Ktx-1029, a Potent, Selective MMSET/NSD2 Inhibitor Is Effective in t(4;14) Multiple Myeloma Preclinical Models
Authors: Flynt Erin , Lewis Chad A. , Winograd Benjamin , Connolly Terry , Xu Lan , Menon Anil 
Published: December 2024

Background: A translocation event between chromosome 4 (MMSET/NSD2 gene) and chromosome 14 (immunoglobulin heavy chain [IgH] locus) (t(4;14)) is a primary, clonal event in 15-20% of newly diagnosed multiple myeloma (MM) patients that is associated with poor clinical prognosis (Morgan, Nat Rev Cancer 2022; Klaff, Blood Cancer J, 2012; Chesi, Blood 1998). Presence of t(4;14) places the transcription of the histone (H3) methyltransferase, MMSET, under the control of the IgH super enhancer resulting in its overexpression. MMSET is responsible for di-methylation of H3 lysine 36 (H3K36me2) and when overexpressed, MMSET results in an abnormal histone code that is associated with activated gene transcription and myelomagenesis. While MMSET has been recognized as an attractive therapeutic target, the development of targeted pharmacological inhibitors has been challenging. KTX-1029 is a potent, selective, small molecule inhibitor of MMSET. KTX-1001 is an orally-available small molecule inhibitor being evaluated in an ongoing Phase 1 trial in relapsed/refractory MM patients (NCT05651932). Here we report the biological characterization and efficacy of KTX-1029 in preclinical MM models.
Results:KTX-1029 inhibited MMSET activity with the half maximal inhibitory concentration (IC50) of 16.0 nM. Testing of KTX-1029 against 25 other histone methyl transferases demonstrated high selectivity for MMSET. Treatment of four t(4;14) MM cell lines (KMS26, KMS34, LP1, OPM2) as well as the MM.1S cell line that contains the E1099K gain-of-function mutation in MMSET demonstrated a consistent decrease in H3K36me2. Analysis of 26 other detectable histone marks in KMS26 and MM.1S cells after treatment with KTX-1029 demonstrated that other than K36 methylation, K27 methylation was also dose-dependently impacted by KTX-1029 while there was no clear dose-dependent effect on other histone marks. Dose-dependent decreases in colony formation were also observed in the KMS11 and KMS26 t(4;14) positive cell lines. The addition of KTX-1029 to bortezomib restored the sensitivity of the KMS11/BTZ bortezomib-resistant cell line to bortezomib (IC50: bortezomib alone=28.4 nM, KTX-1029 alone=1327 nM, 1000 nM KTX-1029+bortezomib= 2.3 nM). The combination of 1 mM KTX-1029 and bortezomib (0.03-30 nM) resulted in a maximum inhibition of cell viability of 73.9% which approached the activity of bortezomib single-agent in the parental bortezomib-sensitive KMS11 cell line (IC50 2.5 nM, maximum inhibition 99.8%). The KMS11/BTZ line is also cross-resistant to carfilzomib and addition of KTX-1029 to carfilzomib resulted in synergistic inhibition of cell viability (IC50: carfilzomib alone=15.27 nM, KTX-1029 alone=5104 nM, 1000 nM KTX-1029+bortezomib= 7.87 nM). In addition to the synergistic effect in the bortezomib-resistant cell line, the combination of KTX-1029 with carfilzomib synergistically inhibited cell growth in the parental KMS11 cell line. The anti-tumor activity of KTX-1029 was tested in vivo in the KMS26 and MM.1S systemic MM mouse models. KTX-1029 treatment significantly extended survival versus vehicle control in the KMS26 model (median 31 days vs 23.5 days, P=0.0007). Similarly, in the MM.1S model, KTX-1029 treatment significantly extended survival (median 60 vs 31 days). In both mouse models, KTX-1001 decreased tumor burden in the bone marrow and reduced human kappa and lambda immunoglobulin levels in the serum.
Conclusion:KTX-1029 is a novel, potent, selective inhibitor of MMSET that demonstrated in vitro and in vivo efficacy in MM preclinical models as a single agent and in combination with the proteasome inhibitors, bortezomib and carfilzomib in both PI-sensitive and -resistant settings. KTX-1029 compliments the orally-available clinical candidate, KTX-1001 that is being evaluated in an ongoing clinical trial for relapsed/refractory MM patients. Preclinical data with KTX-1029 adds to the body of evidence for targeting MMSET in MM patients with t(4;14) and for further exploration of combination regimens with MM standards of care.