Cell Surface Protein Expression Research Articles

Mapping genotypes to chromatin accessibility profiles in single cells.

In somatic tissue differentiation, chromatin accessibility changes govern priming and precursor commitment towards cellular fates1-3. Therefore, somatic mutations are likely to alter chromatin accessibility patterns, as they disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wild-type cells. Here, to chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed genotyping of targeted loci with single-cell chromatin accessibility (GoT-ChA). This high-throughput platform links genotypes to chromatin accessibility at single-cell resolution across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from patients with myeloproliferative neoplasms with JAK2V617F-mutated haematopoiesis. Differential accessibility analysis between wild-type and JAK2V617F-mutant progenitors revealed both cell-intrinsic and cell-state-specific shifts within mutant haematopoietic precursors, including cell-intrinsic pro-inflammatory signatures in haematopoietic stem cells, and a distinct profibrotic inflammatory chromatin landscape in megakaryocytic progenitors. Integration of mitochondrial genome profiling and cell-surface protein expression measurementallowed expansion of genotyping onto DOGMA-seq through imputation, enabling single-cell capture of genotypes, chromatin accessibility, RNA expression and cell-surface protein expression. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner, influencing inflammation states and differentiation trajectories. We envision that GoT-ChA will empower broad future investigations of the critical link between somatic mutations and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

A high-content screen of FDA approved drugs to enhance CAR T cell function: ingenol-3-angelate improves B7-H3-CAR T cell activity by upregulating B7-H3 on the target cell surface via PKCα activation

BackgroundCAR T cell therapy is a promising approach to improve outcomes and decrease toxicities for patients with cancer. While extraordinary success has been achieved using CAR T cells to treat patients with CD19-positive malignancies, multiple obstacles have so far limited the benefit of CAR T cell therapy for patients with solid tumors. Novel manufacturing and engineering approaches show great promise to enhance CAR T cell function against solid tumors. However, similar to single agent chemotherapy approaches, CAR T cell monotherapy may be unable to achieve high cure rates for patients with difficult to treat solid tumors. Thus, combinatorial drug plus CAR T cell approaches are likely required to achieve widespread clinical success.MethodsWe developed a novel, confocal microscopy based, high-content screen to evaluate 1114 FDA approved drugs for the potential to increase expression of the solid tumor antigen B7-H3 on the surface of osteosarcoma cells. Western blot, RT-qPCR, siRNA knockdown and flow cytometry assays were used to validate screening results and identify mechanisms of drug-induced B7-H3 upregulation. Cytokine and cytotoxicity assays were used to determine if drug pre-treatment enhanced B7-H3-CAR T cell effector function.ResultsFifty-five drugs were identified to increase B7-H3 expression on the surface of LM7 osteosarcoma cells using a novel high-content, high-throughput screen. One drug, ingenol-3-angelate (I3A), increased B7-H3 expression by up to 100%, and was evaluated in downstream experiments. Validation assays confirmed I3A increased B7-H3 expression in a biphasic dose response and cell dependent fashion. Mechanistic studies demonstrated that I3A increased B7-H3 (CD276) mRNA, total protein, and cell surface expression via protein kinase C alpha activation. Functionally, I3A induced B7-H3 expression enhanced B7-H3-CAR T cell function in cytokine production and cytotoxicity assays.ConclusionsThis study demonstrates a novel high-content and high-throughput screen can identify drugs to enhance CAR T cell activity. This and other high-content technologies will pave the way to develop clinical trials implementing rational drug plus CAR T cell combinatorial therapies. Importantly, the technique could also be repurposed for an array of basic and translational research applications where drugs are needed to modulate cell surface protein expression.

Abstract 4682: Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma

Abstract The B-cell Receptor (BCR), comprising membrane Ig (mIg) and CD79a/b, controls normal signaling in B-cells, but dysregulation of this signaling is the dominant driver of leukemia/lymphoma. The class-specific mIg subunit of the BCR is a rational drug target for B-cell malignancies. It plays a critical role in the functioning of these cells, and allows for restricted targeting of only B-cells expressing an Ig class, offering a more tumor-specific approach than current pan-B-cell treatments. We developed a first-in-class antibody, WBMP-4, to the mIgM-BCR, which inhibits cell growth and induces apoptosis via widespread down-regulation of tyrosine kinase activity. Given the potent inhibition of intracellular signaling induced upon WBMP-4 binding to the mIgM-BCR, we expect that translation of membrane proteins will be impacted. Here we investigate the expression and phosphorylation profile of cell surface proteins following treatment of malignant B-cells with WBMP-4. Using western blot and flow cytometry, we measure levels of CD79a/b, CD19, CD20, CD21, CD22, CD32a, CD81, and CXCR4 at 6, 24, and 48 hours after low (5ug/mL) and high (20ug/mL) dose WBMP-4 treatment, as compared to control, untreated Burkitt lymphoma (CA46) cells. We analyze phosphorylation levels of those surface proteins that serve as kinases in this system (CD79a/b, CD19, CXCR4, etc.). Using these same methods, we also examine the expression and phosphorylation of key B-cell transcription factors (e.g. NF-kB, c-MYC, STAT5, PAX5, EBF1, FOXO, and BCL-2). Preliminary analyses show a complete absence of CD79a protein (western blot), and decreased phospho-kinase activity of CD79b (Pamgene phosphokinase array) following treatment of Burkitt lymphoma cells with WBMP-4, as compared to untreated cells. While the effect of an antibody binding to mIg may be most pronounced for CD79a/b relative to other proteins, as mIg and CD79a/b form a complex, we expect that the absence of CD79a/b is a result of WBMP-4-induced mIgM inhibition modulating downstream signaling and transcription factor activity. By examining cell surface markers, we may gain insight into how WBMP-4 impacts a cell’s interaction with the tumor microenvironment, and this information is valuable for diagnostic and patient monitoring purposes, and to determine drug combination strategies. This work will be considered in the context of our ongoing research on WBMP-4’s inhibition of intracellular signaling pathways, and the resulting biologic effects, and will contribute to the continued development of WBMP-4 by providing an understanding of the effects achieved (cell growth inhibition, cell differentiation, apoptosis) at different dosages and if these effects are maintained over time. As a novel, efficacious, and low toxicity treatment strategy for B-cell leukemia and lymphoma, WBMP-4 has significant clinical potential. Citation Format: Rachel S. Welt, David Kostyal, Virginia Raymond, Jose M. Lobo, Sydney Welt. Membrane protein expression and activity following inhibition of B-cell receptor-initiated signaling by WBMP-4, a therapeutic antibody for leukemia/lymphoma [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 4682.

