Asciminib resistance of a new BCR::ABL1 p.I293_K294insSSLRD mutant detected in a Ph + ALL patient

Abstract

Chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia patients largely benefit from an expanding tyrosine kinase inhibitors (TKIs) toolbox that has improved the outcome of both diseases. However, TKI success is continuously challenged by mutation-driven acquired resistance and therefore, close monitoring of clonal genetic diversity is necessary to ensure proper clinical management and adequate response to treatment. Here, we report the case of a ponatinib-resistant Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL) patient harboring a BCR::ABL1 p.I293_K294insSLLRD mutation. Using in vitro proliferation assays on newly generated Ba/F3 cell lines, we confirmed that the mutation confers moderate resistance to ponatinib, and to imatinib and nilotinib. In contrast, BCR::ABL1^SLLRD Ba/F3 cells remain highly sensitive to dasatinib. Unexpectedly, the insertion also provides resistance to asciminib with no inhibitory effect up to 1000 nM. Based on predicted structural models, we speculate that the p.I293_K294insSLLRD disrupts the interaction between the SH3 domain and the kinase domain, shifting the equilibrium toward the active conformation. This shift confers resistance to TKIs that preferentially bind to the inactive conformation, as well as to the allosteric asciminib inhibitor. However, the mutation retains sensitivity to dasatinib, which targets the active form of the kinase.

Keywords: ALL; BCR::ABL1; CML; Cancer resistance; Insertion mutation; Tyrosine kinase inhibitor.

Fig. 1

TKI resistance profile induced by the p.I293_K294insSLLRD mutation. (a) Experimental workflow for assessing the impact of the p.I293_K294insSLLRD mutation on proliferation or cell viability following TKI treatment. (b,c) BCR::ABL1^WT or BCR::ABL1^SLLRD cells treated for 72 h were assessed for proliferation with ponatinib at concentrations ranging from 0 to 10 nM (b) and for cell viability at concentrations up to 100 nM (c). (d) Proliferation of BCR::ABL1^WT or BCR::ABL1^SLLRD cells after 72-h treatment with imatinib at concentrations up to 10 µM. (e,f) Proliferation of BCR::ABL1^WT or BCR::ABL1^SLLRD cells treated for 72 h with nilotinib at concentrations ranging from 0 to 100 nM (e) and cell viability assessed at concentrations up to 2000 nM (f). (g,h) Proliferation of BCR::ABL1^WT or BCR::ABL1^SLLRD cells treated for 72 h with dasatinib at concentrations ranging from 0 to 10 nM (g), and cell viability assessed at concentrations as low as 0.05 nM (h). Proliferation data are from two independent experiments (b,d,g) or one experiment (e) with n = 4 replicates for each experiment. Cell viability data are from one independent experiment with n = 1 replicate (c,f,h). Results are presented as mean ± SEM as appropriate

Fig.2

The p.I293_K294insSLLRD mutation induces resistance to asciminib. (a) Comparison of proliferation of BCR::ABL1^WT or BCR::ABL1^SLLRD cells treated for 72 h with 0 to 20 nM asciminib. (b,c) Detection of phosphorylated tyrosine (P-Tyr) by western blot (b) and associated ratio (c) in protein lysates extracted after 2 h of asciminib treatment. Hsp60 protein was used as loading control. (d,e) Detection of Stat5 and phosphorylated Stat5 by western blot (d) and associated ratio (e) in protein lysates extracted after 2 h of asciminib treatment. (f) Cell viability of BCR::ABL1^WT (left panel) or BCR::ABL1^SLLRD (right panel) cells treated for 72 h with 0 to 1000 nM asciminib. Proliferation data are from two independent experiments with n = 4 replicates for each experiment (a). Cell viability data are from one independent experiment with n = 1 replicate (f). Protein level quantification was performed on one (b) or two (d) western blot replicates from the same lysates. Results are presented as mean ± SEM as appropriate

Fig.3

Location of the p.I293_K294insSLLRD mutation. (a,b) AlphaFold-predicted ABL1 structure for (a) BCR::ABL1^WT and (b) BCR::ABL1^SLLRD. (c,d) Focus on N-lobe and SH3 interface. In the BCR::ABL1^WT, p.E98 (located in the SH3 domain) interacts with p.K294 of the N-lobe of the kinase domain (KD). The salt bridge is represented by a black line. In the BCR::ABL1^SLLRD, the p.I293_K294insSLLRD insertion mutation induced a conformational change of p.K294 (≈ 90° rotation). (e,f) Superposition of AlphaFold-predicted ABL1 conformation of BCR::ABL1^WT and BCR::ABL1^SLLRD (e) and focus on the interface between the N-lobe of the kinase domain (KD) and SH3 (f). BCR::ABL1^WT and BCR::ABL1^SLLRD are represented in cyan and orange respectively. In addition, p.I293_K294, p.I293_K294insSLLRD and p.E98 are highlighted in green, purple and blue respectively. The overall conformation of ABL1 is unchanged by the presence of the p.I293_K294insSLLRD mutation, except at the SH3-KD interface