Asciminib antagonizes transplantable BCR::ABL1-positive lymphoid blast crisis in vivo by targeting malignant stem cells

Abstract

Philadelphia chromosome-positive (Ph+) lymphoid blast crisis (BC), emanating from chronic myeloid leukemia (CML), is a fatal disease with limited treatment options. Asciminib (ABL001) is a novel selective allosteric inhibitor of the ABL kinase with high efficacy against TKI-resistant BCR::ABL1. In this study, we demonstrate significant suppression of an aggressive B-lymphoblastic disease and restoration of normal hematopoiesis in an inducible transgenic mouse model of p210-BCR::ABL1-positive CML-BC. Molecularly, asciminib treatment significantly reduced BCR::ABL1 transcripts to background levels, demonstrating its ability to suppress BCR::ABL1-induced disease. Furthermore, asciminib treatment normalized the long-term repopulating hematopoietic stem cell (LT-HSC) population in the BM, suggesting the selective targeting of malignant LT-HSCs. This was supported by secondary transplantation experiments, resulting in absence of BC in a proportion of mice. Importantly, none of the secondary transplanted mice that received further asciminib treatment developed leukemia. Sanger sequencing of the BCR::ABL1 myristoyl pocket region of both treatment-naïve and treated mice demonstrated a high mutational load. However, there was no indication of asciminib-specific mutations. These promising findings highlight the potential of asciminib as a drug that targets BC stem cells and as an alternative stand-alone or combinatorial therapy for first-line treatment of CML BC or Ph+ acute lymphoblastic leukemia.

Fig. 1. Lymphoid blast crisis is blocked by ABL001 treatment.

A Log-rank (Mantel-Cox) test demonstrates significantly (p = 0.0259) higher survival of ABL001-treated mice or single-transgenic (stg) mice (p = 0.0123) in comparison to vehicle controls (n = 5 vehicle and ABL001; n = 3 stg), 49 days after transplantation. B Spleen weight of vehicle- and ABL001-treated, as well as single-transgenic (stg; n = 3) mice, were compared using a One-way ANOVA test (Kruskal–Wallace and Dunn’s multiple comparisons test). C Percentage of B220 medium-high (med-high) cells in peripheral blood, bone marrow, and spleen was analyzed by flow cytometry. One-way ANOVA test (Kruskal–Wallace and Dunn’s multiple comparisons test) was used for statistical analysis. D In the lin- Sca1+ cKIT+ (LSK) cell population, percentage of the LT-HSCs, ST-HSCs, MPP1, and MPP2 cells was analyzed in bone marrow and spleen. One-way ANOVA test (Kruskal–Wallace and Dunn’s multiple comparisons test) was used for statistical analysis. E Absolute cell number of LT-HSC in BM. One-way ANOVA test (Kruskal–Wallace and Dunn’s multiple comparisons test) was used for statistical analysis. F BCR::ABL1 expression, normalized to Gapdh, in the bone marrow and spleen of transplanted mice. Mann–Whitney-U-test was used for evaluating significance using ABL001 as reference. *p < 0.05, **p < 0.01, ns not significant.

Fig. 2. Asciminib retains potent antileukemic activity in secondary transplants.

A Percentage of B220 medium-high (med-high) cells in peripheral blood, bone marrow, and spleen of the secondary transplantations were analyzed by flow cytometry. Colors indicate the treatment in the first transplant. B Peripheral blood, bone marrow, and spleen were analyzed by flow cytometry for the B lymphoblastic population, and vehicle-treated mice were divided into the treatment groups of the first transplant and illustrated in a box-and-whisker plot with highlighted median. C BCR::ABL1 expression, normalized to Gapdh, in the spleen of transplanted mice. For (A–C), Mann–Whitney-U-test was performed. D Overview of mutations found in the kinase domain and myristoyl binding site (aa 268–540) of BCR::ABL1 in secondary transplantations of SCL tTA-BCR::ABL1 mice. The mutations which were detected were assorted into four groups, depending on the treatment of first and secondary recipients, and again into three subgroups, comprising already described mutations in BCR::ABL1 (violet; partly known to provide TKI resistance), previously described mutations of unknown significance in ABL1 (dark red), and undescribed, novel mutations (black) of unknown significance in the kinase domain and myristoyl binding site of BCR::ABL1. aa amino acid, VEH vehicle.