Abstract 335: BD®OMICS-Guard Sample Preservation Buffer preserves mRNA and cell surface epitopes for single-cell sequencing applications

Abstract Multiomics has emerged as a powerful tool to derive insights on the transcriptomic and proteomic data of human cancer samples and preclinical models of blood and tumor malignancies. Due to the sensitivity of sequencing techniques, sample quality and stability can potentially interfere with results. BD® OMICS-Guard Sample Preservation Buffer (SPB), a novel preservation solution, allows cells and bulk tissues to be stored for up to 72h at 4 °C, while reliably capturing the true biology of cells. This reagent gently preserves mRNA and protein integrity without the traditional cross-linking and harsh fixatives, increasing flexibility and expanding options for study designs. Here, we showcase the preservation properties ofthis reagent for multiomic readouts in human cells and mouse tissue across three different timepoints.BD® OMICS-Guard SPB’s preservation of mRNA from human PBMC and bulk mouse spleen tissues wasevidenced by WTA for differentially expressed genes in samples preserved for 24, 48 and 72h comparedto fresh/unpreserved controls. Protein epitope preservation was demonstrated via CITE-Seq assay withthe BD® AbSeq Human Immune Discovery Panel (human PBMC) or a 30-plex panel of anti-mouse BD®AbSeq Antibody-Conjugated Oligonucleotides (mouse splenocytes) and confirmed with multicolor flowcytometry. For both sample types, high correlation R2 values (>0.87) were calculated for the preservation time points versus fresh controls. Concomitant flow cytometry analysis of major cell types and surface protein expression was consistent with sequencing data for all samples. Specificity and sensitivity of major cell type markers and cell surface proteins across cell types and time is visualized in the BD®AbSeq Antibody-generated heatmap for each sample. Additionally, BD® OMICS-Guard SPB maintains mRNA integrity in targeted gene assays and VDJ full-length assays, suggesting robust preservation of select transcripts readily interrogated in single-cell profiling. Batch effect across time and technical replicates for both sample types was limited with the BD Rhapsody™ HT Xpress System and delineated by donor/sample specific tSNEs across time. We demonstrate that the BD® OMICS-Guard Sample Preservation Buffer effectively preserves both mRNA and surface proteins for single-cell experiments. BD® OMICS-Guard SPB allows the convenient preservation of samples such as tumor biopsies to be collected and stored for up to 72h at 4 °C while maintaining mRNA and protein integrity, allowing the flexibility for precious samples to be potentially shipped to a central sequencing site to ensure standardized sequencing workflows. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. Citation Format: Samuel X. Shi, Cynthia Sakofsky, Zhiqi Zhang, Samatha Vadrevu, Xiaoshan Shi, Hye-Won Song, Aruna Ayer. BD®OMICS-Guard Sample Preservation Buffer preserves mRNA and cell surface epitopes for single-cell sequencing applications [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 335.

Abstract 3696: Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate cancer

Abstract Background: Prostate-Specific Membrane Antigen (PSMA) is a highly expressed prostate cancer (PCa) cell surface protein regulated by androgen receptor signaling and the target of the recently approved radioligand therapy 177Lu-PSMA-617. While some patients have dramatic responses to this therapy, rates of upfront or acquired resistance are high even in patients with PSMA-PET-avid disease, and better biomarkers of 177Lu PSMA-617 response and resistance are needed to select patients more effectively for this therapy. To address this need, we evaluated the relationship between longitudinal circulating tumor cell (CTC) androgen signaling and PSMA expression to 177Lu-PSMA-617 response or resistance in patients with metastatic prostate cancer. We leveraged a CTC androgen signaling signature that we have previously shown is prognostic for patients with metastatic prostate cancer treated with AR signaling inhibitors. Methods: Longitudinal blood samples were collected from 20 patients for circulating tumor cell (CTC) analysis at C1D1, C2D1, C4D1, and C6D1. CTCs were isolated with antibodies to EpCAM followed by parallel enumeration, analysis of cell surface expression of PSMA and RNA extraction for AR or neuroendocrine marker transcriptional profiling. Results: CTC samples were assessed for PSMA cell surface protein expression prior to treatment and on treatment. FOLH1 (PSMA) gene expression was detected from CTCs at baseline that persisted through treatment. Gene expression representative of AR pathway activation was present in 70% of the cohort at baseline and 50% of patients at C2D1. Concordant with these observations, no significant decrease in CTC number or PSMA protein expression was seen from baseline to C2D1. However, increased CTC AR pathway activation at C2D1 was associated with increased mean serum PSA (155 vs 21.93 ng/ml) and was associated with decreased duration of response. Conclusions: Improved tools to predict who will respond to 177Lu-PSMA-617 and detect early treatment resistance and disease progression are needed. Here, we show that persistent CTC burden, CTC AR signaling, and CTC PSMA expression are common early in treatment with 177Lu-PSMA-617 and may be associated with lower likelihood of clinical response. Further ongoing studies are needed to elucidate the relationship between these liquid biopsy results and clinical outcomes. Citation Format: Jamie M. Sperger, Marina N. Sharifi, Luke Nunamaker, Viridiana Carreno, Alex H. Chang, Shannon R. Reese, Charlotte Linebarger, Amy K. Taylor, Grace C. Bliitzer, John Floberg, Shuang G. Zhao, Joshua M. Lang. Liquid biopsy biomarker analysis during treatment with 177Lu-PSMA-617 in castrate resistant prostate 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 3696.

