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
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 
Published: November 2019

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 expanded with disease progression. mC3 was enriched for a cytokine signaling, interferon and inflammatory response signature and mC4 was enriched for a cell cycle signature. Unexpectedly, PDCD1 was expressed in MCL cells in all patients before PALIBR, modestly in mC3 in responding patients and strongly in both mC3 and mC4 in primary resistant patients. Loss of MCL cells in a durable CR (Pt 17) correlated with marked expansion of CD8+ T cells, whereas re-emergence of PDCD1- expressing mC3 and mC4 on progression after a durable PR (Pt 11) or CR (Pt 25) was preceded by loss of CD8+T cells (Lee et al Abstract # 122322). Flow cytometry confirmed that PD-1 was expressed on the surface of MCL cells. WTS of purified MCL cells further revealed that PDCD1 was regulated by the cell cycle, being repressed in MCL cells by palbociclib alone in patients responding to a separate sequential palbociclib-bortezomib therapy and elevated in mC3 on progression after a durable PR.
In summary, we have demonstrated, for the first time, that composite gain of CDK4 and hemizygous deletion of CDKN2A and RB1, but not each alone, predisposes MCL to resistance to dual CDK4/6 and BTK inhibition, and identified the resistant MCL clusters. The discovery of PD-1 expression in MCL cells suggests the existence of a clinically relevant tumor-intrinsic program that impinges on immunomodulation. A failure to curb the expansion of PD-1+ MCL clusters may fuel resistance by restricting cytotoxic T cell expansion. Conversely, repressing PD-1 expression in MCL cells and eliminating MCL cells in G1 arrest induced by CDK4/6 inhibition may augment ibrutinib's antitumor immunity by favoring T cell expansion. As the first integrative analysis of longitudinal scRNA-seq, WES and WTS in the context of clinical response to CDK4/6 inhibitor therapy in human cancer, our study provides new insights into tumor-intrinsic and -extrinsic mechanisms for targeting CDK4/6 in cancer.

Disclosures
Bartlett: Merck: Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Consultancy, Research Funding; Affimed: Research Funding; Bristol-Myers Squibb: Research Funding; Genenetech: Research Funding; Gilead: Research Funding; Immune Design: Research Funding; Pfizer: Research Funding; Millenium: Research Funding; Janssen: Research Funding; Celgene: Research Funding; Forty Seven: Research Funding. Maddocks:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; BMS: Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding. Park:Rafael Pharma: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Research Funding; Seattle Genetics: Research Funding, Speakers Bureau; G1 Therapeutics: Consultancy; Teva: Consultancy, Research Funding; Gilead: Speakers Bureau. Ruan:Juno: Consultancy; Kite: Consultancy; Janssen: Consultancy, Honoraria; Pharmacyclics LLC, an AbbVie company: Research Funding; AstraZeneca: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Galluzzi:Inzen: Consultancy; Luke Heller TECPR2 Foundation: Consultancy; Astra Zeneca: Consultancy; OmniSEQ: Consultancy, Membership on an entity's Board of Directors or advisory committees. Leonard:Sutro Biopharma: Consultancy; Bayer Corporation: Consultancy; AstraZeneca: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Bayer Corporation: Consultancy; MorphoSys: Consultancy; Karyopharm Therapeutics: Consultancy; Miltenyi: Consultancy; Akcea Therapeutics: Consultancy; Celgene: Consultancy; Epizyme, Inc: Consultancy; Merck: Consultancy; Akcea Therapeutics: Consultancy; Gilead: Consultancy; Sutro Biopharma: Consultancy; BeiGene: Consultancy; MorphoSys: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; Epizyme, Inc: Consultancy; Karyopharm Therapeutics: Consultancy; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy; Nordic Nanovector: Consultancy; ADC Therapeutics: Consultancy; Sandoz: Consultancy; BeiGene: Consultancy; Sandoz: Consultancy; Gilead: Consultancy; ADC Therapeutics: Consultancy; Nordic Nanovector: Consultancy; Merck: Consultancy; Miltenyi: Consultancy. Martin:Janssen: Consultancy; Teneobio: Consultancy; Karyopharm: Consultancy; Celgene: Consultancy; I-MAB: Consultancy; Sandoz: Consultancy.

OffLabel Disclosure:
Palbociclib is a CDK4/6 inhibitor FDA-approved for breat cancer treatment. It was used off-label in combination with ibrutinib in a phase I clinical trial in patients with relapsed/refractory mantle cell lymphoma.