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Cantor Lab Projects

What is wrong with FA cells?

With the development of several new assays to analyze replication fork dynamics, we are investigating how MSH2 contributes to replication restart defects in Fanconi anemia (FA) patient cells. Given that MSH2-dependent DNA breaks form at stalled replication forks in cells lacking the FANCJ/MLH1 interaction, we are exploring if MSH2 is recruited to a distinct replication stress induced DNA structure. Using proteomics, we also are defining the MSH2 dependent pathway that causes the replication stress in FA cells. Ultimately, the goal will be to determine if leveraging MSH2 or associated functions could provide a therapeutic strategy in FA to reduce bone marrow failure.  Moreover, given that MSH2 loss confers bypass of replication stress, we propose to investigate if MSH2 loss correlates and predicts the onset of leukemia in FA patients. 

Model of FANCJ-MLH1 interaction with MSH2. Replication forks are blocked by barriers, such as secondary structures formed during replication stress. The FANCJ-MLH1 interaction coordinates with the MMR pathway to restart replication forks. MSH2 heterodimers bind replication barriers, such G4 quartets. FANCJ DNA helicase/translocase activity both displaces MSH2 and unwinds secondary structures to enable restart.  In FANCJ null cells, nucleolytic processing removes secondary structures and induces breaks useful for restoring replication. However, MSH2 depletion does not alter this processing because FANCJ is not available to unwind secondary structure.  In cells with no FANCJ-MLH1 interaction, FANCJ fails to displace MSH2 from secondary structure. MSH2 blocks restart and FANCJ blocks nucleolytic processing. Consequently, stalled forks collapse. MLH1 depletion will not rescue because MSH2 is “locked on” the replication barrier. In contrast, MSH2 depletion will rescue because FANCJ gains ability to unwind replication barrier and replication resumes (Peng et al., EMBO 2014).  Model of FANCJ-MLH1 interaction with MSH2.

Does FANCJ prevent or cause cancer?

The idea that FANCJ is not just a tumor suppressor, but also could be oncogenic comes from two findings.  First, in some tumors FANCJ is overexpressed, such as 11% of breast invasive carcinoma (cBioPortal).  Second, in vitro studies reveal FANCJ helicase domain mutants can have enhanced enzyme activity. Moreover, when expressed in Fanconi anemia patient cells (null for FANCJ), these mutants elevate resistance to DNA damaging agents, such as cisplatin and ultraviolet light. Understanding the functional outcomes of FANCJ mutations will be essential for effectively treating associated cancers. Tumors that lack functional FANCJ, and are defective in DNA repair should be sensitive to DNA damaging agents such as cisplatin.  A completely different approach will be needed for tumors in which FANCJ could be an oncogenic factor (i.e. over-expressed or unregulated).

Model depicting loss and gain of function for FANCJ in cancer.

Model depicting loss and gain of function for FANCJ in cancer.


What is the role of FANCJ in replication fork dynamics? 

Our collective findings indicate that to balance genome preservation with survival in the face of replication stress, FANCJ is essential. Whereas a FANCJ-BRCA1 interaction promotes a replication stress checkpoint to favor repair and limit bypass, we find that a FANCJ-MLH1 interaction promotes recovery from replication stress by counteracting toxic fork processing events. Despite this understanding it is unclear how FANCJ interactions direct its function to checkpoint and restart functions.  Thus, we have developed several assays to study replication fork dynamics and are interrogating how FANCJ or its interactions contribute to replication fork associated proteins, preservation of DNA sequences or structures, fork integrity, and recovery from replication stress.  Key questions are if one or more of these functions are disrupted by clinical mutations or could be useful targets for cancer therapy.

Potential role for FANCJ in the replication stress response Figure 

Potential role for FANCJ in the replication stress response: In response to replication arrest, FANCJ interactions with ToBP1 and BRCA1 promote checkpoint activation and inhibit translesion synthesis (TLS).  Following repair processing, FANCJ interaction with MLH1 and helicase/translocase activities promote replication restart through displacement of MSH2 complexes and unwinding of secondary structures, such as G4s as shown. (Cantor and Nayak, Mut Research 2016)  


How Do DNA repair defective tumors become resistant to chemotherapy?

Having performed a whole-genome screen and identified genes, such as CHD4 whose loss promotes cisplatin resistance in BRCA2 mutant cancer, we seek to identify the mechanism of enhanced cisplatin resistance and if this mechanism also provides resistance to other chemotherapies, such as inhibitors of PARP. Resistance through the restoration of replication fork integrity is a current model we are investigating.  Low CHD4 mRNA levels in BRCA2-mutant ovarian cancers significantly correlated with shorter progression free and overall survival. Thus, we will also determine if CHD4-associated factors or other genes found in our screen predict patient survival. Because CHD4 loss uniquely rescues a BRCA2 mutant background and BRCA2-associated cancers typically retain an N-terminal protein, we are determining if this N-terminal mutant protein retains a function important for cisplatin resistance and is therefore a worthy target.  Lastly, we are developing assays to identify novel agents that either enhance the efficacy of current therapies or work effectively as monotherapy to kill otherwise chemoresistant cancer.

Model of selective therapy and chemoresistance in BRCA-cancer.