SUMMARY
BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph+) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1T315I-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP-site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph+ clinical isolates and cell lines. Inclusion of asciminib restores ponatinib’s effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.
While the use of ATP-competitive tyrosine kinase inhibitors of oncoprotein BCR-ABL1 has enabled durable responses in patients with chronic myeloid leukemia (CML), issues of drug resistance and residual leukemic stem cells remain. To test whether the degradation of BCR-ABL1 kinase could offer improved response, we developed a series of proteolysis targeting chimera (PROTACs) that allosterically target BCR-ABL1 protein and recruit the E3 ligase Von Hippel Lindau (VHL), resulting in ubiquitination and subsequent degradation of the oncogenic fusion protein. In both human CML K562 cells and murine Ba/F3 cells expressing BCR-ABL1, lead compound GMB-475 induced rapid proteasomal degradation and inhibition of downstream biomarkers, such as STAT5, and showed increased sensitivity compared to diastereomeric controls lacking degradation activity. Notably, GMB-475 inhibited the proliferation of certain clinically relevant BCR-ABL1 kinase domain point mutants and further sensitized Ba/F3 BCR-ABL1 cells to inhibition by imatinib, while demonstrating no toxicity toward Ba/F3 parental cells. Reverse phase protein array analysis suggested additional differences in levels of phosphorylated SHP2, GAB2, and SHC associated with BCR-ABL1 degradation. Importantly, GMB-475 reduced viability and increased apoptosis in primary CML CD34 + cells, with no effect on healthy CD34 + cells at identical concentrations. GMB-475 degraded BCR-ABL1 and reduced cell viability in primary CML stem cells. Together, these findings suggest that combined BCR-ABL1 kinase inhibition and protein degradation may represent a strategy to address BCR-ABL1-dependent drug
<p>Supplemental Figures and Tables. Figure S1 - Immunoblotting characterization of GMB-101 and GMB-475. Figure S2 - Validation of degradation and compound toxicity via comparison of GMB-475 and GMB-651. Figure S3 - Combinations of ATP competitive TKIs with PROTAC. Figure S4 - Schematic of scaffolding roles, VHL ligand and imatinib controls for VHL and pGAB2. Figure S5 - Patient sample sorting workflow and additional patient samples. Table S2 - Patient characteristics for primary samples</p>
<p>Supplemental Figures and Tables. Figure S1 - Immunoblotting characterization of GMB-101 and GMB-475. Figure S2 - Validation of degradation and compound toxicity via comparison of GMB-475 and GMB-651. Figure S3 - Combinations of ATP competitive TKIs with PROTAC. Figure S4 - Schematic of scaffolding roles, VHL ligand and imatinib controls for VHL and pGAB2. Figure S5 - Patient sample sorting workflow and additional patient samples. Table S2 - Patient characteristics for primary samples</p>
<div>Abstract<p>Although the use of ATP-competitive tyrosine kinase inhibitors of oncoprotein BCR-ABL1 has enabled durable responses in patients with chronic myeloid leukemia (CML), issues of drug resistance and residual leukemic stem cells remain. To test whether the degradation of BCR-ABL1 kinase could offer improved response, we developed a series of proteolysis-targeting chimera (PROTAC) that allosterically target BCR-ABL1 protein and recruit the E3 ligase Von Hippel-Lindau, resulting in ubiquitination and subsequent degradation of the oncogenic fusion protein. In both human CML K562 cells and murine Ba/F3 cells expressing BCR-ABL1, lead compound GMB-475 induced rapid proteasomal degradation and inhibition of downstream biomarkers, such as STAT5, and showed increased sensitivity compared with diastereomeric controls lacking degradation activity. Notably, GMB-475 inhibited the proliferation of certain clinically relevant BCR-ABL1 kinase domain point mutants and further sensitized Ba/F3 BCR-ABL1 cells to inhibition by imatinib, while demonstrating no toxicity toward Ba/F3 parental cells. Reverse phase protein array analysis suggested additional differences in levels of phosphorylated SHP2, GAB2, and SHC associated with BCR-ABL1 degradation. Importantly, GMB-475 reduced viability and increased apoptosis in primary CML CD34<sup>+</sup> cells, with no effect on healthy CD34<sup>+</sup> cells at identical concentrations. GMB-475 degraded BCR-ABL1 and reduced cell viability in primary CML stem cells. Together, these findings suggest that combined BCR-ABL1 kinase inhibition and protein degradation may represent a strategy to address BCR-ABL1–dependent drug resistance, and warrant further investigation into the eradication of persistent leukemic stem cells, which rely on neither the presence nor the activity of the BCR-ABL1 protein for survival.</p>Significance:<p>Small-molecule–induced degradation of BCR-ABL1 in CML provides an advantage over inhibition and provides insights into CML stem cell biology.</p></div>
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