Tafazzin (TAZ) Regulates the Differentiation of AML Cells By Reducing Levels of the Phospholipid Phosphatidylethanolamine
Authors: Seneviratne Ayesh K , Aristizabal Henao Juan J , Xu G Wei , Hurren Rose , Kim Sejin , MacLean Neil , Wang Xioaming , Xu Mingjing , Fajado Val , Hao Zhenyue , Gronda Marcela , Jeyaraju Danny V. , Chouinard-Watkins Raphael , Schafer Caitlin , LeBlanc Paul , Zaza Khuchua , Bazinet Richard , Claypool Steven , Stark Ken , Schimmer Aaron 
Published: April 2021

Abstract
AML cells have unique mitochondrial characteristics with an increased reliance on mitochondrial metabolism and oxidative phosphorylation. To identify new biological vulnerabilities in the mitochondria of AML, we conducted a CRISPR knockout screen. CAS9-overexpressing human OCI-AML2 leukemia cells were transduced with a library of 91,320 sgRNAs in barcoded lentiviral vectors targeting 17,237 nuclear-encoded genes. Cells were harvested, genomic DNA was isolated, and the relative abundance of sgRNAs were determined by sequencing barcodes 14 days after puromycin selection. We focused on the sgRNAs targeting the 1050 mitochondrial proteins to identify targets in the mitochondrial proteome whose knockout reduced AML growth and viability.
The cardiolipin remodeling enzyme tafazzin (TAZ) was among the top 1% of mitochondrial hits. Using individual sgRNA, we confirmed that knockout of TAZ reduced the growth of CAS9-OCI-AML2 cells by &gt;70%, thus validating the findings from our screen. We also knocked down TAZ with 2 independent shRNA and demonstrated reductions in growth and viability of a panel of AML cells: OCI-AML2 (&gt;80%), TEX (&gt;50%), K562 (&gt;50%), and U937 (&gt;40%). Moreover, TAZ knockdown significantly reduced the engraftment of TEX leukemia cells in vivo by 80%, indicating that TAZ-knockdown reduces AML growth in vivo and can target leukemia initiating cells. In contrast, knockdown of TAZ in mouse models did not impair normal hematopoiesis nor reduced the abundance of hematopoietic stem cells, although more subtle defects in the hematopoietic stem cells might explain transient episodes of neutropenia seen in Barth's syndrome, a congenital condition associated with X-linked TAZ mutations.
TAZ is responsible for the majority of Cardiolipin (CL) remodeling under physiological conditions. As expected the knockdown of TAZ in both AML and normal mouse hematopoietic cells increased the substrate (monlysocardiolipin) to product (CL) ratio of TAZ. CL is required for the proper localization, and efficient function of, respiratory chain enzymes. However, in AML cells, knockdown of TAZ did not alter respiratory chain complex activity, basal oxygen consumption, or respiratory chain reserve capacity.
Recent studies have shown that mitochondrial pathways can regulate cell fate and differentiation independent of their effects on oxidative phosphorylation. Therefore, we examined changes in AML cell differentiation after TAZ knockdown. Knockdown of TAZ promoted the differentiation of AML cells as evidenced by increased non-specific esterase staining and increased CD11b expression on the cell surface.
In breast cancer cells decreasing phosphatidylethanolamine (PE) levels, induced the differentiation of these cells. As TAZ regulates phospholipid remodeling, therefore we measured levels of PE and phosphatidylserine (PS) after TAZ-knockdown by spot densitometry. Interestingly, knockdown of TAZ in OCI-AML2 cells decreased PE and increased PS lipid levels. To determine whether alterations in PE and PS phospholipids are functionally important for differentiation of AML cells, we treated AML cells with MMV007285, an inhibitor of the phosphatidylserine decarboxylase (PISD), an enzyme that converts PS to PE. MMV007285 mimicked the effects of TAZ-knockdown and increased differentiation of OCI-AML2 and 8227 AML cells.
In summary, the cardiolipin remodeling enzyme TAZ regulates the differentiation of AML cells by controlling levels of PS and PE, thereby highlighting a new mechanism by which phospholipids and mitochondrial enzymes regulate AML cell fate and differentiation. Moreover, PISD inhibition may be a novel therapeutic strategy to selectively promote the differentiation of AML.

Disclosures
No relevant conflicts of interest to declare.