Single Base Substitution Mutations Research Articles

Abstract PO5-27-03: Mutational impact of APOBEC3A and APOBEC3B in a human cell line

Abstract A major source of mutation in cancer is DNA cytosine deamination by APOBEC3 enzymes resulting in C-to-T and C-to-G mutations in TCA and TCT motifs. Here, we develop a selectable system to quantify genomic mutations and compare the mutagenic activities of three leading APOBEC3 candidates - APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and nearly indistinguishable TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from TK mutant clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs over 70% of the time and APOBEC3B just under 50% of the time (Y=C/T; W=A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes are contributing in a combinatorial manner to the overall mutation landscape. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both enzymes. Citation Format: Michael Carpenter, Nuri Temiz, Mahmoud Ibrahim, Matthew Jarvis, Margaret Brown, Prokopios Argyris, William Brown, Douglas Yee, Reuben Harris. Mutational impact of APOBEC3A and APOBEC3B in a human cell line [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-03.

H3K4me1 recruits DNA repair proteins in plants.

DNA repair proteins can be recruited by their histone reader domains to specific epigenomic features, with consequences on intragenomic mutation rate variation. Here, we investigated H3K4me1-associated hypomutation in plants. We first examined 2 proteins which, in plants, contain Tudor histone reader domains: PRECOCIOUS DISSOCIATION OF SISTERS 5 (PDS5C), involved in homology-directed repair, and MUTS HOMOLOG 6 (MSH6), a mismatch repair protein. The MSH6 Tudor domain of Arabidopsis (Arabidopsis thaliana) binds to H3K4me1 as previously demonstrated for PDS5C, which localizes to H3K4me1-rich gene bodies and essential genes. Mutations revealed by ultradeep sequencing of wild-type and msh6 knockout lines in Arabidopsis show that functional MSH6 is critical for the reduced rate of single-base substitution (SBS) mutations in gene bodies and H3K4me1-rich regions. We explored the breadth of these mechanisms among plants by examining a large rice (Oryza sativa) mutation data set. H3K4me1-associated hypomutation is conserved in rice as are the H3K4me1-binding residues of MSH6 and PDS5C Tudor domains. Recruitment of DNA repair proteins by H3K4me1 in plants reveals convergent, but distinct, epigenome-recruited DNA repair mechanisms from those well described in humans. The emergent model of H3K4me1-recruited repair in plants is consistent with evolutionary theory regarding mutation modifier systems and offers mechanistic insight into intragenomic mutation rate variation in plants.

Abstract 7243: CRISPR-cas9 genetic targeting of metastatic pancreatic cancer

Abstract Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here we test the hypothesis that metastatic cancer can be genetically targeted by exploiting somatic single base substitution mutations that occur as part of normal aging prior to transformation. These passenger mutations are targetable because ~10% of them form novel tumor-specific “NGG” protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9. We studied five patients with pancreatic cancer (pc) who underwent a rapid autopsy to determine areas of evolutionary conservation that were maintained throughout all metastases in each patient’s cancer. Using whole genome sequencing validated by high-depth capture sequencing, we found that 90% of somatic PAM targets were maintained between primary carcinomas and metastases. We identified regions of loss of heterozygosity (LOH) surrounding tumor suppressor genes and oncogenes in the primary tumor where PAMs were 100% conserved in metastases. These regions of truncal LOH represent genetic vulnerabilities in these carcinomas and a CRISPR-based gene therapy approach targeting PAMs in these regions may be a novel way to genetically target metastatic cancer. Using multitarget sgRNA which cut at a known number of sites in the human genome, we determined a relationship between the number of CRISPR-Cas9 cuts and growth inhibition in PC cells. We found that 8-10 target sites in non-coding regions were sufficient to induce >95% cancer cell death. Furthermore, only 2-3 targets were required to achieve a comparable level of cell death in the presence of a DNA double strand break inhibitor. Finally, using cocultures of PC cells transduced with sgRNA targeting multiple tumor specific PAMs, we demonstrate selective cell killing of three different PC cell lines. Importantly, deep sequencing of patient-matched normal lymphocytes lacking the tumor-specific PAMs treated with the same sgRNA array exhibited no editing at the targeted loci. This work establishes a bioinformatic platform for the discovery of tumor-specific CRISPR targets in the context of metastatic disease and demonstrates selective killing of cancer cells while sparing the patient’s normal cells. These data establish the proof of principle for a gene therapy approach targeting metastatic disease. Citation Format: Kirsten Bowland, Jiaying Lai, Selina Shiqing K. Teh, Alexis Bennett, Alyza Skaist, Yan Zhang, Elizabeth Thompson, Ralph H. Hruban, Nicholas J. Roberts, Rachel Karchin, Christine A. Iacobuzio-Donahue, James R. Eshleman. CRISPR-cas9 genetic targeting of metastatic pancreatic cancer [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 7243.

