Clonal Populations Research Articles

The Patterns of Restriction Fragment of Several Enzymes to Distinguish Toxoplasma gondii Isolates Virulent and Avirulent Strains using GRA1 and GRA7 Genetic Marker

Toxoplasma gondii pathogenicity depends on the type derived from a clonal population. A genetic analysis of the locus has been carried out to determine the different genotypes of T. gondii (strain types I, II, and III) that are associated with human toxoplasmosis. The several genotypes of T. gondii (strain types I, II, and III) that are linked to human toxoplasmosis have been identified through genetic study of the locus. In this investigation, PCR-RFLP was found to be a useful, and simple method genotypic characterization. The objective of this study was to genotyping characterize T. gondii RH and BEV strains isolates by PCR-RFLP using several restriction enzymes. T. gondii tachyzoite DNA was extracted and amplified by PCR using dense granule genetic markers (GRA1 and GRA7) designed with Primer3plus. The amplification were digested using the restriction enzymes. The PCR-RFLP amplified dense granule products was used to classify strains into two genotypes of T. gondii (virulent and avirulent). The results demonstrated that the RFLP patterns of the GRA1 and GRA7 gene area digested by DdeI, MvaI, HinfI, RsaI, and Sau96I enzymes can be used to identify virulent or avirulent strains of T. gondii. Toxoplasma gondii RH and BEV strain produced different digestion product which can be used to distinguished the strains.

Production of MSTN Gene-Edited Embryos of Buffalo Using the CRISPR/Cas9 System and SCNT.

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system and somatic cell nuclear transfer (SCNT) have been used to produce genome-edited farm animal species for improved production and health traits; however, these tools are rarely used in the buffalo and can play a pivotal role in milk and meat production in tropical and subtropical countries. In this study, we aimed to produce myostatin (MSTN) gene-edited embryos of the Murrah buffalo using the CRISPR/Cas9 system and SCNT. For this, fibroblast cells were electroporated with sgRNAs carrying all-in-one CRISPR/Cas9 plasmids targeting the first exon of the MSTN gene. Following puromycin selection, single-cell clonal populations were established and screened using the TA cloning and Sanger sequencing methods. Of eight single-cell clonal populations, one with a monoallelic and another with a biallelic heterozygous gene editing event were identified. These two gene-edited clonal cell populations were successfully used to produce blastocyst-stage embryos using the handmade cloning method. This work establishes the technical foundation for generation of genome-edited cloned embryos in the buffalo.

Causes and consequences of linkage disequilibrium among transposable elements within eukaryotic genomes.

Sex and recombination can affect the dynamics of transposable elements (TEs) in various ways: while sex is expected to help TEs to spread within populations, the deleterious effect of ectopic recombination among transposons represents a possible source of purifying selection limiting their number. Furthermore, recombination may also increase the efficiency of selection against TEs by reducing selective interference among loci. In order to better understand the effects of recombination and reproductive systems on TE dynamics, this article provides analytical expressions for the linkage disequilibrium among TEs in a classical model in which TE number is stabilized by synergistic purifying selection. The results show that positive linkage disequilibrium is predicted in infinite populations despite negative epistasis, due to the effect of the transposition process. Positive linkage disequilibrium may substantially inflate the variance in the number of elements per genome in the case of partially selfing or partially clonal populations. Finite population size tends to generate negative linkage disequilibrium (Hill-Robertson effect), the relative importance of this effect increasing with the degree of linkage among loci. The model is then extended in order to explore how TEs may affect selection for recombination. While positive linkage disequilibrium generated by transposition generally disfavors recombination, the Hill-Robertson effect may represent a non-negligible source of indirect selection for recombination when TEs are abundant. However, the direct fitness cost imposed by ectopic recombination among elements generally drives the population towards low-recombination regimes, at which TEs cannot be maintained at a stable equilibrium.

Spectra and characteristics of somatic mutations induced by ionizing radiation in hematopoietic stem cells

Spectra and frequencies of spontaneous and X-ray-induced somatic mutations were revealed with mouse long-term hematopoietic stem cells (LT-HSCs) by whole-genome sequencing of clonal cell populations propagated invitro from single isolated LT-HSCs. SNVs and small indels were the most common types of somatic mutations, and increased up to twofold to threefold by whole-body X-irradiation. Base substitution patterns in the SNVs suggested a role of reactive oxygen species in radiation mutagenesis, and signature analysis of single base substitutions (SBS) revealed a dose-dependent increase of SBS40. Most of spontaneous small deletions were shrinkage of tandem repeats, and X-irradiation specifically induced small deletions out of tandem repeats (non-repeat deletions). Presence of microhomology sequences in non-repeat deletions suggested involvement of microhomology mediated end-joining repair mechanisms as well as nonhomologous end-joining in radiation-induced DNA damages. We also identified multisite mutations and structural variants (SV), i.e., large indels, inversions, reciprocal translocations, and complex variants. The radiation-specificity of each mutation type was evaluated from the spontaneous mutation rate and the per-Gy mutation rate estimated by linear regression, and was highest with non-repeat deletions without microhomology, followed by those with microhomology, SV except retroelement insertions, and multisite mutations; these types were thus revealed as mutational signatures of ionizing radiation. Further analysis of somatic mutations in multiple LT-HSCs indicated that large fractions of postirradiation LT-HSCs originated from single LT-HSCs that survived the irradiation and then expanded invivo to confer marked clonality to the entire hematopoietic system, with varying clonal expansion and dynamics depending on radiation dose and fractionation.

