Background Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. Methods We developed transgenic mice carrying D oxycycline (Dox)- i nducible SB mutag e nesis s y stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8 + T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 + T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively. Results Under chronic stimulation, SB -mutagenized CD8 + T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. Conclusions Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.

Despite advances in screening and treatment, breast cancer remains a leading cause of cancer-related mortality. APOBEC enzymes, particularly APOBEC3B (A3B), are upregulated in many cancers, contributing to a characteristic C-to-T mutational signature found in 30-50% of breast cancers. However, the relationship between A3B mutational signatures and A3B expression across subtypes, and the resulting potential biologic consequences, have not been fully defined. Using TCGA and ICGC datasets, we analyzed DNA and RNA expression data to assess the relationship between A3B mRNA expression and APOBEC enrichment scores. Pathway enrichment analyses (KEGG, GO, Reactome) were performed to identify biological processes associated with high A3B expression, specifically stratifying by breast cancer intrinsic subtypes (HR+/HER2-, HR+/HER2+, HR-/HER2+, and TNBC). Over 64% of tumors with enriched A3B mutational genomic signatures demonstrated above-median A3B mRNA expression (p < 0.001). High A3B-expressing tumors exhibited specific alterations in drug metabolism pathways. Notably, we observed reduced expression of CYP2D6 and CYP3A isoforms which is required for the conversion of tamoxifen to its active metabolites. Conversely, genes involved in pyrimidine metabolism, including IMPDH1, NME1, TK1, and DPYS, were downregulated in high A3B tumors. Elevated A3B expression correlates with mutational signatures and may contribute to impaired tamoxifen activation and endocrine resistance, while concurrently creating metabolic vulnerabilities to pyrimidine-based chemotherapies. Targeting A3B or exploiting these metabolic dependencies may improve therapeutic response in selected patient subsets.

Endocrine therapy resistance remains a major challenge in the treatment of advanced estrogen receptor positive (ER+) breast cancer. This can be driven by acquired mutations in the estrogen receptor gene (ESR1), such as Y537S or D538G, that results in constitutive estrogen-independent ER activity. Progesterone receptors (PR) are important modifiers of ER activity, in part via direct binding. We previously showed that PR mediates expansion of cancer stem-like cell (CSC) populations. In this study, we sought to define whether PR function changes in the context of ESR1 mutations. PR readily interacted with wild type (WT), but not Y537S or D538G ERs. RNA-seq and ChIP-seq studies demonstrated that ER+ breast cancer models expressing Y537S ER exhibited a distinct response to progesterone. CSC populations were enhanced in Y537S ER+ cells compared to WT ER+ cells. PR knockdown demonstrated that this property required PR expression but was unresponsive to antiprogestins. Moreover, we identified PR-dependent transcriptional programs such as the unfolded protein response (UPR) that can be leveraged to target CSCs in Y537S ESR1-mutant breast cancer. Our findings demonstrate an interplay between PR and mutant ER function and provide insight into PR-driven pathways that can be exploited as potential therapeutic avenues in ER+ breast cancer.

Mutations in somatic cells are inflicted by both extrinsic and intrinsic sources and contribute over time to cancer. Tobacco smoke contains chemical carcinogens that have been causatively implicated with cancers of the lung and head & neck1,2. APOBEC family DNA cytosine deaminases have emerged as endogenous sources of mutation in cancer, with hallmark mutational signatures (SBS2/SBS13) that often co-occur in tumors of tobacco smokers with an equally diagnostic mutational signature (SBS4)3,4. Here we challenge the dogma that mutational processes are thought to occur independently and with additive impact by showing that 4-nitroquinoline 1-oxide (NQO), a model carcinogen for tobacco exposure, sensitizes cells to APOBEC3B (A3B) mutagenesis and leads to synergistic increases in both SBS2 mutation loads and oral carcinomas in vivo. NQO-exposed/A3B-expressing animals exhibit twice as many head & neck lesions as carcinogen-exposed wildtype animals. This increase in carcinogenesis is accompanied by a synergistic increase in mutations from APOBEC signature SBS2, but not from NQO signature SBS4. Interestingly, a large proportion of A3B-catalyzed SBS2 mutations occurs as strand-coordinated pairs within 32 nucleotides of each other in transcribed regions, suggesting a mechanism in which removal of NQO-DNA adducts by nucleotide excision repair exposes short single-stranded DNA tracts to enzymatic deamination. These highly enriched pairs of APOBEC signature mutations are termed didyma (Greek for twins) and are mechanistically distinct from other types of clustered mutation (omikli and kataegis). Computational analyses of lung and head & neck tumor genomes show that both APOBEC mutagenesis and didyma are elevated in cancers from smokers compared to non-smokers. APOBEC signature mutations and didyma are also elevated in normal lung tissues in smokers prior to cancer initiation. Collectively, these results indicate that DNA adducting mutagens in tobacco smoke can amplify DNA damage and mutagenesis by endogenous APOBEC enzymes and, more broadly, suggest that mutational mechanisms can interact synergistically in both cancer initiation and promotion.

