A Nuclear Phosphatase-Kinase Signaling Complex That Supports Acute Myeloid Leukemia

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. A tightly regulated balance between phosphorylation and dephosphorylation, sustained by coordinated and competing activities of phosphatases and kinases, lies at the basis of cellular signaling. Given the advances of the past two decades in designing potent and selective inhibitors against phosphatases and kinases, these phosphosignaling enzymes present some of the priority targets in AML. Here, we used domain-focused CRISPR screening to identify the nuclear phosphatase SCP4 as a dependency in AML. We provide evidence that this enzyme is likely dispensable in normal hematopoietic progenitor cells and could constitute a novel therapeutic target in leukemia. Using CRISPR exon scanning and gene complementation assays, we showed that the catalytic function of SCP4 was essential in AML. Our work, for the first time, elucidates a link between SCP4 and human cancer and provides a context for a deeper understanding of the molecular functions of this poorly studied phosphatase. Through mass spectrometry analysis of the SCP4 interactome, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. STK35/PDIK1L signaling roles and biochemical interactions are largely unknown, and this study sheds light on their involvement in leukemogenesis. We showed that STK35 and PDIK1L catalytic activity was required in AML. Moreover, these kinases function redundantly in the same pathway as SCP4 to maintain leukemia cell proliferation. We found that SCP4 regulated STK35/PDIK1L through two distinct mechanisms: promoting kinase stability and kinase catalytic activity. Our study provides the first genetic evidence that the conserved serine residue at the DFG+2 position could serve as the site of inhibitory phosphorylation at the kinases activation loop. Overall, our findings reveal a novel phosphatase-kinase signaling complex that supports the pathogenesis of AML

SCP4-STK35/PDIK1L complex is a dual phospho-catalytic signaling dependency in acute myeloid leukemia

Acute myeloid leukemia (AML) cells rely on phospho-signaling pathways to gain unlimited proliferation potential. Here, we use domain-focused CRISPR screening and identify the nuclear phosphatase SCP4 as a dependency in AML, yet this enzyme is dispensable in normal hematopoietic progenitor cells. Using CRISPR exon scanning and gene complementation assays, we show that the catalytic function of SCP4 is essential in AML. Through mass spectrometry analysis of affinity-purified complexes, we identify the kinase paralogs STK35 and PDIK1L as binding partners and substrates of the SCP4 phosphatase domain. We show that STK35 and PDIK1L function catalytically and redundantly in the same pathway as SCP4 to maintain AML proliferation and to support amino acid biosynthesis and transport. We provide evidence that SCP4 regulates STK35/PDIK1L through two distinct mechanisms: catalytic removal of inhibitory phosphorylation and by promoting kinase stability. Our findings reveal a phosphatase-kinase signaling complex that supports the pathogenesis of AML

SLC5A3-dependent myo-inositol auxotrophy in acute myeloid leukemia.

An enhanced requirement for nutrients is a hallmark property of cancer cells. Here, we optimized an in vivo genetic screening strategy in acute myeloid leukemia (AML), which led to the identification of the myo-inositol transporter SLC5A3 as a dependency in this disease. We demonstrate that SLC5A3 is essential to support a myo-inositol auxotrophy in AML. The commonality among SLC5A3-dependent AML lines is the transcriptional silencing of ISYNA1, which encodes the rate limiting enzyme for myo-inositol biosynthesis, inositol-3-phosphate synthase 1. We use gain- and loss-of-function experiments to reveal a synthetic lethal genetic interaction between ISYNA1 and SLC5A3 in AML, which function redundantly to sustain intracellular myo-inositol. Transcriptional silencing and DNA hyper-methylation of ISYNA1 occur in a recurrent manner in human AML patient samples, in association with IDH1/IDH2 and CEBPA mutations. Our findings reveal myo-inositol as a nutrient dependency in AML caused by the aberrant silencing of a biosynthetic enzyme

Transcriptional silencing of ALDH2 confers a dependency on Fanconi anemia proteins in acute myeloid leukemia.

Hundreds of genes become aberrantly silenced in acute myeloid leukemia (AML), with most of these epigenetic changes being of unknown functional consequence. Here, we demonstrate how gene silencing can lead to an acquired dependency on the DNA repair machinery in AML. We make this observation by profiling the essentiality of the ubiquitination machinery in cancer cell lines using domain-focused CRISPR screening, which revealed Fanconi anemia (FA) proteins UBE2T and FANCL as unique dependencies in AML. We demonstrate that these dependencies are due to a synthetic lethal interaction between FA proteins and Aldehyde Dehydrogenase 2 (ALDH2), which function in parallel pathways to counteract the genotoxicity of endogenous aldehydes. We show that DNA hypermethylation and silencing of ALDH2 occur in a recurrent manner in human AML, which is sufficient to confer FA pathway dependency. Our study suggests that targeting of the ubiquitination reaction catalyzed by FA proteins can eliminate ALDH2-deficient AML