Low Mutational Load Research Articles

SET facilitates immune escape of microsatellite stability colorectal cancer by inhibiting c-Myc degradation.

Microsatellite stability (MSS) colorectal cancer (CRC) exhibits a low mutation load and poor immunogenicity, contributing to immune escape of tumor cells and less benefit from immune checkpoint blockade (ICB) treatment. The mechanisms underlying immunotherapeutic resistance in MSS CRC remain to be elucidated. Here, we identified that nuclear proto-oncogene SET is significantly higher expressed in MSS CRC compared to microsatellite instability (MSI) CRC and facilitates immune escape of MSS CRC. Mechanistically, SET represses the expression of C-C motif chemokine ligand 5 (CCL5) and upregulates mismatch repair (MMR) proteins expression in a c-Myc-dependent manner, which inhibits infiltration and migration of CD8+ T cells to tumor tissues and results in low immunogenicity in MSS CRC. In addition, we found that SET impairs ubiquitination and proteasomal degradation of c-Myc by disrupting the interaction between E3 ligase FBXW7 and c-Myc. Moreover, SET inhibition enhances the response to immunotherapy in MSS CRC invivo. Overall, this study reveals the critical roles and posttranslational regulatory mechanism of SET in immune escape and highlights the SET/c-Myc axis as a potential target for immunotherapy of MSS CRC that have implications for targeting a unique aspect of this disease.

Mutational mechanisms in multiply relapsed pediatric acute lymphoblastic leukemia

Pediatric acute lymphoblastic leukemia (ALL) is marked by low mutational load at initial diagnosis, which increases at relapse. To determine which processes are active in (relapsed) ALL and how they behave during disease progression before and after therapy, we performed whole genome sequencing on 97 tumor samples of 29 multiply relapsed ALL patients. Mutational load increased upon relapse in 28 patients and upon every subsequent relapse in 22 patients. In addition to two clock-like mutational processes, we identified UV-like damage, APOBEC activity, reactive oxygen species, thiopurine-associated damage and an unknown therapy component as drivers of mutagenesis. Mutational processes often affected patients over longer time periods, but could also occur in isolated events, suggesting the requirement of additional triggers. Thiopurine exposure was the most prominent source of new mutations in relapse, affecting over half of the studied patients in first and/or later relapse and causing potential relapse-driving mutations in multiple patients. Our data demonstrate that multiple mutational processes frequently act in parallel as prominent secondary drivers with dynamic activity during ALL development and progression.

Complement C3d enables protective immunity capable of distinguishing spontaneously transformed from non-transformed cells.

We show that intracellular soluble fragment 3d of complement (C3d) induces regression of spontaneous multiple myeloma in mice reducing tumor burden by 10 fold, after 8 weeks. C3d enables cell-mediated immunity to target multiple myeloma clones sparing non-transformed polyclonal B cells and plasma cells with lower mutation loads. We show that C3d increases the expression of ribosomal subunits associated with the translation of defective ribosomal products (DRiPs). C3d also decreases expression of protein arginine methyl transferase (PRMT) 5 which in turn relieves E2f1 repression increasing the expression of Lnc RNAs and derived peptides that evoke anti-tumor cellular immunity. The approach increases MHC-I expression by tumor cells and generates a CMI response that overcomes tumor immune-evasion strategies. Tumors are immunogenic in part because of somatic mutations that originate novel peptides that once presented on MHC engage cell-mediated immunity (CMI). However, in spite of the higher mutation load most tumors evade immunity. We discovered that a component of the complement system (C3d) overcomes tumor immune evasion by augmenting expression of ribosomal proteins and lncRNAs linked to the presentation of novel peptides by tumor cells. C3d induced CMI targets cancer cells sparing non transformed cells uncovering a novel function for complement in immune surveillance.

GMP-compliant iPS cell lines show widespread plasticity in a new set of differentiation workflows for cell replacement and cancer immunotherapy.

Cell therapeutic applications based on induced pluripotent stem cells (iPSCs) appear highly promising and challenging at the same time. Good manufacturing practice (GMP) regulations impose necessary yet demanding requirements for quality and consistency when manufacturing iPSCs and their differentiated progeny. Given the scarcity of accessible GMP iPSC lines, we have established a corresponding production workflow to generate the first set of compliant cell banks. Hence, these lines met a comprehensive set of release specifications and, for instance, displayed a low overall mutation load reflecting their neonatal origin, cord blood. Based on these iPSC lines, we have furthermore developed a set of GMP-compatible workflows enabling improved gene targeting at strongly enhanced efficiencies and directed differentiation into critical cell types: A new protocol for the generation of retinal pigment epithelium (RPE) features a high degree of simplicity and efficiency. Mesenchymal stromal cells (MSCs) derived from iPSCs displayed outstanding expansion capacity. A fully optimized cardiomyocyte differentiation protocol was characterized by a particularly high batch-to-batch consistency at purities above 95%. Finally, we introduce a universal immune cell induction platform that converts iPSCs into multipotent precursor cells. These hematopoietic precursors could selectively be stimulated to become macrophages, T cells, or natural killer (NK) cells. A switch in culture conditions upon NK-cell differentiation induced a several thousand-fold expansion, which opens up perspectives for upscaling this key cell type in a feeder cell-independent approach. Taken together, these cell lines and improved manipulation platforms will have broad utility in cell therapy as well as in basic research.

