consumption through glycolysis even in the presence of oxygen (aerobic glycolysis) was initially hypothesized to be due to defective mitochondria [2]. However, more recent studies have established that many cancer cells harbor and are dependent upon functional mitochondria [3][4][5][6]. The question why cancer cells preferentially use glycolysis, an uneconomical route to produce ATP compared to mitochondrial oxidative phosphorylation, is beginning to be addressed in cancer cells. Glycolysis is not the dominant mechanism for many cancer cells to produce ATP, and ATP production is not the limiting factor for proliferation [7]. Rather, glycolysis offers cancer cells with metabolites essential for rapid division through metabolic pathways that branch away from glycolysis. For example, glucose-6-phosphate can be diverted to the pentose phosphate pathway (PPP) to produce reducing agents NADPH and ribonucleotides [8]. Additionally, 3-phospho-glycerate can enter the one-carbon cycle to generate amino acids, lipids, and reducing agents [9]. Thus, increased glycolysis meets the demand of proliferative cancer cells for anabolism, producing metabolic intermediates through multiple branching pathways. The decision of how cancer cells divert glycolysis intermediates remains elusive, but it is likely to be affected by cellular metabolic state and the environment in which the cancer cells are exposed.The metabolic milieu of the tumor microenvironment dictates the behavior of tumors. Tumors are exposed to nutrient-and oxygen-poor conditions as they grow exponentially due to insufficient vascularization [10]. Metabolic adaptation to these stress conditions is vital for tumor survival and expansion, and as such multiple metabolic regulators that enable metabolic adaptation are dysregulated in cancer. These include the components of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling that feeds into the mechanistic target of rapamycin (mTOR) pathway, Abstract Metabolic homeostasis is a fundamental property of cells that becomes dysregulated in cancer to meet the altered, often heightened, demand for metabolism for increased growth and proliferation. Oncogenic mutations can directly change cellular metabolism in a cell-intrinsic manner, priming cells for malignancy. Additionally, cellextrinsic cues from the microenvironment, such as hypoxia, nutrient availability, oxidative stress, and crosstalk from surrounding cells can also affect cancer cell metabolism, and produce metabolic heterogeneity within the tumor. Here, we highlight recent findings revealing the complexity and adaptability of leukemia cells to coordinate metabolism.

There is increasing evidence that the metabolic regulation of acute myeloid leukemia (AML) cell growth interacts with epigenetic pathways of gene expression and differentiation. Jiang et al link inhibition of glucose metabolism to epigenetic changes and altered transcriptional pathways in leukemic cells and demonstrate synergy between simultaneously targeting metabolism and chromatin modifiers in suppression of AML.

Recent advances in next-generation sequencing have identified novel mutations and revealed complex genetic architectures in human hematological malignancies. Moving forward, new methods to quickly generate animal models that recapitulate the complex genetics of human hematological disorders are needed to transform the genetic information to new therapies. Here, we used a ribonucleoprotein-based CRISPR/Cas9 system to model human clonal hematopoiesis of indeterminate potential and acute myeloid leukemia (AML). We edited multiple genes recurrently mutated in hematological disorders, including those encoding epigenetic regulators, transcriptional regulators, and signaling components in murine hematopoietic stem/progenitor cells. Tracking the clonal dynamics by sequencing the indels induced by CRISPR/Cas9 revealed clonal expansion in some recipient mice that progressed to AML initiated by leukemia-initiating cells. Our results establish that the CRISPR/Cas9-mediated multiplex mutagenesis can be used to engineer a variety of murine models of hematological malignancies with complex genetic architectures seen in human disease.

Metabolic dysregulation underlies malignant phenotypes attributed to cancer stem cells, such as unlimited proliferation and differentiation blockade. Here, we demonstrate that NAD+ metabolism enables acute myeloid leukemia (AML) to evade apoptosis, another hallmark of cancer stem cells. We integrated whole-genome CRISPR screening and pan-cancer genetic dependency mapping to identify NAMPT and NMNAT1 as AML dependencies governing NAD+ biosynthesis. While both NAMPT and NMNAT1 were required for AML, the presence of NAD+ precursors bypassed the dependence of AML on NAMPT but not NMNAT1, pointing to NMNAT1 as a gatekeeper of NAD+ biosynthesis. Deletion of NMNAT1 reduced nuclear NAD+, activated p53, and increased venetoclax sensitivity. Conversely, increased NAD+ biosynthesis promoted venetoclax resistance. Unlike leukemia stem cells (LSCs) in both murine and human AML xenograft models, NMNAT1 was dispensable for hematopoietic stem cells and hematopoiesis. Our findings identify NMNAT1 as a previously unidentified therapeutic target that maintains NAD+ for AML progression and chemoresistance.

Oncogenic mutations confer on cells the ability to propagate indefinitely, but whether oncogenes alter the cell fate of these cells is unknown. Here, we show that the transcriptional regulator PRDM16s causes oncogenic fate conversion by transforming cells fated to form platelets and erythrocytes into myeloid leukemia stem cells (LSCs). Prdm16s expression in megakaryocyte-erythroid progenitors (MEPs), which normally lack the potential to generate granulomonocytic cells, caused AML by converting MEPs into LSCs. Prdm16s blocked megakaryocytic/erythroid potential by interacting with super enhancers and activating myeloid master regulators, including PU.1. A CRISPR dropout screen confirmed that PU.1 is required for Prdm16s-induced leukemia. Ablating PU.1 attenuated leukemogenesis and reinstated the megakaryocytic/erythroid potential of leukemic MEPs in mouse models and human AML with PRDM16 rearrangement. Thus, oncogenic PRDM16s expression gives MEPs an LSC fate by activating myeloid gene regulatory networks.

