The transcription factor STAT5 is constitutively activated in many forms of hematologic malignancies, including chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). STAT5 can be activated by constitutively activated tyrosine kinases or autocrine and paracrine secretion of cytokines signaling through Jak kinases. STAT5 is essential for the pathogenesis of neoplasms induced by BCR-ABL1 and Jak2V617F, as well as for leukemia stem cell self-renewal. Development of tyrosine kinases inhibitors (TKIs), such as imatinib, has greatly improved the outcome of patients with leukemias harboring aberrantly activated oncogenic tyrosine kinases. However, TKIs used as a single agent only achieve significant success in CML, with very limited benefit in the more aggressive ALL. Moreover, patients with CML who initially respond well may acquire resistance to TKIs with the progression of their disease. In fact, increased activity of STAT5 is often associated with CML progression and may underlie resistance to TKIs. Importantly, leukemia cells that are resistant to TKIs remain sensitive to STAT5 inhibition, and dual inhibition of both tyrosine kinases and STAT5 leads to more efficient reduction of leukemia cell viability. Thus targeting STAT5 alone or in combination is a promising therapeutic strategy for many hematological malignancies. While many strategies directly inhibit STAT5, we considered the possibility that STAT5 association with co-regulatory proteins is essential for STAT5 function and therefore targeting this association may be a suitable therapeutic strategy.
Given the importance of BET bromodomain proteins in chromatin remodeling necessary for transcription, we tested the activity of the BET bromodomain inhibitor JQ1 on STAT5-dependent transcriptional activity. Using both heterologous reporter systems and endogenous STAT5 target genes, we found that JQ1, but not its inactive enantiomer, potently and specifically inhibited STAT5-dependent gene expression. Inhibition of STAT5 dependent gene regulation was also replicated by another BET bromodomian inhibitor, iBET, further demonstrating that BET inhibition inhibits STAT5. Since JQ1 inhibits BET family members Brd2, Brd3, Brd4, and BrdT, we asked which BET family member is specifically associated with STAT5 transcriptional function. To do this, we utilized shRNA to knock-down each bromodomain protein and determined the effect on STAT5 activity. We found that knocking-down Brd2, but not Brd3 or Brd4, reduces STAT5 target gene expression, indicating that Brd2 is specifically involved in regulating STAT5 transcriptional function. JQ1 can reduce STAT5 transcriptional activity without inhibiting STAT5 phosphorylation or STAT5 binding to its genomic binding sites. Similarly, knocking-down Brd2 can reduce STAT5 target gene expression without influencing STAT5 phosphorylation. We hypothesize that Brd2 regulates STAT5 transcriptional function by acting as a co-activator for STAT5. Thus through blocking Brd2, JQ1 can inhibit STAT5 transcriptional function without directly targeting STAT5 itself. In a group of aggressive T cell acute lymphoblastic leukemia (T-ALL) cell lines, where constitutively activated STAT5 contributes to leukemia cell survival, knocking-down Brd2 renders leukemia cells more sensitive to TKI induced apoptosis. In addition, combined treatment with TKIs and JQ1 showed strong synergy in inducing T-ALL leukemia cells apoptosis and reducing viability. Overexpressing a constitutively active form of STAT5 rescues these leukemia cells from death induced by TKIs and JQ1, indicating an important role of STAT5 as a target for TKI and JQ1 induced cell death in T-ALL cells.
We found that the BET bromodomain inhibitor JQ1 can reduce STAT5 transcriptional function by blocking Brd2 without reducing STAT5 phosphorylation or STAT5 DNA binding. In addition, the combination of TKIs and JQ1 induces T-ALL leukemia cell apoptosis and reduces survival in a synergistic manner, and represents a rational drug combination for treating this sub-group of highly aggressive leukemias.
Bradner:Tensha Therapeutics: Consultancy, Equity Ownership, Scientific founder Other.
Asterisk with author names denotes non-ASH members.