Targeting CD38 Enhances the Antileukemic Activity of Ibrutinib In

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 2019 American Association for Cancer Research. cell survival. Here, we provide evidence that CD38 engagement

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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 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 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% Clinic Jacksonville Institutional Review Board and in accor- 1.30% (Fig. 1A). Subset analysis in CLL cells from patients þ 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% 3.46%) were not on active anti-CLL treatment or those off anti-CLL versus CD38 cases (14.11% 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% 2.36% of cells. And in CD19 /CD38lo clones, mononuclear cells (PBMC) from human donors were used in ADCC was noted in 16.47% 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% 0.92% (Fig. 1D). þ (CDC) assays, and human macrophages were used in antibody- Marginally higher levels were observed in CLL cells from CD38 dependent cellular phagocytosis (ADCP) assays, as described patients (18.22% 2.41%) versus CD38 patients (13.58% by de Weers and colleagues (8). Apoptosis, mitochondrial 0.95%; Fig. 1E). In flow-sorted cells, CDC was noted in 19.10% þ transmembrane permeability, and Western blotting assays were 1.98% and 9.23% 1.81% of CD19 /CD38hi clones and þ conducted as per prior methods (10–13). All cells were cultured CD19 /CD38lo clones, respectively. (Fig. 1F). Comparative anal- in AIM-V media under conditions previously reported by us (10, ysis in JVM13 and MEC1 cells did not show significant induction 11). CD38 receptor density on CLL cells was quantified as mean of ADCC or CDC with daratumumab alone (Supplementary Fig. fluorescence intensity (MFI) and cell surface antibody bound/ S3A–S3C). Cell death through ADCP has been previously cell (sAbc). For certain experiments, PBMCs were isolated from reported with daratumumab in myeloma cells (18). We noted þ patients 4, 18, 19, 28, and 31 and CD19/CD5 CLL cells were ADCP in 8.52% 0.44% of CLL cells (Fig. 1G); with little þ selected out using magnetic beads, followed by flow sorting difference observed between CLL cells from CD38 (9.37% þ with an anti-CD38 APC antibody for separation of CD19 / 0.77%) versus CD38 patients (8.79% 0.92%). Similar findings þ þ þ CD38hi-andCD19/CD38lo-purified cells. Cells were then were seen in flow-sorted CD19 /CD38hi- or CD19 /CD38lo- treated with trypsin-EDTA for 10 minutes and washed twice purified cells. (Fig. 1H). Comparative analysis in the cell lines followed by culture in AIM-V serum-free media for 24 hours. showed higher ADCP in JVM13 (18.01% 1.0%) versus MEC1 CD38 expression in purified cells was again reassessed using a (3.55% 0.14%; Supplementary Fig. S3D). Correlation between multi-epitope FITC-conjugated anti-CD38 antibody (Cytognos ADCP results, CD38 MFI (r ¼ 0.49; P ¼ 0.006), and CD38 sAbc CD38 multi-epitope-FITC antibody). Our sorting strategy is (r ¼ 0.41; P ¼ 0.021) was significant (Supplementary Fig. S4A and þ presented in Supplementary Figs. S1 and S2. JVM13 (CD38 ) S4B). Similarly, correlation between CDC results and percent of

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Figure 1. Daratumumab induces CLL cell death through immune effector–mediated mechanisms. A, ADCC induced by single-agent daratumumab (Dara, 0.1 mg/mL) was determined in Calcein-AM–labeled primary CLL cells (target) from 30 patients, ex vivo, in the absence or presence of effector (peripheral blood mononuclear) cells from healthy human donors at an effector:target (E:T) ratio of 50:1 for 6 hours. Specific lysis was calculated as described in Supplementary Materials and þ Methods. Spontaneous release was determined using a nonspecific IgG1-b12 isotype antibody at 0.1 mg/mL. B, ADCC in primary CLL cells from CD38 (n ¼ 8) and CD38 (n ¼ 22) patients (Pts) was assessed as a subset analysis and showed significantly higher specific lysis in cells from CD38þ patients. C, ADCC was also assessed in flow-sorted CD19þ/CD38hi and CD19þ/CD38lo CLL cells from five patients (patients 4, 18, 19, 28, and 31) and revealed a similar trend. D, Specific lysis from CDC was measured in CLL cells in the presence of 10% human serum from a single healthy donor for 1 hour. E, Subset analysis of specific lysis from CDC induced in CLL cells from CD38þ (n ¼ 8) versus CD38 (n ¼ 22) patients was also determined. F, Similarly, CDC was determined in flow-purified CD19þ/CD38hi and CD19þ/CD38lo CLL cells. G, Cell death through phagocytosis was assessed in Calcein-AM–labeled primary CLL cells (n ¼ 30), with subset analysis in CD38þ versus CD38 cases and separately in flow-purified CD19þ/CD38hi and CD19þ/CD38lo CLL cells (H) using CD11bþ macrophages differentiated from healthy human donor monocytes at an E:T ratio of 2:1 for 6 hours incubation period. Percent phagocytosis was determined as described in Supplementary Materials and Methods. Results are expressed as mean SEM. Comparative significance analyses between the groups (brackets) show P (, P 0.05; , P < 0.01; , P < 0.001).