O-275 Prenatal genetic screening to assess mtDNA disease occurrence in children

Abstract Study question Can prenatal genetic testing accurately predict mtDNA mutation load anddisease transmission to children for families affected by Leigh Syndrome? Summary answer Whole genome mtDNA sequencing for 13513G>A mutation in chorionic villi and amniotic fluid was similar to those measured in babies suggesting accuracy of disease prediction. What is known already Severity of mtDNA disease in children depends on heteroplasmy levels(proportion of mutant to wild-type mtDNA). There is a minimum threshold for the mutant mtDNA heteroplasmy (typically 60%) above which oxidative metabolism is impaired to cause disease. Predicting the disease based on the mutation load measured during embryonic or fetal development is critically needed. However, preimplantation genetics screening for many mtDNA mutations has limitations due to the potential increase in heteroplasmy levels after implantation. We hypothesized that prenatal screening of chorionic villi and/or amniotic fluid during the first trimester of pregnancy could provide more reliable diagnosis of disease occurrence in children. Study design, size, duration We evaluated mtDNA heteroplasmy in biopsies collected from chorionic villi and amniotic fluid at 10- and 16-weeks of gestation, respectively, for two consecutive, naturally conceived pregnancies for a carrier of 13513G>A mutation. Sequencing results were compared to mutations in the blood, skin, and urine of children after birth. Both children were also extensively monitored for a few years for disease occurrence. Participants/materials, setting, methods A family with maternally transmitted Leigh Syndrome including both parents and two existing children were enrolled during the initial phase. Whole mtDNA sequencing was used to confirm the maternal transmission of the mutation by analysis of blood, skin, and urine from asymptomatic parents and the older son and the severely affected younger son. Furthermore, prenatal diagnostics were performed for two consecutive pregnancies and children after birth. Main results and the role of chance The mother carried 13513G>A mutation in the skin (91% heteroplasmy), urine(39%), and blood (6.8%), while no mutation was detected in the father’s samples. The asymptomatic eldest child revealed a low mutation in the skin (14%), urine(23%), and blood (10%). While the younger son diagnosed with Leigh syndrome carried 56% mutation in blood, 86% in skin, and 97% in urine. Heteroplasmy ranging from 0.6% to 40% was also measured in 4 individual immature oocytes retrieved upon hormonal stimulation of the carrier. The family later naturally conceived 2 consecutive pregnancies and requested help with prenatal diagnostics. In the first pregnancy, mutation loads were 27.5% in chorionic villi and 29.5% in amniotic fluid. The family decided to keep the pregnancy which resulted in the birth of a healthy child with low mutations in blood (0.07%) and skin (14.5%). In the second pregnancy, prenatal diagnostics in CVS showed low mutation at 0.11%, resulting in the birth of an unaffected child with mutation loads of 1.4% in blood and 0% in skin. Limitations, reasons for caution Genetic counseling for mtDNA disease transmission is difficult due tocomplex inheritance of heteroplasmic variants for various pathogenicmutations. Inheritance and distribution of mtDNA mutations also influencedby individual family genetics. Therefore, prenatal diagnostic screeningshould be cautiously considered based on the specific circumstances for eachfamily. Wider implications of the findings Prenatal genetic diagnostics for families with mtDNA disease based on whole mtDNA sequencing by the qualified laboratory may accurately predict heteroplasmy levels and disease occurrence in children. However, careful analysis of family history and the likelihood of having low mutation load oocytes was critical for the success of our case. Trial registration number N/A

A rare case of TFEB/6p21/VEGFA-amplified renal cell carcinoma diagnosed by whole-exome sequencing: clinicopathological and genetic feature report and literature review

BackgroundTFEB/6p21/VEGFA-amplified renal cell carcinoma (RCC) is rare and difficult to diagnose, with diverse histological patterns and immunohistochemical and poorly defined molecular genetic characteristics.Case presentationWe report a case of a 63-year-old male admitted in 2017 with complex histomorphology, three morphological features of clear cell, eosinophilic and papillary RCC and resembling areas of glomerular and tubular formation. The immunophenotype also showed a mixture of CD10 and P504s. RCC with a high suspicion of collision tumors was indicated according to the 2014 WHO classification system; no precise diagnosis was possible. The patient was diagnosed at a different hospital with poorly differentiated lung squamous cell carcinoma one year after RCC surgery. We exploited molecular technology advances to retrospectively investigate the patient’s molecular genetic alterations by whole-exome sequencing. The results revealed a 6p21 amplification in VEGFA and TFEB gene acquisition absent in other RCC subtypes. Clear cell, papillary, chromophobe, TFE3-translocation, eosinophilic solid and cystic RCC were excluded. Strong TFEB and Melan-A protein positivity prompted rediagnosis as TFEB/6p21/VEGFA-amplified RCC as per 2022 WHO classification. TMB-L (low tumor mutational load), CCND3 gene acquisition and MRE11A and ATM gene deletion mutations indicated sensitivity to PD-1/PD-L1 inhibitor combinations and the FDA-approved targeted agents Niraparib (Grade C), Olaparib (Grade C), Rucaparib (Grade C) and Talazoparib (Class C). GO (Gene Ontology) and KEGG enrichment analyses revealed major mutations and abnormal CNVs in genes involved in biological processes such as the TGF-β, Hippo, E-cadherin, lysosomal biogenesis and autophagy signaling pathways, biofilm synthesis cell adhesion substance metabolism regulation and others. We compared TFEB/6p21/VEGFA-amplified with TFEB-translocated RCC; significant differences in disease onset age, histological patterns, pathological stages, clinical prognoses, and genetic characteristics were revealed.ConclusionWe clarified the patient’s challenging diagnosis and discussed the clinicopathology, immunophenotype, differential diagnosis, and molecular genetic information regarding TFEB/6p21/VEGFA-amplified RCC via exome analysis and a literature review.

