The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDH), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHTET2, but not IDH or TET2 AML. Gene sets characteristic for IDH AML were enriched in samples from patients with an IDHTET2BCAT1 status. BCAT1 AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHTET2 AML.

Extensive expression profiling studies have shown that sporadic breast cancer is composed of five clinically relevant molecular subtypes. However, although BRCA1-related tumours are known to be predominantly basal-like, there are few published data on other classes of familial breast tumours. We analysed a cohort of 75 BRCA1, BRCA2 and non-BRCA1/2 breast tumours by gene expression profiling and found that 74% BRCA1 tumours were basal-like, 73% of BRCA2 tumours were luminal A or B, and 52% non-BRCA1/2 tumours were luminal A. Thirty-four tumours were also analysed by single nucleotide polymorphism-comparative genomic hybridization (SNP-CGH) arrays. Copy number data could predict whether a tumour was basal-like or luminal with high accuracy, but could not predict its mutation class. Basal-like BRCA1 and basal-like non-BRCA1 tumours were very similar, and contained the highest number of chromosome aberrations. We identified regions of frequent gain containing potential driver genes in the basal (8q and 12p) and luminal A tumours (1q and 17q). Regions of homozygous loss associated with decreased expression of potential tumour suppressor genes were also detected, including in basal tumours (5q and 9p), and basal and luminal tumours (10q). This study highlights the heterogeneity of familial tumours and the clinical consequences for treatment and prognosis.

IntroductionMetastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.MethodsWe used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.ResultsMost brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.ConclusionsDeregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.

Mutations in the nucleophosmin 1 ( NPM1 ) gene are considered founder mutations in the pathogenesis of acute myeloid leukemia (AML). To characterize the genetic composition of NPM1 mutated ( NPM1 mut ) AML, we assess mutation status of five recurrently mutated oncogenes in 129 paired NPM1 mut samples obtained at diagnosis and relapse. We find a substantial shift in the genetic pattern from diagnosis to relapse including NPM1 mut loss ( n = 11). To better understand these NPM1 mut loss cases, we perform whole exome sequencing (WES) and RNA-Seq. At the time of relapse, NPM1 mut loss patients (pts) feature distinct mutational patterns that share almost no somatic mutation with the corresponding diagnosis sample and impact different signaling pathways. In contrast, profiles of pts with persistent NPM1 mut are reflected by a high overlap of mutations between diagnosis and relapse. Our findings confirm that relapse often originates from persistent leukemic clones, though NPM1 mut loss cases suggest a second “de novo” or treatment-associated AML (tAML) as alternative cause of relapse.

Introduction: The progression of Ductal Carcinoma in situ (DCIS) to Invasive Ductal