Therapeutic effect of menin inhibitors is reversible in AML treatment and could be enhanced by the targeting of differentiation associated chromatin complex Free

Menin, encoded by the MEN1 gene, is a chromatin adaptor critical in AML through its role in the MLL complex. Inhibiting the Menin–MLL interaction promotes differentiation and reduces leukemia burden in MLL-rearranged and NPM1c AMLs, leading to the approval of Menin inhibitors by FDA. However, acquired resistance remains a challenge, highlighting the need to understand long-term effects.

To understand the Menin inhibitors' effect in the long-term treatment, we firstly treated the MLL-r and NPM1c AML cell lines for long term and then withdraw the inhibitors to observe the potential recovery of cells. We found the cell growth inhibition and viability decrease were not sustained and proliferation resumed after the withdrawal of 18-30 days treatment of Menin inhibitor both in MLL-r and NPM1c cell lines and NPM1c primary AML cells. In parallel, differentiation cannot be maintained after Menin inhibitor withdrawal. Differentiation marker CD11b expression also gradually lost after 10-day of withdrawal. To determine whether these regrown cells develop resistance, we compared the regrown cells to untreated cells and found that there was no significant difference in IC50 of Menin inhibitors between the regrown and untreated cells, showing the cells regrown due to the reversibility of Menin inhibitors but not resistance. Furthermore, due to the heterogeneous differentiation induced by Menin inhibitors, we compare the effect of differentiation on the cell growth. After 24 days of treatment, CD11b^hi and CD11b^lo cells were sorted after inhibitor removal, we could observe a significant slower cell growth in CD11b^hi than CD11b^lo cells. We also found a CD11b^hi group gradually lost the differentiation marker and revert the grow rate as CD11b^lo. This indicates that these cells underwent dedifferentiation, suggesting that Menin inhibitors alone induce only reversible differentiation. Interestingly, we found the regrown cells silenced HOX/MEIS1 genes permanently showing the HOXA/MEIS1 genes are not playing a role in the long-term treatment of Menin inhibitors.

To enhance and stabilize differentiation, we conducted a CRISPR screen targeting chromatin regulators in MOLM13 and OCI-AML3 cells treated with Menin inhibitor or DMSO for 2 weeks. CD11b^hi and CD11b^lo cells were sorted by FACS. Hits included members of the SAGA, LSD1/CoREST, MBD2 NuRD complex, ING5, non-canonical BAF, and MLL/Menin complexes. Knockdown of RCOR1, LSD1, MBD2, MEAF6, KAT6A, and KAT2A enhanced Menin inhibitor–induced differentiation and stabilized it after withdrawal for up to 20 days.

We futher screened pharmacological inhibitors of these targets and found that LSD1, KAT6A, and MBD2 inhibitors enhanced differentiation. In particular, combining the experimental MBD2 inhibitor KCC07 with the Menin inhibitor Revumenib accelerated differentiation as early as day 3 (vs. day 7 with Revumenib alone). Long-term co-treatment stabilized differentiation, induced cell death, and suppressed proliferation, indicating chromatin modulation can lock in terminal differentiation, lead to cell death, and potentiate Menin inhibitor effects.

RNA-seq of OCI-AML3 cells treated with DMSO, Reumenib, KCC07, or their combination showed that the MBD2/Menin combination inhibition significantly upregulated differentiated monocytes and neutrophils signatures while reducing immature neutrophil signature. GO analysis revealed strong activation of monocyte differentiation and repression of PI3K-AKT signaling. HOXA and MEIS1 expression were only modestly changed, showing the combination mostly enhanced diffrentiation gene programs but not self-renewal maintaining HOX/MEIS1 axis.

ATAC-seq revealed globally increased chromatin accessibility in the MBD2/Menin combination inhibition group in comparison with Revumenib only treatment group, with enhanced peaks located predominantly in myeloid differentiation genes, suggesting the MBD2 inhibition also leads to derepression of differentiation related genes repressed by MBD2-NuRD complexes.

Our study is the first to demonstrate the reversibility of Menin inhibitor–induced growth inhibition and differentiation. More importantly, we identified differentiation associated chromatin complex targeting (such as MBD2 inhibition) as a low-toxicity strategy that enhances and stabilizes Menin inhibitor–induced differentiation, offering a promising approach to overcome durability limitations in AML therapy.