Single Cell Transcriptomic Evolution and Resistance Mechanisms of BTK and BCL-2 Inhibition in Mantle Cell Lymphoma

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.