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C022 Lenalidomide treatment modulates proliferation and differentiation of human mesenchymal stromal cells from healthy donors and MDS patients
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Oral Communications
Results: We found 59 mutations of the TET2 gene by direct sequencing of exons 3 to 11 (27 frame shifts, 21 nonsense and 11 missense mutations in conserved domains) in 43/206 pts. The frequencies according to the WHO subtypes were 21.8% in RA, 5.2% in RCMD, 21.4% in RARS/RARST/RCMD-RS, 34.9% in RAEB 1, 15.6% in RAEB 2, 18.2% in AML post MDS. Other anomalies of the 4q24 region were found including a deletion in 1/46 pts analyzed by CGH and 3 LOH and 2 deletions in 5/23 pts analyzed SNP arrays. Thus, the overall prevalence of 4q24 anomalies were 22.3% pts (46/206). 20 pts had two anomalies of TET2 identified by direct sequencing (17 pts), or sequencing plus SNP array (3 pts), indicating that the two copies of the gene were targeted in 43.5% of mutated pts. Comparison between the 43 pts with TET2 coding sequence mutations and unmutated patients found no significant differences in initial characteristics for sex, age, previous exposure to chemo or radiotherapy, Hb level, WBC count, ANC, plt count, % bone marrow blasts, multilineage dysplasia, WHO and FAB subtypes, karyotype and IPSS. The impact on prognosis will be evaluated. Conclusion: TET2 mutations are observed in about 20% of MDS, and are seen in all WHO or FAB subtypes.
3. Molecular Mechanisms and Drug Targets C021 The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1 J. Ohyashiki ° , S. Okabe, T. Tauchi, K. Ohyashiki. Tokyo Medical University, Shinjuku-ku, Japan *E-mail: [email protected] Purpose: Recent reports demonstrated that the oral iron chelator, deferasirox, acts as a potent NF-kappa-B inhibitor and improves hematological data in a subset of patients with myelodysplastic syndromes (MDS). The aim of this study is to evaluate the effect of deferasirox in human myeloid leukemia cells, and to identify the molecular pathways responsible for anti-proliferative effects on leukemia cells during chelation therapy. Method: The inhibitory effect of deferasirox on cell growth was assessed in 3 human myeloid leukemia cell lines, K562, U937, and HL-60 and peripheral blood mononuclear cells isolated from 4 patients with acute myeloid leukemia including one with post-MDS AM. Gene expression profiling was done using the GeneChip U133 Plus 2.0 (Affymetrix, Santa Clara, CA, USA). Results and Conclusions: The inhibitory concentration (IC50 ) of deferasirox was 17 to 172 mM in human myeloid cells. Based on the results obtained from gene expression profiling, we found an enhanced expression of REDD1 and its down-stream protein, tuberin (TSC2). Notably,
S6 ribosomal protein, which is known to be a target of mTOR, was significantly repressed in deferasirox-treated K562 cells, and REDD1 siRNA restored phosphorylation of S6. Although iron chelation may affect multiple signaling pathways related to cell survival, our data support the conclusion that REDD1 functions up-stream of tuberin to down-regulate the mTOR pathway in response to deferasirox. Deferasirox might not only have benefit for iron chelation but also be an anti-proliferative agent in some myeloid leukemias, especially patients who need both iron chelation and reduction of leukemia cells. C022 Lenalidomide treatment modulates proliferation and differentiation of human mesenchymal stromal cells from healthy donors and MDS patients M. Wobus ° , G. Duennebier, M. Bornhaeuser, G. Ehninger, U. Platzbecker. Medical Clinic and Polyclinic I, University Hospital Carl Gustav Carus, Dresden, Germany *E-mail: [email protected] Recent studies in patients with MDS have clearly demonstrated the clinical efficacy of lenalidomide. However, its exact mechanisms of action have not been elucidated yet. Myelosuppression is the most common adverse event and seems to be dependent on dose as well as MDS subtype, being rather infrequent in patients other than del5q. We therefore speculated whether lenalidomide affects the microenvironment by modulating mesenchymal stem cells (MSCs). Bone marrow samples were collected from healthy donors (n =5) and patients with MDS (del5q MDS n = 5, RA n = 2, RAEB1/2 n = 3). MSCs were isolated according to the standard adhesion protocol and cultured in the presence or absence of lenalidomide (50 mM). Lenalidomide treatment of MSCs caused no phenotypical changes but increased the cobblestone forming potential of hematopoietic stem cells on MSC layers. MSC typical surface molecules, CD73, CD90, CD105 and CD166, were expressed. Interestingly, lenalidomide caused an upregulation of mean fluorescence intensity of CD29 by 17.8±4.4% and CD73 by 24±5.7%. Furthermore, the secretion of IL-6 and IL-8 is increased in lenalidomide treated cells. We found osteoblastic differentiation to start as early as on day 5 with lenalidomide whereas in the control cells first calcium deposits were visible after 7 days. All described effects were observed in both normal and MDS MSCs. In conclusion, lenalidomide enhances cell proliferation and the potential for osteogenic differentiation of MSCs by a yet unknown mechanism. Whether these invitro effects are associated with the clinical efficacy of this compound in patients with MDS remains to be investigated.
