Composite Copy Number Variation in CDK4, CDKN2A and RB1 Predisposes Mantle Cell Lymphoma to Expansion of PD1+ Tumor Cells and Resistance to CDK4/6 Inhibitor Therapy as Revealed by Integrative Longitudinal scRNA-seq

• Published in: Blood 2019-11, Vol.134 (Supplement_1), p.1492-1492
• Main Authors: Di Liberto, Maurizio, Hu, Yang, Huang, Xiangao, Lee, Christina Y., Wang, Kevin, Bartlett, Nancy L., Ridling, LeAnn, Blum, Kristie A., Maddocks, Kami J., Park, Steven I, Ruan, Jia, Eng, Kenneth, Galluzzi, Lorenzo, Leonard, John P., Martin, Peter, Elemento, Olivier, Chen-Kiang, Selina
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2019-130944

Mantle cell lymphoma (MCL) is incurable due to the development of drug resistance. MCL cells proliferate without constraint on disease progression, fueled by aberrant cyclin D1 expression due to (11;14)(q13;q32) chromosomal translocation and dysregulation of CDK4 (CDK6 is silenced) that accelerate cell cycle progression through G1. Previously, we demonstrated that selective inhibition of CDK4/6 with palbociclib leads to prolonged early G1 arrest that sensitizes MCL cells to partner drugs including the BTK inhibitor ibrutinib. In our phase I clinical trial of palbociclib + ibrutinib (PALIBR) in recurrent MCL, complete response (CR) was achieved in 10/28 patients with only 5/19 responding patients progressing in 5 years. CDK4/6 inhibition thus appears to prolong and deepen the clinical response to ibrutinib. To determine the tumor-intrinsic mechanism that discriminate sensitivity from resistance to PALIBR, we performed integrative analysis of longitudinal whole exome- and whole transcriptome- sequencing (WES/WTS) of MCL cells isolated from the tissue and peripheral blood of patients before, during therapy and on progression. To reveal tumor heterogeneity, clonal evolution, and the tumor-immune interaction during the clinical response, we undertook single cell RNA-sequencing (scRNA-seq) of the corresponding peripheral blood mononuclear cells (PBMCs) in conjunction with flow cytometry. Integrative WES/WTS analysis demonstrated that CR (n=8) was achieved despite copy number gain of CDK4, BCL6 or MYC, or hemizygous deletion (h-del) of RB1, CDKN2A (encoding CDK4/6 inhibitor p16), ATM or TP53. However, composite CDK4 gain + CDKN2A and RB1 h-del, coupled with ATM or TP53 h-del before treatment was associated with loss of cell cycle control in 3 of 4 evaluable primary resistant patients, on progression at cycle 5 after a transient CR (Pt 18) and cycle 30 after a durable partial response (PR) (Pt 2). The other PR patients harbor various copy number variations (CNV) but not the resistance composite CNV. Thus, by perturbing the stoichiometry of CDK4/p16/Rb, composite CNV of CDK4, CDKN2A and RB1 predisposes MCL to resistance to dual CDK4/6 and BTK inhibition. scRNA-seq analysis further revealed 5 transcriptionally distinct MCL clusters (cyclin D1+CD19+) in PBMCs of treatment-naïve patients (n=4) as well as PBMCs, bone marrow and tissue samples of recurrent MCL patients before PALIBR. Cluster 3 (mC3) and cluster 4 (mC4) were absent in PBMCs of normal subjects (n=4) but e