Inhibitor of the CREB Proto-Oncogene Suppresses AML Cell Proliferation and Induces Apoptosis In Vitro and In Vivo

Abstract 3270

The cAMP Response Element Binding Protein, CREB, is a nuclear transcription factor that is critical for cell proliferation, differentiation, and survival in a variety of cell types. We previously demonstrated that CREB is overexpressed in leukemia blasts from greater than 60% of patients with AML. CREB overexpression is also associated with an increased risk of relapse and decreased event free survival in a cohort of AML patients. To understand the role of CREB in leukemogenesis, a transgenic mouse was created by overexpressing CREB under the control of the hMRP8 promoter in Gr-1/Mac-1 + myeloid cells. Bone marrow progenitors from these mice have increased proliferative potential and replating ability. CREB transgenic mice developed myeloproliferative disease after one year, but not acute myeloid leukemia suggesting that CREB is not sufficient to induce complete transformation. Given these results, we hypothesized that CREB could be a potential target for AML therapy. To test this possibility, we knocked down CREB using lentiviral shRNAs in primary mouse hematopoietic stem cells in vitro and in vivo. Downregulation of CREB resulted in significant decrease in myeloid colony numbers in methylcellulose colony assays. However, there was no effect of CREB knockdown in hematopoietic stem cell numbers or long-term engraftment by stem cell transduction and transplantation experiments. In contrast, inhibition of CREB by shRNAs in human myeloid leukemia cells and Ba/F3 cells transduced with T315I mutant BCR-ABL resulted in suppression of growth of leukemia cells in vitro and in NOD-SCID mice. Taken together, these data suggest that CREB preferentially inhibits the growth of leukemia cells without significantly affecting normal hematopoietic stem cell proliferation or function. To target CREB in AML cells, a small molecule, XX-650-23 (MW: 288.3), was developed to inhibit the interaction between CREB and its binding partner, the histone acetyltransferase CBP (CREB Binding Protein). We tested the effects of the drug on AML cell lines Molm-13 and MV-411 in vitro using MTT assays and trypan blue exclusion. The IC50 for both cell lines was between 1–2 micromolar concentration after 72 hours of treatment. Our results also demonstrated that treatment of MV-411 cells with XX-650-23 not only inhibited cell proliferation but also resulted in apoptosis. Treatment of normal human bone marrow progenitors with XX-650-23 had no effect on colony numbers in methylcellulose containing IL-3, IL-6, and Stem Cell Factor up to 10 micromolar concentration. NOD-SCID IL-2Rgamma null (NSG) mice treated with the drug at 10, 15, 17.5, and 20 mg/kg IP once daily for 28 days had no evidence of weight loss or toxicity to organs as measured by peripheral blood counts and chemistries to assess kidney and liver function. We next tested the drug in xenograft models of AML with MV-411 cells (2×10⁶) injected subcutaneously into the flanks of NSG mice. Three groups of 5 mice were treated with either XX-650-23 or vehicle control at 17.5 mg/kg IP daily at the time of injection of MV-411 cells or after the tumors reached a size of 300 mm³. Serum drug concentration 1 hour after first injection was 33 nM. Estimated drug mean residence time was 7.5 hours, and plasma clearance was 9.6 ml/min/kg, assuming complete IP absorption. Our results demonstrated a significant inhibition of tumor growth with treatment of the drug and MV-411 cells at the same time on Day 1. There was also an effect of the drug alone when treated after the tumor reached a size of 300 mm³, although less dramatic. These data suggest that development of a drug that targets CREB is a novel approach to treat AML. We are currently optimizing the treatment of AML cells with XX-650-23 in mice and investigating the downstream signaling pathways to understand the mechanism by which XX-650-23 suppresses proliferation and induces apoptosis of AML cells.