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þ CD38-expressing cells was also significant (r ¼ 0.49; P ¼ 0.006; tivity in CLL cells from CD38 patients (36.41% 3.34%) versus Supplementary Fig. S4C). ADCC cell death assay results did not CD38 patients (23.39% 1.70%; P ¼ 0.002; Fig. 2B and C); with show any significant correlation with either CD38 MFI/sAbc or similar effects in JVM13 (37.58% 1.88%) versus MEC1 cells percent of CD38-expressing cells (Supplementary Table S3). (14.82% 1.37%; Supplementary Fig. S5). In unsorted CLL cells þ from patients with either CD38 or CD38 disease, a significant Daratumumab can directly induce apoptosis of CLL cells correlation was observed between percent apoptosis with CD38 Prior studies in multiple myeloma cells suggest that daratu- MFI (r ¼ 0.39; P ¼ 0.036), CD38 sAbc (r ¼ 0.53; P ¼ 0.003), and mumab can directly induce apoptosis (19). In CLL cells treated percent CD38 positivity of CLL cells (r ¼ 0.45; P ¼ 0.012; with daratumumab, we noted 31.33% 2.56% annexin-V/ Supplementary Table S3). Interestingly, in flow-sorted CLL cells, þ propidium iodide (PI) positivity overall (Fig. 2A). Subset analysis a significant degree of apoptosis (P < 0.001) was noted in CD19 / þ of CD38 and CD38 cases showed greater annexin-V/PI posi- CD38hi cells (70.10% 12.18%); with comparatively lower

Figure 2. Daratumumab (Dara) induces apoptosis in CLL cells, which is partially dependent on FcgR cross-linking. A, Apoptosis was assessed in CLL cells treated with daratumumab or IgG1-b12 isotype antibody (0.1mg/mL) for 24 hours, followed by staining with annexin-V/PI and flow cytometry analysis. B, Subset analysis was also performed in CD38þ (n ¼ 8) and CD38 (n ¼ 22) CLL cases. C, Annexin-V/PIþ cell scatter plots from a representative patient with CD38þ CLL [patient (Pt) 9, 38% CD38þ cells] and a CD38 patient (patient 8, 7.54% CD38þ cells) are shown. Flow-sorted CD19þ/CD38hi and CD19þ/CD38lo CLL cells (n ¼ 5 patients) were treated with daratumumab (0.1 mg/mL; D) or kuromanin (Kuro, 10 mmol/L, flavonoid small-molecule CD38 enzymatic inhibitor; E) and showed significantly more apoptosis in the CD38hi population relative to the CD38lo fraction of CLL cells (representative scatterplot from a single patient shown in F). G, From a subset of patients (n ¼ 7, primarily CD38 cases), CLL cells were treated with daratumumab (D), F(ab')2 (control), or the combination of daratumumab þ F(ab')2 in the presence or absence of a pan-FcgR blocker (Human TruStain FcX). F(ab')2 alone triggered no apoptosis and while daratumumab treatment of CLL cells F(ab')2 without FcgR blocker showed significant cell death (red bars), the addition of an FcgR blocker significantly decreased the degree of apoptosis (black bars). H, A representative patients' scatterplot showing apoptosis. Data are presented as mean SEM (, P 0.05; , P < 0.01; , P < 0.001).

