A major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. ...Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.
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•Luminal breast tumors are auxotrophic for serine•Luminal serine auxotrophy is caused by lineage-specific hypermethylation of PSAT1•PSAT1 suppression preexists in the luminal tumor cells of origin•Serine auxotrophy sensitizes to dietary serine starvation
Many cancer types can synthesize serine de novo, which limits the effectiveness of dietary serine starvation. Choi et al. demonstrate that luminal breast tumors are auxotrophic for serine due to lineage-specific hypermethylation of the PSAT1 gene. This serine auxotrophy may be a targetable vulnerability of luminal breast tumors.
The non-essential amino acid serine is a critical nutrient for cancer cells due to its diverse biosynthetic functions. While some tumors can synthesize serine de novo, others are auxotrophic and ...therefore reliant on serine uptake. Importantly, despite several transporters being known to be capable of transporting serine, the transporters that mediate serine uptake in cancer cells are not known. Here, we characterize the amino acid transporter ASCT2 (SLC1A5) as a major contributor to serine uptake in cancer cells. ASCT2 is well known as a glutamine transporter in cancer, and our work demonstrates that serine and glutamine compete for uptake through ASCT2. We further show that ASCT2-mediated serine uptake is essential for purine nucleotide biosynthesis and that estrogen receptor α (ERα) promotes serine uptake by directly activating SLC1A5 transcription. Collectively, our work defines an additional important role for ASCT2 as a serine transporter in cancer and evaluates ASCT2 as a potential therapeutic target.
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•ASCT2 contributes to serine uptake in cancer cells•Serine and glutamine compete for uptake via ASCT2•ERα promotes serine uptake by directly activating SLC1A5 transcription•Loss of ASCT2 combined with dietary serine starvation shrinks xenograft tumors
Despite significant interest in targeting serine metabolism for cancer therapy, the transporters that mediate serine uptake in cancer cells are not known. Conger et al. describe the amino acid transporter ASCT2 as a major contributor to serine uptake in cancer cells that is required for tumor growth in low-serine conditions.
The non-physiological nutrient levels found in traditional culture media have been shown to affect numerous aspects of cancer cell physiology, including how cells respond to certain therapeutic ...agents. Here, we comprehensively evaluated how physiological nutrient levels impact therapeutic response by performing drug screening in human plasma-like medium (HPLM). We observed dramatic nutrient-dependent changes in sensitivity to a variety of FDA-approved and clinically trialed compounds including rigosertib, an experimental cancer therapeutic that has recently failed in phase 3 clinical trials. Mechanistically, we found that the ability of rigosertib to destabilize microtubules is strongly inhibited by the purine metabolism end product uric acid, which is uniquely abundant in humans relative to traditional in vitro and in vivo cancer models. These results demonstrate the broad and dramatic effects nutrient levels can have on drug response, and how incorporation of human-specific physiological nutrient media might help to identify compounds whose efficacy could be impacted in humans.
Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt ...signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in β-catenin expression. β-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6−/−), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production.
•Wnt signaling is essential for MK proliferation and maturation in addition to profoundly stimulating proplatelet formation.•These observations suggest that mature megakaryocytes may be able to respond to known Wnt gradients in the osteoblastic and vascular niches.
The number and volume of cells in the blood affect a wide range of disorders including cancer and cardiovascular, metabolic, infectious and immune conditions. We consider here the genetic variation ...in eight clinically relevant hematological parameters, including hemoglobin levels, red and white blood cell counts and platelet counts and volume. We describe common variants within 22 genetic loci reproducibly associated with these hematological parameters in 13,943 samples from six European population-based studies, including 6 associated with red blood cell parameters, 15 associated with platelet parameters and 1 associated with total white blood cell count. We further identified a long-range haplotype at 12q24 associated with coronary artery disease and myocardial infarction in 9,479 cases and 10,527 controls. We show that this haplotype demonstrates extensive disease pleiotropy, as it contains known risk loci for type 1 diabetes, hypertension and celiac disease and has been spread by a selective sweep specific to European and geographically nearby populations.
