D-cyclins are universally dysregulated in multiple myeloma and frequently over-expressed in acute leukemia. To better understand the regulation of D-cyclins and the effects of targeting their expression in myeloma and leukemia cells, we conducted a chemical screen of the 56,000 compound Maybridge chemical library for inhibitors of the human cyclin D2 promoter using NIH 3T3 cells stably expressing the Cyclin D2promoter-driving a luciferase reporter gene. From this screen, 11 compounds that reproducibly inhibited transactivation of the cyclin D2 promoter, but did not inhibit transactivation of an unrelated RSV promoter driving luciferase, and did not reduce the growth and viability of NIH3T3 in an MTS assay. Among these 11 compounds, the most active was 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161). Given the identification of S14161 as a potential inhibitor of cyclin D2 transactivation, we evaluated its effects on D-cyclin expression in myeloma and leukemia cell lines. S14161 decreased D-cyclin protein and mRNA levels across a range of events that dysregulated D-cyclins including cyclin D1 translocation (KMS12), c-maf over-expression (RPMI-8226, KMS11, LP1, JJN3), and FGFR3 translocation (KMS11) at low micromolar concentrations. Consistent with its effects on D-cyclin expression, S14161 arrested cells in the G0/G1 phase of the cell cycle at concentrations associated with its ability to decrease D-cyclins. Next, we tested the effects of S14161 on the viability of myeloma and leukemia cells. S14161 induced cell death in 6/7 myeloma and 6/7 leukemia cell lines with an IC50 <10 µM by the MTS assay. Cell death and apoptosis were confirmed by Annexin V-FITC staining. S14161 reduced the growth and viability of 4/5 primary AML samples with an IC50 <10 µM. In contrast, it was less toxic to normal hematopoietic cells with an IC20 > 25 µM. Given the ability of S14161 to induce apoptosis and reduce D-cyclin expression, we evaluated its efficacy in vivo. SCID mice were injected subcutaneously with K562 human chronic leukemia cells. After injection, mice were treated with S14161 (100mg/kg/day) intraperitoneally or vehicle control. Treatment with S14161 delayed tumor growth by up to 90% compared to vehicle control without evidence of gross organ toxicity or weight loss. Similar results were obtained in a U937 xenograft model. Of note, in our acute toxicity experiment, no weight loss or other toxic changes were noted in mice treated with 500 mg/kg/daily for two weeks. S14161 is a novel chemical compound with an unknown mechanism of action. However, the compound contains a chromene motif that is similar to the chromone motif found in the PI3 kinase inhibitor, LY294002. Therefore, we tested whether S14161 could inhibit the PI3 kinase signalling pathway and whether this inhibition was functionally important for its effects on D-cyclin expression and cell viability. Pre-treatment with S14161 blocked the increase in phospho-Akt without altering levels of total Akt. Moreover, S14161 inhibited the activities of Class I PI3 kinase , β, , and with similar efficacy. In contrast, S14161 was less effective in inhibiting the PI3 kinase-associated enzymes PDK1, mTOR and DNA-PKcs with less than 20% inhibition at a concentration of 300 µM. Likewise, S14161 did not inhibit Akt1, Akt2, or Akt3, at a concentration up to 300 µM. To assess the relationship between inhibition of PI3 kinase signalling, decreased D-cyclin expression and cell death, we evaluated a series of chromene-containing compounds structurally related to S14161. Similar to the effects of S14161, the highly structurally related compound 8-methoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14147) inhibited accumulation of phospho-Akt after IGF1 stimulation, decreased D-cyclin expression and induced cell death in KMS11 myeloma cells. In contrast, 4 other related compounds did not decrease cyclin D expression or prevent Akt phosphorylation after IGF1 stimulation. These inactive analogues were also not toxic to myeloma cells. Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation. Moreover, we demonstrated that PI3 kinase inhibition can overcome dysregulation of D-cyclins across a broad array of transforming events in myeloma and leukemia.