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þ apoptosis in CD19 /CD38lo cells (31.57% 11.22%; Fig. 2D and Supplementary Figs. S8A–S8F). Next, to test whether changes in F). A significant correlation between apoptosis results with per- BCR proteins could also be elicited by small-molecule inhibition þ cent CD38 CLL cells (r ¼ 0.45; P ¼ 0.012), CD38 MFI (r ¼ 0.39; of CD38, we treated CLL cells with kuromanin and observed P ¼ 0.036), and CD38 sAbc (r ¼ 0.53; P ¼ 0.003) was observed relatively analogous changes in BCR-signaling proteins, albeit, (Supplementary Fig. S4D–S4F). Other than association between compared with daratumumab, the intensity of inhibition was less degree of CDC induction and patient age (P ¼ 0.036), no other (Supplementary Fig. S9A–S9G). This observation is critical as it significant correlation was observed between daratumumab- demonstrates that targeting CD38 by either an antagonistic mAb mediated cell death and clinical characteristic of patients (Supple- or a small molecule leads to reduction in the signaling capabilities mentary Table S4). of the BCR complex. Given the biological functions of CD38 To test whether small-molecule–based inhibition of CD38 (particularly in regulation of BCR signaling), we reasoned that could induce apoptosis, we used kuromanin, a flavonoid inhib- targeting both CD38 (with daratumumab/kuromanin) and BTK itor of CD38 enzymatic activity (20, 21), which has been previ- (with ibrutinib), would lead to synergistic reduction in phos- ously used to interrogate CD38 biology (22). In flow-sorted phorylated BCR proteins. As anticipated, in tumor cells from þ CLL cells, apoptosis induced by kuromanin was significantly patients with CD38 CLL, the combined effect of ibrutinib þ þ þ greater (P < 0.001) in CD19 /CD38hi versus CD19 /CD38lo cells daratumumab (ID) resulted in a significantly more pronounced (54.97 4.99 vs. 15.60 1.28, respectively; Fig. 2E and F). decrease in p-Lyn, p-BTK, p-PLCg2, p-ERK1/2, and p-AKT levels In a subset of CLL patient cells, we also examined whether compared with either of the agents alone (Fig. 3A–G, combina- daratumumab-induced apoptosis was due to FC-gamma recep- tion treatment highlighted by open bars). In CLL cell lysates from tor (FcgR) cross-linking. CLL cells were noted to express FcgRI CD38 patients treated with the ID combination, only p-BTK, (CD64, 73.67% 3.37%), FcgRII (CD32, 95.18% 1.85%), p-PLCg2, and p-ERK1/2 were significantly downregulated com- and FcgRIII (CD16, 19.31% 3.89%; Supplementary Fig. S6). pared with either agent alone (Fig. 3H–N). Representative immu- When treated with daratumumab þ an FcgRblocker,the noblots are shown in Fig. 3G and N. Likewise, in flow-sorted þ þ percentage of apoptotic CLL cells was significantly lower CD19 /CD38hi and CD19 /CD38lo CLL cells, we probed for (18.7% 0.15%) than cells treated with daratumumab alone p-BTK and p-PLCg2. CD38hi clones appeared to have higher lo (27.21% 1.67%; P ¼ 0.007). Addition of an F(ab')2 fragment BTK expression versus CD38 clones. In addition, all drugs to daratumumab-treated CLL cells triggered further apoptosis (including the ID combination) appeared to more effectively (39.43% 0.56%); however, a decrease was noted when FcgR downregulate p-BTK, as well as p-PLCg2inCD38hi clones blocker was added (29.91% 1.3% apoptosis; P ¼ 0.0079; (Supplementary Fig. S9H). Altogether, these results allowed us Fig. 2G and H). Altogether, these results indicated to us that to conclude that blocking either the receptor function (dara- daratumumab-mediated apoptosis in CLL cells is partially tumumab) or the enzymatic properties (kuromanin) of CD38 dependent on FcgRcross-linking. leads to downregulation of BCR-associated proteins in CLL cells and which can be further amplified through simultaneous Targeting CD38 modulates proteins associated with BCR inhibition of BTK with ibrutinib. signaling CD38 colocalizes with CD19 and CD81 in the lipid rafts at the Ibrutinib significantly augments the cytotoxic activity of cell membrane and this result in amplification of BCR and daratumumab collateral signaling events (23). CLL cells with high proliferative Previous studies have shown that inhibition of BTK can down- potential inherently depend on BCR signaling and consequently regulate cell surface antigens on CLL cells (29). Thus, we first are also reliant on CD38 coreceptorial function (24–26). Indeed, examined whether ibrutinib modulates CD38 expression and prior studies have demonstrated an increase in ERK activity (one noted that ibrutinib did not downregulate CD38 on CLL cells of the terminal effectors of BCR signaling) upon CD38 agonistic (24 hours exposure; Supplementary Fig. S10). As such, we pro- ligation (21–23, 27). Given our interest in targeting CD38; its ceeded toward antitumor testing of the ID combination in pri- established role in BCR signal amplification (23, 27) and the high mary CLL cells. Cytolysis from ADCC in ibrutinib-treated CLL clinical relevance of BCR-targeting agents in CLL (28), we ques- cells was 9.92% 0.88%, which significantly (P < 0.001) tioned whether therapeutic interference of CD38 would modulate increased to 42.81% 1.12%, in ID-treated cells (Fig. 4A). This þ the BCR pathway. Basal expression of BCR-signaling components: effect was more pronounced in cells from CD38 (63.73% (p) p-Lyn, p-Syk, p-BTK, p-PLCg2, p-ERK1/2, and p-AKT in 4.43%) versus CD38 (35.21% 1.61%) patients with CLL þ CLL cells from CD38 versus CD38 patients is shown in Sup- (Fig. 4B). In flow-sorted CD19þ/CD38hi clones treated with the þ plementary Fig. S7A–S7F. We treated (IgM-stimulated) CLL ID combination, ADCC was 45.53% 3.19% and in CD19 / þ cells from both CD38 and CD38 patients with daratumumab, CD38lo cells was 20.29% 0.95% (P < 0.001; Fig. 4C). When we ex vivo, and noted a significant decrease in p-Syk, p-BTK, p-ERK1/2, examined CDC, lysis induced by ibrutinib alone was 6.17% þ and p-AKT (Fig. 3A–G for CD38 CLL patient cells and Fig. 3H–N 0.56% and this was significantly (P < 0.001) higher in cells treated for CD38 CLL patient cells; compare red bars with black bars; with ID combination (32.23% 1.47%; Fig. 4D). Although the þ P < 0.05). As expected, comparative analysis between CLL cells trend in specific lysis was higher in cells from CD38 (40.25% þ from the CD38 versus CD38 patients revealed the percent 2.82%) versus CD38 patients (29.56% 1.60%), the difference þ decrease in proximal BCR-signaling proteins (p-Lyn, p-BTK, and was not significant (Fig. 4E). In flow-sorted CD19 /CD38hi and þ p-PLCg2) elicited by daratumumab was significantly more nota- CD19 /CD38lo CLL cells, specific lysis from ID treatment was þ ble in CLL cells from CD38 patients (P < 0.05). Intriguingly, we 44.13% 2.59% and 14.86% 1.18%, respectively (P < observed the opposite for p-ERK and p-AKT, which decreased 0.01; Fig. 4F). We then determined the effect on ADCP and noted more significantly in daratumumab-treated CLL cells from that ibrutinib alone induced negligible phagocytosis (3.47% þ CD38 patients (relative to CLL cells from CD38 patients; 0.32%), whereas in ID-treated cells, ADCP increased to 21.50%

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Figure 3. Targeting CD38 results in downregulation of BCR-signaling proteins, which are further augmented by ibrutinib treatment. Phosphorylated (p-) and total protein levels for Lyn, Syk, BTK, PLCg2, ERK1/2, and AKT were probed for by western blot analysis in CLL cell lysates (/þ BCR stimulation with anti-IgM for 1 hour), from CD38þ patients (patients 11, 16, 26, 33, and 34; A–G) and CD38 patients (patients 8, 14, 28, 35, and 36; H–N). Primary CLL cells were treated for 2 hours with isotype IgG1-b12 Ab (control, 0.1mg/mL), ibrutinib (Ibr, 1 mmol/L), daratumumab (Dara, 0.1mg/mL), or the combination of ibrutinib þ daratumumab (ID) before þ lysate preparation. G and N, Representative Western blots from a patient with CD38 and CD38 CLL are shown. Results shown are mean SD. , P < 0.01; #, statistically significant (P < 0.05) difference compared with control or single-agent–treated cells; #d, significant (P < 0.05) compared with all single-agent or control-treated cells, except daratumumab-treated cells; #i, significant (P 0.05) compared with all single-agent or control-treated cells, except ibrutinib- treated cells.

