The researchers of a recent study published in Nature present a novel drug combination to treat acute myelogenous leukemia (AML). Drugs that inhibit lysine-specific demethylase 1 (LSD1) and glycogen Synthase Kinase 3 (GSK3) rewire transcriptional genes to promote myeloid cell differentiation while inhibiting cell (tumor) proliferation.
This combination therapy selectively targets leukemia cells while sparing normal bone marrow-derived macrophages to limit disease progression and extend the survival of AML patients.
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Introduction
Acute myeloid leukemia is an aggressive cancer that limits patient survival to eight to nine months. The disease impairs differentiation of myeloid progenitor cells in the bone marrow, accumulation of immature precursors, and impaired blood cell replenishment.
Treatment involves drugs to circumvent this developmental blockade. One way to address this is by targeting dysfunctional gene expression programs in leukemic stem cells. Drugs that inhibit the epigenetic enzyme LSD1 have shown limited success in clinical studies.
To reduce toxicity and enhance efficacy, other drugs need to synergize with LSD1 inhibitors to overcome differentiation arrest and suppress cancer cell proliferation.
About the Study
In the present study, researchers combined inhibitors of LSD1 and GSK3 to induce differentiation of myeloid cells and suppress their self-renewal in AML.
The researchers conducted a small-molecule screen to find synergistic agents with LSD1 inhibitors. The screening yielded an inhibitor of the GSK3α/β enzyme, LY2090314, for further analysis. Dose-response proliferation assays performed using ER-HOXA9 murine bone marrow cells and normal bone marrow-derived macrophages investigated the selectivity of the combination treatment for leukemia cells.
Colony-forming assays quantified colonies formed by primary AML samples treated with the inhibitor to analyze the effects on tumor cell self-renewal. Immunoprecipitation experiments assessed protein interactions to elucidate the mechanisms of drug action and differentiation.
The assay for transposase-accessible chromatin with sequencing (ATAC-Seq) assessed chromatin accessibility in treated cells.
Gene set enrichment analysis (GSEA) revealed transcriptional signatures, and principal component analysis (PCA) investigated chromatin states. Cleavage under targets and release using nuclease-quantitative polymerase chain reaction (CUTRUN-qPCR) analyzed protein occupancy on promoter genes in treated AML cells.
The researchers evaluated the effects of the combination treatment in AML patient samples cultured ex vivo and DNMT3A-mutated patient-derived xenografts (PDXs). They also scored individuals from the OHSU dataset based on the drug combination’s synergy signatures.
They cultured the human colorectal carcinoma cell line 116 (HCT116) carrying T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) reporters to investigate the combination’s effects on the Wingless-related integration site signaling pathway (WNT) that regulates cell development, differentiation, and proliferation.
Results
The concurrent inhibition of LSD1 and GSK3 kinase promotes differentiation of AML cells, reducing tumor burden and extending patient survival. The drug combination activates genes that drive cell differentiation while suppressing genes that promote cell proliferation and cancer growth.
The treatment promotes cell differentiation and maturation by enhancing the expression of type I interferon (IFN) genes that primarily activate two transcription factors, IFN regulatory factor 7 (IRF7) and β-catenin. These transcription factors co-localize at targets like signal transducer and activator of transcription 1 (STAT1).
Treatment with ruxolitinib, a Janus kinase 1/2 (JAK1/JAK2) inhibitor, which reduces the activated phosphorylated form of STAT1, reduced differentiation-associated markers such as the cluster of differentiation 11b (CD11b). CD11b increased within five days of combination treatment, indicating myeloid cell maturation.
The combination inhibits the Wnt pro-oncogenic signaling pathway and suppresses cell cycle-related genes to prevent cell proliferation. An increase in chromatin accessibility facilitates these transcriptional changes at the genetic level.
AML patient datasets suggest an association between the combined drug treatment-induced transcriptional signature and improved prognosis, highlighting the treatment’s clinical translational potential. The action is not unique to LY2090314 and can be observed with other LSD1 and GSK3 inhibitors like IMG-7289 and 9-ING-41, respectively.
Patient synergy signature scores were consistent with experimental study results, correlating positively with type I IFN pathway signature scores and negatively with those of the Wnt signaling pathway. The drug combination repressed the TCF reporter under basal conditions and actively suppressed recombinant WNT3A-induced WNT hyperactivation.
Among the 16 patient samples, 11 exhibited a more than fivefold increase in CD11b+ cells and responded more strongly to the combination treatment than each inhibitor alone. None had deoxyribonucleic acid (DNA) methyltransferase-3 alpha (DNMT3A) mutations, and two non-responding patients had tumor protein p53 (TP53) mutations.
In xenotransplant AML mouse models, the combination significantly reduced tumor burden, with complete leukemia clearance in three of five mice. The treatment has selective action on leukemic cells and did not result in toxicity or body weight loss in treated mice, suggesting a favorable safety profile for this therapeutic approach.
Conclusion
The study highlights concomitant LSD1 inhibition and WNT pathway suppression (by GSK3 kinase) as a promising AML treatment strategy, overcoming the differentiation block commonly observed in AML.
The combination treatment upregulates type I IFN genes to induce cell differentiation and downregulates MYC proto-oncogene and cell cycle-related genes to prevent proliferation.
The study findings provide compelling evidence to support combination therapy testing in AML patients to validate the findings in clinical settings and improve patient outcomes.
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