Inhibitors of Condensin I as Chemotherapy for Breast Cancer

Institution: University of California, Irvine
Investigator(s): Kyoko Yokomori, Ph.D. -
Award Cycle: 2009 (Cycle 15) Grant #: 15IB-0089 Award: $100,000
Award Type: IDEA
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

Initial Award Abstract (2009)

Condensin I is an essential protein complex required for chromosome structural organization in both mitosis and interphase. Depletion of condensin I results in chromosome segregation defects. In addition, condensin I functions in interphase (period of cell cycle between division phases) in DNA repair, although the underlying molecular mechanism is not completely understood. Interestingly, condensin I functionally interacts with critical cancer drug targets, namely topoisomerase II (essential in the separation of daughter strands at the end of replication) and PARP-1 (key protein involved in repairing DNA damage). This raises the important possibility that condensin I may also be an effective target for breast cancer therapy.

Thus, we plan to develop small molecules that disrupt chromosome association and/or the DNA repair function of condensin I and test their efficacy on breast cancer cell proliferation and damage sensitization. Our aims are to: (1) perform high-throughput screening to identify chemical compounds that inhibit condensin I function, and (2) to refine and analyze the biological activities of the identified compounds.

Based on our preliminary data and work by others, we plan to use three assays to screen the small molecule library for possible inhibitors that can compromise condensin I function. Since condensin I must physically interact with chromosomes to mediate higher-order chromatin organization important for mitosis and interphase functions, we will use a cell-based method to monitor the mitotic chromosome association of fluorescently-labeled condensin I components in the presence of the library compounds. The second portion of the project entails a protein binding assay to monitor possible disruption of the condensin I subunit CNAP1/hCAP-D2 HE domain interaction with PARP-1 by the addition of compounds. Our final assay is a reverse yeast two-hybrid screening that will complement the in vitro protein binding assay. By using both cell biological and biochemical assays, we should be able to maximize the chance to identify condensin I inhibitors.

Our strategy of investigating a condensin I pathway directly involving PARP-1 is the most logical approach, since PARP-1 has already shown great promise as a new therapeutic target. In particular, patients having defects in BRCA 1 or 2 pathways (called “BRCAness” in sporadic breast cancers) depend almost exclusively on PARP-1 for DNA repair functions, so inhibitors of PARP-1, or any associated pathways, are very promising avenues for developing novel treatments.

Final Report (2010)

Condensin I is an essential protein complex important for the maintenance of genome integrity during cell division and DNA repair. We found that condensin I is an important binding partner of PARP1, a promising new target of breast cancer therapy, during the DNA repair process. The goal of this project was to identify inhibitors of condensin I and to test whether condensin I can also be a new therapeutic target.

As proposed, we examined 1,320 different small compounds to find candidates that would interfere with condensin I function, and found three compounds that can displace the fluorescent-tagged condensin I subunit from mitotic chromosomes. In vitro assays to monitor the interaction between the recombinant PARP1 and the PARP1-interacting subunit of condensin I were not pursued at this time since the bacterially-expressed proteins did not interact with each other well, suggesting that additional factor(s) or protein modifications are important for this interaction. However, we were able to demonstrate that the minimum chromosome targeting domain of the condensin I subunit was also effectively displaced by all three compounds, suggesting that the compounds act through this small domain.

Next, we analyzed the biological activities of our compounds further using breast cancer cell models. Our initial immunofluorescence staining assays suggest that none of these compounds affect the integrity of the mitotic spindle apparatus. Instead, we found that at least one of these compounds could decrease condensin I’s association with DNA damage sites, suggesting that it may interfere with condensin I’s function in DNA repair. Most importantly, this same compound appears to be very toxic to a BRCA1 mutant breast cancer cell line, which is also sensitive to PARP inhibitors. It also had some radiosensitizing effect against a sporadic breast cancer cell line, supporting our contention that this inhibitor may act as a sensitizer for DNA damage-inducing chemo- and radiotherapies. Although we ran out of time to perform further refinement of these compounds, the results we have obtained during the project period strongly support “proof-of-principle” that it is possible to identify inhibitors of condensin I that may be effective as new agents against breast cancers.

Our future work will be directed to (1) greatly expanding our analyses of these compounds’ activities against both hereditary and sporadic breast cancer cell lines; (2) further structural chemical refinement of one or more of these three candidates to maximize their biological effect; and (3) testing of one or more of the compounds, and any derivatives, in animal models. Furthermore, we plan to screen an additional 1,500 compounds in order to increase the number of candidate compounds to maximize the chance of finding a robust condensin I inhibitor. We hope our study will provide an important basis for future development of one or more compounds into useful anti-cancer agents.