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Alterations of the epigenetic landscape and transcription are hallmarks of acute myeloid leukemia (AML) that drive leukemogenic gene expression and therefore can be exploited for therapeutic intervention. To look for such targets that harbor both an altered epigenetic feature and are genetically essential for AML cells, we performed a multi-database analysis integrating pan-cancer super enhancer landscapes with whole genome CRISPR dropout screens. Among the top targets, we discovered SEPHS2. An enhancer was present upstream of the gene marked by H3K27ac and bound by leukemogenic transcription factors including MYB, Pu.1 and RUNX1. In addition, AML cells with SEPHS2 deletion significantly dropped out in a genome wide CRISPR screen. This gene encodes a critical enzyme in the underappreciated selenoprotein synthesis pathway which was highly upregulated in TCGA AML patients compared to control blood cells from healthy individuals. Collectively, our initial bioinformatic analysis suggested that the selenoprotein synthesis pathway is a new vulnerability in AML. To test the functional requirement of the selenoprotein synthesis pathway in AML and other cells, we performed CRISPR mediated deletion of three key genes in the selenoprotein synthesis pathway, SEPHS2, SEPSECS and EEFSEC. The human AML cell lines (MOLM13, THP1 and Kasumi1), murine AML cells transformed by MLL-AF9 and human AML PDX cells all depicted a significant dependency on these genes, while proliferation of normal cord blood cells and myeloma cells (U266B1) was almost not affected. We then transplanted these cells into recipients. Deletion of SEPHS2, SEPSECS or EEFSEC significantly ameliorated AML progression, indicated by decreased AML burden and extended survival. Since selenoproteins including GPX1 and GPX4 are known antioxidants, we hypothesized that perturbing the selenoprotein synthesis pathway disrupted the redox state in AML cells. We found that deletion of SEPHS2, SEPSECS or EEFSEC elevated ROS in AML cells as demonstrated by Cell-Rox or DCF-DA staining. Western blotting revealed significantly downregulated GPX4 level and upregulated DNA damage marker γ-H2AX. Moreover, the defective proliferation was partially rescued by adding antioxidant TEMPOL. These results suggested selenoprotein synthesis pathway produced key antioxidants to balance the proper redox state and was required for AML cell proliferation. A major source of selenium is diet. Therefore, we hypothesized that consuming selenium low diet could suppress AML. We compared the survival of AML bearing mice on selenium proficient and deficient diet. The selenium deficient diet significantly extended survival, lowered GPX4 level and increased ROS in AML cells. Interestingly, normal mouse on selenium deficient diet for a 3-months period did not develop any abnormalities in CBC or bone marrow hematopoiesis. This suggests selenium deficient diet could be clinically applicable without significant side effects. Altogether, the integration of a pan-cancer enhancer landscape study with CRISPR dropout gene screen offered a powerful tool to dissect cancer targets that possessed unique enhancer features and genetic essentiality. The analysis yielded SEPHS2 and its selenoprotein synthesis pathway to be a new vulnerability in AML. The underappreciated selenoprotein synthesis pathway was key to produce antioxidant selenoproteins such as GPX1 and GPX4 to maintain a proper redox state in AML. Deleting the genes or removing selenium from diet could perturb the pathway and ameliorate the AML disease. Disclosures Lin: Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

Maintaining metabolic homeostasis is a fundamental requirement for cells to survive. One critical requirement is to accomplish a balance between anabolism and catabolism. AMPK regulates this balance by directly sensing AMP-to-ATP ratio and its activation during cell energy deficit promotes ATP production and inhibits ATP usage. Several pieces of evidence point AMPK as a tumor suppressor: the upstream protein, LKB1, is a well-established tumor suppressor; AMPK negatively interacts with the tumor promoting mTOR pathway; anti-diabetic drugs such as metformin with tumor preventive potential are shown to activate AMPK. However, emerging evidence accumulates to support an opposite role of AMPK in promoting tumor growth. AMPK is found to protect tumor cells from metabolic crisis through different mechanisms: autophagy induction; maintaining proper ATP levels and redox environment; histone H2B tail phosphorylation. These two contrasting findings suggested multiple facets of AMPK, thus pointing to the urge to understand mechanisms of AMPK in specific contexts. We examined the role of AMPK by deleting both α1 and α2 subunit in a mouse model of acute myeloid leukemia with t(9;11) translocation. AMPK deficiency depletes the leukemia-initiating-cell population and decreases the leukemogenic potential of these cells. In order to study the metabolic regulatory effects of AMPK, we profiled the metabolites and found AMPK deficiency associates with a decreased level of glycolytic activity and reduction of acetyl-CoA, which is the major donor for histone acetylation. Therefore, we hypothesized that AMPK can affect histone acetylation through regulating the level of acetyl-CoA, and functionally alter leukemogenic potential. We first profiled histone acetylation using western blot with antibodies targeting global histone acetylation and specific histone residues. Intriguingly, we found in AMPK-deficient cells, global histone H3 and H4 acetylation levels are decreased, as well as acetylation at specific histone residues such as H3K9, H3K27 and H4K8. To build a causal link between decreased acetyl-CoA and histone acetylation, we supplemented leukemia-initiating-cells in culture with acetyl-CoA precursors such as acetate and pyruvate. The supplementation successfully increased intracellular acetyl-CoA levels as well as histone acetylation levels. Functionally restoring the intracellular acetyl-CoA and histone acetylation increased the proliferative potential of AMPK-deficient leukemia-initiating-cells and maintained cell at a more undifferentiated state. These results suggest that AMPK regulates a set of leukemogenic genes by maintaining histone acetylation levels. We hypothesize that AMPK links metabolic status to epigenetic gene regulation to promote leukemogenesis. Disclosures No relevant conflicts of interest to declare.