HPV16 E7 modulates the cell surface expression of MET and CD109 via the AP2 complex

Multiple cellular pathways are affected by HPV E6 and E7 oncoproteins, including endocytic and cellular trafficking. HPV-16 E7 can target the adaptor protein (AP) complex, which contains proteins important during endocytosis transport. To further investigate the role of HPV E7 during this process, we analysed the expression of cell surface proteins in NIKS cells expressing HPV-16 E7. We show that different cell surface proteins are regulated by HPV-16 E7 via interaction with AP2. We observed that the expression of MET and CD109 membrane protein seems to be upregulated in cells expressing E7. Moreover, the interaction of MET and CD109 with AP2 proteins is disrupted by HPV-16 E7. In addition, in the absence of HPV-16 E7, there is a downregulation of the cell membrane expression of MET and CD109 in HPV-positive cell lines. These results expand our knowledge of the functions of E7 and open new potential cellular pathways affected by this oncoprotein.

Phenotypic and Functional Characterization of Memory CD4+ and CD8+ T Cells After Antigenic Stimulation.

The encounter of T cells with the antigen through the interaction of T cell receptors with peptides and major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs) can generate effector response and memory T cells. Memory T cells developed following infections or vaccination may persist, leading to the generation of a specific immune response upon reexposure to the same pathogen through rapid clonal proliferation and activation of effector functions. T cell memory subsets can be identified based on the expression of several membrane markers such as CCR7, CD27, and CD45RA. Using fluorescent antibodies against these markers and a flow cytometer, it is possible to perform immunophenotyping via the analysis of cell surface expression of proteins by different subpopulations such as the subsets of naïve, effector, and memory T cells as well as via the analysis of functional markers that further characterize each sample. Intracellular cytokine staining allows for the evaluation of intracellular proteins expressed in T cells in response to antigenic stimulation. This chapter presents the phenotypic and functional characterization of memory T cells after antigenic stimulation, detailing the procedures for identifying intracellular and surface protein markers. Herein, we review and present a reproducible standardized protocol using antibodies for specific markers and applying flow cytometry.

Single-Cell Proteomics by Barcoded Phage-Displayed Screening via an Integrated Microfluidic Chip.

Recent advancements in the profiling of proteomes at the single-cell level necessitate the development of quantitative and versatile platforms, particularly for analyzing rare cells like circulating tumor cells (CTCs). In this chapter, we present an integrated microfluidic chip that utilizes magnetic nanoparticles to capture single tumor cells with exceptional efficiency. This chip enables on-chip incubation and facilitates in situ analysis of cell-surface protein expression. By combining phage-based barcoding with next-generation sequencing technology, we successfully monitored changes in the expression of multiple surface markers induced by CTC adherence. This innovative platform holds significant potential for comprehensive screening of multiple surface antigens simultaneously in rare cells, offering single-cell resolution. Consequently, it will contribute valuable insights into biological heterogeneity and human disease.

SBioSITe enables sensitive identification of the cell surface proteome through direct enrichment of biotinylated peptides

BackgroundCell surface proteins perform critical functions related to immune response, signal transduction, cell–cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite several enrichment methods exploiting their biochemical and biophysical properties.MethodsHere, we report a novel method for enrichment of proteins localized to cell surface. We developed this new approach designated surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. In this strategy, the primary amine groups of lysines on proteins on the surface of live cells are first labeled with biotin, and subsequently, biotinylated peptides are enriched by anti-biotin antibodies and analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS).ResultsBy direct detection of biotinylated lysines from PC-3, a prostate cancer cell line, using sBioSITe, we identified 5851 peptides biotinylated on the cell surface that were derived from 1409 proteins. Of these proteins, 533 were previously shown or predicted to be localized to the cell surface or secreted extracellularly. Several of the identified cell surface markers have known associations with prostate cancer and metastasis including CD59, 4F2 cell-surface antigen heavy chain (SLC3A2) and adhesion G protein-coupled receptor E5 (CD97). Importantly, we identified several biotinylated peptides derived from plectin and nucleolin, both of which are not annotated in surface proteome databases but have been shown to have aberrant surface localization in certain cancers highlighting the utility of this method.ConclusionsDetection of biotinylation sites on cell surface proteins using sBioSITe provides a reliable method for identifying cell surface proteins. This strategy complements existing methods for detection of cell surface expressed proteins especially in discovery-based proteomics approaches.