Platelet response to activation varies widely between individuals but shows interindividual consistency and strong heritability. The genetic basis of this variation has not been properly explored. We ...therefore systematically measured the effect on function of sequence variation in 97 candidate genes in the collagen and adenosine-diphosphate (ADP) signaling pathways. Resequencing of the genes in 48 European DNA samples nearly doubled the number of known single nucleotide polymorphisms (SNPs) and informed the selection of 1327 SNPs for genotyping in 500 healthy Northern European subjects with known platelet responses to collagen-related peptide (CRP-XL) and ADP. This identified 17 novel associations with platelet function (P < .005) accounting for approximately 46% of the variation in response. Further investigations with platelets of known genotype explored the mechanisms behind some of the associations. SNPs in PEAR1 associated with increased platelet response to CRP-XL and increased PEAR1 protein expression after platelet degranulation. The minor allele of a 3′ untranslated region (UTR) SNP (rs2769668) in VAV3 was associated with higher protein expression (P = .03) and increased P-selectin exposure after ADP activation (P = .004). Furthermore the minor allele of the intronic SNP rs17786144 in ITPR1 modified Ca2+ levels after activation with ADP (P < .004). These data provide novel insights into key hubs within platelet signaling networks.
Within the healthy population, there is substantial, heritable, and interindividual variability in the platelet response. We explored whether a proportion of this variability could be accounted for ...by interindividual variation in gene expression. Through a correlative analysis of genome-wide platelet RNA expression data from 37 subjects representing the normal range of platelet responsiveness within a cohort of 500 subjects, we identified 63 genes in which transcript levels correlated with variation in the platelet response to adenosine diphosphate and/or the collagen-mimetic peptide, cross-linked collagen-related peptide. Many of these encode proteins with no reported function in platelets. An association study of 6 of the 63 genes in 4235 cases and 6379 controls showed a putative association with myocardial infarction for COMMD7 (COMM domain-containing protein 7) and a major deviation from the null hypo thesis for LRRFIP1 leucine-rich repeat (in FLII) interacting protein 1. Morpholino-based silencing in Danio rerio identified a modest role for commd7 and a significant effect for lrrfip1 as positive regulators of thrombus formation. Proteomic analysis of human platelet LRRFIP1-interacting proteins indicated that LRRFIP1 functions as a component of the platelet cytoskeleton, where it interacts with the actin-remodeling proteins Flightless-1 and Drebrin. Taken together, these data reveal novel proteins regulating the platelet response.
Abstract
Liquid biopsy for profiling of cell free DNA (cfDNA) in blood holds huge promise to transform how we experience and manage cancer by early detection and identification of residual disease ...and subtype. However, a standard blood draw yields an average of only 10 ng of cfDNA, of which DNA derived from the tumor is a small minority. Therefore, we are faced with a dilemma when utilizing the limited sample to obtain maximum information. Genetic sequencing provides information on actionable somatic mutations but detection of a few loci in a minority of the sample is challenging. Modified cytosine profiles of cancer are differential from non-cancer at many more loci and so provide a stronger signal. Moreover, they can be used to distinguish tissue-of-origin of the tumor. However, methods such as bisulfite sequencing, EM-seq and TAPS sacrifice genetic information (namely C->T mutations, which are the most common mutation in cancer) to measure modified cytosine. Genetic and modified cytosine data together have been shown to be more powerful for the detection of early cancer than either alone. Here we present a technology, which sequences at base resolution the complete genetic sequence integrated with modified cytosine from low nanogram amounts of cfDNA. It consists of (i) a single pre-sequencing workflow, which creates a copy of the original DNA and performs enzymatic base conversions which discriminate genetic and epigenetic states and (ii) post-sequencing data processing which resolves the resultant sequencing data to an information-rich 16-state code and derives genetic variants integrated with modified cytosine levels, within easy-to-use software. It is, in principle, compatible with any sequencing methodology and is shown here optimized for the Illumina fleet. In cfDNA we show accuracy of modC measurement is higher than that of bisulfite sequencing and EM-seq; and the accuracy of genetic sequencing is higher than that of Illumina alone. Accuracy of measurement is extremely important for liquid biopsy where tumor DNA is in the minority and observed in a small number of reads. We demonstrate the impact of varying base calling error rate on limit of detection for rare alleles in cfDNA samples. Further we show derivation of cfDNA fragment characteristics and that this is produced in combination with genetic and modC information. This further increases the signal available from cfDNA in only one workflow. We suggest this method will help advance the field of liquid biopsy towards its promise.
Citation Format: Fabio Puddu, Casper K. Lumby, Nick Harding, David J. Morley, Jamie Scotcher, Robert Crawford, Jens Füllgrabe, Walraj S. Gosal, Shirong Yu, Daniel Brudzewsky, Jane Haywood, Andrada Tomoni, Philippa Burns, Joanna D. Holbrook, Paidi Creed. Refining liquid biopsy: generating more information from cell free DNA abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6602.
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