Subclone Of Cells Research Articles

The use of liquid biopsy comprehensive genomic profiling to identify the molecular landscape of patients with metastatic colorectal cancer who are candidates for anti-EGFR rechallange therapy: Findings from the CAVE-2 GOIM trial.

221 Background: In the era of precision medicine, biomarker guided therapies are necessary for patients with chemorefractory metastatic colorectal cancer (mCRC). Circulating tumor DNA (ctDNA) comprehensive genomic profiling (CGP) may contribute to the development of more effective treatments. Methods: CAVE-2 GOIM trial (ClinicalTrials.gov ID: NCT05291156) is the first randomized phase II study to assess the efficacy of avelumab plus cetuximab compared to cetuximab alone as rechallenge strategy in pre-treated plasma ctDNA RAS, BRAF WT mCRC patients. Before being enrolled in the trial, patients were selected by baseline plasma ctDNA analysis by Foundation One Liquid CDx (F1L CDx) 324 genes to identify ctDNA RAS/BRAF WT cases. Results: As of July 31th 2024, 229 (out of approximately 310) patients have been screened. A total of 1562 pathogenic variants (PV) were detected, with TP53 (86.0%), APC (84.2%), DNMT3A (30.5%) representing the most common altered genes. In these anti-EGFR pretreated patients different acquired mechanisms of resistance were observed. In particular, 72/229 (31.4%) patients had RAS or BRAF V600E PV (102 KRAS , 21 NRAS and 8 BRAF V600E mutations, respectively). Additionally, in these 72 patients, multiple RAS/BRAF V600E gene alterations (median of 1 PV, range 1-7) were observed in 41.6% cases. MAPK, RTK , and PI3K/AKT/mTOR PV were detected in 43.2%, 24.5% and 24.5% patients, respectively. Of note, the variant allele frequency of resistant PV was lower than other gene alterations, suggesting a high tumor heterogeneity as a consequence of the development and/or expansion of EGFR-inhibitor resistant cancer cell sub-clones. All patients were assessed for tumor mutational burden (TMB). Median TMB was 6 (IQR 4-10) mut/Mb, with 29.6% cases showing a TMB ≥10 mut/Mb. Finally, a total of 258 actionable genomic alterations were detected in 52,4% of patients according to the ESMO Scale for Clinical Actionability of Molecular Targets (ESCAT). Eighty-one tier I alterations were detected in 32.3% patients, which included TMB high (29,7%), BRAF V600E (3,5%), KRAS G12C (6,5%), and RET fusions (0,4%). Conclusions: These findings support liquid biopsy-based GCP for individualized therapeutic choices in chemorefractory mCRC. Up-dated final analyses of the entire screened patient population of the CAVE-2 GOIM trial will be presented. Clinical trial information: NCT05291156 .

Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors.

Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.

Advances in spatial multi-omics in tumors.

Single-cell techniques have convincingly demonstrated that tumor tissue usually contains multiple genetically defined cell subclones with different gene mutation sets as well as various transcriptional profiles, but the spatial heterogeneity of the microenvironment and the macrobiological characteristics of the tumor ecosystem have not been described. For the past few years, spatial multi-omics technologies have revealed the cellular interactions, microenvironment, and even systemic tumor-host interactions in the tumor ecosystem at the spatial level, which can not only improve classical therapies such as surgery, radiotherapy, and chemotherapy but also promote the development of emerging targeted therapies in immunotherapy. Here, we review some emerging spatial omics techniques in cancer research and therapeutic applications and propose prospects for their future development.

Liquid biopsy of peripheral blood using mass spectrometry detects primary extramedullary disease in multiple myeloma patients

Multiple myeloma (MM) is the second most prevalent hematological malignancy, characterized by infiltration of the bone marrow by malignant plasma cells. Extramedullary disease (EMD) represents a more aggressive condition involving the migration of a subclone of plasma cells to paraskeletal or extraskeletal sites. Liquid biopsies could improve and speed diagnosis, as they can better capture the disease heterogeneity while lowering patients’ discomfort due to minimal invasiveness. Recent studies have confirmed alterations in the proteome across various malignancies, suggesting specific changes in protein classes. In this study, we show that MALDI-TOF mass spectrometry fingerprinting of peripheral blood can differentiate between MM and primary EMD patients. We constructed a predictive model using a supervised learning method, partial least squares-discriminant analysis (PLS-DA) and evaluated its generalization performance on a test dataset. The outcome of this analysis is a method that predicts specifically primary EMD with high sensitivity (86.4%), accuracy (78.4%), and specificity (72.4%). Given the simplicity of this approach and its minimally invasive character, this method provides rapid identification of primary EMD and could prove helpful in clinical practice.

