Abstract
This project is focused on characterizing the malignant stem cells that drive the pathogenesis of MDS, with the goal of developing improved therapeutic strategies. To this end, we have established methods by which MDS stem cells can be identified, isolated and characterized in xenograft models. These approaches have been employed to permit global metabolomic and transcriptomic analyses, which have subsequently led to modeling novel therapeutic regimens.
Initial studies exploited previous work in acute myelogenous leukemia to identify candidate phenotypic markers of stem cell malignancy. These efforts demonstrated that up-regulation of CD123 in the hematopoietic stem cell compartment identifies MDS stem cells as they transition from low risk to high risk stages of pathogenesis. Thus, using cell sorting, we are able to isolate early (CD123-) vs. late (CD123+) stage stem cells from MDS patient specimens and subject them to the experimental analyses outlined below. We first performed a transcriptomic study that demonstrated a massive increase in cellular protein translation machinery through significant increases in ribosomal proteins as stem cells progress to advanced stages of MDS. To functionally validate these findings, MDS stem cell populations were cultured with the fluorescent protein substrate OP-puro, which detects newly synthesized polypeptide chains. CD123+ stem cells strongly increase protein synthesis levels (~13-fold increase). Given the established role of protein translation homeostasis in hematopoietic stem cells [Signer et al., 2014], this finding indicates a major change in cellular metabolism as well as a potential therapeutic entry point.
To further characterize cellular changes occurring as MDS stem cells evolve, we performed global mass spectrometry-based metabolomic analyses which further demonstrates increased protein biosynthesis as well as altered glutathione metabolism (elevated oxidized glutathione). Taken together, these findings indicate major metabolic changes in MDS stem cells as they acquire increasing malignant phenotypes.
Next, we investigated signaling related to increased ribosomal protein production. Specifically, we examined the hypothesis that ribosomal subunit binding of MDM2, may play a role in pathogenesis. Intriguingly, our data show that the specific subunits known to bind MDM2 are increased in CD123+ MDS stem cells. Consistent with this observation, MDM2 is elevated as well. This finding indicates that increased ribosomal protein levels may function to inhibit p53 activity, thereby enhancing pathogenic outgrowth of MDS stem cells.
Using the mechanistic insights outlined above, we explored novel therapeutic strategies. First, we examined drugs known to selectively target metabolism through altering protein synthesis and targeting ribosomal proteins (e.g. homoharringtonine, HHT)). In addition, given the prevalent role of hypomethylating agents in current MDS treatment regimens, we also examined how these inhibitors interact with 5-azacytidine (5-aza). In vitro studies indicate that multiple protein synthesis inhibitors selectively eradicate MDS stem cells (CD123+). In addition, combination with 5-aza yielded additive to synergistic eradication of MDS stem cells. We have demonstrated that high risk MDS specimens effectively engraft the NSG-S strain of immune deficient mice, when T cell depletion and a busulfan conditioning regimen are employed. Using this model, we transplanted primary MDS specimens and challenged using the regimens above. Treatment with HHT demonstrated selective eradication of MDS stem cells, with a significant differential toxicity observed in multiple samples (50-60% selective ablation). Finally, analysis of protein synthesis inhibitors in combination with the hypomethylating agent 5-aza demonstrated potent efficacy in targeting the MDS stem cell population with greater then additive toxicity when compared to single agent treatment(70-80% selective ablation).
Taken together, these data show that changes in metabolic properties represent a critical inflection point in the pathogenesis and progression of the MDS. Focusing on such changes, we have identified novel pharmacological approaches that may effectively target the MDS stem cell population. Importantly, these approaches function well in conjunction with commonly used agents used in the treatment of MDS.
Disclosures
Pollyea: Alexion: Other: advisory board; Celgene: Other: advisory board, Research Funding; Glycomimetics: Other: DSMB member; Ariad: Other: advisory board; Pfizer: Other: advisory board, Research Funding.
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