Abstract 139: Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma

Introduction: Mantle cell lymphoma (MCL) is a rare and aggressive subtype of B-cell non-Hodgkin's lymphoma with high risk of relapse after frontline therapies. Ibrutinib and venetoclax are two efficacious therapies for refractory/relapsed MCL patients. However, resistance to these therapies occurs frequently and is an urgent unmet clinical need. To understand the underlying mechanism of how intra- and inter-tumor heterogeneity (ITH) and its immune microenvironment contributes to therapeutic resistance, we performed a state-of-art single cell RNA sequencing on longitudinal samples from ibrutinib and venetoclax dual-resistant MCL patients with side-by-side comparison to ibrutinib-sensitive patients in our discovery cohort. To support our novel findings, patient samples from multiple validation cohorts were collected and analyzed via various approaches. Methods: Patient specimens from our discovery cohort that included ibrutinib-sensitive and ibrutinib-venetoclax dual-resistant MCL patients were collected longitudinally and subject to single cell RNA sequencing using 10x genomics. Integrative computational analysis was conducted to uncover the ITH and tumor immune microenvironment at single cell resolution and the underlying mechanism of therapeutic resistance and clonal evolution. To validate the novel findings, additional cohorts of patient samples were collected and subject to bulk RNA sequencing, whole exome sequencing, and multi-color flow cytometry analysis. An orthotopic PDX model was established from one of the ibrutinib-venetoclax dual-resistant MCL patients and was used to validate the novel findings as well as to test the potential therapies in vivo to overcome resistance. Results: To understand the underlying mechanism of heterogeneity and therapeutic relapse, we carried out sequential single cell RNA sequencing on 21 specimens (18,794 cells in total) collected from ibrutinib-sensitive and ibrutinib-venetoclax dual-resistant MCL patients along the course of ibrutinib and/or venetoclax treatments. Integrative computational analysis revealed a high degree of ITH with distinct profiles of cellular and molecular transcriptome. We revealed 15 top cancer hallmarks associated with disease progression and therapeutic resistance, albeit with remarkable clinical, pathological, and genetic-based inter-patient heterogeneity. We observed appearance and clearance of multiple subpopulations in patient blood samples, which likely interprets the clinical ibrutinib-induced lymphocytosis phenomenon at single-cell resolution and disease-progression-associated clonal evolution, which were further validated. Our analysis revealed reprogramming of the tumor microenvironment and tumor immune evasion. Moreover, we revealed multiple actionable targets to help overcome therapeutic resistance as tailored anti-MCL strategies. We found that the 17q gain strongly correlated with this dual resistance and thus targeting survivin located at 17q by YM155 significantly inhibited tumor growth and prolonged mouse survival in the ibrutinib-venetoclax dual-resistant PDX model. Conclusions: This study is the first to describe the mechanisms underlying dual resistance to ibrutinib and venetoclax at the single cell level. We not only identified various pathways underlying this resistance, but also characterized the evolutionary dynamics by using a longitudinal sampling strategy to uncover the underlying mechanisms. We found that the 17q gain highly correlates with ibrutinib-venetoclax dual resistance and showed that inhibition of survivin, located at 17q, overcame this dual resistance. These data provide evidence that 17q gain may be the driving force of disease progression and therapeutic resistance. Moreover, for the first time in MCL, we characterized changes in tumor immune microenvironment and identified a T-cell exhaustion signature correlated with the dual resistance. These changes to the tumor microenvironment strongly suggest the role of immune resistance in mediating dual resistance to ibrutinib and venetoclax in MCL. Disclosures Wang: Lu Daopei Medical Group: Honoraria; Beijing Medical Award Foundation: Honoraria; OncLive: Honoraria; Molecular Templates: Research Funding; Verastem: Research Funding; Dava Oncology: Honoraria; Guidepoint Global: Consultancy; Nobel Insights: Consultancy; Oncternal: Consultancy, Research Funding; InnoCare: Consultancy; Acerta Pharma: Research Funding; VelosBio: Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

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Abstract 3135: Single-cell RNA sequencing identifies macrophage-specific expression signatures associated with phagocytosis of multiple myeloma after treatment with cIAP antagonist

10.1158/1538-7445.am2018-3135 ◽

2018 ◽

Cited By ~ 1

Author(s):

Nicholas Banovich ◽

Martin Boateng ◽

Marta Chesi ◽

Leif Bergsagel ◽

Jonathan Keats

Keyword(s):

Multiple Myeloma ◽

Single Cell ◽

Rna Sequencing ◽

Specific Expression ◽

Single Cell Rna Sequencing

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A Novel Prognostic Model Based on Nine-Targeted Therapeutic Response Relevant Secreted Protein Genes (Trspgs) from Single-Cell Rna Sequencing in Multiple Myeloma

SSRN Electronic Journal ◽

10.2139/ssrn.4001775 ◽

2022 ◽

Author(s):

Liping Zeng ◽

Ting Yin ◽

Linyi Zhang ◽

Sixuan Guo ◽

Chuangqin Pu ◽

...