1.02% (Fig. 4G). And the difference in ADCP was significantly was noted (P < 0.05; Supplementary Table S3). A significant þ appreciable between ID-treated CLL cells from CD38 (29.96% correlation between degree of immune-mediated cytolysis 1.41%) versus CD38 patients (18.42% 0.94%; P < (ADCC, CDC, and ADCP) and CD38 expression (percent þ þ 0.001; Fig. 4H). Examination in flow-sorted CD19 /CD38hi CD38 cells, MFI/sAbc) was observed in ID-treated CLL cells. cells, ADCP was noted in 17.35% 0.90% of ID-treated clones Interestingly, significantly higher ADCC, as well as CDC were þ versus 10.64% 0.88% in CD19 /CD38lo cells (P < noted in ID-treated CLL cells from Del17pþ patients (P ¼ 0.021 0.01;Fig.4I).Ofnote,comparative analyses (for ADCC, CDC, and P ¼ 0.036; Supplementary Table S5). These data demon- and ADCP) in JVM13 and MEC1 cell lines was also performed strate that cotargeting CD38 and BTK results in a significant and showed similar results as in primary CLL cells, with some increase in immune-directed CLL cell killing (vs. either dara- expected variations (Supplementary Fig. S3). A significant cor- tumumab or ibrutinib alone) and although this effect was þ relation between ADCC, CDC, and ADCP in ID-treated CLL perceptible in both CLL cells from CD38 and CD38 patients, þ cells with percent CD38 CLL cells, CD38 MFI and CD38 sAbc it was more pronounced in the latter.

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We also examined apoptosis and observed 36.86% 2.56% plots from two representative patients are shown in Fig. 5C. Using annexin-V/PI positivity in CLL cells treated with ibrutinib, which a different combination of probes (7AAD/annexin-V), we noted increased to 61.90% 2.41% in ID-treated cells (Fig. 5A). Subset a similar trend (56.6% 4.92%; 7AAD/annexin-V positivity) in þ analysis of cells from patients with CD38 versus CD38 CLL ID-treated CLL cells from seven patients (five of who had CD38 þ revealed 45.26% 2.11% versus 33.81% 3.18% apoptosis in disease; Supplementary Fig. S11). In flow-purified CD19 / þ ibrutinib-treated and 70.71% 3.44% versus 58.7% 2.77% CD38hi and CD19 /CD38lo cells, ID combination treatment apoptosis in ID-treated CLL clones, respectively (Fig. 5B). Scatter resulted in apoptosis of 89.53% 2.16% and 49.27%

Figure 4. The immune-mediated cytolytic activity of daratumumab is significantly enhanced by ibrutinib. A, ADCC was examined in Calcein-AM–labeled primary CLL cells (n ¼ 30 patients) treated with either IgG1-b12 isotype antibody (0.1 mg/mL), ibrutinib (Ibr, 1 mmol/L), daratumumab (Dara, 0.1 mg/mL), or the combination of ibrutinib þ daratumumab with or without effector cells at an effector:target (E:T) ratio of 50:1 for 6 hours. Specific lysis was calculated as described in Supplementary Materials and Methods and in the same manner as Fig. 1. B, ADCC in cells from CD38þ (n ¼ 8) and CD38 (n ¼ 22) patients treated with ibrutinib, daratumumab, or ibrutinib þ daratumumab was analyzed. C, Similarly, ADCC was determined in flow-sorted CD19þ/CD38hi and CD19þ/CD38lo CLL cells from five patients and showed a similar trend. D and E, CDC was measured in Calcein-AM–labeled CLL cells incubated with human serum (10%) from a healthy donor for 1 hour; effect of ibrutinib, daratumumab, or combination treatment in CLL cells from CD38þ (n ¼ 8) versus CD38 (n ¼ 22) patients. F, CDC in a similar manner was examined in flow-sorted CD19þ/CD38hi and CD19þ/CD38lo CLL cells (n ¼ 5 patients). G, ADCP was assessed in Calcein-AM–labeled CLL cells treated with either ibrutinib, daratumumab, or ibrutinib þ daratumumab by incubating target tumor cells with human monocyte–derived macrophages (effectors) from a healthy donor at an E:T ratio of 2:1. Flow cytometry analysis to detect CD11bþ macrophage engulfment of Calcein-AM–labeled tumor cells was used to calculate percent phagocytosis. H, ADCP levels were separately analyzed for patients with CD38þ and CD38 CLL. I, ADCP was also determined in flow-sorted CD19þ/ þ CD38hi and CD19 /CD38lo CLL cells from five patients. Data are presented as mean SEM (, P 0.05; , P < 0.01; , P < 0.001).

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Figure 5. Cotargeting CD38 and BTK leads to significantly greater apoptosis of primary CLL cells than compared with singular targeting of either CD38 or BTK. A, Apoptosis was assessed in primary CLL cells treated with daratumumab (Dara, 0.1 mg/mL), kuromanin (Kuro, 10 mmol/L) ibrutinib (Ibr, 1 mmol/L), Ibr alone, or IgG1-b12 isotype antibody for 24 hours, followed by staining with annexin-V/PI and flow cytometry analysis. B, Subset analysis was also performed in primary CLL cells from CD38þ (n ¼ 8) and CD38 (n ¼ 22) patients. C, Annexin-V/PIþ cell scatter plots from a representative CD38þ CLL case (patient 9) and a CD38 case (patient 8) are shown. D and E, Apoptosis was also assessed separately in flow-sorted CD19þ/CD38hi and CD19þ/CD38lo CLL cells (n ¼ 5 patients). F, Annexin-V/PIþ cell scatter plots from a representative patients' (patient 19.) CD38hi CLL clones and CD38lo CLL clones are shown. Results expressed as mean SEM (, P 0.05; , P < 0.01; , P < 0.001).