Abstract 607: CD8+ T cell clones maintain a pool of memory-like Ly108+ cells in the tumor during their contraction phase

Abstract The immune response against cancer is effectuated by the recruitment of expanding CD8+ T cell clones. However, the fate of these clonal populations that have migrated into the tumor environment - whether they persist, contract, and/or produce memory cells - is unknown. An improved understanding of clonal durability and potency will be essential for the development of novel cancer immunotherapies. Here, we applied our previously developed bilateral tumor mouse model to track the fate of endogenous T cell clones responding to tumors in the same individual. This approach relied on the fact that the clonal composition of the two tumors is identical, allowing the sampling and comparison of tumor-infiltrating cells from the same clonal populations on different days in the same individual. Mice were inoculated bilaterally with Lewis lung carcinoma (LLC), and tumors were resected, one after the other, on days 14 and 21 post-inoculation. At each timepoint, sampled CD8+ T cells were gated on PD1, Ly108, and TIM-3 expression and subjected to T-cell receptor (TCR) sequencing to identify clones. We utilized this data to quantify changes in the fraction of memory-like (Ly108+TIM-3-) and exhausted (Ly108-TIM-3+, Ly108+TIM-3+) cells of individual clonal populations over time. The majority (75%) of tumor-infiltrating CD8+ T cells on day 14 belonged to clonal populations that persisted on day 21. These clonal populations were classed as ‘expanding’ or ‘contracting’ based on changes in their clone size. Despite the polyclonal response to the tumor, almost all expanding clones contained a high fraction of memory-like (Ly108+TIM-3-) cells, whereas almost all ‘contracting’ clones contained a high fraction of exhausted (TIM-3+) cells. These characteristics were sufficient to predict whether a clonal population at day 14 would later expand or contract. We further quantified how the makeup of ‘contracting’ clonal populations changed over time. While all ‘contracting’ clonal populations lost a considerable number of exhausted (TIM3+) cells, half of the clonal populations maintained a pool of memory-like (Ly108+TIM-3-) cells in the tumor. These results demonstrate that clonal dynamics can be predicted from static measurements, facilitating the development of novel methods to infer patient immune activity. The results also demonstrate the possibility of altering clonal fate. Reinvigoration of the pool of memory-like cells maintained in the tumor could re-expand contracting clonal populations, and thereby enhance the immune response against the tumor. Citation Format: Munetomo Takahashi, Mikiya Tsunoda, Hiroyasu Aoki, Haru Ogiwara, Shigeyuki Shichino, Kouji Matsushima, Satoshi Ueha. CD8+ T cell clones maintain a pool of memory-like Ly108+ cells in the tumor during their contraction phase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 607.

Abstract 1164: Spatiotemporal profiling defines persister and resistance signatures in targeted treatment of melanoma

Abstract Over half of BRAF mutant melanoma patients with response to targeted therapy recur within 15 months. Resistance is postulated to arise from certain clonal populations that enter a slow cycling persister state, evade treatment with relative dormancy, and repopulate the tumor when reactivated. Via longitudinal profiling we define expression states of clonal populations and track their evolutionary progression. BRAF mutant patient derived melanoma xenografts were treated with BRAF/MEK inhibitors through maximum tumor size reduction and re-growth. Spatial transcriptomics (ST) was performed at 5 timepoints across 94 days of treatment, tracking clonal populations with intact tissue structure. Deep learning on H&E-stained images automatically detected histological features that co-localize with expression levels. A novel computational pipeline performed clustering, differential expression and pathway enrichment analysis, copy number variation detection, and pseudotemporal ordering, allowing spatial recreation of clonal phylogenies and cell fate trajectory inference during the treatment course. Three clonal categories were defined; Sensitive: predominant in treatment-naïve samples, Persister: remaining clusters in maximally shrunken specimens, Resistant: re-emergent, fast-proliferating. Analysis of clonal composition identified GAPDH and CCND1 as highly expressed in sensitive clones, suppressed in persisters, and reactivated in resistant clones; TYRP1, DCT, MITF, and NGFR showed an opposite pattern. Persister clones evolved from oxidative phosphorylation toward glycolysis and MAPK regulation. Spatial analysis showed increased glycolysis:oxidative phosphorylation ratio in centrally located sensitive clusters, with more balanced peripheral expression. Resistant clones showed upregulated DUSP6 expression and enrichment in Orexin signaling and MAPK regulation. Imaging- and expression-based clones were highly concordant. Spatial profiling during melanoma treatment identified transient persister and emergent resistant clones, defining expression profiles and phenotypic features. Altered ratio of oxidative to glycolytic metabolic pathways was a hallmark of clonal evolution in both space and time. Specific MAPK pathway re-entry points were identified as candidate resistance mechanisms, suggesting potential therapeutic targets. Deep learning features derived from histological images showed good correlation with ST profiles, providing a promising method for clonal tracking via images alone. This longitudinal experiment mimics a tumor’s clinical course, inducing persistence and resistance via treatment that parallels that seen by patients. Combining ST and imaging techniques, we provide insight into clonal dynamics with novel spatiotemporal resolution, defining an evolutionary roadmap to acquired treatment resistance. Citation Format: Jill Carol Rubinstein, Sergii Domanskyi, Todd B. Sheridan, Brian J. Sanderson, SungHee Park, Jessica Kaster, Haiyin Li, Olga Anczukow, Meenhard Herlyn, Jeffrey H. Chuang. Spatiotemporal profiling defines persister and resistance signatures in targeted treatment of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1164.

