Immunoproteasome Inhibition to Target AML with Activated RAS Pathways
Authors: Navis Connor , Noble-Orcutt Klara , LaRue Rebecca S , Hansen Conner , Ha Ngoc , Hillesheim Alexandria , Schulz Ryan , Kim Do-Hyung , Largaespada David A , Sachs Zohar 
Published: November 2019

Abstract
Acute myeloid leukemia (AML) is a lethal cancer with a survival of less than 50%. Standard cytotoxic therapies frequently induce complete remission, but patients frequently relapse and die of their disease. Leukemia stem cells (LSCs) are the only leukemia cells with self-renewal potential and ability to recapitulate the disease. Our goal is to develop therapeutics that target and eradicate AML LSCs. We showed that activated NRAS facilitates self-renewal in a murine model of AML; mTORC1 activation by NRAS is important for this effect (Sachs et al. Blood 2014). RAS activates mTORC1 through the PI3K/AKT/mTOR pathway which is highly upregulated in AML (Xu et al. Blood 2003, Altman et al. Oncogtarget 2011). Recently, we found that RAS-activated malignancies, including AML, induce immunoproteasome formation via mTORC1 (Yun et al. Mol Cell 2016). Pan-proteasome inhibitors, such as bortezomib and carfilzomib, are widely used in the treatment of lymphoid malignancies and have some activity in AML. We hypothesize that specific inhibition of the immunoproteasome may target NRAS-mediated self-renewal in LSCs. We use a manipulatable, transgenic mouse model of AML with an Mll-AF9 fusion and a tetracycline repressible, activated NRAS (NRASG12V, Kim et al. Blood 2009). We investigated the possible role of the immunoproteasome in our murine model and found that abolishing NRASG12V transgene expression led to a loss of immunoproteasome component genes expression (p<0.001). We also found that the LSC-enriched subpopulation preferentially upregulated expression of immunoproteasome component genes (p<0.002) in comparison to non-stem cells from this model. Accordingly, we found that primary human AML CD34+ precursors upregulated immunoproteasomal components relative to CD34- AML cells by up to 3.7 fold. mTOR inhibition with everolimus diminished expression of immunoproteasome component genes (p=0.05) suggesting that immunoproteasome activity may be NRASG12V/mTOR-dependent in this model as well. Next, we sought to identify the self-renewing cells among immunophenotypically defined LSC-enriched subgroup in this model. We performed single-cell RNA sequencing on the LSC-enriched population from this model. We identified three discrete trasncriptional profiles among the LSCs. Comparing the single-cell transcriptional profiles of NRASG12V-expressing LSCs to those of LSCs treated with doxycycline to extinguish NRASG12V ("RAS-On" and "RAS-Off" LSCs) revealed that two of these profiles are NRASG12V-dependent. These two profiles can be differentiated by CD36 and CD69 expression. We sorted LSCs based on CD36 and CD69 expression as well as established immunophenotypic markers, Mac1, Kit, Sca-1 (LSC-enriched supopulation in this model is Mac1LowKit+Sca1+). These sorted LSC subsets were transplanted into recipient mice to compare their ability to transfer leukemia as a measure of their self-renewal capacity. We found that CD36-CD69+Mac1LowKit+Sca1+ cells (consistent with Profile 1 gene expression) rapidly transferred leukemia with high penetrance. In contrast, CD36+CD69- Mac1LowKit+Sca1+ cells (consistent with Profile 2 gene expresison) did not transfer leukemia to mice (p < 0.004). These experiments characterize the NRASG12V-mediated self-renewal transcriptional signature at the singel-cell level and suggest that single-cell RNA sequencing data may be an effective tool for delineating the self-renewing subpopluation among immunophenotypically-defined LSCs. We performed single-cell RNA sequencing on CD34+ human AML precursors obtained from a diagnostic bone marrow specimen. Analogous to our murine model, we found that these human AML cells express 2 distinct single-cell transcriptional profiles and that differentially express RAS-activated gene expression profiles. Finally, we found that, PR957, a specific immunoproteasome inhibitor, inhibits in vitro colony formation more effectively than carfilzomib (a second generation pan-proteasome inhibitor) in our murine model of AML suggesting that immunoproteasome inhibition may be specifically target self-renewal in AML. In these experiments, we use single-cell RNA sequencing to identify the self-renewing subset of RAS-driven LSCs at the single-cell level and implicate immunoproteasome inhibition as an approach to target RAS-mediated self-renewal.

Disclosures
No relevant conflicts of interest to declare.