Correction of substitution, deletion, and insertion mutations by 5′-tailed duplexes

Germline and somatic mutations cause various diseases, including cancer. Clinical applications of genome editing are keenly anticipated, since it can cure genetic diseases. Recently, we reported that a 5'-tailed duplex (TD), consisting of an approximately 80-base editor strand oligodeoxyribonucleotide and a 35-base assistant strand oligodeoxyribonucleotide, could edit a target gene on plasmid DNA and correct a single-base substitution mutation without an artificial nuclease in human cells. In this study, we assessed the ability of the TD to correct base substitution mutations located consecutively or separately, and deletion and insertion mutations. A TD with an 80-base editor strand was co-introduced into human U2OS cells with plasmid DNA bearing either a wild-type or mutated copepod green fluorescent protein (copGFP) gene. Among the mutations, three-base consecutive substitutions were efficiently repaired. The correction efficiencies of deletion mutations were similar to those of substitution mutations, and two to three times higher than those of insertion mutations. Up to three-base substitution, deletion, and insertion mutations were excellent targets for correction by TDs. These results suggested that the TDs are useful for editing disease-causing genes with small mutations.

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

Structure-guided inhibition of the cancer DNA-mutating enzyme APOBEC3A

The normally antiviral enzyme APOBEC3A is an endogenous mutagen in human cancer. Its single-stranded DNA C-to-U editing activity results in multiple mutagenic outcomes including signature single-base substitution mutations (isolated and clustered), DNA breakage, and larger-scale chromosomal aberrations. APOBEC3A inhibitors may therefore comprise a unique class of anti-cancer agents that work by blocking mutagenesis, slowing tumor evolvability, and preventing detrimental outcomes such as drug resistance and metastasis. Here we reveal the structural basis of competitive inhibition of wildtype APOBEC3A by hairpin DNA bearing 2′-deoxy-5-fluorozebularine in place of the cytidine in the TC substrate motif that is part of a 3-nucleotide loop. In addition, the structural basis of APOBEC3A’s preference for YTCD motifs (Y = T, C; D = A, G, T) is explained. The nuclease-resistant phosphorothioated derivatives of these inhibitors have nanomolar potency in vitro and block APOBEC3A activity in human cells. These inhibitors may be useful probes for studying APOBEC3A activity in cellular systems and leading toward, potentially as conjuvants, next-generation, combinatorial anti-mutator and anti-cancer therapies.

Human APOBEC3B promotes tumor development in vivo including signature mutations and metastases

The antiviral DNA cytosine deaminase APOBEC3B has been implicated as a source of mutation in many cancers. However, despite years of work, a causal relationship has yet to be established invivo. Here, we report a murine model that expresses tumor-like levels of human APOBEC3B. Animals expressing full-body APOBEC3B appear to develop normally. However, adult males manifest infertility, and older animals of both sexes show accelerated rates of carcinogenesis, visual and molecular tumor heterogeneity, and metastasis. Both primary and metastatic tumors exhibit increased frequencies of C-to-T mutations in TC dinucleotide motifs consistent with the established biochemical activity of APOBEC3B. Enrichment for APOBEC3B-attributable single base substitution mutations also associates with elevated levels of insertion-deletion mutations and structural variations. APOBEC3B catalytic activity is required for all of these phenotypes. Together, these studies provide a cause-and-effect demonstration that human APOBEC3B is capable of driving both tumor initiation and evolution invivo.

