Clinical and pharmacodynamic activity of bortezomib and decitabine in acute myeloid leukemia

Abstract

We recently reported promising clinical activity for a 10-day regimen of decitabine in older AML patients; high miR-29b expression associated with clinical response. Subsequent preclinical studies with bortezomib in AML cells have shown drug-induced miR-29b up-regulation, resulting in loss of transcriptional activation for several genes relevant to myeloid leukemogenesis, including DNA methyltransferases and receptor tyrosine kinases. Thus, a phase 1 trial of bortezomib and decitabine was developed. Nineteen poor-risk AML patients (median age 70 years; range, 32-84 years) enrolled. Induction with decitabine (20 mg/m(2) intravenously on days 1-10) plus bortezomib (escalated up to the target 1.3 mg/m(2) on days 5, 8, 12, and 15) was tolerable, but bortezomib-related neuropathy developed after repetitive cycles. Of previously untreated patients (age ≥ 65 years), 5 of 10 had CR (complete remission, n = 4) or incomplete CR (CRi, n = 1); 7 of 19 overall had CR/CRi. Pharmacodynamic analysis showed FLT3 down-regulation on day 26 of cycle 1 (P = .02). Additional mechanistic studies showed that FLT3 down-regulation was due to bortezomib-induced miR-29b up-regulation; this led to SP1 down-regulation and destruction of the SP1/NF-κB complex that transactivated FLT3. This study demonstrates the feasibility and preliminary clinical activity of decitabine plus bortezomib in AML and identifies FLT3 as a novel pharmacodynamic end point for future trials.

Trial registration: ClinicalTrials.gov NCT00703300.

Figures

Figure 1

Posttreatment down-regulation of FLT3 in AML patients. Pretreatment and day 26 expression levels of miR-29b (A) and FLT3 mRNA (B) in patients with serial bone marrow from the decitabine-bortezomib clinical trial (n = 5).

Figure 2

Regulation of FLT3 expression via the SP1/NF-κB(p65) complex. (A) SP1 and NF-κB(p65) binding sites in the promoter region of the FLT3 gene. (B) EMSA assays of the 2 identified binding sites for SP1 and NF-κB(p65), demonstrating specific binding of SP1 to the first site and binding of NF-κB(p65) to both sites using specific antibody to supershift the DNA-protein complexes. (C) Chromatin (ChIP) for the region containing binding sites of SP1 and of NF-κB(p65). (D) Luciferase promoter activity reporter assay including 700 bp of the promoter region of the FLT3 gene containing all identified SP1 and NF-κB(p65) binding sites, demonstrating increased activity after cotransfection with constructs to overexpress SP1 or NF-κB(p65) and decreased activity after siRNA mediated knockdown of SP1 and NFκB(p65). EV indicates empty vector; and sc, scramble oligo.

Figure 3

Bortezomib-induced FLT3 down-regulation via interaction with the SP1/NF-κB(p65) complex. (A) SiRNA mediated knock-down of SP1 or NFκ-B(p65) down-regulates FLT3 expression in MV4-11 cells that harbor a FLT3-ITD and express FLT3 at high levels. (B) Overexpression of SP1 or NF-κB(p65) increases the expression of FLT3 in the KG1 cell line that usually has low expression of FLT3. (C) Increasing miR-29b in the MV4-11 cell line decreases FLT3 expression in MV4-11 cells. (D) Bortezomib treatment decreases FLT3 expression in a time- and dose-dependent manner in the MV4-11 cell line and in primary patient blasts (obtained from patients not enrolled on the current clinical trial; samples were procured in the OSU Leukemia Tissue Bank).