PELP1 and SRC-3 function as essential coregulatory proteins in normal development and cancer. Overexpression of SRC-3 and PELP1 independently influence cancer cell biology through the regulation of 1) proliferation; 2) apoptosis and autophagy; 3) migration, invasion, and metastasis; and 4) therapy resistance. PELP1 and SRC-3 have both nuclear and cytoplasmic functions, and prior reports indicate that cytoplasmic localization of SRC-3 is necessary for SRC-3 phosphorylation that impacts the transcriptional coregulatory function of SRC-3. Cytoplasmic localization of PELP1 promotes tamoxifen and paclitaxel resistance, and breast cancer stem-like cell (CSC) phenotypes. We previously showed that cytoplasmic and nuclear PELP1/SRC-3 complexes are enriched in ER+ therapy resistant breast cancer. Our published data suggests that altered PELP1/SRC-3 signaling leads to altered gene expression that contributes to the expansion of breast CSC population in therapy resistant ER+ breast cancer. To better understand the transcription and epigenomic landscape of this therapy resistant model, we used a combination of RNA-seq and CUT&RUN. Genes previously shown to be associated with paclitaxel resistance were found upregulated in MCF7 TaxR CSC-enriched tumorspheres (i.e. ABCB1, TUBB2B), with ABCB1 being the most differentially expressed gene compared to parental MCF7 cells. RNA-seq analysis suggests that an upregulation of genes involved in interferon and inflammatory response, as well and Ras signaling and EMT in TaxR cells. We also identified 114 overlapping genes from our TaxR model and our previously published cytoplasmic PELP1 model. Pathway analysis indicates that these genes are regulated by nuclear receptors (i.e. ER and PR), which is supported by an increase in PR expression in TaxR CSC-enriched 3D cultures. CUT&RUN data indicates an enrichment in PELP1/SRC-3 overlapping peaks in TaxR cells. Genes associated with overlapping peaks are associated with androgen response, cholesterol homeostasis, notch signaling, protein secretion and TGF-beta signaling MSigDB Hallmark pathways. Together these data support our hypothesis that PELP1/SRC-3 complexes are reprogrammed to promote therapy resistances and CSC phenotypes. We aim to use these studies to identify pathways that can be targeted within CSCs to eliminate this quiescent population and prevent breast cancer metastasis. Julie Hanson Ostrander, Thu Truong, Marygrace Trousdell, Kyla Hagen, Nuri Alpay Temiz, Michael Ciccone, Fatemah Iman Dewji, Camila dos Santos, Carol A. Lange. Transcriptional and epigenomic landscape of paclitaxel resistant MCF-7 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1425.