An ultrasensitive strip sensor for rapid detection of African swine fever virus

African swine fever (ASF) is a highly contagious disease caused by the African swine fever virus (ASFV) infecting pigs, which has caused huge economic losses in countries around the world. Currently, there is no effective vaccine, and the prevention and control of ASF is mainly through rapid detection, so it is particularly important to carry identify and develop rapid detection methods for ASFV. In this study, recombinant plasmid PET-28a(+)-p30 was constructed, and the recombinant protein was obtained by inducing expression and Ni+ resin affinity column purification. Mice were immunized with recombinant p30 protein, and after three immunizations, ten strains of hybridoma cells that stably secreted monoclonal antibodies (mAbs) against p30 protein were obtained by cell fusion and subcloning. A colloidal gold immunochromatography assay (GICA) based on double antibody sandwich technology was established to screen the paired antibodies, and the trapping and detecting antibodies were mAb-11F11 and mAb-7A8, respectively, with a detection limit of 1 ng/mL, which laid an important material foundation for the early detection of ASF in the future.

Bioactive poly(amino acid)s for multi-modal cancer therapy.

The interplay between the tumor cells and their microenvironments is as inseparable as the relationship between "seeds" and "soil." The tumor microenvironments (TMEs) exacerbate malignancy by enriching malignant cell subclones, generating extracellular matrices, and recruiting immunosuppressive cells, thereby diminishing the efficacy of clinical therapies. Modulating TMEs has emerged as a promising strategy to enhance cancer therapy. However, the existing drugs used in clinical settings do not target the TMEs specifically, underscoring the urgent need for advanced strategies. Bioactive materials present unique opportunities for modulating TMEs. Poly(amino acid)s with precisely controllable structures and properties offer exceptional characteristics, such as diverse structural units, excellent biosafety, ease of modification, sensitive biological responsiveness, and unique secondary structures. These attributes hold significant potential for the modulation of TMEs and clinical applications further. Consequently, developing bioactive poly(amino acid)s capable of modulating the TMEs by elucidating structure-activity relationships and mechanisms is a promising approach for innovative clinical oncology therapy. This review summarizes the recent progress of our research team in developing bioactive poly(amino acid)s for multi-modal tumor therapy. First, a brief overview of poly(amino acid) synthesis and their advantages as nanocarriers is provided. Subsequently, the pioneering research of our research group on synthesizing the biologically responsive, dynamically allosteric, and immunologically effective poly(amino acid)s are highlighted. These poly(amino acid)s are designed to enhance tumor therapy by modulating the intracellular, extracellular matrix, and stromal cell microenvironments. Finally, the future development of poly(amino acid)s is discussed. This review will guide and inspire the construction of bioactive poly(amino acid)s with promising clinical applications in cancer therapy. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease Biology-Inspired Nanomaterials > Peptide-Based Structures.

Optimization of a mouse model of pancreatic cancer to simulate the human phenotypes of metastasis and cachexia

BackgroundPancreatic ductal adenocarcinoma (PDAC) presents with a high mortality rate. Two important features of PDAC contribute to this poor outcome. The first is metastasis which occurs in ~ 80% of PDAC patients. The second is cachexia, which compromises treatment tolerance for patients and reduces their quality of life. Although various mouse models of PDAC exist, recapitulating both metastatic and cachectic features have been challenging.MethodsHere, we optimize an orthotopic mouse model of PDAC by altering several conditions, including the subcloning of parental murine PDAC cells, implantation site, number of transplanted cells, and age of recipient mice. We perform spatial profiling to compare primary and metastatic immune microenvironments and RNA sequencing to gain insight into the mechanisms of muscle wasting in PDAC-induced cachexia, comparing non-metastatic to metastatic conditions.ResultsThese modifications extend the time course of the disease and concurrently increase the rate of metastasis to approximately 70%. Furthermore, reliable cachexia endpoints are achieved in both PDAC mice with and without metastases, which is reminiscent of patients. We also find that cachectic muscles from PDAC mice with metastasis exhibit a similar transcriptional profile to muscles derived from mice and patients without metastasis.ConclusionTogether, this model is likely to be advantageous in both advancing our understanding of the mechanism of PDAC cachexia, as well as in the evaluation of novel therapeutics.

A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Differential Competitive Growth of Transgenic Subclones of Neuroblastoma Cells Expressing Different Levels of Cathepsin D Co-Cultured in 2D and 3D in Response to EGF: Implications in Tumor Heterogeneity and Metastasis.

