PSPC1 bridges cancer stemness and malignancy in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry. We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation. Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.

PSPC1 exerts an oncogenic role in AML by regulating a leukemic transcription program in cooperation with PU.1.

Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the accumulation of malignant myeloid cells that have arrested maturation. Most therapeutic regimens approved or under development are cytotoxics. An alternate, but less explored therapeutic approach, is to induce terminal differentiation of AML cells. Upon differentiation, AML cells cease to proliferate or die. Phosphatidylserine decarboxylase (PISD) is a mitochondrial enzyme that converts phosphatidylserine (PS) to phosphatidylethanolamine (PE). Here, we explored the effects of inhibiting PISD on AML growth, stemness and differentiation. Knockout of PISD by CRISPR reduced the growth and clonogenic growth of OCI-AML2 cells. The reported chemical PISD inhibitor, 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine (aka: MMV007285), reduced growth and viability of OCI-AML2 cells (IC50 = 4.741 μM) and TEX cells (IC50 = 4.868 μM). Using the 8227 primary AML cell culture model, we showed that inhibiting PISD induced cell death in the functionally defined stem cell fraction (CD34+CD38-). MMV007285 also preferentially inhibited the clonogenic growth of primary AML cells (n = 7) over normal hematopoietic cells (n= 3). Moreover, MMV007285 induced AML cell differentiation as evidenced by increased CD11b expression and staining for non-specific esterase. Using high-performance thin layer chromatography (HPTLC), we found that inhibition of PISD with MMV007285 increased intracellular PS. To determine whether increased PS was functionally important, OCI-AML2 cells were treated with PS, resulting in reduced growth and clonogenic growth. Furthermore, PS supplementation targeted AML progenitor cells as it decreased engraftment of TEX cells in mice. Mechanistically, inhibiting PISD induced differentiation and decreased stemness in AML by activating Toll-like receptor (TLR) signaling. Specifically, inhibiting PISD upregulated TLR4 and 8 expression and increased expression of cytokines downstream of TLR activation. We also showed that TLR activation was functionally important to induce AML differentiation. Finally, we evaluated the effects of PISD inhibition in AML mouse models. MMV007285 (300 mg/kg/5 of 7 days orally for 10 days) decreased the growth of OCI-AML2 cells in SCID mice. Moreover, MMV007285 (150 mg/kg/5 of 7 days orally for 5 weeks) impeded the leukemic engraftment of primary AML cell in NOD/SCID mice without toxicity. Using secondary transplants, we showed that MMV007285 also targeted the leukemic stem cells. Taken together, inhibition of PISD altered phospholipid metabolism, inhibited growth and stemness, and increased differentiation in AML cells. Our findings reveal a previously undescribed link between mitochondrial phospholipid metabolism and AML stemness and differentiation, highlighting a potential new therapeutic strategy for AML. Citation Format: Mingjing Xu, Ayesh Seneviratne, Val A. Fajardo, Geethu E. Thomas, G. Wei Xu, Rose Hurren, S. Kim, Neil MacLean, Xiaoming Wang, Marcela Gronda, Danny Jeyaraju, Yulia Jitkova, David Sharon, Ahmed Aman, Rima Al-awar, Steven Chan, Mark D. Minden, Paul LeBlanc, Aaron D. Schimmer. Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3003.

Inhibition of Mitochondrial Translation As a Therapeutic Strategy for Acute Myeloid Leukemia (AML)

IPO11 Regulates the Nuclear Import of BZW1/2 and Is Necessary for AML Cells and Stem Cells

Inhibition of Drp1, a mitochondrial fission GTPase disrupts mitochondrial quality control and impairs stem-like properties in Acute Myeloid Leukemia

CD99 Is Highly Expressed in Acute Myeloid Leukemia (AML) and Presents a Viable Therapeutic Target

Src Kinase Inhibition by Dasatinib Enhances Targeting of Human AML Stem/Progenitor Cells by Chemotherapeutic Agents

Selective Targeting Of Inv(16)+ AML Stem Progenitor Cells By Inhibiting HDAC8

CD99 Is a Therapeutic Target On Disease Stem Cells In Acute Myeloid Leukemia and The Myelodysplastic Syndromes

Mitochondrial Dysfunction in Normal and Malignant Hematopoiesis

The Composition of Acute Myeloid Leukemia Cell Differentiation States Predicts Response to Immune Checkpoint Blockade

Inhibition of BMP-Smad Pathway Reduces Leukemic Stemness in Pediatric AML

Targeted therapies, such as the BCL-2 inhibitor venetoclax, have expanded the treatment options for patients with acute myeloid leukemia (AML), but survival remains poor because of drug resistance and disease relapse. We found that the translation initiation factor EIF4A1, which unwinds complex messenger RNA structures in the 5' untranslated region (UTR) of oncogenic transcripts, is highly expressed in AML stem- and progenitor-like cells relative to healthy hematopoietic stem and progenitor cells. Inhibition of eukaryotic initiation factor 4A (eIF4A) with the first-in-class small molecule zotatifin reduces the translation efficiency of transcripts related to the cell cycle and oncogenic signaling via the PI3K/AKT/mTOR pathway, as shown by ribosome profiling and gene set enrichment analysis. Western blot analysis corroborated these findings and demonstrated the downregulation of AKT, STAT-5, and MCL-1, factors implicated in resistance to venetoclax-based regimens. The combination of zotatifin and venetoclax synergistically kills AML cells in vitro and induces apoptosis across AML genotypes with selectivity toward progenitor-like cells in primary AML bone marrow (BM); however, its effect in primary healthy BM is limited. Using 3 in vivo xenograft models derived from patients with relapsed/refractory AML, the combination significantly suppressed the tumor burden and prolonged survival. These results support eIF4A-mediated protein translation as a therapeutic target in AML and highlight the potential of zotatifin and venetoclax in relapsed/refractory disease.

The Determination of Acute Myeloid Leukemia Stem Cell (AML LSC) in Childhood Acute Leukemia and Its Clinical Significance