Published OnlineFirst April 2, 2019; DOI: 10.1158/1078-0432.CCR-18-3412 Translational Cancer Mechanisms and Therapy Clinical Cancer Research Targeting CD38 Enhances the Antileukemic Activity of Ibrutinib in Chronic Lymphocytic Leukemia Alak Manna1, Sonikpreet Aulakh2, Prachi Jani2, Salman Ahmed2, Sharoon Akhtar1, Marie Coignet3, Michael Heckman4, Zahara Meghji2, Kirtipal Bhatia2, Aarushi Sharma1, TaimurSher2,Victoria Alegria2, Fabio Malavasi5, Eduardo N.Chini6, Asher Chanan-Khan1,2,7, Sikander Ailawadhi2, and Aneel Paulus1,2 Abstract Purpose: CD38 has emerged as a high-impact thera- measured via immunoblotting of Lyn, Syk, BTK, PLCg2, peutic target in multiple myeloma, with the approval ERK1/2, and AKT. of daratumumab (anti-CD38 mAb). The clinical impor- Results: In addition to immune-effector mechanisms; tance of CD38 in patients with chronic lymphocytic daratumumab also induced direct apoptosis of primary leukemia (CLL) has been known for over 2 decades, CLL cells, which was partially dependent on FcgR cross-link- although it's relevance as a therapeutic target in CLL re- ing. For the first time, we demonstrated the influence of mains understudied. CD38 on BCR signaling where interference of CD38 down- Experimental Design: We investigated the biological regulated Syk, BTK, PLCg2, ERK1/2, and AKT; effects that effects and antitumor mechanisms engaged by daratumu- were further enhanced by addition of ibrutinib. In comparison mab in primary CLL cells. Besides its known immune- to single-agent treatment, the combination of ibrutinib and effector mechanisms (antibody-dependent cell-mediated daratumumab resulted in significantly enhanced anti-CLL cytotoxicity, complement-dependent death, and anti- activity in vitro and significantly decreased tumor growth and body-dependent cellular phagocytosis), we also measured prolonged survival in the in vivo CLL xenograft model. direct apoptotic effects of daratumumab alone or in com- Conclusions: Overall, our data demonstrate the antitumor bination with ibrutinib. In vivo antileukemic activity was mechanisms of daratumumab in CLL; furthermore, we show assessed in a partially humanized xenograft model. The how cotargeting BTK and CD38 lead to a robust anti-CLL influence of CD38 on B-cell receptor (BCR) signaling was effect, which has clinical implications. Introduction ation with other cell surface (and cell-type specific) antigens. On B lymphocytes, CD38 associates with the B-cell receptor (BCR) CD38 is a highly conserved transmembrane type II glycopro- complex [(BCR)/CD81/CD19/CD21] and amplifies signal inten- tein expressed on B lymphocytes and other hematopoietic sity transmitted through the complex to drive cell prolifera- cells (1). Physiologically, CD38 functions as an ectoenzyme and tion (2). Patients with CLL with a higher proportion of CLL cells a coreceptor, the latter depending on its spatiotemporal associ- expressing CD38 (30%) have a shorter time to symptomatic disease and a more aggressive clinical course with inferior survival þ 1Department of Cancer Biology, Mayo Clinic, Jacksonville, Florida. 2Division of versus patients who have <30% of CD38 CLL cells (3, 4), Hematology and Oncology, Mayo Clinic, Jacksonville, Florida. 3Department of thus establishing CD38 expression as a marker of poor progno- Cancer Prevention & Control, Roswell Park Cancer Institute, Buffalo, New York. sis (5, 6). Despite its known association with an aggressive CLL 4Department of Health Science Research, Mayo Clinic, Jacksonville, Florida. phenotype, the role of CD38 as a therapeutic target remains 5 Lab of Immunogenetics, Department of Medical Science, University of Torino, unclear. Italy. 6Signal Transduction Laboratory, Kogod Aging Center, Department of Daratumumab is a first-in-class anti-CD38 therapeutic mAb Anesthesiology, Mayo Clinic, Rochester, Minnesota. 7Mayo Clinic Cancer Center at St. Vincent's Hospital, Jacksonville, Florida. approved for the treatment of relapsed/refractory multiple myeloma (7). It comprises a fully human IgG1k mAb, which Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). binds the C-terminus of CD38 at an epitope composed of b-strand–containing amino acids 233–246 and 267–280 (8). A fi A. Manna and S. Aulakh contributed equally and are co- rst authors of this report by Matas-Cespedes and colleagues demonstrated the anti- article. leukemic effects of single-agent daratumumab in ex vivo and in vivo Corresponding Authors: Aneel Paulus, Mayo Clinic, 4500 San Pablo Road CLL models (9). The cytotoxicity reported was modest, with South, Jacksonville, FL 32224. Phone: 904-953-6184; Fax: 904-953-6233; partial insight into the direct killing mechanism of daratumumab E-mail: [email protected]; and Asher Chanan-Khan, E-mail: [email protected] in CLL cells. We hypothesized that CD38 is a high value target in CLL and blocking of its receptorial function can be translated Clin Cancer Res 2019;25:3974–85 into a clinically beneficial therapeutic strategy through improved doi: 10.1158/1078-0432.CCR-18-3412 understanding of the mechanism(s) that