Single Base Substitution Mutation Profiling in Non-Matched and Matched Metastases across Various Tumor Histologies and Anatomic Sites

<h3>Purpose/Objective(s)</h3> As the landscape of oligometastatic care continues to evolve, it has become clear that not all histologies (or anatomic sites) respond equivalently to systemic therapy or radiation. This study aims to gain more insight into metastatic tumor biology by evaluating single base substitution mutations across various metastatic sites and within matched primary-metastatic patient samples. <h3>Materials/Methods</h3> We evaluated our prospectively collected institutional biorepository for metastatic (Met) tumors which underwent targeted exome sequencing for 1,327 cancer-related genes. The patient cohorts included: 1) non-matched Mets (N-Met; n=794 [707 tumors]) and 2) a subset of matched primary (Pri)-Met (M-Pri-Met; n=207 [390 tumors]). Exome data were enriched for somatic non-silent mutations by filtering out artifacts and known germline and silent mutations. Mutation signatures based on 6 nucleotide base alterations (96 subtypes) were generated and compared across samples followed by hierarchical clustering analysis to assess mutation signature correlation to the COSMIC mutation database. The mutation signatures were assessed across unique histology groups from Prim (n=17) and Met (n=16) sites and compared by Kruskal-Wallis test. Also, counts of individual mutations were related to given mutation signatures. A generalized linear mixed model was used to infer the odds ratio (OR) of mutation incidence in Pri vs Met tumors. <h3>Results</h3> Across all Mets, we found that C>T and G>A alterations were identified in 23% and 21% of tumors, respectively, whereas T>A was <2%. Notable differences were seen in the proportions of mutation signatures (n=9) across Pri histology (range p < 0.03 to 1.0e⁻¹⁶) and among different Met sites within a given histology. For example, our 6^th signature was associated with AID/APOBEC family activity and varied across Pri histology (p = 1.0e⁻¹³) and within Met sites (p = 4.3e⁻⁰⁹). In contrast, our 4^th signature was related to <i>ERCC2</i> mutation, but only differed across Pri histology and not Met site. The median number of mutations related with mutation signatures was 27 (range: 1-1404) with the highest counts in melanoma. Within our M-Pri-Met analysis, several genes were identified to be either private or shared among tumors. For example, within the M-Pri-Met breast (n=93) group, <i>EPHB4</i> mutation was concordant in 58% of samples with a LogOR 1.38 (95% CI 1.2-13.2; p=0.02) in Pri vs Met. In contrast, <i>BRAF</i> mutation was 40% concordant with higher odds of being in Met vs Pri (p=0.04). No significant differences were identified in the lung M-Pri-Met cohort (n=45). Though, individual mutations did not appear to be different in lung histology, we did observe differences in the proportions of mutation signatures within individual patient tumors. <h3>Conclusion</h3> A deeper knowledge of similarities and differences among metastases may aid in biologically-informed metastasis-directed therapy. This analysis highlights diversity in mutation type and incidence among metastases.

No evidence of increased mutations in the germline of a group of British nuclear test veterans

The potential germline effects of radiation exposure to military veterans present at British nuclear tests in Australia and the South Pacific is of considerable interest. We analyzed germline mutations in 60 families of UK military personnel comprising 30 control and 30 nuclear test veterans (NTV). Using whole-genome sequencing we studied the frequency and spectra of de novo mutations to investigate the transgenerational effect of veterans’ (potential) exposure to radiation at nuclear bomb test sites. We find no elevation in total de novo single nucleotide variants, small insertion-deletions, structural variants or clustered mutations among the offspring of nuclear test veterans compared to those of control personnel. We did observe an elevated occurrence of single base substitution mutations within mutation signature SBS16, due to a subset of NTV offspring. The relevance of this elevation to potential exposure of veteran fathers and, future health risks, require further investigation. Overall, we find no evidence of increased mutations in the germline of a group of British nuclear test veterans. ISRCTN Registry 17461668.

Mutations in PmUFGT3 contribute to color variation of fruit skin in Japanese apricot (Prunus mume Sieb. et Zucc.)

