Inhibition of Cyclin-Dependent Kinase 9 Rapidly Induces Apoptosis in Acute Lymphoblastic Leukemia and Shows Synergistic Activity with BH3-Mimetics

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Around 85% are derived from B-cell precursors (BCP-ALL) while the remaining 15% are of T-cell lineage (T-ALL). Although successful treatment of ALL patients using cytotoxic therapies has continuously improved, differently characterized subgroups show inferior survival including leukemias with high-risk genetic abnormalities, patients at relapse or with T-ALL. In addition, the use of intensive chemotherapy protocols is associated with high treatment-related morbidity and mortality, underscoring the need for new therapeutic approaches. Cyclin-dependent kinases (CDKs) are central regulators of the cell cycle. Strategies to inhibit CDKs have been investigated as potential anti-cancer therapies leading to clinical application in different cancers. However, CDKs might be involved in regulation of other cellular processes.

In this study, we addressed sensitivities of BCP- and T-ALL samples to CDK inhibitors and analyzed mechanisms of cell death induction, involved regulators and possible strategies to increase anti-leukemia activities.

Using the multi-CDK inhibitor dinaciclib, which targets CDK1, 2, 5 and 9, and the specific CDK9 inhibitor AZD4573, we analyzed the effects of both inhibitors in a series of ALL cell lines (BCP-ALL n=10, T-ALL n=6) and primary, patient-derived xenograft (PDX) samples (BCP-ALL n=15, T-ALL n=9). Both CDK inhibitors led to a rapid induction of cell death at low nanomolar concentrations in BCP- and T-ALL, cell line and PDX samples. Cell death was characterized by Annexin V and propidium iodide positivity, mitochondrial cytochrome-c release, activation of caspase 3 and 7, cleavage of PARP and DNA fragmentation, indicating induction of apoptosis by the CDK inhibitors. Interestingly, an unchanged distribution of ALL cells to cell cycle phases was observed, suggesting that both inhibitors primarily do not affect cellular cycling. CDK9 functions as a regulator of RNA polymerase II (RNAP II) and CDK9 inhibition leads to decreased RNAP II activity and shutdown of the transcriptional machinery. Upon both inhibitors, we found a dose-dependent reduction of RNAP II phosphorylation along with downregulated transcript levels of genes coding for the pro-survival regulators BCL-2, BCL-XL and MCL-1. At the same time, only protein levels of MCL-1 were significantly reduced while BCL-2 and BCL-XL protein remained at unchanged high expression levels. In contrast to the long-lived proteins BCL-2 and BCL-XL, MCL-1 has a very short half-life, in line with our finding of almost completely lost MCL-1 levels upon CDK inhibition. This imbalance of pro-survival molecules might explain the incomplete cell death induction by CDK inhibitors and points to involvement of anti-apoptotic signaling pathways mediated by BCL-2/BCL-XL as mechanism of intrinsic insensitivity of ALL cells to CDK inhibition. Based on this, we investigated combinatorial inhibition of CDK9 (AZD4573) together with inhibitors of BCL-2 or BCL-XL (venetoclax, A-1331852, AZD4320) using dose-response matrix analyses and found enhanced cell death and synergistic activity for both combinations. Thus, incomplete cell death induction observed upon CDK9 inhibition alone can be overcome by co-targeting pro-survival molecules. Interestingly, and in line with our functional data obtained in BH3-profiling showing dependence on BCL-2 in BCP-ALL and dependence on BCL-XL in T-ALL, the most effective combinations in most of the BCP-ALL samples were achieved by combining CDK9 inhibition with the specific BCL-2 inhibitor venetoclax, while the most effective combination for T-ALL was observed upon co-inhibition involving BCL-XL.

Taken together, our study demonstrates that CDK9 inhibitors effectively induce apoptosis in both BCP- and T-ALL at low nanomolar concentrations. However, intrinsic insensitivity due to dysbalanced protein levels of pro-survival proteins might limit efficacy. Combining CDK9 inhibition with inhibitors of the anti-apoptotic molecules BCL-2/BCL-XL significantly enhances cell death, thus overcoming insensitivity and providing an effective, novel therapeutic anti-ALL strategy to be further evaluated for clinical application.