SnoRNAs are highly expressed in AML and have implications in leukemogenesis and leukemic maintenance. SnoRNAs can be further processed into snoRNA-derived RNAs (sdRNAs). The role of sdRNAs in AML and healthy hematopoiesis remains largely elusive. We characterized sdRNA and snoRNA levels in hematopoietic stem and progenitor cells (HSPCs), healthy WBCs, and 159 intensively treated AML patient samples at initial diagnosis. HSPCs, healthy WBCs, and AML blasts could be differentiated by their sdRNA expression pattern in a cell-type-specific manner. In AML, high sd3’-RNA/snoRNA-host gene ratios were associated with an inverse patient outcome. Particularly, in NPM1-mutated patients with favorable risk stratification and good initial therapy response, high sd3’-RNA ratios identified a subgroup with inferior outcome. High sd3’-RNA ratios were associated with altered oncogenic, inflammatory, and immune response signaling. Forced expression of single sdRNAs, such as sd3’-SNORD78, sd3’-SNORD76, and sd5’-SNORD93, enhanced clonogenic potential in AML and drove sdRNA-specific gene expression signatures in both AML and healthy HSPCs. Exemplarily, we propose and characterize NUDT21, an important regulator of alternative polyadenylation and oncogenic gene expression, as a downstream target of sd3’-SNORD78 in AML. Our data introduce sdRNAs as standalone regulatory effector molecules in healthy hematopoiesis and AML.

Accurate quantification of chitin oligosaccharides (COS) is essential for enzymatic hydrolysis research and bioactivity exploration, yet current analytical methods lack harmonization and limit profiling and quantitative accuracy. This study addresses these challenges by optimizing a gas chromatography (GC) method, validated to achieve precise quantification of COS in the range of DP1-DP4. By optimizing the silylation protocol, we increased the signal-to-noise (S/N) ratio in all DP standards, including a sevenfold increase in the S/N ratio of DP4. This GC method demonstrated robust linearity (R2 ≥ 0.99), high sensitivity (DP1 range from 10 to 2000 µg mL−1), strong precision (< 15

SnoRNAs are highly expressed in AML and play a role in leukemogenesis and leukemic maintenance. SnoRNAs can be further processed into snoRNA-derived RNAs (sdRNAs). Expression and implications of sdRNAs in AML and healthy hematopoiesis, however, remain largely elusive. We characterized sdRNA and snoRNA levels in hematopoietic stem cells (HSCs), healthy peripheral blood cells, and 159 AML patient samples at initial diagnosis. HSCs, healthy WBCs and AML blasts could be differentiated by their sdRNA expression pattern in a cell-type specific manner. In AML, high sd3’-RNA/snoRNA-hostgene ratios were associated with inverse patient outcome. Particularly, in NPM1-mutated patients with favorable risk stratification and good initial therapy response, high sd3’-RNA ratios identified a subgroup with inferior outcome, and could therefore represent biomarkers to identify those at-risk patients. High sd3’-RNA ratios were associated with clear alterations in oncogenic, inflammatory and immune response signalling. Forced expression of single sdRNAs, such as sd3’-SNORD78 and sd5’-SNORD93, enhanced clonogenic potential in AML. Total proteome and transcriptome analyses suggested NUDT21, a reported tumor suppressor with implications in inflammatory and immune response signalling, as novel target of sd3’-SNORD78. Our data introduces sdRNAs as effector molecules in healthy hematopoiesis and AML with mechanistic, diagnostic, as well as potential prognostic and therapeutic implications.