Abstract 5222: Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL)

Abstract Low count (LC) MBL is characterized by a circulating population of clonal B-cells and is a precursor state to CLL. Most individuals with LC MBL do not progress to CLL and never come to clinical attention. An active area of research is to identify factors that distinguish those individuals with MBL who progress from those who do not. Currently, 41 single nucleotide polymorphisms (SNPs) are known to be associated with CLL risk, and a portion of these SNPs have also been found to be associated with MBL risk. We hypothesize that these CLL-susceptibility SNPs associated with both MBL and CLL risk are SNPs associated with developing a B-cell clone; whereas the remaining SNPs found not to be associated with MBL risk are associated with progression of the B-cell clone (i.e., progression from MBL to CLL). To test this hypothesis, we evaluated the association of these 41 SNPs with risk of progression to CLL using a case-control study of 1,911 CLL cases and 1,579 individuals with MBL from Mayo Clinic. Individuals with MBL were identified through screening using 8-color flow cytometry assay. Individuals with LC MBL (defined by having a clonal B-cell population with the percent clonal B-cells out of total B-cell count <85%) who had an immunophenotype consistent with CLL were included as controls. CLL cases were obtained through the Mayo Clinic hematology practice. DNA was genotyped from whole blood. We analyzed the 41 CLL susceptibility SNPs with risk of progression to CLL using logistic regression and estimated odds ratio (OR) and 95% confidence interval (CI), adjusting for sex and age. We evaluated the CLL polygenic risk score (CLL-PRS), comprised of a weighted average of the 41 SNPs, with risk of CLL. Out of the 41 established CLL susceptibility SNPs, 15 were associated with CLL progression from MBL to CLL (all P<0.05). Nine of these 15 significant SNPs were not previously found to be associated with risk of MBL. The most significant SNPs were rs9880772 (OR=1.23, CI: 1.11-1.36, P=6.1 × 10−5), rs888096 (OR=1.25, CI: 1.12-1.38, P=3.0 × 10−5), and rs305065 (OR=1.28, CI: 1.14-1.43, P=1.5 × 10−5). One SNP (rs77551289) had a suggestive association (OR=1.21, P=0.069). The remaining 25 SNPs had no evidence of association with progression (ORs ranging between 0.9-1.1 and all P>0.1). The CLL-PRS was also associated with CLL risk (continuous OR = 1.25, CI: 1.15 - 1.35, P = 6.7 × 10−8). In our study accounting for the presence of B-cell clonal population, we identified 15 out of 41 CLL-susceptibility SNPs to be significantly associated with progression from LC MBL to CLL. The remaining 25 non-significant CLL-SNPs were not associated with progression and may instead be associated with development or initiation of the B-cell clone. These findings suggest that the established CLL susceptibility SNPs may play differing role (i.e., development or progression of the clone) in the etiology of CLL. Citation Format: Raphael Mwangi, Geffen Kleinstern, Sara J. Achenbach, Dennis Robinson, Aaron D. Norman, Kari G. Rabe, Janet E. Olson, Neil E. Kay, Rosalie G. Waller, Nicholas J. Boddicker, James R. Cerhan, Esteban Braggio, Sameer A. Parikh, Curtis A. Hanson, Celine M. Vachon, Tait Shanafelt, Susan L. Slager. Genetic variants associated with progression from monoclonal B-cell lymphocytosis (MBL) to chronic lymphocytic leukemia (CLL). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5222.

