A Small Molecule Inhibitor Targeting CREB and CBP Inhibits Proliferation of AML Cells In Vitro and In Vivo,

Abstract 3607

The cAMP Response Element Binding Protein, CREB, is a nuclear transcription factor that is a downstream target of signaling pathways regulating memory, glucose homeostasis, cell proliferation, differentiation, and survival. We previously demonstrated that CREB is overexpressed at both the protein and mRNA levels in leukemia blasts and in leukemia stem cells. AML patients who overexpress CREB in their bone marrow have an increased risk of relapse and decreased event-free survival. To determine whether CREB is sufficient for leukemogenesis, we created a transgenic mouse in which CREB is expressed under the control of a myeloid specific hMRP8 promoter. Bone marrow progenitors from CREB transgenic mice had higher proliferative potential and replating ability. These mice developed myeloproliferative disease after one year but not acute leukemia, suggesting that CREB is not sufficient to induce myeloid transformation. To determine whether CREB is necessary for leukemia cell proliferation, we transduced AML cells with lentiviral CREB shRNA. We observed that downregulation of CREB led to decreased AML cell proliferation and survival in vitro. Furthermore, our results demonstrated that CREB knockdown inhibits the growth of AML cells in vivo without affecting normal hematopoietic stem cell function. Together, these results strongly suggest that CREB acts as a proto-oncogene and is a potential target for AML therapy.

CREB is activated through phosphorylation, leading to the recruitment of the histone acetyltransferase, CREB binding protein (CBP) and subsequent target gene expression. XX-650-23 (MW: 288.3) is a small molecule that was identified through in silico screening methods to inhibit the interaction between CREB and CBP. We tested the effects of the drug on various AML cell lines using MTT assays and trypan blue exclusion. The IC50 ranged from 700 nM to 2 μM after 72 hours of drug treatment. However, treatment of normal human bone marrow progenitors cultured in methylcellulose containing XX-650-23 at a concentration of 10 μM had no effect on colony numbers. We also tested the in vivo effects of XX-650-23 using xenograft NOD-SCID IL-2Rgamma null (NSG) mouse models. To assess toxicity, mice were treated with the drug at various concentrations, ranging from 10 to 20 mg/kg, by intraperitoneal injection (IP) once daily for 28 days. We did not observe any weight loss or hematologic, renal, hepatic, or cardiac toxicity in the mice. We also performed pharmacokinetic analysis to determine the half-life and stability of the drug. After 1 hour of treatment, the serum drug concentration was 33 nM. The estimated drug mean residence time was 7.5 hours. Plasma clearance divided by IP absorption fraction was 9.6 L/min/kg. The mice were treated daily with drug (17.5mg/kg IP) or vehicle control once MV4-11 cells reached a tumor size of 300mm³ or at the time cells were injected. Our results demonstrated significant inhibition of tumor growth with treatment of the drug started on the day of injection of cells compared to waiting until the tumor reached 300mm³. Together, these data suggest that a small molecule inhibitor targeting CREB and CBP interaction is a potential avenue for drug development. We are currently studying the mechanisms by which XX-650-23 inhibits AML cell proliferation and analyzing the effects of combining the drug with chemotherapy.