CREB Increases Chemotherapy Resistance through Regulation of the DNA Damage Repair Pathway in AML Cells

CREB (cAMP Response Element Binding Protein) is a nuclear transcription factor that plays a critical role in regulating myeloid cell proliferation and differentiation. CREB is overexpressed in Acute Myeloid Leukemia (AML) cells from the majority of AML patients at diagnosis, and CREB overexpression is associated with a poor prognosis. Transgenic mice overexpressing CREB in myeloid cells develop myelodysplasia/myeloproliferative neoplasms. CREB also cooperates with other oncogenes, such as Sox4, to induce transformation to AML. Knockdown of CREB inhibits AML proliferation but does not affect normal hematopoietic stem cell activity, establishing the crucial role of CREB in AML cell growth and survival.

In vitro, CREB overexpression leads to increased resistance to apoptosis in AML cells. Thus, we hypothesized that increased CREB expression confers chemoresistance, as this may represent one reason that patients with high CREB levels have worse prognoses and relapse following therapy. Previous studies have demonstrated that chemotherapy resistance can result from increased DNA damage repair activity, but CREB has never been implicated in these DNA damage repair processes, nor has CREB even been described as an important transcriptional regulator of DNA damage repair genes.  The goal of this study was to characterize whether CREB expression confers chemoresistance through regulation of DNA repair genes in AML cells. Firstly, we established that CREB expression levels correlate with chemoresistance by treating KG-1 cells engineered to express lower and higher levels of CREB with etoposide and doxorubicin, both chemotherapy drugs used to treat AML. Cells with CREB overexpression had increased viability compared to CREB knockdown cells after treatment with both chemotherapies at a range of concentrations. To investigate the underlying mechanism, we performed CREB chromatin immunoprecipitation and RNA-seq following small molecule CREB inhibition to identify the sets of genes that are regulated by CREB in AML cells and whose expression levels are sensitive to CREB inhibition. Out of 88 DNA damage repair genes found to be CREB-bound, 41 exhibited at least a 2-fold change in expression after CREB inhibition. qPCR was performed to determine whether the expression of DNA damage repair genes were proportional to CREB levels. Transcription of ATM, ATR, RAD54L, and RAD51, genes important in sensing and repairing DNA damage, were coordinately regulated with CREB expression. ATM, ATR, RAD54L, and RAD51 were reduced by approximately 42.0%±0.1%, 44.8%±0.1%, 40.2%±0.1%, and 27.9%±0.1% respectively in CREB knockdown cells (p≤0.05). Reduced expression of these genes also had a functional consequence. CREB knockdown cells initiated a lesser DNA damage repair response in response to etoposide treatment, as determined by measured phospho-H2AX levels, compared to wild-type CREB-expressing cells.  Conversely, cells with CREB overexpression exhibited the strongest DNA damage repair response following etoposide treatment.  

Taken together, these data demonstrate that CREB overexpression has a protective effect against DNA damage and confers chemoresistance, likely through upregulation of DNA damage repair genes. Future studies will seek to determine if small molecule inhibition of CREB can reduce the transcription of DNA damage repair genes and thus sensitize AML cells to chemotherapeutic agents.