The cellulolytic system of Trichoderma reesei depends on β-glucosidases as central enzymes completing biomass hydrolysis and generating inducers such as sophorose. Three β-glucosidases from Aspergillus clavatus (AC), Penicillium oxalicum (PO) and Talaromyces stipitatus (TS) were expressed in Komagataella phaffii and characterized for parameters relevant to biomass conversion and inducer formation. PO and TS showed higher thermostability between 60 and 70 °C, whereas AC was inactivated above 50 °C. All variants were glucose inhibited, with AC least affected (Ki = 8.4 mM), followed by PO (Ki = 6 mM) and TS (Ki = 2.3 mM). Supplementation of Celluclast® with TS increased glucose release from cellulose and wheat straw and reached levels comparable to Cellic®CTec2 on wheat straw after 24 h. All enzymes catalyzed condensation reactions and produced up to 55 g/L disaccharides, with TS yielding the highest sophorose titers at 700 g/L glucose. Induction performance was assessed using T.reesei RUT-C30 cultivated with glucose, a TS derived disaccharide solution or steam-ex wheat straw hydrolysate. For each enzyme class, the highest activity across all conditions was defined as 100 %. The disaccharide solution generated the strongest cellulase response, while glucose yielded only 26 % BGL and 20 % CBH1 activity relative to these maxima, and hydrolysates produced intermediate levels of 65 % BGL and 24 % CBH1 activity while concurrently generating the highest hemicellulolytic activities, set to 100 %. Proteomics confirmed that the disaccharide solution upregulated core cellulases (EGL1 +2.80, CBH1 +1.66, EGL5 +3.16), whereas hydrolysates enriched CBH2 (-2.02), GH11 xylanases (-3.27) and GH3 β-xylosidases (-6.45, -2.49).

ABSTRACT:Dysregulated RNA modifications contribute to cancer progression and therapy resistance, yet the underlying mechanism often remains unknown. Here, we perform CRISPR-based synthetic lethality screens to systematically explore the role of RNA modifications in mediating resistance to antileukemic drugs. We identify the tRNA methyltransferase 5 (TRMT5)-mediated formation of N1-methylguanosine (m1G) in the transfer RNA (tRNA) anticodon loop as essential for mediating drug tolerance to cytarabine and venetoclax (Ven) in acute myeloid leukemia (AML). TRMT5 methylates nearly all mitochondrial and nuclear tRNAs with a guanosine at position 37, but its role in promoting drug tolerance specifically depends on its mitochondrial function. TRMT5 is essential for the dynamic upregulation of mitochondrial messenger RNA translation and oxidative phosphorylation, which are critical for sustaining drug tolerance in leukemia cells. This mitochondrial dependency correlates with therapy outcomes in patients with leukemia: lower expression of electron transport chain genes is linked to poorer outcomes in a cohort of nearly 100 patients with AML undergoing first induction therapy. Finally, we demonstrate that targeted depletion of the TRMT5 protein using a conditional degron, in conjunction with cytarabine and Ven treatment, synergistically induces cell death in drug-tolerant AML cells. Thus, our study reveals TRMT5 as a promising drug target for therapy-resistant leukemia.

Protein hydrolysis under acidic conditions can improve the product quality, nutrient availability, and cost efficiency, particularly when neutral or alkaline enzymes are ineffective. Six fungal aspartic endopeptidases (FAPs) were recombinantly expressed as active enzymes in Komagataella phaffi, with peak activity between 30-50 degrees C and pH 3.0-4.0. Despite FAP1 yielding a higher degree of hydrolysis for soy protein isolate (SPI) than FAP4, mass spectrometry analysis revealed similar cleavage preferences for the two peptidases. FAP1 and FAP4 experienced competitive product inhibition (Ki : 2.8 mg mL-1, K m : 3.2 mg mL-1 for FAP1 and K i : 9.67 mg mL-1, K m : 6.58 mg mL-1 for FAP4). These findings suggest that K i and K m values, when studied in isolation, do not always predict a peptidase's hydrolytic efficacy. Among the FAPs, FAP6 notably increased soluble protein content in animal feed by similar to 3-fold. FAP1, when combined with pepsin, had a positive effect on the hydrolysis of SPI. These results underscore the potential of FAPs to hydrolyze proteins-specifically, animal feed proteins-in acidic environments.