Genes that enhance T cell function represent promising targets for improving engineered T cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T cell persistence, employing Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-(GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. We developed transgenic mice carrying Doxycycline (Dox)-inducible SB mutagenesis system (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T cell persistence under chronic antigen exposure. Specifically, CD8⁺ T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8⁺ T cells using enhanced-specificity tagmentation sequencing (esTag-seq) and RNA-seq, respectively. Under chronic stimulation, SB -mutagenized CD8⁺ T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation stress. Among these, T2/Onc2 insertions into Bach2 and Elmo1 were repeatedly found at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T cell memory formation and resistance to chronic viral infection but less characterized in engineered T cells for cancer therapy, was found to enhance in vivo tumor persistence in the B16-Ova tumor model. We showed that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2low signature allowed differentiation into both KLRG1⁺ and CD62L⁺ phenotypes, whereas Bach2high restricted differentiation predominantly to the CD62L⁺ subset. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and improved tumor control following chronic cancer stimulation. Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T cell therapeutic phenotypes. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation. ### Competing Interest Statement D.A.L. is the co-founder and co-owner of NeoClone Biotechnologies, Discovery Genomics. (acquired by Immusoft), B-MoGen Biotechnologies (acquired by Bio-Techne), and Luminary Therapeutics. D.A.L consults for Styx Biotechnologies and Genentech. S.S.K. is an inventor on patents in the field of CAR immunotherapy that are licensed to Novartis, MustangBio, Humanigen/Taran, Immix Biopharma, and Chymal Therapeutics. S.S.K. receives research funding from Kite, Gilead, Juno, BMS, Novartis, Humanigen, MorphoSys, Tolero, Sunesis/Viracta, LifEngine Animal Health Laboratories Inc, and Lentigen. S.S.K. has participated in advisory meetings with Kite/Gilead, Humanigen, Juno/BMS, Capstan Bio, and Novartis. S.S.K. consults for Torque, Calibr, Novartis, Kite, Capstan Bio, Carisma, and Humanigen. I.M.S. has served on the scientific advisory boards for Luminary Therapeutics and Immunogenesis, had a sponsored research project with Bonum Therapeutics, and has patents in human T-cell engineering constructs and the TRex mouse model. American Cancer Society Research Professor Award Minnesota Partnership for Biotechnology and Medical Genomics, https://ror.org/0417t4z61 Mayo Clinic Comprehensive Cancer Center (Minnesota) National Institutes of Health, R37CA266344 AIRP Grant

Despite advancements in screening and treatment, breast cancer mortality remains significant, with nearly one-third of patients worldwide succumbing to the disease. APOBECs, a large family of cytosine deaminase enzymes, function endogenously to restrict viral replication. However, their upregulation in many cancers has been linked to a distinct mutational signature characterized by cytosine-to-uracil conversions, leading to C-to-T transitions. Among these enzymes, APOBEC3B (A3B) is notably overexpressed in breast cancer. It remains uncertain, however, whether tumors with an enriched A3B mutational signature consistently exhibit elevated A3B expression across different breast cancer subtypes. Studies estimate the A3B mutational signature is present in 30-50% of breast cancer cases, with higher prevalence in triple-negative breast cancer (TNBC) and HER2-enriched subtypes. Understanding the pathways influencing A3B expression is an area of growing research interest. Using DNA and RNA sequencing data from breast cancer patients in The Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC), we observed that over 64% of tumors with enriched A3B genomic mutational signatures exhibited above-median A3B mRNA expression compared to non-enriched tumors (p < 0.001). Pathway analyses, including KEGG, Gene Ontology (GO), and Reactome, of high versus low A3B-expressing tumors revealed associations with pathways involved in tumor drug metabolism. Notably, the tamoxifen regulatory pathway was altered in high A3B-expressing tumors, which showed reduced expression of CYP2D6 and CYP3A10. This downregulation was consistent in luminal-A-like and luminal-B-like samples. Previous studies in mouse model systems have demonstrated diminished responses to tamoxifen in cells with high A3B expression; our findings suggest a potential relationship between high A3B expression and altered tamoxifen metabolism through CYP2D6. Additionally, pathways related to uracil synthesis, involving drugs such as 5-fluorouracil and 6-mercaptopurine, were downregulated in tumors with high A3B expression. Downregulated genes included IMPDH1, NME1, TK1, and DPYS, many of which are directly associated with pyrimidine synthesis. This pattern of downregulation was consistent across luminal-A-like, luminal-B-like, and basal-like breast cancer subtypes. These findings suggest that tumors with elevated A3B expression may have a heightened ability to develop resistance to therapies such as tamoxifen. Conversely, such tumors might exhibit increased susceptibility to treatments like 5-fluorouracil. Evaluating the functional consequences of A3B expression in breast cancers could pave the way for strategies to modulate A3B activity, enhancing the efficacy of existing therapies and mitigating drug resistance. Joel Pardo, Nuri Temiz, Douglas Yee. Examining APOBEC3B in TCGA and ICGC breast cancer datasets reveals altered drug metabolism pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2409.

