Abstract: Recent studies have documented frequent evolution of clones carrying common cancer mutations in apparently normal tissues, which are implicated in cancer development 1–3 . However, our knowledge is still missing with regard to what additional driver events take place in what order, before one or more of these clones in normal tissues ultimately evolve to cancer. Here, using phylogenetic analyses of multiple microdissected samples from both cancer and non-cancer lesions, we show unique evolutionary histories of breast cancers harbouring der(1;16), a common driver alteration found in roughly 20% of breast cancers. The approximate timing of early evolutionary events was estimated from the mutation rate measured in normal epithelial cells. In der(1;16)(+) cancers, the derivative chromosome was acquired from early puberty to late adolescence, followed by the emergence of a common ancestor by the patient’s early 30s, from which both cancer and non-cancer clones evolved. Replacing the pre-existing mammary epithelium in the following years, these clones occupied a large area within the premenopausal breast tissues by the time of cancer diagnosis. Evolution of multiple independent cancer founders from the non-cancer ancestors was common, contributing to intratumour heterogeneity. The number of driver events did not correlate with histology, suggesting the role of local microenvironments and/or epigenetic driver events. A similar evolutionary pattern was also observed in another case evolving from an AKT1 -mutated founder. Taken together, our findings provide new insight into how breast cancer evolves.

Abstract: [Introduction] Proliferative lesions in the breast have been implicated in the development of breast cancer. Previous studies showed that some proliferative lesions and adjacent breast cancers shared common genetic alterations, suggesting that these originated from the same ancestral cell. However, the clonal structure of normal epithelia and their clonal history during evolution to cancer are poorly understood. In this study, we analyzed genetic profiles of normal epithelia and proliferative lesions in the cancer-borne breast to illustrate the clonal evolution of cancer from a normal epithelial cell. [Methods] Single cell-derived organoids (n=47) were established from breast milk of 4 healthy women aged 22–36 and normal breast tissue of 15 breast cancer patients aged 29–83 to evaluate somatic mutation rate in normal epithelial cells. Multiple normal lobules and proliferative lesions together with cancer lesions were collected using laser-capture micro-dissection (LCM) from fresh frozen (n=3) or formalin-fixed paraffin-embedded (n=5) surgical specimens in 9 premenopausal breast cancer patients. Somatic mutations and copy number alterations were evaluated using whole-genome sequencing. [Results] The mutation profile of single cell-derived organoids suggests that somatic mutations accumulate in normal mammary epithelial cells at a constant rate of 19.4/genome/year before menopause, and the mutation rate decreases to 6.9/genome/year after menopause. Parity was negatively associated with mutation number (-49.3 per life birth). In total, we analyzed 143 LCM samples, including those from 72 normal lobules, 43 proliferative lesions, and 19 non-invasive and 9 invasive cancer samples. Five cases showed a large expansion of proliferative lesions sharing a substantial number of somatic mutations with cancer. These lesions expanded over a distance of 35-90 mm, sharing tens to hundreds of mutations including those in breast cancer-related driver genes, such as PIK3CA, AKT1, GATA3, CBFB and PTEN, while harboring private mutations or copy number alterations of their own. Of interest, the cancers in 4 out of these 5 cases was luminal-A type invasive ducal carcinoma or ER-positive HER2-negative ductal carcinoma in situ, and characterized in common by the presence of der(1;16), concurrent whole-arm 1q gain and 16q loss, in both cancer and proliferative lesions. Phylogenetic analysis adapted with the mutation rate in normal cells predicted that der(1;16) had been acquired between puberty and early 20’s, and the common ancestors of non-cancerous and cancerous lesions emerged by early 30’s, & gt;10 years earlier than at the time of cancer diagnosis. By contrast, analysis of non-cancerous lobules unrelated to cancer showed that der(1;16)-negative non-cancer clones that had emerged after puberty stayed within a single lobule or spatially confined to adjacent lobules