Abstract 1125: Neoantigen immunogenicity landscapes and evolution of tumor ecosystems during immunotherapy with nivolumab

Abstract Immune checkpoint blockade (ICB) therapy is a cornerstone of oncologic treatment for patients with advanced stage non-small cell lung cancer (NSCLC) and other malignancies. Neoantigen immunoediting drives ICB efficacy, yet how tumor evolution and neoantigen immunogenicity shape treatment response remains poorly understood. To address these questions, we conducted a prospective clinical trial of NSCLC patients treated with nivolumab. We assessed genomic alterations in tumors from 58 patients and performed large-scale immune epitope analyses, before and during treatment (CheckMate153, CA209-153). Tumors were analyzed by whole-exome and transcriptome sequencing. In responding patients, loss of mutation and neoantigen burden early during therapy was associated with clinical benefit. We evaluated the immunogenicity of 1,453 candidate neoantigens and identified 502 neopeptides that bound to MHC I and 196 neopeptides that were immunogenic and recognized by T cells in the setting of nivolumab treatment. These T cell reactive neoantigens were differentially present in clonal populations that underwent distinctive evolutionary trajectories across responders and nonresponders. Mapping these neoantigens to tumor clonal dynamics and clinical response revealed strong selection against immunogenic mutations compared to non-immunogenic mutations. We used this large collection of neoantigens to build ImmunoSEEK, a machine learning-based neoantigen prediction model and validated it using an orthogonal set of neoantigens. Changes in the genomic and neoantigen immunogenicity landscapes were accompanied by temporal changes in the tumor microenvironment. Nivolumab-induced microenvironmental evolution in NSCLC shared some similarities with that in melanoma, yet critical differences in immunologic programs were apparent from comparative network analysis between tumor types. This study provides unprecedented molecular portraits of the genomic and neoantigen landscapes underlying nivolumab’s mechanism of action. Citation Format: Tyler Joseph Alban, Nadeem Riaz, Prerana Parthasarathy, Vlad Makarov, Slav Kendall, Seong-Keun Yoo, Rachna Shah, Nils Weinhold, Raghvendra Srivastava, Xiaoxiao Ma, Chirag Krishna, Edward Garon, Wallace Akerley, Ben Creelan, Nivedita Aanuren, Diego Chowell, William Geese, Naiyer Rizvi, Timothy Chan. Neoantigen immunogenicity landscapes and evolution of tumor ecosystems during immunotherapy with nivolumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1125.

Abstract 5932: Clonal mapping phylogenetic and transcriptomic analysis of an M36 PDX amputation lung metastatic osteosarcoma model

Abstract Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. It has been revealed that metastatic cancers including osteosarcoma have a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. However, the lack of a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution has been a challenge. In this study, we used an established model to track and identify the clonal subpopulations and assess the genetic signatures that confer specific advantages selecting for uncontrolled growth, adaptation and colonization in the lungs. Methods: We injected barcoded M36 PDX osteosarcoma cells intratibially in ten severe combined immunodeficiency disease (SCID) mice to track the clonal population/subpopulation of cells that metastasize the established primary tumors to the lungs. Limb amputation was performed on the tumor bearing leg upon detection of palpable tumor. Lung tissue was harvested post-amputation following death from metastasis or euthanasia. DNA and RNA from frozen tissues was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Limb amputation was performed in 7 mice with all mice surviving the surgery. The mean mouse survival following amputation was 46.9 days and the longest survival was 167 days. Four (40%) of the primary tumor bearing mice had lung metastasis. A Shannon-Wiener and Jaccard similarity index show a diversity in clonal architecture between the primary tumors of all mice injected on the same day and a diversity in clonal population in both the primary tumors and their matched lung metastatic nodules. Clonal mapping further revealed 21 unique clones shared in all primary tumors examined. However, the clonal frequencies and distribution varied within each primary tumor and across the tumor samples. Interestingly, clonal frequencies increased in the lung metastatic nodules when compared to their matched primary tumors. Four distinct clones that were present in all the primary tumors were also found in all lung metastatic samples. These unique clones, here in called “seeding clones” may play an important role in the lung metastatic process. Conclusions: We previously showed that the barcoded osteosarcoma PDX models are inter and intratumorally heterogenous both in vitro and in vivo. In the current model, we identify the multiple tumorigenic seeding clones that potentially act as drivers in the lung metastatic process. Furthermore, we show the dynamic evolutionary relationships between the clones by reconstructing a phylogenetic map of the M36 PDX tumor model. Funding: Swim Across America, Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Zhongting Zhang, Zhang Wendong, Zhaohui Xu, Michael Roth, Jonathan Gill, Richard Gorlick. Clonal mapping phylogenetic and transcriptomic analysis of an M36 PDX amputation lung metastatic osteosarcoma model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5932.

