Global proteomic profiling of tgrx-3247，a heterobifunctional degrader for the treatment of BCR::ABL1-driven chronic myeloid leukemia Free

Tyrosine kinase inhibitors (TKIs) and agents Specifically Targeting ABL1 Myristoyl Pocket (STAMP) are currently the standard of care for BCR::ABL1-driven chronic myeloid leukemia (CML). However, durable and deep molecular responses are not consistently achievable with drugs through “catalytic inhibition only” mode of action. Targeted protein degradation (TPD) has emerged as an increasingly promising therapeutic modality. A heterobifunctional degrader typically employs a warhead targeting the protein of interest (POI), an E3 ligase ligand and a linker to hitchhike the ubiquitin-proteosome system for the elimination of POI. As previously reported, TGRX-3247 is a pan-BCR::ABL1 degrader which exhibits extraordinary and broad spectrum TPD activity and cellular potency in a panel of CML cell lines and in KCL22s cells harboring knock-in BCR::ABL1 mutations, regardless of orthosteric, allosteric or even highly resistant compound mutations. Here, we describe a global proteomics approach to systematically interrogate the selectivity and cellular effects of TGRX-3247.

We treated KU812 and KCL22s-BCR::ABL1-Y253H/T315I (YT) cells with TGRX-3247 at 10nM for 6h, and a normal cell line HEK293 for 24h, and performed data-independent acquisition (DIA) mass-spectrometry (MS). Analysis of differentially expressed proteins (DEPs) identified ABL1 and ABL2 as the top down-regulated proteins and the only 2 DEPs shared by all 3 cell lines. We extended the treatment to 24h in KCL22s, KCL22s-BCR::ABL1-T315I (T315I) and YT cells, and ABL1 remained one of the top down-regulated proteins. Expectedly, prolonged treatment resulted in greater changes in DEPs landscape. ABL1 was the only down-regulated protein found both in CML (KCL22s, T315I and YT) and HEK293 cells. The majority of the top 30 down-regulated proteins found exclusively in CML cells are highly associated with cell cycle, mitosis, DNA replication or DNA damage repair, while p27^Kip1 (CDKN1B) appeared as the top up-regulated protein exclusively in CML cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses identified cell cycle, DNA replication and nucleotide metabolism among the most enriched terms. In TGRX-3247 treated CML cells, gene set enrichment analysis (GSEA) also confirmed that biological processes, signaling pathways or cellular components such as DNA replication, cell cycle, pyrimidine metabolism and centrosome protein were negatively enriched, while proteolysis and lysosome were positively enriched. None of these terms were significantly enriched in HEK293 cells.

To validate the findings from the proteomics data, we performed siRNA-mediated knockdown of BCR::ABL1 in KCL22s cells. Cell cycle related proteins such as TACC, CDCA5, SKP2 and PLK1, selected from the top down-regulated proteins, were robustly decreased in the presence of 3 individual siRNA targeting ABL1. We then treated LAMA-84 and KCL22s cells with TGRX-3247 along with the warhead, a non-degrader methyl analog, asciminib and ponatinib. Consistent down-regulation of TACC, CDCA5, SKP2 and PLK1, and up-regulation of p27^Kip1 was observed in all conditions, suggesting that the effect of TGRX-3247 on cell cycle related DEPs is indeed dependent on BCR::ABL1 kinase activity. Furthermore, cell cycle profiling using propidium iodide (PI) staining followed by flow cytometry demonstrated a substantial increase in the sub-G1 population at the expense of S and G2/M phases, indicating DNA fragmentation during apoptosis, which was further confirmed with positive Annexin V/PI staining.

Additionally, CRBN neosubstrates including IKZF1, CK1α, GSPT1, ZFP91 and ZMYM2 were not evidently down-regulated in any of the samples in the proteomics data or in the Western blot analysis.

Overall, derived from a powerful proteomics approach, these data support that TGRX-3247 is a highly selective BCR::ABL1 degrader. The predominant cellular effect of TGRX-3247 is cell cycle perturbation and apoptosis induction in CML cells but not in normal cells, consistent with the central oncogenic role of BCR::ABL1. Of note, our proteomics study reconsolidated previous discoveries on BCR::ABL1 functions, such as in p27^Kip1regulationthrough the SKP2 pathway, as well as in lysosome and autophagy. More information of TGRX-3247 is worth mining to bring new insights to the biology of CML, which may ultimately lead to novel treatments for CML patients.