BackgroundJapanese apricot (Prunus mume Sieb. et Zucc.) is popular for both ornamental and processing value, fruit color affects the processing quality, and red pigmentation is the most obvious phenotype associated with fruit color variation in Japanese apricot, mutations in structural genes in the anthocyanin pathway can disrupt the red pigmentation, while the formation mechanism of the red color trait in Japanese apricot is still unclear. ResultsOne SNP marker (PmuSNP_27) located within PmUFGT3 gene coding region was found highly polymorphic among 44 different fruit skin color cultivars and relative to anthocyanin biosynthesis in Japanese apricot. Meantime, critical mutations were identified in two alleles of PmUFGT3 in the green-skinned type is inactivated by seven nonsense mutations in the coding region, which leads to seven amino acid substitution, resulting in an inactive UFGT enzyme. Overexpression of the PmUFGT3 allele from red-skinned Japanese apricot in green-skinned fruit lines resulted in greater anthocyanin accumulation in fruit skin. Expression of same allele in an Arabidopsis T-DNA mutant deficient in anthocyanidin activity the accumulation of anthocyanins. In addition, using site-directed mutagenesis, we created a single-base substitution mutation (G to T) of PmUFGT3 isolated from green-skinned cultivar, which caused an E to D amino acid substitution and restored the function of the inactive allele of PmUFGT3 from a green-skinned individual.ConclusionThis study confirms the function of PmUFGT3, and provides insight into the mechanism underlying fruit color determination in Japanese apricot, and possible approaches towards genetic engineering of fruit color.

Detection and Significance of Cell-Free DNA Mutation in Pleural Effusion in Patients with Advanced NSCLC.

Objective To detect EGFR/KRAS genes in pleural effusion cell-free DNA in patients with advanced non-small-cell lung cancer (NSCLC) and to explore the clinical significance of EGFR/KRAS mutation status in pleural effusion. Methods A retrospective collection was performed on the specimens of pleural effusion and matched tissues from 50 patients with advanced NSCLC admitted to the hospital between January 2019 and January 2021. DNA mutation status of EGFR/KRAS in different specimens was detected and compared by pyrosequencing. The clinicopathological data and follow-up data of survival were collected. The relationship between DNA mutation and clinicopathological characteristics and prognosis was analyzed. Results In the 50 pleural effusion specimens, there were 22 cases (44.00%) with EGFR mutations (19/21 exon mutations), including 12 cases with EGFR19 deletion mutation and 10 cases with EGFR21 exon L858R mutation. There were 6 cases (12.00%) with KRAS mutations (single-base substitution mutations), including 4 cases with 12-codon mutation and 2 cases with 13-codon mutation. In the 50 tissue specimens, there were 24 cases (48.00%) with EGFR mutations and 4 cases (8.00%) with KRAS mutations. There was no significant difference between pleural effusion specimens and tissue specimens, with good consistency (kappa = 0.920–0.779, P > 0.05). EGFR mutation in pleural effusion was related to smoking history, types of pathological tissues, and lymph node metastasis (P < 0.05). The incidence of EGFR mutation was higher in nonsmokers, patients with lung adenocarcinoma, and patients with lymph node metastasis. The carcinoembryonic antigen (CEA) in patients with EGFR mutation was higher than that with wild-type EGFR, while the level of cytokeratin 19 fragment (Cy21-1) was lower than that with wild-type EGFR (P < 0.05). The 1-year overall survival rate in the EGFR mutation group was significantly higher than that in the EGFR wild group (68.18% vs. 42.86%) (HR = 0.419, 95% CI = 0.178–0.989, and P < 0.001). Conclusion For the detection of EGFR gene mutation, the results of the pleural effusion specimens and the tumor pathological tissue specimens were well consistent and the detection of pleural effusion could be used as an alternative method when tissue specimens cannot be obtained. EGFR gene mutations are present in majority in patients with advanced NSCLC. The incidence of EGFR mutation is higher in nonsmokers, patients with lung adenocarcinoma, those with lymph node metastasis, those with high-expression CEA, and those with low-expression Cy21-1. The prognosis is better in patients with EGFR mutation.

Abstract P5-12-01: Apobec mutation signature in breast cancer explained by combinatorial action of apobec3a and apobec3b