BRAF-Mutated Acute Myeloid Leukemia (AML) Represents a Distinct, Prognostically Poor Subgroup Enriched in Myelodysplasia-Related (MR-)AML

Background RAS signaling pathway mutations, mainly NRAS, KRAS, and/or PTPN11 variants, are common somatic events in de novo and MR-AML. BRAF mutations occur with lower frequency in AML and are reported to be associated with poor treatment outcome. However, no comprehensive analysis exists that firmly delineates clinical and molecular characteristics of BRAF-mutated AML, including insights into clonal architecture and co-occurring mutations. Methods We identified 42 patients (pts) with BRAF-mutated AML among pts with molecularly profiled disease in the setting of large comprehensive cancer centers [enriched for relapsed/refractory (R/R) and secondary AML] and cooperative group frontline trials (Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology, enriched for de novo AML). All pts had NGS-based targeted sequencing data, cytogenetic information, and detailed clinical annotations available. To fully evaluate the molecular landscape, 6 pts underwent integrated genomic profiling including paired tumor/normal whole exome sequencing and total transcriptome sequencing. We also analyzed pt samples using simultaneous single-cell molecular profiling and cell surface protein expression (DNA+Protein) sequencing to assess cell states and clonal make-up at single-cell resolution in a subset of pts (n=8). Results The 42 pts with BRAF-mutated AML had a median age of 67 years (range, 19-84); 57% were male. BRAF mutations were most common in cases of MR-AML (as defined by WHO 5th edition), accounting for 32 (76%) pts. BRAF mutations were found in newly diagnosed (n=19, 45%), R/R (n=8, 19%) and secondary AML (n=15, 36%) pts. As detected by bulk NGS sequencing, BRAF mutations were found both as dominant clonal and subclonal events, present at variant allele fractions (VAF) of 1-83% ( Figure 1, BRAF VAF indicated via color scale). Most (n=30, 71%) pts had non-V600 BRAF mutations (including G469, n=12; D594, n=7 and other, n=12). The most frequent co-occurring mutations were in the TET2 (36%), ASXL1 (33%), NRAS (29%), KRAS (26%), RUNX1 (19%), DNMT3A, FLT3-ITD/TKD, NPM1 and SRSF2 (all 17%) genes. By whole exome sequencing, BRAF-mutated AML samples carried a median mutational burden of 15 non-synonymous coding variants (range, 9-30), including 6 RAS-pathway and 3 MR-AML mutations. In 2 pts with paired relapse material available, the BRAF mutation was either stable at relapse or lost and the pt instead acquired a IQGAP3 mutation, supporting the RAS-pathway “addiction” of this leukemia. Single-cell multi-omic sequencing studies uncovering unique genotype-immunophenotype relationships and confirming clonal hierarchies are ongoing and will be presented at the meeting. Clinically, the BRAF-mutated pts had extremely poor survival, regardless of therapy. Pts were treated with low intensity [LI, n=10 (29%); LI+venetoclax (VEN, n=10 (29%)], high-intensity [HI, n=12 (34%) or HI+VEN, n=3 (9%)] regimens. The overall composite remission (CR+CRi) rate was 39%. Median overall survival (OS) for the entire cohort was 7 months, with 31 (89%) of pts deceased at the time of last follow-up. There were no significant differences in OS based on treatment regimens ( Figure 2). Importantly, there was no impact of BRAF variant allele burden on OS, with pts wiith dominant clonal and those with subclonal mutations having similar outcomes. Given the enrichment of BRAF mutations in MR-AML, we assessed whether RAS pathway mutations generally represented poor outcome prognosticators in this subgroup. A comparison of a control cohort of MR-AML pts with (n=129) and without (n=403) RAS pathway mutations treated on cytarabine/daunorubicin-based frontline protocols, revealed no survival difference between RAS-mutated and wild-type pts, suggesting a negative survival impact specific to BRAF-carrying leukemic clones. Conclusions BRAF mutations are rare, but recurrent molecular alterations in AML that are enriched in MR-AML. They associate with distinctly poor prognosis regardless of their clonal burden, without significant therapeutic advantage of currently available treatment regimens. This suggests the need to assess the utility of BRAF inhibitors and/or RAS pathway-targeting regimens such as MEK inhibitors in pts with AML carrying BRAF mutations. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202)

Minimal Residual Disease Detection By Single Cell DNA Sequencing Technology: A Feasible Approach for Clinical Application and Identification of the Landscape of MRD Clones

Introduction:Despite recent advances in AML treatment, patients continue to relapse and become chemotherapy resistant, leading to poor long-term overall survival. The identification of this measurable residual disease (MRD), defined as the post-therapy presence of leukemic cells, currently stands as one of the most well-established risk factors. The abnormal phenotypic and molecular characteristics of AML cells offer an opportunity for disease monitoring using techniques like multiparameter flow cytometry (MFC) and qPCR, which are currently considered the gold standard for MRD detection. However, these techniques have limitations. The aim of this study was to validate a different approach using single-cell DNA sequencing (scDNAseq) technology to detect MRD in patients achieving complete response (CR) and to characterize the genomic landscape of MRD clones and the potential identification of clonal evolution. Methods:We selected 24 cryopreserved bone marrow samples from 15 AML patients who participated in the QUIWI-PETHEMA clinical trial (NCT04107727) and achieved CR after induction and consolidation (n=20). 4 diagnosis samples were analyzed to determine genomic differences between the MRD clone and the initial leukemic population. All patients had bulk targeted NGS data available at diagnosis. Selection of CD34+ and/or CD117+ cells was performed using magnetic beads for enrichment of blasts and Mission Bio multiome single cell DNA+protein was performed using an AML-related 469 amplicon panel and 19 surface antibody mix. Multiplex of 3 independent samples was performed in each library preparation. Analysis was done according to manufacturer's instructions. MRD was also analyzed using MFC (n=14) EuroFlow panel with a limit of detection of 0.1% aberrant cells. RNA qRT-PCR was used in cases with NPM1 mutations (n=6). Quantification of MRD by scDNAseq was calculated according to the enrichment performance obtained from the manufacturer and the percentage of mutant cells. Results: The concordance between gold standard techniques for MRD and scDNAseq was 75% (15/20) (Table 1). Concordance between MFC and scDNAseq was 78% (11/14). The 3 discordant cases, positives by scDNAseq, MRD levels ranged between 0.04-0.09%, below the consensus cutoff of 0.1% to define MRD+. These results suggest that scDNAseq may complement MFC in the detection of very low levels of MRD. Concordance with qRT-PCR was 66% (4/6), but we only detected one patient with a persistent NPM1 clone. Taking advantage of the single cell approach, we were able to assess the genomic landscape of MRD clones and the clonal evolution in sequential samples. The number of clones/subclones and the number of variants per clone varied between patients with positive MRD as shown in Table 1. Interestingly, we analyzed 4 samples at diagnosis by scDNAseq and observed 2 cases in which MRD mutation was already present at diagnosis but was not informed as VAF was < 1%. In 4 patients, we detected small clones (around 1%) that remained unchanged in size despite treatment, suggesting that they likely represent clonal hematopoiesis. In 6 cases, consecutive samples were obtained showing clearance of some clones with other ones remaining stable (UPN2), progressive clearance of clones (UPN3, UPN4), acquisition of new clones (UPN9) and clearance of some clones and acquisition of new ones (UPN15) (Figure 1). Integration of scDNA and cell surface protein expression in the same cell allowed us to perform mutation-clone specific immunophenotypic analysis. Some cases showed a clear pattern in which one of the mutant clones had a significantly higher expression of some markers that was correlated with previous flow cytometry data (e.g. UPN1). Conclusions: Our study suggests that the use of scDNA technology is a feasible approach for detection of MRD in AML patients. Moreover, the use of this approach in sequential samples may allow deciphering clonal evolution, the co-occurrence of different mutations including potential clonal hematopoiesis mutations and identifying winner clones potentially responsible for disease persistence and relapse. Finally, the integration of mutations and surface antibody markers in the same cell provides a means for identifying the presence of mutations in different cell populations. Validation of these results in larger series of patients and correlation with clinical outcome are the next steps for validation of this technology.

