Abstract
Introduction: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of Non-Hodgkin Lymphoma. Double Hit Lymphomas (DHL) and Double Expresser Lymphomas (DEL) are high grade B-cell lymphomas (HGBLs) that have high MYC and BCL2 dependency. DHL harbor translocations of MYC and BCL2 (or BCL6 less frequently), which lead to increased cell growth and survival. Standard R-CHOP therapy is inadequate and results in poor patient outcomes. Novel targeted agents, which inhibit MYC and BCL2 are currently under investigation; however, there is no established standard-of-care for these highly aggressive lymphomas.
Menin is a scaffold protein that is essential for cancers driven by oncogenic MLL-fusions, such as in acute leukemias. At present, the role of menin in DLBCL is not well described. A recent study demonstrated that MYC and menin co-localize on chromatin, leading to menin-mediated enhancement of MYC target gene expression in cancer cells. However, the relationship between menin and MYC and the direct effect of menin inhibition on MYC in liquid tumors has not been examined. Here, we demonstrate the ability of BMF-219, a novel, selective, orally bioavailable, irreversible inhibitor of menin, to modulate MYC expression in leukemia cells as the basis for exploring activity of the molecule in DLBCL cells.
Methods: MOLM-13 cells incubated with BMF-219 for 6 and 24 hours at 0.5 μM and 1 μM were analyzed by RNA-seq on the Illumina NextSeq 550 platform. Differentially expressed genes were identified through DeSeq2 negative binomial model analysis. Transcription factor activity inference of differentially expressed genes was calculated using a published statistical framework and algorithm. DLBCL DHL cell lines, DB and Toledo, were treated with BMF-219 as monotherapy, and in combination with ABT-199, at a concentration range of 0.01 μM to 3 μM for a 4-day duration. Cell viability was measured using CellTiter Glo and IC 50 values were calculated.
Results: RNA-seq analysis of MOLM-13 cells treated with BMF-219 showed a marked reduction of MYC transcript levels by ~25-fold at 6 hours of treatment at both concentrations tested, and ~110-fold and ~220-fold at 0.5 μM and 1 μM BMF-219 treatment, respectively, at 24 hours. Additionally, MYC and its co-factor, MAX, emerged as top candidates in transcription factor activity inference analysis based on ChIP-seq GEO repository datasets and differential expression of MOLM-13 cells treated with BMF-219 at 24 hours. These results prompted an examination of the effects of BMF-219 on lymphoid malignancies known to be highly dependent on MYC aberrations.
Single-agent BMF-219 reduced >90% of cell viability in both DB and Toledo cells, at 1.0 μM and 0.36 μM, respectively. The IC 50 values of BMF-219 were calculated as 0.32 μM and 0.29 μM for DB and Toledo, respectively. Two reversible menin inhibitors tested were less effective in reducing growth of DHL cell lines. One reversible menin inhibitor exhibited IC 50 values of 3 μM and 1.5 μM in DB and Toledo, respectively, while neither cell line was sensitive to the other reversible compound.
Given that a 20-fold reduction in BCL2 transcript occurred in MOLM-13 cells at 24 hours of 0.5 μM BMF-219 exposure, we investigated whether a combination of BCL2 inhibitor, ABT-199, with BMF-219 could further reduce cell viability of DHL cells. ABT-199 as a single agent reduced viability of Toledo cells, but not DB cells, at 0.33 μM. By contrast, the combination of BMF-219 and ABT-199 synergistically killed both DB and Toledo cells. Combination treatment using both inhibitors at 0.33 μM killed >80% of DB cells and 100% of Toledo cells. These results demonstrate that DLBCL DHL cells are sensitive to menin inhibition. Of note, initial data has indicated that multiple myeloma cells also manifest responses to BMF-219 similar to DHL DLBCL.
Conclusions: Collectively, our study demonstrates the unique ability of BMF-219's irreversible menin inhibition, which leads to marked killing of DHL cells. BMF-219, as a single agent, substantially reduces MYC and BCL2 gene expression in liquid tumor cells, providing a molecular basis for targeting DLBCL and other tumors with these aberrations. Additionally, BMF-219 offers single agent as well as treatment synergy when combined with ABT-199 in DHL cells. These data serve as initial pre-clinical evidence for employing irreversible menin inhibition as a promising therapeutic strategy in DHL DLBCL.
Somanath: Biomea Fusion, Inc.: Consultancy, Current Employment, Current equity holder in publicly-traded company. Lu: Biomea Fusion Inc.: Current Employment, Current equity holder in publicly-traded company. Law: Biomea Fusion Inc.: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Archer: Biomea Fusion: Current Employment, Current holder of stock options in a privately-held company. Cacovean: Abbvie: Current equity holder in publicly-traded company; Lyell Imunopharma: Current equity holder in publicly-traded company; CytomX: Current equity holder in publicly-traded company; Iovance: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Biomea Fusion: Current Employment, Current equity holder in publicly-traded company. Palmer: Nyrada, Inc.: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Exopharm: Consultancy; Biomea Fusion Inc.: Consultancy, Current equity holder in publicly-traded company. Kinoshita: Biomea Fusion INC.,: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Butler: Biomea Fusion Inc.: Current Employment, Current equity holder in publicly-traded company.