Neuroblastoma (NB) is an embryonal tumor arising from the sympathetic central nervous system. The epidermal growth factor (EGF) plays a role in NB growth and metastatic behavior. Recently, we have demonstrated that cathepsin D (CD) contrasts EGF-induced NB cell growth in 2D by downregulating EGFR/MAPK signaling. Aggressive NB is highly metastatic to the bone and the brain. In the metastatic process, adherent cells detach to form clusters of suspended cells that adhere once they reach the metastatic site and form secondary colonies. Whether CD is involved in the survival of metastatic NB clones is not known. Therefore, in this study, we addressed how CD differentially affects cell growth in suspension versus the adherent condition. To mimic tumor heterogeneity, we co-cultured transgenic clones silenced for or overexpressing CD. We compared the growth kinetics of such mixed clones in 2D and 3D models in response to EGF, and we found that the Over CD clone had an advantage for growth in suspension, while the CD knocked-down clone was favored for the adherent growth in 2D. Interestingly, on switching from 3D to 2D culture conditions, the expression of E-cadherin and of N-cadherin increased in the KD-CD and Over CD clones, respectively. The fact that CD plays a dual role in cancer cell growth in 2D and 3D conditions indicates that during clonal evolution, subclones expressing different level of CD may arise, which confers survival and growth advantages depending on the metastatic step. By searching the TCGA database, we found up to 38 miRNAs capable of downregulating CD. Interestingly, these miRNAs are associated with biological processes controlling cell adhesion and cell migration. The present findings support the view that during NB growth on a substrate or when spreading as floating neurospheres, CD expression is epigenetically modulated to confer survival advantage. Thus, epigenetic targeting of CD could represent an additional strategy to prevent NB metastases.

Abstract 1214: Investigating therapeutic strategies to promote immune rejection of KRASG12C inhibitor-resistant subpopulations in lung cancer

Abstract Oncogenic KRAS signaling not only promotes progression and survival of cancer cells, but it can also play a major role in suppressing anti-tumor immunity through several mechanisms, including secretion of cytokines, reduction of antigen presentation and suppression of tumor-intrinsic interferon responses. Consequently, treatment with KRASG12C inhibitors alleviates oncogenic KRAS driven immune suppression, resulting in a profound remodeling of the tumor microenvironment (TME). In immunogenic mouse models, these changes can lead to the generation of some complete responses which are dependent on the engagement of the immune system. In this project we aim to investigate whether the induction of immune responses triggered by KRASG12C inhibition can effectively target inhibitor-resistant subclones. To achieve this, we mimic the development of drug resistance by combining reporter-traced isogenic KRASG12C and KRASG12D mutant lung cancer cells, where the KRASG12D mutant cells are inherently resistant to KRASG12C inhibitors. Growth of resistant cells was monitored via in vivo luciferase imaging and end point flow cytometry. KRASG12C inhibition as monotherapy, either with the active-state KRASG12C inhibitor RMC-4998 or the inactive-state inhibitor adagrasib, led to a proliferative advantage of the drug-resistant subpopulation, suggesting that any immune response to KRASG12C inhibition in the bulk tumor population was insufficient to target the resistant cell subclones. To investigate if immune mediated elimination of resistant cells could be achieved using combinations, we assessed two strategies commonly evaluated in clinical trials with KRASG12C inhibitors: anti-PD1 blockade and SHP2 inhibition. Of note, KRASG12D mutant cells do not respond to these therapies when grown subcutaneously. Treatment of RMC-4998 in combination with either anti-PD1 or the SHP2 inhibitor RMC-4550 was able to induce complete eradication of both KRASG12C and KRASG12D mutant lung cancer cells, despite the resistant cells being insensitive to these therapies when treated in the absence of sensitive cells. Furthermore, both combinations induced immune memory towards the resistant cells. These long-term effects were dependent on the immune system as they were not observed in immune deficient mice. Immune profiling of treated tumors revealed that both combinations can result in a more profound remodeling of the TME and potentiate the immune responses. Overall, our preclinical results demonstrate that the anti-tumor responses generated by the combination of KRASG12C inhibitors with SHP2 inhibition and/or immune checkpoint blockade can result in the bystander immune mediated killing of subclones of KRASG12C inhibitor resistant cells, providing a paradigm for the development of therapeutic combinations that may be more able to counter the development of inhibitor resistance. Citation Format: Mona Tomaschko, Christopher Moore, Claire E. Pillsbury, Kangbo Ng, Sophie de Carne, Sareena Rana, Andrea de Castro, Panayiotis Anastasiou, Miriam Molina-Arcas, Julian Downward. Investigating therapeutic strategies to promote immune rejection of KRASG12C inhibitor-resistant subpopulations in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1214.