Transcranial Agonism of CD40 and ICOS Promotes Meningeal Immunity and Control of Glioblastoma

INTRODUCTION: Glioblastomas generate an immunosuppressive microenvironment that limits antitumor immune responses. METHODS: We used the SB28 model of glioblastoma in C57BL/6J (B6) mice, which is a poorly immunogenic tumor with a lower mutational load than other many glioblastoma cell lines used in rodents. We stereotactically injected 7,000 SB28 cells at a depth of 1 mm into the neocortex of 8- to 10-week-old mice. At day 7, a carboxymethylcellulose gel containing 100 µg of agonistic anti-ICOS and 200 µg anti-CD40 were injected transcranially (i.e., under the scalp) of mice. Control mice received corresponding isotype control antibodies. Meningeal whole mounts were then stained using immunohistochemistry and imaged by confocal microscopy. RESULTS: Transcranial agonism of CD40 and ICOS generated a markedly enhanced and more localized meningeal immune response against SB28 tumors than intravenous agonism. Specifically, transcranial treatment promoted formation of CD4 T cell and B cell clusters in the meninges as well as increased production of class-switched immunoglobulins. These clusters were also associated with reduced tumor size at day 14. CONCLUSIONS: Our results demonstrate that transcranial delivery of antibodies against CD40 and ICOS can drive a local humoral immune response against an immunologically silent SB28 glioblastoma. We postulate that application of these agonistic antibodies in combination with a tumor neoantigen will further enhance tumor control and may offer a possible treatment modality in human glioblastoma patients.

TM4SF19 controls GABP-dependent YAP transcription in head and neck cancer under oxidative stress conditions

Tobacco and alcohol are risk factors for human papillomavirus-negative head and neck squamous cell carcinoma (HPV- HNSCC), which arises from the mucosal epithelium of the upper aerodigestive tract. Notably, despite the mutagenic potential of smoking, HPV- HNSCC exhibits a low mutational load directly attributed to smoking, which implies an undefined role of smoking in HPV- HNSCC. Elevated YAP (Yes-associated protein) mRNA is prevalent in HPV- HNSCC, irrespective of the YAP gene amplification status, and the mechanism behind this upregulation remains elusive. Here, we report that oxidative stress, induced by major risk factors for HPV- HNSCC such as tobacco and alcohol, promotes YAP transcription via TM4SF19 (transmembrane 4 L six family member 19). TM4SF19 modulates YAP transcription by interacting with the GABP (Guanine and adenine-binding protein) transcription factor complex. Mechanistically, oxidative stress induces TM4SF19 dimerization and topology inversion in the endoplasmic reticulum membrane, which in turn protects the GABPβ1 subunit from proteasomal degradation. Conversely, depletion of TM4SF19 impairs the survival, proliferation, and migration of HPV- HNSCC cells, highlighting the potential therapeutic relevance of targeting TM4SF19. Our findings reveal the roles of the key risk factors of HPV- HNSCC in tumor development via oxidative stress, offering implications for upcoming therapeutic approaches in HPV- HNSCC.

Identification of molecular subtypes and diagnostic model in clear cell renal cell carcinoma based on collagen-related genes may predict the response of immunotherapy.