link CD38 to CLL Ó2019 American Association for Cancer Research. cell survival. Here, we provide evidence that CD38 engagement 3974 Clin Cancer Res; 25(13) July 1, 2019 Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst April 2, 2019; DOI: 10.1158/1078-0432.CCR-18-3412 Daratumumab and Ibrutinib in CLL À and MEC1 (CD38 ) cell lines were also used in experiments. Translational Relevance An in vivo model of disseminated disease (14, 15) was estab- Increased CD38 expression on chronic lymphocytic leuke- lished using luciferase-labeled JVM13 (JVM13-Luc) cells, mia (CLL) cells is linked to aggressive disease features and injected via tail vein intravenously into 6- to 8-week old NSG poor clinical outcome. Biologically, CD38 promotes CLL cell (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice, following a proto- proliferation through association with multiple cell surface col approved by the Mayo Clinic Institutional Animal Care and receptors, including the B-cell receptor (BCR). As therapeutic Use Committee. Ibrutinib and kuromanin were purchased from opportunities to disrupt CD38 function become increasingly Selleckchem. Daratumumab was acquired through the Mayo available, we investigated the antitumor activity of daratumu- Clinic pharmacy and came predissolved/diluted. Statistical mab (anti-CD38 human mAb) in patient-derived CLL cells. analyses were performed using R Statistical Software (version Daratumumab was noted to promote immune-effector– 3.2.3; R Foundation for Statistical Computing), further detailed mediated cytolysis, as well as direct apoptosis of CLL cells. in figure legends. Data are represented as mean Æ SEM, unless Inhibition of CD38 (with daratumumab or a small-molecule otherwise stated in the figure legend. inhibitor) decreased activation of BCR signaling proteins and A full description of all assays is presented in Supplementary this effect was enhanced by ibrutinib. In vivo, the combination Materials and Methods. of ibrutinib and daratumumab significantly delayed tumor growth in B-cell leukemia–bearing mice and prolonged their survival. Altogether, our results suggest that combination of Results ibrutinib and daratumumab yields greater anti-CLL activity Patient and sample characteristics than either agent alone and support clinical evaluation of Patients with CLL (n ¼ 36) representing all clinical and genetic this regimen in patients with CLL. subsets were included in the study (clinical characteristics in Supplementary Tables S1 and S2). Relatively consistent with prior þ reports, we noted 27.7% (n ¼ 10) of patients had CD38 disease (using standard 30% cutoff; refs. 16, 17), whereas the remaining À by daratumumab modulates BCR signaling and enhances the patients (n ¼ 26, 72.2%) had CD38 disease. Notably, 16.6% anti-CLL activity of ibrutinib. Our observations provide impor- (n ¼ 6) of patients carried a deletion in chromosome 17p positive tant preclinical evidence for clinical exploitation of CD38 as a (Del17pþ). target for treatment of CLL. Daratumumab induces immune-mediated cytotoxicity in CLL cells Materials and Methods We first investigated the ability of daratumumab to induce Written informed consent wasobtainedfromallpatients CLL-specific lysis through immune-effector mechanisms (ADCC, whose samples were used in this study, approved by the Mayo CDC, and ADCP). Mean specific lysis from ADCC was 17.59% Æ Clinic Jacksonville Institutional Review Board and in accor- 1.30% (Fig. 1A). Subset analysis in CLL cells from patients þ À dance with the Declaration of Helsinki. Peripheral blood was defined as having CD38 and CD38 disease showed significant- þ collected from patients with a confirmed diagnosis of CLL who ly greater ADCC (P ¼ 0.023) in CD38 cases (24.63% Æ 3.46%) À were not on active anti-CLL treatment or those off anti-CLL versus CD38 cases (14.11% Æ 1.11%; Fig. 1B). In flow-sorted þ therapy for 1 month. This was followed by isolation of CD19 /CD38hi clones treated with daratumumab, ADCC was þ þ þ CD19 /CD5 B cells (primary CLL cells). Peripheral blood noted in 22.45% Æ 2.36% of cells. And in CD19 /CD38lo clones, mononuclear cells (PBMC) from human donors were used in ADCC was noted in 16.47% Æ 1.03% of cells (Fig. 1C). We then antibody-dependent cell-mediated cytotoxicity (ADCC) assays, assessed cell death via CDC in all CLL cells (n ¼ 30) and noted that 10% human serum was used in complement-dependent death mean specific lysis, was in the order of 14.88% Æ 0.92% (Fig. 1D). þ (CDC) assays, and human macrophages were used in antibody- Marginally higher levels were observed in CLL cells from CD38 À dependent cellular phagocytosis (ADCP) assays, as described patients (18.22% Æ 2.41%) versus CD38 patients (13.58% Æ by de Weers and colleagues (8). Apoptosis, mitochondrial 0.95%; Fig. 1E). In flow-sorted cells, CDC was noted in 19.10% Æ þ transmembrane permeability, and Western blotting assays were 1.98% and 9.23% Æ 1.81% of CD19 /CD38hi clones and þ conducted as per prior methods (10–13). All cells were cultured CD19 /CD38lo clones, respectively.