The bone marrow microenvironmental components interleukin (IL)-6 and fibronectin (FN) individually influence the proliferation and survival of multiple myeloma (MM) cells; however, in vivo, these effectors most likely work together. We examined signaling events, cell cycle progression, and levels of drug response in MM cells either adhered to FN via B1 integrins, stimulated with IL-6, or treated with the two combined. Although G 1 -S cell cycle arrest associated with FN adhesion was overcome when IL-6 was added, the cell adhesion-mediated drug resistance (CAM-DR) was maintained in the presence of IL-6. Concomitant exposure of MM cells to IL-6 and FN adhesion revealed a dramatic increase in signal transducers and activators of transcription 3 (STAT3) phosphorylation, nuclear translocation, and DNA binding, compared with either IL-6 or FN adhesion alone in four MM cell lines. Importantly, this increase in STAT3 activation correlated with a novel association between STAT3 and gp130 in cells adhered to FN before stimulation with IL-6, relative to nonadherent cells. Taken together, these results suggest a mechanism by which collaborative signaling by B1 integrin and gp130 confers an increased survival advantage to MM cells. [Cancer Res 2009;69(3):1009-15]

The Fanconi Anemia (FA)/BRCA DNA damage repair pathway plays a pivotal role in the cellular response to replicative stress induced by DNA alkylating agents and greatly influences drug response in cancer treatment. We recently reported that FA/BRCA genes are overexpressed and causative for drug resistance in human melphalan-resistant multiple myeloma (MM) cell lines. However, the transcriptional regulation of the FA/BRCA pathway is not understood. In this report, we describe for the first time a novel function of the NF-κB subunits, RelB/p50, as transcriptional activators of the FA/BRCA pathway. Specifically, our findings point to constitutive phosphorylation of IκB Kinase IKKα and subsequent alterations in FANCD2 expression and function as underlying events leading to melphalan resistance in repeatedly exposed MM cells. Inhibiting NF-κB by siRNA, blocking the IKK complex with BMS-345541, or using the proteasome inhibitor bortezomib drastically reduced FA/BRCA gene expression and FANCD2 protein expression in myeloma cells, resulting in diminished DNA damage repair and enhanced melphalan sensitivity. Importantly, we also found that bortezomib decreases FA/BRCA gene expression in multiple myeloma patients. These results show for the first time that NF-κB transcriptionally regulates the FA/BRCA pathway, and provide evidence for targeting FA-mediated DNA repair to enhance chemotherapeutic response and circumvent drug resistance in myeloma patients.

The emergence of acquired drug resistance results from multiple compensatory mechanisms acting to prevent cell death. Simultaneous monitoring of proteins involved in drug resistance is a major challenge for both elucidation of the underlying biology and development of candidate biomarkers for assessment of personalized cancer therapy. Here, we have utilized an integrated analytical platform based on SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring mass spectrometry, a versatile and powerful tool for targeted quantification of proteins in complex matrices, to evaluate a well-characterized model system of melphalan resistance in multiple myeloma (MM). Quantitative assays were developed to measure protein expression related to signaling events and biological processes relevant to melphalan resistance in multiple myeloma, specifically: nuclear factor-B subunits, members of the Bcl-2 family of apoptosis-regulating proteins, and Fanconi Anemia DNA repair components. SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring methods were developed for quantification of these selected target proteins in amounts of material compatible with direct translation to clinical specimens (i.e. less than 50,000 cells). As proof of principle, both relative and absolute quantification were Multiple myeloma (MM)1 is an incurable malignancy of plasma cells harbored in the bone marrow, which is clinically characterized by secretion of monoclonal antibodies, calcium dysregulation, anemia, lytic bone lesions, and kidney damage. For five decades, therapeutic regimens for MM have included the alkylating agent, melphalan (p-di-2-chloroethylamino-1-phenylalanine, L-phenylalanine mustard, or L-PAM) (1). Therapy with L-PAM (alone or in combination with novel agents) remains the standard of care for transplant-ineligible patients and is the backbone of high-dose therapy associated with autologous stem cell transplant. Although MM patients initially respond to chemotherapy, treatment failure is inevitable because of the emergence of drug resistance. Because of the importance of detecting acquired drug resistance (ADR) for patient assessment and treatment, numerous mechanisms of ADR have been elucidated for MM in vitro, utilizing cell line models developed by chronic exposure to chemotherapy (2-7). These models were designed to identify potential drug targets to reverse ADR as well as to discover prognostic or chemopredictive biomarkers that could be translated to assessment of MM patients. These isogenic model systems clearly show that drug resistance is derived from multiple factors and that prediction of response can not depend on the measurement of one pathway.The biology of one such model of ADR, the 8226/LR5 cell line, selected by chronic exposure of RPMI-8226 MM cells to the DNA-damaging agent, melphalan, has been extensively characterized, leading to the identification of causative mechFrom the ‡Molecular Oncology, §Experimental Therapeutics,