10.97% of cells, respectively (Fig. 5D and E). Scatter plots from a combination þ effector cells (Fig. 6A). On postimplantation day representative experiment are shown in Fig. 5F. Akin to primary 28, treatment was concluded in all groups before onset of any CLL cells, similar results were seen in ID-treated CLL cell lines signs/symptoms of xeno-GVHD (typically occurring at week 4, (JVM13, 70.88% 4.23% and MEC1, 41.03 1.62; Supplemen- postimplantation of human effector cells; ref. 15) and final tary Fig. S5). We also assessed apoptosis in CLL cells exposed to antitumor response was assessed. Compared with vehicle- or kuromanin ibrutinib, where the combination of these two effector cell–only groups, all other groups showed significantly agents showed significantly greater annexin-V/PI positivity reduced tumor burden (P < 0.01). Tumor burden in mice treated (49.53% 2.39%; Fig. 5A), compared with either kuromanin with daratumumab þ effector cells versus ibrutinib þ effector cells or ibrutinib alone. This validates the fact that the cytotoxicity of was not significantly different (P ¼ 0.063). However, in mice ibrutinib is enhanced with concurrent targeting of CD38 and this treated with ID therapy ( effector cells), significantly lower effect is independent of whether a mAb or an anti-CD38 small disease burden was noted, approximately 3.5- and 2.8-fold lower molecule is used. Apoptosis was mirrored by a loss of mitochon- than that observed in vehicle (P < 0.01) or effector cell alone– drial membrane permeability by 69.22% 2.49% in CLL cells (n treated (P < 0.01) mice, respectively (Fig. 6B and C). No significant ¼ 30 patients), the percent change of which was marginally higher changes in weight were noted in any of the treatment groups in cells from patients that were CD38 (Dilc15 MFI 1,100 193.7 (Fig. 6D). Mice in groups 2, 5, 6, and 8, which were administered vs. 4,340 608.2 in control cells) compared with those who were effector cells, were sacrificed on day 30; whereas mice remaining þ clinically categorized as CD38 (Dilc15 MFI 687.3 111.11 vs. in the other groups (that did not receive effector cells) were 2,145 289.8 in controls; Supplementary Fig. S12A–S12C). In followed up to 107 days for survival analysis (Fig. 6E). A trend þ þ flow-sorted CD19 /CD38hi and CD19 /CD38lo cells, a similar for longer survival in the daratumumab monotherapy versus trend was noted (Supplementary Fig. S12D–S12G). vehicle-treated cohort was noted (median overall survival 89 vs. 59 days, respectively, P ¼ 0.057). By day 107, 50% of the mice in The combination of daratumumab and ibrutinib reduces tumor the ID combination–treated arm were alive and healthy. burden in a mouse model of CLL We tested the anti-CLL activity of daratumumab ibrutinib in an in vivo disseminated disease model system. NSG mice were Discussion injected with JVM13-Luc cells via tail vein injection and disease Targeting CD38 for therapeutic purposes has been largely burden was monitored by bioluminescent signal. On day 7 examined in multiple myeloma (30). In CLL, preclinical proof- postimplantation, mice were randomly divided into eight groups of-concept for disrupting CD38 function was first reported by (6 mice/group) to receive either; (i) vehicle, (ii) effector cells only Vaisitti and colleagues where inhibition of its enzymatic activity on the days that daratumumab was given, (iii) daratumumab with the flavonoid kuromanin slowed CLL cell homing and alone, (iv) ibrutinib, (v) daratumumab þ effector cells, (vi) adhesion in vitro and in a murine model (22). Subsequently, ibrutinib þ effector cells, (vii) ID combination, and (viii) ID Matas-Cespedes and colleagues reported on the anti-CLL activity

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of daratumumab showing its mechanism of action to be through Although the enzymatic functions of CD38 and its inhibition in ADCC and ADCP, with a trend for higher cytolytic activity in CLL CLL cells have been described previously (1), the receptorial þ cells from CD38 patients (9). Our results herein support these properties of CD38 and particularly their role in signal transmis- ﬁndings and show that in addition to immune-effector–mediated sion through the BCR complex are less understood. Studies by cell kill, daratumumab induces apoptosis in CLL cells; partially Deaglio and colleagues and Malavasi and colleagues have pro- dependent on FcgR-mediated cross-linking and which is actual- vided signiﬁcant insight on localization of CD38/BCR complexes ized through destabilization of the mitochondria. in cell membrane lipid rafts. Indeed, CD38 ligation with an

Figure 6. Daratumumab (Dara) and ibrutinib (Ibr) significantly delay tumor growth and extend survival of leukemia-bearing mice. A, NSG mice were implanted with JVM13-Luc (8 106) cells via intravenous tail vein injection and postimplantation day 7, mice were randomized in 8 groups to receive either (i) vehicle (control, intraperitoneal), (ii) effector cells only (healthy donor PBMCs, 8 106 cells, i.v.), (iii) daratumumab alone on a weekly schedule (20/10/10/10/10 mg/kg, i.p.), (iv) ibrutinib (6 mg/kg, i.p.), (v) daratumumab þ effector cells, (vi) ibrutinib þ effectors, (vii) ibrutinib and daratumumab (ID) combination, and (viii) ID combination þ effectors. B and C, By day 28, tumor burden (average photon radiance, p/s/cm/sr x 105) in vehicle-treated mice reached a median of 9.18 versus 7.59 in effector-only–treated mice, 4.87 in daratumumab-treated mice, 4.03 in daratumumab þ effector-treated mice, 3.81 in ibrutinib-treated mice, 3.90 in ibrutinib þ effector–treated mice, 2.89 in ID-treated mice, and 2.57 in ID þ effector–treated mice. D, Minor variances in weight of mice over duration of treatment were noted, however these differences were not significant. E, Survival study was performed in mice from treatment groups 1, 3, 4, and 7 (that did not receive effector cells). Pairwise comparison of survival is shown. Notably, ID-treated mice had significantly longer survival compared with vehicle-treated mice.