and rarely expanded to a large area as observed for those carrying der(1;16), even if the clones had acquired mutations in driver genes such as PIK3CA and PIK3R1, which highlighted the role of der(1;16) in wide clonal expansion. [Conclusions] Our results suggest that in some breast cancer cases, particularly in those with der(1;16), a highly recurrent translocation accounting for the major subset of Luminal A breast cancer, the clones with the funder driver alterations expanded macroscopically long before the onset of cancer, in which further clonal evolutions recursively occur multi-focally, giving rise to multiple proliferative lesions and ultimately, invasive cancers. Our findings provide new insight into the early development of breast cancer. Citation Format: Tomomi Nishimura, Nobuyuki Kakiuchi, Kenichi Yoshida, Takaki Sakurai, Tatsuki R. Kataoka, Eiji Kondoh, Yoshitsugu Chigusa, Masahiko Kawai, Morio Sawada, Takuya Inoue, Yasuhide Takeuchi, Hirona Maeda, Satoko Baba, Yusuke Shiozawa, Ryunosuke Saiki, Masahiro M. Nakagawa, Yasuhito Nannya, Yotaro Ochi, Tomonori Hirano, Yukiko Inagaki-Kawata, Kosuke Aoki, Masahiro Hirata, Eiji Suzuki, Masahiro Takada, Masahiro Kawashima, Kosuke Kawaguchi, Kenichi Chiba, Yuichi Shiraishi, Junko Takita, Satoru Miyano, Masaki Mandai, Kengo Takeuchi, Hironori Haga, Masakazu Toi, Seishi Ogawa. Clonal evolution of mammary epithelial cells into breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl) :Abstract nr P5-13-04.

Abstract: The genetic and clinical characteristics of breast tumors with germline variants, including their association with biallelic inactivation through loss-of-heterozygosity (LOH) and second somatic mutations, remain elusive. We analyzed germline variants of 11 breast cancer susceptibility genes for 1,995 Japanese breast cancer patients, and identified 101 (5.1%) pathogenic variants, including 62 BRCA2 and 15 BRCA1 mutations. Genetic analysis of 64 BRCA1/2 -mutated tumors including TCGA dataset tumors, revealed an association of biallelic inactivation with more extensive deletions, copy neutral LOH, gain with LOH and younger onset. Strikingly, TP53 and RB1 mutations were frequently observed in BRCA1- (94%) and BRCA2- (9.7%) mutated tumors with biallelic inactivation. Inactivation of TP53 and RB1 together with BRCA1 and BRCA2 , respectively, involved LOH of chromosomes 17 and 13. Notably, BRCA1/2 tumors without biallelic inactivation were indistinguishable from those without germline variants. Our study highlights the heterogeneity and unique clonal selection pattern in breast cancers with germline variants.

Abstract: DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele (most commonly p.A500fs or p.D140fs, depending on the ethnicity) is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms have not been elucidated. To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles. Vav1-Cre mediated homozygous deletion of Ddx41 resulted in embryonic lethality, suggesting that Ddx41 is indispensable for normal hematopoiesis. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 (Ddx41+/+), heterozygous Ddx41 KO (Ddx41+/-), heterozygous for the Ddx41 R525H mutation (Ddx41R525H/+), or hemizygous for the Ddx41 R525H mutation (Ddx41R525H/-), in which expression of the mutant allele was induced by tamoxifen administration. First, we assessed cell intrinsic effects of these Ddx41 alleles, using noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were reconstituted with BM from Ddx41R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF) with no development of myeloid neoplasms. However, about 20% of mice transplanted with BM derived from Ddx41R525H/- mice survived longer without showing BMF. These mice exhibited macrocytic anemia and increased platelet counts four months after tamoxifen-induction. In contrast, mice transplanted with BM from Ddx41+/- and Ddx41R525H/+ animals showed increased white blood cell counts compared to those with BM from Ddx41+/+ mice. In flow cytometry, Ddx41R525H/--derived BM-transplanted mice showed a significant increase in the number of long-term and short-term hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs) and granulocyte/macrophage lineage-restricted progenitors (GMPs), compared to those transplanted with BM from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice. Single cell RNA-seq of lineage negative cell fractions from these mice also revealed expanded stem cell fractions in mice transplanted with BM from Ddx41R525H/- mice, even though there was