Abstract 314: Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer

Abstract The high incidence of colon cancer (CC) worldwide is a major health concern. Although surgery and adjuvant chemotherapy are effective to a high extent in lymph-node negative colon cancers, there are still some 10-15% of patients that show disease relapse within the next 5-year period after intended curative surgery. We hypothesize that genomic instability and intratumor heterogeneity fuel the formation of subclonal populations in the primary tumor that might promote metastasis. In the current study, we assessed the presence of intratumor heterogeneity in CC using multiplex interphase FISH (miFISH) and whole-exome sequencing (WES) to evaluate samples obtained from primary stage II colon tumors and their patient-matched associated liver metastases using multi-region sampling (N=9). In combination with genome-wide copy-number changes obtained from WES analysis, miFISH allowed the simultaneous quantification of copy-numbers for nine CC relevant genes and a centromeric ploidy control probe in intact tumor nuclei derived from archival patient material. Our preliminary data on the four cases analyzed so far, revealed high similarities regarding copy-number changes and ploidy between primary tumor and metastatic samples for three of the cases. While two of these cases showed either triploid or highly aneuploid genomes with different degrees of genomic instability and subclonality among them, the third case displayed a diploid content for both matched lesions. Intriguingly, as seen by miFISH and WES, loss of CDX2 in the primary tumor might have originated the main clonal population giving rise to metastasis in this latter case. Conversely, the fourth case analyzed exhibited a triploid baseline for the primary tumor while the metastasis revealed a diploid population. Interestingly, despite higher absolute copy numbers in the primary tumor, there was an overlap of five of the gains and losses observed for the major clone populations of the tumor and metastasis by miFISH. However, two of the highly clonal gains observed in the tumor, namely the EGFR and MYC gain, were not present in the metastasis. Altogether, in addition to the ploidy change, results indicated major discrete copy-number changes from primary tumor to metastasis in this patient. In summary, we show here that the combined analysis of WES and miFISH is able to describe the subclonal composition of copy-number alterations in primary tumors and their patientmatched liver metastases. Future perspectives also include the assessment of subclonality affecting single-nucleotide variants to complete the genomic landscape of tumor evolution and clonal development in this patient cohort with the goal to better understand the nature of these early metastases. Citation Format: Sanam Yaghoubi, Kerstin Heselmeyer-Haddad, Ivan Archilla, Carolina Parra, Darawalee Wangsa, Giancarlo Castellano, Sara Lahoz, Veronica Pablo-Fontecha, Daniela Hirsch, Wei-Dong Chen, Thomas Ried, Miriam Cuatrecasas, Paul S. Meltzer, Jordi Camps. Intratumor genetic heterogeneity dictates metastatic subclones in stage II colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 314.

Abstract 1594: SMARCA2 (BRM) degraders promotes differentiation and inhibit proliferation in AML models

Abstract Dysregulated cellular differentiation is a major pathological feature of myeloid malignancies such as acute myeloid leukemia (AML). Targeting cellular differentiation programs has emerged as a novel therapeutic approach to treat patients with AML. Advantages of such differentiation therapy may include fewer systemic side-effects as well as opportunities to target leukemic stem cells (LSCs) and a broader range of clonal populations, likely resulting in lower frequencies of resistance and relapse in AML patients. The success of ATRA and decitabine in subsets of AML patients has proven that inducing differentiation can play a critical role in long-term durable responses. More agents targeting epigenetic regulators have been increasingly studied as differentiation inducers, including LSD1, DNMT1, Menin, and BET inhibitors. Recently, targeting SWI/SNF chromatin remodeling complexes has also been shown to regulate key leukemic gene expression signatures and induce AML differentiation. Small molecule inhibitors as well as gene knockdown for ATP-dependent SWI/SNF subunits SMARCA2 (BRM) and SMARCA4 (BRG1) are associated with re-direction of oncogenic transcriptional regulation to drive cellular differentiation and apoptosis in AML models. We have previously described the activity of highly potent, bispecific SMARCA2 degraders that efficiently promote SMARCA2 protein degradation in preclinical models. In the present study, we investigated the effects of our SMARCA2 selective degraders in AML models. Treatment with SMARCA2 degraders significantly inhibits AML cell line proliferation in vitro with IC50 ranging from 10 to 50 nM. In the SMARCA2 degrader treated cells, expression of PU.1 (SPI1), a key transcription factor in myeloid leukemias, was downregulated. In an in vivo OCI-AML3 xenograft model, treatment with a SMARCA2 selective degrader showed moderate tumor growth inhibition accompanied by robust increases in monocytic maturation markers CD11b and CD14. Further analyses of SMARCA2 degrader effects on global transcriptome and AML immunophenotypes as well as combination effects with other therapies are currently in progress. These findings highlight the potential of SMARCA2 degraders to target AML differentiation blocks and to improve the effectiveness of other therapeutic agents such as decitabine and venetoclax in AML patients. Citation Format: Anjana Agarwal, Olusola Peace Osinubi, Komali Vykuntam, Norman Fultang, Neha Bhagwat, Diane Heiser, Kris Vaddi, Koichi Ito, Peggy Scherle. SMARCA2 (BRM) degraders promotes differentiation and inhibit proliferation in AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1594.

