Arno Velds

Metastatic breast cancer represents a major societal burden and is a main cause of cancer-related death in women, with limited rationale-based treatment options and often dismal outcomes. Intra-tumor heterogeneity, tumor-stromal interactions and clonal selection are considered to play a major role in metastasis formation and disease progression, but these processes remain incompletely understood. Leveraging spatial transcriptomics as cost-effective method for generating transcriptomics data at single-cell resolution, we present a single-cell spatial transcriptomics atlas of 126 tumor samples from 44 metastatic breast cancer patients, encompassing 520,850 cells from primary and metastatic lesions, with 171,766 cancer cells and 349,084 cells belonging to the tumor microenvironment. For most patients, multiple time-points of sample collection throughout metastatic disease progression were analyzed, allowing for detection of treatment-induced transcriptional changes driving therapy resistance within the tumor cell compartment. Distinct immune cell subpopulations were enriched upon progression following specific therapeutics and were predictive for hormone receptor loss. By inferring DNA copy number alterations from the single-cell transcriptomics data, we tracked subclonal tumor cell selection upon progression and identified critical transcriptomic features of outcompeting clones with future metastatic potential. Cumulatively, we present a unique single-cell spatial framework on metastatic breast cancer development and progression, in a highly complex systemic treatment landscape.

Arginine biosynthesis is frequently suppressed in cancer because of the loss of argininosuccinate synthase 1 (ASS1) expression, rendering cancer cells reliant on extracellular arginine. This feature has driven the development of systemic arginine-depleting strategies, which are clinically safe but offer limited clinical benefit. In this study, we demonstrated that under arginine scarcity, cancer cells with low ASS1 expression resort to aberrant mRNA translation, characterized by ribosomal frameshifts and amino acid misincorporations. Although aberrant proteins originated from most arginine codons, the predominant effect was observed at AGA. This codon preference was caused by a selective decrease in tRNAArg (UCU) levels following arginine deprivation, linked to methyltransferase-like 1 (METTL1)–mediated tRNA modification. Proteomics and immunopeptidomics analyses validated that arginine shortage induced aberrant protein production at the endogenous level. T-cell receptor (TCR) T cells that specifically recognize these HLA-presented mistranslated peptides efficiently killed cancer cells after arginine deprivation. These results lay the foundation for improved cancer therapies by combining systemic arginine-depleting strategies with TCR-based targeting of nonclassical neoantigens. Significance: Aberrant protein production induced by arginine deprivation in ASS1-low cancers leads to production of neoantigens that represent promising targets for TCR-T cell therapies.

Perturbation of protein phosphorylation represents an attractive approach to cancer treatment. Besides kinase inhibitors, protein phosphatase inhibitors have been shown to have anti-cancer activity. A prime example is the small molecule LB-100, an inhibitor of protein phosphatases 2A/5 (PP2A/PP5), enzymes that affect cellular physiology. LB-100 has proven effective in pre-clinical models in combination with immunotherapy, but the molecular underpinnings of this synergy remain understood poorly. We report here a sensitivity of the mRNA splicing machinery to phosphorylation changes in response to LB-100 in colorectal adenocarcinoma. We observe enrichment for differentially phosphorylated sites within cancer-critical splicing nodes of U2 snRNP, SRSF and hnRNP proteins. Altered phosphorylation endows LB-100-treated colorectal adenocarcinoma cells with differential splicing patterns. In PP2A-inhibited cells, over 1000 events of exon skipping and intron retention affect regulators of genomic integrity. Finally, we show that LB-100-evoked alternative splicing leads to neoantigens that are presented by MHC class 1 at the cell surface. Our findings provide a potential explanation for the pre-clinical and clinical observations that LB-100 sensitizes cancer cells to immune checkpoint blockade.

Summary: While neoantigen depletion, a form of immunoediting due to Darwinian pressure exerted by the T cell based immune system during tumor evolution, has been clearly described in murine models, its prevalence in treatment-naive, developing human tumors remains controversial. We developed two novel methodologies to test for depletion of predicted neoantigens in patient cohorts, which both compare patients in terms of their expected number of neoantigens per mutational event. Application of these strategies to TCGA patient cohorts showed that neither basic nor more extensive versions of the methodologies, controlling for confounding factors such as genomic loss of the HLA locus, provided statistically significant evidence for neoantigen depletion. In the subset of analyses that did show a trend towards neoantigen depletion, statistical significance was not reached and depletion was not consistently observed across HLA alleles. Our results challenge the notion that neoantigen depletion is detectable in cohorts of unmatched patient samples using HLA binding prediction-based methodology.

Ageing is associated with changes in the cellular composition of the immune system. During ageing, hematopoietic stem and progenitor cells (HSPCs) that produce immune cells are thought to decline in their regenerative capacity. However, HSPC function has been mostly assessed using transplantation assays, and it remains unclear how HSPCs age in the native bone marrow niche. To address this issue, we present an in situ single cell lineage tracing technology to quantify the clonal composition and cell production of single cells in their native niche. Our results demonstrate that a pool of HSPCs with unequal output maintains myelopoiesis through overlapping waves of cell production throughout adult life. During ageing, the increased frequency of myeloid cells is explained by greater numbers of HSPCs contributing to myelopoiesis rather than the increased myeloid output of individual HSPCs. Strikingly, the myeloid output of HSPCs remains constant over time despite accumulating significant transcriptomic changes throughout adulthood. Together, these results show that, unlike emergency myelopoiesis post-transplantation, aged HSPCs in their native microenvironment do not functionally decline in their regenerative capacity.

