Simultaneous Sessions I: Mechanisms and strategies to address TKI resistance and stem cell persistence
The BCR-ABL1 chimeric oncoprotein drives chronic myeloid leukaemia (CML) pathogenesis. The introduction of tyrosine kinase inhibitors (TKIs) has transformed clinical outcomes for patients with CML, with over 80% of those treated with imatinib (IM) surviving for more than 10 years. Second and third-generation TKIs are more potent and can impede the emergence of resistance, inducing deep molecular responses in both untreated and IM-resistant CML patients. Despite the success of TKIs, up to 35% of patients discontinue their TKI due to intolerance or develop resistance and this remains a key area of unmet clinical need. Asciminib (formerly ABL001) is a potent and selective allosteric inhibitor of ABL1 that binds to the myristoyl binding pocket of ABL1 to hold BCR-ABL1 in an inactive conformation. Preclinical studies have shown that asciminib selectively inhibits the growth of BCR-ABL1 positive (+) cells regardless of the presence of BCR-ABL1 point mutations1. Clinical trials for patients with CML or Ph+ acute lymphoblastic leukaemia, testing asciminib alone and in combination with TKIs are currently underway and preliminary results are promising.
To assess if dual inhibition of BCR-ABL1 leads to improved treatment outcomes in preclinical studies.
Here, we assess the effects of asciminib, alone and in combination with ATP-competitive TKIs (IM, nilotinib (NIL) and ponatinib (PON)) in CML cell lines and primary CD34+ chronic phase (CP)-CML stem/progenitor cells (HSPC). We assessed synergy using resazurin readouts using CompuSyn Software. We performed cell counts, apoptosis, cell-cycle and proliferation assays to determine the effect of drug combinations in CML cell lines and primary samples; and confirmed effects on primitive cells using colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays in vitro.
Dose-response studies using the resazurin assays in CML cell lines (Bv173, K562, KCL22) indicate that asciminib is potent at low nanomolar concentration, even in cells that express the BCR-ABL1 T315I point mutation (KCL22). Apoptosis and cell cycle assays' assessed by FACS showed that the inhibitory effects of asciminib were maintained in KCL22T315I-expressing cells when asciminib was used in combination with PON. Washout studies with asciminib in KCL22WT/T315I-expressing cells demonstrated a prolonged phenotypic response using low-nanomolar doses of asciminib as the cells failed to regrow and had irreversible cell-cycle damage.
Primary CD34+ CML HSPCs demonstrated proliferation arrest and increased apoptosis (70-100% increase relative to control; p < 0.001) when treated for up to 72 hours with asciminib, alone and in combination with IM or NIL. LTC-IC and CFC assays, determining the functional activity of primitive CML HSPCs in vitro, demonstrated that the combination of asciminib with IM or NIL reduced colony outputs (60-90% decrease relative to controll, p < 0.001), beyond that achieved with each drug alone (40-80% relative to control, p < 0.001), and in separate experiments, with minimal effect on normal HSPC.
These results suggest that asciminib represents a novel therapeutic approach with effects on primitive CP-CML HSPCs both as a single agent and in combination with TKI and has efficacy in cells expressing the multi-TKI resistant T315I mutation. We are now investigating the mechanism of action for asciminib, alone and in combination with NIL, by RNA-sequencing.