The single-stranded DNA cytosine deaminase enzyme, APOBEC3B (A3B), has been implicated as a mutational driver in multiple human cancers1,2. Breast cancer in particular shows high levels of A3B expression and positive associations with poor clinical outcomes3. Recent studies have also demonstrated that human A3B expression in mice is carcinogenic with significantly shortened life expectancies4. The only genetic factor in breast cancer thus far associated with A3B expression and APOBEC signature single base substitutions is p53 mutation5. Therefore, here we test the hypothesis in vivo that human A3B expression will show pathlogical synergy in the form of accelerated mammary tumor penetrance upon combination with p53 haploinsufficiency due to a heterozygous deletion mutation spanning exons 2-10. Custom and commercially available animals were subjected to standard breeding practices to generate experimental (A3B, p53Delta exons 2-10/+) and control groups (p53Deltanexons 2-10/+). Surprisingly, in contrast to A3B-accelerated tumor development on a wildtype genetic background, A3B had no effect on the rates of tumor development in p53 heterozygous animals (median 13.5 months). The observed genetic epistasis was not due to a lack of A3B expression or activity, as evidenced by strong IHC positivity and a clear acculumlation of APOBEC signature single base substitution mutation in tumors. We conclude that A3B-accelerated tumor development requires full p53 function, and that a haploinsufficiency in p53 enables tumor cells (or their precursors) to better tolerate DNA damage lesions induced by A3B. Selected References: 1. Petljak, M. et al. Addressing the benefits of inhibiting APOBEC3-dependent mutagenesis in cancer. Nat Genet 54, 1599-1608 (2022). 2. Butler, K. & Banday, A. R. APOBEC3-mediated mutagenesis in cancer: causes, clinical significance and therapeutic potential. J Hematol Oncol 16, 31 (2023). 3. Roelofs, P. A. et al. Clinical implications of APOBEC3-mediated mutagenesis in breast cancer. Clin Cancer Res 29, 1658-1669 (2023). 4. Durfee, C. et al. Human APOBEC3B promotes tumor development in vivo including signature mutations and metastases. Cell Rep Med 4, 101211 (2023). 5. Burns, M. B. et al. APOBEC3B is an enzymatic source of mutation in breast cancer. Nature 494, 366–370 (2013).\ Citation Format: Joshua Proehl, Cameron Durfee, Yuan Zhao, Nuri Alpay Temiz, Reuben Harris. p53 Haploinsufficiency Epistatically Masks A3B Tumor Phenotype in vivo [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-06-26.

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.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by 28 cellular APOBEC3 enzymes, which results in C-to-T and C-to-G mutations in TCA and TCT motifs. 29 Although multiple enzymes have been implicated, reports conflict and it is unclear which 30 enzyme(s) are responsible. Here we develop a selectable system to quantify genome mutation 31 and compare the mutagenic activities of three leading candidates - APOBEC3A, APOBEC3B, 32 and APOBEC3H. The human cell line, HAP1, was engineered to express the thymidine kinase 33 ( TK ) gene of HSV-1, which confers sensitivity to ganciclovir. Clonal expression of APOBEC3A 34 and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggered elevated DNA 35 damage responses and increased frequencies of TK mutation. Mutant TK DNA sequences 36 revealed nearly indistinguishable cytosine mutation patterns. Whole genome sequences from TK 37 mutant clones confirmed these results and enabled broader bioinformatic analyses. Most 38 importantly, comparisons of “pure” APOBEC3A- and APOBEC3B-inflicted mutation signatures 39 from this system and the actual APOBEC3 signature from breast cancer indicated that most 40 tumors manifest a composite signature. These studies help resolve a long-standing etiologic 41 debate in the cancer field and indicate that future diagnostic and therapeutic efforts should focus 42 on both APOBEC3A and APOBEC3B. ( g -H2AX positivity), DNA breakage (alkaline COMETs), and R frequencies. Sanger sequences from panels of individual TK mutant clones revealed a clear 5’- TC-biased mutation pattern including two hotspots, which had no obvious stem-loop secondary structures. A global perspective was obtained by analyzing the full genomic DNA sequences of TK mutant clones. These WGS results demonstrated that both A3A and A3B can generate the APOBEC3 mutational signatures SBS2 and SBS13. Moreover, although these “pure” A3A- and A3B-inflicted mutation signatures each cluster with the APOBEC3 signature extracted from different subsets of breast tumors, neither the A3A- nor the A3B-preferred motifs from the HAP1- TK system are able to fully explain the observed composite APOBEC3 mutation signature in cancer suggesting that both enzymes may be acting in concert in most tumors.

