Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research
Using a high-throughput screen, we discovered LPA-81A as a highly potent BCR::ABL1 degrader based on asciminib as warhead and cereblon as E3 ubiquitin ligase ligand. For validation, we developed a NanoBiT protein complementation assay consisting of BaF3 cells expressing HiBiT-BCR::ABL1 and the LgBiT subunit, allowing for accurate quantitation of degradation potency in intact cells. LPA81 at 0.5 µM reduced BCR::ABL1 protein in K562 cells by >90% within 12 hours. We next designed LPA-81S, a derivative with increased stability and potency. LPA-81S inhibits growth and viability of cells expressing native or kinase domain mutant BCR::ABL1 at low nanomolar concentrations and strongly reduces BCR::ABL1 protein in primary CML CD34+ cells. NanoBRET assays confirmed ternary complex formation between BCR::ABL1, CRBN, and LPA-81S, and computational modeling identified lysine 202 in the BCR::ABL1 SH2 domain as the lysine critical for degradation, which was confirmed by site-directed mutagenesis.
To distinguish between effects of BCR::ABL1 degradation and kinase inhibition, we generated a degradation-deficient but kinase inhibitory methylated derivative (LPA-81S-Me). BCR::ABL1 degradation activity dramatically enhances growth inhibition compared to kinase inhibition alone, reducing IC50 for CML cell lines up to 200-fold and up to 10-fold for LSPCs. Phosphorylation of BCR::ABLY177 and ERK1/2 signaling is abrogated only by LPA-81S but not LPA-81S-Me, while mutation of tyrosine 177 to phenylalanine reduces the difference between LPA-81A and LPA-81S-Me, implicating mitogen activated kinase signaling as a scaffold function of BCR::ABL1 that may maintain LSPC viability in the presence of TKIs.
Unbiased proteomics studies reveal that LPA-81 is exceptionally selective with no discernible off-target protein degradation. Consistent with this, in preliminary studies we see no toxicity in mice. Lastly we demonstrate that LAP-81S inhibits CML LSPCs in vitro and BCR::ABL1+ leukemia in a retroviral CML model in vivo. Ongoing structure activity relation (SAR)-based optimization has already yielded derivatives with 1-log increased potency over parent LPA-81S.
In summary, we have identified LPA81S is an exceptionally potent PROTAC capable of killing CML cells by degrading both native and mutant BCR::ABL1 via the ubiquitin-proteasomal system. Additionally, our data strongly suggest that BCR::ABL1 degradation kills CML cells more effectively than BCR::ABL1 kinase inhibition and implicate BCR::ABL1Y177 as a crucial scaffold function of BCR::ABL1. BCR::ABL1 PROTACs may be more effective than TKIs in targeting CML LSPCs.
Disclosures: Rouhimoghadam: Abbvie: Current Employment. Deininger: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Grants, travel, clinical trial support, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Grants, Travel, , Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Other: Part of a study management committee, Research Funding; Incyte: Honoraria, Research Funding; Medscape: Honoraria, Other: Case Author ; Sangamo: Consultancy, Honoraria; Takeda: Honoraria, Other: Part of a study management committee, Research Funding; DisperSol: Consultancy; Fusion Pharma: Consultancy; Leukemia & Lymphoma Society: Research Funding; SPARC: Research Funding.
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