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Blast cells surviving acute myeloid leukemia induction therapy are in cycle with a signature of FOXM1 activity

ABSTRACT:

Background

Disease relapse remains common following treatment of acute myeloid leukemia (AML) and is due to chemoresistance of leukemia cells with disease repopulating potential. To date, attempts to define the characteristics of in vivo resistant blasts have focused on comparisons between leukemic cells at presentation and relapse. However, further treatment responses are often seen following relapse, suggesting that most blasts remain chemosensitive. We sought to characterise in vivo chemoresistant blasts by studying the transcriptional and genetic features of blasts from before and shortly after induction chemotherapy using paired samples from six patients with primary refractory AML.

Methods

Leukemic blasts were isolated by fluorescence-activated cell sorting. Fluorescence in situ hybridization (FISH), targeted genetic sequencing and detailed immunophenotypic analysis were used to confirm that sorted cells were leukemic. Sorted blasts were subjected to RNA sequencing. Lentiviral vectors expressing short hairpin RNAs were used to assess the effect of FOXM1 knockdown on colony forming capacity, proliferative capacity and apoptosis in cell lines, primary AML cells and CD34+ cells from healthy donors.

Results

Molecular genetic analysis revealed early clonal selection occurring after induction chemotherapy. Immunophenotypic characterisation found leukemia-associated immunophenotypes in all cases that persisted following treatment. Despite the genetic heterogeneity of the leukemias studied, transcriptional analysis found concerted changes in gene expression in resistant blasts. Remarkably, the gene expression signature suggested that post-chemotherapy blasts were more proliferative than those at presentation. Resistant blasts also appeared less differentiated and expressed leukemia stem cell (LSC) maintenance genes. However, the proportion of immunophenotypically defined LSCs appeared to decrease following treatment, with implications for the targeting of these cells on the basis of cell surface antigen expression. The refractory gene signature was highly enriched with targets of the transcription factor FOXM1. shRNA knockdown experiments demonstrated that the viability of primary AML cells, but not normal CD34+ cells, depended on FOXM1 expression.

Conclusions

We found that chemorefractory blasts from leukemias with varied genetic backgrounds expressed a common transcriptional program. In contrast to the notion that LSC quiescence confers resistance to chemotherapy we find that refractory blasts are both actively proliferating and enriched with LSC maintenance genes. Using primary patient material from a relevant clinical context we also provide further support for the role of FOXM1 in chemotherapy resistance, proliferation and stem cell function in AML.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-021-08839-9.

SUBMITTER: Williams M

PROVIDER: S-EPMC8554867 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ImportanceTests that predict outcomes for patients with acute myeloid leukemia (AML) are imprecise, especially for those with intermediate risk AML.ObjectivesTo determine whether genomic approaches can provide novel prognostic information for adult patients with de novo AML.Design, setting, and participantsWhole-genome or exome sequencing was performed on samples obtained at disease presentation from 71 patients with AML (mean age, 50.8 years) treated with standard induction chemotherapy at a single site starting in March 2002, with follow-up through January 2015. In addition, deep digital sequencing was performed on paired diagnosis and remission samples from 50 patients (including 32 with intermediate-risk AML), approximately 30 days after successful induction therapy. Twenty-five of the 50 were from the cohort of 71 patients, and 25 were new, additional cases.ExposuresWhole-genome or exome sequencing and targeted deep sequencing. Risk of identification based on genetic data.Main outcomes and measuresMutation patterns (including clearance of leukemia-associated variants after chemotherapy) and their association with event-free survival and overall survival.ResultsAnalysis of comprehensive genomic data from the 71 patients did not improve outcome assessment over current standard-of-care metrics. In an analysis of 50 patients with both presentation and documented remission samples, 24 (48%) had persistent leukemia-associated mutations in at least 5% of bone marrow cells at remission. The 24 with persistent mutations had significantly reduced event-free and overall survival vs the 26 who cleared all mutations. Patients with intermediate cytogenetic risk profiles had similar findings. [table: see text].Conclusions and relevanceThe detection of persistent leukemia-associated mutations in at least 5% of bone marrow cells in day 30 remission samples was associated with a significantly increased risk of relapse, and reduced overall survival. These data suggest that this genomic approach may improve risk stratification for patients with AML.