SINGLE-CELL TRANSCRIPTOMICS IDENTIFIES INTRA-TUMOR HETEROGENEITY IN HUMAN MYELOMA CELL LINES Amit Kumar Mitra1, Holly Stessman2, Michael A. Linden3 and Brian G. Van Ness1,4 1. Department of Genetics, Cell Biology & Development, University of Minnesota, Minneapolis, MN; 2. Department of Genome Sciences, University of Washington, Seattle, WA; 3. Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN; 4. PUMA-Institute of Personalized Medicine, College of Pharmacy, University of Minnesota, Minneapolis, MN.
MMM1 Global Z Score • Stessman et al identified a 23-gene expression signature of baseline Heatmap of Expression (Global Z-Score) PCA Score Plot BRIEF OVERVIEW -2 -1 0 1 2 responsiveness to PI that was successful in stratifying good versus poor 6
outcomes in human MM subjects from MM TT3 trial and APEX MM 4 Multiple myeloma (MM) is a difficult disease to cure with median survival CCND1_5572...
clinical trials (both using PI therapy). RB1_55727_... rate of around 7 years. In a recently published study, we confirmed the 2 LGALS1_598...
• Shaughnessy et al identified a 17-gene model from newly diagnosed MM FOS_56350_... PC2
presence of residual proteasome inhibitor (PI)-resistant sub-population in 0 patients on TT3 trial that could discriminate between low and high-risk JUN_12227_...
drug-naïve MM tumors. We hypothesize that this pre-existing resistant CD53_25138... -2 subclone may give rise to emerging resistance in course of PI treatment. myeloma with accuracies of over 95%. KIF14_3018...
LTBP1_1369... -4 In the current study, we used single cell transcriptomics to identify tumor • We used a GEP signature-based model derived from combination of CCNE1_4353... SMOC1_2794... -5 0 5 10
subclones within human myeloma cell lines (HMCLs) based on a 48-gene these gene panels of baseline PI response (and a set of internal control PC1
cDNA_002_C43
cDNA_002_C41
cDNA_002_C29
cDNA_002_C32
cDNA_002_C37
cDNA_002_C25
cDNA_002_C70
cDNA_002_C45
cDNA_002_C63
cDNA_002_C08
cDNA_002_C94
cDNA_002_C96
cDNA_002_C27
cDNA_002_C65
cDNA_002_C85
cDNA_002_C71
cDNA_002_C62
cDNA_002_C75
cDNA_002_C69
cDNA_002_C76
cDNA_002_C52
cDNA_002_C87
cDNA_002_C79
cDNA_002_C26
cDNA_002_C16
cDNA_002_C81
cDNA_002_C11
cDNA_002_C36
cDNA_002_C30
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cDNA_002_C72
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cDNA_002_C49
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cDNA_002_C80 cDNA_002_C01 model of predictive genetic signature for baseline PI response. genes) to identify pre-existing PI-resistant subclones within untreated bulk Figure: Heat map generated using unsupervised hierarchical clustering (HC) analysis and Principal Component Our results demonstrated the presence of pre-existing subclones of cells myeloma tumor cell populations. Analysis (PCA) score plot for MMM1 cells based on top GEP signatures of PI resistance. within untreated myeloma cells with a characteristic genetic signature profile SSBP4 HIST1H1C CXCR4 KIF20B CTBS E2F1 CDK4 SNAI3 DDX3Y TSPAN32 TMPO CLCC1 TP53 CDKN1A Primary Myeloma cells (from Patient) distinct from the pre-treatment overall profile. SMOC1 PMPCB SCD5 NADK LTBP1 FOXO4 GAPDH CBS TAP2 SCD LARS2 JUN CCNA1 ACTB Global Z Score Heatmap of Expression (Global Z-Score) PCA Score Plot FZD6 CD53 KIF14 TBRG4 FOS CCNB1 -2 -1 0 1 2 This may help identify the presence and extent of intra-tumor heterogeneity IFITM1 SLC35B1 SLC19A1 AIM2 MYC CCND1 RGS16 LGALS1 CKS1B ASPM MYB CCNE1 6 in MM and may define residual pre-existing subclones resistant to PI ENO1 SRR YWHAZ AHCYL1 RB1 CDK2
4 therapies. • Statistical analysis was performed using a combination of Fluidigm’s LGALS1_598... CD53_25138...
