We and others have shown that Mcl-1 was essential for the survival of human myeloma cells in vitro. Furthermore, this antiapoptotic protein is upregulated by interleukin-6, which plays a critical role in multiple myeloma (MM). For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC), that is, myeloma cells from 51 patients with MM and 21 human myeloma cell lines (HMCL) using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. In total, 52% of patients with MM at diagnosis (P ¼ 0.017) and 81% at relapse (P ¼ 0.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only HMCL but not reactive plasmacytoses have abnormal Mcl-1 expression, although both PC expansions share similar high proliferation rates. Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. Finally, the level of Mcl-1 expression is related to disease severity, the highest values at diagnosis being associated with the shortest eventfree survival (P ¼ 0.002). In conclusion, Mcl-1, which has been shown to be essential for the survival of human myeloma cells in vitro, is overexpressed in vivo in MM in relation with relapse and shorter survival. Mcl-1 represents a potential therapeutical target in MM.

Targeting the ubiquitin-proteasome pathway has emerged as a potent anticancer strategy. Bortezomib, a specific proteasome inhibitor, has been approved for the treatment of relapsed or refractory multiple myeloma. Multiple myeloma cell survival is highly dependent on Mcl-1 antiapoptotic molecules. In a recent study, proteasome inhibitors induced Mcl-1 accumulation that slowed down their proapoptotic effects. Consequently, we investigated the role of Bcl-2 family members in bortezomib-induced apoptosis. We found that bortezomib induced apoptosis in five of seven human myeloma cell lines (HMCL). Bortezomib-induced apoptosis was associated with Mcl-1 cleavage regardless of Mcl-1L accumulation. Furthermore, RNA interference mediated Mcl-1 decrease and sensitized RPMI-8226 HMCL to bortezomib, highlighting the contribution of Mcl-1 in bortezomib-induced apoptosis. Interestingly, an important induction of Noxa was found in all sensitive HMCL both at protein and mRNA level. Concomitant to Mcl-1 cleavage and Noxa induction, we also found caspase-3, caspase-8, and caspase-9 activation. Under bortezomib treatment, Mcl-1L/Noxa complexes were highly increased, Mcl-1/Bak complexes were disrupted, and there was an accumulation of free Noxa. Finally, we observed a dissociation of Mcl-1/Bim complexes that may be due to a displacement of Bim induced by Noxa. Thus, in myeloma cells, the mechanistic basis for bortezomib sensitivity can be explained mainly by the model in which the sensitizer Noxa can displace Bim, a BH3-only activator, from Mcl-1, thus leading to Bax/Bak activation. [Cancer Res 2007;67(11):5418-24]

Insulin-like growth factor 1 (IGF-1) is a well-known growth factor for myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for multiple myeloma treatment. In this study, we investigated the effect of the antagonistic anti-IGF-1R murineAVE1642 Ab (mAVE1642). We show that mAVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines. Since we have previously shown the functional relevance of CD45 expression in the growth of myeloma cells and the association of CD45-negative (CD45neg) status with a less favorable clinical outcome, both CD45-positive (CD45pos) and CD45neg myeloma cell lines were selected for our study. We found that mAVE1642 strongly inhibits the growth of CD45neg myeloma cell lines, leading to a G1 growth arrest, whereas it has almost no effect on the growth of CD45pos myeloma cell lines. Furthermore, mAVE1642 binding induced a significant reduction of IGF-1R expression. We next demonstrated that the overexpression of IGF-1R in the CD45pos myeloma cell line increased Akt phosphorylation but was not sufficient to sensitize these cells to mAVE1642. In contrast, we generated a stable CD45-silencing XG-1 cell line and showed that it became sensitive to mAVE1642. Thus, for the first time, we provided direct evidence that the expression of CD45 renders cells resistant to mAVE1642. Taken together, these results support that therapy directed against IGF-1R can be beneficial in treating CD45neg patients.

No abstract

Summary Survivin is selectively expressed in most of common human cancers and is now viewed as a potent modulator of the cell death/proliferation balance in tumour cells. We previously found that myeloma cells expressed high levels of Survivin protein in correlation with disease progression and that Survivin knock‐down by RNA interference decreased myeloma cell growth. We now demonstrate that Survivin overexpression promotes the proliferation and survival of human myeloma cells both in vitro and in vivo in the absence of their major growth factor, interleukin 6. Of particular interest, this effect correlates with the down regulation of Bim, a critical BH3‐only cell death activator during cytokine deprivation, mainly at transcriptional level. The tight link between Survivin and Bim expression, reported for the first time here in myeloma cells and in other cell lines, is further confirmed in a panel of newly diagnosed patients with myeloma, and BIRC5 is validated as a gene significantly associated with short survival in these patients. Altogether, our findings provide evidence that Survivin directly contributes to malignant progression of myeloma and strongly suggest that targeting Survivin may disrupt the delicate balance controlling cell survival and proliferation, opening new avenues for the therapy of this still difficult‐to‐treat cancer.