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agonistic anti-CD38 mAb (IB4) results in calcium flux and should be cautiously interpreted as they were determined in increased ERK1/2 activity (22, 27); however, antagonism of CD38 ex vivo assays, whereas the effect of ibrutinib or daratumumab in and its effects on BCR signaling have not been previously humans is remarkably enhanced through engagement and reported. We show for the first time that mAb-based engagement reshaping of the innate and adaptive immune environ- of CD38 (with daratumumab, which minimally inhibits CD38 ment (35, 37). In addition, as CD38 status has not been reported ectoenzymatic function) or small molecule–based targeting of to be a determinant of clinical response to ibrutinib, it is plausible þ CD38 (with kuromanin, which primarily inhibits enzymatic that patients with CD38 and CD38 CLL alike would demon- activity of CD38) results in significant downregulation of prox- strate equivalent response to ibrutinib þ daratumumab combi- imal (Syk and BTK), terminal (PLCg2 and ERK), and collateral nation treatment. (AKT) proteins involved in BCR signaling. Our observation that As daratumumab does not bind murine CD38 (thus precluding CD38 enzymatic inhibition can to a large extent mimic CD38 use of transgenic Eu-TCL1 mice), we established a disseminated receptorial block in terms of apoptosis induction and BCR sig- disease xenograft model to study the anti-CLL activity profile of þ naling attenuation (mostly in CD38 /CD38hi CLL cells) opens daratumumab ( ibrutinib). In this short-course study, the goal avenues of investigation for the use of highly specific small- was to measure time to tumor growth before onset of xeno-GVHD molecule inhibitors of CD38 such as 78c, reported by Tarrago could impact the results. We noted significant activity of daratu- and colleagues (31). Moreover, highly specific inhibition of CD38 mumab relative to mice treated with effector cells only (serving as (receptor and NADase activity) may also be able to shift the T-cell a control for all mice groups that received drug þ effectors). repertoire from a pro- to antitumor disposition as eloquently Survival analysis was only performed on mice that received drug shown by Chatterjee and colleagues using CD38 knockout without effector cells. Although underpowered, this analysis sug- mice (32). gested that the ID combination was superior in prolonging the The effects of individually targeting CD38 and BTK yielded survival of mice compared with vehicle-treated mice (P ¼ 0.013). modest downregulation of BCR components. Thus, we con- Although the median survival of mice treated with single agent sidered whether disrupting CD38 and BTK simultaneously daratumumab or ibrutinib was lower than mice that received could further decrease the aforementioned proteins; translating combination ID treatment, the differences were not statistically to enhanced lethality in CLL cells. As expected, cotargeting of significant. Further experiments that can incorporate the immune- CD38 (with daratumumab/kuromanin) and BTK (ibrutinib) effector activity of daratumumab in an appropriate mouse model significantly reduced most of the phosphorylated BCR-signal- system are needed to comprehensively evaluate the survival ing proteins and was associated with not only increased apo- advantage conferred with use of daratumumab ibrutinib. ptosis and mitochondrial disturbance, but also significantly In summary, our data highlights that: (i) daratumumab greater immune-effector–mediated cell death. In the case of induces cell death in primary CLL cells through various ADCC, this is not entirely surprising as ibrutinib shifts Th1, mechanisms ex vivo (ADCC, CDC, ADCP, and apoptosis); (ii) þ Th2, and CD8 T-cell populations toward an overall antitumor statistically significant correlations between CD38 receptor disposition (33, 34). This immunomodulation in turn poten- density (MFI or sAbc) with ADCC, ADCP, and apoptosis were tially synergizes with the T-cell–modulating properties of dar- observed; (iii) targeting CD38 with either daratumumab or atumumab in both the multiple myeloma (35) and CLL kuromanin can significantly modulate BCR-associated and þ microenvironment (36). Although this may explain enhanced theseeffectsaremoreprominentinCD38 cases, and (iv) the ADCC, it does not explain improved CDC or ADCP. Further combination of daratumumab and ibrutinib induces signifi- studies on complement inhibitor protein expression changes, cantly more immune-effector–mediated and direct apoptosis of þ as well as the effects of BTK CD38 inhibition on macro- CLL cells from CD38 and CD38 patients alike. On the basis phages are being conducted by us under both ex vivo and in vivo of our results, although single-agent daratumumab may be þ conditions. Altogether, the overall antitumor activity of dar- more effective in patients with CLL with CD38 disease, the atumumab is significantly enhanced when partnered with combination of daratumumab and ibrutinib may be highly ibrutinib and this is reflected by increased CLL cytolysis in effective in all treatment requiring patients irrespective of CD38 every underlying assay: ADCC, CDC, ADCP, and apoptosis expression status. Our data provide the framework for future (Supplementary Table S6). clinical investigations entailing therapeutic strategies targeting To more accurately gauge CLL cell sensitivity toward daratu- CD38 and BTK together. mumab, kuromanin, and/or ibrutinib based on CD38 expression/receptor density, we used FACS-purified CD38hi and CD38lo Disclosure of Potential Conflicts of Interest þ þ CD19 /CD5 CLL cells in our workflow. Although results mea- F. Malavasi reports receiving speakers bureau honoraria from Takeda and fi fi suring ADCC, CDC, and ADCP showed a similar trend as seen in Sano , and is a consultant/advisory board member for Takeda, Sano , and Tusk þ þ þ Therapeutics. S. Ailawadhi reports receiving other commercial research support unsorted CD19 /CD5 CLL cells from patients with CD38 and from Pharmacyclics, Inc., and is a consultant/advisory board member for CD38 CLL, the results from the apoptosis assays were more Amgen, Celgene, Takeda, and Janssen. No potential conflicts of interest were hi noteworthy. In CD38 CLL clones, we detected significant apo- disclosed by the other authors. ptosis from ibrutinib alone, which did not increase with the addition of daratumumab. In contrast, albeit lower overall com- Authors' Contributions pared with CD38hi cells, the magnitude of apoptosis induced by Conception and design: A. Manna, S. Aulakh, T. Sher, E.N. Chini, ID therapy was more prominent in CD38lo clones. The signifi- A. Chanan-Khan, S. Ailawadhi, A. Paulus cance of these findings is unclear; however, overall our data Development of methodology: A. Manna, S. Aulakh, S. Akhtar, T. Sher, E.N. Chini, A. Chanan-Khan, A. Paulus suggest that CD38 receptor levels and survival dependency on Acquisition of data (provided animals, acquired and managed patients, BCR signaling are intricately linked and associated with response provided facilities, etc.): A. Manna, S. Aulakh, P. Jani, S. Ahmed, S. Akhtar, to BCR/CD38-targeting agents. These associations, however, Z. Meghji, V. Alegria, A. Paulus