2 Singular Analysis Toolset and the R Statistical analysis package. AIM2_21352...
RB1_55727_... 0
PC2
LTBP1_1369... OBJECTIVE RESULTS KIF14_3018... -2 JUN_12227_...
-4 FOS_56350_...
CCND1_5572... U266 Parental vs U266 PI-resistant cells -6 CCNE1_4353...
Violin Plot of Gene Expression By the Order of PCA Gene Scores SMOC1_2794... -10 -5 0 5
PC1
SMOC1_27944_i8 CD53_25138_i4 AIM2_21352_i2 FOS_56350_i0
cDNA_003_C67
cDNA_003_C57
cDNA_003_C63
cDNA_003_C05
cDNA_003_C65
cDNA_003_C70
cDNA_003_C75
cDNA_003_C11
cDNA_003_C85
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cDNA_003_C06
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cDNA_003_C09
cDNA_003_C31
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cDNA_003_C84
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cDNA_003_C33
cDNA_003_C74
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cDNA_003_C51
cDNA_003_C24
cDNA_003_C17
cDNA_003_C26
cDNA_003_C79
cDNA_003_C28
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cDNA_003_C53 cDNA_003_C08 Does GEP profile help in 10 10 Figure: Heat map generated using unsupervised hierarchical clustering (HC) analysis and Principal Component identifying pre-existing drug 5 5 Analysis (PCA) score plot for primary myeloma cells based on top GEP signatures of PI resistance. 0 0 CCND1_55725_i1 RB1_55727_i15 JUN_12227_e0 LTBP1_13699_i24
resistant clonal subpopulations survival % 10 10 CONCLUSIONS within sensitive population? 5 5
Expression(log2) 0 0 KIF14_30185_i5 LGALS1_59844_i1 CCNE1_4353_i3 • Multiple subclones of cells are present within untreated myeloma cells with a characteristic genetic 10 10 signature profile distinct from the pre-treatment overall profile. 5 5 • PI-resistant sub-populations represent a pre-existing subset of PI-sensitive cell lines based on GEP 0 0
To perform automated single-cell capture & cDNA synthesis from cellular profile that may give rise to emerging resistance in course of treatment with PIs.
U266.VR U266.VR
RNA in individual cells and single-Cell qRT-PCR-based gene expression U266.VR
U266.Parental U266.Parental U266.Parental • Similar findings have also been observed using multicolor flow cytometry. Immunophenotypic analysis of human myeloma tumor prior to exposure to PI treatment to profiling of human myeloma cell lines (HMCLs) demonstrated unique signatures representing identify resistant pre-existing sub-clones using a GEP-based model (48-gene Figure: Violin plot representing the top significant genes (p<0.05) that distinguish U266 parental cells from clonally-derived PI-resistant U266 cells. sub-clonal populations. panel of baseline responsiveness to PIs). Global Z Score Heatmap of Expression (Global Z-Score) PCA Score Plot -2 -1 0 1 2 FUTURE PERSPECTIVES Sample Group U266.Parental U266.Parental U266.VR
U266.VR 6
METHODS • Our work will help identify the presence and extent of intra-tumor heterogeneity in MM and may 4
LGALS1_598...
1:annotation_width define residual pre-existing subclones resistant to PI therapies. 1:nc
LTBP1_1369... 2 FOS_56350_... • This will establish the use of microfluidics technology-based single-cell approaches as novel
JUN_12227_... 0 PC2 strategy to detect therapy-resistant subclones within bulk tumor populations from patient samples. SMOC1_2794...
CCNE1_4353... -2
KIF14_3018...
