TP53mutations and drug sensitivity in acute myeloid leukaemia cells with acquired MDM2 inhibitor resistance

Abstract

Background:MDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AML TP53 wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.Methods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. The TP53 status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.Results:All MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existing TP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varying TP53 mutations or wild type TP53, indicating the induction of de novo TP53 mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.Conclusion:We introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existing TP53-mutant subpopulations or induce de novo TP53 mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.

The RS4;11 cell line as a model for leukaemia with t(4;11)(q21;q23): Revised characterisation of cytogenetic features

“…For instance, the absence of the der(4) in the B‐1 cell line, which is however present in MV‐4‐11, RS4;11, AN4;11, and SEM, suggests that it is the der(11) to detain a pivotal role in the promotion and maintenance of the malignancy . Differences in other molecular features, such as mutations of known cancer‐related genes TP53 and FLT3 , can prove useful in investigating the role of point mutations in leukaemogenesis (Table ).…”

Mutation Evaluation in P53 exon 5 in Iraqi AML Patients with 4 Growth Levels

“…The purification of DNA template was performed in the thermocycler (Eppendorf, German) according to the conditions indicated in Table (3).…”

“…Choosing the type of causative mutation of AML, helps decide treatment type . A previous study (3) investigated genes and DNA errors associated with TP 53 mutations and found that (hub genes) are responsible for it which are : LEP,BMP 2 , ITGA 2 B,MNX 1 , TRH,NMU,CDH 1 , KDR, CASR and APOE. These genes may change the type of treatment received by patient and may increase his healing opportunity.…”

Review 2: Long-term cultivation using ineffective MDM2 inhibitor concentrations alters leukaemia cell drug sensitivity profiles

“…Resistance formation of TP53 wild-type cancer cells to MDM2 inhibitors commonly results in the formation of TP53 mutations as resistance mechanism [2][3][4][5][6][7][8]. TP53 mutations may be the consequence of the selection of pre-existing TP53-mutant cell subpopulations or the induction of de novo TP53 mutations [3,[5][6][7].…”

“…Resistance formation of TP53 wild-type cancer cells to MDM2 inhibitors commonly results in the formation of TP53 mutations as resistance mechanism [2][3][4][5][6][7][8]. TP53 mutations may be the consequence of the selection of pre-existing TP53-mutant cell subpopulations or the induction of de novo TP53 mutations [3,[5][6][7]. De novo TP53 mutations may be the consequence of the selection of cells in which TP53 mutations have occurred by chance and which would have disappeared in the absence of the selection pressure induced by an MDM2 inhibitor.…”