Abstract 5387: Host-derived CCL2 drives prostate cancer bone metastasis via CCND2/STAT3 pathway

Abstract Overexpression of tumor-derived CCL2 has been shown to partially promote prostate cancer (PCa) bone metastasis, but the function of host-derived CCL2 is not fully understood. We first developed a mouse model using intracardiac injection and obtained highly metastatic PCa cell subclones through in vivo selection. The highly metastatic tumor cells were injected intracardially into CCL2 knockout (CCL2 KO) mice and wild-type (WT) controls, and the tumor cell growth and metastasis were monitored weekly by bioluminescence imaging. The osteolytic lesions were evaluated by radiography, micro-computed tomography (microCT) and immunohistochemistry (IHC) staining. The cells in the bone microenvironment were analyzed by flow cytometry. Also, RNA sequencing (RNA-seq) analysis was performed on the cells either from CCL2 KO mice or WT controls. We found that a loss of host-derived CCL2 significantly retarded tumor growth in bone and prolonged mouse survival. In addition, bone density analysis revealed a decrease in osteolytic lesion in CCL2 KO mice, compared to WT controls. Systemically, CCL2 deficiency reduced the proportion of granulocyte-myeloid-derived suppressor cell (G-MDSC) populations with immunosuppressive function and decreased the PCa conditioned medium (CM)-induced osteoclast formation. The enrichment analysis revealed that the dysregulated genes in bone metastatic cells from CCL2 KO mice were significantly enriched in several biological processes, including vitamin metabolism, monoatomic anion homeostasis, immune response, and bone growth. Additionally, we also confirmed that the CCND2/STAT3 pathway is apparently suppressed in the cells from CCL2 KO mice. This study demonstrated that targeting the host-derived CCL2 might be a novel potential anti-metastatic approach. Supported by the NSFC projects (81972766, 81972420, 82173336), as well as grants from the Science and Technology Project of Shenzhen (JCYJ20190809161811237, JCYJ20210324104214040). Key words Host-derived CCL2; Prostate cancer; Bone metastasis; osteoclast; CCND2 Citation Format: Jie Meng, Ming Chang, Siyuan Qin, Xin Huang, Weiping Liang, Haibo Tong, Jian Zhang. Host-derived CCL2 drives prostate cancer bone metastasis via CCND2/STAT3 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5387.

Abstract 5661: Identifying and intervening rare resistant subclones to BRAF/MEK inhibitors in metastatic melanoma

Abstract Acquired drug resistance poses a significant challenge in the treatment of cancer. This study focuses on melanoma as a model system to understand and address pre-existing subclonal resistance. The use of BRAF and MEK inhibitors (BRAFi/MEKi) in patients with metastatic melanoma harboring BRAF activating mutations has resulted in remarkable responses in the majority of cases. However, most patients have disease relapse after one year, and there are limited subsequent treatment options. One possible cause of relapse is the existence of a subpopulation of cells resistant to BRAFi/MEKi prior to treatment, raising the possibility that commencing counter-resistance treatment earlier may delay or abolish resistance to BRAFi/MEKi. We aim to enhance the detection and treatment of low-frequency resistant mutations, addressing the current oversight by existing technologies. This research is crucial due to a lack of consensus on the clinical management of these mutations. By using quantitative blocker displacement amplification (qBDA) technology and integrating patient samples with cell line models, our project seeks to determine 1) the prevalence of low-frequency resistant subclones by measuring the Variant Allele Frequency (VAF) of known resistance mutations along the MAPK/PI3K pathways in FFPE biopsies from 149 metastatic melanoma patients and 2) determine the functional impact of different VAF levels on the outgrowth of resistant subclones. We assessed 14 loci in 6 known resistance genes: NRAS, KRAS, PIK3CA, AKT1, MAP2K1, and MAP2K2, as well as BRAF itself as a positive control. As expected, all 55 patients analyzed harbored BRAF activating mutations, while 17 patients had at least one resistant subclone above our preliminary cutoff of 60% VAF. To simulate the behavior of low-VAF resistance mutations, we conducted in vitro experiments by spiking in labeled BRAF/MEK inhibitor-resistant melanoma cells into their isogenic sensitive parental cell line. Our initial data demonstrated that, at least in this model system, a 10% VAF spike-in leads to the eventual outgrowth of resistant cells, but not in a 1% VAF spike-in, suggesting that either the density or the absolute number of the pre-existing resistant cells can dictate resistance evolution. In future experiments, we will assess multiple VAFs in an independent cell line as well as in vivo to identify the nature of its correlation with resistance outcomes and expand to additional models. In summary, our data suggest that a substantial proportion of metastatic melanoma patients with BRAF mutations have pre-existing resistant cell subclones, and that accurately assessing its VAF could provide clinically-relevant information. Ultimately, results from this work would be the basis for future personalized counter-resistance therapies, potentially extending treatment effectiveness for patients with pre-existing resistant mutations. Citation Format: Han Wang, Michael Nakazawa, Omid Veiseh, Lawrence Kwong, David Zhang. Identifying and intervening rare resistant subclones to BRAF/MEK inhibitors in metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5661.