Background: Collagen represents a prominent constituent of the tumor's extracellular matrix (ECM). Nonetheless, its correlation with the molecular subtype attributes of clear cell renal cell carcinoma (ccRCC) remains elusive. Our objective is to delineate collagen-associated molecular subtypes and further construct diagnostic model, offering insights conducive to the precise selection of ccRCC patients for immunotherapeutic interventions. Methods: We performed unsupervised non-negative matrix factorization (NMF) analysis on TCGA-KIRC samples, utilizing a set of 33 collagen-related differentially expressed genes (33CRDs) for clustering. Our analysis encompassed evaluations of subtype-associated differences in pathways, immune profiles, and somatic mutations. Through weighted gene co-expression network analysis (WGCNA) and four machine learning algorithms, two core genes were found and a diagnostic model was constructed. This was subsequently validated in a clinical immunotherapy cohort. Single cell sequencing analysis and experiments demonstrated the role of core genes in ccRCC. Finally, we also analyzed the roles of MMP9 and SCGN in pan-cancer. Results: We described two novel collagen related molecular subtypes in ccRCC, designated subtype 1 and subtype 2. Compared with subtype 1, subtype 2 showed more infiltration of immune components, but had a higher TIDE (tumor immunedysfunctionandexclusion) score and increased levels of immune checkpoint molecules. Furthermore, reduced prognosis for subtype 2 was a consistent finding in both high and low mutation load subgroups. MMP9 and SCGN were identified as key genes for distinguishing subtype 1 and subtype 2. The diagnostic model based on them could better distinguish the subtype of patients, and the differentiated patients had different progression free survival (PFS) in the clinical immunotherapy cohort. MMP9 was predominantly expressed in macrophages and has been extensively documented in the literature. Meanwhile, SCGN, which was overexpressed in tumor cells, underwent experimental validation, emphasizing its role in ccRCC. In various cancers, MMP9 and SCGN were associated with immune-related molecules and immune cells. Conclusion: Our study identifies two collagen-related molecular subtypes of ccRCC and constructs a diagnostic model to help select appropriate patients for immunotherapy.

Current State-of-the-Art Therapy for Malignant Pleural Mesothelioma and Future Options Centered on Immunotherapy.

Malignant pleural mesothelioma (MPM) is a locally aggressive disease related to asbestos exposure with a median survival for untreated patients of 4-8 months. The combination of chemotherapy based on platinum and antifolate is the standard treatment, and the addition of bevacizumab adds two months to median survival. Recently, in first-line treatment, immunotherapy combining nivolumab with ipilimumab has been shown to be superior to chemotherapy in the CheckMate-743 study in terms of overall survival (18.1 months), leading to its approval by the FDA and EMA. The positive results of this study represent a new standard of treatment for patients with MPM; however, not all patients will benefit from immunotherapy treatment. In an effort to improve the selection of patient candidates for immunotherapy for different tumors, biomarkers that have been associated with a greater possibility of response to treatment have been described. MPM is a type of tumor with low mutational load and neo-antigens, making it a relatively non-immunogenic tumor for T cells and possibly less susceptible to responding to immunotherapy. Different retrospective studies have shown that PD-L1 expression occurs in 20-40% of patients and is associated with a poor prognosis; however, the predictive value of PD-L1 in response to immunotherapy has not been confirmed. The purpose of this work is to review the state of the art of MPM treatment in the year 2023, focusing on the efficacy results of first-line or subsequent immunotherapy studies on patients with MPM and possible chemo-immunotherapy combination strategies. Additionally, potential biomarkers of response to immunotherapy will be reviewed, such as histology, PD-L1, lymphocyte populations, and TMB.

Single-Cell Whole-Genome Sequencing after Chemotherapeutic Treatment Reveals Cell Type-Specific Mutational Burdens

Introduction The hematopoietic system is especially vulnerable to chemotherapeutic treatment-related long-term consequences, including clonal and malignant hematopoiesis. These diseases are caused by the acquisition of driver mutations in hematopoietic stem and progenitor cells (HSPCs). An increased rate of mutation accumulation, as identified in HSPCs of patients who receive chemotherapeutic treatment, can therefore result in disrupted blood production. Accordingly, childhood cancer survivors have a five-fold increased risk of secondary cancers, amongst which therapy-related myeloid neoplasms (t-MN) are one of the most prevalent. Here, we aim to increase the understanding of the origin of t-MN through the characterization of the short- and long-term mutational consequences of chemotherapy in different hematopoietic stem and progenitor cell types. Methods We studied the genome-wide mutation burden in the HSPC compartment at the time of an initial cancer diagnosis, following chemotherapeutic treatment, and upon a second cancer diagnosis. We performed whole-genome sequencing (WGS) of single HSPCs, including hematopoietic stem cells (HSCs, CD34+CD38-CD45RA-CD90+), multipotent progenitor cells (MPPs, CD34+CD38-CD45RA-CD90-), common myeloid progenitor cells (CMPs, CD34+CD38+CD45RA-), and granulocyte-monocyte progenitor cells (GMPs, CD34+CD38+CD45RA+). In order to obtain enough DNA for WGS, we performed clonal expansion of single cells or primary template-directed amplification of the DNA of one cell. Next, we normalized the mutation burden in each cell to the expected number of mutations (Osorio et al. Cell Reports 2018). In addition, we performed both whole genome and transcriptome sequencing of individual HSCs/MPPs. Results First, we investigated pediatric ten t-MN patients with a latency between the initial and second cancer diagnosis spanning 1-12 years. We applied WGS to clonally expanded HSPCs (CD34+CD38-CD45RA-) and found that the relative increase in mutation burden in HSPCs is restricted to the first five years after initial treatment. To gain more insight into the heterogeneous HSPC compartment, we subsequently investigated cell-type specific mutational loads based on immunophenotypic markers. So far, we have included three pediatric cancer patients with comparable treatment regimens and diagnoses, namely medium risk acute lymphoblastic leukemia. Here, we found that the mutation burden is lower in HSCs when compared to the progenitor cell types (Figure 1A). When comparing the mutation load in the HSCs to the linear mutation accumulation observed in HSCs and MPPs of healthy donors, we identified a fraction of cells overlapping with the expected mutation load based on the age of the patient (Figure 1B). These data suggest that HSCs are more protected against chemotherapy-induced mutagenesis than progenitor cells, although this protective effect varies between individual cells. Therefore, we also performed both whole genome and transcriptome sequencing from a single cell, to characterize the transcriptional state of HSCs/MPPs showing a relatively high or low mutation load within their respective cell type. Conclusions Our results indicate that primitive hematopoietic stem cells are more protected against chemotherapy-induced mutations than the more proliferative progenitor cells. In addition, we find that in t-MN patients with a long latency (>5 years), the mutation load in HSPCs returns to the expected baseline. Based on our data, we hypothesize that the more damaged progenitor cells may be replaced over time, fueled by the undamaged primitive stem cell pool. Thus, our results may explain why the risk of t-MN drops 5-7 years after treatment end.