Abstract Background: Mutations drive the initiation and progression of cancer. A leading druggable source of mutation in cancer is enzymatic deamination of single-stranded DNA cytosines by cellular APOBEC3 enzymes. Cytosine-to-uracil deamination can result in a variety of different mutational outcomes including DNA breakage and chromosomal aberrations as well as single base substitution mutations. The latter are comprised of C-to-T and C-to-G mutations in TCA or TCT trinucleotides and attributable to the intrinsic preference of several APOBEC3 family members for binding to these motifs. This mutation pattern, commonly called the “APOBEC signature”, is evident in approximately one-quarter of primary breast tumors and one-third of metastatic breast tumors. Although multiple APOBEC3 enzymes have been implicated as a source of this signature in breast cancer (namely, APOBEC3A, APOBEC3B, and APOBEC3H), the literature is full of conflicting views and it is not clear which of these enzymes contributes most significantly to the mutational landscape of breast cancer. Methods: The near-haploid human cell line, HAP1, was engineered to express the HSV-1 TK gene as a mutation reporter. Candidate APOBEC3 enzymes were expressed individually and confirmed by immunoblotting and activity assays. DNA breakage was measured directly by COMET assays and DNA damage responses indirectly by phosphorylated gamma-H2AX staining. Mutation frequencies were quantified by assaying rates of drug resistance, and mutation patterns were analyzed by sequencing locally in TK and globally across whole genomes. Results: APOBEC3A and APOBEC3B both caused significant increases in chromosomal DNA breakage and DNA damage responses. These enzymes also elevated drug resistance mutation frequencies. In contrast, expression of active APOBEC3H or catalytic mutant derivatives of APOBEC3A and APOBEC3B failed to trigger increases beyond normal spontaneous levels. Interestingly, APOBEC3A and APOBEC3B both inflicted mutation signatures that were indistinguishable locally in TK and globally across whole genomes. The vast majority of these APOBEC signature mutations were dispersed (non-kataegic) and not associated with obvious mesoscale chromosomal features such as single-stranded loop regions of stem-loop structures. Computational comparisons of the broader pentanucleotide APOBEC3A and APOBEC3B mutation signatures and those extracted from 794 primary breast tumor genomes (ICGC cohort) revealed an APOBEC3A-biased subset, an APOBEC3B-biased subset, and a larger group of tumors best explained by combinatorial action of both of these enzymes. Conclusions: Our results indicate that APOBEC3A and APOBEC3B contribute combinatorially in most instances to the observed APOBEC mutation signature in breast cancer. These results provide a framework for developing diagnostic and therapeutic approaches for APOBEC-positive breast cancer. Citation Format: Reuben S Harris, Matthew C Jarvis, Michael A Carpenter, Margaret R Brown, Prokopios P Argyris, William Brown, Douglas Yee. Apobec mutation signature in breast cancer explained by combinatorial action of apobec3a and apobec3b [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-01.

Abstract P5-01-10: A panel of murine mammary tumor cell lines to study immune responses to epitopes created by different mutagens including human APOBEC3 enzymes

Abstract Background: Tumor mutation loads are a strong predictor of immunotherapy benefit. This is evidenced by studies on skin, lung, and colon cancer, where well-defined processes elicit high mutation burdens and checkpoint therapies often provoke strong anti-tumor immune responses. In comparison, only a small number of breast cancer patients have responded to checkpoint therapies such as anti-PD-L1 and anti-PD-1 monoclonal antibodies. Approximately one-fourth of primary breast cancer and one-third of metastatic tumors have a significant APOBEC mutation burden defined as C-to-T and C-to-G mutations in TCA or TCT trinucleotide motifs, and many tumors have &amp;gt;10,000 of these APOBEC signature single base substitution mutations. However, to our knowledge, systematic immunotherapy studies focusing on this unique, hypermutated subset of breast cancers have yet to be done. Methods: To address the potential relationship between different checkpoint therapies and APOBEC mutation burden, we created a panel of &amp;gt;30 mammary tumor cell lines derived from fully immune-proficient C57BL/6 animals. At least 6 of these cell lines are immortalized, readily engineered using standard molecular biology procedures, and capable of re-engraftment and tumor formation in immune proficient C57BL/6 animals. Two lines were engineered to inducibly express human APOBEC3A, subjected to 1- to 20-rounds of sublethal mutagenesis, and tested systematically for checkpoint therapy responsiveness. Results: Two cell lines were engineered to inducibly express APOBEC3A or a catalytic mutant (E72A). Clones were characterized for inducible APOBEC3A expression and associated DNA damage responses. After varying rounds of mutagenesis, clones were engrafted into the subcutaneous flank or mammary fat pad of C57BL/6 animals. Immunohistochemical and flow cytometry analysis revealed a robust intratumoral CD8 T cell population including a subset with PD-1 expression. Additionally, engrafted tumor cells expressed high levels PD-L1. Experiments are ongoing and immunotherapy responses will be presented. Conclusions: We have isolated a panel of C57BL/6 mammary tumor-derived cell lines and demonstrated re-engraftment into immunocompetent animals. These cell lines constitute a platform for studying the relationship between different mutagens including APOBEC and immunotherapy responses. Citation Format: Jordan Naumann, Michael Carpenter, Prokopios Argyris, William Brown, Reuben Harris. A panel of murine mammary tumor cell lines to study immune responses to epitopes created by different mutagens including human APOBEC3 enzymes [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-01-10.

