Overcoming midostaurin resistance via dual MYC and GSPT1 degradation by the protac GT19715 in FLT3-amplified AML cells Free

In acute myeloid leukemia (AML) carrying FLT3 mutations, resistance to the multikinase inhibitor midostaurin arises via clonal evolution from microclones to macroclones under drug selection pressure (Joudinaud, Blood Adv., 2024, PMID: 39418643), and incomplete suppression of FLT3 signaling appears to allow the emergence and expansion of resistant clones (Schmalbrock, Blood, 2021, PMID: 33598693).

To model this process, we established the AML cell line with midostaurin resistance (MR) by culturing FLT3-ITD–positive MV4;11 cells in a stepwise increase in midostaurin concentration over eight months. MR cells, which retained the original FLT3-ITD mutation, exhibited notably elevated FLT3 mRNA (2.8 fold) and protein expression (30 fold) and reduced sensitivity to midostaurin compared to MV4;11 cells (IC₅₀: 4.4 ± 0.5 nM vs. 39.4 ± 4.2 nM; ED₅₀: 35.5 ± 6.4 nM vs. 177.4 ± 1.5 nM for MV4;11 parental and MR cells, respectively).

Compared to MV4;11 cells, MR cells displayed enhanced activation of FLT3 downstream signaling pathways, including STAT5, AKT, S6, mTOR, and ERK, and elevated c-Myc protein levels. The reduced ability of midostaurin to inhibit FLT3 downstream signaling in MR cells suggests that FLT3 amplification plays a role in diminishing drug efficacy.

Given the elevated c-Myc levels observed in MR cells, we hypothesized that c-Myc, a transcription factor downstream of FLT3 with tumor-promoting functions, may also be involved in the transcriptional regulation of FLT3 itself. Based on this premise, we evaluated the antitumor efficacy of GT19715, a PROTAC (proteolysis-targeting chimera) that induces degradation of both c-Myc and GSPT1, one of the key translation termination factors (Nishida et al. biorxiv 650490). GSPT1 degradation promotes stop codon readthrough (SCR) and interferes with translation termination, thereby suppressing the production of tumor-associated proteins (Nishida, ASH 2024).

GT19715 demonstrated strong antitumor activity in both MV4;11 and MR cells, with IC₅₀ values of 0.3 ± 0.01 nM and 1.1 ± 0.1 nM, and ED₅₀ values of 0.4 ± 0.02 nM and 1.9 ± 0.3 nM for MV4;11 and MR cells, respectively. GSPT1 protein expression levels in MR cells were markedly higher than those in MV4;11 cells. Notably, GT19715 significantly reduced protein levels of not only c-Myc and GSPT1 but also FLT3 in both cell lines, thereby suppressing downstream FLT3 signaling. Additionally, it induced ATF4 and cleaved caspase-3 expression, indicative of integrated stress response activation and apoptotic response. In MR cells, however, AKT activity remained elevated despite FLT3 suppression by GT19715, suggesting the engagement of alternative pathways that may underlie FLT3-independent resistance.

Combination treatment with GT19715 and midostaurin exerted an additive antiproliferative effect in MV4;11 cells and a synergistic effect accompanied by apoptosis induction in MR cells (ZIP synergy scores: 8.3 and 45.4 for MV4;11 and MR cells, respectively), indicating that GT19715 may overcome midostaurin-mediated restoration of FLT3 signaling.

Time-course analysis (0–22 h) revealed that GSPT1 protein levels began to decline at 4–6 h, followed by FLT3 at 6 h and c-Myc at 8–10 h post-treatment. At the mRNA level, FLT3transiently decreased and then recovered, MYC transiently increased and then declined, and GSPT1 showed a slight increase. These results suggest that c-Myc does not act as a transcriptional mediator of GT19715-induced FLT3 suppression.

To further explore translational regulation of FLT3 via GSPT1 degradation, we developed an FLT3-stop-EGFP reporter to detect SCR at the FLT3 stop codon. GT19715 induced GFP expression in constructs containing the native FLT3 TAG stop codon, confirming SCR occurrence. Together with temporal and quantitative changes in FLT3 and GSPT1 mRNA/protein levels, these data indicate that GSPT1 degradation promotes SCR and disruptsFLT3 translation termination.

Taken together, chronic exposure to midostaurin enhanced FLT3 signaling as well as c-Myc and GSPT1 expression in FLT3-ITD AML cells. To counteract this, the c-Myc/GSPT1 degrader GT19715 induced synergistic cell death in midostaurin-resistant MR cells by suppressing FLT3 expression through GSPT1 degradation-mediated SCR. Meanwhile, AKT escape from FLT3 regulation in MR cells under drug selection pressure may contribute to the development of FLT3-independent resistance.