly pathway. First, novel non-canonical complexes comprised of proteasome subunit alpha4 were identified in vivo, revealing proteasome subunits can assemble into complexes outside of the proteasome. Second, Hsp70 proteins, Ssa1/2, were shown to assist in the assembly of 20S CPs, adding to the growing list of proteins guiding proteasome assembly. Third, a novel complex was identified which is believed to represent a new proteasome assembly intermediate.

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