Stratification and selective targeting based on anti-apoptotic protein expression in chronic lymphocytic leukaemia (CLL)

Abstract

Chronic lymphocytic leukaemia (CLL) remains a largely incurable haematological malignancy, with variable responses to targeted therapies such as the BCL2 inhibitor venetoclax. Richter's transformation (RT) involves the evolution of CLL into an aggressive high-grade lymphoma and is associated with particularly poor outcomes. This highlights the urgent need for more effective therapeutic strategies. Resistance to venetoclax is linked to compensatory upregulation of alternative anti-apoptotic BCL2 family members, particularly MCL1, which is at least partially driven by aberrant NF-kB signalling within the lymphoid microenvironment. Although direct MCL1 inhibitors have shown promising preclinical activity in CLL, these results have failed to translate to the clinic due to the labile nature of MCL1 protein and cardiotoxicity concerns.
Cyclin-dependent kinase 9 (CDK9) is a key regulator of transcription elongation through its phosphorylation of RNA Polymerase II. Acute inhibition of CDK9 preferentially suppresses transcription and rapidly depletes short-lived proteins including MCL1.Selective CDK9 inhibitors have demonstrated strong anti-tumour activity and favourable tolerability in haematological malignancies including acute myeloid leukaemia and diffuse large B-cell lymphoma, supporting their potential use in MCL1-high CLL and RT.
In this thesis, CLL patient samples were stratified based on anti-apoptotic BCL2 family abundances to guide targeted therapeutic strategies. Flow cytometry analysis revealed significant inter- and intra-tumoral heterogeneity of basal anti-apoptotic BCL2 family proteins (BCL2, BCL-XL, MCL1) and NF-kB subunits (p65/RelA, RelB, c-Rel) in primary CLL cells. In vitro models that mimic microenvironmental signalling (B-cell receptor and Toll-like receptor 9 activation, and co-culture with CD40L-expressing NIH/3T3 fibroblasts) were subsequently employed. These resulted in modest increases in BCL2 yet substantially greater induction of BCL-XL and, most strikingly, MCL1, identifying a subset of patients with apparent reliance on MCL1-mediated survival.
Two orally bioavailable, selective second-generation CDK9 inhibitors, AU4-53 and AU11-8, were evaluated in the MCL1-high CLL subset, as well as in the RT cell line U-RT1 and ex vivo RT patient-derived xenograft (PDX) cells. For primary CLL and RT-PDX RS9737 cells, this was determined under cytoprotective CD40L co-culture conditions. AU11-8consistently demonstrated superior cytotoxic activity as well as greater selectivity for primary CLL cells over autologous T-cells via Annexin V and 7-AAD staining. Mechanistically, this inhibitor suppressed Serine 2 phosphorylation of RNA Polymerase II and caused a rapid loss of MCL1 protein after 4 hours. Importantly, sensitivity to AU11-8correlated strongly with MCL1 expression, and combination treatment with venetoclax consistently revealed marked synergy at defined concentrations, highlighting the potential of selective CDK9 inhibitors to resensitise MCL1-dependent tumour cells to venetoclax induced apoptosis.
Taken together, these findings support selective CDK9 inhibition as a promising therapeutic strategy for MCL1-high CLL and RT. By depleting labile MCL1 protein through suppression of transcriptional elongation, this approach may help overcome microenvironment-mediated treatment resistance and enhance sensitivity to venetoclax. Ultimately, this work highlights MCL1 expression and functional MCL1 dependence as candidate biomarkers for patient stratification while supporting the rationale for diagnosticBH3 profiling to inform more precise, personalised treatment strategies.

Date of Award	Jul 2026
Original language	English
Awarding Institution	University of Brighton
Supervisor	Andrea Andrea Pepper (Supervisor), Chris Pepper (Supervisor) & Simon Mitchell (Supervisor)