Identification and characterisation of a novel GHR defect disrupting the polypyrimidine tract and resulting in GH insensitivity

Objective GH insensitivity (GHI) is caused in the majority of cases by impaired function of the GH receptor (GHR). All but one known GHR mutation are in the coding sequence or the exon/intron boundaries. We identified and characterised the first intronic defect occurring in the polypyrimidine tract of the GHR in a patient with severe GHI. Design We investigated the effect of the novel defect on mRNA splicing using an in vitro splicing assay and a cell transfection system. Methods GHR was analysed by direct sequencing. To assess the effect of the novel defect, two heterologous minigenes (wild-type and mutant L1-GHR8-L2) were generated by inserting GHR exon 8 and its flanking wild-type or mutant intronic sequences into a well-characterised splicing reporter (Adml-par L1–L2). 32P-labelled pre-mRNA was generated from the two constructs and incubated in HeLa nuclear extracts or HEK293 cells. Results Sequencing of the GHR revealed a novel homozygous defect in the polypyrimidine tract of intron 7 (IVS7-6T>A). This base change does not involve the highly conserved splice site sequences, and is not predicted in silico to affect GHR mRNA splicing. Nevertheless, skipping of exon 8 from the mutant L1-GHR8-L2 mRNA was clearly demonstrated in the in vitro splicing assay and in transfected HEK293 cells. Conclusion Disruption of the GHR polypyrimidine tract causes aberrant mRNA splicing leading to a mutant GHR protein. This is predicted to lack its transmembrane and intracellular domains and, thus, be incapable of transducing a GH signal

Adipocytes disrupt the translational programme of acute lymphoblastic leukaemia to favour tumour survival and persistence

The specific niche adaptations that facilitate primary disease and Acute Lymphoblastic Leukaemia (ALL) survival after induction chemotherapy remain unclear. Here, we show that Bone Marrow (BM) adipocytes dynamically evolve during ALL pathogenesis and therapy, transitioning from cellular depletion in the primary leukaemia niche to a fully reconstituted state upon remission induction. Functionally, adipocyte niches elicit a fate switch in ALL cells towards slow-proliferation and cellular quiescence, highlighting the critical contribution of the adipocyte dynamic to disease establishment and chemotherapy resistance. Mechanistically, adipocyte niche interaction targets posttranscriptional networks and suppresses protein biosynthesis in ALL cells. Treatment with general control nonderepressible 2 inhibitor (GCN2ib) alleviates adipocyte-mediated translational repression and rescues ALL cell quiescence thereby significantly reducing the cytoprotective effect of adipocytes against chemotherapy and other extrinsic stressors. These data establish how adipocyte driven restrictions of the ALL proteome benefit ALL tumours, preventing their elimination, and suggest ways to manipulate adipocyte-mediated ALL resistance

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens. Keywords: AML; RUNX1; CKMT1; cyclocreatine; arginine metabolismNational Cancer Institute (U.S.) (NIH 1R35 CA210030-01)Stand Up To CancerBridge ProjectNational Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Grant P30-CA14051