A Mass Cytometry-Based Clinical Pharmacodynamic Assay for Histone 3 Lysine 36 Dimethyl (H3K36me2) Levels in Myeloma Cells and Peripheral Blood for Implementation in NSD2 Inhibitor Ktx-1001 First in Human Trial in Relapsed and Refractory Multiple Myeloma

Background:

Translocation of chromosomes 4 and 14 (t4;14) is a primary, clonal event in 15-20% of newly diagnosed multiple myeloma (MM) patients. T(4;14) translocation places the transcription of the histone (H3) methyltransferase, MMSET/NSD2, under the control of the IgH super enhancer resulting in its overexpression. MMSET is responsible for di-methylation of H3 lysine 36 (H3K36me2) resulting in an abnormal histone code that is associated with activated gene transcription and myelomagenesis. KTX-1001 is an oral, first-in-class, selective, MMSET inhibitor that is being evaluated in an ongoing Phase 1 trial in patients with relapsed/refractory MM (NCT05651932). Reliable pharmacodynamic (PD) biomarker assays are needed to interrogate clinical exposure with the enzymatic inhibition of MMSET, the target of KTX-1001. Here we describe the development of a custom mass-cytometry time of flight (CyTOF) panel for investigation of PD effects of KTX-1001 on the level of H3K36 dimethyl mark, and report data from ex vivo treated peripheral blood mononuclear cells (PBMCs) and a MM cell line. Analysis of samples from the ongoing KTX-1001 clinical trial is planned and will be presented

Methods:

Antibodies were either purchased pre-conjugated or were conjugated by OTMC and were titrated and validated on healthy donor PBMCs as previously described (van Oekelen O, et al. Cell Reports Medicine. 2024). The H3K36me2 rabbit monoclonal antibody was provided by K36 Therapeutics and was titrated in a series of 10-fold dilutions to determine optimal concentration for mass cytometry analysis. Healthy donor blood was obtained from Oxford Hospital and processed using standard methods to isolate and viably preserve PBMCs. Healthy donor PBMCs and MM cells were treated ex-vivo for 5 days with DMSO or indicated concentration of KTX-1001. The KMS11/BTZ (JCRB1642) t(4;14)+ MM cell line is a derivative of KMS11 cells that are resistant to the proteasome inhibitor bortezomib (Ri M, et al. Leukemia 2010;24:1506-12). The PD effects of KTX-1001 in healthy donor PBMCs were evaluated by ex vivo treatment of cells with either DMSO or KTX-1001 (0.5 mM-3mM). Following treatment, PBMC/MM cells were stained with antibody cocktail, fixed/permeabilized, and analyzed by CyTOF (Helios). Samples were run using biological and technical replicates and data were averaged across replicates. Analysis was performed using CytoBank software. KTX-1001 treated patient samples were provided from K36 Therapeutics and all patients provided consent for correlative studies. Samples were collected pre-dose at screening or Cycle 1 Day 1 or on-treatment at the start of subsequent 28-day cycles from the dose-escalation phase of the study.

Results:

A custom CyTOF panel included a mixture of markers for identification of immune and tumor cell subsets (CD45, CD3, CD19, CD4, CD11c, CD16, CD66b, CD8, CD25, CD38, CD56) along with markers of interest for primary (H3K36me2) and secondary PD effects (not shown) of KTX-1001 in MM patient samples. H3K36me2 signal was analyzed across PBMC cell subsets (DMSO or 0.5-3uM KTX-1001) and percent change was calculated versus DMSO control. The maximum decrease of H3K36me2 level was observed in CD19+ B cells (97.2%), followed by CD11c +monocytes (74.8%), CD56 + NK cells (51.0%), CD3 +T cells (33.9%), and CD38+ cells (32.9%). A dose-dependent decrease was observed within each cell subset versus DMSO control: B cells (0.5 uM -5.3% [increase] and 3 uM 97.2% decrease), monocytes (0.5 uM 31.8% decrease, 3 uM 74.8% decrease), NK cells (0.5 uM 15.2% decrease, 3 uM 50.9% decrease), T cells (0.5 uM 6.4% decrease, 3 uM 33.9% decrease), and CD38+ cells (0.5 uM 4.8% decrease, 3.0 uM 32.9% decrease). In KMS11/BTZ MM cells, a decrease in H3K36me2 signal was also observed with KTX-1001 treatment versus DMSO treated cells (1 uM 37.6% decrease; 3.0 uM 38.6% decrease).

Conclusions:

We found that the anti-H3K36me2 antibody is highly sensitive and specific in detecting Lysine 36 dimethyl Histone 3 marks in healthy PBMC and a MM cell line in vitro. In addition, the ability to detect the signal in multiple cell types in PBMC (B, T or NK cells and monocytes) and in MM cell lines in a range of drug concentrations suggest the potential utility of the assay to test planned clinical samples (eg, PBMC or bone marrow aspirates) to interrogate the correlation between dose, pharmacokinetics and pharmacodynamic effects of KTX-1001 in the dose escalation phase of the clinical trial.

Flynt:K36 Therapeutics, Bristol Myers Squibb, JnJ, Abbvie, Pfizer, Amgen: Current Employment, Current equity holder in private company, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Oppermann:Bristol Myers Squibb: Research Funding. Winograd:K36 Therapeutics: Current Employment, Current equity holder in private company, Current holder of stock options in a privately-held company.