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4157 Targeting Chronic Myeloid Leukemia with Potent and Specific BCR::ABL1 Degraders

Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster III
Hematology Disease Topics & Pathways:
Research, Translational Research
Monday, December 9, 2024, 6:00 PM-8:00 PM

Nataly Cruz-Rodriguez, PhD1, Hua Tang, PhD2*, Milad Rouhimoghadam, PhD1*, Junzhuo Liao, PhD2*, Benjamin Bateman1*, Briana Bates, MS1*, Mario Pinzas1*, Xuhui Huang, PhD2*, Weiping Tang, PhD2* and Michael W. Deininger, MD, PhD1,3

1Versiti Blood Research Institute, Milwaukee, WI
2University of Wisconsin-Madison, Madison, WI
3Medical College of Wisconsin, Milwaukee, WI

Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm driven by BCR-ABL1, a chimeric tyrosine kinase derived from the t(9;22)(q11;q34.1) reciprocal translocation, cytogenetically visible as the Philadelphia chromosome (Ph). Successive generations of BCR::ABL1 tyrosine kinase inhibitors (TKIs) have improved the outcome of CML, resulting in an estimated increase of US CML prevalence from 70,000 in 2010 to 181,000 in 2050. However, only one-quarter of patients are able to discontinue TKI therapy and achieve a state of functional cure termed treatment free remission (TFR). Additionally, 10-15% of patients progress to blast phase (BP)-CML, an aggressive acute leukemia with poor prognosis. There is evidence that kinase-independent functions of BCR-ABL1 contribute to leukemogenesis, reflecting scaffold functions of BCR-ABL1 that maintain activation of survival signaling in leukemic stem and progenitor cells (LSPCs) in the presence of TKIs. Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that recruit an E3 ligase to a target protein to facilitate its ubiquitination and subsequent proteasomal degradation. Most previously reported BCR::ABL1 PROTACs exhibit significant limitations, including low potency, off-target degradation, and the absence of in vivo efficacy data, and no BCR::ABL1-targeting PROTAC has entered clinical trials to-date.

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.

*signifies non-member of ASH