PPPR15A, a stress response gene is upregulated in malignant myeloid progenitors in AML and higher risk MDS. Free

Introduction: Acute myeloid leukemia (AML) is an aggressive blood cancer driven by rapid immature myeloid cell proliferation and genomic instability. This instability, leading to DNA damage, fuels AML development through mutation accumulation. Myelodysplastic Syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis and an increased risk of progression to AML. The DNA damage response (DDR) in AML and MDS is often dysregulated, either by impairment, which allows persistent DNA damage, or by exploitation of DDR pathways for survival and therapy resistance. Our group previously showed that PPP1R15A, CDKN1A, GADD45A and GADD45G are differentially expressed in AML cell lines and especially PPP1R15A is upregulated in patients with de novo AML with a median age of 30 years, receiving intensive chemotherapy. Now, we expanded the evaluation of DDR genes expression in older AML patients , treated with a combination of hypomethylating agents (HMAs) and Venetoclax and high risk MDS treated with HMAs.

Materials & Methods: Peripheral Blood Mononuclear Cells (PBMCs) were isolated with the standard Ficoll-Paque procedure from 3 ml of aspirated Bone Marrow in EDTA tubes from AML patients (n=21) with a median age of 70 years and a median BM blast percentage 40% (21-60%), from high risk MDS patients (n=21) and from lymphoma patients (n=10) without bone marrow involvement, which were used as controls. All samples obtained in accordance with the Declaration of Helsinki and in agreement with Attikon University Hospital Ethics Committee. CD34+ cells were isolated with FACS Aria™ III sorter (BD Biosciences) after proper immunostaining with mouse anti-human CD34-PE antibody (clone: 4H11) (exbio). Total RNA extraction or CD34+ RNA extraction was performed either by using the PureLink™ RNA Mini Kit (Thermofischer Scientific) or via TRIzol® (Thermofischer Scientific). Relative quantitation of DDR genes : PPP1R15A, CDKN1A, GADD45A and GADD45G was performed with QuantiTect® SYBR® Green PCR Kit (Qiagen) and primers from QuantiTect® Primer Assay (Qiagen) for each gene of interest. GAPDH was used as report gene. Box and whiskers plots and statistical analysis were performed in GraphPad Prism v8.0.1™. Statistical analysis performed using unpaired t test with statistical significance p<0,05.

Results & Discussion:In elderly AML patients, PPP1R15A, CDKN1A, and GADD45A were significantly downregulated compared to controls (p<0,05), while GADD45G exhibited similar expression levels (p=0,275). This contrasts with our previous data, which showed PPP1R15A to be upregulated in younger, de novo AML patients, who had higher BM infiltration (median 80%, r: 70-90%). Intriguisgly, in CD34+ cells from high-risk MDS patients, PPP1R15A is significantly upregulated (p<0,05) compared to controls, similar to its expression pattern in young AML patients. Concurrently, CDKN1A, GADD45A, and GADD45G showed a tendency towards downregulation in high-risk MDS CD34+ cells. These findings suggest PPP1R15A's higher expression in CD34+ hematopoietic progenitors in myeloid malignancies. Regarding treatment response in elderly AML patients receiving HMAs plus Venetoclax, PPP1R15A showed a tendency for lower expression in patients achieving CR (n=13) versus non-CR (n=8) (p=0,361). Conversely, CDKN1A and GADD45G tended to be highly expressed in CR patients (p=0,376 and p=0,289, respectively). Notably, in CD34+ cells from high-risk MDS patients treated with HMAs, GADD45G showed a tendency for higher expression in CR (n=9) versus non-CR (n=12) (p=0,159), while PPP1R15A, CDKN1A, and GADD45A did not exhibit differential expression between the two groups.

Conclusion: Our findings indicate a significant role for PPP1R15A expression in AML, with upregulation in younger AML and CD34+ cells in high-risk MDS patients, contrasting with downregulation in elderly AML. This suggests PPP1R15A expression is closely associated with abnormal CD34+ hematopoietic progenitors in myeloid malignancies. The differential gene expression pattern between young and elderly AML could be attributed to the lower blast infiltration in elderly patients. While preliminary, the expression patterns of PPP1R15A, CDKN1A, and GADD45G also show tendencies that may correlate with response to HMAs plus Venetoclax in elderly AML, and to HMAs in high-risk MDS, warranting further investigation into their potential as prognostic biomarkers and therapeutic targets.