CLL Cell-Derived Exosomes Impair Immune Function and Normal Hematopoiesis in CLL

CLL is characterized by immune and hematopoietic abnormalities such as T-cell dysfunction, hypogammaglobulinemia, increased levels of inflammatory cytokines, aberrant monocyte polarity, anemia, neutropenia, and low-grade bone marrow fibrosis. As a result, CLL patients become susceptible to increased rates of infection and/or second neoplasms. Exosomes are nanovesicles that participate in bidirectional communication between neoplastic cells and their microenvironment. Using electron microscopy, we found that CLL cells produce and release CD19 +/CD5 +exosomes, and in agreement with other investigators, we detected high levels of CLL cell-derived exosomes in the plasma of patients with CLL. By using bead-assisted flow cytometry, we found that at least 50% of circulating extracellular vesicles are CLL cell-derived exosomes, identified as CD63 +/CD41a - particles co-expressing CD19 +/CD5 +, whereas only a small proportion of CLL patients' plasma-derived exosomes expressed T-cell or endothelial cell surface markers. Furthermore, we found a positive correlation between levels of CLL cell-derived exosomes in the plasma of patients with CLL and the patients' peripheral white blood cell counts, indicating that CLL cells are the primary source of circulating exosomes. To investigate the effects of CLL cell-derived exosomes, we conducted an analysis of CLL cells and CLL cell-derived exosomes from a cohort of 78 previously untreated patients. To identify the molecular profiles of CLL cell-derived exosomes, we analyzed their RNA transcripts using RNA-seq and qRT-PCR. Our data revealed that CLL cell-derived exosomes expressed significant levels of oncogenic micro-RNAs. Additionally, we identified 1052 differentially expressed genes, and enrichment analysis further unveiled the significant upregulation of gene clusters associated with p53, NF-κB, MAPK, mTOR, and interleukin signal transduction pathways, consistent with their neoplastic cell origin. The distinctive transcriptional profile of CLL cell-derived exosomes led us to explore their effects on normal cell populations. We labeled ultra-centrifuged CLL cell-derived exosomes with fluorescent markers and incubated them with various cell types in culture conditions conducive to their physiological activity. Using confocal microscopy and flow cytometry, we observed significant uptake of CLL cell-derived exosomes by normal monocytes, fibrocytes, B cells, T cells, and CLL cells. Furthermore, incubation with CLL exosomes resulted in notable alterations in normal CD14 + monocyte polarity and subset-specific shifts in the expression of inflammatory cytokines. Remarkably, CLL cell-derived exosomes induced overexpression of the T-cell makers CD5 and CD8 in normal CD19 + cells, suggesting that CLL exosomes may promote immune-evasive reprogramming of normal B cells. Moreover, when normal T cells were incubated with CLL cell-derived exosomes, CyTOF analysis using 46 markers revealed elevated levels of immune checkpoints, including PD1, CTLA4, and LAG3, in both CD4 + and CD8 + subsets, indicating that exosomes derived from CLL cells induce T-cell dysfunction. Given their profound effect on immune cells, we wondered whether CLL exosomes affect hematopoietic cells. We previously found that normal monocyte derived fibrocytes stimulate hematopoietic colony proliferation. In sharp contrast to control fibrocytes, fibrocytes preincubated with CLL exosomes and CLL cell-derived exosomes inhibited normal BFU-E and CFU-GM colony formation, suggesting that CLL exosomes might contribute to CLL patients' anemia or neutropenia. Wondering whether CLL cell-derived exosomes affect the viability of other cell types, we found that they induced a dose-dependent apoptosis of CD19 + B cells, CLL cells but not normal T cells. In summary, CLL cell-derived exosomes exert a profound effect on gene and cell surface protein expression in immune and tissue-remodeling cells, leading to functional modifications, alteration of inflammatory cytokine levels, and suppression of normal hematopoiesis. The impact of CLL exosomes may vary across different cell types; however, as the leukemic cell mass increases, exosome levels escalate, invariably affecting CLL cell environment. The identification of exosomes with distinct molecular signatures holds promising potential for novel therapeutic interventions in future treatment of CLL.

