Novel Inhibitors of MLLT1/3, a Critical Component of the Super-Elongation Complex (SEC), Target a Range of Molecularly Defined Acute Leukemias, with a Differential Profile to Menin Inhibition

Introduction: The SEC is a developmental transcriptional regulator which is often hijacked by cancer. The SEC regulates RNA Polymerase II dependent transcription of major oncogenes e.g., Myc, Bcl2, CDK6, along with developmental genes e.g. HOXA9 and MEIS1. SEC-dependent transcription is recognised as a driver in multiple acute myeloid leukemia (AML) sub-populations, such as those defined by Mixed Lineage Leukemia 1 (MLL1)-rearrangements (MLLr), which are prevalent in >10% of acute leukemias, and NPM1 mutations, which account for ~30% of leukemias. MLLT1 is known to be a key nucleating component of the SEC, and CRISPR/Cas-9 screens have previously highlighted a critical dependency for MLLT1 in MLLr driven leukemias. Clinical proof-of-concept for targeting the SEC has recently been demonstrated with inhibitors of menin, a protein that also impacts SEC activity in certain contexts. However, whilst the clinical data with menin inhibitors have been encouraging, there are a number of AML cell lines and patients that do not respond, and a growing number of examples of acquired clinical resistance. Hence there is a need for additional approaches which may benefit a broader spectrum of patients. Targeting MLLT1/3 provides one such promising approach. We have discovered novel MLLT1/3 inhibitors which have broad therapeutic potential across a range of molecularly defined acute leukemias, with a differential profile to menin inhibitors.

Methods: In this study, a FRET assay was used to measure the ability of MLLT inhibitors to prevent the interaction of the MLLT1 and MLLT3 YEATS domains with acetylated histones. Selectivity was assessed against other YEATS domain proteins. MLLT target gene expression was measured by qPCR in leukemia cell lines. The anti-proliferative potential of MLLT1/3 inhibitors was assessed across a broad range of leukemia and normal cell lines and compared with menin inhibitors and other standard of care compounds using a cell viability readout (total ATP quantification). Combination studies were also performed in leukemia cell lines with MLLT1/3 and menin inhibitors. The clonogenic potential of MLLT1/3 inhibitors was determined using colony formation assays. Apoptosis was measured using caspase 3/7 readout. In vivo activity was assessed in a MV4;11 subcutaneous model.

Results: We have identified novel MLLT1/3 inhibitors which block the interaction of the MLLT1 and 3 YEATS domains with acylated histones with IC₅₀ values <100 nM. They were shown to have excellent selectivity for MLLT1/3, with no inhibition observed against two other YEATS domain proteins, YEATS2 and 4. The MLLT1/3 inhibitors were shown to inhibit the expression of MLLT target genes known to be regulated via the SEC e.g., MYC and HOXA9. These compounds profoundly inhibited the growth of MLLr fusion and NPM1 mutant leukemias, with minimal activity seen against normal cell lines. Notably, potent inhibition of cell growth in non-MLLr fusion leukemia cell lines was observed, which were not sensitive to menin inhibition. MLLT1/3 inhibitors were shown to induce apoptosis and block the clonogenic potential of leukemia cell lines. Synergy between MLLT1/3 and menin inhibitors was also observed in leukemic cell lines. MLLT1/3 inhibitors have been tested in an MV4;11 model with substantial tumour growth inhibition observed.

Conclusion: Targeting MLLT1/3 is an attractive approach and has the potential for single agent activity across a broad range of molecular defined acute leukemias, with a differential profile to menin inhibitors.