Orally Bioavailable CDK9 Inhibitor Voruciclib Downregulates Oncogenic Pathways in Leukemic Cells from Patients Treated on a Clinical Trial
Authors: Dominguez Edward C. , Roleder Carly , Chen Canping , Xia Zheng , Wiley Sandra , Davids Matthew S. , Danilov Alexey 
Published: December 2024

Background

BCL2 family proteins are essential regulators of apoptosis. MCL1 is dysregulated in both lymphoid and myeloid blood malignancies. Its overexpression predicts inferior outcomes with standard therapies and contributes to resistance to targeted agents, including BTK and BCL2 inhibitors. MYC is a transcription factor that drives oncogenesis across hematologic cancers. Our preclinical work demonstrated that inhibitors of cyclin-dependent kinase-9 (CDK9i) downregulate MCL1 and disrupt MYC transcriptional program causing anti-tumor effects in preclinical studies (Thieme et al, 2023). Here we investigated in vivo effects of voruciclib, an orally bioavailable CDK9i, in patients with acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL).

Methods

Patients with AML or CLL were treated on a Phase I dose-escalation trial with 100 mg, 150 mg, or 200 mg of voruciclib on an intermittent dosing schedule (2 weeks on, 2 weeks off; NCT03547115). Peripheral blood samples were collected at cycle 1 day 1 (C1D1) pre-dose, C1D1 6 hr post-dose, C1D8 (pre-dose) and (pre-dose, AML) or C2D1 (pre-dose, CLL). PBMCs were isolated using Ficoll technique; RNA from 6 AML patients and 3 NHL patients was subjected to bulk RNA-Seq. Single-cell RNA-Seq (scRNA-Seq) was performed using samples from 2 AML patients.

Results

Immunoblotting analysis of voruciclib-treated samples confirmed downregulation of phosphorylated Ser2 of RNA Pol II, consistent with CDK9 inhibition. Principal component analysis of RNA-Seq samples from 6 AML patients dosed with 100-200 mg of voruciclib revealed that samples clustered by patient rather than by time point, indicating significant heterogeneity in baseline activity of signaling pathways between individual patients. PBMCs expressed 22,055 protein coding genes. Expression of 952 genes was affected by voruciclib (&gt;1.5-fold change). Eleven genes were deregulated at 6 hr and 50 genes were deregulated after 8 days of treatment (padj&lt;0.05). MYC transcriptional targets, TNF signaling via NFKB, cell cycle E2F targets, and G2M checkpoint were the top oncogenic pathways significantly associated with the downregulated genes after voruciclib exposure. Expression of genes known to be regulated by MYC was significantly affected by voruciclib as early as 6 hr (p&lt;0.05). AML demonstrated a trend towards reduction in MYC, and MCL1 mRNA transcript levels, while CDK9 mRNA remained unchanged. Consistent with this, RT-PCR analysis of on-treatment samples demonstrated a decrease in Mcl-1 mRNA levels following voruciclib exposure compared to pre-treatment. RNA-Seq analysis was also performed on longitudinal PBMC samples from subjects with CLL, revealing a downregulation of TNF signaling via NFKB and a decrease in the OxPhos gene set following voruciclib treatment. We also observed a trend towards reduced mRNAs for MYC, MCL-1, and BCL2A1 in CLL cells.

Longitudinal samples from 2 AML patients with high baseline blast counts were submitted for sc-RNA-Seq analysis. Patient 1 received treatment with 200 mg voruciclib. In total, we identified 11 clusters across several distinct lineages: myeloid, T cells, B cells and platelets. There was no clustering by collection timepoints, suggesting negligible batch effects because of our multiplexing strategy. We identified several distinct myeloid cell clusters, including HSC_MPP, “monocytes” and “dendritic cells”, all exhibiting distinct gene expression patterns. InferCNV analysis was used to identify malignant myeloid cells to enable evaluation of genes and pathways deregulated by voruciclib in the myeloid cell clusters. Similar to bulk RNA-Seq data, MYC gene targets and OxPhos pathways were the most downregulated in the malignant cell cluster by day 15 compared to baseline. We also observed MYC pathway downregulation within the CD4+ and CD8+ T-cell clusters. These results were broadly upheld in sample 2, obtained from a patient treated with 100 mg voruciclib, which also demonstrated MYC and OxPhos downregulation in malignant AML cells.

Conclusions

Here we report that AML and CLL samples from patients treated with the CDK9i voruciclib on a clinical trial demonstrated downregulation of multiple oncogenic pathways, including MYC transcriptional targets and OxPhos, consistent with the predicted mechanism of action. Analysis of additional samples is ongoing.