Abstract 5473: Genomic profiling of chemotherapy-related clonal hematopoiesis in patients with high-grade serous ovarian cancer

Abstract Background: Clonal hematopoiesis (CH) is identified as the presence of clonal populations of hematopoietic stem cells (HSC). Hematopoietic lineage differentiation is subjected to genetic mutations that, due to fitness advantages, might give rise to clonally expanded populations. Clonal hematopoiesis of indeterminate potential (CHIP) is defined as the outgrowth of a single clone driven by acquired somatic mutation(s) in HSCs, in the absence of hematological abnormalities. Previous studies have been shown the association of CH with aging and a higher risk of developing secondary hematologic malignancies in cancer patients treated with chemotherapy agents. It is therefore of great interest to study CH incidence prior to and post chemotherapy exposure and its association with the evolution of hematologic malignancies. We aimed to characterize CHIP variants of a highly selected group of patients with high-grade serous ovarian cancer (HGSC) who underwent neoadjuvant chemotherapy; moreover, while previous CHIP studies are largely knowledge based and are limited to known hematologic genes or target gene panels, our study discovers novel CHIP mutations. Methods: Comprehensive ultra-high-depth whole exome sequencing using unique molecular barcode technologies was performed using plasma-derived cell-free DNA and matched white blood cells DNA and tumor DNA from pre-NACT (n=12) and post-NACT (n=12) samples of patients with HGSC who have excellent or poor response to NACT. Using spike-in mutated DNAs as positive controls, we detected variant alleles at 1% variant allele frequency. Results: We identified on average about 3,000 candidate CHIP variants in one patient. Among these, 1,977 variants affecting 1,375 genes were recurrently found in more than one patient. These CHIP genes include not only previous reported CHIP genes (e.g. DNMT3A, JAK2, TET2, and KMT2D) but also many novel CHIP candidates (such as RPTN, MTCH2, FAM186A, CACNA1A, FCGBP, and MUC3A). A number of CHIP mutations were uniquely found enriched in post-chemotherapy samples (e.g. ARID1A T290P, TP53 G245D, SMARCA4 G495D, and CIC T2456P). Interestingly, there is a strong enrichment of COSMIC cancer census genes in CHIP genes identified in every patient. Conclusions: Our findings corroborate the notion that CHIP mutations are present in nonmalignant blood cells of patients with HGSC, and some are enriched after chemotherapy. Moreover, our innovative sequencing approach allowed discovery of novel candidate CHIP genes. The systematic identification of CHIP in patients with HGSC might be an important new clinical consideration when establishing chemotherapy protocols. Citation Format: Sara Corvigno, Jun Yao, Li Zhao, Amma Asare, Joseph Celestino, Richard Hajek, Ency Arboleda Goette, Elaine Stur, Emine Bayraktar, Mark S Kim, Ping Song, Qingxiu Zhang, Xingzhi Song, Mohammad Mohammad, Kenna Shaw, Jianhua Zhang, Karen Lu, Amir Jazaeri, Shannon Westin, Sanghoon Lee, Anil Sood. Genomic profiling of chemotherapy-related clonal hematopoiesis in patients with high-grade serous ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5473.

Probe-based bacterial single-cell RNA sequencing predicts toxin regulation

Clonal bacterial populations rely on transcriptional variation across individual cells to produce specialized states that increase fitness. Understanding all cell states requires studying isogenic bacterial populations at the single-cell level. Here we developed probe-based bacterial sequencing (ProBac-seq), a method that uses libraries of DNA probes and an existing commercial microfluidic platform to conduct bacterial single-cell RNA sequencing. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. Applied to Bacillus subtilis and Escherichia coli, ProBac-seq correctly identifies known cell states and uncovers previously unreported transcriptional heterogeneity. In the context of bacterial pathogenesis, application of the approach to Clostridium perfringens reveals heterogeneous expression of toxin by a subpopulation that can be controlled by acetate, a short-chain fatty acid highly prevalent in the gut. Overall, ProBac-seq can be used to uncover heterogeneity in isogenic microbial populations and identify perturbations that affect pathogenicity.

