CT1113, a Potent, Oral Small Molecule Inhibitor of USP25, Demonstrates Anti-Tumor Activity in Ph-Positive Acute Lymphoblastic Leukaemia

Background: Patients with Ph-positive acute lymphoblastic leukaemia (Ph+ALL) have a prolonged clinical benefit from the application of tyrosine kinase inhibitors (TKIs). However, patients are at the risk of experiencing recurrence attributing to kinase domain mutation and treatment discontinuation, emphasizing the need for alternative therapeutics. It has established that ubiquitin-specific protease 25 (USP25) plays a role in the stability of BCR::ABL1. Here, we report that using CT1113, a potent small molecule targeting USP25, exerts anti-tumor activity in Ph+ALL.

Methods: Human cell lines (Sup-B15, BV-173), BCR-ABL1-expressing Ba/F3 cells harboring T315I and T315I-including compound mutant, and primary Ph+ALL cells treated with up to 600nM CT1113. Cell viability and apoptosis were assessed via MTS assay and Annexin V-FITC/PI double-staining kit. BCR-ABL1 and downstream signaling pathways changes were evaluated by change in target protein phosphorylation or total protein by immunoblot following drugging. Changes in BCR-ABL1 mRNA and ubiquitylation level were interrogated by RT-qPCR and a immunoprecipitation assay, respectively. In vivo survival studies were performed using the cell-derived allograft models of Ph+ALL, with subsequent randomization into vehicle, TKIs or CT1113 treatment arms.

Results: Following 72 hours of drugging, CT1113 displayed potent growth inhibitory effects on human Ph+ALL cell lines and all tested types of mutant BCR-ABL1-expressing Ba/F3 cells with IC50 values approximately 200 nM (Figure 1A). Furthermore, CT1113 demonstrated potent anti-leukemic activity in primary cultures ex vivo, independent of refractoriness to prior TKI therapies. And also, CT1113 treatment significantly increased the level of apoptosis. More importantly, CT1113 not only suppressed the phosphorylation of BCR-ABL1 and its downstream target STAT5, but also reduced the total protein of BCR-ABL1 and STAT5, a mechanism distinct from that of TKIs. However, CT1113 did not impact the levels of BCR-ABL1 mRNA. Additionally, the proteasome-specific inhibitor MG132 blocked CT1113-induced BCR-ABL1 downregulation in human Ph+ALL cell lines. As a proof of concept, inhibition of USP25 by CT1113 resulted in increased ubiquitination of the BCR-ABL1 protein, leading to its degradation. In in vivo models, intraperitoneal injection of CT1113 at tolerated doses demonstrated a significant growth inhibitory effect on both wild type and mutant BCR::ABL1-expressing Ba/F3 cells (Figure 1B). Flow cytometry analysis of bone marrow and spleen samples showed that CT1113 effectively eradicated leukemia cells from both compartments. Consequently, the survival of mice treated with CT1113 was prolonged compared to those in the vehicle and TKI arms.

Conclusion: Our data demonstrate the efficacy of the USP25 inhibitor CT1113 in preclinical models of Ph+ALL including that with BCR-ABL1 mutations conferring TKI resistance.These findings support the clinical investigation of CT1113 in Ph+ALL.