Keyword(s):

Multiple Myeloma ◽

Single Cell ◽

Rna Sequencing ◽

Prognostic Model ◽

Therapeutic Response ◽

Secreted Protein ◽

Model Based ◽

Single Cell Rna Sequencing ◽

Targeted Therapeutic

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Abstract LB-012: Single cell RNA-sequencing of multiple myeloma clinical patients for identifying novel immuno-oncology drug targets

10.1158/1538-7445.am2018-lb-012 ◽

2018 ◽

Author(s):

Namit Kumar ◽

Vanessa M. Peterson ◽

Kelvin X. Zhang ◽

Lixia Li ◽

Philip W. Garrett-Engele ◽

...

Keyword(s):

Multiple Myeloma ◽

Single Cell ◽

Rna Sequencing ◽

Drug Targets ◽

Oncology Drug ◽

Single Cell Rna Sequencing

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Single-Cell RNA Sequencing Reveals Compromised Immune Microenvironment in Precursor Stages of Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-114145 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2603-2603 ◽

Cited By ~ 1

Author(s):

Oksana Zavidij ◽

Nicholas Haradhvala ◽

Tarek H Mouhieddine ◽

Jihye Park ◽

Romanos Pistofidis ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

T Cells ◽

Single Cell ◽

Rna Sequencing ◽

Plasma Cells ◽

Cellular Composition ◽

Mhc Ii ◽

Healthy Donors ◽

Single Cell Rna Sequencing

Abstract Introduction: In multiple myeloma (MM), despite well-characterized precursor states such as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM), there is a lack of sufficient biomarkers to predict mechanisms of disease progression. Most genomic analyses have sought biomarkers by study of the malignant plasma cells, however, cancers form a complex ecosystem with the immune and stromal microenvironment. Thus, to characterize the cellular composition and transcriptional programs of each component of the tumor and microenvironment at different stages of MM progression, we employed a single-cell RNA sequencing on a cohort of 22 patients and 9 healthy donors. Methods: We performed 10X droplet-based single-cell RNA sequencing using CD138-expressing plasma cells and microenvironmental populations isolated from bone marrow (BM) aspirates of patients with MGUS (n=6), low-risk SMM (n=3), high-risk SMM (n=13), newly diagnosed MM (n=8) and from 9 healthy donors (NBM). We collected a total of ~88.8K cells, comprising ~48K CD138+ cells (~36.4 from MM stages) and ~40.8K CD45+/CD138- cells (~30.8 from MM stages).Raw read data was processed using the Cell Ranger pipeline to obtain a gene-by-cell expression matrix, which was used to identify cell types and transcriptional programs by clustering and non-negative matrix factorization. Results: Expression profiles of plasma cells revealed clear tumor-specific differences including known oncogenic drivers in MM (MMSET/FGFR3, CCND1 and MAFB) as well as Lysosome-associated Membrane Protein 5 (LAMP5),Histone Cluster 1 H1 Family Member C (HIST1H1C) and Amphiregulin (AREG) distinguishing them from healthy plasma cells. We identified a subset of cycling plasma cells, observing a range of proliferative activity of the malignant fraction. Furthermore, our approach allowed a unique head-to-head comparison of gene expression changes in normal and malignant plasma cells in the MGUS and SMM patients within an individual, excluding inter-individual variation. We were able to discriminate malignant from non-malignant plasma cells and identify transcriptional alterations including known drivers, genes related to immune modulation (NKBIA) or controlling transcription and differentiation (EID1).Some alterations were patient-specific, while others, such as MHC I overexpression and CD27 loss, were recurrently observed across subsets of the cohort. Analysis of BM microenvironment in several stages of MM progression demonstrated a striking shift in the composition of immune cells with significant infiltration of natural killer cells, non-classical monocytes/macrophages, and T cells, enriched even in the earliest stages of the disease. Further investigation revealed significant upregulation of HLA expression at the mRNA level in CD14+ monocytes/macrophages. Intriguingly, comparison of healthy and patient samples by CyTOF showed downregulation of surface MHC II representation in the corresponding cell type, and moreover, co-culture with MM cell lines induced a sharp decrease of extracellular MHC II. This provided strong evidence for compromised antigen presentation by macrophages in the disease setting, hinting at a mechanism of immune evasion. Additionally, expression signatures in cytotoxic T-cells indicated a substantial skewing towards either granzyme B/H- or granzyme K-expressing memory cell-like transcriptional program. In a subgroup of patients, we found a strong simultaneous enrichment of the anti-viral/anti-bacterial gene expression signature for interferon type-1 activated genes in CD14+ monocytes/macrophages and T cells. Together, our results provide a comprehensive view at the complex interplay of the immune and malignant cells in different stages of the disease. We, for the first time, demonstrate the immune response beginning in premalignant conditions to be heterogeneous, including compromised antigen presentation as well as alterations in cellular composition and signaling. Consideration of the type of immunological response may prove valuable in determination of progression risk, as well as open up potential strategies for therapy. Disclosures Bustoros: Dava Oncology: Honoraria. Ghobrial:Celgene: Consultancy; Janssen: Consultancy; BMS: Consultancy; Takeda: Consultancy.

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