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Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras -mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC. Colorectal tumors with mutated KRAS and APC require the amino acid transporter SLC7A5 to drive tumorigenesis. Mechanistically, SLC7A5 drives transcriptional and metabolic reprogramming by maintaining intracellular amino acid levels, leading to enhanced protein synthesis.

The genomic landscape of colorectal cancer (CRC) is shaped by inactivating mutations in tumour suppressors such as APC , and oncogenic mutations such as mutant KRAS . Here we used genetically engineered mouse models, and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that untargeted metabolic profiling can be applied to stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-deficient CRC. Loss of Apc in the mouse intestine was found to be sufficient to drive expression of one of its enzymes, adenosylhomocysteinase (AHCY), which was also found to be transcriptionally upregulated in human CRC. Targeting of AHCY function impaired growth of APC-deficient organoids in vitro, and prevented the characteristic hyperproliferative/crypt progenitor phenotype driven by acute deletion of Apc in vivo, even in the context of mutant Kras . Finally, pharmacological inhibition of AHCY reduced intestinal tumour burden in Apc Min/+ mice indicating its potential as a metabolic drug target in CRC. In this study, Vande Voorde et al. investigate the potential of untargeted metabolomics as a stratification tool for colorectal cancer (CRC). They present a comprehensive pipeline to uncover metabolic vulnerabilities in CRC based on its genetic origin. With this approach, they show perturbations in methionine metabolism linked to APC deficiency, and identify adenosylhomocysteinase as an actionable therapeutic target.

With colorectal cancer (CRC) being the second most common cause of cancer-related deaths worldwide, there is an urgent need for better diagnostic tools and new, more targeted therapies. Here we used genetically engineered mouse models (GEMMs), and multimodal mass spectrometry-based metabolomics to study the impact of common genetic drivers of CRC on the metabolic landscape of the intestine. We show that unsupervised metabolic profiling can stratify intestinal tissues according to underlying genetic alterations, and use mass spectrometry imaging (MSI) to identify tumour, stromal and normal adjacent tissues. By identifying ions that drive variation between normal and transformed tissues, we found dysregulation of the methionine cycle to be a hallmark of APC-mutant CRC, and propose one of its enzymes, i.e. adenosylhomocysteinase (AHCY), as a new therapeutic target. Collectively, we show that the profound genotype-dependent alterations in both lipid and small molecule metabolism in CRC may be exploited for tissue classification with no need for ion identification, and we applied further data analysis to expose a novel metabolic vulnerability of CRC.Competing Interest StatementO.D.K.M. is a co-founder, shareholder and board member of Faeth Therapeutics Inc.

Four different histone proteins comprise the octameric histone core, a key component of DNA compaction into chromatin. The N-terminal tails of each histone protein contain a variety of post-translational modifications that can help modulate gene expression. Mutations are rare in these key proteins, but when found they are often linked to very serious and lethal diseases. In 2012, mutations in histone genes HIST1H2B and H3F3A were found to be implicated in brain cancer. The protein products of these genes produced four point mutants in two proteins: H3.1K27M, H3.3K27M, and H3.3G34R/V. The positions of these mutations are located at or adjacent to known sites of post-translational modification. Trimethylation of H3K27 is a known mark of gene repression and tumors harboring the K27M mutation have been found to have globally reduced levels of this mark. G34R/V mutations have been shown to produce locally reduced levels of H3K36 trimethylation as well. H3K36 trimethylation has been tied to transcription regulation and DNA repair. While these mutations are clearly disrupting the histone post-translational landscape they may also be perturbing the nucleosome structure itself. Histone proteins interact with DNA through basic residues. As these mutations are directly changing the basic residue content of the proteins, DNA-histone interactions may be altered. Research examining these mutations thus far have focused on secondary interactions between protein complexes and the nucleosome. No studies have examined how these mutations and changes in post-translational modifications could be effecting overall nucleosome structure. The main purpose of this research was to develop a system to examine the effects that these mutations have on nucleosome structure. To address this, Aim 1 of the project involved cloning eleven genes to produce the four canonical histone proteins (H2A, H2B, H3, and H4), an H3 variant (H3.3), three H3.3 point mutants (H3.3 K27M, H3.3 G34R, H3.3 G34V), one tailless H3.1 mutant (H3.1 Δ5), and two tailless H3.3 mutants (H3.3 Δ32 and H3.3 Δ45). A new, 2-step purification method was developed for the simple and inexpensive purification of histone proteins. Purified histones were then reconstituted into nucleosomes using a salt-gradient method. Four nucleosome constructs were reconstituted, differing only in the H3 protein included (H3.1, H3.3, H3.3 K27M, or H3.3 Δ32). Atomic Force Microscopy images of the four nucleosome constructs were acquired and analyzed. The location of these histone mutations is at or adjacent to known sites of post-translational modification. Due to this, it was necessary to determine how modifications at these sites effected the nucleosome structure as well to get a full scope of the impact of the mutations. Studying individual post-translational modifications is difficult as extracting histone proteins from tissue samples produces a heterogeneous population of modifications. To generate a homogenous population of modified proteins native chemical ligation methods were explored with the goal of producing histone proteins containing site-specific modifications. Preliminary ligation studies successfully created peptide dimers and trimers through a salicylaldehyde ester ligation technique.

The colocation of elemental species with host biomolecules such as lipids and metabolites may shed new light on the dysregulation of metabolic pathways and how these affect disease pathogeneses. Alkali metals have been the subject of extensive research, are implicated in various neurodegenerative and infectious diseases and are known to disrupt lipid metabolism. Desorption electrospray ionisation (DESI) is a widely used approach for molecular imaging, but previous work has shown that DESI delocalises ions such as potassium (K) and chlorine (Cl), precluding the subsequent elemental analysis of the same section of tissue. The solvent typically used for the DESI electrospray is a combination of methanol and water. Here we show that a novel solvent system, (50:50 (%v/v) MeOH:EtOH) does not delocalise elemental species and thus enables elemental mapping to be performed on the same tissue section post-DESI. Benchmarking the MeOH:EtOH electrospray solvent against the widely used MeOH:H2O electrospray solvent revealed that the MeOH:EtOH solvent yielded increased signal-to-noise ratios for selected lipids. The developed multimodal imaging workflow was applied to a lung tissue section containing a tuberculosis granuloma, showcasing its applicability to elementally rich samples displaying defined structural information.

Abstract High dimensionality omics and hyperspectral imaging datasets present difficult challenges for feature extraction and data mining due to huge numbers of features that cannot be simultaneously examined. The sample numbers and variables of these methods are constantly growing as new technologies are developed, and computational analysis needs to evolve to keep up with growing demand. Current state of the art algorithms can handle some routine datasets but struggle when datasets grow above a certain size. We present a training deep learning via neural networks on non-linear dimensionality reduction, in particular t-distributed stochastic neighbour embedding (t-SNE), to overcome prior limitations of these methods. One Sentence Summary Analysis of prohibitively large datasets by combining deep learning via neural networks with non-linear dimensionality reduction. Competing Interest Statement The authors have declared no competing interest.