CDKN2A Upregulation Induces Senescence in AML Cell Lines and Leads to Treatment Resistance

Background: Acute myeloid leukemia (AML) is a heterogenous disease with >80% patients achieving complete remission (CR). But it has a particularly high relapse rate of ~50% with a concomitant overall survival rate of <30%. Considerable research is currently focused on uncovering the putative mechanisms which lead to relapse and subsequent treatment resistance in AML. One such aspect being investigated is the role of treatment-related senescence, especially its impact on promoting persistence of leukemic cells by protecting them from immune surveillance and resistance to subsequent rounds of therapy upon relapse. The standard chemotherapy regimen for AML includes an intensive regimen of genotoxic agents such as cytarabine (Ara-C), daunorubicin, and etoposide. A potential impact of overwhelming DNA damage induced by these agents is the activation of senescence pathways and reduced apoptosis. Studies have shown that exposure to these drugs can induce AML cell senescence mediated by upregulation of p16, a cyclin-dependent kinase inhibitor encoded by CDKN2A. p16 is an inhibitor of cyclin-dependent kinases (CDKs), particularly CDK4 and CDK6, which regulates transition from the G1 phase to the S phase of the cell cycle. Upregulation of CDKN2A can lead to a G1 arrest and given that the anti-leukemic activity of the main AML chemotherapeutics rely on active proliferation of cells, this could result in therapy resistance. Furthermore, we have observed high CDKN2A expression levels to be associated with poor outcomes in AML patients. In this study, we use a CDKN2A overexpression model in AML cell lines to provide in vitro evidence of drug treatment resistance upon senescence induction.

Methods: AML cell lines THP-1 and HL-60, identified to have low basal CDKN2A expression across various gene expression databases, were transduced with pLenti-C-Myc-DDK-IRES-Neo lentiviral particles encoding CDKN2A/p16 to generate stable cells overexpressing CDKN2A. Cell cycle profiling was performed with cells cultured for 7 days using FxCycle™ PI/RNase staining solution. At the same time point, ß-galactosidase activity was used an index of senescence induction and was assayed by flow cytometry using CellEvent senescence assay kit. Drug treatment response was assessed via CellTiter-Glo 2.

Results: To test any potential impact of CDKN2A overexpression by itself, cell cycle profiling was performed using both parental and CDKN2A overexpressing THP-1 and HL-60 cells cultured for 7 days. Compared to the respective isogenic parental cell lines, both THP-1 and HL-60 cells overexpressing CDKN2A showed a ~45% increase in the G1 population, a ~15% decrease in S phase population, and 75-78% decrease in G2/M. Cells from the same time point were used to test whether induction of senescence could be responsible for this accumulation of G1 arrest in cells overexpressing CDKN2A. Change in ß-galactosidase activity was measured as an index of senescence. CDKN2A overexpressing cells showed a 39%-45% increase in ß-galactosidase MFI along with a 7.5-9 fold increase in the % positive cells compared the parental cells. Finally, the IC₅₀ for cells overexpressing CDKN2A compared to parental cells was 1.5-1.7 fold higher treated with etoposide, 1.6-1.75 fold higher with daunorubicin, and 2.8-3.6 fold higher with Ara-C.

Conclusion: We show that elevated CDKN2A expression induces G1 arrest accompanied by senescence induction in unperturbed AML cells and leads to reduced drug sensitivity in vitro. Further study is underway to determine the impact of drug-treatment induced senescence using AML cell lines as well as patient cells and subsequent identification of senescence associated genetic signatures. Our overall goal is to provide translational rationale for developing prognostic and therapeutic strategies that address senescent cells to improve AML treatment outcomes.

No relevant conflicts of interest to declare.