KT-253, a Highly Potent and Selective MDM2 Protein Degrader, Eliminates Malignant Myelofibrosis Stem/Progenitor Cells

Although first generation JAK1/2 inhibitor (JAK2i) therapy has clearly improved the quality of life of myelofibrosis (MF) patients and to a lesser extent prolonged their survival, this approach has not substantially altered the overall disease trajectory. Approximately 96% of chronic myeloproliferative neoplasms (MPNs) express wild-type (WT) p53 and, irrespective of the patient's driver mutational status, MF hematopoietic stem cells (HSCs) are characterized by upregulation of MDM2, an E3 ubiquitin ligase that catalyzes proteasomal degradation and inactivation of WT p53 (Mayo M et al, ASH 2022, Lu M et al. Leukemia, 2022). These observations highlight the importance of modulating MDM2 activity to upregulate WT p53 activity to deplete chronic MF HSCs. Subsequent trials of MDM2 small molecule inhibitors (SMIs) in chronic MPN patients have resulted in reductions of spleen size, total symptom scores and r the variant allele frequencies (VAF) of not only MPN driver mutations but also other associated myeloid gene mutations (Vachani P et al, ASH 2021). The clinical use of MDM2 SMIs, however, has been limited to date to achieving partial clinical responses and associated gastrointestinal (GI) and hematological toxicities. KT-253, a highly selective MDM2 degrader, is currently being evaluated in clinical trials in high grade myeloid malignancies, solid tumors and lymphomas (NCT05775406). In contrast to MDM2 SMIs that block the p53/MDM2 interaction, KT-253 catalytically degrades MDM2 overcoming the p53-dependent feedback loop that upregulates MDM2. We hypothesized that, MDM2 degradation with KT-253 would be a more effective approach than MDM2 SMIs in eliminating malignant MF HSCs. Using UKE-1 cells, a p53 WT JAK2V617F+ cell line, KT-253 treatment was shown to lead to >10-fold increase in the degree of cell growth inhibition and apoptosis as compared to the MDM2 SMI AMG-232 (100nM vs 1000nM). KT-253 treatment also led to significant increase in fraction of cells in sub-G1 phase as well as reduction in cells in S and G2/M phases. In contrast, KT-253 had no effect on p53 mutated JAK2V617F+ SET-2 cells indicating that KT-253 induced cell growth inhibition and apoptosis was p53 dependent. Furthermore, treatment of primary MF CD34+ cells with various doses of the KT-253 led to significant reduction of CD34+ cell viability and induction of apoptosis, while 10-40 fold higher concentrations of AMG-232 were required to suppress MF CD34+ cell growth or induce apoptosis to a similar degree. These effects of KT-253 were associated with upregulation of the p53 transcriptional targets p21, NOXA and PUMA. Treatment of primary MF CD34+ cells at nanomolar concentrations (12.5nM) of KT-253 led to a 60% reduction in hematopoietic colony formation, while this same dose did not affect the colony forming potential of normal CD34+ cells. In contrast, 125nM of AMG-232 treatment led to only 20% reduction in MF hematopoietic colony formation. Since KT-253 is administered intermittently, we also evaluated the effect of a 4-hour exposure of MF and normal donor CD34+ cells to KT-253 followed by a washout period and then assaying hematopoietic colony formation potential. Short term exposure to KT-253 (12.5, 25, 50 and 100nM) significantly decreased MF colony numbers by 30-60 % (p=0.04, p=0.05, p=0.016 and p=0.002, respectively) but had no effect on normal donor colony numbers. By contrast, short term treatment with AMG-232 (125, 250 500 and 1000nM) did not affect either MF or normal colony numbers. Importantly, treatment of MF CD34+ cells with 12.5 nM of KT-253 decreased by 71% the numbers of JAK2V617F+ colonies assayed, while sparing JAK2 WT colonies. By contrast, 125nM of AMG-232 reduced the number of JAK2V617F+ colonies by only 28%, at >10-fold higher concentrations. Selective depletion of malignant MF CD34+ cells by KT-253 strongly suggests that this degrader has a substantial therapeutic window which could eliminate MF CD34+ cells but spare non-mutated CD34+ cells and limit hematological toxicities associated with MDM2 SMI therapy. In conclusion, KT-253 is a highly potent and effective MDM2 degrader which upregulates p53 activity in MF CD34+ cells and can selectively reduce the numbers of JAK2V617F+ colonies formed while sparing the reservoir of colonies with WT JAK2. These data provide a compelling rationale for evaluating the therapeutic potential KT 253 in MF patients.