Mammalian ribosomal RNA (rRNA) molecules are highly abundant RNAs, decorated with over 220 rRNA modifications. Previous works have shown that some rRNA modification types can be dynamically regulated; however, how and when the mammalian rRNA modification landscape is remodeled remains largely unexplored. Here, we employ direct RNA sequencing to chart the human and mouse rRNA epitranscriptome across tissues, developmental stages, cell types, and disease. Our analyses reveal multiple rRNA sites that are differentially modified in a tissue- and/or developmental stage-specific manner, including previously unannotated modified sites. We demonstrate that rRNA modification patterns can be used for tissue and cell-type identification, which we hereby term “epitranscriptomic fingerprinting.” We then explore rRNA modification patterns in normal-tumor matched samples from lung cancer patients, finding that epitranscriptomic fingerprinting accurately classifies clinical samples into normal and tumor groups from only 250 reads per sample, demonstrating the potential of rRNA modifications as diagnostic biomarkers.

Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and - for AcrIIA5 - indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.

This study presents the development of ion chromatography coupled with inductively coupled plasma optical emission spectrometry (IC-ICP-OES) for the simultaneous determination and quantification of inositol phosphates (InsPx). Using a CarboPac PA100 column with a nitric acid-water gradient, 28 InsPx isomers (InsP6 to InsP2) were separated within 33 min. The method eliminates baseline drift and post-column derivatization thereby simplifying detection and quantification. It achieves low detection limits of 63 μg/L P across a range of 63-3200 μg/L P. Various extraction and sample preparation methods for food and feed matrices were tested, including acidic and alkaline agents, C18 SPE and spin concentrators. The analysis shows intra-day and intra-laboratory reproducibility with deviations smaller than 1 % for standard solutions and under 4 % for feed samples (80 % recovery rate of phytate). This methodology is applicable to explore enzymatic degradation pathways and the analysis of InsPx in complex food and animal feed matrices.

A novel peptidyl-lys metalloendopeptidase ( Tc -LysN) from Tramates coccinea was recombinantly expressed in Komagataella phaffii using the native pro-protein sequence. The peptidase was secreted into the culture broth as zymogen (~38 kDa) and mature enzyme (~19.8 kDa) simultaneously. The mature Tc -LysN was purified to homogeneity with a single step anion-exchange chromatography at pH 7.2. N-terminal sequencing using TMTpro Zero and mass spectrometry of the mature Tc- LysN indicated that the pro-peptide was cleaved between the amino acid positions 184 and 185 at the Kex2 cleavage site present in the native pro-protein sequence. The pH optimum of Tc -LysN was determined to be 5.0 while it maintained ≥60% activity between pH values 4.5—7.5 and ≥30% activity between pH values 8.5—10.0, indicating its broad applicability. The temperature maximum of Tc -LysN was determined to be 60 °C. After 18 h of incubation at 80 °C, Tc -LysN still retained ~20% activity. Organic solvents such as methanol and acetonitrile, at concentrations as high as 40% (v/v), were found to enhance Tc -LysN’s activity up to ~100% and ~50%, respectively. Tc -LysN’s thermostability, ability to withstand up to 8 M urea, tolerance to high concentrations of organic solvents, and an acidic pH optimum make it a viable candidate to be employed in proteomics workflows in which alkaline conditions might pose a challenge. The nano-LC-MS/MS analysis revealed bovine serum albumin (BSA)’s sequence coverage of 84% using Tc -LysN which was comparable to the sequence coverage of 90% by trypsin peptides. Key points • A novel LysN from Trametes coccinea (Tc-LysN) was expressed in Komagataella phaffii and purified to homogeneity • Tc-LysN is thermostable, applicable over a broad pH range, and tolerates high concentrations of denaturants • Tc-LysN was successfully applied for protein digestion and mass spectrometry fingerprinting