Malignant peripheral nerve sheath tumors (MPNSTs) are cancers that affect the protective covering of nerves. In these cancers, a protein complex called PRC2, which is important for normal cell behavior, is often absent. The authors of this study wanted to figure out how the loss of the PRC2 may cause tumors to grow. To do this, they studied normal cells that cover nerves that lack PRC2 and compared them with MPNSTs. Their results showed that losing PRC2 changes many genes, including some that are linked to cancer development. Two important pathways, called Notch and SHH, were found to play a key role. Blocking these pathways helped slow down tumor growth.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets singlestranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have been fully established, and the specific influence of the DNA sequence on APOBEC3A APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B selectively targets DNA stem-loop structures, and they are distinct from those subjected deamination by APOBEC3A. We develop Oligo-seq, a novel in vitro sequencing-based to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A an APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Myelodysplastic syndrome (MDS) is characterized by bone marrow failure and a highly variable clinical course. The most catastrophic complication of MDS is transformation to secondary acute myeloid leukemia (sAML). Notably, mutations in TP53 confer the single highest risk of transformation to sAML and death. However, some patients with TP53 mutated MDS do not develop sAML, suggesting that additional genetic events cooperate with TP53 mutations to transform MDS to sAML. Understanding the mechanisms of transformation of MDS to sAML could provide targets for therapeutic intervention. To model the genetics of MDS, we crossed mice bearing Trp53 R270H ( Trp53 is the murine TP53 gene) and deletion of genes syntenic with human chromosome 5q (del(5q)). To discover how additional mutations contribute to disease progression, we utilized Sleeping Beauty (SB) transposon mutagenesis in Trp53 R270H/del(5q) mice. SB transposase mobilized SB mutagenic T2/Onc transposons which randomly insert within the genome. T2/Onc transposons are designed to induce gain or loss of function alterations depending on the site and orientation of insertion with respect to targeted genes. We used the Mx1-Cre transgene to activate SB transposase and T2/Onc transposition in hematopoietic progenitors. Trp53 R270H anddel(5q)(or cytogenetically normal, CN) mice were crossed to SB mice to generate donor mice of the following genotypes: Trp53 R270H/del(5q)/ SB, Trp53 R270H/CN /SB, Trp53 WT/del(5q)/ SB, Trp53 WT/CN/ SB mice, and mice without SB transposition, (no transposition, NT: Trp53 R270H/del(5q) /NT). Bone marrow cells were transplanted into recipients, and SB insertional mutagenesis was activated using pI-pC to activate Cre. Mice receiving Trp53 WT/CN/ SB bone marrow developed more frequent T-cell leukemia (n=3/10) than myeloid leukemia (n=1/10). In contrast, mice receiving Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB bone marrow developed predominantly myeloid leukemia (n=14/28) more commonly than T-cell leukemia (1/28). Mixed phenotype leukemia was seen in 7/28 of these mice. Together, these data demonstrate a strong bias towards myeloid disease in SB-mutagenized Trp53 R270H bone marrow. To identify genes with SB insertions, we performed RNA sequencing to detect SB T2/Onc transposon-endogenous genefusion transcripts. Among Trp53 WT/CN/ SB leukemias, the most common recurrent SB fusions involved Notch1 and Ikzf1 as has previously reported for SB-associated T-cell leukemias . Among Trp53 R270H/del(5q)/SB and Trp53 R270H/CN /SB leukemias, the most common recurrent SB-fusions involved Erg, Eras and Il2rb with Erg fusions detected 85% of Trp53 R270H leukemias (n=17/20). SB inserted upstream of Erg promoter indicating that these fusions likely upregulate expression of Erg. Indeed, Erg levels are significantly higher in leukemias that express SB-Erg fusions relative to leukemias that do not (p<0.0023). ERG is not recurrently mutated in human AML, but the ERG gene locus is commonly amplified, especially TP53 mutant AML. ERG is known to support normal hematopoietic stem cell self-renewal. Notably, Erg-insertions were also detected in a model of MDS expressing stabilized cyclin E with SB-mediated progression to erythroleukemia (Loeb 2019). Using gene set enrichment analysis, we found that hematopoietic stem cell and leukemic stem cell signatures are enriched in Erg-SB fusion leukemias. In our analyses of two independent data sets (TCGA and BEAT AML), stem cell signatures are also among the most highly enriched pathways in human AMLs expressing high ERG levels. Furthermore, in a human AML single cell RNA sequencing dataset (van Galen 2019), we found that ERG expression is highest in AML cells with the most immature stem and progenitor-like features. Together, these findings implicate a role for ERG as a driver of progression of MDS to AML by enhancing aberrant self-renewal. In summary, we present a novel murine model of Trp53/del(5q) MDS. In this model, Erg upregulation is associated with progression to AML and upregulation of leukemia stem cell gene expression profiles. These data implicate ERG as a major contributor to progression of MDS to secondary AML in the setting of mutant p53. Understanding the mechanisms of disease progression and self-renewal in myeloid malignancies with p53 mutations is critical to define effective therapeutic strategies in these rapidly fatal, treatment resistant diseases.