Abstract 6941: Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA)

Abstract Retinoblastoma is a pediatric retinal cancer that initiates in response to biallelic RB1 loss or MYCN amplification in a cone precursor cell of origin. After initial emergence, RB1-/- retinoblastomas progress to more aggressive forms by accumulation of single nucleotide variants (SNVs) and highly recurrent somatic copy number alterations including 1q+, 2p+, 6p+ and 16q- (RB SCNAs), resulting in heterogeneous mixtures of subclonal cell populations. SCNAs are enriched in highly evolved and aggressive tumor subtypes and show prognostic value in retinoblastoma liquid biopsies. However, the transcriptomic effects of retinoblastoma SCNAs are poorly understood. Single cell methods may enable comparison of tumor subclones within emerging retinoblastoma tumors and thereby provide insight into tumor progression. We retrieved single cell RNA sequencing data for 25 retinoblastoma tumors and inferred SCNAs and subclone phylogenies from scRNAseq information using a recently published tool, Numbat. We identified tumors with clonal variation in RB SCNAs and excluded samples with poor transcriptome quality or excessive additional SCNAs. We proceeded with analysis of 11 tumors with subclonal heterogeneity for an SCNA of interest, including 10 with 1q+, eight with 2p+, four with 6p+, and six with 16q-. After removal of nontumor cells, we defined cell clusters, cluster marker genes, and cell cycle phase gene signatures, and we compared cell state distributions for subclones with an acquired SCNA relative to their immediate antecedents. These analyses revealed recurrently discriminated transcriptomic clusters corresponding to six discrete cell cycle states (post-mitotic G1, G1, G1/S, S, S/G2, G2/M) and variable cell stress states, including a cluster with hypoxia and heat shock related marker gene expression in all samples. In each tumor, SCNA-defined tumor subclones had a distinct cell state distribution. Tumor subclones with acquired 1q+ were enriched in G2 states and depleted in G1 states. Subclones with 2p+ were enriched in G2 states. Subclones with 6p+ were enriched in S phase states. Subclones with 16q- were diminished in post-mitotic cell states. Tumor clones with 1q+ and 16q- were most strongly enriched for G2 phase cells. Such 1q+/16q- tumor cells were significantly differentially distributed in three tumor replicates. Tumor clones with other RB SCNAs were enriched over diploid clones in proliferating G2 cells without SCNA specific effects. To identify candidate drivers of SCNA-specific transcriptomic effects we measured RB SCNA clonal variation in gene expression within and across transcriptomic states. We identified recurrently differentially expressed in-segment genes in 1q+, 2p+, 6p+, and 16q-, as well as out-of-segment genes. Finally, we propose molecular mechanisms underlying SCNA recurrence that may enable tumor evolution in retinoblastoma. Citation Format: Kevin Stachelek, David E. Cobrinik. Transcriptomic effects of retinoblastoma progression-related somatic copy number alteration (SCNA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6941.

Morphological profiling in human neural progenitor cells classifies hits in a pilot drug screen for Alzheimer's disease.

Alzheimer's disease accounts for 60-70% of dementia cases. Current treatments are inadequate and there is a need to develop new approaches to drug discovery. Recently, in cancer, morphological profiling has been used in combination with high-throughput screening of small-molecule libraries in human cells in vitro. To test feasibility of this approach for Alzheimer's disease, we developed a cell morphology-based drug screen centred on the risk gene, SORL1 (which encodes the protein SORLA). Increased Alzheimer's disease risk has been repeatedly linked to variants in SORL1, particularly those conferring loss or decreased expression of SORLA, and lower SORL1 levels are observed in post-mortem brain samples from individuals with Alzheimer's disease. Consistent with its role in the endolysosomal pathway, SORL1 deletion is associated with enlarged endosomes in neural progenitor cells and neurons. We, therefore, hypothesized that multi-parametric, image-based cell phenotyping would identify features characteristic of SORL1 deletion. An automated morphological profiling method (Cell Painting) was adapted to neural progenitor cells and used to determine the phenotypic response of SORL1-/- neural progenitor cells to treatment with compounds from a small internationally approved drug library (TargetMol, 330 compounds). We detected distinct phenotypic signatures for SORL1-/- neural progenitor cells compared to isogenic wild-type controls. Furthermore, we identified 16 compounds (representing 14 drugs) that reversed the mutant morphological signatures in neural progenitor cells derived from three SORL1-/- induced pluripotent stem cell sub-clones. Network pharmacology analysis revealed the 16 compounds belonged to five mechanistic groups: 20S proteasome, aldehyde dehydrogenase, topoisomerase I and II, and DNA synthesis inhibitors. Enrichment analysis identified DNA synthesis/damage/repair, proteases/proteasome and metabolism as key pathways/biological processes. Prediction of novel targets revealed enrichment in pathways associated with neural cell function and Alzheimer's disease. Overall, this work suggests that (i) a quantitative phenotypic metric can distinguish induced pluripotent stem cell-derived SORL1-/- neural progenitor cells from isogenic wild-type controls and (ii) phenotypic screening combined with multi-parametric high-content image analysis is a viable option for drug repurposing and discovery in this human neural cell model of Alzheimer's disease.