Abstract Non-coding RNAs drive cancer phenotypes and are associated with patients’ outcome. SnoRNAs are required for the 2’-O-methylation (2’-O-Me) and pseudouridylation of ribosomal RNA (rRNA) thus being essential for ribosomal biogenesis and function. So far, the role of rRNA methylation (ribomethylation) for non-small cell lung cancer (NSCLC) pathogenesis is unknown. To investigate the role of ribomethylation we included samples from 92 patients with lung adenocarcinoma (pathological stage IA to IIB) for comprehensive multi-omics profiling. Amongst transcriptomics, small RNA sequencing, proteomics, and whole exome sequencing, ribomethylation as epitranscriptomic dimension was analyzed using RiboMethSeq. We focused the analysis on complete tumor resection samples. Here 47 out of 106 2’-O-Me sites were fully methylated in all tumor samples suggesting a crucial role for methylation at this position. Surprisingly, 59 sites had a dynamic methylation pattern. By combining ribomethylation sequencing data of dynamic sites with whole-exome-sequencing as well as transcriptome and proteome analyses using a multi-omics factor analysis (MOFA), we discovered a 2’-O-Me signature in a subset of NSCLC patients with a high risk of metastasis and poor prognosis. We termed the patient subset, with underlying ribomethylation signature, epitranscriptomic pro-metastatic phenotype (EPROMET). This phenotype was not associated with genetic mutations as analyzed by exome sequencing. Analysis of gene sets in both transcriptomics and proteomics showed an upregulation of secreted and extracellular matrix proteins in EPROMET patients. Alongside, the ribomethylation site with major contribution to the EPROMET signature; 18S-Um799, mediated through SNORD105/105B, was found to be the most dynamic within the dataset. It also showed the largest difference between EPROMET and non-EPROMET patients. For functional validation, lung cancer cell lines with knockout of snoRNAs contributing to EPROMET signature were generated using CRISPR/Cas9. Lung cancer cells lacking the corresponding rRNA modifications migrated slower in vitro, failed to grow at a distant site in vivo and were impaired in metastasis. Functionally, it was shown that the modulated ribomethylation signature effected translation of secreted proteins by altered mRNA binding to ribosomes. This study indicates the presence of a ribomethylome-associated phenotype of NSCLC related to metastasis and poor prognosis. Altered ribomethylation affects the ribosome function towards differential expression patterns of secreted proteins. Taken together, an epitranscriptomic pattern of 2’-O-Me is associated with invasion/migration properties of NSCLC cells and with development of metastasis. The ribomethylome may present a suitable biomarker and a potential therapeutic target. Citation Format: Cornelius Pauli, Daniel Heid, Christian Rohde, Nadja Krall, Sylvain Delaunay, Michael Kienhoefer, Christian Tischer, Michael Allgäuer, Maximilian Felix Blank, Fengbiao Zhou, Michael Kardorff, Michael Thomas, Hauke Winter, Sarah Sandmann, Marc Kriegsmann, Marc Schneider, Thomas Muley, Alexander Brobeil, Nicole Bäumer, Sebastian Bäumer, Simon Raffel, Albrecht Stenzinger, Peter Schirmacher, Junyan Lu, Judith Zaugg, Michaela Frye, Carsten Müller-Tidow. A specific ribomethylome pattern in lung cancer is associated with increased risk of metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2763.

The natural polymer chitin is an abundant source for valuable N-acetylchitooligosaccharides and N-acetylglucosamine applicable in several industries. The endochitinase Chit36-TA from Trichoderma asperellum was recombinantly expressed in Komagataella phaffii for the enzymatic degradation of chitin from unused insect exuviae into N-acetylchitooligosaccharides. Chit36-TA was purified by Ni–NTA affinity chromatography and subsequently biochemically characterized. After deglycosylation, the endochitinase had a molecular weight of 36 kDa. The optimum pH for Chit36-TA was 4.5. The temperature maximum of Chit36-TA was determined to be 50 °C, while it maintained > 93

