Transcriptomic Heterogeneity and Clonal Evolution Associated with Therapeutic Resistance in Mantle Cell Lymphoma Revealed By Single Cell RNA-Seq
Authors: Jiang Changying , Zhang Shaojun , Wang Yuanxin , Zhang Rongjia , Liu Yang , McIntosh Joseph Mitchell , Han Guangchun , Wang Ruiping , Santos David , Badillo Maria , Leeming Angela , Chen Zhihong , Hartig Kimberly , Bigcal John , Lee Hun Ju , Steiner Raphael E , Romaguera Jorge Enrique , Jain Preetesh , Nomie Krystle , Futreal Andy , Wang Linghua , Wang Michael L. 
Published: November 2019

Introduction:
Both inter- and intra-tumoral heterogeneity are obstacles to improving oncology clinical outcomes. Mantle cell lymphoma (MCL) is an extremely heterogeneous disease in clinical, pathological, genetic, and transcriptomic profiling. Furthermore, MCL patients frequently develop therapeutic resistance after frontline therapies. In this study, we performed longitudinal transcriptomic analysis on primary patient MCL specimens at single-cell resolution, aiming to understand the dynamic and complex cellular and molecular changes underlying therapeutic resistance and identify potential targets to overcome dual resistance to ibrutinib and venetoclax.
Methods:
Sequential single-cell transcriptome sequencing (scRNA-seq) was performed on patient specimens collected during the course of treatment(s) from 5 MCL patients (3 ibrutinib responders and 2 ibrutinib-venetoclax non-responders). Integrative computational approaches were employed to characterize the cellular and molecular basis of therapeutic resistance and clonal evolution. An orthotopic PDX model derived from one of the non-responders was established and used to validate the novel findings and to investigate the in vivo efficacies of multiple novel potential targets.
Results:
The 3 ibrutinib responders and 2 ibrutinib-venetoclax non-responders were highly heterogeneous in clinical and pathological profiling. To dissect the inter- and intra-tumor heterogeneity underlying the therapeutic resistance, we performed sequential scRNA-seq analysis of 21 specimens collected at baseline, during treatment, and/or at disease remission/progression. The scRNA-seq analysis revealed a high degree of inter- and intra-tumor heterogeneity with distinct cellular and transcriptomic profiling within and across ibrutinib-responders and ibrutinib-venetoclax non-responders. Unsupervised pathway enrichment analysis identified more than 15 cancer hallmarks significantly upregulated in ibrutinib-venetoclax non-responders. We tracked the clinical ibrutinib-induced lymphocytosis at a single-cell transcriptomic level in ibrutinib responders and disease-progression-associated clonal evolution in non-responders. Multiple actionable targets were identified, and targeting these showed effective anti-MCL activity in the orthotopic PDX model derived from one of the ibrutinib-venetoclax non-responders.
Conclusions: This study demonstrates the potential of longitudinal single-cell transcriptomic analysis to reveal the molecular mechanisms underlying tumor heterogeneity, clonal evolution, disease progress, and therapeutic resistance, and to identify potential novel targets to circumvent therapeutic resistance in mantle cell lymphoma and other diseases.

Disclosures
Wang: Pharmacyclics: Honoraria, Research Funding; Juno Therapeutics: Research Funding; Celgene: Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Guidepoint Global: Consultancy; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; Loxo Oncology: Research Funding; VelosBio: Research Funding; BioInvent: Consultancy, Research Funding; Dava Oncology: Honoraria; Aviara: Research Funding.