Absence of ABL1 exon 2-encoded SH3 residues in BCR::ABL1 destabilizes the autoinhibited kinase conformation and confers resistance to asciminib

Ariel Leyte-Vidal^{1

2}, RosaAnna DeFilippis¹, Ian R Outhwaite³, Isabelle Kwan^{3

4

5}, Ji Young Lee⁴, Carlyn Leavitt¹, Kaeli B Miller¹, Delphine Rea⁶, Aziz M Rangwala³, Kevin Lou^{7

8}, Suhana Patel¹, Ailin Alvarez¹, Kevan M Shokat^{7

8

9}, Ivet Bahar^{4

5}, Markus A Seeliger^{3

4}, Neil P Shah¹⁰

Affiliations

¹ Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, CA, 94143, USA.
² Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, 33101, USA.
³ Department of Pharmacological Sciences, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
⁴ Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, 11794, USA.
⁵ Department of Biochemistry and Cell Biology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
⁶ Adult Hematology Department, Hôpital Saint-Louis, Paris, France.
⁷ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94158, USA.
⁸ Howard Hughes Medical Institute, University of California, San Francisco, CA, 94158, USA.
⁹ Department of Chemistry, University of California, Berkeley, CA, 94720, USA.
¹⁰ Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, CA, 94143, USA. neil.shah@ucsf.edu.

PMID: 39085402
PMCID: PMC11347358
DOI: 10.1038/s41375-024-02353-0

Absence of ABL1 exon 2-encoded SH3 residues in BCR::ABL1 destabilizes the autoinhibited kinase conformation and confers resistance to asciminib

Ariel Leyte-Vidal et al. Leukemia. 2024 Sep.

. 2024 Sep;38(9):2046-2050.

doi: 10.1038/s41375-024-02353-0. Epub 2024 Jul 31.

Authors

Affiliations

¹ Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, CA, 94143, USA.
² Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL, 33101, USA.
³ Department of Pharmacological Sciences, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
⁴ Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY, 11794, USA.
⁵ Department of Biochemistry and Cell Biology, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY, 11794, USA.
⁶ Adult Hematology Department, Hôpital Saint-Louis, Paris, France.
⁷ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94158, USA.
⁸ Howard Hughes Medical Institute, University of California, San Francisco, CA, 94158, USA.
⁹ Department of Chemistry, University of California, Berkeley, CA, 94720, USA.
¹⁰ Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, CA, 94143, USA. neil.shah@ucsf.edu.

PMID: 39085402
PMCID: PMC11347358
DOI: 10.1038/s41375-024-02353-0

No abstract available

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Conflict of interest statement

NPS has received funding from Bristol-Myers Squibb Oncology for the conduct of clinical research. DR has served on an Advisory Board, Steering Committee and as a Consultant for Novartis Pharmaceuticals. KMS has consulting agreements with the following companies, which involve monetary and/or stock compensation: Revolution Medicines, Black Diamond Therapeutics, BridGene Biosciences, Denali Therapeutics, Dice Molecules, eFFECTOR Therapeutics, Erasca, Genentech/Roche, Kumquat Biosciences, Kura Oncology, Mitokinin, Nested, Novartis, Type6 Therapeutics, Wellspring Biosciences (Araxes Pharma), Initial Therapeutics, Vevo and BioTheryX. Kin of K.L. hold stock in and are employed by Pharmaron. The remaining authors have no conflicts of interest to disclose.