Tumor Reactive T Cells Reside in the Bone Marrow and Are Associated with Clinical Response to Hematological Malignancies

T cell receptors (TCRs) play a crucial role in orchestrating cellular immunity in both health and disease, particularly in the context of cancer. Although immunotherapy and stem cell transplantation have shown promising clinical responses in hematological malignancies, these have not been linked to the presence of endogenous tumor reactive T cells. Moreover, the specificities and phenotypes of such tumor recognizing TCRs in these largely low immunogenic entities are unknown. In this study, we addressed this gap by developing an approach to map TCR specificities and transcriptional phenotypes of human bone marrow-resident T cells directly ex vivo, focusing on patients who were treatment-naïve at the time of biopsy. This allowed us to generalize our findings beyond immune checkpoint inhibitor-treated patients, a limitation of previous studies in solid cancers. To discover T cells that specifically recognize cancer cells or viral epitopes, we developed a multiplexed profiling approach using microfluidic reaction chambers, optical cytokine detection and TCR sequencing of patient-derived primary T cells. We were able to confidently map TCR recognition of 25,957 bone marrow-resident T cells from n=20 hematological cancer patients. Based on the acquired functional data and reverse phenotyping of screened TCRs to their original cell state in situ, we built a bone marrow T cell atlas charting >300,000 expression profiles in human bone marrow-resident T cells, their TCR specificities as well as their longitudinal clonal dynamics upon therapeutic perturbations in n=40 patients with newly diagnosed multiple myeloma (MM) and acute myeloid leukemia (AML). Our approach detected tumor-reactive T cell clones in the bone marrow of all screened individuals with varying frequency (1-5% of T cells), which were largely found to display a CD8+ effector-memory or progenitor-exhausted phenotype. In contrast, non-tumor reactive clones were either CD4+ or enriched for viral specificities. We identified a conserved signature of anti-tumor reactivity across patients. This signature included the ITGB1 gene, which encodes CD29 and was uniformly expressed in tumor reactive T cells. Importantly, ITGB1 expression was specifically restricted to tumor reactive, compared to virus-specific or bystander T cells. In two independent mouse tumor models, we found that CD29 protein was significantly upregulated on homed T cells carrying tumor reactive TCRs compared to non-reactive TCRs. A causal link between tumor reactive T cells and clinical response to hematological cancers would be strengthened by finding that these cells specifically recognize autologous cancer epitopes and clonally expand as part of a clinical anti-tumor response. We therefore cloned patient-individualized TCR libraries and performed immunoprecipitation of MHC class I : peptide complexes from cancer cells. We validated TCR binding against ~1-20 epitopes per patient, which were mostly derived from cancer/testis antigens (CTAs) and novel open reading frames (nORFs). Finally, we classified tumor reactive TCRs in three clinical cohorts: Notably, we observed selective expansion of tumor reactive TCRs upon autologous stem cell transplantation, which was associated with deep clinical response in newly diagnosed MM patients. Conversely, the failure of tumor reactive TCRs to clonally expand on-treatment underlined clinical non-response to bispecific BCMAxCD3 antibody therapy (MM) or combination therapy with immune checkpoint inhibitors and Azacytidine (AML). We show here that, unexpectedly, clinically relevant endogenous anti-tumor reactivity lies in a rare subset of bone marrow-resident T cells. The recurrent detection of these immunotherapy-responsive TCRs in patients with MM or AML revealed the selective clonal expansion of tumor-reactive TCRs following autologous stem cell transplantation. We describe a conserved gene signature of tumor reactivity that diverges from lymphocytes infiltrating solid tumors and includes CD29 as an indicator of antigen-specific T cell homing. Our findings facilitate the antigen-agnostic identification of tumor-reactive TCRs with potential for future patient-individualized cell therapies and suggest the relevance of immune responses against alternative epitopes, such as cancer/testis antigens, in hematological malignancies with low mutational loads.