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Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Predolin Foundation (to A. Chanan-Khan), the University of Iowa/Mayo computational analysis): A. Manna, S. Aulakh, M. Coignet, M. Heckman, Z. Clinic Lymphoma SPORE Developmental Research program (P50 Meghji, E.N. Chini, A. Chanan-Khan, S. Ailawadhi, A. Paulus CA097274, to A. Paulus), and the Mayo Clinic Cancer Center (CA015083, Writing, review, and/or revision of the manuscript: A. Manna, S. Aulakh, to A. Chanan-Khan). P. Jani, S. Ahmed, S. Akhtar, M. Coignet, M. Heckman, Z. Meghji, K. Bhatia, We thank Dr. Laura Lewis-Tufﬁn for her support on cell sorting, which A. Sharma, T. Sher, F. Malavasi, E.N. Chini, A. Chanan-Khan, S. Ailawadhi, was performed using a BD FACS Aria II cell sorter in the Mayo Florida A. Paulus Cellular Imaging and Flow Cytometry Shared Resource. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Manna, P. Jani, S. Ahmed, Z. Meghji, K. Bhatia Study supervision: A. Manna, A. Chanan-Khan, A. Paulus The costs of publication of this article were defrayed in part by the Other (analyzed the data, performed critical revision for important payment of page charges. This article must therefore be hereby marked advertisement intellectual content, contributed to writing of the manuscript and in accordance with 18 U.S.C. Section 1734 solely to indicate approved the ﬁnal draft): F. Malavasi this fact.

Acknowledgments The experiments and analysis carried out in this article were supported in Received October 24, 2018; revised January 28, 2019; accepted March 28, part by the Daniel Foundation of Alabama (to A. Chanan-Khan), the 2019; published ﬁrst April 2, 2019.

References 1. Malavasi F, Deaglio S, Funaro A, Ferrero E, Horenstein AL, Ortolan E, et al. shows antitumor Activity and hampers leukemia-microenvironment inter- Evolution and function of the ADP ribosyl cyclase/CD38 gene family in actions in chronic lymphocytic leukemia. Clin Cancer Res 2017;23: physiology and pathology. Physiol Rev 2008;88:841–86. 1493–505. 2. Funaro A, De Monte LB, Dianzani U, Forni M, Malavasi F. Human CD38 is 15. Herman SE, Wiestner A. Preclinical modeling of novel therapeutics in associated to distinct molecules which mediate transmembrane signaling chronic lymphocytic leukemia: the tools of the trade. Semin Oncol 2016; in different lineages. Eur J Immunol 1993;23:2407–11. 43:222–32. 3. Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, et al. Ig V gene 16. Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840–7. chronic lymphocytic leukemia. Blood 1999;94:1840–7. 4. Jelinek DF, Tschumper RC, Geyer SM, Bone ND, Dewald GW, Hanson CA, 17. Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW, Stevenson FK, et al. Analysis of clonal B-cell CD38 and immunoglobulin variable region et al. CD38 expression and immunoglobulin variable region mutations are sequence status in relation to clinical outcome for B-chronic lymphocytic independent prognostic variables in chronic lymphocytic leukemia, but leukaemia. Br J Haematol 2001;115:854–61. CD38 expression may vary during the course of the disease. Blood 2002;99: 5. Morabito F, Mangiola M, Stelitano C, Deaglio S, Callea V, Malavasi F. 1023–9. Peripheral blood CD38 expression predicts time to progression in B-cell 18. Overdijk MB, Verploegen S, Bogels M, van Egmond M, Lammerts van chronic lymphocytic leukemia after first-line therapy with high-dose chlor- Bueren JJ, Mutis T, et al. Antibody-mediated phagocytosis contributes to ambucil. Haematologica 2002;87:217–8. the anti-tumor activity of the therapeutic antibody daratumumab in 6. Durig J, Naschar M, Schmucker U, Renzing-Kohler€ K, Holter€ T, Huttmann€ lymphoma and multiple myeloma. MAbs 2015;7:311–21. A, et al. CD38 expression is an important prognostic marker in chronic 19. Overdijk MB, Jansen JH, Nederend M, Lammerts van Bueren JJ, Groen RW, lymphocytic leukaemia. Leukemia 2002;16:30–5. Parren PW, et al. The therapeutic CD38 monoclonal Antibody daratumu- 7. Costello C. An update on the role of daratumumab in the treatment of mab induces programmed cell death via Fcg receptor-mediated cross- multiple myeloma. Ther Adv Hematol 2017;8:28–37. linking. J Immunol 2016;197:807–13. 8. De Weers M, Tai Y-T, van der Veer MS, Bakker JM, Vink T, Jacobs DC, et al. 20. Kellenberger E, Kuhn I, Schuber F, Muller-Steffner H. Flavonoids as Daratumumab, a novel therapeutic human CD38 monoclonal antibody, inhibitors of human CD38. Bioorg Med Chem Lett 2011;21:3939–42. induces killing of multiple myeloma and other hematological tumors. 21. Vaisitti T, Aydin S, Rossi D, Cottino F, Bergui L, D'Arena G, et al. CD38 J Immunol 2011;186:1840–8. increases CXCL12-mediated signals and homing of chronic lymphocytic 9. Matas-Cespedes A, Vidal-Crespo A, Rodriguez V, Villamor N, Delgado J, leukemia cells. Leukemia 2010;24:958–69. Gine E, et al. The Human CD38 monoclonal antibody daratumumab 22. Vaisitti T, Audrito V, Serra S, Buonincontri R, Sociali G, Mannino E, shows antitumor activity and hampers leukemia–microenvironment inter- et al. The enzymatic activities of CD38 enhance CLL growth and actions in chronic lymphocytic leukemia. Clin Cancer Res 2017;23: trafficking: implications for therapeutic targeting. Leukemia 2015; 1493–505. 29:356–68. 10. Chitta KS, Khan AN, Ersing N, Swaika A, Masood A, Paulus A, et al. Neem 23. Deaglio S, Capobianco A, Bergui L, Durig€ J, Morabito F, Duhrsen€ U, et al. leaf extract induces cell death by apoptosis and autophagy in B-chronic CD38 is a signaling molecule in B-cell chronic lymphocytic leukemia cells. lymphocytic leukemia cells. Leuk Lymphoma 2014;55:652–61. Blood 2003;102:2146–55. 11. Paulus A, Masood A, Miller KC, Khan AN, Akhtar D, Advani P, et al. The 24. Zupo S, Isnardi L, Megna M, Massara R, Malavasi F, Dono M, et al. CD38 investigational agent MLN2238 induces apoptosis and is cytotoxic to CLL expression distinguishes two groups of B-cell chronic lymphocytic leuke- cells in vitro, as a single agent and in combination with other drugs. Br J mias with different responses to anti-IgM antibodies and propensity to Haematol 2014;165:78–88. apoptosis. Blood 1996;88:1365–74. 12. Paulus A, Akhtar S, Caulfield TR, Samuel K, Yousaf H, Bashir Y, 25. Lanham S, Hamblin T, Oscier D, Ibbotson R, Stevenson F, Packham G. et al. Coinhibition of the deubiquitinating enzymes, USP14 and Differential signaling via surface IgM is associated with VH gene mutational UCHL5, with VLX1570 is lethal to ibrutinib- or bortezomib-resistant status and CD38 expression in chronic lymphocytic leukemia. Blood 2003; Waldenstrom macroglobulinemia tumor cells. Blood Cancer J 2016; 101:1087–93. 6:e492. 26. Malavasi F, Deaglio S, Damle R, Cutrona G, Ferrarini M, Chiorazzi N. CD38 13. Paulus A, Akhtar S, Yousaf H, Manna A, Paulus SM, Bashir Y, et al. and chronic lymphocytic leukemia: a decade later. Blood 2011;118: Waldenstrom macroglobulinemia cells devoid of BTKC481S or 3470–8. CXCR4WHIM-like mutations acquire resistance to ibrutinib through upre- 27. Deaglio S, Vaisitti T, Billington R, Bergui L, Genazzani AA, Malavasi F. gulation of Bcl-2 and AKT resulting in vulnerability towards venetoclax or CD38/CD19: a lipid raft–dependent signaling complex in human B cells. MK2206 treatment. Blood Cancer J 2017;7:e565. Blood 2007;109:5390–8. 14. Matas-Cespedes A, Vidal-Crespo A, Rodriguez V, Villamor N, Delgado J, 28. Jeyakumar D, O'Brien S. B cell receptor inhibition as a target for CLL Gine E, et al. The human CD38 monoclonal antibody daratumumab therapy. Best Pract Res Clin Haematol 2016;29:2–14.