Single Cell Multi-Omic Correlation of Single Nucleotide Variants, Copy Number Variation and Surface Epitopes for Clonal Profiling of Myeloma

Introduction Multiple myeloma (MM) is a cancer of plasma cells with approximately 200,000 new diagnoses each year and a 54% 5-year overall survival rate. As myeloma cells expand, clonal genetic differences lead to relapse due to acquired resistance in 100% of patients, mandating regular, long-term surveillance. Improvements in precision medicine for MM have come from monoclonal antibodies and antibody-drug conjugates that specifically target the offending cells. However, while existing diagnostic modalities offer exquisite sensitivity, they are unable to correlate subclonal genetic changes and putative antibody targets on refractory myeloma cells, leaving the clinician to use their best judgment on salvage therapy which can lead to unnecessary expense and toxicity to patients due to ineffective treatment. Specifically knowing actual targets on refractory MM cells would better facilitate precision medicine and potentially improve outcomes. Methods Cryopreserved, human MM patient samples were processed on the Mission Bio Tapestri platform, enabling simultaneous single cell quantification of subclones by single nucleotide variants (SNV), copy number variants (CNV), and surface protein analysis. These single time point samples were thawed from frozen bone marrow mononuclear cells previously enriched for CD38, stained with a 45-plex antibody-oligo cocktail to label common heme-specific surface markers for sequencing analysis, and processed with a 733-plex DNA primer panel that combined whole-genome CNV coverage with MM gene hotspots. Sequencing was performed on an Illumina system with the raw data being analyzed using Mission Bio proprietary algorithms. Results We show complex clonal evolution of MM in individual samples as copy gains and losses that were sequentially acquired and correlated with expression changes of MM markers. Across the cases analyzed, complex branching phylogenetic trees were reconstructed with as many as five subclones and clear associated shifts in MM-marker expression. When averaged across subclones, each sample had CNV profiles that matched patient bulk genome-wide array records, ranging from a single gene-level copy gain to arm-level gains and losses across the majority. The progression of protein expression within different samples could be mapped together on a single plot and correlated with generally higher MM-markers as subclonal genetic variants were acquired, though occasional branches saw reversal. The fraction of other cell phenotypes, such as T-cells and those with low viability, decreased within subclones as mutational burden grew. Conclusion This high-resolution, single cell assay offers a potential new modality for the diagnosis and surveillance of patients with suspected MGUS, SGUS or high-risk MM. We have demonstrated: 1) exceptional results from cryopreserved human specimens, 2) the ability to use genetic lesion profiling to positively identify subclonal MM and, most importantly, 3) correlate cell surface protein expression of potential therapeutic targets with each clonal and subclonal population

Multifactorial Basis of T Cell Dysfunction in CLL: Disrupted Mitochondrial Metabolism Induces T Cell Senescence

Despite being a B-cell malignancy, dysfunction of the T-cell compartment is a key feature of chronic lymphocytic leukemia (CLL). This leads to complications in acquired immunity and low success rates in autologous T cell-based therapies in these patients, particularly chimeric antigen receptor T cells (CAR-T). Despite exhibiting features of exhaustion, T cells from CLL patients are not reinvigorated by immune checkpoint inhibitors. Although T-cell dysfunction in CLL is heterogeneous, a consistent feature among all CLL patients is T-cell subset skewing towards effector T cells at the expenses of the central memory compartment (Martens et al., Leukemia 2023) and intact ability to produce inflammatory cytokines. A common feature of senescent T cells includes this maintained ability to produce pro-inflammatory cytokines alongside dysfunctions such as cell-cycle arrest, a decline in proliferation, and reduced activation. Notably, senescence often coincides with impaired mitochondrial function which is known to play a crucial role in the development of memory phenotypes and the efficacy of CAR-T cells (Van der Windt et al., Immunity 2012; Van Bruggen et al., Blood 2019). Given these associations, our aim is to comprehensively investigate the interplay between T-cell dysfunction, senescence, and mitochondrial impairment in CLL patients through a multifaceted approach encompassing analysis of T-cell metabolism, (epi)genetics, and functional phenotyping. Metabolic profiling of T cells from CLL patients and age-matched healthy donors (HD) was performed by extracellular flux analysis, flow cytometry, metabolomics and 13C tracing. A decreased mitochondrial respiration upon T-cell receptor stimulation was observed in all CLL patients. On the other hand, glycolytic activity correlated with T-cell activation status, which varied among patients. We identified alterations in the utilization of the three main mitochondrial fuels in T cells from CLL patients: i) induction of pseudohypoxia by CLL cells, leading to a re-routing of glucose-derived pyruvate into lactate rather than into the mitochondria; ii) a reduced rate of conversion of glutamine into glutamate resulting in decreased utilization of this fuel within the mitochondria; and iii) impaired lipid uptake, along with diminished fatty acid oxidation. Accordingly, mitochondria from CLL T cells showed intrinsic features of dysfunction including depolarization and increased production of reactive oxygen species (ROS). So far, these findings strongly demonstrate a misbalance in metabolic fueling resulting in mitochondrial dysfunction, aligning more closely with senescence rather than classical T-cell exhaustion. To further reinforce this observation, we assessed the expression of cell-surface proteins that characterize senescent T cells (e.g., absence of CD27/CD28 and presence of PD1/CD57/KLRG1). Indeed, we found an increased proportion of senescent cells in the T-cell compartment of CLL patients, as compared to HD. In response to stimulation, CLL T cells showed a strikingly distinct cell-cycle pattern whereby a larger proportion of cells resided in S and G2/M phases, indicating cell-cycle arrest. Cell cycle arrest is often induced by DNA damage. Transcriptomic and epigenomic profiling of T cells of CLL patients indeed revealed a molecular profile consistent with inflammation and an increased p53/DNA damage response upon stimulation. These features are known to be induced by dysfunctional mitochondria and ROS in senescent cells, underscoring a direct link between mitochondrial metabolism and senescence. Altogether, our results strongly point towards senescence as the dominant dysfunctional T-cell state in CLL patients, with impaired mitochondrial metabolism at its basis. The distinctive mechanisms leading to T-cell senescence, as opposed to classical exhaustion, underscore the necessity for different strategies to reinvigorate T cells in the context of immunotherapy. Therefore, interventions aimed at ameliorating T-cell dysfunction in CLL should prioritize enhancement of mitochondrial metabolism to attenuate DNA damage and the induction of senescence. This would be especially beneficial to improve the fitness of autologous CAR-T cells for these patients.