Low Mutation Load in a Supergene Underpinning Alternative Male Mating Strategies in Ruff (Calidris pugnax).

A paradox in evolutionary biology is how supergenes can maintain high fitness despite reduced effective population size, the suppression of recombination, and the expected accumulation of mutational load. The ruff supergene involves 2 rare inversion haplotypes (satellite and faeder). These are recessive lethals but with dominant effects on male mating strategies, plumage, and body size. Sequence divergence to the wild-type (independent) haplotype indicates that the inversion could be as old as 4 million years. Here, we have constructed a highly contiguous genome assembly of the inversion region for both the independent and satellite haplotypes. Based on the new data, we estimate that the recombination event(s) creating the satellite haplotype occurred only about 70,000 yr ago. Contrary to expectations for supergenes, we find no substantial expansion of repeats and only a modest mutation load on the satellite and faeder haplotypes despite high sequence divergence to the non-inverted haplotype (1.46%). The essential centromere protein N (CENPN) gene is disrupted by the inversion and is as well conserved on the inversion haplotypes as on the noninversion haplotype. These results suggest that the inversion may be much younger than previously thought. The low mutation load, despite recessive lethality, may be explained by the introgression of the inversion from a now extinct lineage.

EPEN-05. TRANSCRIPTIONAL FACTOR-DIRECTED EPIGENETIC REGULATION IN CANCER NEUROSCIENCE OF ZFTA-RELA EPENDYMOMA

Abstract ZFTA-RELA (ZRFUS) ependymoma is a pediatric brain tumor with dismal 10-year progression free survival rates. Prior studies revealed low somatic mutation load in pediatric ependymoma compared to adult glioma, suggesting that developmental mechanisms play a key role during disease development. Using human tissues and mouse tumors generated from our autochthonous in utero electroporation (IUE) model, we identified a cohort of developmental transcription factors (TFs) linked to ZRFUS by investigating histone serotonylation changes with other epigenetic datasets. We further performed in vivo barcode-screening and identified the ETS variant transcription factor 5 (ETV5) is sufficient and required for disease progression. To further reveal the disease mechanism driven by ETV5, we applied transcriptomic, epigenetic, and proteomic analyses, and found ETV5 suppresses neuropeptide Y (NPY) expression in mouse ZRFUS tumors by binding with epigenetic modulator, chromobox 3 (CBX3). We further found NPY overexpression delays tumor progression and suppressed tumor-driven neuronal activity in mice. This study suggests developmental TF can shape tumor-promoting microenvironment during disease progression through epigenetic regulation.

Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation

BackgroundAmong six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers.ResultsIn this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious mutations, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records.ConclusionsThe identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos.

Abstract 1395: Genomic and transcriptomic analyses identify distinct features of triple-negative inflammatory breast cancer

Abstract Background: Triple-negative inflammatory breast cancer (TN-IBC) is the most aggressive breast cancer. It is important to identify its unique genetic and molecular features. In this study we performed comprehensive genomic and transcriptomic analyses of TN-IBC patient samples and identified TN-IBC unique features. Methods: We collected matched pretreatment blood and tumor samples from 19 patients with primary TN-IBC enrolled in a phase II clinical trial (NCT01036087). Patients were treated with neoadjuvant chemotherapy (NAC) or the anti-EGFR antibody panitumumab (PmAb) plus NAC (PmAb/NAC). Whole-exome sequencing, RNA sequencing, and multiplexed immunofluorescent staining were used to compare genetic and molecular profiles of these TN-IBC samples with 60 similarly analyzed stage III TN-non-IBC patient samples. We also compared the genetic and molecular characteristics of TN-IBC in patients who had a pathologic complete response (pCR) to NAC or PmAb/NAC treatment, a suitable surrogate end point for IBC, with those who did not have pCR (non-pCR). Results: Most TN-IBC patients (90%) had somatic gene alterations, most frequently in TP53 (42%), GATA3 (37%), and PI3KCA (26%). TN-IBC had distinct molecular and cellular differences from TN-non-IBC: a higher frequency of alteration of GATA3 (adj-p=0.001), KEAP1 (adj-p=0.05), CDKN1B (adj-p=0.05), amplification of ARNT, DDR2, BCL9, LMNA, and NDRG1, lower mutation load, and fewer copy number variations (p<0.0001). TN-IBC had significantly higher expression of BRCA1, BRCA2, PIK3CA, PALB2, and RB1 (log2FC≥1, adj-p<0.0001), enriched mitotic spindle and protein secretion hallmark gene sets, fewer CD8 T cells (p=0.01), and more γδ T cells (p=0.004) and M1 macrophages (p=0.01) than TN-non-IBC. TN-IBC patients in the NAC arm who had pCR had significantly higher somatic mutation load (p=0.04), upregulated expression of KRT36, KRT43P, and OR6K3 (log2FC≥1, adj-p<0.0001), enriched MTORC1 signaling and E2F targets, and a significantly higher percentage of M2 macrophages (p=0.04) than those without pCR. Patients with pCR to PmAb/NAC had upregulated expression of SERPINA9, OR52N5, and CLEC3A (log2FC≥1, adj-p<0.01), enriched interferon-α and -γ responses, and significantly lower percentages of monocytes (p=0.03) and M0 macrophages (p=0.05) compared to non-pCR patients. Conclusion: Our study demonstrated distinct genetic abnormalities, immune responses, and molecular characteristics associated with TN-IBC as well as differences related to patients’ responses to NAC and PmAb/NAC. This first comprehensive genomic, transcriptomic, and immune profiling of the largest TN-IBC patient cohort improves our understanding of the molecular landscape of the most aggressive subtype of breast cancer. Our analysis could lead to the discovery of novel prognostic biomarkers and druggable targets for TN-IBC. Citation Format: Xiaoping Wang, Li Zhao, Takashi Semba, Xingzhi Song, Mark Knafl, Savitri Krishnamurthy, Shan Shao, Larry W. Coffer II, Angela Alexander, Anita Wood, Swetha Bopparaju, Wendy A. Woodward, Randy Chu, Jianhua Zhang, Scott Woodman, Andrew Futreal, Clinton Yam, Debu Tripathy, Naoto T. Ueno. Genomic and transcriptomic analyses identify distinct features of triple-negative inflammatory breast cancer [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 1395.

