The Fanconi anemia signaling network regulates the mitotic spindle assembly checkpoint

Indiana University-Purdue University Indianapolis (IUPUI)Fanconi anemia (FA) is a heterogenous genetic syndrome characterized by progressive bone marrow failure, aneuploidy, and cancer predisposition. It is incompletely understood why FA-deficient cells develop gross aneuploidy leading to cancer. Since the mitotic spindle assembly checkpoint (SAC) prevents aneuploidy by ensuring proper chromosome segregation during mitosis, we hypothesized that the FA signaling network regulates the mitotic SAC. A genome-wide RNAi screen and studies in primary cells were performed to systematically evaluate SAC activity in FA-deficient cells. In these experiments, taxol was used to activate the mitotic SAC. Following taxol challenge, negative control siRNA-transfected cells appropriately arrested at the SAC. However, knockdown of fourteen FA gene products resulted in a weakened SAC, evidenced by increased formation of multinucleated, aneuploid cells. The screen was independently validated utilizing primary fibroblasts from patients with characterized mutations in twelve different FA genes. When treated with taxol, fibroblasts from healthy controls arrested at the mitotic SAC, while all FA patient fibroblasts tested exhibited weakened SAC activity, evidenced by increased multinucleated cells. Rescue of the SAC was achieved in FANCA patient fibroblasts by genetic correction. Importantly, SAC activity of FANCA was confirmed in primary CD34+ hematopoietic cells. Furthermore, analysis of untreated primary fibroblasts from FA patients revealed micronuclei and multinuclei, reflecting abnormal chromosome segregation. Next, microscopy-based studies revealed that many FA proteins localize to the mitotic spindle and centrosomes, and that disruption of the FA pathway results in supernumerary centrosomes, establishing a role for the FA signaling network in centrosome maintenance. A mass spectrometry-based screen quantifying the proteome and phospho-proteome was performed to identify candidates which may functionally interact with FANCA in the regulation of mitosis. Finally, video microscopy-based experiments were performed to further characterize the mitotic defects in FANCA-deficient cells, confirming weakened SAC activity in FANCA-deficient cells and revealing accelerated mitosis and abnormal spindle orientation in the absence of FANCA. These findings conclusively demonstrate that the FA signaling network regulates the mitotic SAC, providing a mechanistic explanation for the development of aneuploidy and cancer in FA patients. Thus, our study establishes a novel role for the FA signaling network as a guardian of genomic integrity

FANCA safeguards interphase and mitosis during hematopoiesis in vivo

The Fanconi anemia (FA/BRCA) signaling network controls multiple genome-housekeeping checkpoints, from interphase DNA repair to mitosis. The in vivo role of abnormal cell division in FA remains unknown. Here, we quantified the origins of genomic instability in FA patients and mice in vivo and ex vivo. We found that both mitotic errors and interphase DNA damage significantly contribute to genomic instability during FA-deficient hematopoiesis and in nonhematopoietic human and murine FA primary cells. Super-resolution microscopy coupled with functional assays revealed that FANCA shuttles to the pericentriolar material to regulate spindle assembly at mitotic entry. Loss of FA signaling rendered cells hypersensitive to spindle chemotherapeutics and allowed escape from the chemotherapy-induced spindle assembly checkpoint. In support of these findings, direct comparison of DNA crosslinking and anti-mitotic chemotherapeutics in primary FANCA−/− cells revealed genomic instability originating through divergent cell cycle checkpoint aberrations. Our data indicate that FA/BRCA signaling functions as an in vivo gatekeeper of genomic integrity throughout interphase and mitosis, which may have implications for future targeted therapies in FA and FA-deficient cancers

The tumor suppressor CDKN3 controls mitosis

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2(pThr-161) at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKβ phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics