Abstract
The nucleoside analogues 8-amino-adenosine and 8-chloro-adenosine have been investigated in the context of B-lineage lymphoid malignancies by our laboratories due to the selective cytotoxicity they exhibit toward multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and mantle cell lymphoma (MCL) cell lines and primary cells. Encouraging pharmacokinetic and pharmacodynamic properties of 8-chloro-adenosine being documented in an ongoing Phase I trial in CLL provide additional impetus for the study of these promising drugs. In order to foster a deeper understanding of the commonalities between their mechanisms of action and gain insight into specific patient cohorts positioned to achieve maximal benefit from treatment, we devised a novel two-tiered chemoinformatic screen to identify molecular determinants of responsiveness to these compounds. This screen entailed: 1) the elucidation of gene expression patterns highly associated with the anti-tumor activity of 8-chloro-adenosine in the NCI-60 cell line panel, 2) characterization of altered transcript abundances between paired MM and MCL cell lines exhibiting differential susceptibility to 8-amino-adenosine, and 3) integration of the resulting datasets. This approach generated a signature of seven unique genes including G6PD which encodes the rate-determining enzyme of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase. Bioinformatic analysis of primary cell gene expression data demonstrated that G6PD is frequently overexpressed in MM and CLL, highlighting the potential clinical implications of this finding. Utilizing the paired sensitive and resistant MM and MCL cell lines as a model system, we go on to demonstrate through loss-of-function and gain-of-function studies that elevated G6PD expression is necessary to maintain resistance to 8-amino- and 8-chloro-adenosine but insufficient to induce de novo resistance in sensitive cells. Taken together, these results indicate that G6PD activity antagonizes the cytotoxicity of 8-substituted adenosine analogues and suggests that administration of these agents to patients with B-cell malignancies exhibiting normal levels of G6PD expression may be particularly efficacious.
Highlights
The novel nucleoside analogues 8-amino-adenosine (8-NH2Ado) and 8-chloro-adenosine (8-Cl-Ado) have undergone intensive preclinical development for cancer treatment by our laboratories due to the unique RNA-directed effects they elicit in tumor cells
This data was generated through the Rapid Access to Intervention Development (RAID) initiative offered through the Developmental Therapeutics Program at the National Cancer Institute
In all microarray-based transcriptomic gene expression studies, incongruence between mRNA and protein expression levels of a given gene can result in the manifestation of false positive and false negative errors, necessitating caution in deriving conclusions from data pertaining only to mRNA abundance. These results suggest that the t(11;14) genotype and high CCND1 expression are associated with resistance to 8-NH2-Ado/8-Cl-Ado in the context of myeloma while the discrepancies between cyclin D1 transcript and protein abundances noted above suggest that this association is not meaningful in the context of mantle cell lymphoma
Summary
The novel nucleoside analogues 8-amino-adenosine (8-NH2Ado) and 8-chloro-adenosine (8-Cl-Ado) have undergone intensive preclinical development for cancer treatment by our laboratories due to the unique RNA-directed effects they elicit in tumor cells. 8-NH2-Ado and 8-Cl-Ado show strong activity against indolent hematological malignancies characterized by intrinsically low rates of DNA replication and poor responsiveness to classical nucleoside analogues Cellular conversion of these antimetabolites to their respective triphosphorylated forms is a prerequisite for induction of their pleiotropic activities leading to cell killing. MRNA and protein quantities of the receptor tyrosine kinase c-Met fall rapidly in multiple myeloma cells exposed to 8Cl-Ado [8] and Mcl-1 expression declines within hours of treatment initiation with either analogue in CLL cells [5,9] In addition to these shared properties, 8-NH2-Ado exhibits compound-specific attributes which may account for its increased potency in relation to 8-Cl-Ado. 8-NH2-Ado acutely suppresses glucose consumption in multiple myeloma cells [10] (which is associated with intracellular sequestration of GLUT4 and activation of autophagy) and elicits dephosphorylation and inactivation of Akt, mTOR and Erk kinases in a cancer-specific manner [3]. Accumulation of high micromolar to low millimolar levels of 8-Cl-ATP in peripheral blood mononuclear cells (PBMC) following administration of 8-Cl-Ado to mice and rats [15] provides in vivo evidence supporting the auspicious clinical prospects of 8-substituted adenosine analogues to treat lymphoid neoplasms
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