Mini-Chromosome Maintenance (MCM) DNA Helicase Genes Influence Acute Myeloid Leukemia (AML) Replication and Response to Chemotherapy-Induced DNA Damage

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic stem/progenitor cells (HSCs). For older adults (≥60 yo) with AML, the prognosis is poor. The functional decline of HSCs with aging has been linked to increased replication stress from decreased expression of mini-chromosome maintenance (MCM) DNA helicase complex proteins. As AML incidence sharply rises with age, we explored age-related differences in gene expression of MCM and RECQ DNA helicases and DNA damage response (DDR) genes in AML patient blood and bone marrow samples. We hypothesized that older AML patients would show differences in DNA replication and DDR pathways compared to younger patients.

We began with an analysis of the TCGA AML database for MCM and RECQ helicase gene aberrations and found these in 37% (61/166) of the cases, with a median age of 60 years. There was reduced 5-year overall survival, 8.2 vs. 26.3 months, for those with vs. without such defects. Only a few mutations occur in these genes, with majority of aberrations due to mRNA up-regulation.

30 AML patient samples were obtained at diagnosis (n = 24) or relapse (n = 6). Total RNA was extracted from AML blasts and gene expression examined with the Affymetrix U133 Plus 2 arrays. Patients were categorized as older (≥65 yo), middle-aged (50-64 yo) and younger (<50 yo). Ingenuity Pathway Analysis (IPA) was performed on differentially expressed genes between the age-groups. FACS was used to analyze the effect of cytokine stimulation (G-CSF, IL-3, SCF) +/- 5 µM mitomycin C (induces double stranded DNA breaks) on levels of BrdU, γ-H2AX and cleaved-PARP as measures of cell cycle activity, DNA damage and apoptosis, respectively, in AML blasts.

Older AML patients showed increased expression of the MCM and RECQ DNA helicases and in multiple genes involved in the DDR response, including Ataxia telangiectasia mutated (ATM)/ATM- and RAD3-related (ATR) signaling, homologous recombination (HR), nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR) and Fanconi anemia (FA) families. Increased expression of the MCM, RECQ helicase and DDR genes in AML blast cells conferred a significantly worse prognosis. Basal γ-H2AX levels were increased in AML patients with abnormal or complex karyotype vs. normal karyotype. IPA showed age-related changes in ERG transcription factor activity, NFκB signaling and histone H3 modification. AML cells with high vs. low MCM gene expression differed in their response to growth factor stimulation and MMC treatment, in that the low MCM3 gene expressors did not progress through cell cycle after treatment with myeloid growth factors, and thus were spared of DNA damage and induction of apoptosis with MMC treatment. By Western blot analysis compared to actin control, the low MCM3 gene expressors also exhibited a trend toward significant positive correlation with MCM3 protein levels (Pearson r = 0.7, p = 0.06)

In summary, older AML patients exhibited increased expression of MCM and RECQ helicases and multiple DDR genes compared to middle-aged and younger patients, and there were age-associated changes in ERG transcriptional activity, NFkB signaling and histone H3 epigenetic regulation of gene expression. These elements are thus potential targets for future drug development, particularly for older adults with AML.