impaired formation of mature peripheral blood cells, which was suggestive of impaired HSPC differentiation. We also assessed the reconstitution capacity of whole BM cells from different Ddx41 mutant mice in competitive transplantation experiments. The donor chimerism of Ddx41R525H/- mice-derived cells in PB was reduced compared to that of cells derived from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice. Transcriptome analysis of stem cells (Kit+Sca-1-Linlow cells) from different Ddx41 mutant mice revealed significant changes in gene expression and splicing patterns in many genes in stem cells from all the mutant mice, with larger changes for Ddx41R525H/- than Ddx41+/- or Ddx41 R525H/+ cells. Notably, Ddx41R525H/- cells exhibited a significant upregulation of genes involved in innate immunity, whereas there was a downregulation of genes related to RNA metabolism and ribosome biogenesis. Proteomics analysis confirmed the significant downregulation of ribosomal proteins in hematopoietic cells derived from Ddx41R525H/- mice. In summary, our results revealed an essential role of Ddx41 in normal hematopoiesis. While both heterozygous Ddx41 KO and heterozygous R525H knock-in did not develop myeloid neoplasm, compound biallelic loss-of function and R525 alleles led to a compromised function of hematopoietic stem cells, which was evident from reduced competitive repopulation capacity and impaired hematopoietic differentiation, where activated innate immunity and impaired ribosome functions may play important roles. Their roles in myeloid neoplasms need further evaluation. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Current Employment. Kataoka:Takeda Pharmaceutical Company: Research Funding; Asahi Genomics: Current equity holder in private company; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Otsuka Pharmaceutical: Research Funding. Ogawa:KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company.

Abstract: TP53 and RAS-pathway mutations predict very poor survival, when seen with CK and MDS/MPNs, respectively. For patients with mutated TP53 or CK alone, long-term survival could be obtained with stem cell transplantation.

Abstract: Recent genetic studies have revealed frequent and specific pathway mutations involving multiple components of the RNA splicing machinery in myelodysplasia. Among these, U2AF1 mutations are more prevalent in MDS without increased ring sideroblasts and AML with myelodysplasia-related changes and are associated with a poor prognosis. Also found in approximately 4% of lung adenocarcinoma, U2AF1 mutations exclusively involved two highly conserved amino acid positions (S34 or Q157) within the amino- and the carboxyl-terminal zinc finger motifs flanking the U2AF homology motif (UHM) domain. Comprehensive analysis in a large cohort of MDS showed that U2AF1 mutations showed a significant trend to coexist with ASXL1. The molecular mechanism by which U2AF1 mutations lead to myelodysplasia have not fully been elucidated. To elucidate the role of U2AF1 mutations in the development of myelodysplasia, we generated heterozygous conditional knock-in mice for the U2af1 S34F mutation, which were crossed them with Vav1-Cre transgenic mice. Vav1-Cre mediated U2af1 S34F knock-in mice exhibited severe leukopenia and macrocytic anemia at 8-20 weeks after birth. Although there was no significant difference in blood cell morphology between wild-type and mutant mice in bone marrow (BM) and peripheral blood (PB) cells, there was strong myeloid skewing in lineage composition both in U2af1 mutant BM and PB cells compared to wild-type controls. Flow cytometry of U2af1 S34F BM cells showed a significant decrease in the number of hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs) and megakaryocyte/erythrocyte lineage-restricted progenitors (MEPs), and an increase of the granulocyte/macrophage lineage-restricted progenitors (GMPs) compared to wild-type BM cells. These observations suggest that heterozygous U2af1 mutation leads to differentiation defects of HSCs, which however, is not sufficient for the development of MDS. We next assessed the phenotype of U2af1-mutated BM cells in transplantation settings to evaluate the effect of increasing replicative stress, which has been shown to substantially affect the behavior of normal and abnormal stem cells. In PB, progressive leukopenia, macrocytic anemia and decreased platelet counts were observed in mutant mice in transplantation settings. Surprisingly, all of the U2af1 mutant-transplanted