The Potential of Using DNA Damage Repair Deficiency As a Biomarker for Cytarabine Response in AML Patients

Acute Myeloid Leukemia (AML) is most commonly seen in people over the age of 65 and has a median age of 63. Globally there is an increasingly elderly population so the rate of incidence of AML is set to increase. The therapy landscape for AML has changed little over the past four decades. Cytarabine, first approved in 1969, is still the standard of care induction therapy for AML. There has been only modest improvements in survival rates during this time and there is currently no method of determining which patients will or will not respond to Cytarabine treatment.

An assay, developed in 2014, used microarray data to determine which breast cancer patients had a DNA Damage Repair Deficiency (DDRD) and therefore would be more susceptible to DNA damaging agents. A negative DDRD (DDRD^-) score predicts that patients do not to have a DNA Repair Deficiency whilst patients with a positive DDRD (DDRD⁺) score are predicted to have a DNA Repair Deficiency. This assay has been adapted to different solid cancer types such as ovarian and oesophageal cancer. This project has assessed the potential of using the DDRD assay for AML patients.

The assay was applied to publically available microarray data of >600 AML patients (TCGA AML data &GSE6891), who were classed as DDRD^- or DDRD^+. Excluding patients not treated with Cytarabine, this left 639 patients, 405 DDRD⁺ and 234 DDRD^-. Kaplan Meier analysis showed the DDRD⁺ patients survived significantly (p=0.00047) worse than the DDRD^- cohort.

Whole exome sequencing was available for 183 patients (131 DDRD⁺) and the mutations associated with each group were identified. As the DDRD⁺ patients had the worst outcome, we focused on group. The list of genes more commonly mutated in the DDRD⁺ patients (>2 instances and >50% occurring in this group) were subjected to pathway analysis. Deregulated pathways included "leukemogenisis" and "cell proliferation and regulation"; however, the most deregulated pathway was "metabolism of nucleobase containing compounds". As Cytarabine is a nucleobase-containing compound, this is potentially a contributing factor as to why these patients responded poorly to this treatment.

The assay was applied to microarray data of a panel of myeloid cell lines, and DDRD^-(NB4 & SKM1) and a DDRD⁺(HL-60) cell line were chosen as experimental models.

Clonogenic assays, used to analyse the effect of Cytarabine on these cell lines, showed that the DDRD^- cell lines were more sensitive with a lower colony growth rate than the DDRD⁺cell line.

DNA damage induction and repair, following cytarabine treatment or 2gy radiation, were measured using RAD51 foci counts. Whilst foci counts were high in all cell lines 2hrs and 4hrs following radiation, the DDRD⁺ cell line continued to show high levels after 24hrs whereas the levels in the DDRD^- cell lines returned to a basal level. RAD51 response to radiation treatment showed that a repair defect is present in DDRD⁺ cells as they fail to repair the damage induced by radiation. Following treatment with Cytarabine however, few foci were seen in the DDRD⁺ cell line 2hrs, 4hrs or 24hrs following treatment whereas the DDRD^- cell lines responded in a similar fashion to radiation treatment. That RAD51 foci are not present following Cytarabine treatment indicates that Cytarabine fails to induce damage in these cells.

The DDRD assay has shown to be an effective method for determining cellular response to Cytarabine in vivo. The non-response of the DDRD⁺ cell line to Cytarabine suggests that these cells do not elicit a DNA damage or an apoptotic response. This perhaps contributes to their poorer outcome and suggests that Cytarabine is not an effective treatment plan for patients deemed to be DDRD⁺. Although alternative induction treatment options are currently unavailable for DDRD⁺ AML patients, this DDRD assay could be used as a biomarker for Cytarabine response in the future.