Enhanced sensitivity to glucocorticoids in cytarabine-resistant AML.

We sought to identify drugs that could counteract cytarabine resistance in acute myeloid leukemia (AML) by generating eight resistant variants from MOLM-13 and SHI-1 AML cell lines by long-term drug treatment. These cells were compared with 66 ex vivo chemorefractory samples from cytarabine-treated AML patients. The models and patient cells were subjected to genomic and transcriptomic profiling and high-throughput testing with 250 emerging and clinical oncology compounds. Genomic profiling uncovered deletion of the deoxycytidine kinase (DCK) gene in both MOLM-13- and SHI-1-derived cytarabine-resistant variants and in an AML patient sample. Cytarabine-resistant SHI-1 variants and a subset of chemorefractory AML patient samples showed increased sensitivity to glucocorticoids that are often used in treatment of lymphoid leukemia but not AML. Paired samples taken from AML patients before treatment and at relapse also showed acquisition of glucocorticoid sensitivity. Enhanced glucocorticoid sensitivity was only seen in AML patient samples that were negative for the FLT3 mutation (P=0.0006). Our study shows that development of cytarabine resistance is associated with increased sensitivity to glucocorticoids in a subset of AML, suggesting a new therapeutic strategy that should be explored in a clinical trial of chemorefractory AML patients carrying wild-type FLT3.

Monitoring therapy responses at the leukemic subclone level by ultra-deep amplicon resequencing in acute myeloid leukemia.

In our individualized systems medicine program, personalized treatment options are identified and administered to chemorefractory acute myeloid leukemia (AML) patients based on exome sequencing and ex vivo drug sensitivity and resistance testing data. Here, we analyzed how clonal heterogeneity affects the responses of 13 AML patients to chemotherapy or targeted treatments using ultra-deep (average 68 000 × coverage) amplicon resequencing. Using amplicon resequencing, we identified 16 variants from 4 patients (frequency 0.54-2%) that were not detected previously by exome sequencing. A correlation-based method was developed to detect mutation-specific responses in serial samples across multiple time points. Significant subclone-specific responses were observed for both chemotherapy and targeted therapy. We detected subclonal responses in patients where clinical European LeukemiaNet (ELN) criteria showed no response. Subclonal responses also helped to identify putative mechanisms underlying drug sensitivities, such as sensitivity to azacitidine in DNMT3A mutated cell clones and resistance to cytarabine in a subclone with loss of NF1 gene. In summary, ultra-deep amplicon resequencing method enables sensitive quantification of subclonal variants and their responses to therapies. This approach provides new opportunities for designing combinatorial therapies blocking multiple subclones as well as for real-time assessment of such treatments.