Mutations of members of the ras family are among the most common oncogene mutations found in multiple myeloma (MM). We have examined the mutational status of the N- and K-ras genes at codons 12, 13, and 61 in 160 newly diagnosed MM patients enrolled on the Eastern Cooperative Oncology Group (ECOG) phase III clinical trial E9486. The total incidence of ras mutations was found to be 39% of the samples analyzed. Five patients showed evidence of more than one mutation. We obtained 22 marrow samples from patients at the time of disease progression or relapse, for whom a ras mutation was identified at diagnosis. In all cases, the ras mutation of the disease progression sample was identical to that found at diagnosis. In contrast, three of 25 patients who did not show any ras mutation at diagnosis acquired a ras mutation at the time of disease progression. No significant association was observed between any ras mutation and stage of disease, beta 2-microglobulin levels, labelling index, or protein type. The mean tumor burden and median survival for patients with mutations of N-ras was indistinguishable from patients with no ras mutations. However, patients with K-ras mutations had a significantly higher mean bone marrow tumor burden at diagnosis than patients with no ras mutations (57% v 36%, P ‼ .02); and the median survival of patients with a K-ras mutation was significantly shorter (2.0 v 3.7 years, P ‼ .02). To determine if the status of ras mutations could affect treatment response, we examined patient survival on the three treatment arms of E9486. Although the presence of a ras mutation in the multidrug treatment, VBMCP alone, showed a marginal significance, neither the VBMCP, nor the addition of interferon-alpha showed statistically significant survival differences between mutant and wildtype ras status. Interestingly, there appeared to be a statistically significant difference in survival of patients treated with VBMCP and alternating high doses of cyclophosphamide + prednisone. Patients with ras mutations had a median survival of 2.1 years; patients with wild-type ras had a median survival of 4.0 years (P ‼ .01).

O-class forkhead box (FOXO) transcription factors are critical regulators of diverse cellular processes, including apoptosis, cell-cycle arrest, DNA damage repair and oxidative stress resistance. Here, we show that FOXO1 and FOXO3a have an essential function in promoting cell detachment-induced anoikis, resistance to which is implicated in cancer development and metastasis. In contrast, the oncoprotein cyclin D1 inhibits anoikis. We further show that cyclin D1 interacts with FOXO proteins and impedes their transcriptional regulatory and anoikis-promoting functions. This effect of cyclin D1 requires its transcription repression domain but is independent of cyclin-dependent kinases CDK4 and CDK6. Moreover, we show that cancer-derived mutants of cyclin D1 are much more stable than wild-type cyclin D1 under anchorage-independent conditions and possess a greater antagonistic effect on FOXO-regulated anoikis and anchorage-independent growth of cancer cells. These data suggest that cyclin D1 may have a critical function in tumorigenesis and cancer metastasis by inhibiting the anoikis-promoting function of FOXO proteins.

Because of the increasing use of IFN-␣ in both induction and maintenance therapy for multiple myeloma (MM), its effect on growth and apoptosis of myeloma cells is important to consider. To investigate the role of IFN-␣ on the growth of myeloma cells, we have studied its effects on the response of interleukin 6 (IL-6)-dependent myeloma cell line (ANBL6) and IL-6-independent myeloma cell line (C2E3) in the presence of IL-6 and dexamethasone (Dex). We found that although IFN-␣ is a potent inhibitor of proliferation, it has only a minimal effect on induction of apoptosis. Moreover, we found IFN-␣ as well as IL-6 can significantly suppress dexamethasone-induced apoptosis. The suppression of apoptosis is concurrent with the induction of both AP-1 and STAT binding activity. We also found that IL-6 but not IFN-␣ up-regulates Bcl-X L expression. However, IL-6-mediated Bcl-X L expression is suppressed in the presence of Dex. Therefore, the expression of Bcl-X L does not account for the protection of Dex-induced apoptosis by IFN-␣ and IL-6. Taken together, our results suggest that IFN-␣ may counteract the beneficial effects of corticosteroids or perhaps other apoptosis inducing agents in the treatment of myeloma.