Single-Cell Proteogenomics Analysis of AML Samples Treated with Decitabine and Venetoclax Reveals Divergent Treatment Escape Mechanisms

Background: The combination therapy of hypomethylating agents and venetoclax (HMA+VEN) has emerged as a standard of care for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. Nonetheless, significant clinical challenges persist, including relapse and treatment resistance. Previous studies have revealed genetic ( RAS/RTK pathway mutations and TP53 mutations) and phenotypic (monocytic differentiation) resistance mechanisms in the context of HMA+VEN therapy. Despite these advancements, our comprehensive understanding of the HMA+VEN resistance mechanisms remains incomplete. To gain further insights into the resistance mechanism in HMA+VEN therapy, we implemented single-cell proteogenomics analysis on longitudinally collected samples from AML patients treated with decitabine+venetoclax (DAC+VEN) trial. Methods: We performed a high-throughput, single-cell targeted DNA-antibody sequencing (DAb-seq) using Tapestri platform from Mission Bio Inc. Sixty-three AML samples sequentially collected from 33 unique patients who participated in DAC+VEN clinical trial (NCT03404193) were analyzed. Our custom DNA panel covered 37 recurrently mutated genes in AML, whereas the custom antibody panel included either 15 cell surface proteins (N = 20 patients) or 48 proteins (N = 13 patients). Cellular phenotypes were inferred based on the patterns of cell surface protein expression. Median 2,767 cells were sequenced per sample, with total 196,719 cells sequenced for this study. Results: The median age of the cohort was 70 years (range 21-84). Twenty-four (73%) patients had primary AML and 9 (27%) had secondary or therapy-related AML. Based on the FAB classification, 13 patients had M0/M1/M2, 6 had M4/M5, one had M7, and the rest had unknown subtypes. The most frequent gene mutation at baseline was DNMT3A (45%), followed by NPM1 (39%), TET2 (30%), and TP53 (24%). Among the 33 patients, 32 had evaluable treatment response; 27 (82%) were defined as responders by achieving CR or CRi, whereas 5 (15%) were non-responders. Six patients experienced relapse after responding to the therapy, and their baseline-relapse pairs were analyzed by the single-cell platform. Using the composition of cellular phenotype data, AML samples were classified into either stem-like (N = 5), progenitor-like (N = 1), predominantly monocytic (N = 4), predominantly erythroid (N = 2) and mixed population AML (mixed population of stem, progenitor, monocyte or erythroid-like population, N = 21). Genotype-phenotype analysis showed a significant correlation between the KRAS mutation and the mixed phenotype (p= 0.0403), as well as between the U2AF1 mutation and the mixed phenotype (p= 0.0471). There was no significant association between treatment response and AML phenotype. Analysis of six baseline-relapse pairs showed that relapse was associated with phenotypic shift of AML cells. Stem-like cells were largely eradicated at relapse and replaced by more differentiated cells. Among the six baseline-relapse pairs, three showed monocytic shift, one showed erythroid shift and two showed mixed monocytic/erythroid shift. Phenotypic shifts at relapse were not always associated with expansion of new genetic mutations, except for in two cases: one had monocytic shift accompanied with expansion of KRAS mutation, and another had erythroid shift accompanied with expansion of NRAS mutation. Conclusions: Phenotypic transition from stem-like cells to more differentiated cells (either monocytic or erythroid) was frequently observed in AML relapse following DAC+VEN therapy. These findings are consistent with the known dependency of these cells on non-BCL-2 anti-apoptotic proteins such as MCL-1 and BCL-xL. Close monitoring of these phenotypic alterations throughout the course of HMA+VEN treatment may potentially facilitate the precise identification of cellular populations predisposed to relapse.

Abstract 13203: CD36 Regulates Factor VIII Secretion From Liver Endothelial Cells

Introduction: Abnormal levels of the coagulation factor VIII (FVIII) are a risk factor for venous thromboembolism (VTE). Genome-wide association studies have identified novel candidate gene associations that may regulate FVIII levels in humans, including CD36. Hypothesis: We hypothesized that CD36 regulates FVIII release from endothelial cells. Methods and Results: We identified a subset of human liver endothelial cells (HLEC) expressing FVIII and purified them using a differentially expressed cell surface protein CD32. CD32+ HLEC expressed FVIII, transported FVIII in intracellular vesicles, and secreted FVIII. We stimulated CD36 signaling or silenced CD36 to test the effect of CD36 on FVIII release. Oxidized LDL (oxLDL), a CD36 agonist, increased endothelial release of FVIII. Conversely, silencing CD36 decreased FVIII release. We searched for an intracellular signaling pathway that mediates CD36 stimulation of FVIII secretion and found that oxLDL increased p38 activity and a p38 inhibitor decreased FVIII release. Conclusions: Taken together, our data show that CD36 stimulates FVIII release through a p38 dependent pathway in specialized liver endothelial cells. These results validate genetic epidemiology data linking CD36 to FVIII levels in humans and provide new insights into the regulation of FVIII levels.

