Dissecting Heterogeneity of Tumor Cells and Their Microenvironment in Refractory Multiple Myeloma
Authors: Tirier Stephan M , Mallm Jan-Philipp , Casiraghi Nicola , Susak Hana , Giesen Nicola , Poos Alexandra M , Bauer Katharina , Baumann Anja , John Lukas , Xu Jing , Klett Lara , Müller-Tidow Carsten , Brors Benedikt , Goldschmidt Hartmut , Weinhold Niels , Stegle Oliver , Rippe Karsten , Raab Marc S. 
Published: February 2020

Introduction: Despite significant improvements in therapy during the last decade, most multiple myeloma (MM) patients develop refractory disease over time. Treatment of refractory MM is a major challenge, likely due to the still poorly characterized inter- and intratumor heterogeneity at this stage of the disease, and the complex interplay of MM cells with the microenvironment (ME). In particular, there is an urgent need to unravel how these features of MM are linked to molecular mechanisms of drug resistance.
Methods: We resolved the cellular composition, underlying transcriptional inter- and intra-patient heterogeneity and molecular treatment response of relapsed/refractory MM by single cell RNA sequencing (scRNA-seq). Using droplet-based microfluidics, ~230,000 single cell gene expression profiles from bone marrow (BM) aspirates of 21 patients sorted into CD138+and CD138- fractions were acquired, allowing for a comprehensive analysis of both MM cells as well as their ME. Patients had a median of 4 prior lines of therapy including both a proteasome inhibitor and an immunomodulator and were refractory to their immediate prior line of therapy at time of sampling. In addition, paired samples before either pomalidomide- or carfilzomib-based therapies were analyzed for 16/21 patients. Genomic aberrations in individual patients were mapped by interphase fluorescence in situ hybridization. Cells were clustered and CD138+ MM subtypes as well as immune cell-types of the ME were identified from their single cell transcriptomes and a copy number variation (CNV) analysis. As a reference for non-malignant cells and to construct a developmental B-cell trajectory the Human Cell Atlas BM scRNA-seq reference dataset was used. To characterize interactions of MM cells with their ME, the correlated expression of ligand-receptor pairs was exploited.
Results: The analysis of inter- and intra-tumor heterogeneity of molecular MM subgroups revealed distinct transcriptome signatures with contributions that could be assigned to differences in heavy and light chain immunoglobulin expression as well as known genomic alterations, including t(11;14), t(4;14) and hyperdiploidy. MM cells from individual patients largely maintained a plasma cell specific gene expression profile but a partial loss of plasma cell identity was detected based on mapping to a developmental B-cell trajectory. It was characterized by the upregulation of subgroup transcriptome signatures associated with earlier stages of B-cell development in almost 50% of patients, such as a pre-B or mature B cell-like phenotype.
Within individual samples, subclonal MM cell populations with specific gene expression programs were resolved based on the CNV analysis and included those characterized by expression of the immune-activator CD27 and the modulator of WNT signaling FRZB. The analysis of longitudinally collected samples revealed both changes in the cell subtype cluster structure as well as drug-specific adaptation of gene expression programs in distinct subpopulations persisting or emerging at relapse. These profile changes were characterized by e.g. downregulation of Myc target genes upon pomalidomide treatment or induction of heat shock proteins under carfilzomib.
Within the ME of refractory MM patients, we observed that the fraction of B cells and CD4+ T cells was strongly reduced while CD14+ and CD16+ monocytes as well as dendritic cells expanded. Notably, the immune checkpoint protein PD-1H (aka VISTA) that inhibits T cell activation was highly expressed in cell types from the myeloid compartment in contrast to healthy donors. Further, a ligand-receptor analysis revealed that MM cells displayed the strongest interactions with monocytes, which were mediated by MIF, BAFF and other cytokines.
Conclusions: Our study demonstrates the value of scRNA-seq analysis for identifying crucial transcriptome features that classify refractory MM subtypes and their evolution in response to treatment including regulation of drug resistance associated signaling pathways. Our data suggest that refractory MM cells shape the myeloid compartment in the BM to generate an immune suppressive ME. Understanding the evolution of MM cell heterogeneity and the bone marrow milieu in refractory disease will lead to novel treatment approaches and eventually improve patient outcome.

Disclosures
Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Goldschmidt:John-Hopkins University: Research Funding; Molecular Partners: Research Funding; Amgen: Consultancy, Research Funding; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MSD: Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Dietmar-Hopp-Stiftung: Research Funding; Janssen: Consultancy, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; John-Hopkins University: Research Funding; Chugai: Honoraria, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnology: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Mundipharma: Research Funding.