P1207: serial single-cell profiling of mantle cell lymphoma reveals functional and molecular correlates of resistance to btk inhibitor-inclusive triplet therapy

Topic: 20. Lymphoma Biology & Translational Research Background: Most patients with mantle cell lymphoma (MCL) achieve complete remission with front line therapy, but relapses invariably occur due to the persistence, evolution, and expansion of drug-tolerant lymphoma cell subpopulations. This biologic heterogeneity has hindered efforts to identify tolerable precision therapies capable of overcoming residual disease and curing patients with MCL. Aims: We aim to resolve biological heterogeneity in MCL with single-cell resolution by integrating functional biophysical properties (mass and stiffness) with transcriptomic and genetic features. Specifically, we seek to define ex vivo biomarkers of clinical response or resistance in primary biospecimens from patients with MCL treated on an ongoing phase I/II trial of acalabrutinib, venetoclax, and obinutuzumab (AVO). Methods: We acquired synchronous blood, bone marrow and lymph node biopsies from patients with MCL treated with AVO on clinical trial NCT04855695 prior to treatment, followed when available by blood and bone marrow at clinical remission and post-progression. Biophysical properties of individual cells were measured using suspended microchannel resonators (SMR) in serial with single-cell transcriptomics via Seq-Well. Somatic copy-number variants (SCNVs) were inferred from transcriptome read counts. Additionally, we linked biophysical measurements of individual MCL cells from three patient-derived xenograft (PDX) models of MCL to single-cell transcriptomes via Smart-Seq2 (scSMR/RNA-seq). Results: Within the first four enrolled patients, we observed inter- and intra-tumor heterogeneity in mass and stiffness. In parallel, 33,171 single cells were sequenced across 17 samples spanning the pre-treatment to progression continuum. Notably, one patient with blastoid/pleomorphic histology and a TP53 mutation progressed on AVO treatment. Serial analyses demonstrated biophysical correlates of histologic transformation, with a significant shift toward higher single-cell mass distributions in blastoid/pleomorphic cells. Linked single-cell transcriptomes suggested the acquisition of new SCNVs in the malignant cells at progression. These cells were also distinguished by increased mTORC1 and oxidative phosphorylation signaling, as previously described in BTK-inhibitor resistance, but independently of TLR/CD40-mediated activation, suggesting potential metabolic rewiring. Finally, tumor cells at progression were strongly associated with an active cell proliferation state and enriched in genes involved in ATM signaling, DNA damage response and B cell receptor (BCR) signaling pathway, suggesting aberrant activation of BTK-mediated proliferation while on acalabrutinib therapy. These results corroborate prior data from in vitro activation assays and preclinical models linking biophysical phenotypes with B cell transcriptional states, including developmental stage, histological subtype, cell cycle and BCR activity. Here, application of scSMR/RNA-seq to three PDXs models of MCL revealed that mass and stiffness correlate with genes annotated for cell division, cell cycle, and BCR signaling pathway. Summary/Conclusion: The combination of serial orthogonal biophysical and multi-omic molecular profiling at single-cell resolution can nominate potential mechanisms of drug resistance. Our long-term goal is to leverage biophysical properties to resolve functionally resistant cellular subclones in which to focus single-cell multi-omics to nominate candidate targets for precision therapeutic strategies. Keywords: Clinical trial, Bruton’s tyrosine kinase inhibitor (BTKi), Mantle cell lymphoma, Drug resistance