Dynamic Immune Surveillance in Durable Clinical Response to Combined BTK and BCL2 Inhibition in MCL at Longitudinal Single-Cell Resolution
Authors: Wang Kevin , Di Liberto Maurizio , Hu Yang , Huang Xiangao , Koldej Rachel M , Ritchie David , Martin Peter , Inghirami Giorgio , Elemento Olivier , Tam Constantine S. , Chen-Kiang Selina 
Published: November 2021

Abstract
Combined inhibition of BTK with ibrutinib and BCL2 with venetoclax is one of the most promising therapies for B cell malignancies, especially mantle cell lymphoma (MCL), where durable complete remission continued after therapy cessation in some patients (Tam et al, NEJM 2018, Handunnetti ASH 2019). The MCL-intrinsic and extrinsic mechanisms underlying this deep and durable clinical response are unknown, nor have resistance mechanisms been identified. Since BTK is expressed mainly in B lineage cells and venetoclax inhibits BCL2 universally, we hypothesize that BTK inhibition selectively primes MCL cells for vulnerability to BCL2 inhibition while maintaining immune cell homeostasis, leading to differential elimination of MCL cells through immune surveillance.
To test this, we undertook integrative longitudinal single-cell RNA-sequencing analysis (scRNA-seq) of PBMCs from sequential tissue and blood specimens (n=32) of 8 MCL patients before and during ibrutinib-venetoclax combination therapy, after therapy cessation or progression, as well as 4 treatment-naïve MCL patients and 4 normal subjects as controls. High dimensional analysis using a unique MCL RNA reference library that we built from bulk RNA-seq data of MCL cells from 57 patients reveals that MCL cells comprise 4 transcriptomically distinct clusters. Cluster 1 (C1) is similar to quiescent normal B cells; C2 resembles hyper-activated B cells enriched for signatures of BCR and cytokine signaling and proinflammatory pathways; C3 represents non-proliferating, long-lived MCL cells that accumulate as disease progresses; and C4 is highly proliferative, expanding with disease progression in untreated patients or on therapy.
Integrative analysis of scRNA-seq and CBC with differential showed that homeostasis of all immune cells was maintained throughout ibrutinib-venetoclax therapy and after therapy cessation in 6 MCL patients with a complete response (CR). CD8+T and NK cells were functional, evidenced by the expression of cytotoxic genes such as GNLY, FGFBP2, and GZMH. In contrast, these genes were profoundly suppressed in CD8+T cells that were rapidly depleted on MCL progression after transient response in 2 patients. NK cells were also depleted on progression. In one patient, this was preceded by suppression of cytotoxic genes and loss of MHC-I and MHC-II in MCL cells. Exhaustion did not appear to be the cause. Rather, TSC22D3 upregulation suggests that inhibition of TCR-induced IL2 and IL2R expression and NF-kB activation may underlie the loss of CD8+T and NK cells in ibrutinib-venetoclax resistance.
To determine whether BTK inhibition primes MCL cells for subsequent venetoclax killing while sparing other immune cells in a clinical response to ibrutinib-venetoclax, we found that BCL2 expression was higher in MCL cells than in other immune cells pre-therapy. Ibrutinib selectively downregulated the anti-apoptotic MCL1 and upregulated PMAIP1 encoding the pro-apoptotic NOXA in MCL cells, concurrent with TSC22D3 induction and inhibition of the NF-kB signaling pathway. As expected from the lack of BTK expression, CD4+ and CD8+T cells and NK cells expressed MCL1 over PMAIP1 in CR patients, but greater PMAIP1 in patients who developed progressive MCL. Collectively, these data support the hypothesis that BTK inhibition selectively primes MCL cells for venetoclax sensitivity.
scRNA-seq analysis further showed that B cells emerging after therapy cessation in CR patients were nearly identical to the bone marrow B cells pre-therapy, as quiescent B cells in cluster 1 and activated B cells in cluster 2. None were detected in clusters 3 or 4. The restoration of B cell immunity confirms CR at the single-cell transcriptome level, speaking to the power of dual BTK and BCL2 inhibition, and supporting the role of immune surveillance in clinical response in targeted therapy.
In summary, by unbiased longitudinal scRNA-seq analysis of sequential patient specimens from the ibrutinib-venetoclax clinical trial, we have provided the first evidence that priming of MCL cells for venetoclax vulnerability by BTK inhibition cooperates with immune surveillance to determine the depth and durability in combined targeting of BTK and BCL2 in lymphoma in humans. These findings parallel our longitudinal scRNA-seq analysis of dual BTK and CDK4/6 inhibition, suggesting priming of cancer cells and cooperation with immune surveillance underpin targeted therapy.

Disclosures
Koldej: CRISPR Therapeutics: Research Funding. Ritchie: CRISPR Therapeutics: Research Funding; Takeda: Research Funding; BMS: Research Funding; Novartis: Honoraria; Amgen Inc: Honoraria, Research Funding; CSL: Honoraria. Martin: ADCT: Consultancy. Elemento: One Three Biotech: Consultancy, Other: Current equity holder; Owkin: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; AstraZeneca: Research Funding; Johnson and Johnson: Research Funding; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Janssen: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Champions Oncology: Consultancy. Tam: Beigene: Research Funding; Loxo: Honoraria; Beigene: Honoraria; Janssen: Honoraria; Abbvie: Honoraria; Abbvie: Research Funding; Janssen: Research Funding.

OffLabel Disclosure:
Venetoclax is a BCL2 inhibitor FDA-approved for chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), and acute myeloid leukemia (AML). It was used off-label in combination with ibrutinib in a phase II clinical trial in patients with mantle cell lymphoma.