Oral Communications
Results: We found 59 mutations of the TET2 gene by direct sequencing of exons 3 to 11 (27 frame shifts, 21 nonsense and 11 missense mutations in conserved domains) in 43/206 pts. The frequencies according to the WHO subtypes were 21.8% in RA, 5.2% in RCMD, 21.4% in RARS/RARST/RCMD-RS, 34.9% in RAEB 1, 15.6% in RAEB 2, 18.2% in AML post MDS. Other anomalies of the 4q24 region were found including a deletion in 1/46 pts analyzed by CGH and 3 LOH and 2 deletions in 5/23 pts analyzed SNP arrays. Thus, the overall prevalence of 4q24 anomalies were 22.3% pts (46/206). 20 pts had two anomalies of TET2 identified by direct sequencing (17 pts), or sequencing plus SNP array (3 pts), indicating that the two copies of the gene were targeted in 43.5% of mutated pts. Comparison between the 43 pts with TET2 coding sequence mutations and unmutated patients found no significant differences in initial characteristics for sex, age, previous exposure to chemo or radiotherapy, Hb level, WBC count, ANC, plt count, % bone marrow blasts, multilineage dysplasia, WHO and FAB subtypes, karyotype and IPSS. The impact on prognosis will be evaluated. Conclusion: TET2 mutations are observed in about 20% of MDS, and are seen in all WHO or FAB subtypes.
3. Molecular Mechanisms and Drug Targets C021 The oral iron chelator deferasirox represses signaling through the mTOR in myeloid leukemia cells by enhancing expression of REDD1 J. Ohyashiki ° , S. Okabe, T. Tauchi, K. Ohyashiki. Tokyo Medical University, Shinjuku-ku, Japan *E-mail: [email protected] Purpose: Recent reports demonstrated that the oral iron chelator, deferasirox, acts as a potent NF-kappa-B inhibitor and improves hematological data in a subset of patients with myelodysplastic syndromes (MDS). The aim of this study is to evaluate the effect of deferasirox in human myeloid leukemia cells, and to identify the molecular pathways responsible for anti-proliferative effects on leukemia cells during chelation therapy. Method: The inhibitory effect of deferasirox on cell growth was assessed in 3 human myeloid leukemia cell lines, K562, U937, and HL-60 and peripheral blood mononuclear cells isolated from 4 patients with acute myeloid leukemia including one with post-MDS AM. Gene expression profiling was done using the GeneChip U133 Plus 2.0 (Affymetrix, Santa Clara, CA, USA). Results and Conclusions: The inhibitory concentration (IC50 ) of deferasirox was 17 to 172 mM in human myeloid cells. Based on the results obtained from gene expression profiling, we found an enhanced expression of REDD1 and its down-stream protein, tuberin (TSC2). Notably,
S6 ribosomal protein, which is known to be a target of mTOR, was significantly repressed in deferasirox-treated K562 cells, and REDD1 siRNA restored phosphorylation of S6. Although iron chelation may affect multiple signaling pathways related to cell survival, our data support the conclusion that REDD1 functions up-stream of tuberin to down-regulate the mTOR pathway in response to deferasirox. Deferasirox might not only have benefit for iron chelation but also be an anti-proliferative agent in some myeloid leukemias, especially patients who need both iron chelation and reduction of leukemia cells. C022 Lenalidomide treatment modulates proliferation and differentiation of human mesenchymal stromal cells from healthy donors and MDS patients M. Wobus ° , G. Duennebier, M. Bornhaeuser, G. Ehninger, U. Platzbecker. Medical Clinic and Polyclinic I, University Hospital Carl Gustav Carus, Dresden, Germany *E-mail: [email protected] Recent studies in patients with MDS have clearly demonstrated the clinical efficacy of lenalidomide. However, its exact mechanisms of action have not been elucidated yet. Myelosuppression is the most common adverse event and seems to be dependent on dose as well as MDS subtype, being rather infrequent in patients other than del5q. We therefore speculated whether lenalidomide affects the microenvironment by modulating mesenchymal stem cells (MSCs). Bone marrow samples were collected from healthy donors (n =5) and patients with MDS (del5q MDS n = 5, RA n = 2, RAEB1/2 n = 3). MSCs were isolated according to the standard adhesion protocol and cultured in the presence or absence of lenalidomide (50 mM). Lenalidomide treatment of MSCs caused no phenotypical changes but increased the cobblestone forming potential of hematopoietic stem cells on MSC layers. MSC typical surface molecules, CD73, CD90, CD105 and CD166, were expressed. Interestingly, lenalidomide caused an upregulation of mean fluorescence intensity of CD29 by 17.8±4.4% and CD73 by 24±5.7%. Furthermore, the secretion of IL-6 and IL-8 is increased in lenalidomide treated cells. We found osteoblastic differentiation to start as early as on day 5 with lenalidomide whereas in the control cells first calcium deposits were visible after 7 days. All described effects were observed in both normal and MDS MSCs. In conclusion, lenalidomide enhances cell proliferation and the potential for osteogenic differentiation of MSCs by a yet unknown mechanism. Whether these invitro effects are associated with the clinical efficacy of this compound in patients with MDS remains to be investigated.