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Multiple myeloma (MM) is a highly refractory hematologic cancer. Targeted immunotherapy has shown promise in MM but remains hindered by the challenge of identifying specific yet broadly representative tumor markers. We analyzed 53 bone marrow (BM) aspirates from 41 MM patients using an unbiased, high-throughput pipeline for therapeutic target discovery via single-cell transcriptomic profiling, yielding 38 MM marker genes encoding cell-surface proteins and 15 encoding intracellular proteins. Of these, 20 candidate genes were highlighted that are not yet under clinical study, 11 of which were previously uncharacterized as therapeutic targets. The findings were cross-validated using bulk RNA sequencing, flow cytometry, and proteomic mass spectrometry of MM cell lines and patient BM, demonstrating high overall concordance across data types. Independent discovery using bulk RNA sequencing reiterated top candidates, further affirming the ability of single-cell transcriptomics to accurately capture marker expression despite limitations in sample size or sequencing depth. Target dynamics and heterogeneity were further examined using both transcriptomic and immuno-imaging methods. In summary, this study presents a robust and broadly applicable strategy for identifying tumor markers to better inform the development of targeted cancer therapy. Significance: Single-cell transcriptomic profiling and multiomic cross-validation to uncover therapeutic targets identifies 38 myeloma marker genes, including 11 transcribing surface proteins with previously uncharacterized potential for targeted antitumor therapy.

Multiple myeloma (MM) is a highly refractory hematologic cancer. Targeted immunotherapy has shown promise in MM but remains hindered by the challenge of identifying specific yet broadly representative tumor markers. We analyzed 53 bone marrow (BM) aspirates from 41 MM patients using an unbiased, high-throughput pipeline for therapeutic target discovery via single-cell transcriptomic profiling, yielding 38 MM marker genes encoding cell-surface proteins and 15 encoding intracellular proteins. Of these, 20 candidate genes were highlighted that are not yet under clinical study, 11 of which were previously uncharacterized as therapeutic targets. The findings were cross-validated using bulk RNA sequencing, flow cytometry, and proteomic mass spectrometry of MM cell lines and patient BM, demonstrating high overall concordance across data types. Independent discovery using bulk RNA sequencing reiterated top candidates, further affirming the ability of single-cell transcriptomics to accurately capture marker expression despite limitations in sample size or sequencing depth. Target dynamics and heterogeneity were further examined using both transcriptomic and immuno-imaging methods. In summary, this study presents a robust and broadly applicable strategy for identifying tumor markers to better inform the development of targeted cancer therapy.Significance:Single-cell transcriptomic profiling and multiomic cross-validation to uncover therapeutic targets identifies 38 myeloma marker genes, including 11 transcribing surface proteins with previously uncharacterized potential for targeted antitumor therapy.

Pearson R correlation values between bulk RNA expression and scRNA expression in PCs for target genes.

