Zalypsis Possess Potent Antileukemic Effect through the Induction of a DNA Damage Response, Independently of the p53 Status.

p53 mutation is the most frequent single genetic abnormality found in therapy related AML and is also quite frequent in de novo AML with an incidence between 10% and 25%; Moreover patients with p53 mutations characteristically present complex karyotypes and complicated chromosome rearrangements, leading to an adverse prognosis and resistance to conventional chemotherapeutic agents. Consequently, there is a need for novel antileukemic drugs that could work on both p53-wild type and p53-mutated AML patients. In this study we have evaluated the activity of Zalypsis, a novel alkaloid from marine origin, in several AML cell lines and patients samples with different p53 status.

The efficacy of Zalypsis was analyzed in four AML cell lines (HL60, HEL, MV4.11 and KG1) and in cells from fresh bone marrow samples from ten newly diagnosed AML patients. The cytotoxicity was analyzed by means of MTT assay and with a multiparametric flow cytometry (MFC) technique that allowed us to study the efficacy of the drug in both mature and immature blast cell compartments. Proteomic and genomic changes induced after treatment with Zalypsis were analyzed in two AML cell lines with different p53 status (HEL and HL60) by Western-Blot and gene expression profile studies.

Zalypsis showed a very potent antileukemic activity in the four AML cell lines tested, with IC50s at 48 hours below 1 nM. When compared with the in vitro activity of conventional antileukemic agents, such as cytarabine, doxorubicine or fludarabine, Zalypsis turned out to be 10-100 times more potent. It also showed remarkable ex vivo potency in freshly isolated blasts from ten AML patients. In these patients samples, we had the opportunity to separately analyze by multiparametric flow cytometry, the activity of Zalypsis in the different blasts populations, and we constantly observed similar activity in the most mature blast population and in the most immature one (CD34+, CD38- Lineage-), which is thought to include the leukemic stem cells and which is usually more resistant to conventional chemotherapy. Regarding toxicity, Zalypsis preserved the CD34+ non tumoral hematopoietic progenitor cells. The combination of this novel drug with conventional antileukemic agents indicated that Zalypsis is a good partner for combination with all of them, being the combination with cytarabine and daunorubicin particularly attractive for the clinic. Interestingly, as already said, all cell lines were extremely sensitive to Zalypsis, independently of the p53 status, as some of them displayed high basal levels of this protein by Western-Blot and had mutated p53 (HEL and MV 4.11), whereas its protein expression in the remaining cell lines was low, with KG1 bearing a mutation of the gene and HL60 a p53 deletion. Nevertheless, these last cells with low levels of the protein were a bit more sensitive to the drug, pointing out to a role of p53 in Zalypsis induced cell death. This was in agreement with a clear induction of DNA double strand breaks after treatment with Zalypsis, which was evidenced by an increase in phospho Histone H2AX, phospho CHK1 and phospho CHK2 after treatment of both HEL and HL60 with this compound. This was followed by the overexpression of p53 in the AML cell line bearing low basal levels of this protein (HL60). These results were further confirmed in the gene expression profile studies, in which treatment of AML cells with Zalypsis resulted in an important deregulation of genes involved in DNA damage response, including genes implicated in the ATM repair pathway and other mRNAs related to DNA repair, such as TLK2, ATR, ATMIN, CHEK2, RAD5, RAD52, RAD54L, BRCA1, BRCA2 and GADD45B among others. This response ended up in a clear induction of apoptosis as assessed by annexin V studies, caspase and PARP cleavage with partial rescue of cell death with the caspase inhibitor ZVAD-FMK, DNA laddering and loss of mitochondrial membrane potential analyzed by DiOC6 with the subsequent release of cytochrome C and AIF into the cytosol.

The potent and selective antileukemic effect observed with Zalypsis in AML cell lines and in patient's samples through a DNA damage response, together with its activity in both p53 mutated- and deleted-cells, provides the rationale for the investigation of Zalypsis in clinical trials for patients with AML.

Galmarini:Pharmamar: Employment. Avilés:Pharmamar: Employment. Cuevas:Pharmamar: Employment. Pandiella:Pharmamar: Research Funding. San-Miguel:Pharmamar: Research Funding.