-4 REFERENCES CCND1_5572...
CD53_25138... • Rajkumar SV. Multiple myeloma: 2013 update on diagnosis, risk-stratification, and management. Am J Hematol. 2013;88(3):226-235. -6 AIM2_21352... • Shaughnessy JD,Jr, Zhan F, Burington BE, et al. A validated gene expression model of high-risk multiple myeloma is defined by deregulated RB1_55727_... -5 0 5 10 15 expression of genes mapping to chromosome 1. Blood. 2007;109(6):2276-2284.
PC1 • Stessman HA, Baughn LB, Sarver A, et al. Profiling bortezomib resistance identifies secondary therapies in a mouse myeloma model. Mol Cancer
cDNA_002_C7 cDNA_001_C7 cDNA_001_C9 cDNA_001_C2 cDNA_001_C3 cDNA_002_C2 cDNA_002_C1 cDNA_002_C5
• Automated single-cell capture and cDNA synthesis from cellular RNA cDNA_001_C1
cDNA_001_C25 cDNA_002_C45 cDNA_001_C39 cDNA_002_C24 cDNA_002_C70 cDNA_002_C35 cDNA_002_C58 cDNA_001_C12 cDNA_002_C95 cDNA_001_C43 cDNA_002_C82 cDNA_001_C53 cDNA_001_C56 cDNA_001_C81 cDNA_002_C19 cDNA_001_C17 cDNA_001_C10 cDNA_001_C15 cDNA_001_C73 cDNA_001_C23 cDNA_001_C35 cDNA_001_C48 cDNA_001_C64 cDNA_001_C44 cDNA_001_C69 cDNA_001_C80 cDNA_001_C91 cDNA_001_C31 cDNA_001_C94 cDNA_001_C87 cDNA_001_C18 cDNA_001_C86 cDNA_001_C72 cDNA_001_C20 cDNA_001_C66 cDNA_001_C36 cDNA_001_C82 cDNA_001_C47 cDNA_001_C89 cDNA_001_C75 cDNA_001_C51 cDNA_001_C40 cDNA_001_C37 cDNA_001_C95 cDNA_001_C93 cDNA_001_C45 cDNA_001_C38 cDNA_002_C13 cDNA_001_C71 cDNA_001_C74 cDNA_002_C52 cDNA_001_C92 cDNA_002_C71 cDNA_002_C59 cDNA_002_C92 cDNA_002_C91 cDNA_002_C87 cDNA_002_C34 cDNA_002_C29 cDNA_002_C88 cDNA_002_C25 cDNA_002_C79 cDNA_002_C56 cDNA_002_C74 cDNA_002_C78 cDNA_002_C94 cDNA_002_C21 cDNA_002_C43 cDNA_002_C15 cDNA_002_C84 cDNA_002_C93 cDNA_002_C20 cDNA_002_C28 cDNA_002_C75 cDNA_002_C47 cDNA_002_C30 cDNA_002_C48 cDNA_002_C63 cDNA_002_C81 cDNA_002_C89 cDNA_002_C80 cDNA_002_C85 cDNA_002_C44 cDNA_002_C64 cDNA_002_C11 Ther. 2013;12(6):1140-1150. TM were performed using Fluidigm’s C1 Single-Cell Auto Prep System. Figure: Heat map generated using unsupervised hierarchical clustering (HC) analysis • The cDNA was harvested and transferred to BioMark HD System for and Principal Component Analysis (PCA) score plot based on significantly associated ACKNOWLEDGEMENTS genes (p<0.05) displaying pre-existing subclones of parental U266 cells single-cell targeted high-throughput qPCR-based gene expression analysis corresponding to the expression patterns of PI-resistant U266 cells, distinct from the This research was supported in part by University of Minnesota Genomics Center (UMGC) Fluidigm C1 Single-cell Genomics Grant. of a gene-panel using Fluidigm DELTAgene assays. pre-treatment overall profile of U266. There are no relevant conflicts of interest to disclose.