Gene Expression Studies May Identify Lower Risk Myelodysplastic Syndrome Patients Likely to Respond to Therapy with Ezatiostat Hydrochloride (TLK199)

Abstract 2779

Interpretation of gene expression studies in MDS have been especially challenging due to the heterogeneity of the cell lineages that comprise the malignant clone. In attempting to overcome these difficulties we have used a bedside-to-bench approach to define an expression signature that may identify patients likely to respond. Ezatiostat hydrochloride (TLK199) is an inhibitor of glutathione S-transferase, an enzyme that is over expressed in many cancers, and has been shown in vitro to stimulate growth and differentiation of hematopoietic progenitor cells and to induce apoptosis in leukemia cells. Based on multilineage responses in low-Int1 MDS patients in our phase 2 study of oral TLK199, a multi institutional phase 2 study was conducted in low-Int1 patients. Response was evaluated by International Working Group (IWG 2006) criteria. Pre-therapy bone marrow mononuclear cells of patients treated with TLK199 were analyzed for gene expression on the Illumina HT12v4 whole genome array with IRB approval. RNA isolated from the marrow mononuclear cells was available on 9 responders (R) and 21 non-responders (NR). Five R and 13 NR were randomly chosen to create a training set with the intent to later use the remaining samples for model testing. We identified the top 100 differentially expressed genes using a sensitive metric based on the normalized mutual information. We also performed single-sample Gene Set Enrichment Analysis to find the most salient differences in terms of pathways and biological processes between R/NR. Of special note are the 4 microRNA s differentially expressed between R/NR. Three miRNAs are under-expressed (miR-129, 802 and 548e) and one (miR-155) is over-expressed in R. Reduced expression of miR-129 has been reported in solid tumors when over-expressed has been shown to have anti-proliferative activity in cell lines. SOX4 is a target gene for miR129 and reduced expression of miR-129 results in concomitant up-regulation of SOX4 mRNA which can function as both an oncogene and a tumor suppressor gene depending on tumor lineage. Over-expression of SOX4 inhibited cytokine induced granulocyte maturation in the myeloid 32Dcl3 cell line suggesting a possible role in MDS. MiR-802 targets the receptor for angiotensin II and when expression is decreased there is increased angiotensin II activity. It has recently been shown that angiotensin is a pro-inflammatory mediator that participates in apoptosis, angiogenesis and promotes mitochondrial dysfunction, all characteristics of MDS. In addition, the transcription factor ZFHX3, a predicted target of miR-802, is a negative regulator of c-MYB which has been shown to be up-regulated in all subtypes of MDS. Similarly, c-MYB is a predicted target of miR-155, which is over-expressed in TLK199 responders. MiR-155 was shown to be over-expressed in marrow cells of a subset of human AML patients. Of particular note are the studies showing that sustained expression of miR-155 in mouse hematopoietic stem cells cause a myeloproliferative/myelodysplastic disorder.

Subsequent pathway analysis of this expression data revealed that a JNK gene set as defined from the GEO dataset GDSS8081 was consistently under-expressed in responders and over-expressed in non-responders. TLK199 has been shown to induce JUN/JNK by binding to glutathione S-transferase, a key inhibitor of this pathway. The expression data confirms that patients whose pre-therapy marrow shows under-expression of the JNK gene set are precisely those who benefit from this drug therapy and those patients who already over-express these genes are unlikely to respond. This study highlights two important points: 1) Using a bedside-to-bench strategy yielded a signature that distinguished responders from non-responders 2) The signature identified genes and signaling pathways that shed light on both the biology of the disease and the mechanism of action of the drug. In conclusion, if these results are confirmed in the test set, we will use the signature in a future prospective study to preselect MDS patients for therapy with this promising drug.