372 HMGB1 Localization as a Driver of Carcinogenesis in RDEB-Associated Squamous Cell Carcinoma

OBJECTIVES/GOALS: This study investigates whether localization of high mobility group box 1 (HMGB1) controls inflammatory signaling and DNA damage response in human keratinocytes, the cell of origin for squamous cell carcinoma (SCC). SCC is especially metastatic in chronic wounds, burns, and in patients with recessive dystrophic epidermolysis bullosa (RDEB). METHODS/STUDY POPULATION: We used CRISPR/Cas9 gene-editing to knock out HMGB1 in a keratinocyte line, p16INK4a-negative keratinocytes immortalized by ectopic hTERT expression (N/TERT-2G [46,XY]). Following gene editing, clonal keratinocyte populations were screened for knockout by PCR followed by TIDE analysis (Tracking of Indels by Decomposition) to identify indels that would result in a frameshift mutation. Total cell lysates for each clonal population were analyzed by immunoblot and immunofluorescence for HMGB1 protein. These cells will be used to assay for DNA damage sensitivity in the presence of genotoxic agents (etoposide, ultraviolet radiation, γ-irradiation). A lentiviral vector will then be used to express mutant forms of HMGB1 that localize to the nucleus (C23/45S) or cytoplasm (C106S) and DNA damage assays repeated. RESULTS/ANTICIPATED RESULTS: We have confirmed by sequencing, immunoblot, and immunofluorescence that HMGB1 is knocked out in a clonal population of N/TERT-2G human keratinocyte cells. We anticipate that cells with a complete absence of HMGB1 will have high sensitivity to DNA damaging agents, but little change in inflammatory signaling. We also expect that cells expressing mutant HMGB1 that localizes exclusively to the cytoplasm will demonstrate an increased sensitivity to DNA damage relative to wild-type controls, while mutant HMGB1 that localizes exclusively to the nucleus will be protected from DNA damage caused by exposure to genotoxic agents. DISCUSSION/SIGNIFICANCE: HMGB1 is a nuclear protein and damage associated molecular pattern that is elevated systemically in patients with RDEB, many of whom will go on to develop metastatic squamous cell carcinoma. The experiments described here investigate whether HMGB1 plays a mechanistic role in skin carcinogenesis via regulation of the DNA damage response.

Persistent Molecular Disease in Adult Patients With AML Evaluated With Whole-Exome and Targeted Error-Corrected DNA Sequencing.

Persistent molecular disease (PMD) after induction chemotherapy predicts relapse in AML. In this study, we used whole-exome sequencing (WES) and targeted error-corrected sequencing to assess the frequency and mutational patterns of PMD in 30 patients with AML. The study cohort included 30 patients with adult AML younger than 65 years who were uniformly treated with standard induction chemotherapy. Tumor/normal WES was performed for all patients at presentation. PMD analysis was evaluated in bone marrow samples obtained during clinicopathologic remission using repeat WES and analysis of patient-specific mutations and error-corrected sequencing of 40 recurrently mutated AML genes (MyeloSeq). WES for patient-specific mutations detected PMD in 63% of patients (19/30) using a minimum variant allele fraction (VAF) of 2.5%. In comparison, MyeloSeq identified persistent mutations above 0.1% VAF in 77% of patients (23/30). PMD was usually present at relatively high levels (>2.5% VAFs), such that WES and MyeloSeq agreed for 73% of patients despite differences in detection limits. Mutations in DNMT3A, ASXL1, and TET2 (ie, DTA mutations) were persistent in 16 of 17 patients, but WES also detected non-DTA mutations in 14 of these patients, which for some patients distinguished residual AML cells from clonal hematopoiesis. Surprisingly, MyeloSeq detected additional variants not identified at presentation in 73% of patients that were consistent with new clonal cell populations after chemotherapy. PMD and clonal hematopoiesis are both common in patients with AML in first remission. These findings demonstrate the importance of baseline testing for accurate interpretation of mutation-based tumor monitoring assays for patients with AML and highlight the need for clinical trials to determine whether these complex mutation patterns correlate with clinical outcomes in AML.

Statistical theory of asymmetric damage segregation in clonal cell populations.

Asymmetric damage segregation (ADS) is ubiquitous among unicellular organisms: After a mother cell divides, its two daughter cells receive sometimes slightly, sometimes strongly different fractions of damaged proteins accumulated in the mother cell. Previous studies demonstrated that ADS provides a selective advantage over symmetrically dividing cells by rejuvenating and perpetuating the population as a whole. In this work we focus on the statistical properties of damage in individual lineages and the overall damage distributions in growing populations for a variety of ADS models with different rules governing damage accumulation, segregation, and the lifetime dependence on damage. We show that for a large class of deterministic ADS rules the trajectories of damage along the lineages are chaotic, and the distributions of damage in cells born at a given time asymptotically becomes fractal. By exploiting the analogy of linear ADS models with the Iterated Function Systems known in chaos theory, we derive the Frobenius-Perron equation for the stationary damage density distribution and analytically compute the damage distribution moments and fractal dimensions. We also investigate nonlinear and stochastic variants of ADS models and show the robustness of the salient features of the damage distributions.

Hyperextended telomeres promote formation of C-circle DNA in telomerase positive human cells

