FANCA Fine-Tunes Chromosome Segregation By Controlling BUBR1^K250 Acetylation at the Kinetochores

Fanconi anemia (FA) is an inherited bone marrow failure syndrome associated with genomic instability, high risk of acute myeloid leukemia (AML) and other malignancies. Somatic mutations within the FA/BRCA signaling network occur in AML in the general population, reflecting the importance of FA genes in tumor suppression. While the role of FA signaling in DNA damage repair and replication is well-established, we and others found that the FA network is essential for error-free chromosome segregation during cell division. Both interphase and mitotic errors contribute to the evolution of genomic instability during FA-/- human and murine hematopoiesis in vivo. However, the molecular mechanisms of FA pathway-dependent genome housekeeping during mitosis are incompletely understood.

Through a synthetic lethal kinome-wide shRNA screen in FANCA patient cells, we discovered interphase and mitotic phosphosignaling networks that FANCA-/- cells depend on for survival, including the BUB1-BUBR1 axis of the spindle assembly checkpoint (SAC). BUB1 and BUBR1 are essential SAC kinases that prevent premature anaphase onset and chromosome mis-segregation by inhibiting the APC (anaphase-promoting complex) ubiquitin ligase at the centromeres until all kinetochores achieve correct attachment to the spindle microtubules. Our super-resolution microscopy and biochemistry experiments revealed that FANCA shuttles to kinetochores upon mitotic entry and physically interacts with BUB1 and BUBR1 at the kinetochore-microtubule attachment sites in attachment- and tension-dependent manner. Consistent with impaired SAC, we found that that anaphase onset as well as APC-mediated degradation of cyclin B1, BUBR1 and CDC20 all occur prematurely in FANCA-/- cells. We found that FANCA is essential for BUBR1 lysine 250 (K-250) acetylation at prometaphase kinetochores, and we confirmed that endogenous BUBR1^K250 acetylation is disrupted in FANCA-/- primary patient cells using a validated acetyl-specific antibody. BUBR1^K250 acetylation event works as a molecular switch in which BUBR1 is converted from a degradation target to a potent inhibitor of the APC ligase. Further, we observed that loss of FANCA disrupts kinetochore recruitment of the BUBR1^K250 acetyltransferase PCAF and its upstream regulator, FANCD1/BRCA2.

Our findings establish the first mechanistic connection between FANCA, the canonical SAC tumor suppressor cascade and the FA effector FANCD1/BRCA2. These findings further our understanding of the mechanisms of genomic instability and carcinogenesis resulting from loss of FA signaling. Since impaired BUBR1^K250 acetylation causes chromosomal instability and cancer in vivo, our results have a direct translational relevance.

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