Abstract PD1-06: Apobec mutagenesis is a pervasive feature of poor prognosis breast cancer associating with ESR1 wild type, endocrine resistant disease

Abstract Background: Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes drive genomic instability in breast cancer (BC) characterized by specific single base substitution mutation signatures (COSMIC S2 and S13). Computational algorithms have the potential to derive signatures from targeted sequencing assays with relatively small genomic footprints. We sought to develop a computational approach to identify APOBEC mutagenesis in samples subjected to targeted sequencing and to characterize the genomic and clinical features of APOBEC-positive BC. Methods: We retrieved clinical and genomic data from 4,595 consecutive BCs that had been subjected to targeted sequencing using the FDA-approved MSK-IMPACT assay. Additional whole exome sequencing (WES) data were retrieved from publicly available datasets, including TCGA (primary BC, n=1019) and Bertucci et al. cohorts (metastatic BC, n=617). APOBEC-related mutational signatures were computed by using the SigMA algorithm (Gulhan, Nat Genet 2019) for MSK-IMPACT samples with at least 5 single nucleotide variants (SNV) (n=2,983). Survival analyses were performed by using Kaplan Meier method with log-rank test and Cox proportional-hazards models. Results: A significant positive correlation between APOBEC exposure calculated by SigMA vs other computational tools (e.g., DeconstructSig, SigProfiler, MutationalPatterns) was found (r=0.99). After down-sampling the WES data from the TCGA BC dataset to the genomic footprint of MSK-IMPACT, SigMA showed high concordance in determining APOBEC exposures (r=0.78). BCs with high APOBEC exposure were enriched for pathogenic somatic mutations affecting several genes, including PIK3CA, CDH1 and GATA3 (q&amp;lt;0.05), as compared to tumors with no/low APOBEC. In the MSK-IMPACT cohort, median APOBEC mutational exposure was significantly higher in metastatic than primary samples (0.27 vs 0.19, p&amp;lt;0.001) and was the dominant mutational signature in 26% of primary and 34% of metastatic BC. A significant enrichment for APOBEC exposure in the metastatic setting was observed in both hormone receptor-positive (HR+) (p&amp;lt;0.001) and triple-negative (TN) (p&amp;lt;0.001) subtypes. Invasive lobular BCs displayed a higher proportion of cases with high APOBEC mutagenesis when compared to invasive ductal BCs in both primary and metastatic setting (p&amp;lt;0.001). Enrichment of APOBEC in metastatic samples could be due to signature acquisition or enrichment in poor prognosis tumors, to address this, we assessed the presence of APOBEC mutational exposure in patients with paired samples, which included 352 primary/metastasis (P/M) cases and 514 metastasis/metastasis (M/M) cases. Strikingly, no statistically significant increment of APOBEC exposure was found in the P/M nor in the M/M cohort (Wilcoxon signed-rank test p&amp;gt;0.05), overall and in different BC subtypes. We also assessed the association between APOBEC and response to endocrine therapy (ET). Patients with metastatic HR+ BC and APOBEC as dominant signature had a statistically significant lower progression-free survival (PFS) on first-line single-agent ET (aromatase inhibitors or SERDs; median PFS of 9.7 versus 14.7 months, p&amp;lt;0.001). This difference was more pronounced in patients treated with SERD with a median PFS of 4.7 and 14.3 months for APOBEC-dominant and APOBEC non-dominant tumors (p&amp;lt;0.001), respectively. Conclusions: Evidence for APOBEC mutagenesis is present in &amp;gt;30% of metastatic breast cancers and associated with resistance to ET. Unlike ESR1 mutations, which are acquired as a late event in metastatic HR+ BCs, APOBEC mutagenesis is commonly present in paired primary and metastatic BCs, indicating its relatively early onset and potential role in driving the poor prognosis of these cancers. Citation Format: Antonio Marra, Andrea Gazzo, Pier Selenica, Xin Pei, Avantika Gupta, Fresia Pareja, Giuseppe Curigliano, Reuben Harris, Nadeem Riaz, Jorge S. Reis-Filho, Sarat Chandarlapaty. Apobec mutagenesis is a pervasive feature of poor prognosis breast cancer associating with ESR1 wild type, endocrine resistant disease [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-06.