mice died within two months after transplantation due to severe bone marrow failure. Cytological analysis of BM cells revealed morphological abnormalities in U2af1 mutant-transplanted mice, including hypersegmentation in neutrophils and erythroid dysplasia. Flow cytometrical analysis revealed decreased numbers of HSCs, CMPs and MEPs, and increased number of GMPs. These observations suggest that the U2af1 mutation leads to ineffective hematopoiesis and morphological abnormalities, which seems to recapitulate the phenotype of MDS in transplantation settings. Subsequently, we assessed the reconstitution capacity of whole BM cells from U2af1 mutant mice in competitive transplantation experiments. The donor chimerism of U2af1 S34F-derived cells in PB was remarkably reduced compared to that of wild-type cells. At four months post transplantation, the chimerism of U2af1 S34F-derived cells was markedly lower than that of wild-type cells in the fractions of HSCs, CMPs, MEPs, GMPs and common lymphoid progenitors (CLPs) in BM. RNA sequencing analysis of HSCs defined as Kit+Sca-1+Linlow (KSL) cells and CMPs from the mutant mice showed significant changes in alternative splicing and expression levels in many genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies. In summary, our results demonstrated that heterozygous U2af1 S34F mutation led to impaired HSC functions that was evident from reduced competitive repopulation and deregulated hematopoietic differentiation, which were augmented in transplantation settings. Our mice model provides a valuable tool to understand the molecular pathogenesis of U2af1-mutated myeloid neoplasms. Disclosures Nakagawa: Sumitomo Dainippon Pharma Co., Ltd.: Research Funding.

Abstract: Background: Acute erythroid leukemia (AEL) is a rare subtype of AML characterized by erythroid predominant proliferation and classified into two subtypes with pure erythroid (PEL) and myeloid/erythroid (MEL) phenotypes. Although gene mutations in AEL have been described in several reports, genotype phenotype correlations are not fully understood with little knowledge about the feasible molecular targets for therapy. Methods: To understand the mechanism of the erythroid dominant phenotype of AEL and identify potential therapeutic targets for AEL, we analyzed a total of 105 AEL cases with the median age of 60 (23-86), using targeted-capture sequencing of commonly mutated genes in myeloid neoplasms, together with 1,279 SNPs for copy number measurements. Among these 105 cases, 13 were also analyzed by RNA sequencing. Genetic profiles of these 105 AEL cases were compared to those of 775 cases with non-erythroid AML (NEL) including 561 cases from The Cancer Genome Atlas and Beat AML study. An immature erythroid cell line (TF1) and three patient-derived xenografts (PDX) established from AEL with JAK2 and/or EPOR amplification. Cell line and samples from patients were inoculated into immune-deficient mice and tested for their response to JAK1/2 inhibitor. Results: According to unique genetic alterations, AEL was classified into 4 subgroups (A-D). Characterized by TP53 mutations and complex karyotype, Group A was the most common subtype and showed very poor prognosis. Remarkably, all PEL cases were categorized into Group A. Conspicuously, 80% of PEL cases had amplifications of JAK2 (6/10; 60%), EPOR (7/10;70%), and ERG (6/10;60%) loci on chromosomes 9p, 19q, and 21q, respectively, frequently in combination, although they were rarely seen in NEL cases. All cases in Group B (n=19, 18%), another prevalent form of AEL, had STAG2 mutations and classified in MEL. To further characterize this subgroup, we compared genetic profiles of STAG2-mutated AEL and NEL. Prominently, 70% (14/20) of STAG2-mutated cases in AEL had KMT2A-PTD, whereas it was found only in 8.8% (3/34) of NEL. CEBPA mutations were also more common in AEL (6/21; 29%) than NEL (4/34; 12%). While Group C was characterized by frequent NPM1 mutations, in contrast to the frequent co-mutation of FLT3 in the corresponding subgroup of NPM1-mutated cases in NEL, NPM1-mutated patents in this subgroup lacked FLT3 mutations but had frequent PTPN11 mutations (8/16; 50%), which were much less common in NEL (25/209; 12%). The remaining cases were categorized into Group D, which was enriched for mutations in ASXL1, BCOR, PHF6, U2AF1 and KMT2C. Recurrent loss-of-function mutations in USP9X were unique to this subtype, although USP9X mutations have been reported in ALL with upregulation of JAK-STAT pathway. In RNA