A Small Molecule Inhibitor of D-Cyclin Transactivation Displays Preclinical Efficacy in Myeloma and Leukemia
DATTI, Alessandro;
2009
Abstract
D-cyclins are universally dysregulated in multiple myeloma and frequently over-expressed in acute leukemia. To better understand the regulation of D-cyclins and the effects of targeting their expression in myeloma and leukemia cells, we conducted a chemical screen of the 56,000 compound Maybridge chemical library for inhibitors of the human cyclin D2 promoter using NIH 3T3 cells stably expressing the Cyclin D2promoter-driving a luciferase reporter gene. From this screen, 11 compounds that reproducibly inhibited transactivation of the cyclin D2 promoter, but did not inhibit transactivation of an unrelated RSV promoter driving luciferase, and did not reduce the growth and viability of NIH3T3 in an MTS assay. Among these 11 compounds, the most active was 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161). Given the identification of S14161 as a potential inhibitor of cyclin D2 transactivation, we evaluated its effects on D-cyclin expression in myeloma and leukemia cell lines. S14161 decreased D-cyclin protein and mRNA levels across a range of events that dysregulated D-cyclins including cyclin D1 translocation (KMS12), c-maf over-expression (RPMI-8226, KMS11, LP1, JJN3), and FGFR3 translocation (KMS11) at low micromolar concentrations. Consistent with its effects on D-cyclin expression, S14161 arrested cells in the G0/G1 phase of the cell cycle at concentrations associated with its ability to decrease D-cyclins. Next, we tested the effects of S14161 on the viability of myeloma and leukemia cells. S14161 induced cell death in 6/7 myeloma and 6/7 leukemia cell lines with an IC50 <10 µM by the MTS assay. Cell death and apoptosis were confirmed by Annexin V-FITC staining. S14161 reduced the growth and viability of 4/5 primary AML samples with an IC50 <10 µM. In contrast, it was less toxic to normal hematopoietic cells with an IC20 > 25 µM. Given the ability of S14161 to induce apoptosis and reduce D-cyclin expression, we evaluated its efficacy in vivo. SCID mice were injected subcutaneously with K562 human chronic leukemia cells. After injection, mice were treated with S14161 (100mg/kg/day) intraperitoneally or vehicle control. Treatment with S14161 delayed tumor growth by up to 90% compared to vehicle control without evidence of gross organ toxicity or weight loss. Similar results were obtained in a U937 xenograft model. Of note, in our acute toxicity experiment, no weight loss or other toxic changes were noted in mice treated with 500 mg/kg/daily for two weeks. S14161 is a novel chemical compound with an unknown mechanism of action. However, the compound contains a chromene motif that is similar to the chromone motif found in the PI3 kinase inhibitor, LY294002. Therefore, we tested whether S14161 could inhibit the PI3 kinase signalling pathway and whether this inhibition was functionally important for its effects on D-cyclin expression and cell viability. Pre-treatment with S14161 blocked the increase in phospho-Akt without altering levels of total Akt. Moreover, S14161 inhibited the activities of Class I PI3 kinase , β, , and with similar efficacy. In contrast, S14161 was less effective in inhibiting the PI3 kinase-associated enzymes PDK1, mTOR and DNA-PKcs with less than 20% inhibition at a concentration of 300 µM. Likewise, S14161 did not inhibit Akt1, Akt2, or Akt3, at a concentration up to 300 µM. To assess the relationship between inhibition of PI3 kinase signalling, decreased D-cyclin expression and cell death, we evaluated a series of chromene-containing compounds structurally related to S14161. Similar to the effects of S14161, the highly structurally related compound 8-methoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14147) inhibited accumulation of phospho-Akt after IGF1 stimulation, decreased D-cyclin expression and induced cell death in KMS11 myeloma cells. In contrast, 4 other related compounds did not decrease cyclin D expression or prevent Akt phosphorylation after IGF1 stimulation. These inactive analogues were also not toxic to myeloma cells. Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation. Moreover, we demonstrated that PI3 kinase inhibition can overcome dysregulation of D-cyclins across a broad array of transforming events in myeloma and leukemia.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