Venetoclax/azacitidine combination therapy is effective in acute myeloid leukemia (AML) and tolerable for older, multimorbid patients. Despite promising response rates, many patients do not achieve sustained remission or are upfront refractory. Identification of resistance mechanisms and additional therapeutic targets represent unmet clinical needs. By using a genome-wide CRISPR/Cas9 library screen targeting 18,053 protein- coding genes in a human AML cell line, various genes conferring resistance to combined venetoclax/azacitidine treatment were identified. The ribosomal protein S6 kinase A1 (RPS6KA1) was among the most significantly depleted sgRNA-genes in venetoclax/azacitidine- treated AML cells. Addition of the RPS6KA1 inhibitor BI-D1870 to venetoclax/azacitidine decreased proliferation and colony forming potential compared to venetoclax/azacitidine alone. Furthermore, BI-D1870 was able to completely restore the sensitivity of OCI-AML2 cells with acquired resistance to venetoclax/azacitidine. Analysis of cell surface markers revealed that RPS6KA1 inhibition efficiently targeted monocytic blast subclones as a potential source of relapse upon venetoclax/azacitidine treatment. Taken together, our results suggest RPS6KA1 as mediator of resistance towards venetoclax/azacitidine and additional RPS6KA1 inhibition as strategy to prevent or overcome resistance.

FLT3 tyrosine kinase inhibitor (TKI) therapy evolved into a standard therapy in FLT3-mutated AML. TKI resistance, however, develops frequently with poor outcomes. We analyzed acquired TKI resistance in AML cell lines by multilayered proteome analyses. Leupaxin (LPXN), a regulator of cell migration and adhesion, was induced during early resistance development, alongside the tyrosine kinase PTK2B which phosphorylated LPXN. Resistant cells differed in cell adhesion and migration, indicating altered niche interactions. PTK2B and LPXN were highly expressed in leukemic stem cells in FLT3-ITD patients. PTK2B/FAK inhibition abrogated resistance-associated phenotypes, such as enhanced cell migration. Altered pathways in resistant cells, assessed by nascent proteomics, were largely reverted upon PTK2B/FAK inhibition. PTK2B/FAK inhibitors PF-431396 and defactinib synergized with different TKIs or daunorubicin in FLT3-mutated AML. Midostaurin-resistant and AML cells co-cultured with mesenchymal stroma cells responded particularly well to PTK2B/FAK inhibitor addition. Xenograft mouse models showed significant longer time to leukemia symptom-related endpoint upon gilteritinib/defactinib combination treatment in comparison to treatment with either drug alone. Our data suggest that the leupaxin-PTK2B axis plays an important role in acquired TKI resistance in AML. PTK2B/FAK inhibitors act synergistically with currently used therapeutics and may overcome emerging TKI resistance in FLT3-mutated AML at an early timepoint.

BCL-2 inhibition has been shown to be effective in acute myeloid leukemia (AML) in combination with hypomethylating agents or low-dose cytarabine. However, resistance and relapse represent major clinical challenges. Therefore, there is an unmet need to overcome resistance to current venetoclax-based strategies. We performed high-throughput drug screening to identify effective combination partners for venetoclax in AML. Overall, 64 antileukemic drugs were screened in 31 primary high-risk AML samples with or without venetoclax. Gilteritinib exhibited the highest synergy with venetoclax in FLT3 wild-type AML. The combination of gilteritinib and venetoclax increased apoptosis, reduced viability, and was active in venetoclax-azacitidine-resistant cell lines and primary patient samples. Proteomics revealed increased FLT3 wild-type signaling in specimens with low in vitro response to the currently used venetoclax-azacitidine combination. Mechanistically, venetoclax with gilteritinib decreased phosphorylation of ERK and GSK3B via combined AXL and FLT3 inhibition with subsequent suppression of the antiapoptotic protein MCL-1. MCL-1 downregulation was associated with increased MCL-1 phosphorylation of serine 159, decreased phosphorylation of threonine 161, and proteasomal degradation. Gilteritinib and venetoclax were active in an FLT3 wild-type AML patient-derived xenograft model with TP53 mutation and reduced leukemic burden in 4 patients with FLT3 wild-type AML receiving venetoclax-gilteritinib off label after developing refractory disease under venetoclax-azacitidine. In summary, our results suggest that combined inhibition of FLT3/AXL potentiates venetoclax response in FLT3 wild-type AML by inducing MCL-1 degradation. Therefore, the venetoclax-gilteritinib combination merits testing as a potentially active regimen in patients with high-risk FLT3 wild-type AML.

T cell–dependent reprogramming of intratumoral macrophages by cIAP1/2 inhibition leads to control of MHC class I–negative pancreatic cancer in mice.