Driving effect of P16 methylation on telomerase reverse transcriptase-mediated immortalization and transformation of normal human fibroblasts.

P16 inactivation is frequently accompanied by telomerase reverse transcriptase (TERT) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT. However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. A zinc finger protein-based P16-specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. P16 methylation drives TERT-mediated immortalization and transformation of normal human cells that may contribute to cancer development.

Abstract A019: The role of tumor transcriptional variability in evading acute CD8+ T cell exposure in melanoma

Abstract Throughout melanoma progression and immunotherapy treatment, melanoma cells often develop an ability to evade the immune system. Thus, there is a pressing need to identify and target the melanoma cell-intrinsic mechanisms underlying immune evasion. Our laboratory has pioneered the identification of transcriptional pathways that underlie melanoma progression. We have found that within even clonal melanoma cell populations, subclones of cells demonstrate tremendous heterogeneity in their transcriptional state. We previously demonstrated that these varying transcriptional states correspond to varying sensitivities of melanoma cells to stressors such as targeted therapies, and that these states can be stable across multiple cell divisions. We hypothesize that varying transcriptional states also correspond to cells that have differing abilities to evade CD8+ T cells, which are major effector cells against melanoma. To study the interactions between melanoma cells and CD8+ T cells, we engineered an ovalbumin antigen and OT-I T cell receptor system using mouse melanoma cell lines. We combined this system with DNA barcoding, which enables us to track subclones of cells undergoing immune selection. When cocultured together in vitro, ovalbumin-expressing melanoma cells are effectively killed by OT-I cells. However, we find that subclones of melanoma cells consistently possess different sensitivity to OT-I cells, and these differences are not due to their genetic background nor their ability to express and present ovalbumin. We then leveraged our DNA barcoding system with single cell RNA sequencing (scRNA-seq) to link the lineage and transcriptional state of single melanoma cells that persist through OT-I coculture. By expanding on these preliminary results, we aim to identify novel melanoma cell targets to decrease immune evasion. Citation Format: Raymond W.S. Ng, Sydney M Shaffer. The role of tumor transcriptional variability in evading acute CD8+ T cell exposure in melanoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A019.

Reciprocal interactions between malignant cells and macrophages enhance cancer stemness and M2 polarization in HBV-associated hepatocellular carcinoma.

Background: The tumor microenvironment of cancers has emerged as a crucial component in regulating cancer stemness and plays a pivotal role in cell-cell communication. However, the specific mechanisms underlying these phenomena remain poorly understood. Methods: We performed the single-cell RNA sequencing (scRNA-seq) on nine HBV-associated hepatocellular carcinoma (HCC) patients. The heterogeneity of the malignant cells in pathway functions, transcription factors (TFs) regulation, overall survival, stemness, as well as ligand-receptor-based intercellular communication with macrophages were characterized. The aggressive and stemness feature for the target tumor subclone was validated by the conduction of in vitro assays including sphere formation, proliferation, Annexin V apoptosis, flow cytometry, siRNA library screening assays, and multiple in vivo preclinical mouse models including mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection. Results: Our analysis yielded a comprehensive atlas of 31,664 cells, revealing a diverse array of malignant cell subpopulations. Notably, we identified a stemness-related subclone of HCC cells with concurrent upregulation of CD24, CD47, and ICAM1 expression that correlated with poorer overall survival. Functional characterization both in vitro and in vivo validated S100A11 as one of the top downstream mediators for tumor initiation and stemness maintenance of this subclone. Further investigation of cell-cell communication within the tumor microenvironment revealed a propensity for bi-directional crosstalk between this stemness-related subclone and tumor-associated macrophages (TAMs). Co-culture study showed that this interaction resulted in the maintenance of the expression of cancer stem cell markers and driving M2-like TAM polarization towards a pro-tumorigenic niche. We also consolidated an inverse relationship between the proportions of TAMs and tumor-infiltrating T cells. Conclusions: Our study highlighted the critical role of stemness-related cancer cell populations in driving an immunosuppressive tumor microenvironment and identified the S100A11 gene as a key mediator for stemness maintenance in HCC. Moreover, our study provides support that the maintenance of cancer stemness is more attributed to M2 polarization than the recruitment of the TAMs.

A Novel Role for the Adenovirus L4 Region 22K and 33K Proteins in Adeno-Associated Virus Production.