Telomere length maintenance is crucial to cancer cell immortality. Up to 15% of cancers utilize a telomerase-independent, recombination-based mechanism termed alternative lengthening of telomeres (ALT). Currently, the primary ALT biomarker is the C-circle, a type of circular DNA with extrachromosomal telomere repeats (cECTRs). How C-circles form is not well characterized. We investigated C-circle formation in the human cen3tel cell line, a long-telomere, telomerase+ (LTT+) cell line with progressively hyper-elongated telomeres (up to ∼100 kb). cECTR signal was observed in 2D gels and C-circle assays but not t-circle assays, which also detect circular DNA with extrachromosomal telomere repeats. Telomerase activity and C-circle signal were not separable in the analysis of clonal populations, consistent with C-circle production occurring within telomerase+ cells. We observed similar cECTR results in two other LTT+ cell lines, HeLa1.3 (∼23 kb telomeres) and HeLaE1 (∼50 kb telomeres). In LTT+ cells, telomerase activity did not directly impact C-circle signal; instead, C-circle signal correlated with telomere length. LTT+ cell lines were less sensitive to hydroxyurea than ALT+ cell lines, suggesting that ALT status is a stronger contributor to replication stress levels than telomere length. Additionally, the DNA repair-associated protein FANCM did not suppress C-circles in LTT+ cells as it does in ALT+ cells. Thus, C-circle formation may be driven by telomere length, independently of telomerase and replication stress, highlighting limitations of C-circles as a stand-alone ALT biomarker.

Analysis of Salmonella lineage-specific traits upon cell sorting.

Microbial cell individuality is receiving increasing interest in the scientific community. Individual cells within clonal populations exhibit noticeable phenotypic heterogeneity. The advent of fluorescent protein technology and advances in single-cell analysis has revealed phenotypic cell variant in bacterial populations. This heterogeneity is evident in a wide range of phenotypes, for example, individual cells display variable degrees of gene expression and survival under selective conditions and stresses, and can exhibit differing propensities to host interactions. Last few years, numerous cell sorting approaches have been employed for resolving the properties of bacterial subpopulations. This review provides an overview of applications of cell sorting to analyze Salmonella lineage-specific traits, including bacterial evolution studies, gene expression analysis, response to diverse cellular stresses and characterization of diverse bacterial phenotypic variants.

Clonal isolates of Treponema pallidum subsp. pallidum Nichols provide evidence for the occurrence of microevolution during experimental rabbit infection and in vitro culture.

The recent development of a system for long-term in vitro culture of the syphilis spirochete, Treponema pallidum subsp. pallidum, has introduced the possibility of detailed genetic analysis of this bacterium. In this study, the in vitro culture system was used to isolate and characterize clonal populations of T. pallidum subsp. pallidum Nichols, the most widely studied strain. In limiting dilutions experiments, it was possible to establish cultures with inocula as low as 0.5 T. pallidum per well despite the long generation time (~35 to 40 hours) of this organism. Six Nichols strain clones isolated by limiting dilution were characterized in detail. All clones exhibited indistinguishable morphology and motility, highly similar in vitro multiplication rates, and comparable infectivity in the rabbit model (ID50 ≤ 100 bacteria). Genomic sequencing revealed sequence heterogeneity in the form of insertions or deletions at 5 sites, single nucleotide variations at 20 sites, and polynucleotide (polyG/C) tract length differences at 22 locations. Genomic sequences of the uncloned Nichols strain preparations propagated in rabbits or in vitro cultures exhibited substantial heterogeneity at these locations, indicating coexistence of many varied 'clonotypes' within these populations. Nearly all genetic variations were specific for the Nichols strain and were not detected in the >280 T. pallidum genomic sequences that are currently available. We hypothesize that these Nichols strain-specific sequence variations arose independently either during human infection or within the 110 years since the strain's initial isolation, and thus represent examples of microevolution and divergence.

Virulence Phenotyping and Molecular Genotyping Reveal High Diversity Within and Strong Gene Flow Between the Puccinia striiformis f. sp. tritici Populations Collected from Barberry and Wheat in Shaanxi Province of China.

Emergence of new Puccinia striiformis f. sp. tritici races that overcome resistance of wheat cultivars is a challenging issue for wheat production. Although sexual reproduction of the fungus on barberry plants under field conditions in the spring in China has been reported, the diversity of the pathogen on barberry plants and the relationship to the population in wheat fields have not been determined. In the present study, two P. striiformis f. sp. tritici populations collected in western Shaanxi Province in May 2016, one from barberry plants (103 isolates) and the other from nearby wheat crops (107 isolates), were phenotyped for virulence and genotyped with simple sequence repeat (SSR) markers. The phenotypic and genotypic data of the two populations were compared to determine their relationships. A total of 120 races, including 29 previously known races (seven were shared by the two populations) and 91 new races (35 from barberry and 56 from wheat), were identified. Similarly, a total of 132 multilocus genotypes, including 51 only from barberry, 77 only from wheat, and four from both, were detected using the SSR markers. Analyses of molecular variance identified high (93%) genetic variance within populations and low but still significant variance (7%) between the populations. Nonparametric multivariate discriminant analysis of principal components and STRUCTURE analysis showed that the two populations had a close relationship with little genetic differentiation (FST = 0.038) and strong gene flow (Nm = 6.34, P = 0.001) between them. Although the analysis of linkage disequilibrium indicated clonal populations, the isolation of P. striiformis f. sp. tritici from barberry plants and the high genetic diversities in the barberry and wheat populations suggest that barberry plants provide aeciospores to infect wheat crops in the area. The information is useful for understanding stripe rust epidemiology and management of the disease.