CRISPR/Cas9 System-Mediated Gene Editing in the Fujian Oysters (Crassostrea angulate) by Electroporation

The Fujian oyster (Crassostrea angulate) is an important marine bivalve mollusk with high economic value. Gene function research and gene editing techniques have broad application prospects in oyster. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for genome engineering in many species. CRISPR-mediated gene editing has also been used successfully in the Pacific oyster through direct delivery of the CRISPR/Cas9 components into oyster embryos by microinjection. However, the low throughput and operational difficulties associated with microinjection is one of the factors limiting the widespread application of CRISPR/Cas9 in oysters. In this study, we attempted to deliver the CRISPR/Cas9-system into the embryos of C. angulate by electroporation. An all-in-one CRISPR/Cas9 vector plasmid was used as CRISPR/Cas9 system in this study. Electroporation was carried out using both eggs and blastula larvae. A large number of larvae became malformed or die after electroporation. A single base substitution mutation was detected in the D-larvae developed from electroporated eggs. Our results demonstrate that the CRISPR/Cas9 system can be delivered into embryos of C. angulate for gene editing by electroporation, which provides a reference and will further contribute to the future application of electroporation in mollusks.

Abstract IA04: Drug resistance in end-stage high-grade serous ovarian cancer

Abstract Background: Acquired drug resistance is the major obstacle in controlling high-grade serous ovarian cancer (HGSC) and leads to poor overall survival. Comparatively little massively parallel sequencing data exist from HGSC patients with recurrent or end-stage disease who have been extensively treated with chemotherapy or newer targeted agents, such as antiangiogenics or PARP inhibitors (PARPi). We previously identified four mechanisms of acquired resistance from whole-genome sequencing (WGS) of 23 HGSC patients with recurrent disease; a resistance mechanism of any kind was only identified in half of the patients. Aim: To characterize genomic features and identify mechanisms of acquired drug resistance in end-stage HGSC patients. Methods: Since 2012, we have collected tumor samples from 19 HGSC patients through our rapid-autopsy program. On average, we collect 17 tumor sites per patient during the autopsy, which is performed a median 6 hours after death. SNP array, WGS, and RNA sequencing were performed to detect germline and somatic mutations, copy number alterations and gene expression patterns. Results: A total of 89 end-stage tumor samples from 15 patients with germline or somatic mutations in components of the homologous recombination (HR) DNA repair pathway have been analyzed by SNP array, and a subset of those have undergone WGS (n=44) and RNAseq (n=47). As expected, all samples had a somatic pathogenic TP53 mutation and have highly aberrant somatic copy number profiles. Prominent mutational signatures (single- and double-base substitutions) are associated with defective HR and prior treatment with platinum drugs. The total single-base substitution mutation burden is similar across tumor samples within patients; however, many mutations are unique to individual tumor samples. Currently, reversion mutations, which are secondary mutations in HR genes, are the most common resistance mechanism identified in our cohort of end-stage patients. Conclusion: Our data are allowing us to understand HGSC at end stage, by examining the catalogue of resistance mechanisms within an individual patient and reconstructing the natural history of HGSC. Citation Format: Elizabeth L. Christie, Kathryn Alsop, George Au-Yeung, Ahwan Pandey, Australian Ovarian Cancer Study, David D.L. Bowtell. Drug resistance in end-stage high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA04.

Whole-Exome Sequencing Analysis Identified Novel Mutations in the TSPAN12 Gene in Chinese Families with Familial Exudative Vitreoretinopathy.

Background: Familial exudative vitreoretinopathy (FEVR, OMIM 133780), characterized by incomplete retinal vascular development and pathological neovascularization, is a severe inherited retinal disorder. Mutations in 10 genes have been reported to be associated with FEVR, but this still leaves ∼50% of FEVR cases to be genetically explained. Purpose: The purpose of this study was to identify novel FEVR-causing mutations and explore the causative mutations in Chinese FEVR families. Methods: Whole-exome sequencing was performed to analyze the genomic DNA of the probands from 121 Chinese FEVR families. Sanger sequencing was carried out to verify all identified mutations. Luciferase assays were used to test the activity of a mutant protein in the Norrin-β-catenin signaling pathway. Results: Four novel heterozygous TSPAN12 (Tetraspanin 12) mutations, including two single-base substitution mutations and two small-deletion mutations, were identified in these FEVR families: c.1A>G (p.0), c.614G>A (p.G205D), c.695delT (p.V232Gfs*7), and c.833_842del (p.L278Qfs*25). Conclusion: This study revealed the causative mutations in four Chinese FEVR families and identified four novel FEVR-causing mutations, thus expanding the mutation spectrum of FEVR in the Chinese population.