Abstract 4734: BRD8-driven EP400 complex hijacks H2AZ to maintain proliferation in glioblastoma

Abstract Glioblastoma (GBM) is the most notorious primary brain tumor. The median survival of GBM patients is only about one year and about 95% patients succumb after five years. This gloomy picture has not been improved for decades, even with extensive treatments including surgery, radiotherapy, and chemotherapy. Hence, a better understanding of the mechanisms underlying GBM development may provide new therapeutic opportunities. Primary GBM generally harbors a low mutational load when compared to other human cancers. Even P53—the guardian of the genome that is essentially disabled in nearly all human cancers, remains unmutated in about 71% of GBM cases. Moreover, these P53 wildtype GBM cases are as aggressive as those with P53 mutations. To explore the mechanism by which P53 wildtype GBM thrive in the presence of intact P53, we performed CRISPR screens in a panel of human cancer cell lines using an sgRNA library specifically targeting the functional domains of ~200 chromatin regulators. We discovered that BRD8’s bromodomain—a druggable domain as shown by the tremendous success in preclinical and clinical trial in diverse cancers—is a vulnerability specifically in P53 wildtype GBM cases. Our mechanistic studies demonstrate that BRD8 functions through the EP400 chromatin remodeling complex and hijacks the histone variant H2AZ at P53 target loci, enforcing a compact chromatin state that blocks P53’s accessibility to its targets. We show that targeting the BRD8 bromodomain releases H2AZ, opens up chromatin, engages P53-mediated transactivation, and triggers growth arrest. Consistent with these findings, BRD8 is highly expressed with H2AZ in patient-derived proliferating GBM cells, and is inversely correlated with the expression of P53 targets. Our work solves a long-standing mystery in P53 wildtype GBM, and presents a promising therapeutic target for the majority of GBM patients. Citation Format: Sherine Xueqin Sun, Olaf Klingbeil, Christopher Vakoc, Alea Mills. BRD8-driven EP400 complex hijacks H2AZ to maintain proliferation in glioblastoma. [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 4734.

Immunophenotyping of Circulating and Intratumoral Myeloid and T Cells in Glioblastoma Patients

Glioblastoma is the most common and lethal primary brain malignancy that almost inevitably recurs as therapy-refractory cancer. While the success of immune checkpoint blockade (ICB) revealed the immense potential of immune-targeted therapies in several types of cancers outside the central nervous system, it failed to show objective responses in glioblastoma patients as of now. The ability of glioblastoma cells to drive multiple modes of T cell dysfunction while exhibiting low-quality neoepitopes, low-mutational load, and poor antigen priming limits anti-tumor immunity and efficacy of antigen-unspecific immunotherapies such as ICB. An in-depth understanding of the GBM immune landscape is essential to delineate and reprogram such immunosuppressive circuits during disease progression. In this view, the present study aimed to characterize the peripheral and intratumoral immune compartments of 35 glioblastoma patients compared to age- and sex-matched healthy control probands, particularly focusing on exhaustion signatures on myeloid and T cell subsets. Compared to healthy control participants, different immune signatures were already found in the peripheral circulation, partially related to the steroid medication the patients received. Intratumoral CD4+ and CD8+ TEM cells (CD62Llow/CD45ROhigh) revealed a high expression of PD1, which was also increased on intratumoral, pro-tumorigenic macrophages/microglia. Histopathological analysis further identified high PSGL-1 expression levels of the latter, which has recently been linked to increased metastasis in melanoma and colon cancer via P-selectin-mediated platelet activation. Overall, the present study comprises immunophenotyping of a patient cohort to give implications for eligible immunotherapeutic targets in neurooncology in the future.