sequencing analysis, AEL cases exhibited gene expression profiles implicated in an upregulated STAT5 signaling pathway, which was seen not only those cases with JAK2 or EPOR amplification, but also those without, suggesting that aberrantly upregulated STAT5 activation might represent a common defect in AEL. Based on this finding, we evaluated the effect of a JAK inhibitior, ruxolitinib, on an AEL-derived cell line and three PDX models established from AEL having TP53 mutations and JAK2 and EPOR mutation/amplification. Of interest, ruxolitinib significantly suppressed cell growth and prolonged overall survival in mice engrafted with TF1 and 2 PDX models with STAT5 downregulation, although the other model was resistant to JAK2 inhibition with persistent STAT5 activation. Conclusion: AEL is a heterogeneous group of AML, of which PEL is characterized by frequent amplifications/mutations in JAK2, EPOR and/or ERG. Frequent involvement of EPOR/JAK/STAT pathway is a common feature of AEL, in which a role of JAK inhibition was suggested. Disclosures Yoda: Chordia Therapeutics Inc.: Research Funding. Shih:Novartis: Research Funding; Celgene: Research Funding; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ishiyama:Alexion: Research Funding; Novartis: Honoraria. Miyazaki:Astellas Pharma Inc.: Honoraria; Sumitomo Dainippon Pharma Co., Ltd.: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Celgene: Honoraria; Otsuka Pharmaceutical: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Novartis Pharma KK: Honoraria; Kyowa Kirin Co., Ltd.: Honoraria. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Takaori-Kondo:Celgene: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Thyas Co. Ltd.: Research Funding; Takeda: Research Funding; CHUGAI: Research Funding; OHARA Pharmaceutical: Research Funding; Sanofi: Research Funding; Novartis Pharma: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Research Funding; Otsuka Pharmaceutical: Research Funding; Eisai: Research Funding; Astellas Pharma: Honoraria, Research Funding; Kyowa Kirin: Honoraria, Research Funding; Nippon Shinyaku: Research Funding; MSD: Honoraria. Kataoka:Asahi Genomics: Current equity holder in private company; Otsuka Pharmaceutical: Research Funding; Takeda Pharmaceutical Company: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding. Usuki:Alexion: Research Funding, Speakers Bureau; Apellis: Research Funding; Novartis: Research Funding, Speakers Bureau; Chugai: Research Funding. Maciejewski:Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau. Ganser:Novartis: Consultancy; Celgene: Consultancy. Thol:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Ogawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Asahi Genomics Co., Ltd.: Current equity holder in private company; Eisai Co., Ltd.: Research Funding; Chordia Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; KAN Research Institute, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Ruxolitinib is used for drug efficacy test using patient-derived xenografts established from acute erythroid leukemia.

Abstract: Functional deregulation of transcription factors has been found in many types of tumors. Transcription factor AML1/RUNX1 is one of the most frequent targets of chromosomal abnormalities in human leukemia and altered function of AML1 is closely associated with malignant transformation of hematopoietic cells. However, the molecular basis and therapeutic targets of AML1-related leukemia are still elusive. Here, we explored immediate target pathways of AML1 by in vitro synchronous inactivation in hematopoietic cells. We found that AML1 inhibits NF-κB signaling through interaction with IκB kinase complex in the cytoplasm. Remarkably, AML1 mutants found in myeloid tumors lack the ability to inhibit NF-κB signaling, and human cases with AML1-related leukemia exhibits distinctly activated NF-κB signaling. Furthermore, inhibition of NF-κB signaling in leukemic cells with mutated AML1 efficiently blocks their growth and development of leukemia. These findings reveal a novel role for AML1 as a cytoplasmic attenuator of NF-κB signaling and indicate that NF-κB signaling is one of the promising therapeutic targets of hematologic malignancies with AML1 abnormality.

Abstract: Blood-specific expression of the Srsf2 P95H mutant results in decreased stem/progenitor cell numbers and a reduced repopulation capacity. Srsf2 P95H mutation by itself is not sufficient to develop MDS but contributes to the MDS phenotype in transplantation settings.