Scientists from the Cancer Science Institute of Singapore (CSI Singapore) at the National University of Singapore report that RUNX proteins are integral to efficient DNA repair via the Fanconi Anemia (FA) pathway. The team undertook a thorough molecular characterization of RUNX proteins in the FA pathway that showed that the proteins had substantial physical association with DNA repair complexes and co-regulated the recruitment of DNA repair protein FANCD2 to sites of DNA damage.
The findings open opportunities to target RUNX-deficient cancers such as breast cancer and leukemia, according to the researchers whose study (“RUNX Poly(ADP-Ribosyl)ation and BLM Interaction Facilitate the Fanconi Anemia Pathway of DNA Repair”) appears in Cell Reports.
“The Fanconi anemia (FA) pathway is a pivotal genome maintenance network that orchestrates the repair of DNA interstrand crosslinks (ICLs). The tumor suppressors RUNX1 and RUNX3 were shown to regulate the FA pathway independent of their canonical transcription activities, by controlling the DNA damage-dependent chromatin association of FANCD2. Here, in further biochemical characterization, we demonstrate that RUNX3 is modified by PARP-dependent poly(ADP-ribosyl)ation (PARylation), which in turn allows RUNX binding to DNA repair structures lacking transcription-related RUNX consensus motifs. SILAC-based mass spectrometric analysis revealed significant association of RUNX3 with core DNA repair complexes, including PARP1, even in unstressed cells,” write the investigators.
“After DNA damage, the increased interaction between RUNX3 and BLM facilitates efficient FANCD2 chromatin localization. RUNX-Walker motif mutations from breast cancers are impaired for DNA damage-inducible PARylation, unveiling a potential mechanism for FA pathway inactivation in cancers. Our results reinforce the emerging paradigm that RUNX proteins are tumor suppressors with genome gatekeeper function.”
The study was led by Yoshiaki Ito, Ph.D., senior principal investigator at CSI Singapore, and involved senior research scientist Vaidehi Krishnan, Ph.D., and former CSI Singapore researcher Lavina Tay, Ph.D.
"Humans are constantly exposed to mutagenic DNA damaging lesions called DNA interstrand crosslinks (ICLs) which can be induced by environmental sources such as formaldehyde, and chemotherapeutics such as cisplatin. Left unrepaired, ICLs could drastically increase predisposition to cancer," explains Dr. Ito.
People with an impaired FA pathway have shown to have a dramatic increase in cancer incidence. Hence, identifying the mechanisms by which the pathway is regulated could have far-reaching clinical potential.
RUNX proteins are tumor suppressors in many cancers. "RUNX1 is aberrant in several hematopoietic cancers while RUNX3 expression is turned off in many solid cancers. How and under what contexts RUNX proteins exert their role in tumor suppression is still very much an unanswered question," notes Dr. Ito.
To determine if RUNX proteins bind to DNA during repair, the researchers tested RUNX binding to DNA repair intermediates. Observations showed the RUNX proteins preferentially bound to structures that mimicked replication fork or DNA repair intermediates such as single-strand DNA and splayed arm DNA.
Cells were treated with different concentrations of the crosslinker Mitomycin C (MMC) which induces DNA damage. The researchers observed that RUNX proteins bound to DNA repair intermediates in a concentration-dependent manner after MMC exposure. Hence, this indicates that DNA damage specifically increases the binding of RUNX proteins to DNA repair intermediates.
The team further discovered that these repair functions were enabled by a modification induced by the PARP1 enzyme, called poly-ADP-ribosylation.
The team also observed protein-protein interaction between RUNX proteins and BLM -- a protein that is impaired in the genetic disorder Bloom's syndrome -- during the process of DNA repair. In unstressed cells, RUNX3 and BLM interacted at a low level; however, the interaction increased upon exposure to MMC, indicating that the formation of the RUNX3 and BLM complex is dependent on DNA damage.
The combination of RUNX and BLM were depleted and cells were exposed to MMC. Recruitment of DNA repair protein FANCD2 after the depletion of RUNX and BLM were seen to be significantly impaired after exposure to MMC. In contrast, control cells with normal levels of RUNX and BLM exhibited robust recruitment of FANCD2.
The data observed thus showed that RUNX and BLM co-regulated the recruitment of FANCD2 to DNA damage sites.
The study adds an extra layer of complexity of FA pathway regulation, and could make it more possible to develop synthetic-lethal approaches to attack RUNX-deficient cancers, adds Dr. Ito. "By showing how RUNX inactivation weakens DNA repair, the study has unveiled newer opportunities to target RUNX-deficient epithelial cancers at the early stages of carcinogenesis.”
Examples of cancers where RUNX proteins are inactivated include solid tumors of the breast, lung, and stomach and RUNX-deficient leukemias like acute myeloid leukemia.