Alvocidib Potentiates the Activity of Cytarabine and Mitoxantrone through the Targeting of MCL-1 When Administered in a Time Sequential Regimen in AML

Alvocidib has demonstrated a significant improvement in the complete response rates of newly diagnosed acute myeloid leukemia (AML) patients when administered before cytarabine and mitoxantrone (FLAM regimen) in a randomized Phase 2 study compared to 7+3, the current standard of care. Although the mechanism of action of alvocidib as a single agent is documented, the mechanism underlying synergy found in the FLAM regimen is still not fully understood. The FLAM regimen was originally developed based on the perceived benefit of time-sequential cell cycle arrest (alvocidib) followed by release of the cells from cell cycle arrest and inhibition of DNA replication (cytarabine) during S-phase. However, recent reports suggest that the transcriptional repression of key anti-apoptotic proteins (eg., MCL-1) mediated by alvocidib's CDK9 inhibition, drive the activity in the FLAM regimen. We, therefore, hypothesized that MCL-1 transcriptional repression constitutes the primary mechanism for the synergism observed with the treatment of the FLAM regimen. Here, we demonstrate that treatment with alvocidib, followed by treatment with cytarabine and mitoxantrone, is synergistic in vitro and correlates with the downregulation of MCL-1 expression. The FLAM regimen results in significant increases in caspase activity in comparison to any single agent within the combination. As has been previously reported, we also observe that increased activity of cytarabine in alvocidib-treated cells corresponds with progression into the S-phase of the cell cycle, following the washout of alvocidib. However, this observation accounts for only a small portion of the inhibition of cell proliferation. This is further confirmed by the observation that CDK4/6 (cell cycle) specific inhibitors, such as palbociclib, do not show synergistic increases in caspase activity following treatment in the same setting. In various AML cell lines treated with MCL-1 siRNA, followed by cytarabine and mitoxantrone treatment, we also observe a synergistic increase in the inhibition of cell proliferation. Therefore, considering our earlier work showing that MCL-1 dependence predicts AML patient response to the FLAM regimen, we propose that MCL-1 repression is the primary mechanism of alvocidib's biological activity and also a primary mechanism conferring resistance to cytarabine. We also conclude that the FLAM regimen is an effective regimen, clinically, in treating patients with high-risk AML, as a consequence of its inhibition of transcription via CDK9.