Translational Control of Leukemia Stem Cells - Resubmission - 1

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Approximately 13,000 new cases of adult AML are diagnosed each year in the U.S. Unfortunately for these patients, treatment options have remained essentially unchanged for 30 years, and clinical outcomes remain poor. Moreover, little is known about the genes that regulate leukemia stem cells (LSCs), which represent the population of blasts that is resistant to chemotherapy and are critical for maintaining disease and re-initiating disease after therapy. Thus, eradication of the LSC is a prerequisite for cure. We recently identified a novel LSC antigen, CD99, that is expressed in the vast majority (~85%) of human AMLs [3]. We have shown that CD99 is expressed on leukemic blasts and can be used to prospectively separate leukemic blasts from residual hematopoiesis, similar to LSC antigens such as TIM3 and CD47, but CD99 exhibits several unique features: 1) CD99 is the most commonly expressed LSC antigen; 2) CD99 does not just mark LSCs, but regulates blast growth and survival; 3) CD99 allows isolation of LSCs from blasts with CD99hi blasts enriched ~10-100 fold for leukemia initiating-cell activity over CD99low cells; 4) CD99 is the only LSC antigen that can identify LSCs in both CD34+ and CD34- AMLs; and, 5) Novel monoclonal antibodies (mAbs) against CD99 induce cell death directly by activating Src-family kinases (SFKs). We have evaluated the consequences of CD99 loss in both LSCs and hematopoietic stem cells (HSCs). Our studies indicate that decreased expression of CD99 in both LSCs and HSCs results in global upregulation of protein synthesis and loss of self-renewal. Moreover, cytotoxic mAbs against CD99 mimic the effects of CD99 loss, activating protein synthesis and inducing similar gene expression changes to those observed in CD99 null HSCs or CD99low blasts in most genetic subtypes of AML tested. Collectively, these data support a model in which LSCs require highly regulated levels of protein synthesis, similar to HSCs, and based on our work in normal HSCs, we expect these alterations in translation to result in selective recruitment of mRNA’s to active translating ribosomes (polysomes). Overall, we hypothesize that CD99 constrains the translation of specific mRNA’s, thereby promoting a translational program required for LSC self-renewal. Given our ability to enrich for LSCs, our group is in a unique position to investigate the role of mRNA translation in LSC function. We have painstakingly optimized methods to perform polysome profiling and RNA- sequencing from polysome fractions from small numbers of cells, and we have developed a computational pipeline to identify mRNA’s that are preferentially translated in LSCs. These tools, in combination with unique reagents such as our CD99 KO mice and cytotoxic CD99 mAbs that mimic CD99 loss, place us in an excellent position to investigate how CD99 regulates translation in LSCs. Understanding the molecular pathways that regulate protein synthesis has the potential to help better characterize a poorly understood process in AML biology and to credential targeting translation using mAbs as a potential therapeutic strategy in AML.