Abstract 1355: Mutation signatures including APOBEC in cancer cell lines

Abstract Background: Multiple endogenous and exogenous sources of DNA damage contribute to the overall mutation burden in cancer, with distinct and overlapping combinations contributing to each cancer type. Many mutation sources result in diagnostic mutation signatures, which can be deduced from tumor genomic DNA sequences. Examples include spontaneous hydrolytic deamination of methyl-cytosine bases in CG motifs (AGEING signature) and C-to-T and C-to-G mutations in 5'-TC(A/T) motifs (APOBEC signature). Methods: The deconstructSigs R package was used to analyze single base substitution mutation signatures in over 1000 cancer cell lines. Two additional approaches were used to analyze the APOBEC mutation signature. Results: Most cell lines show evidence for multiple mutation signatures. For instance, the AGEING signature, which is the largest source of mutation in most primary tumors, predominates in the majority of cancer cell lines. The APOBEC mutation signature is enriched in cancer cell lines from breast, lung, head/neck, bladder, and cervical cancers, where this signature also comprises a large fraction of all mutations. Conclusions: The single base substitution mutation signatures of cancer cell lines often reflect those of the original tumors from which they are derived. Cancer cell lines with enrichments for distinct mutation signatures such as APOBEC have the potential to become model systems for fundamental research on the underlying mechanisms and for advancing clinical strategies to target these processes and, ultimately, constrain tumor evolvability. Citation Format: Reuben S. Harris. Mutation signatures including APOBEC in cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1355.

Mechanistic insights into staphylopine-mediated metal acquisition

Metal acquisition is vital to pathogens for successful infection within hosts. Staphylopine (StP), a broad-spectrum metallophore biosynthesized by the major human pathogen, Staphylococcus aureus, plays a central role in transition-metal acquisition and bacterial virulence. The StP-like biosynthesis loci are present in various pathogens, and the proteins responsible for StP/metal transportation have been determined. However, the molecular mechanisms of how StP/metal complexes are recognized and transported remain unknown. We report multiple structures of the extracytoplasmic solute-binding protein CntA from the StP/metal transportation system in apo form and in complex with StP and three different metals. We elucidated a sophisticated metal-bound StP recognition mechanism and determined that StP/metal binding triggers a notable interdomain conformational change in CntA. Furthermore, CRISPR/Cas9-mediated single-base substitution mutations and biochemical analysis highlight the importance of StP/metal recognition for StP/metal acquisition. These discoveries provide critical insights into the study of novel metal-acquisition mechanisms in microbes.

Mutation Signatures Including APOBEC in Cancer Cell Lines.

Background:Multiple endogenous and exogenous sources of DNA damage contribute to the overall mutation burden in cancer, with distinct and overlapping combinations contributing to each cancer type. Many mutation sources result in characteristic mutation signatures, which can be deduced from tumor genomic DNA sequences. Examples include spontaneous hydrolytic deamination of methyl-cytosine bases in CG motifs (AGEING signature) and C-to-T and C-to-G mutations in 5'-TC(A/T) motifs (APOBEC signature).Methods:The deconstructSigs R package was used to analyze single-base substitution mutation signatures in more than 1000 cancer cell lines. Two additional approaches were used to analyze the APOBEC mutation signature.Results:Most cell lines show evidence for multiple mutation signatures. For instance, the AGEING signature, which is the largest source of mutation in most primary tumors, predominates in the majority of cancer cell lines. The APOBEC mutation signature is enriched in cancer cell lines from breast, lung, head/neck, bladder, and cervical cancers, where this signature also comprises a large fraction of all mutations.Conclusions:The single-base substitution mutation signatures of cancer cell lines often reflect those of the original tumors from which they are derived. Cancer cell lines with enrichments for distinct mutation signatures such as APOBEC have the potential to become model systems for fundamental research on the underlying mechanisms and for advancing clinical strategies to exploit these processes.