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers.

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neo antigens. Such neo antigens are particularly abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neo antigen-inducing translational frameshifts. The abundance of mutational neo antigens renders MSI cancers sensitive to immune checkpoint blockade. However, the neoantigen landscape of MMR-deficient cancers has not yet been systematically mapped. In the present study, we used a novel tool to monitor neo antigen-inducing indel mutations in MSI colorectal and endometrial cancer. Our results show that MSI cancers share several highly immunogenic neo antigens that result from specific, recurrent indel mutation events. Notably, the frequency of such indel mutations was negatively correlated to the predicted immunogenicity of the resulting neo antigens. These observations suggest continuous immunoediting of emerging MMR-deficient cells during tumor evolution. One sentence summary Quantitative indel mutation analysis reveals evidence of immune selection in mismatch repair-deficient cancers * ELS : Epitope likelihood score GELS : General epitope likelihood score IRS : Immune relevance score, based on the mutation frequency (ReFrame) and the GELS M1 : Reading frame resulting from the deletion of one nucleotide or insertions of two nucleotides M2 : Reading frame resulting from the deletions of two nucleotides or insertion of one nucleotide m1 , m2 , m3 , etc. : Minus one, two, three base pair deletions p1 , p2 , p3 , etc. : Plus one, two, three base pair insertions ReFrame : REgression-based FRAMEshift quantification

Abstract The immune system can recognize and attack cancer cells and their precursors, especially those with a high load of mutation-induced neoantigens. Such neoantigens are particularly abundant in DNA mismatch repair (MMR)-deficient cancers. MMR deficiency results in microsatellite instability (MSI), which leads to multiple insertion/deletion mutations at coding microsatellites and to neoantigen-inducing translational frameshifts. The significance of immune selection and immunoediting potentially shaping the neoantigen landscape during the progression from premalignant MMR-deficient lesions into cancers has not yet been analyzed. We hypothesized that the neoantigen landscape of MSI cancers may reflect the impact of immunoediting. We developed a novel tool for quantitative analysis of microsatellite mutations to explore the neoantigen landscape of MSI colorectal (CRC, n=139) cancers. Frameshift mutations were examined in 41 coding microsatellite (cMS) regions using our new algorithm. We predicted the resulting frameshift neoantigen sequences and used the publicly available prediction tool NetMHCpan 4.0 for prediction of MHC binding sequences. Immunological scores were generated to quantify the likelihood of defined cMS mutations to generate immunogenic neoantigens in different populations with defined HLA allele distributions. Across the 41 cMS analyzed, 77% of all mutations were in the reading frame of 1 nucleotide deletions (m1). The cMS mutation frequency and FSP epitope distribution across HLA genotypes (described by a general epitope likelihood score, GELS) showed a significant negative correlation (Pearson’s r=-0.42, p=0.0149). Some cMS presented with high mutation frequencies despite a high GELS (i.e. TGFBR2: pmut = 88%, GELS = 78.9%), suggesting mutation-induced driver effects, which may outweigh the increased immunogenicity. Our results show that MSI cancers share several highly immunogenic neoantigens. Importantly, a negative correlation between the antigenic strength of neoepitopes and their mutation frequency in MMR-deficient cancers points towards continuous immunoediting during their evolution. These findings will have substantial impact on the optimization of vaccines designed to potentially prevent or treat MSI-driven cancers. Citation Format: Matthias Kloor, Alexej Ballhausen, Moritz Przybilla, Michael Jendrusch, Elisabeth Pfaffendorf, Markus Draxlbauer, Florian Seidler, Sonja Krausert, Aysel Ahadova, Simon Kalteis, Daniel Heid, Johannes Gebert, Maria Bonsack, Sarah Schott, Hendrik Bläker, Toni Seppälä, Jukka-Pekka Mecklin, Sanne Ten Broeke, Maartje Nielsen, Julia Krzykalla, Axel Benner, Angelika Riemer, Magnus von Knebel Doeberitz. The shared mutation and neoantigen landscape of MMR-deficient colorectal cancers suggests immunoediting during tumor evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 571.