Multiple myeloma (MM) is a cancer characterized by the unchecked proliferation of antibody-secreting plasma cells (PCs), which to date remains incurable despite therapeutic advancements. The idea that deeper tumor clearance leads to better outcomes is widely accepted in MM treatment: minimal residual disease (MRD) testing, whereby ultra-sensitive sequencing or fluorescence-based cytometric detection of the clonal immunoglobulin secreted by tumor PCs, has been found to be the strongest independent prognostic factor when predicting survival. However, MRD status remains an imperfect predictor. As PCs have a well-defined physiological niche within the bone marrow (BM), and patients often exhibit immunosuppressive BM dysregulation, we investigate the presence of BM microenvironment signatures present at remission that may influence clinical outcomes insufficiently explained by tumor risk strata or MRD status. We conducted 3’ single-cell RNA sequencing of 36 remission, 8 primary diagnosis, and 3 relapse whole BM aspirates taken from 35 patients enrolled in an ongoing clinical trial evaluating a treatment regimen consisting of ixazomib, lenalidomide, and dexamethasone (IRD). 19 of 35 patients were MRD+ at remission; among these, 6 have been reported as having poor outcome (PO; progression-free survival 2.5yrs), and 1 is awaiting assessment. The remaining 16 patients were MRD- at remission, 6 of whom had poor outcome, 7 average, and 3 awaiting assessment. Preliminary analysis of 43 samples have yielded an average capture of 8,620 cells per sample with 1,247 median genes per cell. In comparing 3 PO to 8 AO MRD- remission samples, we see clear polarity between PO and AO cells in every major immune lineage, with a majority of clusters being predominantly (>80%) made of either PO or AO cells. Surprisingly, T, NK, and B cells from AO samples exhibit FOS/JUN upregulation relative to those from PO samples, indicative of AP-1 transcription factor activity that is typically associated with cell proliferation. PO samples also exhibited signs of active cytotoxicity, including upregulated granzymes H and B and IL7R in T and NK cells. Sizeable PC populations were detected in several MRD- remission samples with subpopulation heterogeneity in the expression of MM marker genes; comparison against PCs isolated from healthy donor BM suggests possible persistence of disease phenotypes not captured by MRD testing. We further investigate BM microenvironment evolution between primary diagnosis, remission, and relapse timepoints to elucidate changes in response to tumor clearance. Overall, this study enhances our understanding of how immune surveillance and interaction may influence treatment response in MM. Citation Format: Julia T. Wang, Mark Fiala, Julie Fortier, Ruiyang Liu, Reyka Jayasinghe, Ravi Vij, Li Ding. Investigating the role of bone marrow microenvironment dysregulation at remission in disease outlook of multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3815.

Transposable elements (TEs) make up half of mammalian genomes and shape genome regulation by harboring binding sites for regulatory factors. These include binding sites for architectural proteins, such as CTCF, RAD21, and SMC3, that are involved in tethering chromatin loops and marking domain boundaries. The 3D organization of the mammalian genome is intimately linked to its function and is remarkably conserved. However, the mechanisms by which these structural intricacies emerge and evolve have not been thoroughly probed.Here, we show that TEs contribute extensively to both the formation of species-specific loops in humans and mice through deposition of novel anchoring motifs, as well as to the maintenance of conserved loops across both species through CTCF binding site turnover. The latter function demonstrates the ability of TEs to contribute to genome plasticity and reinforce conserved genome architecture as redundant loop anchors. Deleting such candidate TEs in human cells leads to the collapse of conserved loop and domain structures. These TEs are also marked by reduced DNA methylation and bear mutational signatures of hypomethylation through evolutionary time.TEs have long been considered a source of genetic innovation. By examining their contribution to genome topology, we show that TEs can contribute to regulatory plasticity by inducing redundancy and potentiating genetic drift locally while conserving genome architecture globally, revealing a paradigm for defining regulatory conservation in the noncoding genome beyond classic sequence-level conservation.

BackgroundTransposable elements (TEs) make up half of mammalian genomes and shape genome regulation by harboring binding sites for regulatory factors. These include binding sites for architectural proteins, such as CTCF, RAD21, and SMC3, that are involved in tethering chromatin loops and marking domain boundaries. The 3D organization of the mammalian genome is intimately linked to its function and is remarkably conserved. However, the mechanisms by which these structural intricacies emerge and evolve have not been thoroughly probed.ResultsHere, we show that TEs contribute extensively to both the formation of species-specific loops in humans and mice through deposition of novel anchoring motifs, as well as to the maintenance of conserved loops across both species through CTCF binding site turnover. The latter function demonstrates the ability of TEs to contribute to genome plasticity and reinforce conserved genome architecture as redundant loop anchors. Deleting such candidate TEs in human cells leads to the collapse of conserved loop and domain structures. These TEs are also marked by reduced DNA methylation and bear mutational signatures of hypomethylation through evolutionary time.ConclusionsTEs have long been considered a source of genetic innovation. By examining their contribution to genome topology, we show that TEs can contribute to regulatory plasticity by inducing redundancy and potentiating genetic drift locally while conserving genome architecture globally, revealing a paradigm for defining regulatory conservation in the noncoding genome beyond classic sequence-level conservation.