Despite decades of research in adeno-associated virus (AAV) and the role of adenovirus in production, the interplay of AAV and adenovirus is not fully understood. Specific regions of the adenoviral genome containing E1, E2a, E4 open reading frame (ORF), and VA RNA have been demonstrated as necessary for AAV production; however, incorporating these regions into either a producer cell line or subcloning into an Ad helper plasmid may lead to inclusion of neighboring adenoviral sequence or ORFs with unknown function. Because AAV is frequently used in gene therapies, removing excessive adenovirus sequences improves the Ad helper plasmid size and manufacturability, and may lead to safer vectors for patients. Furthermore, deepening our understanding of the helper virus genes required for recombinant AAV (rAAV) production has the potential to increase yields and manufacturability of rAAV for clinical and commercial applications. One region continuously included in various Ad helper plasmid iterations is the adenoviral E2a promoter region that appears to be necessary for E2a expression. Due to the compact nature of viral genomes, the E2a promoter region overlaps with the Hexon Assembly/100K protein and the L4 region. The L4 region, which contains the coding sequences for 22K and 33K proteins, had not been thought to be necessary for AAV production. Through molecular techniques, this study demonstrates that the adenoviral 22K protein is essential for rAAV production in HEK293 cells by triple transfection and that the 33K protein synergistically increases rAAV yield.

Early Subclones Showing Aberrant Co-Expression of Stem Cell, T Cell and Myeloid Genes Can be Detected By Flow Cytometry in MYD88 Mutated Waldenström's Macroglobulinemia Patients

Background: Our RNA-Seq analysis of 249 untreated MYD88 mutated WM patients showed aberrant expression of stem cell, pre-B-cell, T-cell and myeloid genes in early WM B clones. This expression pattern was associated with inferior progression free survival with BTK inhibitor therapy and diminished with advancing disease. We therefore sought to validate this early signature at the protein level using flow cytometry and evaluate its potential as a clinical assay to monitor WM disease evolution. Methods: RNA-Seq predictions were validated on an independent cohort of treatment naïve MYD88 mutated WM patients and healthy (HD) samples. We designed a 13-color panel for flow cytometry detection of surface markers CD19, CD138, k and l immunoglobulin (Ig) light chains, CD34, CD117 (KIT), CD4, CD8A, CD33, FCGR3A (CD16), CEACAM8 (CD66b), pre-BCR (using an antibody that recognizes a unique conformational epitope of the BCR complex formed by the association of IGLL1 and VPREB1); Zombie Yellow was used to label dead cells. Peripheral B (PBMC) and/or BM mononuclear cells (BMMC) were isolated from WM and HD samples by density gradient centrifugation using Ficoll-Paque medium. Data acquisition was performed for at least 10^6 nucleated cells per tube in BDLSR Fortessa flow cytometer using the FACSDiva software. Results: RNA-Seq analysis showed high expression of genes atypical of mature B-cells that significantly diminished with increasing disease burden. For validation purposes, we selected genes reaching at least a 10-fold drop in their expression levels between the earliest and the most advanced disease cohort. In particular, the T-cell and myeloid genes including CD4, CD8A, FCGR3A, CD33, CEACAM8 as well as the prePCR surrogate light chain genes IGLL1 and VREPB1 respectively decreased from 67.6, 7.1, 80.5, 23.2, 8.3, 114, 38.3 to 2.4, 0.9, 1.7, 0.7, 0.4, 12.2 and 4.3 transcripts per million (TpM). A subclonal stem cell signature was detected, including CD34 and KIT, which dropped from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively. It is well established that WM represents a mature, post germinal center clone. Consistent with this notion, expression of memory B-cell and plasma blast genes in our RNASeq data set, including PRDM1, XBP1 and SPIB, was high in all patients regardless of disease progression and expression of the studied atypical genes. Our independent patient validation cohort had a median age of 68 (range 50-88 years), BM involvement by immunohistochemistry of 40% (range 10-95%), hemoglobin of 13.2 (range 8.1-13.4 g/dL) and serum IgM of 686 (range 536-1,151 mg/dL). An IgMk monoclonal spike was detected in all cases. All patients were mutated for MYD88. Within the CD19+k+-selected cells, we detected a subpopulation of cells co-expressing CD34, KIT, CD4, CD8A, FCGR3A, CD33, CEACAM8 and preBCR in all the patients, including in PBMC. As predicted, these markers were relatively enriched in samples with lower BM disease burden ( Figure 1, 2). Even in those with the highest BM involvement, the subclone was still detectable in both cellular compartments. Conversely, HD controls showed normal B cell populations and no evidence of the aberrant subclone. Conclusions: In WM primary cells, we observed the existence of a clonal B cell population that appears to re-activate stem cell programming early on and progressively is replaced by clones bearing more typical B cell markers with disease expansion. This study lays the foundation to develop a widely accessible methodology to accurately diagnose IgM MGUS/early-stage WM and monitor disease evolution. Flow-based sorting of these subclones will allow targeted multi-omic studies with insights into disease pathogenesis and targeted therapeutic applications.

