Daratumumab Mitigates B-Cell Receptor Signaling and Enhances Antitumor Activity of Ibrutinib in Chronic Lymphocytic Leukemia (CLL) Cells

Introduction: B-cell receptor (BCR) signaling is a central driver of proliferation in chronic lymphocytic (CLL) cells. BCR engagement leads to recruitment of several intracellular kinases such Lyn, Syk and BTK, that activate ERK, NFkB and induce CLL cell proliferation. While this pathway is effectively targeted with the BTK inhibitor, ibrutinib, various combination treatment strategies that result in greater tumor cell killing are under investigation. On B-cells, the BCR interacts with the CD19 and CD38 cell surface antigens; where the later amplifies intracellular pro-survival signal transmission through the BCR. CD38 is a multifunctional protein, which possess both enzymatic and receptor functions. As such, we hypothesized that concurrent targeting of CD38 and BTK would lead to enhanced mitigation of BCR signaling as well as direct cell death than use of either agent alone.

Materials: CD19⁺ cells were isolated from CLL patients (n=30, collected under an IRB-approved protocol) by magnetic bead sorting. JVM13 (CD38+) and MEC1 (CD38-) cell lines were also used. CD38 expression was measured by flow cytometry with quantification of MFI and surface antibody binding (sAbc). Drug-induced antibody-dependent cellular cytotoxicity (ADCC) in target Calcein AM labeled CLL cells was assessed by co-culturing them with healthy donor PBMCs as effectors (effector: target [E:T] ratio, 50:1) and complement dependent cytotoxicity (CDC) was measured using complement-containing serum from a healthy donor. Antibody dependent cellular phagocytosis (ADCP) was measured by co-culturing calcein AM labeled CLL cells with healthy human macrophages at a 2:1 ratio. Apoptosis by annexin-V/PI dual staining along with mitochondrial outer membrane potential (MOMP) using MitoProbe^TMDilC₁(5) were assessed by flow cytometric analysis. Immunoblotting was performed using antibodies against Lyn, Syk, BTK, PLCγ2, ERK1/2, AKT, Ikβα, NFAT2 and their phosphorylated isoforms along with GAPDH followed by densitometry analysis.

Results: Using a cutoff of 30% (gated CD38 expressing cells), we observed 26.6% (n=8) of CLL pts. to be CD38+ and 73.3% of pts. to be CD38- (n=22). As some pts. deemed CD38- had high CD38 MFI or sAbc, we applied an interquartile range (IQR with 95% confidence interval) for CD38 MFI; re-classifying pts. as CD38^lo (56.75 - 199) vs. CD38^hi (199 - 355). Using this system, specific-lysis of CLL cells from Dara-mediated ADCC was 23.74±2.55% vs. 14.17±1.27, CDC: 15.93±1.68% vs. 12.29±0.88, ADCP: 8.630.96% vs. 9.50±0.97% and apoptosis: 32.42±3.42 vs. 23.65±1.82 in CD38^hi vs. CD38^lo pts., respectively. Apoptosis was accompanied by a significant shift in MOMP [21.65±7.90% (CD38^hi) vs. 32.52±7.42% (CD38^lo), p<0.05]. In CLL cells treated with the combination of Ibr + Dara (ID), ADCC increased to 54.21±4.09% vs. 37.66±2.16; CDC: 34.35±2.47% vs. 28.88±1.51%, ADCP 25.56±2.30% vs. 16.69±1.97%, apoptosis: 61.63±3.14% vs 62.06±3.41% and MOMP: 66.22±3.68% vs 71.13±3.31%: in CD38^hi vs. CD38^lo pts., respectively. Next we examined the effect of Dara +/- Ibr on BCR signaling in CD38^hi and CD38^lo CLL cells. In CD38^hi cells, single agent Dara significantly decreased phosphorylation Lyn, Syk, BTK and PLCγ2 as well as ERK1/2, AKT, Ikβα and NFAT; the ID combination decreasing these further (except Ikβα and NFAT2) (p<0.01). In CD38^lo cells, Dara significantly downregulated phosphorylation of Lyn and PLCγ2, however, insignificant changes were noted in all other proteins. In ID combination treated CD38^lo cells, Lyn, Syk, BTK, PLCγ2 and AKT were further decreased.

Conclusions: Herein, we provide first evidence on the ability of Dara to modulate BCR signaling and associated proteins in primary CLL cells and that this can be further augmented with Ibr. Dara-induced protein changes were more prominent in CD38^hi cells; accompanied by higher % apoptosis vs. that observed in CD38^lo cells, yet the ID combination significantly enhanced mitochondrial-mediated apoptosis in both CD38^hi and CD38^lo cells. This indicates that the combination can potentially overcome lower induction of apoptosis and ADCC in CD38^lo cells treated with single agent Dara or Ibr. Studies on how Dara and the ID combination induce direct programmed cell death (independent of modulating BCR signaling) are underway. Overall, our investigations provide rationale for clinical testing of Ibr combined with Dara in CLL.