CDK9 Inhibition Overcomes Ibrutinib Resistance in Mantle Cell Lymphoma (MCL)

Introduction: Bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib [ibr]) have shown clinical efficacy in MCL. However, resistance to BTK inhibitors is inevitable. Cyclin-dependent kinases (CDKs) are Ser/Thr kinases that associate with specific cyclins during the cell cycle to regulate its progression. We and others have shown that selective targeting of CDK9, a pTEFb component which facilitates RNA transcription, downmodulates MYC and MCL1 in malignant B-cells. Here, we investigated the effects of a selective CDK9 inhibitor, AZD4573, in ibr-naive and ibr-resistant MCL in vitro and in vivo.

Methods: MCL cell lines (Mino, JeKo-1) were cultured in increasing doses of ibr for ~6 months to generate resistance. MCL cell lines were treated with AZD4573 and assayed for apoptosis/proliferation, metabolic phenotype (Seahorse), and protein and RNA expression. Primary MCL cells were incubated for 24 h in CD40L/BAFF-conditioned media prior to drug treatment. Two MCL PDX models were used (chemo-resistant and ibr-resistant). MCL cells were injected IV in NSG mice and tracked by flow cytometry (CD5⁺ CD19­­⁺ CD45⁺). Upon MCL detection in the peripheral blood (~1-2%), mice began treatment with AZD4573 (25 mg/kg IP once or twice weekly) or vehicle control. Splenocytes were harvested and RNA was isolated and analyzed by RNA-Seq/RT-PCR. Additionally, primary circulating MCL cells were collected from a patient treated with one dose of AZD4573 (at baseline; 4 and 7 h post-infusion and day 7) and analyzed by scRNA-Seq.

Results: Treatment with AZD4573 potently inhibited proliferation and induced apoptosis of both ibr-sensitive and resistant MCL cell lines in vitro (IC₅₀ ~ 2-15 nM). AZD4573 also induced apoptosis of primary MCL cells in stromal conditions. Loss of RNA polymerase phosphorylation at Ser2 residue, a CDK9-dependent site, preceded apoptosis as measured by cleaved PARP. AZD4573 did not inhibit CDKs 2, 4/6, and 7 (as measured by pRb and pRNApol [Ser5]), confirming its selective effect. CDK9 inhibition resulted in rapid and dose-dependent downregulation of MYC and MCL1 mRNA and protein levels in MCL cell lines and primary cells.

Treatment with AZD4573 but not with ibr extended survival of ibr-resistant PDX mice without significant toxic events. RNA-Seq analysis of the MCL splenocytes harvested from these mice after 3 weeks of AZD4573 treatment demonstrated that gene sets related to transcription-factor activity were most enriched in the treated samples, consistent with known drug mechanism. TNFa/NFκB, TGF-β and PI3K/AKT/mTOR signaling pathways were most downregulated. We have shown that CDK9 inhibition leads to epigenetic reprogramming in neoplastic B-cells, resulting in upregulation of a subset of superenhancer-associated oncogenes, i.e. MYC, PIM kinase and others (Thieme et al, 2023). Consistent with this notion, splenocytes derived from AZD4573-treated mice exhibited upregulation of MYC-dependent targets and OxPhos at the time of sacrifice. A number of genes relating to components of the electron transport chain were upregulated (ATP5F1D, NDUFS7, ATP5MC1 as the top three). This suggested that these pathways may govern resistance to CDK9 inhibition in MCL over time. To further explore this, parental and ibr-resistant MCL cell lines were treated with low doses of AZD4573 (~2 nM). After 48 h, CDK9 inhibition was associated with increased oxygen consumption rates (OCR) in Seahorse analysis. This was not accompanied by increased cell proliferation by cell cycle analysis or Ki-67 staining. IACS-010759, an inhibitor of complex I of the ETC which targets OxPhos, demonstrated synergy with AZD4573 in parental and ibr-resistant MCL cell lines.

Single-cell RNA-Seq analysis of primary MCL cells obtained from a patient treated with AZD4573 on a clinical trial revealed rapid downmodulation of NFκB signaling (at 4 h) and MYC transcriptional targets and PI3K/AKT/mTOR signaling pathways (at 7 h). Interestingly, we observed partial recovery of MYC, mTOR pathways and induction of OxPhos by day 7 (prior to next planned dose), validating our pre-clinical findings.

Conclusion: CDK9 inhibition with AZD4573 induced apoptosis, downregulated MYC and MCL1 and NFκB signaling, and overcame ibr resistance in preclinical MCL models. This was also noted in a sample obtained from a clinical trial. Prolonged CDK9 inhibition led to metabolic reprogramming towards OxPhos, which thus can serve as a therapeutic target in MCL.