Application of bioluminescence resonance energy transfer to quantitate cell-surface expression of membrane proteins

We report a bioluminescence resonance energy transfer (BRET) assay to quantitate the fraction of an engineered membrane protein at the cell surface versus inside the cell. As test cases, we engineered two different G protein-coupled receptors (GPCRs) in which a NanoLuc luciferase (NLuc) and a HaloTag are fused to the extracellular amino-terminal tail of the receptors. We then employed a pulse-chase labeling approach relying on two different fluorescent dyes with distinctive cell permeability properties. The dyes are efficiently excited by luminescence from NLuc, but are spectrally distinct. Measuring BRET from the chemiluminescence of the NLuc to the fluorophores bound to the HaloTag minimizes the limitations of in-cell fluorescence resonance energy transfer (FRET)-based approaches such as photobleaching and autofluorescence. The BRET surface expression assay can quantitatively differentiate between the labeling of receptors at the cell surface and receptors inside of the cell. The assay is shown to be quantitative and robust compared with other approaches to measure cell surface expression of membrane proteins such as enzyme-linked immunosorbent assay or immunoblotting, and significantly increases the throughput because the assay is designed to be carried out in microtiter plate format.

Using combined single-cell gene expression, TCR sequencing and cell surface protein barcoding to characterize and track CD4+ T cell clones from murine tissues.

Single-cell gene expression analysis using sequencing (scRNA-seq) has gained increased attention in the past decades for studying cellular transcriptional programs and their heterogeneity in an unbiased manner, and novel protocols allow the simultaneous measurement of gene expression, T-cell receptor clonality and cell surface protein expression. In this article, we describe the methods to isolate scRNA/TCR-seq-compatible CD4+ T cells from murine tissues, such as skin, spleen, and lymph nodes. We describe the processing of cells and quality control parameters during library preparation, protocols for multiplexing of samples, and strategies for sequencing. Moreover, we describe a step-by-step bioinformatic analysis pipeline from sequencing data generated using these protocols. This includes quality control, preprocessing of sequencing data and demultiplexing of individual samples. We perform quantification of gene expression and extraction of T-cell receptor alpha and beta chain sequences, followed by quality control and doublet detection, and methods for harmonization and integration of datasets. Next, we describe the identification of highly variable genes and dimensionality reduction, clustering and pseudotemporal ordering of data, and we demonstrate how to visualize the results with interactive and reproducible dashboards. We will combine different analytic R-based frameworks such as Bioconductor and Seurat, illustrating how these can be interoperable to optimally analyze scRNA/TCR-seq data of CD4+ T cells from murine tissues.

ADP-Ribosylation Factor 6 Pathway Acts as a Key Executor of Mesenchymal Tumor Plasticity.

Despite the "big data" on cancer from recent breakthroughs in high-throughput technology and the development of new therapeutic modalities, it remains unclear as to how intra-tumor heterogeneity and phenotypic plasticity created by various somatic abnormalities and epigenetic and metabolic adaptations orchestrate therapy resistance, immune evasiveness, and metastatic ability. Tumors are formed by various cells, including immune cells, cancer-associated fibroblasts, and endothelial cells, and their tumor microenvironment (TME) plays a crucial role in malignant tumor progression and responses to therapy. ADP-ribosylation factor 6 (ARF6) and AMAP1 are often overexpressed in cancers, which statistically correlates with poor outcomes. The ARF6-AMAP1 pathway promotes the intracellular dynamics and cell-surface expression of various proteins. This pathway is also a major target for KRAS/TP53 mutations to cooperatively promote malignancy in pancreatic ductal adenocarcinoma (PDAC), and is closely associated with immune evasion. Additionally, this pathway is important in angiogenesis, acidosis, and fibrosis associated with tumor malignancy in the TME, and its inhibition in PDAC cells results in therapeutic synergy with an anti-PD-1 antibody in vivo. Thus, the ARF6-based pathway affects the TME and the intrinsic function of tumors, leading to malignancy. Here, we discuss the potential mechanisms of this ARF6-based pathway in tumorigenesis, and novel therapeutic strategies.

Flow cytometry analysis of protein expression using antibody-derived tags followed by CITE-Seq.

Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) is a single-cell phenotyping method that uses antibody-derived tags (ADTs) to quantitatively detect cell surface protein expression and generate transcriptomic data at the single-cell level. Despite the increased popularity of this technique to study cellular heterogeneity and dynamics, detailed methods on how to choose ADT markers and ensuring reagent performance in biological relevant systems prior to sequencing is not available. Here we describe a novel and easy-to-use multiplex flow proxy assay in which multiple protein markers can be measured simultaneously using a combination of ADT reagents and dye-oligo conjugates by flow cytometry. Using dye-oligo conjugates with sequences complementary to the ADT reagents, we can achieve specific binding and evaluate protein marker expression in a multiplex way. This quality control assay is useful for guiding ADT marker choice and confirming protein expression prior to sequencing. Importantly, the labeled cells can be directly isolated based on the specific fluorescence from dye-oligo conjugates using a flow cytometry cell sorter and processed for downstream single-cell multiomics. Using this streamlined workflow, we sorted natural killer cells and T cells efficiently using only ADT and dye-oligo reagents, avoiding the possibility of decreased marker resolution from co-staining cells with ADT and fluorescent antibodies. This novel workflow provides a viable option for improving ADT marker choice and cell sorting efficiency, allowing subsequent CITE-Seq.