Abstract
CDK9 plays an integral role throughout the cell cycle as a regulator of transcription elongation through RNA polymerase II. CDK9 is essential to the synthesis of key proteins that drive acute myeloid leukemia (AML), like MEIS1, MCL-1, and MYC. The downregulation of protein synthesis that occurs when CDK9 is inhibited provides a rationale for a potential therapeutic window in the treatment of AML with CDK9 inhibitors.
Tambiciclib (SLS009) is a novel and highly selective CDK9 inhibitor, making it an attractive candidate for use in combination with a standard of care AML treatment. Tambiciclib has steadily advanced in early-phase clinical trials and has already shown promising clinical benefits when combined with azacitidine, a hypomethylating agent, and venetoclax, a BCL-2 inhibitor; however, there is still a need for optimization of the treatment window for maximal benefit.
Here, we aim to examine the mechanism through which tambiciclib induces cell death and further examine the therapeutic window for treatment of AML with tambiciclib, as a single-agent and when combined with standard therapeutic agents, azacitidine and venetoclax. To thoroughly examine the effects of tambiciclib, we examined cell viability after tambiciclib exposure of AML cell lines harboring mutations that drive leukemia resistance (TP53 and ASXL1). We assessed how the expression of several key leukemia-driving proteins changes throughout exposure to tambiciclib, and how those levels recovered over time following treatment.
An 8-hour exposure to tambiciclib showed dose-dependent cytotoxicity in the low nanomolar range for various AML cell lines. To examine how a mutation in the ASXL1 gene affects the leukemia response to CDK9 inhibition, we used the TP53 and ASXL1 mutated cell line, NOMO-1, and the TP53 mutated and ASXL1 wildtype cell line, THP-1, and compared cell viability after an 8-hour exposure to tambiciclib. The IC50 was 43 nM for the NOMO-1 cell line and 42 nM for the THP-1 cell line. To isolate the effect of TP53 on CDK9 inhibition, we compared the effects of tambiciclib on the wild-type TP53 MOLM-13 cell line and the Cas12a-edited TP53 knockout MOLM-13 cell line. Their IC50s were 23 nM and 46 nM, respectively. These low nanomolar IC50s suggest CDK9 inhibition could potentially be an effective treatment for leukemias characterized by ASXL1 and TP53 mutations. After determining the IC50s from one 8-hour exposure, we performed repeated 8-hour exposures of tambiciclib, 24 hours apart, for up to three days. Additional treatments seem to result in lower IC50s, warranting further investigation into the mechanism through which this occurs.
After establishing the cytotoxic effects of tambiciclib as a single agent, we investigated the combinatorial effects of tambiciclib with standard of care chemotherapy agents, azacitidine and venetoclax. Using dose-response matrices, we measured cell viability after an 8-hour exposure to tambiciclib, azacitidine, and venetoclax in two-drug combinations. We found that when combined with venetoclax, there is a substantial decrease in cell viability. At 1.5 uM venetoclax, well below our observed IC50 of roughly 10 uM, the addition of 40 nM tambiciclib decreases the viability of THP1 cells from 83% to 7%. We hypothesize this could be due to the combined effects of tambiciclib and venetoclax on MCL-1. We plan to further investigate this by examining the protein expression of MCL-1, MEIS1, and c-MYC through immunoblotting.
Our data suggests CDK9 inhibition by tambiciclib can be used to induce cell death in AML cell lines with known leukemia-driving mutations. Inhibition of CDK9 with tambiciclib is a promising and effective approach for inducing cell death in AML. Our preliminary data warrants further investigation to optimize the therapeutic window for tambiciclib treatment in repeated doses and combination therapies.