Atypical Stem Cell, Pre-B-Cell, T-Cell and Myeloid Gene Expression Characterizes Early Waldenstrom's Macroglobulinemia Clones Which Diminishes with Advancing Disease and Has Therapeutic Implications

Background: In previous studies using multi-omic data for 253 treatment-naive patients with Waldenstrom's Macroglobulinemia (WM), we identified three subtypes of WM: B-cell like (BCL), Plasma cell like (PCL), and an intermediate clone enriched for early/smoldering WM from which the other two evolved (Hunter et al, ASH 2022). The BCL and PCL subtypes show distinct mutation and transcriptional signatures, as well as clinical characteristics. Diffusion pseudo-time (DPT) analysis suggests all patient tumors, regardless of subtype have a shared evolutionary path and can be divided into Early and Late DPT determined stages that mirror disease progression. Additional analysis of DPT suggests it represents a continuous process akin to clonal evolution. We therefore sought to further characterize the DPT signal in MYD88 mutated WM. Methods: RNASeq was performed on CD19-selected bone marrow (BM) samples from 249 of the 253 treatment-naive WM patients who were MYD88 mutated, as well as 13 paired CD19+CD27- and CD19+CD27+ selected healthy donor (HD) peripheral blood samples. Whole exome sequencing of CD19-selected BM cells along with CD19-depleted peripheral blood mononuclear controls was also performed for 215 of the WM samples. To assess BM histology, slides from 66 BM paraffin blocks from these WM patients were analyzed. Results: WM patients had median age of 66 (range 31-95 years), BM involvement of 50% (5-95%) and serum IgM level of 3,162 (104-10,321 mg/dL). The median follow-up for the study cohort was 8.8 (range 0.2 - 33.8 years). Patients were stratified into 5 cohorts by DPT values. Analysis of BM involvement by DPT cohorts revealed a stepwise increase with the median involvement rising from 20% in the lowest to 80% in the highest cohort (p<0.0001). Increasing DPT predicted for shorter time from biopsy to first therapy (p=0.0003) but was not predictive of time from diagnosis to first therapy consistent with DPT alignment by stage of WM development. RNASeq analysis revealed high expression of genes atypical of mature B-cells that diminished with increasing DPT ( Figure 1). Among these were the myeloid chemo-attractants CXCL1, CXCL8 and CXCL12 which fell from a median of 4.4, 67.2 and 41.1 to 0.1, 3.6 and 0.8 transcripts per million (TpM) in the lowest and highest DPT cohorts, respectively (p<0.0001). Accordingly, all the top 20 gene ontology (GO) terms associated with DPT were related to activation, degranulation, and migration of myeloid cells and leukocytes. Pathological review of the 66 BM slides ranked by DPT, showed increased early myeloid cells and eosinophils clustering near small lymphoid aggregates, and as DPT increased, increased lymphocytes and mast cells with decreased myeloid cell involvement was observed. By RNASeq, pre-B-cell genes including MYB, the VDJ recombination genes RAG1, RAG2 and DNTT as well as the surrogate light chain genes IGLL1 and VPREB1 fell from 22.5, 10, 13.5, 61.1, 144 and 38.3 to 2.2, 1, 1.1, 9.1, 12.2 and 4.3 TpM, respectively (p<0.0001). T-cell and myeloid genes including CD4, CD8A, FCGR3A (CD16), CD14 and CD33 also decreased with increased DPT from 67.6, 7.1, 80.5, 200, and 23.2 to 2.4, 0.9, 1.7, 3.6 and 0.7 TpM, respectively (p<0.0005). A subclonal stem cell signature was also detected, including CD34 and KIT (CD117), which fell from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively (0<0.0001). CD34, KIT, preBCR, CD4, CD8A, FCGR3A, CEACAM8 (CD66b), and CD33 expression was confirmed by CD19+ light chain restricted flow cytometry in WM but not in HD samples. In addition, the MB and plasma blast genes PRDM1 and XBP1 were elevated in all samples regardless of DPT, consistent with a putative memory B-cell origin. Progression free survival (PFS) in response to proteasome inhibitors revealed strong stratification by subtype (p=0.001), as well as inferior PFS with BTK-inhibitors in low DPT patients (p= 0.044; Figure 2), suggesting less BTK dependence in the early DPT clones. Consistent with this finding, gene set enrichment identified increased BCR signaling with increased DPT. Conclusions: This analysis suggests that stem cell program reactivation and atypical marker expression characterize early WM clones that are ultimately replaced by clones exhibiting more typical B-cell markers with disease progression. The findings may be relevant to treatment selection and provide a framework for investigating subtype and early/late evolutionary staging in the treatment approach to WM.