Figures

**Fig. 1. Deletion of SH3, SH2, SH3/SH2 and ABL1 exon2 (resulting in the BCR::ABL1/b3a3 isoform) in BCR::ABL1 confers asciminib resistance.**
A (Left) Schematic depiction of ABL1-SH3 (gold), SH2 (maroon) and SH1 (kinase; blue) domains in an active, disassembled conformation and in an assembled autoinhibited conformation upon binding of asciminib (red). *(Right)* Schematic depiction of sequences encoded by exon 2 [*pink; (“b3a3”)]* in the SH3 domain and hypothesized resultant disruption of autoinhibited conformation despite asciminib binding. B (Upper) Schematic representation of BCR::ABL1 deletions. Location of SH3 (“3”), SH2 (“2”) domains is depicted. (Lower) Proliferation assays of pools of Ba/F3 cells transformed to IL-3 independence by BCR::ABL1 isoforms in varying concentrations of TKIs for 48 h and assessed by CellTiter-Glo. Ba/F3 parental cells were grown in the presence of IL-3. Results were performed in technical and biological triplicate. Mean values and standard errors are depicted. Table provides calculated EC₅₀ values. C Western immunoblot analysis of lysates of Ba/F3 cells transformed by BCR::ABL1 or BCR::ABL1/b3a3 and exposed to TKIs at the concentrations indicated for two hours. D Molecular response of a CML patient with BCR::ABL1/b2a3 while on treatment with imatinib and asciminib.

**Fig. 2. Impact of ABL1 exon 2 deletion on BCR::ABL1 TKI affinity and the stability of the ABL1 closed conformation.**
A Absence of ABL1 exon 2-encoded sequences in BCR::ABL1 decouples asciminib binding from allosteric regulation of the orthosteric site. (*Left*) dasatinib and (*Center*) asciminib bind both constructs with equal potency. (*Right*) asciminib is unable to displace the das-tracer in the context of BCR::ABL1/b3a3. Mean values and standard deviations are depicted. Experiments performed with the asc-tracer were conducted at 4x the IC₅₀ of the tracer for each construct; experiments with the das-tracer were performed at 2x the IC₅₀ for each construct. Table provides calculated IC₅₀ values. Results were performed in technical triplicate. B Comparison of the dynamics of ABL1 in the presence and absence of exon 2. a, b Time evolution of the overall structure RMSD from the initial structure is shown for five MD runs of 100 ns each, conducted for (a) ABL1 structure composed of the kinase, SH2, and SH3 domains, and (b) structural model for the ABL1 Δexon 2. The density plot of the RMSDs is shown along the *right ordinate* in both cases. c The time evolution of the SH3 domain RMSD in the same runs as in (b). Careful examination shows that the fluctuations in RMSD observed in (b) originate from those occurring at the SH3 domain, shown in (c). d, e Residue RMSFs observed in the five runs shown in the respective (a, b). SH3 domain is shadowed in *blue*. *Lower panels* provide a closeup view of the change in residue dynamics within the SH3 domain. Three regions exhibiting high fluctuations in ABL1 Δexon 2 are highlighted: G92- N96 (*red*), T104-Q108 (*orange*), and I116-N120 (*magenta*), using the canonical ABL1 sequence residue numbers. f Three conformers (snapshots 1, 2, and 3) sampled by ABL1-Δexon2 at 0, 50, and 100 ns in *run 5* (see b). The SH3, SH2, and kinase domains are colored *orange*, *bordeaux*, and *dark blue*, respectively. Asciminib (not included in simulations) is displayed in snapshot 1 by *cyan* spheres so as to indicate its binding site, and the spatial neighborhood of the asciminib binding site is highlighted by semi-transparent *yellow circles* in all three snapshots. The linker connecting SH2 and kinase domains as well as that between SH2 and SH3 are colored *gray*.

See this image and copyright information in PMC

References

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1. Leyte-Vidal A, Garrido Ruiz D, DeFilippis R, Leske IB, Rea D, Phan S, et al. BCR::ABL1 Kinase N-lobe mutants confer moderate to high degrees of resistance to asciminib. Blood. 2024. - PubMed

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Absence of ABL1 exon 2-encoded SH3 residues in BCR::ABL1 destabilizes the autoinhibited kinase conformation and confers resistance to asciminib

Affiliations

Absence of ABL1 exon 2-encoded SH3 residues in BCR::ABL1 destabilizes the autoinhibited kinase conformation and confers resistance to asciminib

Authors

Affiliations

Conflict of interest statement

Figures

References

Publication types

MeSH terms

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Grants and funding

LinkOut - more resources

Full Text Sources

Miscellaneous