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Daratumumab and Ibrutinib in CLL

29. Pavlasova G, Borsky M, Seda V, Cerna K, Osickova J, Doubek M, et al. driving a Th1-selective pressure in T lymphocytes. Blood 2013;122: Ibrutinib inhibits CD20 upregulation on CLL B cells mediated by the 2539–49. CXCR4/SDF-1 axis. Blood 2016;128:1609–13. 34. Yin Q, Sivina M, Robins H, Yusko E, Vignali M, O'Brien S, et al. Ibrutinib 30. van de Donk NW, Janmaat ML, Mutis T, Lammerts van Bueren JJ, Ahmadi T, therapy increases T cell repertoire diversity in patients with chronic lym- Sasser AK, et al. Monoclonal antibodies targeting CD38 in hematological phocytic leukemia. J Immunol 2017;198:1740–7. malignancies and beyond. Immunol Rev 2016;270:95–112. 35. Krejcik J, Casneuf T, Nijhof IS, Verbist B, Bald J, Plesner T, et al. Daratu- 31. Tarrago MG, Chini CCS, Kanamori KS, Warner GM, Caride A, de Oliveira mumab depletes CD38þ immune regulatory cells, promotes T-cell expan- GC, et al. A potent and speciﬁc CD38 inhibitor ameliorates age-related sion, and skews T-cell repertoire in multiple myeloma. Blood 2016;128: Metabolic Dysfunction by reversing tissue NAD(þ) decline. Cell Metab 384–94. 2018;27:1081–95. 36. Manna A, Lewis-Tufﬁn LJ, Ailawadhi S, Chanan-Khan AA, Paulus A. Using 32. Chatterjee S, Daenthanasanmak A, Chakraborty P, Wyatt MW, Dhar P, anti-CD38 immunotherapy to enhance anti-tumor T-cell immunity in þ Selvam SP, et al. CD38-NAD axis regulates immunotherapeutic anti- chronic lymphocytic leukemia (CLL). J Immunol 2018;200:58.17–58.17. tumor T cell response. Cell Metab 2018;27:85–100. 37. Long M, Beckwith K, Do P, Mundy BL, Gordon A, Lehman AM, et al. 33. Dubovsky JA, Beckwith KA, Natarajan G, Woyach JA, Jaglowski S, Ibrutinib treatment improves T cell number and function in CLL patients. Zhong Y, et al. Ibrutinib is an irreversible molecular inhibitor of ITK J Clin Invest 2017;127:3052–64.

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Targeting CD38 Enhances the Antileukemic Activity of Ibrutinib in Chronic Lymphocytic Leukemia

Alak Manna, Sonikpreet Aulakh, Prachi Jani, et al.

Clin Cancer Res 2019;25:3974-3985. Published OnlineFirst April 2, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3412

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2019/04/02/1078-0432.CCR-18-3412.DC1

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