A Novel Upregulation of Fanconi Anemia/BRCA DNA Repair Pathway Is Observed Upon Co-Culture of Bone Marrow Stroma with Multiple Myeloma Cell Lines: A Potential New Participant(s) in Environment Mediated Drug Resistance.

Abstract 2798

Poster Board II-774

Multiple Myeloma (MM) is an incurable malignancy of mature clonal B cells. The refractory nature of MM has long been attributed to the acquisition of drug resistance. Traditionally, mechanisms of drug resistance have been defined by acquired changes in the expression or function of specific gene products. To this end, we have recently demonstrated that selected resistance to the cytotoxic agent melphalan correlated with increased expression of components of the Fanconi Anemia (FA)/BRCA DNA repair pathway and a concomitant increase in repair of DNA interstrand cross-links (ICLs).(Hazlehurst et al Cancer Res 2003; Chen et al Blood 2005) Further, the exogenous expression of specific FANC components in RPMI 8226 cell lines enhanced ICL repair, favored the release from melphalan-induced S-phase delay, and rendered these cells partially resistant to melphalan treatment. Together, these results suggest a causal relationship between increased expression of FA DNA repair components, increased DNA repair, and acquired resistance to melphalan. Over the past decade a large body of evidence has emerged demonstrating that in addition to drug resistance mechanisms intrinsic to the cancer cell, there exist dynamic, de novo mechanisms coordinated by the tumor microenvironment resulting in an environment-mediated drug resistance (EM-DR). As such, we examined the potential role of the microenvironment in regulating the FA/BRCA DNA repair pathway. FA pathway protein expression was evaluated with anti-sera to FANCD1/BRCA2, FANCC, FANCD2, FANCI, FANCG and BRCA1 in drug sensitive RPMI 8226 cells and melphalan resistant 8226/LR5 cells in co-culture with the HS-5 bone marrow stromal cell line. With these preliminary results we present three novel findings. First, we demonstrate that expression of FA/BRCA pathway components is regulated by intracellular interactions in both MM cells and bone marrow stromal cells (BMSCs). Second, we show that the acquisition of drug resistance alters FANC protein expression profiles upon co-culture. Third, in the HS-5 BMSCs, mono-ubiquitinated FANCD2 is observed in the absence of detectable FANCG. In RPMI 8226 cells, Western blot analysis demonstrated an acute (within 30minutes) and prolonged (up to 48hours) time-dependent increase in expression of FANCD2/BRCA2, FANCC, FANCD2, and BRCA1 upon incubation with BMSCs relative to MM cells incubated alone. However, no appreciated increases in FANCI or FANCG were noted under the same conditions. Incubation of 8226/LR5s with HS-5 BMSCs demonstrated a slightly different up-regulation of FA/BRCA pathway protein expression with addition of increased FANCI expression and no increase in FANCD2 or FANCC expression. We also examined FANC protein expression in the HS-5 cells. Interestingly, in the BMSCs significant differences were noted in the FANC expression profiles. Co-culture of RPMI 8226 cells with HS-5 cells demonstrated only modest elevations in FANCD2; however, co-culture with drug-resistant 8226/LR5s resulted in increased levels of FANCD2, FANCI and BRCA1. These data indicate that different tumor cells may alternately influence FA/BRCA-mediated DNA repair and potentially drug resistance in juxtaposed bone marrow stroma. Curiously, we also observed mono-ubiquitinated FANCD2 in the absence of any detectable levels of FANCG protein under co-culture conditions. As the FA/BRCA DNA repair pathway has been associated with cell cycle progression, we evaluated cell cycle kinetics under the co-culture conditions. The results of BrdU analysis demonstrated that the observed changes in FA/BRCA protein expression in MM and BMSC could not be fully explained by cell-cycle distribution. Therefore, within this report we demonstrate for the first time that microenvironmental interactions can modulate the FA/BRCA DNA repair pathway in MM and BMSCs. These results suggest that the FA/BRCA DNA damage repair pathway may be an important modulatory component of EM-DR. Importantly, the potential de novo drug resistance likely involves both the MM tumor cell and adjacent stromal cells. Current and future studies will attempt to examine a causal relationship between increased FANC expression and melphalan (and other drug) resistance seen in co-culture conditions, as well as to identify specific signaling molecules and mechanisms controlling the enhanced expression in both cell models.