Mutational Mechanisms in Pediatric Acute Lymphoblastic Leukemia with Multiple Relapses

Pediatric acute lymphoblastic leukemia (ALL) is generally marked by a low mutational load at initial diagnosis, which increases in subsequent relapses, partly because of hypermutation in a subset of cases (Waanders et al., 2020, Blood Canc. Disc., 1:96). This increased mutational load can be driven by intrinsic mutational processes or could be therapy related. In this study, we have investigated these mutational processes in pediatric ALL patients who suffered multiple relapses, to determine which processes are active in (relapsed) ALL and how they behave during disease progression before and after therapy. We identified 63 historical cases of ALL with multiple relapses, and selected patients by availability of material and blast percentage above 70% in multiple leukemic samples. If viable material with less than 70% blasts was available, mononuclear cells were enriched by sorting for CD10 positivity. We collected and whole genome sequenced 97 tumor samples from 28 ALL patients (27 precursor B-ALL and 1 T-ALL) with 2 (n=17) or more (n=11) relapses, and 44 remission samples. Somatic single nucleotide variants (SNVs) were called by Mutect2. SNVs were clustered per sample based on their dynamics in time and all clusters in all patients were included for de novo signature extraction using the R package MutationalPatterns. A mutational process was deemed active in a sample if >150 SNVs and >10% of SNVs in a cluster were attributed to a mutational process by de novo signature extraction. The majority of ALL patients with multiple relapses had a mutational load above average, compared to an unselected diagnosis cohort, which often increased during disease progression. Six cases (21%) passed the hypermutation threshold of 1.3 mutations/Mb as previously defined (Waanders et al., 2020). However, mutational signature analysis revealed that mutational processes were active in a much larger proportion of cases. We extracted 7 single base substitution (SBS) signatures, of which 4 were known COSMIC signatures (cosine similarity >0.95) and 2 were similar to known COSMIC signatures (cosine similarity >0.8). At time of diagnosis 4 distinct mutational processes were found to be active alone or in combination in 10 cases (36%), which is much higher than an unselected diagnosis cohort (13%). These processes included APOBEC-associated mutagenesis (SBS2 & SBS13), a mutational pattern resembling UV-like damage (SBS7a), a process that may be caused by reactive oxygen species (SBS18-like) and an unknown process (Fig. 1). By clustering SNVs per patient in time we followed the activity of mutational processes and identified rising and falling (sub)clones. APOBEC mutagenesis was found to be an ongoing process, sometimes appearing for the first time at relapse (Fig. 1B), although when xenografted in mice (n=2) no continued mutagenesis was observed. SBS7a or SBS18-like mutagenesis always occurred already at diagnosis. One patient, who relapsed 5 times, acquired at each presentation between 500-1500 new SNVs with a specific pattern that did not match any known cosmic mutational signatures, and may represent a novel intrinsic mutational mechanism. At time of relapse, additional (mostly treatment-related) mutational processes became active, eventually affecting 17/28 (61%) patients (Fig. 1A), much higher than observed in cases with single relapses (~40%; Li et al., 2020, Blood, 135:41). We observed the thiopurine-associated SBS87 in 9 (32%) patients, and an unexplained SBS86-like mutational signature in 2 patients (Fig. 1B). Furthermore, we noticed SBS17-like mutagenesis in one patient. The etiology of SBS17 is as yet not established, but it has been proposed to be caused by reactive oxygen species. To conclude, ALL patients with multiple relapses showed an increased prevalence and large variety of interplaying mutational processes at diagnosis (36%) and final relapse (61%), with intrinsic or therapy induced origins. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genomic trajectories of a near-extinction event in the Chatham Island black robin

BackgroundUnderstanding the micro-­evolutionary response of populations to demographic declines is a major goal in evolutionary and conservation biology. In small populations, genetic drift can lead to an accumulation of deleterious mutations, which will increase the risk of extinction. However, demographic recovery can still occur after extreme declines, suggesting that natural selection may purge deleterious mutations, even in extremely small populations. The Chatham Island black robin (Petroica traversi) is arguably the most inbred bird species in the world. It avoided imminent extinction in the early 1980s and after a remarkable recovery from a single pair, a second population was established and the two extant populations have evolved in complete isolation since then. Here, we analysed 52 modern and historical genomes to examine the genomic consequences of this extreme bottleneck and the subsequent translocation.ResultsWe found evidence for two-fold decline in heterozygosity and three- to four-fold increase in inbreeding in modern genomes. Moreover, there was partial support for temporal reduction in total load for detrimental variation. In contrast, compared to historical genomes, modern genomes showed a significantly higher realised load, reflecting the temporal increase in inbreeding. Furthermore, the translocation induced only small changes in the frequency of deleterious alleles, with the majority of detrimental variation being shared between the two populations.ConclusionOur results highlight the dynamics of mutational load in a species that recovered from the brink of extinction, and show rather limited temporal changes in mutational load. We hypothesise that ancestral purging may have been facilitated by population fragmentation and isolation on several islands for thousands of generations and may have already reduced much of the highly deleterious load well before human arrival and introduction of pests to the archipelago. The majority of fixed deleterious variation was shared between the modern populations, but translocation of individuals with low mutational load could possibly mitigate further fixation of high-frequency deleterious variation.