Discovering biomarkers of drug response and finding powerful drug combinations can support the reuse of previously abandoned cancer drugs in the clinic. Indisulam is an abandoned drug that acts as a molecular glue, inducing degradation of splicing factor RBM39 through interaction with CRL4^DCAF15. Here, we performed genetic and compound screens to uncover factors mediating indisulam sensitivity and resistance. First, a dropout CRISPR screen identified SRPK1 loss as a synthetic lethal interaction with indisulam that can be exploited therapeutically by the SRPK1 inhibitor SPHINX31. Moreover, a CRISPR resistance screen identified components of the degradation complex that mediate resistance to indisulam: DCAF15, DDA1, and CAND1. Last, we show that cancer cells readily acquire spontaneous resistance to indisulam. Upon acquiring indisulam resistance, pancreatic cancer (Panc10.05) cells still degrade RBM39 and are vulnerable to BCL-xL inhibition. The better understanding of the factors that influence the response to indisulam can assist rational reuse of this drug in the clinic.

Mouse xenografts from (patient-derived) tumors (PDX) or tumor cell lines are widely used as models to study various biological and preclinical aspects of cancer. However, analyses of their RNA and DNA profiles are challenging, because they comprise reads not only from the grafted human cancer but also from the murine host. The reads of murine origin result in false positives in mutation analysis of DNA samples and obscure gene expression levels when sequencing RNA. However, currently available algorithms are limited and improvements in accuracy and ease of use are necessary. We developed the R-package XenofilteR, which separates mouse from human sequence reads based on the edit-distance between a sequence read and reference genome. To assess the accuracy of XenofilteR, we generated sequence data by in silico mixing of mouse and human DNA sequence data. These analyses revealed that XenofilteR removes > 99.9% of sequence reads of mouse origin while retaining human sequences. This allowed for mutation analysis of xenograft samples with accurate variant allele frequencies, and retrieved all non-synonymous somatic tumor mutations. XenofilteR accurately dissects RNA and DNA sequences from mouse and human origin, thereby outperforming currently available tools. XenofilteR is open source and available at https://github.com/PeeperLab/XenofilteR .

Background: Chromosomal instability (CIN) is a common trait of cancer characterised by the continuous gain and loss of chromosomes during mitosis. Excessive levels of CIN can suppress tumour growth, providing a possible therapeutic strategy. The Mps1/TTK kinase has been one of the prime targets to explore this concept, and indeed Mps1 inhibitors synergise with the spindle poison docetaxel in inhibiting the growth of tumours in mice. Methods: To investigate how the combination of docetaxel and a Mps1 inhibitor (Cpd-5) promote tumour cell death, we treated mice transplanted with BRCA1^−/−;TP53^−/− mammary tumours with docetaxel and/or Cpd-5. The tumours were analysed regarding their histopathology, chromosome segregation errors, copy number variations and cell death to understand the mechanism of action of the drug combination. Results: The enhanced efficacy of combining an Mps1 inhibitor with clinically relevant doses of docetaxel is associated with an increase in multipolar anaphases, aberrant nuclear morphologies and cell death. Tumours treated with docetaxel and Cpd-5 displayed more genomic deviations, indicating that chromosome stability is affected mostly in the combinatorial treatment. Conclusions: Our study shows that the synergy between taxanes and Mps1 inhibitors depends on increased errors in cell division, allowing further optimisation of this treatment regimen for cancer therapy.

Mutations in Fanconi Anemia or Homologous Recombination (FA/HR) genes can cause DNA repair defects and could therefore impact cancer treatment response and patient outcome. Their functional impact and clinical relevance in head and neck squamous cell carcinoma (HNSCC) is unknown. We therefore questioned whether functional FA/HR defects occurred in HNSCC and whether they are associated with FA/HR variants. We assayed a panel of 29 patient-derived HNSCC cell lines and found that a considerable fraction is hypersensitive to the crosslinker Mitomycin C and PARP inhibitors, a functional measure of FA/HR defects. DNA sequencing showed that these hypersensitivities are associated with the presence of bi-allelic rare germline and somatic FA/HR gene variants. We next questioned whether such variants are associated with prognosis and treatment response in HNSCC patients. DNA sequencing of 77 advanced stage HNSCC tumors revealed a 19% incidence of such variants. Importantly, these variants were associated with a poor prognosis (p = 0.027; HR = 2.6, 1.1-6.0) but favorable response to high cumulative cisplatin dose. We show how an integrated in vitro functional repair and genomic analysis can improve the prognostic value of genetic biomarkers. We conclude that repair defects are marked and frequent in HNSCC and are associated with clinical outcome.