Supplemental Table 7. Immunohistochemistry validation of Immune 1 and Immune 2 GCESS

<p>Supplementary methods contains additional information describing methods used and description of all supplemental data included. Supplemental Figures Supplemental Figure 1. Real human data clustered Supplemental Figure 2. Random human data clustered Supplemental Figure 3. Permuted human data clustered Supplemental Figure 4. Real mouse data clustered Supplemental Figure 5. Random mouse data clustered Supplemental Figure 6. Permuted mouse data clustered Supplemental Figure 7. Real dog data clustered Supplemental Figure 8. Random dog data clustered Supplemental Figure 9. Permuted dog data clustered Supplemental Figure 10. Examples of 4 immunohistochemistry groups Supplemental Figure 11. KM significance in real, random, and permuted human and dog datasets. Supplemental Figure 12. KM human cluster-1 Supplemental Figure 13. KM human cluster-4 Supplemental Figure 14. KM human cluster-8 Supplemental Figure 15. KM dog cluster-3 Supplemental Figure 16. GSE212257 Array cluster- 3 association with Outcome Supplemental Figure 17. GSE212257 Array cluster-5 association with Outcome Supplemental Figure 18. GSE212257 Array cluster-3 association with Metastasis Supplemental Figure 19. GSE212257 Array cluster-5 association with Metastasis Supplemental Figure 20. GSE212257 Array cluster-7 association with Metastasis Supplemental Figure 21. Cell Cycle GCESS plotted against Immune-1 and Immune-2 GCESS Supplemental Figure 22. Model depicting how immune cell components may be preventing the occurrence of metastasis in OS patients. Supplemental Figure 23. Flowchart of OS samples utilized in this work</p>

<p>Supplementary tables detailing qRT-PCR primer sequences (S1), IHC antibodies (S2), and parameters for histological stain quantification (S3-6) described in methods.</p>

The single-stranded DNA cytosine-to-uracil deaminase APOBEC3B is an antiviral protein implicated in cancer. However, its substrates in cells are not fully delineated. Here APOBEC3B proteomics reveal interactions with a surprising number of R-loop factors. Biochemical experiments show APOBEC3B binding to R-loops in cells and in vitro. Genetic experiments demonstrate R-loop increases in cells lacking APOBEC3B and decreases in cells overexpressing APOBEC3B. Genome-wide analyses show major changes in the overall landscape of physiological and stimulus-induced R-loops with thousands of differentially altered regions, as well as binding of APOBEC3B to many of these sites. APOBEC3 mutagenesis impacts genes overexpressed in tumors and splice factor mutant tumors preferentially, and APOBEC3-attributed kataegis are enriched in RTCW motifs consistent with APOBEC3B deamination. Taken together with the fact that APOBEC3B binds single-stranded DNA and RNA and preferentially deaminates DNA, these results support a mechanism in which APOBEC3B regulates R-loops and contributes to R-loop mutagenesis in cancer.

Supplemental Table 6. Gene Cluster Expression Summary Scores. Tab 1 Human GCESS values Tab 2 Mouse GCESS values Tab 3 Dog GCESS values Tab 4 GSE212257 GCESS values

Sample clinical, outcome and sequencing metadata for human mouse and dog samples.