The availability of Herceptin® ,an humanized equivalent of the murine 4D5 monoclonal antibody targeted against the Her-2/neu cell-surface receptor, as a therapeutic agent, incited us to re-evaluate the incidence of Her-2/neu expression in some malignant hematological diseases.Her2/neu expression has been evaluated retrospectively in 186 patients with different hematological disorders (AML: n= 31; B-ALL: n= 87; T-ALL: n=13; CLL: n=23; Multiple Myeloma: n=18; Macroglobulinemia: n=2; Lymphoma: n=12) including 173 at diagnosis and 13 at relapse, and 132 adults and 54 children (<18 years). Cells were analyzed on a FACSCALIBUR flow cytometer (Becton Dickinson) with the Her-2/neu Neu 24.7 antibody (BD). The mean fluorescence intensity (MFI) ratio was obtained by dividing the MFI of Her-2/neu antigen by the MFI of its isotypic control. The threshold of positivity for Her-2/neu expression was defined by a ratio > or = 2. All the patients with Her-2/neu expression detected by FACS analysis were analyzed by FISH using the Her-2/neu DNA probe kit (Vysis, Downers Grove, IL). Two breast tumor cell lines were used as positive controls: MCF-7 and BT-474, obtained from the DSMZ (Braunschweig, Germany). The two control specimens demonstrated Her-2/neu surface expression for 96% (BT-474) and 93% (MCF7) of the cell line population with a ratio intensity of 46 and 26 respectively. Only BT-474 showed an amplification of the Her-2/neu oncogene by FISH. Only 15 B-ALL patients were found positive for Her2/neu surface expression, including 2 children and 13 adults, 11 male and 4 female. Median percentage of Her-2/neu positive blasts population was 94% (range: 11–99%). Median ratio intensity was 7,7 (range: 3,5–54,5). Considering only B-ALL patients (n=87), incidence in children was only of 4% (2 patients/48) compared to 33% in adults (13 patients/39) (p=0,001). None of the positive B-ALL patients showed gene amplification by FISH analysis, suggesting that an other mechanism is involved, such as transcriptional activation or post-translational modifications. Considering only adult B-ALL patients (n=38) and without significant differences for main prognostic parameters and treatment (70% of patients were treated according to or in the GOELAL2 trial) between Her2/neu positive and negative patients, we observed that Her2/neu positive patients (n=12, 1 patient was not informed) are significantly associated with chemoresistance (50% versus 11%, p=0,03). Trends for correlation with refractory disease (41% versus 11%, p=0,08) and disease relapse (55% versus 36%, p=0,08) were also observed, suggesting that Her-2/neu surface expression could be a prognostic marker of poor clinical outcome in B-ALL. OS and DFS were similar between Her2/neu positive and negative patients (median 9 months versus 18 months, p=0,17; median 11 months versus 39 months, p=0,27, respectively), maybe due to a small number of patients in the series, but also because, in the same proportion of Her-2/neu negative patients, some patients received autologous or allogeneic stem cell transplants because of the poor results of the first chemotherapy. In conclusion, our results highlight Her2/neu surface expression only on blasts of one third of adult B-ALL patients. Therapy using anti her2neu monoclonal antibody may be a possibility in this selected group of poor-prognostic adult B-ALL patients.

Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (&gt;20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

IGF-1 is a well-known growth factor for human myeloma cells. Thus, therapeutic strategies targeting IGF-1R have been proposed for the treatment of multiple myeloma. In this study, we investigated the effect of a humanized antagonistic monoclonal antibody to the IGF-1R (AVE1642). We showed that AVE1642 selectively inhibits IGF-1R but not insulin signaling in human myeloma cell lines (HMCL). Since we had previously shown the functional relevance of CD45 expression in the growth of myeloma cells, and the association of CD45 negative (CD45neg) status with a less favorable clinical outcome, both CD45 positive (CD45pos) and CD45neg HMCL were studied. We found that AVE1642 strongly inhibited the growth of CD45neg HMCL, leading to a G1 growth arrest, whereas it had almost no effect on the growth of CD45pos HMCL. Indeed, by cell counting over four days in standard culture conditions, AVE1642 induced a significant growth inhibition of the CD45neg LP-1 HMCL of 90% whereas it had no effect on the growth of the CD45pos XG-1 HMCL. Furthermore, AVE1642 binding induced a significant reduction of IGF-1R expression. In western blot, a decrease of 60% the IGF-1R expression of the LP-1 CD45neg HMCL was observed. This decrease was also confirmed by flow cytometry analysis. Finally, a stable CD45 silencing XG-1 cell line was generated and became sensitive to AVE1642. By cell counting over four days in standard culture conditions, AVE1642 inhibited the growth of the shCD45neg XG-1 HMCL of 56% whereas it had almost no effect on the growth of the shLuci-XG-1 HMCL. Thus, for the first time, we provided direct evidence that the expression of CD45 renders myeloma cells resistant to IGF1R inhibition. Furthermore, AVE1642 in combination with Bortezomib strongly increased the apoptosis induced by Bortezomib alone on CD45negHMCL. The CD45neg LP-1 HMCL was considered as a modestly sensitive cell line since 10nM of Bortezomib, which is in the range of therapeutic concentrations, induced 40±16% of apoptosis. Bortezomib in combination with AVE1642 was found to induce 80±1% of apoptosis in the CD45neg LP-1 HMCL. Apoptosis induction by AVE1642/Bortezomib combination was associated with an important induction of Noxa, a BH3-only pro-apoptotic protein that was increased by the addition of AVE1642. We also found a strong increase of caspase-3, -8 activation under this combination compared to Bortezomib alone. Taken together, these results support that therapy directed against IGF-1R associated with Bortezomib, could be beneficial in treating CD45neg patients, a population known for poor clinical outcome.